Prokaryotes

Essays Biochem, 1997, 32, 101 - 11
Ethylene and cytokinin signalling in plants: the role of two-component systems; Schaller GE; Two-component systems involve the use of histidine kinases and response-regulator proteins, and allow for the perception of a variety of signals . ETR1 is an ethylene receptor in plants, and contains domains with sequence similarity to histidine kinases and response regulators . Ethylene signalling involves a phosphorylation cascade, employing both histidine kinases and serine/threonine kinases . CKI1 is involved in the cytokinin signalling pathway in plants, and contains domains with sequence similarity to histidine kinases and response regulators . The identification of two-component systems in eukaryotes indicates that this is a general signalling mechanism, not limited solely to prokaryotes.

Eur J Biochem, 1998 Jan 15, 251(1-2), 81 - 90
Cloning, purification and characterization of DNA polymerase beta from Xenopus laevis--studies on its potential role in DNA-end joining; Reichenberger S et al.; Double-strand breaks in the DNA of vertebrate cells are joined by mechanisms of non-homologous DNA-end joining (NEJ) . In extracts from Xenopus eggs, NEJ is inhibited by dideoxynucleotides, indicating a possible involvement of DNA polymerase beta (Pol beta) . Since some types of NEJ products were shown to be formed in vitro by prokaryotic DNA polymerases lacking exonuclease activity, we were interested in whether Pol beta alone would be capable of catalyzing NEJ reactions . Therefore we have cloned the full-length cDNA of the Xenopus laevis Pol beta . The cDNA, predicting a highly conserved 39-kDa protein of 334 amino acids, was tagged with six histidine residues at its N-terminus for overexpression in Escherichia coli, purified to near homogeneity, and shown to have the same catalytic properties as the previously cloned rat and human enzymes . Using oligonucleotides as substrates we show that the recombinant Xenopus Pol beta adds single untemplated nucleotides to blunt ends . However, under conditions that permit efficient NEJ in Xenopus egg extracts, Pol beta does not form those types of NEJ products formed by the prokaryotic polymerases indicating that Pol beta alone is not able to mediate the complex NEJ process in vitro . Using substrates with 3' protruding single strands of increasing length (6-16 nucleotides) we show that Pol beta initiates fill-in DNA synthesis on fold-back structures formed by the longest 3' protruding stand . This unusual feature of beta-type polymerases requires that the loop of the fold-back structure consists of at least six bases and the stem be paired by at least 2 bp to facilitate priming of DNA synthesis.

Mol Gen Genet, 1998 Jan, 257(2), 124 - 31
Characterization of a novel small RNA encoded by Dictyostelium discoideum mitochondrial DNA; Pi M et al.; In this study, we analyzed a mitochondrial small (ms) RNA in Dictyostelium discoideum, which is 129 nucleotides long and has a GC content of only 22.5% . In the mitochondrial DNA, a single-copy gene (msr) for the ms RNA was located downstream of the gene for large-subunit rRNA . The location of msr was similar to that of the 5S rRNA gene in prokaryotes and chloroplasts, but clearly different from that in mitochondria of plants, liverwort and the chlorophycean alga Prototheca wikerhamii, in which small-subunit rRNA and 5S rRNA genes are closely linked . THe primary sequence of ms rRNA showed low homology with mitochondrial 5S rRNA from plants, liverwort and the chlorophycean alga, but the proposed secondary structure of ms RNA was similar to that of cytoplasmic 5S rRNA . In addition, ms RNA showed a highly conserved GAAC sequence in the same loop as in common 5S rRNA . However, ms RNA was detected mainly in the mitochondrial 25,000 x g supernatant fraction which was devoid of ribosomes . It is possible that ms RNA is an evolutionary derivative of mitochondrial 5S rRNA.

Am J Hum Genet, 1998 Jan, 62(1), 111 - 21
The GAA triplet-repeat expansion in Friedreich ataxia interferes with transcription and may be associated with an unusual DNA structure; Bidichandani SI et al.; Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative disease is the most common inherited ataxia . The vast majority of patients are homozygous for an abnormal expansion of a polymorphic GAA triplet repeat in the first intron of the X25 gene, which encodes a mitochondrial protein, frataxin . Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulation, as observed in yeast cells deficient for a frataxin homologue . Using RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA . The mechanism(s) by which the intronic GAA triplet expansion results in this reduction of X25 mRNA is presently unknown . No evidence was found for abnormal splicing of the expanded intron 1 . Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes with in vitro transcription in a length-dependent manner, with both prokaryotic and eukaryotic enzymes . This interference was most pronounced in the physiological orientation of transcription, when synthesis of the GAA-rich transcript was attempted . These results are consistent with the observed negative correlation between triplet-repeat length and the age at onset of disease . Using in vitro chemical probing strategies, we also show that the GAA triplet repeat adopts an unusual DNA structure, demonstrated by hyperreactivity to osmium tetroxide, hydroxylamine, and diethyl pyrocarbonate . These results raise the possibility that the GAA triplet-repeat expansion may result in an unusual yet stable DNA structure that interferes with transcription, ultimately leading to a cellular deficiency of frataxin.

Eur J Biochem, 1998 Feb 1, 251(3), 830 - 8
Mutation of the distal arginine in Coprinus cinereus peroxidase--structural implications; Neri F et al.; Heme peroxidases of prokaryotic, plant and fungal origin share the essential His and Arg catalytic residues of the distal cavity and a proximal His bound to heme iron . Spectroscopic techniques, in contrast to X-ray crystallography, are well suited to detect the precise structure, spin and coordination states of the heme as influenced by its near environment . Resonance Raman and electronic absorption spectra obtained at various pH values for Fe3+ and Fe2+ forms of distal Arg51 mutants of the fungal Coprinus cinereus peroxidase are reported, together with the fluoride adducts at pH 5.0 . This basic catalytic residue has been replaced by the aliphatic residue Leu, the polar residues Asn and Gln and the basic residue Lys (Arg51-->Leu, Asn, Gln, and Lys, respectively) . These mutations cause changes in the coordination and spin states of the heme iron, and in the v(Fe-Im) stretching frequency . The variations are explained in terms of pH-dependent changes, charge location, size and hydrogen-bonding acceptor/donor properties of the residue at position 51 . The present work indicates that the hydrogen-bond capability of the residue in position 51 influences the occupancy of water molecules in the distal cavity and the ability to form stable complexes between anionic ligands and the heme Fe atom.

Eur J Biochem, 1998 Feb 1, 251(3), 821 - 9
Stop-transfer function of pseudo-random amino acid segments during translocation across prokaryotic and eukaryotic membranes; Saaf A et al.; We have measured the efficiency of stop-transfer function for a set of pseudo-random, 18-residue amino acid segments, both in Escherichia coli and in mammalian microsomes . In general, stop-transfer function correlates well with the mean hydrophobicity of the segment, though exceptions exist . Kinetic studies suggest that polar segments are rapidly translocated through the E . coli inner membrane and that strongly hydrophobic segments become permanently anchored, while sequences with an intermediate mean hydrophobicity become partly trapped in a transmembrane disposition for a considerable time before being released to the periplasm or degraded.

FEBS Lett, 1998 Jan 30, 422(2), 137 - 40
Large-scale preparation of biologically active recombinant ovine obese protein (leptin); Gertler A et al.; Prokaryotic expression vector pMON3401 encoding full size A(-1) ovine leptin was prepared by polymerase chain reaction (PCR) of previously described cDNA . E . coli cells transformed with this vector overexpressed large amounts of ovine leptin upon induction with nalidixic acid . The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (leptin-Q and leptin-SP), composed respectively of 90 and 95% of monomeric protein of the expected molecular mass of 16 kDa . The purified protein was capable of interacting with antibodies raised against (GST-ovine leptin and to bind specifically to ventromedial hypothalamus of ewes . The biological activity of both fractions resulting from proper renaturation was further evidenced by their ability to stimulate DNA synthesis in leptin-sensitive BAF/3 cells transfected with a long form of human leptin receptor construct.

Biochemistry, 1998 Feb 3, 37(5), 1221 - 6
Functional evaluation of invariant arginines situated in the mobile lid domain of phosphoribulokinase; Runquist JA et al.; Rhodobacter sphaeroides phosphoribulokinase contains four invariant arginines (R49, R168, R173, and R187) . The high-resolution structure of this enzyme {Harrison, D . H . T., Runquist, J . A., Holub, A., and Miziorko, H . M . (1998) Biochemistry (submitted for publication)} reveals that it folds in a manner similar to that of adenylate kinase . Three invariant arginines (R168, R173, and R187) as well as arginine-186, which is conserved in prokaryotic phosphoribulokinases, have not been previously functionally evaluated . These arginine residues map within the mobile lid domain that is a distinctive feature of the adenylate kinase family of proteins . Precedent for the significant function of arginines in phosphotransferase reactions prompted substitution of glutamine for each of these three invariant arginines . Solution state characterization of the isolated mutant proteins indicated that they retained a high degree of structural integrity, as indicated by their stoichiometric binding of an alternative nucleotide substrate (trinitrophenyl-ATP) as well as the allosteric effector (NADH) . Kinetic characterization indicated > 10(4)-fold diminution in V/KRu5P for R168Q, attributable to a > 300-fold decrease in catalytic efficiency and an increase (approximately 50-fold) in Km Ru5P . For R173Q, a 15-fold diminution in Vmax and a 100-fold increase in Km Ru5P were observed . These observations implicate new components of the ribulose 5-phosphate binding site . Additionally, they confirm assignment of the mobile lid domain as part of the phosphoribulokinase active site, even though this region is well separated from other active site elements in the structure of the open form of the protein . Characterization of R186Q and R187Q mutants suggests that they influence the cooperativity of substrate binding.

J Mol Evol, 1998 Feb, 46(2), 188 - 201
Rapid diversification of marine picophytoplankton with dissimilar light-harvesting structures inferred from sequences of Prochlorococcus and Synechococcus (Cyanobacteria); Urbach E et al.; Cultured isolates of the unicellular planktonic cyanobacteria Prochlorococcus and marine Synechococcus belong to a single marine picophytoplankton clade . Within this clade, two deeply branching lineages of Prochlorococcus, two lineages of marine A Synechococcus and one lineage of marine B Synechococcus exhibit closely spaced divergence points with low bootstrap support . This pattern is consistent with a near-simultaneous diversification of marine lineages with divinyl chlorophyll b and phycobilisomes as photosynthetic antennae . Inferences from 16S ribosomal RNA sequences including data for 18 marine picophytoplankton clade members were congruent with results of psbB and petB and D sequence analyses focusing on five strains of Prochlorococcus and one strain of marine A Synechococcus . Third codon position and intergenic region nucleotide frequencies vary widely among members of the marine picophytoplankton group, suggesting that substitution biases differ among the lineages . Nonetheless, standard phylogenetic methods and newer algorithms insensitive to such biases did not recover different branching patterns within the group, and failed to cluster Prochlorococcus with chloroplasts or other chlorophyll b-containing prokaryotes . Prochlorococcus isolated from surface waters of stratified, oligotrophic ocean provinces predominate in a lineage exhibiting low G + C nucleotide frequencies at highly variable positions.

J Biol Chem, 1998 Feb 6, 273(6), 3574 - 81
The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro; Evdokimova VM et al.; The major core protein of cytoplasmic messenger ribonucleoprotein particles (p50) has been shown previously to inhibit protein synthesis in vitro and in vivo . Furthermore, p50 is highly homologous to the Y-box-binding transcription factor family of proteins, binds DNA containing the Y-box motif, and thus may have a dual function in cells as a regulator of both transcription and translation . Here we show that binding or removal of p50 from rabbit reticulocyte lysate by monospecific antibodies to p50 strongly inhibits translation of endogenous and exogenous globin mRNAs as well as prokaryotic beta-galactosidase mRNA in a rabbit reticulocyte cell-free system . Thus, depending on the conditions, p50 not only may act as a translational repressor, but may also be required for protein synthesis . Translation inhibition with anti-p50 antibodies is not a result of mRNA degradation or its functional inactivation . The inhibition does not change the ribosome transit time, and therefore, it does not affect elongation/termination of polypeptide chains . The inhibition with anti-p50 antibodies is followed by a decay of polysomes and accumulation of the 48 S preinitiation complex . These results suggest that p50 participates in initiation of protein biosynthesis . Although uninvolved in the formation of the 48 S preinitiation complex, p50 is necessary either for attachment of the 60 S ribosomal subunit or for previous 5'-untranslated region scanning by the 43 S preinitiation complex.

J Mol Evol, 1998 Jan, 46(1), 107 - 14
Toward a better knowledge of the molecular evolution of phosphoenolpyruvate carboxylase by comparison of partial cDNA sequences; Gehrig HH et al.; To get deeper insight into the evolution of phosphoenolpyruvate carboxylase we have identified PEPC fragments (about 1,100 bp) of another 12 plants species not yet investigated in this context . The selected plants include one Chlorophyta, two Bryophyta, four Pteridophyta, and five Spermatophyta species . The obtained phylogenetic trees on PEPC isoforms are the most complete ones up to now available . Independent of their manner of construction, the resulting dendrograms are very similar and fully consistent with the main topology as it is postulated for the evolution of the higher terrestrial plants . We found a distinct clustering of the PEPC sequences of the prokaryotes, the algae, and the spermatophytes . PEPC isoforms of the archegoniates are located in the phylogenetic trees between the algae and spermatophytes . Our results strengthen the view that the PEPC is a very useful molecular marker with which to visualize phylogenetic trends both on the metabolic and organismic levels.

Trends Biotechnol, 1998 Feb, 16(2), 54 - 60
Strategies for optimizing heterologous protein expression in Escherichia coli; Hannig G et al.; The many advantages of Escherichia coli have ensured that it remains a valuable host for the efficient, cost-effective and high-level production of heterologous proteins . Here, we describe the current status of this prokaryotic expression system and focus on strategies designed to maximize the yields of recombinant proteins . Major challenges facing this expression system are also outlined.

Mol Microbiol, 1998 Jan, 27(2), 311 - 22
Genetic analysis of zwittermicin A resistance in Escherichia coli: effects on membrane potential and RNA polymerase; Stabb EV et al.; Zwittermicin A is a novel aminopolyol antibiotic that represents a new structural class of antibiotic and has diverse biological activities, including the suppression of plant disease and the ability to inhibit prokaryotic and eukaryotic cells . To enhance our fundamental understanding and applications of zwittermicin A, we elucidated mechanisms of zwittermicin A resistance in Escherichia coli . Two classes of zwittermicin A-resistant mutants of E . coli were selected and characterized . One class included mutants altered in hemA, hemB, hemL, ubi, cydAB or atp, which were defective in generating a proton motive force (PMF) and resistant to aminoglycosides . The mutant analysis, coupled with physiological data, indicated an association between the electrical membrane potential (deltapsi) component of PMF and zwittermicin A sensitivity . A second class of zwittermicin A-resistant mutants was aminoglycoside sensitive and was affected in rpoB and rpoC, genes that encode subunits of RNA polymerase . The rpoB and rpoC mutants suggested that zwittermicin A might inhibit transcription, DNA replication, DNA gyrase or topoisomerase I; however, we found no further evidence to support any of these as the target for zwittermicin A . This study elucidated the genetic mechanisms of zwittermicin A resistance in E . coli . The results suggest that deltapsi drives zwittermicin A uptake, and that, unlike other antibiotics for which resistance maps in rpoB or rpoC, zwittermicin A does not cause the rapid cessation of DNA or RNA synthesis, suggesting a unique mechanism of antibiosis.

Mol Microbiol, 1998 Jan, 27(2), 247 - 55
The CspA family in Escherichia coli: multiple gene duplication for stress adaptation; Yamanaka K et al.; CspA was originally found as the major cold-shock protein in Escherichia coli, consisting of 70-amino-acid residues . It forms a beta-barrel structure with five anti-parallel beta-strands and functions as an RNA chaperone . Its dramatic but transient induction upon cold shock is regulated at the level of transcription, mRNA stability and translation . Surprisingly, E . coli contains a large CspA family, consisting of nine genes from cspA to cspI . Phylogenetic analysis of these gene products and the cold-shock domain of human YB-1 protein reveals that there are two major branches in the evolution of CspA homologues: one branch for CspF and CspH, and another for all the other known CspA homologues from both prokaryotes and eukaryotes . The locations of these genes on the E . coli chromosome suggest that the large CspA family probably resulted from a number of gene duplications and, after subsequent adaptation, resulted in specific groups of genes that respond to different environmental stresses; for example, cspA, cspB and cspG for cold-shock stress and cspD for nutritional deprivation . The E . coli CspA family will be discussed in terms of their structures and functions, and their gene structures and regulation.

Biochemistry, 1998 Feb 10, 37(6), 1478 - 84
Role of arginine 439 in substrate binding of 5-aminolevulinate synthase; Tan D et al.; 5-Aminolevulinate synthase (EC 2.3.1.37) catalyzes the first reaction in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes . Homology sequence modeling between 5-aminolevulinate synthase and some other alpha-family pyridoxal 5'-phosphate-dependent enzymes indicated that the residue corresponding to the Arg-439 of murine erythroid 5-aminolevulinate synthase is a conserved residue in this family of pyridoxal 5'-phosphate-dependent enzymes . Further, this conserved arginine residue in several enzymes, e.g., aspartate aminotransferase, for which the three-dimensional structure is known, has been shown to interact with the substrate carboxyl group . To test whether Arg-439 is involved in substrate binding in murine erythroid 5-aminolevulinate synthase, Arg-439 and Arg-433 of murine erythroid 5-aminolevulinate synthase were each replaced by Lys and Leu using site-directed mutagenesis . The R439K mutant retained 77% of the wild-type activity; its K(m) values for both substrates increased 9-13-fold, while the activity of R433K increased 2-fold and the K(m) values for both substrates remained unchanged . R439L had no measurable activity as determined using a standard 5-aminolevulinate synthase enzyme-coupled activity assay . In contrast, the kinetic parameters for R433L were comparable to those of the wild-type . Dissociation constants (Kd) for glycine increased 5-fold for R439K and at least 30-fold for R439L, while Kd values for glycine for both R433K and R433L mutants were similar to those of the wild-type . However, there was not much difference in methylamine binding among the mutants and the wild-type, excepting of a 10-fold increase in K(d)methylamine for R439L . R439K proved much less thermostable than the wild-type enzyme, with the thermotransition temperature, T1/2, determined to be 8.3 degrees C lower than that of the wild-type enzyme . In addition, in vivo complementation analysis demonstrated that in the active site of murine erythroid 5-aminolevulinate synthase, R439 is contributed from the same subunit as K313 (which is involved in the Schiff base linkage of the pyridoxal 5'-phosphate cofactor) and D279 (which interacts electrostatically with the ring nitrogen of the cofactor), while another subunit provides R149 . Taken together, these findings suggest that Arg-439 plays an important role in substrate binding of murine erythroid 5-aminolevulinate synthase.

Tsitol Genet, 1997 Sep-Oct, 31(5), 79 - 87
{Experimental and calculated spectra of the amplicons UBC-85 and UBC-126 (RAPD-PCR)}; Glazko GV et al.; The comparative analysis of experimental amplification spectrum in 13 Ungulata species and counting ones in DNA sequences of different taxa in GenBank (mammalian, other vertebrate, invertebrate, viruses, prokaryote) with the uses of RAPD-PCR primers UBC-85 and UBC-126 was carried out . The particularities of the distribution of amplicons' frequencies in experimental and counting spectrums were revealed, for some of them the similar increased frequencies in mammalian and prokaryotic species were observed.

J Biol Chem, 1998 Jan 30, 273(5), 3051 - 9
Mechanistic studies on the impact of transcription on sequence-specific termination of DNA replication and vice versa; Mohanty BK et al.; Since DNA replication and transcription often temporally and spatially overlap each other, the impact of one process on the other is of considerable interest . We have reported previously that transcription is impeded at the replication termini of Escherichia coli and Bacillus subtilis in a polar mode and that, when transcription is allowed to invade a replication terminus from the permissive direction, arrest of replication fork at the terminus is abrogated . In the present report, we have addressed four significant questions pertaining to the mechanism of transcription impedance by the replication terminator proteins . Is transcription arrested at the replication terminus or does RNA polymerase dissociate from the DNA causing authentic transcription termination? How does transcription cause abrogation of replication fork arrest at the terminus? Are the points of arrest of the replication fork and transcription the same or are these different? Are eukaryotic RNA polymerases also arrested at prokaryotic replication termini? Our results show that replication terminator proteins of E . coli and B . subtilis arrest but do not terminate transcription . Passage of an RNA transcript through the replication terminus causes the dissociation of the terminator protein from the terminus DNA, thus causing abrogation of replication fork arrest . DNA and RNA chain elongation are arrested at different locations on the terminator sites . Finally, although bacterial replication terminator proteins blocked yeast RNA polymerases in a polar fashion, a yeast transcription terminator protein (Reb1p) was unable to block T7 RNA polymerase and E . coli DnaB helicase.

J Biol Chem, 1998 Jan 30, 273(5), 2905 - 9
The novel fibronectin-binding motif and key residues of mycobacteria; Naito M et al.; The binding motifs of the immunodominant antigen (Ag) alpha-Ag (Ag 85 complex B) of Mycobacterium kansasii for human fibronectin were examined using digested fragments . We defined two fibronectin-binding epitopes on 27 amino acids from 84 to 110 and on 20 amino acids from 211 to 230 . The epitopes were almost conserved in the closely related Ag 85 complex of other mycobacteria species . Inhibition of fibronectin binding to intact alpha-Ag molecules was observed with peptide-(84-110), but not with peptide-(211-230) . Peptide-(84-110) could also inhibit fibronectin binding to all components of the Ag 85 complex of Bacillus Calmette-Guerin (Ag 85A, Ag 85B, and Ag 85C) . Further study with synthetic peptides defined 11 residues from 98 to 108 as the minimum motif . Six residues (98FEWYYQ103) were critical for interacting with fibronectin . The motif revealed no homology to other known prokaryotic and eukaryotic fibronectin-binding proteins . The defined motif of alpha-Ag is novel and unique for mycobacteria.

EMBO J, 1998 Jan 15, 17(2), 462 - 9
Dynamic assembly of FtsZ regulated by GTP hydrolysis; Mukherjee A et al.; FtsZ forms a cytokinetic ring, designated the Z ring, that directs cytokinesis in prokaryotes . It has limited sequence similarity to eukaryotic tubulins and, like tubulin, it has GTPase activity and the ability to assemble into various structures including protofilaments, bundles and minirings . By using both electron microscopy and sedimentation, we demonstrate that FtsZ from Escherichia coli undergoes a strictly GTP-dependent polymerization and the polymers disappear as the GTP is consumed . Thus, FtsZ polymerization, like that of tubulin, is dynamic and regulated by GTP hydrolysis . These results provide the basis for the dynamics of the Z ring and favor a model in which the Z ring is formed by a nucleation event.

Nucleic Acids Res, 1998 Jan 15, 26(2), 427 - 30
Evidence for an early prokaryotic origin of histones H2A and H4 prior to the emergence of eukaryotes; Slesarev AI et al.; Histones have been identified recently in many prokaryotes . These histones, unlike their eukaryotic homologs, are of a single uniform type that is thought to resemble the archetypal ancestor of the eukaryotic histone family . In this paper we report the finding, the cloning and the phylogenetic analysis of the sequence of a prokaryotic histone from the hyperthermophile Methanopyrus kandleri . Unlike previously described prokaryotic histones, the Methanopyrus sequence has a novel structure consisting of two tandemly repeated histone fold motifs in a single polypeptide . Sequence analyses indicate that the N-terminal repeat is most closely related to eukaryotic H2A and H4 histones, whereas the C-terminal repeat resembles that found in prokaryotic histones . These results imply an early divergence within the histone gene family prior to the emergence of eukaryotes and may represent an evolutionary step leading to eukaryotic histones.

J Theor Biol, 1997 Nov 21, 189(2), 195 - 209
Mechanistic modeling of prokaryotic mRNA decay; Carrier TA et al.; A mechanistic model of gene expression was developed to test three prevailing and sliding prokaryotic mRNA decay theories: ribosome protection of mRNA from endonucleases, 5' binding and sliding of endonucleases on mRNA, and hybrid 5' binding/ribosome protection . The discrete event simulation incorporates the molecular events that determine both cellular mRNA and protein levels . A Monte Carlo technique was used to approximate the inherent randomness of the molecular processes involved in gene expression . Each of the decay theories was tested for the ability to predict the effects of ribosome loading and translation rate on mRNA stability as well as the observed 5' to 3' directionality of mRNA decay . The modeling results show that the hybrid decay mechanism best predicts the experimentally-observed mRNA decay behaviors . The 5' binding mechanism fails to adequately predict the sensitivity of mRNA stability to changes in translation rate and ribosome loading, while the ribosome protection mechanism does not correctly predict 5' to 3' decay directionality . In addition to discriminating between the three decay theories, the simulations provide insights into hybrid decay mechanism specific details such as RNase binding and cleavage characteristics . Finally, we discuss the application of the current mechanistic model for analysing and predicting expression from more complex genetic systems .

Genes Dev, 1998 Jan 1, 12(1), 67 - 83
A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initiation of hepatitis C and classical swine fever virus RNAs; Pestova TV et al.; Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry . We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing . Ribosomal subunits (40S) formed stable binary complexes on both mRNAs . The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal "P" site in binary complexes . Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant . Addition of Met-tRNAiMet, eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes . The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed.

Proc Natl Acad Sci U S A, 1998 Jan 6, 95(1), 108 - 13
5' end maturation and RNA editing have to precede tRNA 3' processing in plant mitochondria; Kunzmann A et al.; We report the characterization and partial purification of potato mitochondrial RNase Z, an endonuclease that generates mature tRNA 3' ends . The enzyme consists of one (or more) protein(s) without RNA subunits . Products of the processing reaction are tRNA molecules with 3' terminal hydroxyl groups and 3' trailers with 5' terminal phosphates . The main processing sites are located immediately 3' to the discriminator and one nucleotide further downstream . This endonucleolytic processing at and close to the tRNA 3' end in potato mitochondria suggests a higher similarity to the eukaryotic than to the prokaryotic tRNA 3' processing pathway . Partial purification and separation of RNase Z from the 5' processing activity RNase P allowed us to determine biochemical characteristics of the enzyme . The activity is stable over broad pH and temperature ranges, with peak activity at pH 8 and 30 degrees C . Optimal concentrations for MgCl2 and KCl are 5 mM and 30 mM, respectively . The potato mitochondrial RNase Z accepts only tRNA precursors with mature 5' ends . The precursor for tRNAPhe requires RNA editing for efficient processing by RNase Z.

Nucleic Acids Res, 1998 Jan 1, 26(1), 360 - 2
OOTFD (Object-Oriented Transcription Factors Database): an object-oriented successor to TFD; Ghosh D; ooTFD (object-oriented Transcription Factors Database) is a successor to TFD (Transcription Factors Database) . ooTFD contains information represented in TFD but also allows the representation of containment, composite, and interaction relationships between transcription factor polypeptides . ooTFD is designed to represent information about all transcription factors, both eukaryotic and prokaryotic, basal as well as regulatory factors, and multiprotein complexes as well as monomers . ooTFD and associated tools and services can be accessed at http://www.isbi.net/

Nucleic Acids Res, 1998 Jan 1, 26(1), 160 - 2
Small RNA database; Gu J et al.; The small RNA database is a compilation of all the small size RNA sequences available to date, including nuclear, nucleolar, cytoplasmic and mitochondria small RNAs from eukaryotic organisms and small RNAs from prokaryotic cells as well as viruses . Currently, approximately 600 small RNA sequences are in our database . It also gives the sources of individual RNAs and their GenBank accession numbers . The small RNA database can be accessed through the WWW (World Wide Web) . Our WWW URL address is: edu/smallRNA/smallrna.html . The new small RNA sequences published since our last compilation are listed in this paper (Table 1).

Curr Opin Genet Dev, 1997 Dec, 7(6), 757 - 63
Prokaryotic genomes: the emerging paradigm of genome-based microbiology; Koonin EV et al.; Comparative analysis of the complete sequences of seven bacterial and three archaeal genomes leads to the first generalizations of emerging genome-based microbiology . Protein sequences are, generally, highly conserved, with -70% of the gene products in bacteria and archaea containing ancient conserved regions . In contrast, there is little conservation of genome organization, except for a few essential operons . The most striking conclusions derived by comparison of multiple genomes from phylogenetically distant species are that the number of universally conserved gene families is very small and that multiple events of horizontal gene transfer and genome fusion are major forces in evolution.

Biol Cell, 1997 Aug, 89(5-6), 321 - 9
The aquaporin-Z water channel gene of Escherichia coli: structure, organization and phylogeny; Calamita G et al.; Aquaporin water channel proteins are found throughout the plant and animal kingdoms, but the first prokaryotic water channel gene, aqpZ, was only recently identified in wild type Escherichia coli (Calamita G et al (1995) J Biol Chem 270, 29063-29066) . Here we define the organization of aqpZ in E coli, produce the AqpZ protein and compare the AqpZ phylogeny to that of some known bacterial homologs . Physical mapping and sequence analyses confirmed the location of aqpZ at minute 19.7 on the E coli chromosome where it is transcribed counterclockwise . The monocistronic nature of aqpZ was clearly indicated by the structural organization of its surrounding genes, ybjD and ybjE' and by the presence of a typical Rho-independent transcriptional terminator following the aqpZ stop codon . Computer sequence analysis indicated the -35/-10 region located 72 bases upstream of the aqpZ start codon as the most likely aqpZ promoter . A series of potential cis-regulatory elements were found in the 400 bp region preceding the aqpZ ORF . The AqpZ protein, produced under T7 phi 10 control, showed a size of about 20 kDa by SDS-PAGE . Striking similarities were found between the E coli aqpZ and a gene included in the genome of the cyanobacterium Synechocystis sp PCC6803, a species permanently living a fresh water . These results may represent a fundamental step to characterize the regulation and the physiological features of the AqpZ water channel in prokaryotes.

J Mol Biol, 1998 Jan 30, 275(4), 635 - 50
Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C; Heinz DW et al.; Phosphoinositide-specific phospholipases C (PI-PLCs) are ubiquitous enzymes that catalyse the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol (DAG) . Whereas the eukaryotic PI-PLCs play a central role in most signal transduction cascades by producing two second messengers, inositol-1,4,5-trisphosphate and DAG, prokaryotic PI-PLCs are of interest because they act as virulence factors in some pathogenic bacteria . Bacterial PI-PLCs consist of a single domain of 30 to 35 kDa, while the much larger eukaryotic enzymes (85 to 150 kDa) are organized in several distinct domains . The catalytic domain of eukaryotic PI-PLCs is assembled from two highly conserved polypeptide stretches, called regions X and Y, that are separated by a divergent linker sequence . There is only marginal sequence similarity between the catalytic domain of eukaryotic and prokaryotic PI-PLCs . Recently the crystal structures of a bacterial and a eukaryotic PI-PLC have been determined, both in complexes with substrate analogues thus enabling a comparison of these enzymes in structural and mechanistic terms . Eukaryotic and prokaryotic PI-PLCs contain a distorted (beta alpha)8-barrel as a structural motif with a surprisingly large structural similarity for the first half of the (beta alpha)8-barrel and a much weaker similarity for the second half . The higher degree of structure conservation in the first half of the barrel correlates with the presence of all catalytic residues, in particular two catalytic histidine residues, in this portion of the enzyme . The second half contributes mainly to the features of the substrate binding pocket that result in the distinct substrate preferences exhibited by the prokaryotic and eukaryotic enzymes . A striking difference between the enzymes is the utilization of a catalytic calcium ion that electrostatically stabilizes the transition state in eukaryotic enzymes, whereas this role is filled by an analogously positioned arginine in bacterial PI-PLCs . The catalytic domains of all PI-PLCs may share not only a common fold but also a similar catalytic mechanism utilizing general base/acid catalysis . The conservation of the topology and parts of the active site suggests a divergent evolution from a common ancestral protein.

Biochimie, 1997 Oct, 79(9-10), 559 - 66
Repair of oxidized DNA bases in the yeast Saccharomyces cerevisiae; Girard PM et al.; An essential requirement for all organisms is to maintain its genomic integrity . Failure to do so, in multicellular organisms such as man, can lead to degenerative pathologies such as cancer and aging . Indeed, a very low spontaneous mutation rate is observed in eukaryotes, suggesting either an inherent stability of the genome or efficient DNA repair mechanisms . In fact, DNA is subjected to unceasing attacks by a variety of endogenous and environmental reactive chemical species yielding a multiplicity of DNA damage, the deleterious action of which is counteracted by efficient repair enzymes . Reactive oxygen species formed in cell as by-products of normal metabolism are probably the major source of endogenous DNA damage . Amongst oxidative damage, base modifications constitute an important class of lesions whose lethal or mutagenic action has been established . Oxidatively damaged DNA bases are mostly repaired by the base excision repair pathway (BER) in prokaryotes and eukaryotes . However, the nucleotide excision repair pathway (NER) may also play a role in the repair of some oxidized bases in DNA . Here, we describe repair pathways implicated in the removal of oxidized bases in Saccharomyces cerevisiae . Yeast is a simple organism that can be used as a paradigm for DNA repair in all eukaryotic cells . S cerevisiae possesses three DNA glycosylases that catalyze the excision of oxidized bases from damaged DNA: the Ogg1, Ntg1 and Ntg2 proteins . The aim of this review is to summarize recent findings dealing with the formation, the biological consequences and the repair of oxidized DNA bases in S cerevisiae.

Appl Environ Microbiol, 1998 Feb, 64(2), 385 - 91
Cloning and targeted disruption of MLG1, a gene encoding two of three extracellular mixed-linked glucanases of Cochliobolus carbonum; Gorlach JM et al.; Mixed-linked glucanases (MLGases), which are extracellular enzymes able to hydrolyze beta 1,3-1,4-glucans (also known as mixed-linked glucans or cereal beta-glucans), were identified in culture filtrates of the plant-pathogenic fungus Cochliobolus carbonum . Three peaks of MLGase activity, designated Mlg1a, Mlg1b, and Mlg2, were resolved by cation-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC) . Mlg1a and Mlg1b also hydrolyze beta 1,3-glucan (laminarin), whereas Mlg2 does not degrade beta 1,3-glucan but does degrade beta 1,4-glucan to a slight extent . Mlg1a, Mlg1b, and Mlg2 have monomer molecular masses of 33.5, 31, and 29.5 kDa, respectively . The N-terminal amino acid sequences of Mlg1a and Mlg1b are identical (AAYNLI) . Mlg1a is glycosylated, whereas Mlg1b is not . The gene encoding Mlg1b, MLG1, was isolated by using PCR primers based on amino acid sequences of Mlg1b . The product of MLG1 has no close similarity to any known protein but does contain a motif (EIDI) that occurs at the active site of MLGases from several prokaryotes . An internal fragment of MLG1 was used to create mlg1 mutants by transformation-mediated gene disruption . The total MLGase and beta 1,3-glucanase activities in culture filtrates of the mutants were reduced by approximately 50 and 40%, respectively . When analyzed by cation-exchange HPLC, the mutants were missing the two peaks of MLGase activity corresponding to Mlg1a and Mlg1b . Together, the data indicate that Mlg1a and Mlg1b are products of the same gene, MLG1 . The growth of mlg1 mutants in culture medium supplemented with macerated maize cell walls or maize bran and the disease symptoms on maize were identical to the growth and disease symptoms of the wild type.

J Mol Evol, 1998 Feb, 46(2), 147 - 54
Microbial relatives of seed storage proteins: conservation of motifs in a functionally diverse superfamily of enzymes
Dunwell JM, Gane PJ.
Plant storage proteins comprise a major part of the human diet . Sequence analysis has revealed that these proteins probably share a common ancestor with a fungal oxalate decarboxylase and/or related bacterial genes . Additionally, all these proteins share a central core sequence with several other functionally diverse enzymes and binding proteins, many of which are associated with synthesis of the extracellular matrix during sporulation/encystment . A possible prokaryotic relative of this sequence is a bacterial protein (SASP) known to bind to DNA and thereby protect spores from extreme environmental conditions . This ability to maintain cell viability during periods of dehydration in spores and seeds may relate to absolute conservation of residues involved in structure determination.

FEBS Lett, 1998 Jan 2, 421(1), 23 - 6
Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII; Frank P et al.; We cloned the Saccharomyces cerevisiae homologue of mammalian RNase HI, which itself is related to the prokaryotic RNase HII, an enzyme of unknown function and previously described as having minor activity in Escherichia coli . Expression of the corresponding yeast 35 kDa protein (named by us RNase H(35)) in E . coli and immunological analysis proves a close evolutionary relationship to mammalian RNase HI . Deletion of the gene (called RNH35) from the yeast genome leads to an about 75% decrease of RNase H activity in preparations from the mutated, still viable cells . Sequence comparison discriminates this new yeast RNase H from earlier described yeast enzymes, RNase H(70) and RNase HI.

Gene, 1997 Dec 31, 205(1-2), 141 - 4
The correlation between introns and the three-dimensional structure of proteins; de Souza SJ et al.; We test the hypothesis that introns were used to construct the first genes from small exons, whose protein products represent compact elements of structure . For any three-dimensional structure, a computer program analyzes the structure into a set of modules, segments of the polypeptide chain bounded in space by a maximum diameter, separated by a set of 'boundary regions' . The 'boundary regions' are such that if the gene were divided by an intron in each 'boundary region', the protein would be divided into modules less than the specified diameter . Using a set of 32 ancient proteins, which have no introns in prokaryotes, we examine the intron positions in their eukaryotic homologs and show that the introns are correlated with modules of diameter 21, 28 and 33 A, with P values below 0.001.

J Biol Chem, 1998 Jan 9, 273(2), 689 - 92
Characterization of a cDNA encoding the thylakoidal processing peptidase from Arabidopsis thaliana . Implications for the origin and catalytic mechanism of the enzyme; Chaal BK et al.; We have identified and sequenced a cDNA containing a complete open reading frame for a putative 340-amino acid precursor of the thylakoidal processing peptidase from Arabidopsis thaliana . The predicted amino acid sequence of the protein includes regions highly conserved among Type I leader peptidases and indicates that the enzyme uses a serine-lysine catalytic dyad mechanism . Phylogenetic analysis indicated a common ancestry of the enzyme with those from oxygenic photosynthetic prokaryotes, suggesting that the cDNA encoded the chloroplast enzyme . The catalytic domain was overexpressed in Escherichia coli, generating a product capable of cleaving the thylakoid-transfer domain from a chloroplast protein . Antibodies to the overexpressed polypeptide cross-reacted with a 30-kDa thylakoid membrane protein.

J Photochem Photobiol B, 1997 Sep, 40(2), 141 - 8
Porphyrin sensitization and intracellular calcium changes in the prokaryote Propionibacterium acnes; Ramstad S et al.; Photosensitization induces intracellular free calcium changes ({Ca2+}i) in some eukaryotic cell systems which either contribute to or protect against cell inactivation . We have investigated whether or not similar changes can be induced in prokaryotes . The skin bacterium Propionibacterium acnes was sensitized using protoporphyrin IX (PP IX) or 5-aminolevulinic acid (ALA) . Exogenous ALA resulted in either a preferential accumulation of protoporphyrin (ALA-PP) or of coproporphyrin and/or uroporphyrin (ALA-CP/UP) in P . acnes . For PP IX or ALA-PP sensitization, exposure to broad-band red light resulted in an increase in {Ca2+}i . For ALA-PP sensitization, this increase was transient and {Ca2+}i returned to basal levels within 5-10 min after irradiation . However, the elevated {Ca2+}i levels obtained after PP IX sensitization were maintained for at least 1 h after irradiation . In both cases, the reduction in the external calcium concentration led to an enhancement in the cell survival, indicating that induced {Ca2+}i changes may participate in photoinactivation . Sensitization by hydrophilic coproporphyrin and/or uroporphyrin (ALA-CP/UP) did not affect the {Ca2+}i levels, but higher levels of cell inactivation were obtained . It therefore appears that damage to membrane-associated components is at least partly responsible for {Ca2+}i alterations after photosensitization.

Helicobacter, 1997 Sep, 2(3), 127 - 31
Rapid polymerase chain reaction screening of Helicobacter pylori chromosomal point mutations; Ge Z et al.; BACKGROUND: Microdiversity (within individual genes) in the genomes of different Helicobacter pylori strains has been demonstrated to be more frequent than that seen in other prokaryotes . Point mutations in some genes, such as the vacA and 23S ribosomal RNA genes could result in the alteration of pathogenicity or antibiotic susceptibility of individual H . pylori strains . Development of a simple, rapid, and reliable screening method would be useful in the molecular characterization of genetic variation among different H . pylori strains . MATERIALS AND METHODS: The copP gene from H . pylori UA802 was used as a model for developing a mutation screening method . Four point mutations were introduced into the copP gene by in vitro site-directed mutagenesis and were verified by DNA sequencing . The mutated copP gene replaced the wild-type locus by natural transformation and homologous recombination . The site-specific mutants were screened by polymerase chain reaction (PCR) using 3'-end mismatched primers . The origins of the PCR fragments were demonstrated by Southern hybridization with the copP-derived DNA probe . RESULTS: Three of these four mutations were characterized by PCR with the specific primers that contained the 3'-terminal nucleotide complementary only to the mutated nucleotide on both plasmid and chromosomal DNA templates . One mutation was able to be identified with the foregoing primer containing an additional wild-type nucleotide at its 3'-end . Point mutant screening with these specific primers offers 100% sensitivity in the aforementioned conditions . To achieve optimal screening, the concentration of magnesium and the annealing temperature have to be adjusted . CONCLUSION: The procedure reported in this study is a simple, economical, rapid, and efficient approach in the identification of site-specific mutations on both plasmids and chromosomal DNA . Although the method was developed by using a specified H . pylori gene, it can be extended easily to other genes of interest in H . pylori or other organisms.

Methods, 1997 Oct, 13(2), 103 - 11
Targeting the secretory pathway of Toxoplasma gondii; Karsten V et al.; Little is known about the extent of conservation in the organization of the secretory pathway in organisms as different as prokaryotes, eukaryotes, and humans . The protozoan parasite Toxoplasma gondii allows easy genetic manipulations, and numerous vectors for selection of transgenic parasites have been developed . One approach to study the molecular mechanism of protein sorting and trafficking is the expression of foreign proteins . Here we describe the design and application of a vector that targets proteins to the secretory pathway of T . gondii and yields high-level expression of Escherichia coli reporter proteins . The general strategies and potential problems in expressing foreign proteins in T . gondii are discussed .

Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13979 - 84
Loss of HMW1 and HMW3 in noncytadhering mutants of Mycoplasma pneumoniae occurs post-translationally; Popham PL et al.; The genomic sequence of Mycoplasma pneumoniae establish this cell-wall-less prokaryote as among the smallest known microorganisms capable of self-replication . However, this genomic simplicity and corresponding biosynthetic austerity are sharply contrasted by the complex terminal structure found in this species . This tip structure (attachment organelle) directs colonization of the human respiratory mucosa, leading to bronchitis and atypical pneumonia . Furthermore, formation of a second tip structure appears to precede cell division, implying temporal regulation . However, the organization, regulation, and assembly of the attachment organelle in M . pneumoniae are poorly understood, and no counterparts have been identified among the walled bacteria . M . pneumoniae possesses a cytoskeleton-like structure required to localize adhesin proteins to the attachment organelle . The cytadherence-associated proteins HMW1, HMW2, and HMW3 are components of the mycoplasma cytoskeleton, with HMW1 localizing strictly along the filamentous extensions from the cell body and HMW3 being a key structural element of the terminal organelle . Disruptions in hmw2 result in the loss of HMW1 and HMW3 . However, the hmw1 and hmw3 genes were transcribed and translated at wild-type levels in hmw2 mutants . HMW1 and HMW3 were relatively stable in the wild-type background over 8 h but disappeared in the mutants over this time period . Evaluation of recombinant HMW1 levels in mycoplasma mutants suggested a requirement for the C-terminal domain of HMW1 for turnover . Finally, an apparent defect in the processing of the precursor for the adhesin protein P1 was noted in the HMW- mutants.

Chem Biol, 1995 Aug, 2(8), 543 - 52
A novel RNA motif for neomycin recognition; Wallis MG et al.; BACKGROUND: Antibiotics can interfere with RNA activity . Translation of RNA by the prokaryotic ribosome, self-splicing of group I introns, HIV replication and hammerhead ribozyme cleavage are inhibited by the aminoglycoside neomycin B . To explore the molecular basis by which small molecules such as antibiotics inhibit RNA function, we undertook an in vitro selection to obtain a variety of RNA molecules with the capacity to recognize neomycin . RESULTS: The majority of the RNA molecules selected to specifically bind neomycin share a region of nucleotide sequence homology . From chemical probing and covariations among different clones we show that in all sequences this region folds into a hairpin structure, which from footprinting and partial alkaline hydrolysis experiments is shown to be the neomycin-binding site . Neomycin is recognized with high affinity (Kd approximately equal to 100 nM) and high specificity (> 100-fold higher affinity for neomycin than for paromomycin) . CONCLUSIONS: The fact that RNAs containing the consensus sequence, as well as sequences that display variations within this region, specifically recognize neomycin suggests that a structural motif rather than a particular nucleotide sequence is required for neomycin recognition . We propose that a hairpin stem-loop structural motif, which might feature a widened major groove, may be a prerequisite for neomycin recognition . This structural pattern can be extrapolated to other natural neomycin-responsive RNAs.

Mol Microbiol, 1997 Oct, 26(2), 361 - 72
Transcriptional analysis of the divergent cagAB genes encoded by the pathogenicity island of Helicobacter pylori; Spohn G et al.; Helicobacter pylori strains isolated from most patients with peptic ulcer disease and adenocarcinoma express the vacuolating toxin VacA and contain a pathogenicity island named cag . The cag pathogenicity island codes for more than 40 putative proteins with features similar to bacterial secretion systems . One of these proteins, CagA, is an immunodominant antigen with unknown function encoded by the cagA gene . In the present study, we have analysed the functional promoter elements of the H . pylori cagA gene as well as of the divergently transcribed cagB gene . Primer extension analyses identified a single 5' end of the cagA mRNA, while two initiation sites were mapped in the case of the cagB mRNA . The promoters deduced upstream of these start points of transcription contained conserved -10 regions but no -35 regions with respect to the Escherichia coli sigma70 consensus sequence . Nevertheless, they could be activated in E . coli and in vitro by purified E . coli RNA polymerase . Deletion analyses indicated that the cagA and cagB genes are transcribed by overlapping promoters and that full activation requires sequences up to -70 and -96 respectively . Instead, basal transcription is likely to be mediated by -10 extended promoter-like sequences . RNA polymerase is able to bind the -40 to -60 region of the cagA promoter, and its binding is mediated by the alpha-subunit . This region resembles the UP elements of prokaryotic promoters in location, sequence and mechanism of interaction with the RNA polymerase . We discuss the features of these promoters and propose that they could represent a class of minimum promoters, which ensures a basic level of transcription, while full activation requires regulatory elements or a defined promoter context.

Arch Virol, 1997, 142(3), 601 - 10
Recombinant human antibodies specific for hepatitis C virus proteins; Esposito G et al.; Human antibodies to hepatitis C virus core, NS4A and NS3 were cloned in a prokaryotic vector and expressed as soluble Fab fragments and as phage-displayed Fabs . The recombinant Fabs were shown to be a suitable tool for immunohistochemistry, since they recognize the cognate antigen expressed in mammalian cells . The nucleotide sequence of the cDNA for the variable domains of these antibodies was determined and the V-gene usage was derived . On the basis of the deduced amino acid sequence, a structural model of the V domains of the Fabs was constructed.

Plant Mol Biol, 1997 Nov, 35(4), 417 - 24
Regulated expression of plant tRNA genes by the prokaryotic tet and lac repressors; Ulmasov B et al.; The prokaryotic tet operator (tetO) sequence was inserted at positions upstream and downstream of sequences encoding the Arabidopsis thaliana tRNA(Lys)AUC or tRNA(Trp)AUC suppressor tRNAs, and tRNA expression in carrot protoplasts was measured by translational suppression of a nonsense codon in a luciferase reporter gene . Regulation of tRNA expression by the tetracycline repressor (tetR) occurred from genes with the tetO inserted at position -1 (for the tRNA(Trp)AUC gene), or at positions -2, -6 and -10 (for the tRNA(Lys)AUC gene), and repression reached 90% . The inducer tetracycline (Tc) restored tRNA expression . Similarly, carrot protoplasts transfected with human tRNA(Ser)AUC genes containing the lac operator (lacO) in their 5'-flanking sequence with or without the lac repressor (lacI) gene, conditionally expressed tRNAs which suppressed the luciferase reporter . Up to 30-fold repression occurred by the lactose repressor when lacO was located at position -1 of the tRNA(Ser)AUC coding sequence . In the presence of the inducer isopropyl-beta-thiogalactoside (IPTG), repression was relieved . These results demonstrate that sequences flanking tRNA genes can strongly influence tRNA expression in plants, and in a conditional fashion when bound by inducible proteins.

J Bacteriol, 1998 Feb, 180(3), 514 - 8
Intracellular immunization of prokaryotic cells against a bacteriotoxin; Chames P et al.; Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells . Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain . Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli . To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope . The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system . To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E . coli . The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins . Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding . Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery . This indicates that the N-terminal domain of the toxin is accessible in the periplasm . Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

J Gen Virol, 1997 Oct, 78 ( Pt 10), 2665 - 73
The gene encoding the capsid protein P82 of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus: sequencing, transcription and characterization by immunoblot analysis; Li X et al.; A gene encoding a capsid-associated viral structural protein has been identified and sequenced in the genome of the Choristoneura fumiferana multicapsid nucleopolyhedrovirus (CfMNPV) . The gene has a 1872 nucleotide open reading frame (ORF) encoding 624 amino acids with a predicted molecular mass of 71.4 kDa . Transcription, which appeared to be initiated from a conserved GTAAG motif of baculovirus late genes, was detected at 12 h, reached a maximum at 48 h and declined at 72 h post-infection (p.i.) . Part of the ORF was cloned in frame into a prokaryotic expression vector, pMAL-c2, and the fusion protein was used to generate antibodies in rabbits . It was shown, with the aid of the polyclonal antiserum, that this viral protein was detectable at 24 h p.i . in infected cells . The protein appeared as an 82 kDa band in occlusion-derived virus and as an 82 kDa band and a 72 kDa band in budded virus . Amino acid sequence comparisons revealed that this ORF had high homology with the ORF p87 (77% similarity) of Orgyia pseudotsugata (Op) MNPV and the ORF p80 (60% similarity) of Autographa californica (Ac) MNPV . Immunoblots confirmed that the CfMNPV protein had antigenic similarities to the P87 protein of OpMNPV, but not to the P80 of AcMNPV.

Biotechniques . 1998 Jan;24(1):116, 118, 120, 122 passim.
Novel vector for generating RNAs with defined 3' ends and its use in antiviral strategies; Schwienhorst A et al.; A novel transcription system was constructed that allows trimming of 3' termini of RNA transcripts in E . coli by endogenous RNase P . Here, the sequence of tRNASer from E . coli fused downstream of the target sequence directs posttranscriptional cleavage 3' of the target sequence . As a first-target MNV11(+), a self-replicating RNA from the QB system was subjected to transcription in vivo . Northern blotting experiments revealed that the primary transcript was indeed successfully processed to an RNA of expected length . The RNA released proved to function as an active template for QB replicase . Moreover, E . coli cells producing these short-chain replicator molecules no longer supported multiplication of QB phages upon infection . Since the novel transcript-trimming system utilizes the endogenous RNase P activity and does not depend on any particular 3'-terminal RNA sequence of target molecules, it may have wide applications for a number of different targets in prokaryotes . Further applications, including those in eukaryotes, are discussed.

Biochemistry, 1998 Jan 27, 37(4), 1101 - 8
Influence of pre-existing methylation on the de novo activity of eukaryotic DNA methyltransferase; Carotti D et al.; Aberrant de novo methylation of CpG island DNA sequences has been observed in cultured cell lines or upon malignant transformation, but the mechanisms underlying this phenomenon are poorly understood . Using eukaryotic DNA (cytosine-5)-methyltransferase (of both human and murine origin), we have studied the in vitro methylation pattern of three CpG islands . Such sequences are intrinsically poor substrates of the enzyme, yet are efficiently methylated when a small amount of 5-methylcytosine is randomly introduced by the M.SssI prokaryotic DNA (cytosine-5)-methyltransferase prior to in vitro methylation by the eukaryotic enzyme . A stimulation was also found with several other double-stranded DNA substrates, either natural or of synthetic origin, such as poly(dG-dC).poly(dG-dC) . An A + T-rich plasmid, pHb beta 1S, showed an initial stimulation, followed by a severe inhibition of the activity of DNA (cytosine-5)-methyltransferase . Methylation of poly(dI-dC).poly(dI-dC) was instead inhibited by pre-existing 5-methylcytosines . The extent of stimulation observed with poly(dG-dC).poly(dG-dC) depends on both the number and the distribution of the 5-methylcytosine residues, which probably must not be too closely spaced for the stimulatory effect to be exerted . The activity of the M.SssI prokaryotic DNA methyltransferase was not stimulated, but was inhibited by pre-methylation on either poly(dG-dC).poly(dG-dC) or poly(dI-dC).poly(dI-dC) . The prokaryotic and eukaryotic DNA methyltransferases also differed in sensitivity to poly(dG-m5dC).poly(dG-m5dC), which is highly inhibitory for eukaryotic enzymes and almost ineffective on prokaryotic enzymes.

Eur J Biochem, 1997 Nov 15, 250(1), 212 - 21
Isolation of a rat histidase cDNA sequence and expression in Escherichia coli--evidence of extrahepatic/epidermal distribution; Sano H et al.; Histidase (histidine ammonia-lyase) is a cytosolic enzyme responsible for catalyzing the non-oxidative deamination of histidine to urocanic acid . Full-length cDNAs encoding rat histidase have been isolated from a lambdaZAP liver cDNA library using a partial cDNA fragment obtained by PCR . Whereas the initial description of the rat histidase 3' untranslated sequence contained a rare polyadenylation signal sequence, the data presented encompass a more distant 28-bp region, possessing a nucleotide stretch (AATATAAA), identical to that in the mouse histidase cDNA . Dideoxynucleotide chain-termination sequencing of two clones obtained by in vivo excision yielded an additional 376 bp and 105 bp of 5' and 3' untranslated sequences, respectively . A selected rat histidase cDNA clone was introduced into the pET-16b prokaryotic vector and expressed in BL21(DE3)pLysS Escherichia coli . After purification by nickel-chelation chromatography, recombinant histidine-tagged protein was employed to raise anti-(rat histidase) immunoglobulin in a Japanese white rabbit . The polyclonal rabbit antibody recognized and formed immune complexes with rat and recombinant human histidase proteins . Immunoblots of crude rat organ extracts detected a spectrum of histidase expression extending beyond that observed in liver and skin . Among other histidase-positive cells were those of the renal cortex tubular epithelium, fundic mucosal glands of stomach, gastric intramuscular (Auerbach's) plexus, and adrenal cortex . Immunohistochemical studies of histidase in rat liver produced discrete staining of hepatocytes in association with portal triads (Rappaport zone I) . Furthermore, in contrast with previous reports of activity confined to epidermal stratum corneum, our findings demonstrate immunoreactive protein within and limited to the adjacent stratum granulosum.

Eur J Biochem, 1997 Nov 15, 250(1), 77 - 84
Genomic organization, cDNA sequence, bacterial expression, and purification of human seryl-tRNA synthase; Vincent C et al.; In this paper, we report the cDNA sequence and deduced primary sequence for human cytosolic seryl-tRNA synthetase, and its expression in Escherichia coli . Two human brain cDNA clones of different origin, containing overlapping fragments coding for human seryl-tRNA synthetase were sequenced: HFBDN14 (fetal brain clone); and IB48 (infant brain clone) . For both clones the 5' region of the cDNA was missing . This 5' region was obtained via PCR methods using a human brain 5' RACE-Ready cDNA library . The complete cDNA sequence allowed us to define primers to isolate and characterize the intron/exon structure of the serS gene, consisting of 10 introns and 11 exons . The introns' sizes range from 283 bp to more than 3000 bp and the size of the exons from 71 bp to 222 bp . The availability of the gene structure of the human enzyme could help to clarify some aspects of the molecular evolution of class-II aminoacyl-tRNA synthetases . The human seryl-tRNA synthetase has been expressed in E . coli, purified (95% pure as determined by SDS/PAGE) and kinetic parameters have been measured for its substrate tRNA . The human seryl-tRNA synthetase sequence (514 amino acid residues) shows significant sequence identity with seryl-tRNA synthetases from E . coli (25%), Saccharomyces cerevisiae (40%), Arabidopsis thaliana (41%) and Caenorhabditis elegans (60%) . The partial sequences from published mammalian seryl-tRNA synthetases are very similar to the human enzyme (94% and 92% identity for mouse and Chinese hamster seryl-tRNA synthetase, respectively) . Human seryl-tRNA synthetase, similar to several other class-I and class-II human aminoacyl-tRNA synthetases, is clearly related to its bacterial counterparts, independent of an additional C-terminal domain and a N-terminal insertion identified in the human enzyme . In functional studies, the enzyme aminoacylates calf liver tRNA and prokaryotic E . coli tRNA.

Genes Dev, 1997 Dec 15, 11(24), 3423 - 31
The preference for GT-rich DNA by the yeast Rad51 protein defines a set of universal pairing sequences; Tracy RB et al.; The Rad51 protein of Saccharomyces cerevisiae is a eukaryotic homolog of the RecA protein, the prototypic DNA strand-exchange protein of Escherichia coli . RAD51 gene function is required for efficient genetic recombination and for DNA double-strand break repair . Recently, we demonstrated that RecA protein has a preferential affinity for GT-rich DNA sequences-several of which exhibit enhanced RecA protein-promoted homologous pairing activity . The fundamental similarity between the RecA and Rad51 proteins suggests that Rad51 might display an analogous bias . Using in vitro selection, here we show that the yeast Rad51 protein shares the same preference for GT-rich sequences as its prokaryotic counterpart . This bias is also manifest as an increased ability of Rad51 protein to promote the invasion of supercoiled DNA by homologous GT-rich single-stranded DNA, an activity not previously described for the eukaryotic pairing protein . We propose that the preferred utilization of GT-rich sequences is a conserved feature among all homologs of RecA protein, and that GT-rich regions are loci for increased genetic exchange in both prokaryotes and eukaryotes.

FEMS Microbiol Rev, 1997 Nov, 21(3), 213 - 29
Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis; von Wintzingerode F et al.; After nearly 10 years of PCR-based analysis of prokaryotic small-subunit ribosomal RNAs for ecological studies it seems necessary to summarize reported pitfalls of this approach which will most likely lead to an erroneous description on the microbial diversity of a given habitat . The following article will cover specific aspects of sample collection, cell lysis, nucleic acid extraction, PCR amplification, separation of amplified DNA, application of nucleic probes and data analysis.

Hum Mutat, 1998, 11(1), 4 - 17
In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function; Waters PJ et al.; Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600) . In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell-free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype . Corresponding analysis of site-directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase . Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here . The core data are accessible at the PAH Mutation Analysis Consortium Web site .

Cytometry, 1998 Jan 1, 31(1), 29 - 36
Flow cytometry of bacterial cells: comparison between different flow cytometers and different DNA stains; Bernander R et al.; The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations . The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources . In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument . Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide . All three stains yielded DNA histograms of similar quality in both flow cytometers . Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample . The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.

J Biol Chem, 1997 Dec 26, 272(52), 33211 - 9
Lack of correlation between in vitro and in vivo replication of precisely defined benz-a-anthracene adducted DNAs; McNees AG et al.; Like other polycyclic aromatic hydrocarbons, certain metabolites of benz{a}anthracene have been implicated as potent carcinogens . These effects are thought to be caused by the covalent binding of these species to nucleophilic groups on the bases of DNA . To address the molecular mechanisms by which these molecules induce mutations, this study employed oligonucleotides containing four site-specific N6 adenine-benz{a}anthracene diol epoxide adducts . Using a prokaryotic in vivo replication system, we have shown that both non-bay region anti-trans-benz{a}anthracene adducts are essentially nonmutagenic . In contrast, the bay region anti-trans-benz{a}anthracene lesions do induce point mutations at the adduct site . The mutagenic frequency of these bay region lesions is dependent on the stereochemistry about the adduct-forming bond, as well as the strain of Escherichia coli in which they are replicated . The ability of the bacterial replication machinery to bypass the lesions does not correlate with the differences observed in their mutagenesis . While both non-bay region adducts are readily bypassed in vivo, the bay region adducts are both blocking to approximately the same degree . In vitro studies of the interactions of E . coli DNA polymerase III with these adducts have also been undertaken to further dissect the relationship between adduct structure and biological activity.

Mol Microbiol, 1997 Oct, 26(1), 37 - 48
Operator interactions by the Bacillus subtilis arginine repressor/activator, AhrC: novel positioning and DNA-mediated assembly of a transcriptional activator at catabolic sites; Miller CM et al.; We have previously reported the initial characterization of a catabolic operator site (O{rocA}) for the Bacillus subtilis arginine repressor/activator protein AhrC . Here, we present the characterization by gel retardation and DNase I footprinting of both O(rocA) and a second catabolic operator site, O(rocD) . Both operator sites encompass a single recognition site, an ARG box, located immediately upstream of the transcriptional start points, a unique positioning for a transcriptional activator protein . Although there is considerable sequence homology between the two catabolic operator sites, they vary significantly, around twofold, in their apparent affinities for the protein (K'd approximately 90 nM for O{rocA} and approximtaely 190nM for O{rocD}) . This difference may result from the lower match to the ARG box consensus of the O(rocD) site . Both catabolic operators show evidence for co-operative binding with respect to protein concentration . Determination of the sequences of two AhrC catabolic operator sites, in combination with the three such biosynthetic sites, has allowed the derivation of an improved B . subtilis ARG box consensus sequence, CATGAATAAAAATg/tCAAg/t . This is not identical to the Escherichia coli consensus operator for the AhrC homologue, ArgR, which may explain the only partial cross-functioning of these proteins in vivo . The O(rocA) site is adjacent to a sharp, stable bend located 5' to the catabolic operator . Circular permutation analysis has been used to determine the relative angle of bend (approximately 50 degrees), its location and the effect of adding magnesium ions and/or AhrC protein . Protein binding increases the relative bend angle to approximately 85 degrees . Bending is shown to be associated with a number of A-tracts in the upstream sequence . However, altering the phasing of the A-tracts has little effect on the affinity for AhrC . Truncation and competition experiments have been used to investigate the possible role of sequences flanking the operator on affinity . Very surprisingly, the affinity of the O(rocA) site appears to increase in the presence of excess, specific competitor fragment, i.e . the system shows anti-competitive effects . Competition is restored at high molar excesses of specific fragment over the protein . We propose a novel model for the assembly of a higher affinity form of AhrC at operator sites that is consistent with both the apparent co-operativity of binding and the anti-competitive effects . These data suggest that the molecular interactions occurring between the prokaryotic arginine-regulatory proteins and their operators may be more complex than is generally appreciated.

Annu Rev Genet, 1997, 31, 91 - 111
Gene amplification and genomic plasticity in prokaryotes; Romero D et al.; Gene amplification is a common feature of the genome of prokaryotic organisms . In this review, we analyze different instances of gene amplification in a variety of prokaryotes, including their mechanisms of generation and biological role . Growing evidence supports the concept that gene amplification be considered not as a mutation but rather as a dynamic genomic state related to the adaptation of bacterial populations to changing environmental conditions or biological interactions . In this context, the potentially amplifiable DNA regions impose a defined dynamic structure on the genome . If such structure has indeed been selected during evolution, it is a particularly challenging hypothesis.

Annu Rev Cell Dev Biol, 1997, 13, 697 - 743
Plant cell morphogenesis: plasma membrane interactions with the cytoskeleton and cell wall; Fowler JE et al.; Because plants are composed of immobile cells, plant morphogenesis requires mechanisms allowing precise control of cell expansion and cell division patterns . Cortical domains, localized in response to directional cues, are of central importance in establishing cell polarity, orienting cell division, and determining daughter cell fates in a wide variety of prokaryotic and eukaryotic organisms . Such domains consist of localized macromolecular complexes that, in plant cells, provide spatial control of cell expansion and cell division functions . The role of the cytoskeleton, plasma membrane, and targeted secretion to the cell wall in the spatial regulation of cell morphogenesis in plants is discussed in light of recent results from model organisms, including brown algal zygotes (e.g . Fucus) . A general model, emphasizing the importance of cortical sites and targeted secretion, is proposed for morphogenesis in higher plant cells based on current knowledge and principles derived from analysis of the establishment of a stable cortical asymmetry in Fucus . The model illustrates mechanisms to direct the orientation of an asymmetric division resulting in daughter cells with different fates.

Semin Cancer Biol, 1997 Jun, 8(3), 183 - 91
Multidrug transporters from bacteria to man: similarities in structure and function; van Veen HW et al.; Organisms ranging from bacteria to man possess transmembrane transporters which confer resistance to toxic compounds . Underlining their biological significance, prokaryotic and eukaryotic multidrug transport proteins are very similar in structure and function . Therefore, a study of the factors which determine the substrate specificity and energy coupling to drug translocation in 'simple' microorganisms has significance for multidrug resistance of mammalian cells . This chapter represents a comprehensive review in which we will summarize the current state of knowledge on three major aspects of drug efflux-based multidrug resistance: (i) the functional and structural similarities among secondary and ABC-type drug transporters encountered in prokaryotic and eukaryotic cells; (ii) the molecular mechanism of these transporters; and (iii) their potential physiological role.

J Biol Chem, 1998 Jan 16, 273(3), 1435 - 43
The R1 subunit of herpes simplex virus ribonucleotide reductase is a good substrate for host cell protein kinases but is not itself a protein kinase; Langelier Y et al.; The N terminus of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is believed to be a protein kinase domain mainly because the R1 protein was phosphorylated in a protein kinase assay on blot . Using Escherichia coli and adenovirus expression vectors to produce R1, we found that, whereas the reductase activity of both recombinant proteins was similar, efficient phosphorylation of R1 and casein in the presence of Mg2+ was obtained only with the R1 purified from eukaryotic cells . Phosphorylation of this R1, in solution or on blot, results mainly from the activity of casein kinase II (CKII), a co-purifying protein kinase . Labeling on blot occurs from CKII leakage off the membrane and its subsequent high affinity binding to in vivo CKII-phosphorylated R1 . CKII target sites were mapped to an acidic serine-rich segment of the R1 N terminus . Improvement in purification of the R1 expressed in eukaryotic cells nearly completely abolished its phosphorylation potential . An extremely low level of phosphorylation observed in the presence of Mn2+ with the R1 produced in E . coli was probably due to an unidentified prokaryotic protein kinase . These results provide evidence that the herpes simplex virus type 2 R1 does not possess an intrinsic protein kinase activity.

Biochem Biophys Res Commun, 1998 Jan 6, 242(1), 39 - 45
Suppression of cell growth by heavy chain ferritin; Guo JH et al.; While producing recombinant rat liver H and L chain ferritin homopolymers using the baculovirus expression system, we noticed that rat liver H chain ferritin, but not L chain ferritin, had a suppressive effect on the growth of Spodoptera frugiperda (Sf-21) cells . Suppression was observed immediately after infection with recombinant H chain ferritin baculovirus prepared from lysed infected cells . Immediate suppression was observed when purified with either recombinant H chain apoferritin or various holoferritins (loaded with 1,970 +/- 50 or 2,520 +/- 90 atoms of iron/ferritin) indicating that suppression was not due to sequestration of iron required for cell growth . Suppression by H chain ferritin was also observed upon attempting to express the protein in Escherichia coli . Strategies for expression of recombinant rat liver H and L ferritin homopolymers in both prokaryotic and eukaryotic expression systems were developed.

Science, 1998 Jan 2, 279(5347), 102 - 5
Ultraviolet-induced cell death blocked by a selenoprotein from a human dermatotropic poxvirus; Shisler JL et al.; Selenium, an essential trace element, is a component of prokaryotic and eukaryotic antioxidant proteins . A candidate selenoprotein homologous to glutathione peroxidase was deduced from the sequence of molluscum contagiosum, a poxvirus that causes persistent skin neoplasms in children and acquired immunodeficiency syndrome (AIDS) patients . Selenium was incorporated into this protein during biosynthesis, and a characteristic stem-loop structure near the end of the messenger RNA was required for alternative selenocysteine decoding of a potential UGA stop codon within the open reading frame . The selenoprotein protected human keratinocytes against cytotoxic effects of ultraviolet irradiation and hydrogen peroxide, providing a mechanism for a virus to defend itself against environmental stress.

J Biol Chem, 1997 Dec 19, 272(51), 32280 - 5
A ubiquitin-specific protease that efficiently cleaves the ubiquitin-proline bond; Gilchrist CA et al.; Ubiquitin is a small eukaryotic protein that is synthesized naturally as one of several fusion proteins, which are processed by ubiquitin-specific proteases to release free ubiquitin . The expression of heterologous proteins as fusions to ubiquitin in either prokaryotic or eukaryotic hosts often dramatically enhances their yield, and allows the exposure of any amino acid following cleavage of ubiquitin . The single exception is when proline is the amino acid immediately following ubiquitin; the ubiquitin-proline bond is poorly cleaved by presently studied ubiquitin-specific proteases . We show that the mouse ubiquitin-specific protease Unp, and its human homolog Unph, can efficiently cleave the ubiquitin-proline bond in ubiquitin fusion proteins of different sizes . N-terminal sequencing of the cleavage products reveals that cleavage occurs precisely at the ubiquitin-proline junction . The biological significance of this cleavage activity is unclear, as ubiquitin-proline fusions do not occur naturally . However, it may indicate a different catalytic mechanism for these ubiquitin-specific proteases and/or that they can cleave ubiquitin-like proteins . Unp and Unph thus represent versatile ubiquitin-specific proteases for cleaving ubiquitin-fusion proteins in biotechnology and basic research, regardless of both the amino acid immediately following ubiquitin, and the size of the fusion partner.

J Biol Chem, 1997 Dec 19, 272(51), 32254 - 9
The gene encoding the elongation factor P protein is essential for viability and is required for protein synthesis; Aoki H et al.; Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine . This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A . Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability . A kanamycin resistance (KanR) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication . After transformation into a recA+ E . coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced . Mutants containing the efp-KanR gene in the chromosome grew at 33 degrees C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44 degrees C . Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44 degrees C . At 44 degrees C, mutant cells are severely defective in peptide-bond formation . We conclude that the efp gene is essential for cell viability and is required for protein synthesis.

Nat Struct Biol, 1998 Jan, 5(1), 31 - 6
Crystal structure of GyrA intein from Mycobacterium xenopi reveals structural basis of protein splicing; Klabunde T et al.; Several genes from prokaryotes and lower eukaryotes have been found to contain an in-frame open reading frame, which encodes for an internal protein (intein) . Post-translationally, the internal polypeptide auto-splices and ligates the external sequences to yield a functional external protein (extein) and an intein . Most, but not all inteins, contain, apart from a splicing domain, a separate endonucleolytic domain that enables them to maintain their presence by a homing mechanism . We report here the crystal structure of an intein found in the gyrase A subunit from Mycobacterium xenopi at 2.2 A resolution . The structure contains an unusual beta-fold with the catalytic splice junctions at the ends of two adjacent antiparallel beta-strands . The arrangement of the active site residues Ser 1, Thr 72, His 75, His 197, and Asn 198 is consistent with a four-step mechanism for the cleavage-ligation reaction . Using site-directed mutagenesis, the N-terminal cysteine, proposed as the nucleophile in the first step of the splicing reaction, was changed to a Ser 1 and Ala 0, thus capturing the intein in a pre-spliced state.

Environ Mol Mutagen, 1997, 30(4), 385 - 95
Nature of mutation in the human hprt gene following in vivo exposure to ionizing radiation of cesium-137; da Cruz AD et al.; The current study comprises the analysis of mutations in 10 individuals accidentally exposed to cesium-137 during the 1987 radiological accident in Goiania, Brazil . Their exposures were among the highest experienced, ranging from 1 to 7 Gy . Peripheral T-lymphocyte samples were obtained 3.3 years after the original exposure and mutation was studied at the hprt locus using the 6-thioguanine-resistance selection assay . The mutational spectrum for the exposed population is comprised of 90 independent mutants . Based on T-cell receptor analysis, only 5% (5/95) were clonally related . Mutants were initially studied using RT-PCR and directly sequenced using an automated laser fluorescent DNA sequencer . Mutants that repeatedly failed to produce cDNAs were studied using a multiplex PCR assay with genomic DNA . Missense mutations were the most frequent event recovered, comprising 40% (23/57) of the spectral sample . An excess of events involving A:T base pairs was observed, exhibiting a significant difference (chi 2 = 12.7, P = 0.0004) when compared to the spontaneous spectrum . This finding may reflect the effect of ionizing radiation-induced damage, suggesting a potential similarity to radiation effects in prokaryotes . At the genomic level, 36.7% (33/90) of the mutants exhibited gross structural alterations, as detected by multiplex PCR . Deletion events were over-represented in our spectral sample, displaying a twofold increase when compared to the frequency observed in the spontaneous mutation database.

Proc Natl Acad Sci U S A, 1998 Jan 20, 95(2), 505 - 9
Production of a polyketide natural product in nonpolyketide-producing prokaryotic and eukaryotic hosts; Kealey JT et al.; The polyketides are a diverse group of natural products with great significance as human and veterinary pharmaceuticals . A significant barrier to the production of novel genetically engineered polyketides has been the lack of available heterologous expression systems for functional polyketide synthases (PKSs) . Herein, we report the expression of an intact functional PKS in Escherichia coli and Saccharomyces cerevisiae . The fungal gene encoding 6-methylsalicylic acid synthase from Penicillium patulum was expressed in E . coli and S . cerevisiae and the polyketide 6-methylsalicylic acid (6-MSA) was produced . In both bacterial and yeast hosts, polyketide production required coexpression of 6-methylsalicylic acid synthase and a heterologous phosphopantetheinyl transferase that was required to convert the expressed apo-PKS to its holo form . Production of 6-MSA in E . coli was both temperature- and glycerol-dependent and levels of production were lower than those of P . patulum, the native host . In yeast, however, 6-MSA levels greater than 2-fold higher than the native host were observed . The heterologous expression systems described will facilitate the manipulation of PKS genes and consequent production of novel engineered polyketides and polyketide libraries.

Appl Environ Microbiol, 1998 Jan, 64(1), 98 - 105
Construction and characterization of a 1,3-propanediol operon; Skraly FA et al.; The genes for the production of 1,3-propanediol (1,3-PD) in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, are naturally under the control of two different promoters and are transcribed in different directions . These genes were reconfigured into an operon containing dhaB followed by dhaT under the control of a single promoter . The operon contains unique restriction sites to facilitate replacement of the promoter and other modifications . In a fed-batch cofermentation of glycerol and glucose . Escherichia coli containing the operon consumed 9.3 g of glycerol per liter and produced 6.3 g of 1,3-PD per liter . The fermentation had two distinct phases . In the first phase, significant cell growth occurred and the products were mainly 1,3-PD and acetate . In the second phase, very little growth occurred and the main products were 1,3-PD and pyruvate . The first enzyme in the 1,3-PD pathway, glycerol dehydratase, requires coenzyme B12, which must be provided in E . coli fermentations . However, the amount of coenzyme B12 needed was quite small, with 10 nM sufficient for good 1,3-PD production in batch cofermentations . 1,3-PD is a useful intermediate in the production of polyesters . The 1,3-PD operon was designed so that it can be readily modified for expression in other prokaryotic hosts; therefore, it is useful for metabolic engineering of 1,3-PD pathways from glycerol and other substrates such as glucose.

Biochem Biophys Res Commun, 1997 Dec 29, 241(3), 714 - 9
cDNA cloning and characterization of a rat spermatogenesis-associated protein RSP29; Ji X et al.; RSP29, a protein secreted by rat round spermatids, stimulates the secretory function of Sertoli cells in the testis . By making use of the N-terminal sequence homology of RSP29 and a human protein hDP1 that we had previously isolated, we cloned the full length cDNA sequence that encodes RSP29 . The entire amino acid sequence of RSP29 showed significant homology with that of hDP1, which was later identified as glyoxalase II . Southern analysis showed that the RSP29 protein sequence is highly conserved in eukaryotes and possibly in prokaryotes . The RSP29 mRNA is expressed in many tissues but has an extremely high abundance in testis . These data suggest that RSP29 may have an important function in most tissues of enkaryotic organisms . The high expression of RSP29 in testis and its stimulatory effects on Sertoli cells suggest that RSP29 could be especially important in the regulation of spermatogenesis.

Gene, 1997 Dec 19, 204(1-2), 85 - 9
cDNA cloning of L-dopa decarboxylase from the eclosion stage of the insect Ceratitis capitata . Evolutionary relationship to other species decarboxylases; Mantzouridis TD et al.; The cDNA encoding the L-dopa decarboxylase (ddc) from the eclosion stage of the insect Ceratitis capitata was isolated by PCR and a molecular cloning strategy . The isolated cDNA clone encoded a protein of 431 amino acids with a calculated molecular weight of 47,843 Da . Northern blot analysis of poly(A)+ RNA showed an approximately 2 kb transcript . The deduced protein sequence shares a high percentage of homology with Ddc protein sequences of other species . Furthermore, the molecular weight of the deduced protein agreed well with that of the purified Ddc from the same insect . Data base search revealed significant and extensive sequence similarities among prokaryotic and eukaryotic PLP-dependent decarboxylases including Ceratitis capitata and bacterial histidine decarboxylase (HDC), strongly suggesting an ancient and common origin for all PLP-dependent decarboxylases.

Trends Biochem Sci, 1997 Dec, 22(12), 453 - 7
Maintaining genetic code through adaptations of tRNA synthetases to taxonomic domains; Shiba K et al.; The universal genetic code is determined by the aminoacylation of tRNAs . In spite of the universality of the code, there are barriers to aminoacylation across taxonomic domains . These barriers are thought to correlate with the co-segregation of sequences of synthetases and tRNAs into distinct taxonomic domains . By contrast, we show here examples of eukaryote-like synthetases that are found in certain prokaryotes . The associated tRNAs have retained their prokaryote-like character in each instance . Thus, co-segregation of domain-specific synthetases and tRNAs does not always occur . Instead, synthetases make adaptations of tRNA-protein contacts to cross taxonomic domains.

Mol Immunol, 1997 Jul, 34(10), 709 - 17
Human IgG is substrate for the thioredoxin system: differential cleavage pattern of interchain disulfide bridges in IgG subclasses; Magnusson CG et al.; Thioredoxin, a 12,000 mol . wt protein with two redox-active cysteine residues, together with thioredoxin reductase and NADPH, may reduce protein disulfides and thereby act as a molecular probe of their structure and reactivity . Interchain and intrachain disulfides are structural elements in all immunoglobulins and therefore potential substrates for the reduced thioredoxin, Trx(SH)2 . It was investigated whether such disulfides are cleaved in human polyclonal IgG and IgG subclass myeloma proteins by both the human and the Escherichia coli thioredoxin systems . The reactions were monitored spectrophotometrically as oxidation of NADPH at 340 nm, and by following the kinetics of the cleavage patterns with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) . Human IgG was a substrate for both prokaryotic and eukaryotic Trx(SH)2, which directly reduced IgG disulfides in a time and dose-dependent manner . Stoichiometric analyses indicated near-complete reduction of mainly inter-heavy light chain and inter-heavy chain disulfides, and SDS PAGE corroborated that the buried intrachain disulfides were left intact . The kinetic studies showed that IgG1, IgG3 and IgG4 were readily reduced into heavy and light chains via the formation of half-molecules with slightly slower kinetics for IgG4 . In sharp contrast, IgG2 was not cleaved at all, even with increased thioredoxin concentrations or reduction times . A small but significant NADPH consumption by IgG2 myeloma proteins suggested reduction of a labile interchain or surface-exposed mixed disulfide . Consistent results were obtained for several IgG myeloma proteins within each subclass . The structural and functional importance of interchain disulfides in immunoglobulins suggests physiological implications of the thioredoxin system.

Biosystems, 1997, 44(2), 107 - 34
A code in the protein coding genes; Arques DG et al.; A statistical analysis with 12,288 autocorrelation functions applied in protein (coding) genes of prokaryotes and eukaryotes identifies three subsets of trinucleotides in their three frames: T0 = X0 {symbol: see text} inverted question markAAA, TTT inverted question mark with X0 = inverted question markAAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC inverted question mark in frame 0 (the reading frame established by the ATG start trinucleotide), T1 = X1 {symbol: see text} inverted question markCCC inverted question mark in frame 1 and T2 = X2 {symbol: see text} inverted question markGGG inverted question mark in frame 2 (the frames 1 and 2 being the frame 0 shifted by one and two nucleotides, respectively, to the right) . These three subsets are identical in these two gene populations and have five important properties: (i) the property of maximal (20 trinucleotides) circular code for X0 (resp . X1, X2) allowing to retrieve automatically the frame 0 (resp . 1, 2) in any region of the gene without start codon; (ii) the DNA complementarity property C (e.g . C(AAC) = GTT): C(T0) = T0, C(T1) = T2 and C(T2) = T1 allowing the two paired reading frames of a DNA double helix simultaneously to code for amino acids; (iii) the circular permutation property P (e.g . P(AAC) = ACA): P(X0) = X1 and P(X1) = X2 implying that the two subsets X1 and X2 can be deduced from X0; (iv) the rarity property with an occurrence probability of X0 = 6 x 10(-8); and (v) the concatenation properties in favour of an evolutionary code: a high frequency (27.5%) of misplaced trinucleotides in the shifted frames, a maximum (13 nucleotides) length of the minimal window to retrieve automatically the frame and an occurrence of the four types of nucleotides in the three trinucleotide sites . In Discussion, a simulation based on an independent mixing of the trinucleotides of T0 allows to retrieve the two subsets T1 and T2 . Then, the identified subsets T0, T1 and T2 replaced in the 2-letter genetic alphabet inverted question markR, Y inverted question mark (R = purine = A or G, Y = pyrimidine = C or T) allow to retrieve the RNY model (N = R or Y) and to explain previous works in the alphabet inverted question markR, Y inverted question mark . Then, these three subsets are related to the genetic code . The trinucleotides of T0 code for 13 amino acids: Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Thr, Tyr and Val . Finally, a strong correlation between the usage of the trinucleotides of T0 in protein genes and the amino acid frequencies in proteins is observed as six among seven amino acids not coded by T0, have as expected the lowest frequencies in proteins of both prokaryotes and eukaryotes.

FEBS Lett, 1997 Dec 15, 419(2-3), 157 - 60
The origin and utility of histone deacetylases; Khochbin S et al.; A large region of two distinct yeast histone deacetylases, RPD3 and HDA1, is highly homologous to several prokaryotic enzymes that catalyze reactions involving various acetylated substrates . Proteins sharing this homology domain are found also in many higher eukaryotes and they all appear to be related to the RPD3 family of histone deacetylases . In each member of the family, the 'prokaryotic homology' domain covers almost two thirds of the protein, with the remaining portion containing the most divergent sequences . These sequences are located at the C-terminal region allowing for a clear definition of variants . Since the involvement of deacetylase members in different distinct regulatory complexes is now well established, the above observation suggests that the C-terminal domain may confer specificity to different members of the family . The RPD3 histone deacetylases thus appear as members of a family with a large conserved domain involved in enzymatic activity targeted to a short C-terminal domain, which probably confers functional specificity . The potential for deacetylases to be involved in multiple regulatory pathways provides an attractive counterpoint to the role of multiple histone acetyltransferases as coactivators.

Plant Mol Biol, 1997 Dec, 35(6), 873 - 92
Characterization of the cyclophilin gene family of Arabidopsis thaliana and phylogenetic analysis of known cyclophilin proteins; Chou IT et al.; We have isolated four members of the Arabidopsis cyclophilin (CyP) gene family, designated ROC1 to ROC4 (rotamase CyP) . Deduced peptides of ROC1, 2 and 3 are 75% to 91% identical to Brassica napus cytosolic CyP, contain no leader peptides and include a conserved seven amino-acid insertion relative to mammalian cytosolic CyPs . Two other Arabidopsis CyPs, ROC5 (43H1; ATCYP1) and ROC6 (ATCYP2), share these features . ROC1, ROC2, ROC3 and ROC5 are expressed in all tested organs of light-grown plants . ROC2 and ROC5 show elevated expression in flowers . Expression of ROC1, ROC2, and ROC3 decreases in darkness and these genes also exhibit small elevations in expression upon wouding . The five Arabidopsis genes encoding putative cytosolic CyPs (ROC1, 2, 3, 5 and 6) contain no introns . In contrast, ROC4, which encodes a chloroplast stromal CyP, is interrupted by six introns . ROC4 is not expressed in roots, and is strongly induced by light . Phylogenetic trees of all known CyPs and CyP-related proteins provide evidence of possible horizontal transfer of CyP genes between prokaryotes and eukaryotes and of a possible polyphyletic origin of these proteins within eukaryotes . These trees also show significant grouping of eukaryotic CyPs on the basis of subcellular localization and structure . Mitochondrial CyPs are closely related to cytosolic CyPs of the source organism, but endoplasmic reticulum CyPs form separate clades . Known plant CyPs fall into three clades, one including the majority of higher-plant cytosolic CyPs, one including only ROC2 and a related rice CyP, and one including only chlorplast CyPs.

Mol Microbiol, 1997 Dec, 26(5), 1109 - 23
ATP-dependent ferric hydroxamate transport system in Escherichia coli: periplasmic FhuD interacts with a periplasmic and with a transmembrane/cytoplasmic region of the integral membrane protein FhuB, as revealed by competitive peptide mapping; Mademidis A et al.; The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes . Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cytoplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane . Interaction of FhuD with FhuB was studied in vitro with biotinylated synthetic 10 residue and 20-24 residue peptides of FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin . Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and part of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD . Decapeptides were transferred into the periplasm of cells through a FhuA deletion derivative that forms permanently open channels three times as large as the porins in the outer membrane . FhuB peptides that bound to FhuD inhibited ferrichrome transport, while peptides that did not bind to FhuD did not affect transport . These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytoplasmic segment 7 of FhuB . The transmembrane region may be part of a pore through which a portion of FhuD inserts into the cytoplasmic membrane during transport . The cytoplasmic segment 7 of FhuB contains the conserved amino acid sequence EAA...G (in FhuB DTA ...G) found in ABC transporters, which is predicted to interact with the cytoplasmic FhuC ATPase . Triggering of ATP hydrolysis by substrate-loaded FhuD may occur by physical interaction between FhuD and FhuC, which bind close to each other on loop 7 . Although FhuB consists of two homologous halves, FhuB(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome transport are asymmetrically arranged.

Infect Immun, 1998 Jan, 66(1), 83 - 8
Phage antibodies obtained by competitive selection on complement-resistant Moraxella (Branhamella) catarrhalis recognize the high-molecular-weight outer membrane protein; Boel E et al.; We used competitive panning to select a panel of 10 different human antibodies from a large semisynthetic phage display library that distinguish between serum complement-resistant and complement-sensitive strains of the gram-negative diplococcus Moraxella (Branhamella) catarrhalis . Western blotting analyses and inhibition enzyme-linked immunosorbent assays showed that all phage antibodies were directed against the same or closely spaced epitopes on the target protein, which is the high-molecular-weight outer membrane protein (HMW-OMP) of M . catarrhalis . HMW-OMP was found in multiple isolates of complement-resistant but not complement-sensitive M . catarrhalis strains . Nucleotide sequence analysis demonstrated that the immunoglobulin heavy- and light-chain variable-region genes encoding the 10 phage antibodies were remarkably similar, with a strong preference for basic amino acid residues in the heavy-chain CDR3 regions . This is the first report showing that competitive panning is a successful procedure to obtain phage antibodies against differentially expressed structures on phenotypically dissimilar strains of prokaryotic cells.

J Biol Chem, 1997 Dec 5, 272(49), 31029 - 35
The role of the pro sequence of Bacillus subtilis sigmaK in controlling activity in transcription initiation; Johnson BD et al.; The sigma (sigma) subunit of prokaryotic RNA polymerase is required for specific recognition of promoter DNA sequences and transcription initiation . Regulation of gene expression can therefore be achieved by modulating the activity of the sigma subunit . In Bacillus subtilis the mother cell-specific sporulation sigma factor, sigmaK, is synthesized as a precursor protein, pro-sigmaK, with a 20-amino acid pro sequence . This pro sequence renders sigmaK inactive for directing transcription of sigmaK-dependent genes in vivo until the pro sequence is proteolytically removed . To understand the role of the pro sequence in controlling sigmaK activity, we have constructed NH2-terminal truncations of pro-sigmaK and characterized their behavior in vitro at the gerE promoter . In this report we show that the pro sequence inactivates sigmaK by interfering with the ability of sigmaK to associate with the core subunits of polymerase and also influences the interactions between holoenzyme and promoter DNA . Additionally, removal of as few as 6 amino acids (pro-sigmaKDelta6) is sufficient to activate pro-sigmaK for DNA binding and transcription initiation . Surprisingly, pro-sigmaKDelta6 binds to DNA with higher affinity and stimulates transcription 30-fold more efficiently than sigmaK, under certain conditions.

J Biol Chem, 1997 Dec 5, 272(49), 30952 - 61
Regulatory role for a novel human thioredoxin peroxidase in NF-kappaB activation; Jin DY et al.; Reduction-oxidation (redox) plays a critical role in NF-kappaB activation . Diverse stimuli appear to utilize reactive oxygen species (e.g . hydrogen peroxide) as common effectors for activating NF-kappaB . Antioxidants govern intracellular redox status, and many such molecules can reduce H2O2 . However, functionally, it does appear that different antioxidants are variously selective for redox regulation of certain transcription factors such as NF-kappaB . For NF-kappaB, thioredoxin has been described to be a more potent antioxidant than either glutathione or N-acetylcysteine . Thioredoxin peroxidase is the immediate enzyme that links reduction of H2O2 to thioredoxin . Several putative human thioredoxin peroxidases have been identified using recursive sequence searches/alignments with yeast or prokaryotic enzymes . None has been characterized in detail for intracellular function(s) . Here, we describe a new human thioredoxin peroxidase, antioxidant enzyme AOE372, identified by virtue of its protein-protein interaction with the product of a proliferation association gene, pag, which is also a thiol-specific antioxidant . In human cells, AOE372 defines a redox pathway that specifically regulates NF-kappaB activity via a modulation of IkappaB-alpha phosphorylation in the cytoplasm . We show that AOE372 activity is regulated through either homo- or heterodimerization with other thiol peroxidases, implicating subunit assortment as a mechanism for regulating antioxidant specificities . AOE372 function suggests thioredoxin peroxidase as an immediate regulator of H2O2-mediated activation of NF-kappaB.

J Biol Chem, 1997 Dec 5, 272(49), 30841 - 7
Cloning, expression, and characterization of a novel Escherichia coli thioredoxin; Miranda-Vizuete A et al.; Thioredoxin (Trx) is a small ubiquitous protein that displays different functions mainly via redox-mediated processes . We here report the cloning of a gene (trxC) coding for a novel thioredoxin in Escherichia coli as well as the expression and characterization of its product . The gene encodes a protein of 139 amino acids (Trx2) with a calculated molecular mass of 15.5 kDa . Trx2 contains two distinct domains: an N-terminal domain of 32 amino acids including two CXXC motifs and a C-terminal domain, with the conserved active site, Trp-Cys-Gly-Pro-Cys, showing high homology to the prokaryotic thioredoxins . Trx2 together with thioredoxin reductase and NADPH is an efficient electron donor for the essential enzyme ribonucleotide reductase and is also able to reduce the interchain disulfide bridges of insulin . The apparent Km value of Trx2 for thioredoxin reductase is similar to that of the previously characterized E . coli thioredoxin (Trx1) . The enzymatic activity of Trx2 as a protein-disulfide reductase is increased by preincubation with dithiothreitol, suggesting that oxidation of cysteine residues other than the ones in the active site might regulate its activity . A truncated form of the protein, lacking the N-terminal domain, is insensitive to the presence of dithiothreitol, further confirming the involvement of the additional cysteine residues in modulating Trx2 activity . In addition, the presence of the N-terminal domain appears to confer heat sensitivity to Trx2, unlike Trx1 . Finally, Trx2 is present normally in growing E . coli cells as shown by Western blot analysis.

Microbiology, 1997 Dec, 143 ( Pt 12), 3683 - 92
A mutation that affects fibril protein, development, cohesion and gene expression in Myxococcus xanthus; Smith DR et al.; Extracellular matrix fibrils are involved in the cell-cell interactions of the social prokaryote, Myxococcus xanthus . The fibrils are composed of a carbohydrate backbone and a set of five integral fibrillar proteins (IFPs) ranging from 14 to 66 kDa . As part of an attempt to understand the function(s) of the IFPs, a mutant (ifp-1:20) was generated that lacks IFP-1:20, one of the fibril proteins, as shown by Western blot analysis of both whole cells and isolated fibrils . Unlike those of the parent strain, the fibrils of the mutant were removed easily from the cells by shear forces . Development in ifp-1:20 was aberrant--aggregation and early mound formation were delayed by 6-10 h and mature fruiting bodies never formed . Myxospore production was also greatly reduced . Additionally, fibril-mediated cohesion in ifp-1:20 was changed . Cohesion resulted in chains of cells rather than the characteristic clumps of cells seen for the parent strain . Isolated ifp-1:20 fibrils, unlike wild-type fibrils, could not rescue cohesion of non-cohesive, fibril-negative dsp cells, supporting the notion that the fibrils were functionally altered . The mutation also reduced developmental gene expression by three- to fourfold in omega 4521, a transposon insertion mutant expressed early in development . Expression of a later developmental gene fusion was not affected, suggesting that the fibrils may not be required for later developmental gene expression . These data suggest that intact fibrils may function early in development to facilitate close cell proximity for signal exchange.

J Soc Gynecol Investig, 1994 Apr-Jun, 1(2), 143 - 9
Expression of engineered human 17 beta-estradiol dehydrogenase in a prokaryotic system; O'Shea DL et al.; OBJECTIVE: 17 beta estradiol dehydrogenase (17 beta DH) is a model for pyridine-dependent steroid-converting enzymes . To define the structural and functional parameters of 17 beta DH, we created an expression system for production of abundant, homogeneous enzyme . METHODS: A full-length 17 beta DH cDNA clone was engineered into the inducible expression vector pMON 5839 . After induction of plasmid-bearing Escherichia coli JM109 cells, the authenticity of the recombinant human placental 17 beta DH (r17 beta DH) was evaluated . RESULTS: Protein electrophoresis and Western blot analysis confirmed the immunologic identity of r17 beta DH with native human placental enzyme . The amino acid sequence, enzyme activity, Vmax, K(m), and kcat of r17 beta DH matched that of the native enzyme . CONCLUSION: Prokaryotic cell lines offer the opportunity to create an unlimited supply of recombinant human placental 17 beta DH without the expense and time commitment of baculoviral or eukaryotic cell lines . We are now able to use r17 beta DH and its mutants to elucidate the mechanisms of action of this class of enzyme.

J Ind Microbiol Biotechnol, 1997 Sep, 19(3), 202 - 19
Bacillus thuringiensis: from biodiversity to biotechnology; Prieto-Samsonov DL et al.; Bacillus thuringiensis is a Gram-positive bacterium, widely used in agriculture as a biological pesticide . The biocidal activity mainly resides in a parasporal protein inclusion body, or crystal . The inclusion is composed of one or more types of delta-endotoxins (Cry and Cyt proteins) . Cry proteins are selectively toxic to different species from several invertebrate phyla: arthropods (mainly insects), nematodes, flatworms and protozoa . The mode of action of the insecticidal proteins is still a matter of investigation; generally, the active toxin is supposed to bind specific membrane receptors on the insect midgut brush-border epithelium, leading to intestinal cell lysis and subsequent insect death by starvation or septicemia . The toxin-encoding cry genes have been extensively studied and expressed in a large number of prokaryotic and eukaryotic organisms . The expression of such genes in transgenic plants has provided a powerful alternative for crop protection.

Science, 1997 Dec 12, 278(5345), 1960 - 3
Mechanism of transcription through the nucleosome by eukaryotic RNA polymerase; Studitsky VM et al.; Nucleosomes, the nucleohistone subunits of chromatin, are present on transcribed eukaryotic genes but do not prevent transcription . It is shown here that the large yeast RNA polymerase III transcribes through a single nucleosome . This takes place through a direct internal nucleosome transfer in which histones never leave the DNA template . During this process, the polymerase pauses with a pronounced periodicity of 10 to 11 base pairs, which is consistent with restricted rotation in the DNA loop formed during transfer . Transcription through nucleosomes by the eukaryotic enzyme and by much smaller prokaryotic RNA polymerases thus shares many features, reflecting an important property of nucleosomes.

Biotechnol Prog, 1997 Nov-Dec, 13(6), 699 - 708
Controlling messenger RNA stability in bacteria: strategies for engineering gene expression; Carrier TA et al.; Recent advances in the understanding of prokaryotic gene expression have led scientists to look beyond traditional promoter control for new methods of regulating gene expression . A promising, new technique centers on controlling the stability of messenger RNA . To exploit the potential of mRNA stability for gene expression control, it is important to understand the mechanisms of prokaryotic mRNA decay as well as the cellular factors that can be used to enhance bacterial gene expression through mRNA stabilization . Factors involved in controlling prokaryotic mRNA stability such as nucleases, secondary structures, translation influences, and transcription effects are discussed and analyzed within the context of three prevailing mRNA decay theories . Several strategies for manipulating mRNA stability in genetically-engineered cells are developed from these discussions and presented as a future direction in gene expression control . In the near future, it should be possible to use these strategies to control mRNA stability in such applications as pharmaceutical protein production and metabolic pathway design.

Genetics, 1997 Dec, 147(4), 1557 - 68
The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism; McDonald JP et al.; Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins . Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S . cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis . Similar to its prokaryotic homologues, RAD30 was found to be damage inducible . Like many S . cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants . However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains . While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group . These findings suggest that RD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5.

RNA, 1997 Nov, 3(11), 1233 - 47
Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase; Bokar JA et al.; The methylation of internal adenosine residues in eukaryotic mRNA, forming N6-methyladenosine (m6A), is catalyzed by a complex multicomponent enzyme . Previous studies suggested that m6A affects the efficiency of mRNA processing or transport, although the mechanism by which this occurs is not known . As a step toward better understanding the mechanism and function of this ubiquitous posttranscriptional modification, we have shown that HeLa mRNA (N6-adenosine)-methyltransferase requires at least two separate protein factors, MT-A and MT-B, and MT-A contains the AdoMet binding site on a 70-kDa subunit (MT-A70) . MT-A70 was purified by conventional chromatography and electrophoresis, and was microsequenced . The peptide sequence was used to design a degenerate oligodeoxynucleotide that in turn was used to isolate the cDNA clone coding for MT-A70 from a HeLa cDNA library . Recombinant MT-A70 was expressed as a fusion protein in bacteria and was used to generate anti-MT-A70 antisera in rabbits . These antisera recognize MT-A70 in HeLa nuclear extracts by western blot and are capable of depleting (N6-adenosine)-methyltransferase activity from HeLa nuclear extract, confirming that MT-A70 is a critical subunit of (N6-adenosine)-methyltransferase . Northern blot analysis reveals that MT-A70 mRNA is present in a wide variety of human tissues and may undergo alternative splicing . MT-A70 cDNA probe hybridizes to a 2.0-kilobase (kb) polyadenylated RNA isolated from HeLa cells, whereas it hybridizes to two predominant RNA species (approximately 2.0 kb and 3.0 kb) using mRNA isolated from six different human tissues . Analysis of the cDNA sequence indicates that it codes for a 580-amino acid protein with a predicted MW = 65 kDa . The predicted protein contains sequences similar to consensus methylation motifs I and II identified in prokaryotic DNA (N6-adenosine)-methyltransferases, suggesting the functional conservation of peptide motifs . MT-A70 also contains a long region of homology to the yeast protein SPO8, which is involved in induction of sporulation by an unknown mechanism.

Microbiol Mol Biol Rev, 1997 Dec, 61(4), 393 - 410
Arac/XylS family of transcriptional regulators; Gallegos MT et al.; The ArC/XylS family of prokaryotic positive transcriptional regulators includes more than 100 proteins and polypeptides derived from open reading frames translated from DNA sequences . Members of this family are widely distributed and have been found in the gamma subgroup of the proteobacteria, low- and high-G + C-content gram-positive bacteria, and cyanobacteria . These proteins are defined by a profile that can be accessed from PROSITE PS01124 . Members of the family are about 300 amino acids long and have three main regulatory functions in common: carbon metabolism, stress response, and pathogenesis . Multiple alignments of the proteins of the family define a conserved stretch of 99 amino acids usually located at the C-terminal region of the regulator and connected to a nonconserved region via a linker . The conserved stretch contains all the elements required to bind DNA target sequences and to activate transcription from cognate promoters . Secondary analysis of the conserved region suggests that it contains two potential alpha-helix-turn-alpha-helix DNA binding motifs . The first, and better-fitting motif is supported by biochemical data, whereas existing biochemical data neither support nor refute the proposal that the second region possesses this structure . The phylogenetic relationship suggests that members of the family have recruited the nonconserved domain(s) into a series of existing domains involved in DNA recognition and transcription stimulation and that this recruited domain governs the role that the regulator carries out . For some regulators, it has been demonstrated that the nonconserved region contains the dimerization domain . For the regulators involved in carbon metabolism, the effector binding determinants are also in this region . Most regulators belonging to the AraC/XylS family recognize multiple binding sites in the regulated promoters . One of the motifs usually overlaps or is adjacent to the -35 region of the cognate promoters . Footprinting assays have suggested that these regulators protect a stretch of up to 20 bp in the target promoters, and multiple alignments of binding sites for a number of regulators have shown that the proteins recognize short motifs within the protected region.

J Biol Chem, 1997 Nov 28, 272(48), 30350 - 5
The cysteine-peptidase bleomycin hydrolase is a member of the galactose regulon in yeast; Zheng W et al.; Bleomycin hydrolase is a cysteine peptidase discovered through its ability to detoxify the anti-cancer glycopeptide, bleomycin . Although found in all tissues in mammals and in both eukaryotes and prokaryotes, the normal cellular function of this peptidase is not known . We had previously reported the purification of bleomycin hydrolase from yeast based on its unexpected ability to bind DNA . Recently we collaborated in solving the crystal structure of this protein, revealing a hexameric ring organization . We now report that the molecular characterization of the gene encoding yeast bleomycin hydrolase is also surprising . The transcription of the gene is regulated by galactose . Furthermore, this regulation is conveyed by a binding site for the Gal4 regulatory protein in its promoter, prompting the designation of this gene as GAL6 . Gal6p also appears to have a negative effect on the GAL system as a deletion of the gene leads to a 2-5-fold higher expression of the GAL1, GAL2, GAL7, and MEL1 genes . The GAL6 deletion does not affect the expression of another inducible gene, HSP26 . Neither the peptidase nor the nucleic acid binding activity of Gal6p as assayed is apparently required to convey this regulation, implying yet another function for this new member of the GAL regulon.

J Biol Chem, 1997 Nov 21, 272(47), 29584 - 9
Identification and characterization of the functional amino acids at the active site of the large thioredoxin reductase from Plasmodium falciparum; Gilberger TW et al.; The thioredoxin system, composed of the pyridine nucleotide-disulfide oxidoreductase thioredoxin reductase, the small peptide thioredoxin, and NADPH as a reducing cofactor, is one of the major thiol-reducing systems of the cell . Recent studies revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, called large thioredoxin reductases . The large thioredoxin reductases are substantially different from the isofunctional prokaryotic Escherichia coli enzyme . The putative essential amino acids at the catalytic center of large thioredoxin reductase from P . falciparum were determined by using site-directed mutagenesis techniques . To analyze the putative active site cysteines (Cys88 and Cys93) three mutant proteins were constructed substituting alanine or serine residues for cysteine residues . Further, to evaluate the function of His509 as a putative proton donor/acceptor of large thioredoxin reductase this residue was replaced by either glutamine or alanine . All mutants were expressed in the E . coli system and characterized . Steady state kinetic analysis revealed that the replacement of Cys88 by either alanine or serine and Cys93 by alanine resulted in a total loss of enzymatic activity . These results clearly identify Cys88 and Cys93 as the active site thiols of large thioredoxin reductase . The replacement of His509 by glutamine yielded in a 95% loss of thioredoxin reductase activity; replacement by alanine provoked a loss of 97% of enzymatic activity . These results identify His509 as active site base, but imply that its function can be substituted, although inefficiently, by an alternative proton donor, similar to glutathione reductase . Spectral analysis of wild-type P . falciparum thioredoxin reductase revealed a 550-nm absorption band upon reduction which resembles the EH2 form of glutathione reductase and lipoamide dehydrogenase . This spectral feature, recently also reported for the human placenta protein (Arscott, L . D., Gromer, S., Schirmer, R . H., Becker K., and Williams, C . H., Jr . (1997) Proc . Natl . Acad . Sci . U . S . A . 94, 3621-3626), further illustrates the similarity between large thioredoxin reductases and glutathione reductases and stresses the profound differences to small E . coli thioredoxin reductase.

Science, 1997 Nov 21, 278(5342), 1467 - 70
Sec-independent protein translocation by the maize Hcf106 protein; Settles AM et al.; The bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts . A third translocation mechanism in chloroplasts {the proton concentration difference (DeltapH) pathway} was previously thought to be unique . The hcf106 mutation of maize disrupts the localization of proteins transported through this DeltapH pathway in isolated chloroplasts . The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the DeltapH pathway has been conserved since the endosymbiotic origin of chloroplasts . Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.

FEMS Microbiol Rev, 1997 Sep, 21(2), 157 - 78
Transposition and site-specific recombination: adapting DNA cut-and-paste mechanisms to a variety of genetic rearrangements; Hallet B et al.; In bacteria, two categories of specialised recombination promote a variety of DNA rearrangements . Transposition is the process by which genetic elements move between different locations of the genome, whereas site-specific recombination is a reaction in which DNA strands are broken and exchanged at precise positions of two target DNA loci to achieve determined biological function . Both types of recombination are represented by diverse genetic systems which generally encode their own recombination enzymes . These enzymes, generically called transposases and site-specific recombinases, can be grouped into several families on the basis of amino acid sequence similarities, which, in some cases, are limited to a signature of a few residues involved in catalysis . The well characterised site-specific recombinases are found to belong to two distinct groups whereas the transposases form a large super-family of enzymes encompassing recombinases from both prokaryotes and eukaryotes . In spite of important differences in the catalytic mechanisms used by these three classes of enzymes to cut and rejoin DNA molecules, similar strategies are used to coordinate the biochemical steps of the recombination reaction and to control its outcome . This review summarises our current understanding of transposition and site-specific recombination, attempting to illustrate how relatively conserved DNA cut-and-paste mechanisms can be used to bring about a variety of complex DNA rearrangements.

Appl Environ Microbiol, 1997 Dec, 63(12), 4859 - 65
Detection of stable pre-rRNA in toxigenic Pseudo-nitzschia species; Cangelosi GA et al.; Nucleotide sequence analysis of ribosomal DNA (rDNA) spacer regions is useful for taxonomic comparisons of closely related microorganisms . These regions have been less useful for routine microbial identification and detection, partly because rRNA precursors (pre-rRNAs) in microbial cells are assumed to be too labile to be detectable by high-throughput probe hybridization methods . We characterized the sequence diversity and physiological stability of pre-rRNA in the toxigenic marine diatoms Pseudo-nitzschia australis, P . multiseries, and P . pungens . As with nucleotide sequences of the first internal transcribed spacer (ITS1) reported previously, sequences of ITS2 and the 5' external transcribed spacer (ETS1) exhibited considerable divergence among these species, including large insertions-deletions detectable by PCR-based spacer length analysis . In slot blot hybridization assays on RNA extracted from lysates of Pseudo-nitzschia cells, oligonucleotide probes directed to pre-rRNA spacers generated much stronger signals than did complementary probes directed to the coding strands of the rDNAs, indicating that the pre-rRNA-targeted probes detected multicopy transcripts . A group of probes directed to a discrete 90-base region within the ITS1 pre-rRNA gave no detectable signal, suggesting that this region is degraded early in the rRNA maturation pathway . Other pre-rRNA regions were always detectable and, in marked contrast to prokaryotic systems analyzed in this manner, were stable and abundant in both actively dividing and nondividing cells . Long, multilabeled RNA probes, which would exhibit considerable cross-reactivity if directed to mature rRNA sequences, detected species-specific pre-rRNA sequences from as few as 1,000 cells . Pre-rRNA is a potentially useful molecular target for detecting and identifying Pseudo-nitzschia species and possibly other unicellular eukaryotes as well.

Acta Biol Hung, 1997, 48(3), 339 - 58
The physiology and biosynthesis of secondary metabolites; Vanek Z et al.; Recently, several excellent reviews appeared /9, 12, 15, 29/ summarizing new findings of the last years and describing the topic in the new interconnections . The signalling systems through which changes in environmental conditions affecting the growth of microorganism are sensed, transmitted and converted into mechanisms controlling the production of antibiotic can now be investigated with the techniques of molecular genetics . Evidence has been accumulated that demonstrated the key roles played by diffusible molecules in regulating cellular differentiation even among prokaryotic microorganisms . This is exemplified by A-factor and its analogues, which act as autoregulators for morphological differentiation and secondary metabolism in Streptomyces . In our article we have concentrated on the physiological changes, which can occur during the growth in natural environment or during cultivation under laboratory conditions . Recent evidence for the presence of molecular signalling systems in Streptomyces is reviewed, along with the inherent implications . The constitution of the metabolic type during the first hours of cultivation has been previously reviewed /36/.

Comp Biochem Physiol A Physiol, 1997 Nov, 118(3), 475 - 9
Acidostable and acidophilic proteins: the example of the alpha-amylase from Alicyclobacillus acidocaldarius; Matzke J et al.; Acidophilic microorganisms grow optimally at pH values between 1-4 . They have adapted to the acid condition by maintaining their cytoplasmic pH at a value close to neutrality . Hence, only those (macro)-molecules, which face the acid medium, have had to adapt to this extreme condition . Literature data show that several exoproteins from thermoacidophilic prokaryotes are characterized by a low charge density . It is proposed that this property contributes to the stability of these proteins both below and above the pKa-values of their glutamate and aspartate residues . As an example of an acidophilic protein, the alpha-amylase from the Gram-positive Alicyclobacillus acidocaldarius ATCC27009 was studied . The enzyme is thermoacidophilic, with optima of temperature and pH of 75 degrees C and pH 3, respectively . The nucleotide sequence of the cloned gene (8) indicates that the alpha-amylase belongs to a large family of starch-degrading enzymes with a characteristic catalytic (beta alpha)8-domain . Three essential and probably catalytic acidic residues have been conserved, suggesting that the acidophilic alpha-amylase degrades starch with essentially the same mechanism as do its neutrophilic relatives . Still, the acidophilic protein contains three exchanges in residues uniformally or almost uniformally conserved among all members of the enzyme family . In order to test whether these exchanges contribute to the acidic pH optimum, the alpha-amylase gene was expressed in Escherichia coli . Sonication of the enzyme-producing cells released alpha-amylase activity associated with a 140 kDa protein . The optima of temperature and pH for the protein produced in E . coli were similar to those of the native enzyme . Experiments are underway in which it is tested which residues contribute to the acid pH optimum of the alpha-amylase.

Med Microbiol Immunol (Berl), 1997 Oct, 186(2-3), 125 - 34
Cloning and functional characterization of the genes encoding 3-dehydroquinate synthase (aroB) and tRNA-guanine transglycosylase (tgt) from Helicobacter pylori; Bereswill S et al.; The aroB gene from Helicobacter pylori strain P1 was cloned and further characterized by sequence analysis and by functional complementation of the aroB mutation in Escherichia coli . The aroB gene encodes the enzyme 3-dehydroquinate synthase which catalyzes one of the early steps in the shikimate pathway . This pathway, which creates aromatic molecules from sugar precursors, is present in prokaryotes, fungi and plants but is absent from mammalian cells . The predicted amino acid sequence of the H . pylori aroB gene product showed significant homology (30-40% identity and 50-60% similarity) to 3-dehydroquinate synthases from various other prokaryotes and eukaryotes . The single gene on a plasmid was biologically active in E . coli . It suppressed the specific phenotype of aroB mutants by restoring the shikimate pathway-dependent synthesis of aromatic amino acids and the production of the siderophore enterobactin . Two other reading frames were found adjacent to the aroB gene . The first, designated as orf1, had no significant homology to proteins and genes present in databases, whereas the second was found to share a significant degree of homology with the tgt gene encoding tRNA-guanine transglycosylase from a variety of other bacteria (40-50% identity and 60-70% similarity) . The function of the tgt gene was confirmed by heterologous complementation . The gene on a plasmid was shown to complement the queuosine biosynthesis defect in a genetically defined tgt- strain of E . coli . The presence of the aroB gene and the putative tgt homologue in unrelated H . pylori strains was confirmed by Southern blot hybridization and by polymerase chain reaction with specific primers.

Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 251 - 9
Molecular evolution and optimization; Trevors JT; Microbial populations (and life) not only evolve, they optimize . The transition from a random, unorganized, lifeless Earth to the present situation, where the Earth is virtually covered with nucleic acids and diverse and complex species, required numerous molecular changes and the integration of metabolic pathways over billions of years . Primitive prokaryotic life was dependent on and constrained by the physical-chemical conditions on the Earth, while slowly reshaping conditions present . In this review, molecular evolution and molecular optimization are examined with an emphasis on the order in which evolutionary events occurred.

Antonie Van Leeuwenhoek, 1997 Oct, 72(3), 209 - 18
Evolutionary advances in the higher fungi; Moore RT; In this report the phrase 'evolutionary advances' is used in three ways: 1 . to describe monophyletic changes perceived within a lineage; 2 . to describe evolutionary sequences that appear to have become parallel/convergent; 3 . to describe major transitions inferred between primary taxa . In monophyletic evolution the changes occur within a specific lineage that arises from a common ancestor, e.g., modern Man, horses, rusts . In parallel/convergent evolution different lineages respond similarly over time to environmental challenges and opportunities and come to acquire a great deal of comparability (e.g., Webster, 1987) . Such lineages may be designated as separate taxa, e.g., similarities in marsupial and placental carnivores, or, if the polyphyleticism is cryptic, as a collective taxon, e.g., Aves, the class of birds, the obsolete Amentiferae for catkin bearing plants, the Gasteromycetes, and lichens . In major transitions there are significant paradigm shifts in which evolutionary changes from one predominant life style pattern to another are accompanied by increases in complexity (see Smith & Szathary, 1995), e.g., symbiosis, the water to land transition, the changes between the phyla of land fungi . Three particular terms are used in evaluating evolutionary relationships (Moore, 1996a): homology, paramology, and analogy . Homology, from Darwin's theory of common descent, is the phenomenon of having a common historical origin but not necessarily the same final structure or function (e.g., vertebrate forelimbs) . Paramology (Moore, 1971) applies to inferred relationships in evolutionary schemes based on contemporary forms that lack fossil antecedents, e.g., the various phylogenetic interpretations of prokaryotes, algae, and fungi; Boekhout et al . (1993) have evaluated the taxonomic resolution of a variety of morphologic, biochemical, physiological, and molecular characters (Table 1) . Analogy is generally applied to similar forms that are unrelated, e.g., insect/vertebrate wings; prokaryote/eukaryote flagella . It should also be borne in mind that, in a given taxon, biotrophism (Coffey, 1975) is an advanced character (Health, 1987) over, respectively, weaker parasitism, symbiotism, commensalism, and freeliving and that seemingly simple or less differentiated forms can be, and more than likely than not are, reduced, polyphyletic, and specialized rather than ancient and rudimentary, e.g., yeasts (Hoog et al., 1988; Kurtzman & Fell, 1996; Moore, 1988b; 1996a).

Mol Microbiol, 1997 Nov, 26(3), 423 - 31
The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation?
Santos MA, Ueda T, Watanabe K, Tuite MF.
A number of yeasts of the genus Candida translate the standard leucine-CUG codon as serine . This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon . Translation of CUG as serine in these species is mediated by a novel serine-tRNA (ser-tRNACAG), which uniquely has a guanosine at position 33, 5' to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs . We propose that G-33 has two important functions: lowering the decoding efficiency of the ser-tRNACAG and preventing binding of the leucyl-tRNA synthetase . This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon . In addition, the novel ser-tRNACAG has 1-methylguanosine (m1G-37) at position 37, 3' to the anticodon, which is characteristic of leucine, but not serine tRNAs . Remarkably, m1G-37 causes leucylation of the ser-tRNACAG both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon . This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment . We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.

J Bacteriol, 1997 Dec, 179(24), 7705 - 11
Cloning, functional analysis, and transcriptional regulation of the Bacillus subtilis araE gene involved in L-arabinose utilization; Sa-Nogueira I et al.; The Bacillus subtilis araR locus (mapped at about 294 degrees on the genetic map) comprises two open reading frames with divergently arranged promoters, the regulatory gene, araR, encoding a repressor, and a partially cloned gene, termed araE by analogy to the Escherichia coli L-arabinose permease gene . Here, we report the cloning and sequencing of the entire araE gene encoding a 50.4-kDa polypeptide . The araE gene is monocistronic (as determined by Northern blot analysis), and its putative product is very similar to a number of prokaryotic proton-linked monosaccharide transporters (the group I family of membrane transport proteins) . Insertional inactivation of the araE gene leads to a conditional Ara- phenotype dependent on the concentration of L-arabinose in the medium . Therefore, we assume that araE encodes a permease involved in L-arabinose transport into the cell . The araE promoter region contains -10 and -35 regions (as determined by primer extension analysis) very similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigmaA, and the -35 region of the transcription start point for araE is located 2 bp from the -35 region of the araR gene . Transcriptional studies demonstrated that the expression from the araE promoter is induced by L-arabinose, repressed by glucose, and negatively regulated by AraR . These observations are consistent with a model according to which in the absence of L-arabinose, AraR binds to a site(s) within the araE/araR promoter, preventing transcription from the araE promoter and simultaneously limiting the frequency of initiation from its own promoter; the addition of L-arabinose will allow transcription from the araE promoter and increase the frequency of initiation from the araR promoter.

J Gen Virol, 1997 Dec, 78 ( Pt 12), 3141 - 5
Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y; Jakab G et al.; A full-length cDNA clone of the potato virus Y (PVY) genome was obtained after cloning difficulties in Escherichia coli were overcome . These difficulties were mainly due to the expression of the CI gene from upstream prokaryotic promoter-like elements within the PVY genome . To overcome this problem, PVY cDNA was maintained in two subclones which were ligated before infection . A plasmid in which these two fragments were contained could be propagated in some E . coli strains but was unstable and yielded only low levels of plasmid DNA . Replacement of the 35S promoter by the SP6 promoter slightly increased the stability of the plasmid and its RNA transcripts were infectious when capped with m7G(5')ppp(5')G . Using two inoculation methods (mechanical or biolistic) the cDNA and its RNA transcript proved infectious on three Nicotiana species and on Solanum tuberosum.

J Gen Virol, 1997 Dec, 78 ( Pt 12), 3125 - 34
Excision of the polydnavirus chromosomal integrated EP1 sequence of the parasitoid wasp Cotesia congregata (Braconidae, Microgastinae) at potential recombinase binding sites; Savary S et al.; Cotesia congregata polydnavirus (CcPDV) is essential for successful parasitism of Manduca sexta larvae by the braconid wasp Cotesia congregata . To determine the molecular mechanisms for the vertical transmission of CcPDV in the wasps, we analysed the different forms of the virus sequences containing the gene encoding the early parasitism-specific protein 1 (EP1) . By a detailed molecular analysis, we demonstrated that the EP1 sequences are present in wasp DNA in two forms: a circular form as seen in the virus particles and a form integrated into the wasp genome . Moreover, we showed that the integrated form of the EP1 sequences is able to excise in the ovary cells . A fragment corresponding to an EP1 'empty locus' (without the viral sequence) was PCR-amplified from ovarian DNA . Comparison of the sequences isolated from the EP1 circle, the integrated form and the empty locus revealed that the extremities of the EP1 genomic sequences constitute a direct repeat . Strikingly, these sequences contain a potential binding site for a recombinase of the Hin family located in close vicinity to the position where the DNA strand exchange occurs . Thus, the data bear upon the possibility that the bracovirus circles are excised via a mechanism related to the Hin mediated Conservative Specific-Specific Recombination (CSSR) of prokaryotes.

Biochemistry, 1997 Dec 2, 36(48), 14827 - 35
Structural and mechanistic studies on chloroplast translational initiation factor 3 from Euglena gracilis; Yu NJ et al.; Chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis contains a central region (homology domain) that is homologous to prokaryotic IF3 . The homology domain is preceded by a long NH2-terminal extension (head), and followed by a 64 amino acid COOH-terminal extension (tail) . Sequences in these extensions reduce the activity of the homology domain . To gain insight into these effects, a possible three-dimensional structure for the homology region of IF3chl has been modeled using the X-ray coordinates from the N- and C-domains of Bacillus stearothermophilus IF3 . In B . stearothermophilus IF3, these two compact domains are thought to fold independently and are separated by a helical lysine-rich linker . The modeled structure suggests that IF3chl has a similar overall fold although some subtle differences are predicted to occur . Both the head and tail regions of IF3chl are oriented toward the linker region in the homology domain where they may potentially interfere with its function . Circular dichroism spectropolarimetry (CD) indicates that the lysine-rich linker region in IF3chl is not in a helical conformation and is probably a random coil . CD analysis indicates that a portion of the tail region of IF3chl is helical and that the tail has a direct interaction with the linker region in the homology domain . Site-directed mutagenesis of the linker indicates that two conserved lysine residues are important for the function of IF3chl and play a role in the binding of IF3chl to the 30S ribosomal subunit . Mutation of these residues affects the interaction of the homology domain with the tail.

Proc Natl Acad Sci U S A, 1997 Nov 25, 94(24), 12803 - 8
The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis; Durbecq V et al.; Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia . The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK) . In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine . We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000 . Its thermostability is considerable, 50% activity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C . The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs . The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P . furiosus CPS from ordinary CKs . Thus the CPS of P . furiosus could represent a primeval step in the evolution of CPS from CK . Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs . The gene coding for this CK-like CPS was called cpkA.

J Bacteriol, 1997 Dec, 179(23), 7233 - 42
Host cell phospholipids are trafficked to and then modified by Chlamydia trachomatis; Wylie JL et al.; There is little information on the trafficking of eukaryotic lipids from a host cell to either the cytoplasmic membrane of or the vacuolar membrane surrounding intracellular pathogens . Purified Chlamydia trachomatis, an obligate intracellular bacterial parasite, contains several eukaryotic glycerophospholipids, yet attempts to demonstrate transfer of these lipids to the chlamydial cell membrane have not been successful . In this report, we demonstrate that eukaryotic glycerophospholipids are trafficked from the host cell to C . trachomatis . Phospholipid trafficking was assessed by monitoring the incorporation of radiolabelled isoleucine, a precursor of C . trachomatis specific branched-chain fatty acids, into host-derived glycerophospholipids and by monitoring the transfer of host phosphatidylserine to chlamydiae and its subsequent decarboxylation to form phosphatidylethanolamine . Phospholipid trafficking to chlamydiae was unaffected by brefeldin A, an inhibitor of Golgi function . Furthermore, no changes in trafficking were observed when C . trachomatis was grown in a mutant cell line with a nonfunctional, nonspecific phospholipid transfer protein . Host glycerophospholipids are modified by C . trachomatis, such that a host-synthesized straight-chain fatty acid is replaced with a chlamydia-synthesized branched-chain fatty acid . We also demonstrate that despite the acquisition of host-derived phospholipids, C . trachomatis is capable of de novo synthesis of phospholipids typically synthesized by prokaryotic cells . Our results provide novel information on chlamydial phospholipid metabolism and eukaryotic cell lipid trafficking, and they increase our understanding of the evolutionary steps leading to the establishment of an intimate metabolic association between an obligate intracellular bacterial parasite and a eukaryotic host cell.

Yeast, 1997 Nov, 13(14), 1363 - 74
Characterization of new proteins found by analysis of short open reading frames from the full yeast genome; Andrade MA et al.; We have analysed short open reading frames (between 150 and 300 base pairs long) of the yeast genome (Saccharomyces cerevisiae) with a two-step strategy . The first step selects a candidate set of open reading frames from the DNA sequence based on statistical evaluation of DNA and protein sequence properties . The second step filters the candidate set by selecting open reading frames with high similarity to other known sequences (from any organism) . As a result, we report ten new predicted proteins not present in the current sequence databases . These include a new alcohol dehydrogenase, a protein probably related to the cell cycle, as well as a homolog of the prokaryotic ribosomal protein L36 likely to be a mitochondrial ribosomal protein coded in the nuclear genome . We conclude that the analysis of short open reading frames leads to biologically interesting discoveries, even though the quantitative yield of new proteins is relatively low.

Pac Symp Biocomput . 1996;:461-71.
Sequence sizes of eukaryotic enzymes; Kolker E et al.; We have shown in earlier studies that an appreciable fraction of proteins display sequence size periodicity with periods of approximately 123 aa and approximately 152 aa for eukaryotes and prokaryotes, respectively . For any firm conclusions to be made, the issue of possible bias due to an overabundance of some protein families should be addressed in more than one way . Here we present the size distributions for various sequence ensembles of eukaryotic enzymes that differ by level of data bank cleaning . The sequences were purged by applying several successive thresholds of relatedness irrespective of the sequence lengths . The previously observed preference to typical sizes is confirmed . Possible reasons for the observed excess of the typical size sequences are discussed.

Biochim Biophys Acta, 1997 Sep 4, 1348(1-2), 236 - 44
Phosphatidylserine decarboxylase; Voelker DR; Phosphatidylserine decarboxylase (PSD) is an important enzyme in the synthesis of phosphatidylethanolamine in both prokaryotes and eukaryotes . The cloned bacterial gene encodes an integral membrane protein that is first made as a proenzyme, and subsequently proteolyzed to an alpha subunit, containing a pyruvoyl prosthetic group, and a beta subunit . Two types of decarboxylases are found in yeast, PSD1 and PSD2, that localize to the inner mitochondrial membrane and the Golgi/vacuole membrane, respectively . The mammalian enzyme is also found in the inner mitochondrial membrane . The yeast genes and mammalian cDNA have been cloned and sequenced . The yeast genes contain 5' sequences associated with regulation of expression by inositol and choline . The yeast PSD1 and the mammalian PSD both contain an LGST amino acid motif that identifies the site of proteolysis and pyruvoyl prosthetic group attachment in the bacterial enzyme . The yeast PSD1 and mammalian PSD also have mitochondrial targeting and inner membrane sorting sequences . Processing intermediates have been defined in the mammalian enzyme that correspond to the sequential removal of the mitochondrial targeting and inner membrane sorting sequence, followed by formation of the alpha and beta subunits . In contrast, the PSD2 enzyme contains a putative Golgi localization/retention sequence and a C2 homology domain, in addition to predicted alpha and beta subunits . The transport requirements for substrate access to the PSD enzymes have provided important information about lipid trafficking, and the availability of yeast mutants is likely to provide important new genetic selections in the future.

Gene, 1997 Oct 1, 198(1-2), 289 - 96
A genetic selection for isolating cDNAs encoding secreted proteins; Jacobs KA et al.; We describe a simple, rapid technique for simultaneously isolating large numbers of cDNAs encoding secreted proteins . The technique makes use of a facile genetic selection performed in a strain of Saccharomyces cerevisiae deleted for its endogenous invertase gene . A cDNA cloning vector which carries a modified invertase gene lacking its leader sequence is used in conjunction with this strain . Heterologous secreted genes fused appropriately upstream of this defective invertase provide the necessary signals to restore secretion, allowing the yeast to grow on sugars such as sucrose or raffinose . This microbial growth selection facilitates scanning cDNA libraries containing millions of clones, enabling the wholesale identification of novel secreted proteins without the need for specific bioassays . The technique is similar to one previously described (Klein et al . (1996) Proc . Natl . Acad . Sci . USA 93, 7108-7113) . We describe results using a cDNA library derived from activated human peripheral blood mononuclear cells (PBMC) . Genes identified from this library encoded signal sequences of proteins of diverse structure, function, and cellular location such as cytokines, type 1 and type 2 transmembrane proteins, and proteins found in intracellular organelles . In addition, a number of novel secreted proteins were identified, including a chemokine and a novel G-protein-coupled receptor . Since signal sequences possess features conserved throughout evolution, the procedure can be used to isolate genes encoding secreted proteins from both eukaryotes and prokaryotes.

FEBS Lett, 1997 Oct 13, 416(1), 93 - 8
Slight sequence variations of a common fold explain the substrate specificities of tRNA-guanine transglycosylases from the three kingdoms; Romier C et al.; tRNA-guanine transglycosylases (TGTs) are the enzymes catalyzing the base exchange required for the synthesis of the modified bases derived from 7-deazaguanine in prokaryotic, archaebacterial, and eukaryotic tRNAs . Unlike the eukaryotic and archaebacterial enzymes, the prokaryotic TGTs have been clearly identified and highly characterized both biochemically and structurally . The recent occurrence in sequence databases of archaebacterial and eukaryotic proteins homologous to the prokaryotic TGTs reveals that all TGTs unexpectedly adopt a common fold . Observed sequence variations at the active site correlate well with their specificities for the various 7-deazaguanine derivatives and the total conservation of the catalytic residues strongly favors a common catalytic mechanism for all TGTs.

Curr Biol, 1997 Oct 1, 7(10), R656 - 9
Genome sequences: genome sequence of a model prokaryote; Koonin EV; The complete Escherichia coli genome sequence is now known; it should greatly facilitate the analysis of other genomes, but a lot remains to be learnt about E . coli itself . About half the genes were previously uncharacterized, but expanding databases and improving analysis methods will help predict their functions.

Mol Microbiol, 1997 Sep, 25(5), 859 - 69
Localized reversible frameshift mutation in an adhesin gene confers a phase-variable adherence phenotype in mycoplasma; Zhang Q et al.; The variable adherence-associated (Vaa) antigen of Mycoplasma hominis is an abundant surface lipoprotein adhesin that may mediate important interactions of this wall-less prokaryotic pathogen with the human host . Extensive mutational variation of Vaa size, as well as sequence and antigenic divergence, has been described previously . Using a series of clonal isolates representing an isogenic lineage of variants oscillating in Vaa expression, Vaa is further shown in this study to undergo high-frequency phase variation in expression, which correlated precisely with the ability of M . hominis to adhere to cultured human cells . Although no DNA rearrangements or sequence differences in the 5' regions flanking vaa alleles were detected between Vaa+ and Vaa variants, intragenic vaa sequences from this lineage revealed an oscillating mutation involving a single nucleotide deletion/insertion in a short tract of adenine residues near the 5' end of the mature Vaa coding sequence, which created a translational frameshift resulting in either a complete Vaa ORF or an in-frame UAG stop codon immediately downstream of the poly-A tract . Evidence for the occurrence of this high-frequency frameshift mutation in vivo was obtained from analysis of PCR-generated vaa sequences amplified from the joint synovial fluid of a patient with M . hominis-associated arthritis, which indicated that Vaa phase variation occurs during M . hominis infection in the natural host . These results identify a distinctive frameshift mutator element in the vaa gene that governs M . hominis adherence and highlight the importance of mutational alteration of primary gene products on the mycoplasma surface as a means of generating and maintaining functional diversity in the host.

Appl Environ Microbiol, 1997 Nov, 63(11), 4608 - 11
Use of nucleic acid dyes SYTO-13, TOTO-1, and YOYO-1 in the study of Escherichia coli and marine prokaryotic populations by flow cytometry; Guindulain T et al.; Three nucleic acid dyes (SYTO-13, TOTO-1, and YOYO-1) were tested on cultures of Escherichia coli and marine prokaryote populations . These dyes stain the RNA and DNA in E . coli but only respond to DNA in marine populations, according to the histograms obtained after DNase and RNase treatments.

Appl Environ Microbiol, 1997 Nov, 63(11), 4298 - 303
Synechococcus diversity in the California current as seen by RNA polymerase (rpoC1) gene sequences of isolated strains; Toledo G et al.; Because they are ubiquitous in a range of aquatic environments and culture methods are relatively advanced, cyanobacteria may be useful models for understanding the extent of evolutionary adaptation of prokaryotes in general to environmental gradients . The roles of environmental variables such as light and nutrients in influencing cyanobacterial genetic diversity are still poorly characterized, however . In this study, a total of 15 Synechococcus strains were isolated from the oligotrophic edge of the California Current from two depths (5 and 95 m) with large differences in light intensity, light quality, and nutrient concentrations . RNA polymerase gene (rpoC1) fragment sequences of the strains revealed two major genetic lineages, distinct from other marine or freshwater cyanobacterial isolates or groups seen in shotgun-cloned sequences from the oligotrophic Atlantic Ocean . The California Current low-phycourobilin (CCLPUB) group represented by six isolates in a single lineage was less diverse than the California Current high-phycourobilin (CCHPUB) group with nine isolates in three relatively divergent lineages . The former was found to be the closest known genetic group to Prochlorococcus spp., a chlorophyll b-containing cyanobacterial group . Having an isolate from this group will be valuable for looking at the molecular changes necessary for the transition from the use of phycobiliproteins to chlorophyll b as light-harvesting pigments . Both of the CCHPUB and CCLPUB groups included strains obtained from surface (5 m) and deep (95 m) samples . Thus, contrary to expectations, there was no clear correlation between sampling depth and isolation of genetic groups, despite the large environmental gradients present . To our knowledge, this is the first demonstration with isolates that genetically divergent Synechococcus groups coexist in the same seawater sample.

Biochim Biophys Acta, 1997 Sep 5, 1341(2), 137 - 56
Structure and function of the low Mr phosphotyrosine protein phosphatases; Ramponi G et al.; Phosphotyrosine protein phosphatases (PTPases) catalyse the hydrolysis of phosphotyrosine residues in proteins and are hence implicated in the complex mechanism of the control of cell proliferation and differentiation . The low Mr PTPases are a group of soluble PTPases displaying a reduced molecular mass; in addition, a group of low molecular mass dual specificity (ds)PTPases which hydrolyse phosphotyrosine and phosphoserine/threonine residues in proteins are known . The enzymes belonging to the two groups are unrelated to each other and to other PTPase classes except for the presence of a CXXXXXRS/T sequence motif containing some of the catalytic residues (active site signature) and for the common catalytic mechanism, clearly indicating convergent evolution . The low Mr PTPases have a long evolutionary history since microbial (prokaryotic and eukaryotic) counterparts of both tyrosine-specific and dsPTPases have been described . Despite the relevant number of data reported on the structural and catalytic features of a number of low Mr PTPases, only limited information is presently available on the substrate specificity and the true biological roles of these enzymes, in prokaryotic, yeast and eukaryotic cells.

Biochem Pharmacol, 1997 Oct 15, 54(8), 927 - 36
Role of nitroreductases but not cytochromes P450 in the metabolic activation of 1-nitropyrene in the HepG2 human hepatoblastoma cell line; Silvers KJ et al.; 1-Nitropyrene is an environmental contaminant that is mutagenic in many prokaryotic and eukaryotic systems, including the hypoxanthine-guanosine phosphoribosyl transferase (HGPRT) locus in the human hepatoma cell line HepG2 . Metabolism and DNA adduct formation of {3H}1-nitropyrene in the HepG2 were quantified to understand the role of nitroreduction and/or cytochrome P450-mediated C-oxidation of 1-nitropyrene in DNA adduct formation and mutagenicity . In uninduced HepG2 cells, 10 microM {3H}1-nitropyrene was metabolized principally by nitroreduction to 1-aminopyrene (516 pmol/24 hr/10(6) cells), and by cytochrome P450-mediated C-oxidation to K-region trans-dihydrodiols (37 pmol/24 hr/10(6) cells), 1-nitropyren-3-ol (51 pmol/24 hr/10(6) cells), and 1-nitropyren-6-ol and 1-nitropyren-8-ol (77 pmol/24 hr/10(6) cells) . Pretreatment of the HepG2 cells for 24 hr with 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in a complete change in the metabolism of {3H}1-nitropyrene, with 1-nitropyren-6-ol and 1-nitropyren-8-ol formation (449 pmol/24 hr/10(6) cells) being 80-fold greater than 1-aminopyrene formation (6 pmol/24 hr/10(6) cells) . This increase in C-oxidation of 1-nitropyrene was consistent with increased levels of cytochrome P450 1A . The only DNA adduct detected using the 32P-postlabeling assay in the HepG2 cells administered 1-nitropyrene was N-(2'-deoxyguanosin-8-yl)-1-aminopyrene (dG-C8-AP) . Induction of C-oxidative metabolism through TCDD treatment resulted in a concomitant decrease in dG-C8-AP formation . DNA adducts for oxidized 1-nitropyrene metabolites were not detected in the TCDD-treated HepG2 cells administered 1-nitropyrene, which indicates that cytochrome P450-mediated C-oxidative pathways are detoxification pathways in HepG2 cells.

J Biol Chem, 1997 Nov 7, 272(45), 28531 - 8
Identification of residues of T4 RNase H required for catalysis and DNA binding; Bhagwat M et al.; Bacteriophage T4 RNase H, which removes the RNA primers that initiate lagging strand fragments, has a 5'- to 3'-exonuclease activity on DNA.DNA and RNA.DNA duplexes and an endonuclease activity on flap or forked DNA structures (Bhagwat, M., Hobbs, L . J., and Nossal, N . J . (1997) J . Biol . Chem . 272, 28523-28530) . It is a member of the RAD2 family of prokaryotic and eukaryotic replication and repair nucleases . The crystal structure of T4 RNase H, in the absence of DNA, shows two Mg2+ ions coordinated to the amino acids highly conserved in this family . It also shows a disordered region proposed to be involved in DNA binding (Mueser, T . C., Nossal, N . G., and Hyde, C . C . Cell (1996) 85, 1101-1112) . To identify the amino acids essential for catalysis and DNA binding, we have constructed and characterized three kinds of T4 RNase H mutant proteins based on the possible roles of the amino acid residues: mutants of acidic residues coordinated to each of the two Mg2+ ions (Mg2+-1: D19N, D71N, D132N, and D155N; and Mg2+-2: D157N and D200N); mutants of conserved basic residues in or near the disordered region (K87A and R90A); and mutants of residues with hydroxyl side chains involved in the hydrogen bonding network (Y86F and S153A) . Our studies show that Mg2+-1 and the residues surrounding it are important for catalysis and that Lys87 is necessary for DNA binding.

Genes Dev, 1997 Nov 1, 11(21), 2897 - 909
Identification of three regions essential for interaction between a sigma-like factor and core RNA polymerase; Cliften PF et al.; The cyclic interactions that occur between the subunits of the yeast mitochondrial RNA polymerase can serve as a simple model for the more complex enzymes in prokaryotes and the eukaryotic nucleus . We have used two-hybrid and fusion protein constructs to analyze the requirements for interaction between the single subunit core polymerase (Rpo41p), and the sigma-like promoter specificity factor (Mtf1p) . We were unable to define any protein truncations that retained the ability to interact, indicating that multiple regions encompassing the entire length of the proteins are involved in interactions . We found that 9 of 15 nonfunctional (petite) point mutations in Mtf1p isolated in a plasmid shuffle strategy had lost the ability to interact . Some of the noninteracting mutations are temperature-sensitive petite (ts petite); this phenotype correlates with a precipitous drop in mitochondrial transcript abundance when cells are shifted to the nonpermissive temperature . One temperature-sensitive mutant demonstrated a striking pH dependence for core binding in vitro, consistent with the physical properties of the amino acid substitution . The noninteracting mutations fall into three widely spaced clusters of amino acids . Two of the clusters are in regions with amino acid sequence similarity to conserved regions 2 and 3 of sigma factors and related proteins; these regions have been implicated in core binding by both prokaryotic and eukaryotic sigma-like factors . By modeling the location of the mutations using the partial structure of Escherichia coli sigma70, we find that two of the clusters are potentially juxtaposed in the three-dimensional structure . Our results demonstrate that interactions between sigma-like specificity factors and core RNA polymerases require multiple regions from both components of the holoenzymes.

Trends Biotechnol, 1997 Oct, 15(10), 390 - 4
Biopolymers from marine prokaryotes; Weiner RM; Biopolymers from marine prokaryotes, both Bacteria and Archaea, offer a number of novel material properties and commercial opportunities . The characteristics of marine exopolysaccharides and melanins that enhance the survival ability of the organisms producing them can be exploited for a number of products ranging from emulsifiers to adhesives . In the prokaryotes, the polyhydroxyalkanoates form carbon-storage molecules, but their technological application is entirely different, serving as a potential base material for biodegradable plastics . Marine biopolymers are a significant and undeveloped biological resource.

Plant J, 1997 Sep, 12(3), 697 - 701
A negative selection scheme based on the expression of cytosine deaminase in plastids; Serino G et al.; The enzyme cytosine deaminase (CD) encoded by codA catalyzes deamination of cytosine to uracil . CD is present in prokaryotes and in many eukaryotic micro-organisms, but is absent in higher plants . 5-fluorocytosine (5FC) is metabolized in CD-expressing cells, causing cellular death . A chimeric codA has been introduced into the tobacco plastid genome and 5FC was used to select against tissue culture cells and seedlings expressing CD . This negative selection scheme will be useful in identifying nuclear genes which control plastid gene expression in higher plants.

Int J Vitam Nutr Res, 1997, 67(5), 336 - 42
Oxidative stress and signal transduction; Schulze-Osthoff K et al.; Reactive oxygen intermediates (ROIs) are an evolutionarily ancient threat to all organisms . Both prokaryotic and higher eukaryotic cells are able to alter their genetic program in response to changes in the intracellular levels of ROIs . In bacteria and yeast, this response leads to the new synthesis of proteins that protect cells from the consequences of oxidative damage, such as DNA strand breaks, lipid peroxidation and oxidative damage of proteins . Intriguingly, higher organisms have also evolved cellular mechanisms to actively produce ROIs . There is increasing evidence that ROIs fulfil an important role as second messengers involved in signal transduction . We have proposed that ROIs have been conserved throughout evolution as universal pathogen messengers turning on genes with important functions in the immune response and cell proliferation . The higher eukaryotic transcription factors NF-kappa B and AP-1 will be described as proteins which are regulated by ROIs under a great variety of pathogenic conditions and initiate the new expression of genes with important roles in immune, inflammatory and other pathogen-related genetic responses.

Biochemistry, 1997 Oct 14, 36(41), 12526 - 34
Mechanism of enolase: the crystal structure of asymmetric dimer enolase-2-phospho-D-glycerate/enolase-phosphoenolpyruvate at 2.0 A resolution; Zhang E et al.; Enolase, a glycolytic enzyme that catalyzes the dehydration of 2-phospho-d-glycerate (PGA) to form phosphoenolpyruvate (PEP), is a homodimer in all eukaryotes and many prokaryotes . Here, we report the crystal structure of a complex between yeast enolase and an equilibrium mixture of PGA and PEP . The structure has been refined using 29 854 reflections with an F/sigma(F) of >/=3 to an R of 0.137 with average deviations of bond lengths and bond angles from ideal values of 0.013 A and 3.1 degrees , respectively . In this structure, the dimer constitutes the crystallographic asymmetric unit . The two subunits are similar, and their superposition gives a rms distance between Calpha atoms of 0.91 A . The exceptions to this are the catalytic loop Val153-Phe169 where the atomic positions in the two subunits differ by up to 4 A and the loop Ser250-Gln277, which follows the catalytic loop Val153-Phe169 . In the first subunit, the imidazole side chain of His159 is in contact with the phosphate group of the substrate/product molecule; in the other it is separated by water molecules . A series of hydrogen bonds leading to a neighboring enolase dimer can be identified as being responsible for ordering and stabilization of the conformationally different subunits in the crystal lattice . The electron density present in the active site suggests that in the active site with the direct ligand-His159 hydrogen bond PGA is predominantly bound while in the active site where water molecules separate His159 from the ligand the binding of PEP dominates . The structure indicates that the water molecule hydrating carbon-3 of PEP in the PEP --> PGA reaction is activated by the carboxylates of Glu168 and Glu211 . The crystals are unique because they have resolved two intermediates on the opposite sides of the transition state.

Genes Cells, 1997 Aug, 2(8), 499 - 512
A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization; Uemori T et al.; BACKGROUND: In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication . So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase a have been found in Archaea . The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms . RESULTS: We have identified a novel DNA polymerase composed of two proteins, DP1 and DP2, with molecular weights of 69294 Da and 143161 Da, respectively, in the hyperthermophilic archaeon, Pyrococcus furiosus, and have cloned the corresponding genes which are tandemly arranged on the Pyrococcus genome . No significant sequence homology was found between these two proteins and other known DNA polymerases . The pol genes were transcribed as part of a single operon that additionally contained genes homologous to the cdc18+/CDC6 and Dmc1/Rad51 family of proteins . We purified the Pyrococcus DNA polymerase from Escherichia coli strains expressing the cloned genes and characterized its activity . It possesses strong 3' --> 5' exonucleolytic activity and has a template-primer preference which is characteristic of a replicative DNA polymerase . CONCLUSION: In P . furiosus, we identified a second DNA polymerase encoded by two genes, neither of which display significant homology to any other known DNA polymerase . Both the enzymatic properties of the enzyme and the gene organization raise the possibility that this enzyme might be the replicative DNA polymerase of P . furiosus.

Eur J Biochem, 1997 Sep 1, 248(2), 282 - 9
Mycothiol-dependent formaldehyde dehydrogenase, a prokaryotic medium-chain dehydrogenase/reductase, phylogenetically links different eukaroytic alcohol dehydrogenases--primary structure, conformational modelling and functional correlations; Norin A et al.; Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing alcohol dehydrogenases . The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric alcohol dehydrogenases of all classes (including the human forms), and several tetrameric reductases/dehydrogenases . Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR alcohol dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure . Residues typical of glutathione binding in class-III alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione . The molecular architecture is different from that of the 'constant' alcohol dehydrogenases (of class-III type) and the 'variable' alcohol dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase . Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.

J Mol Biol, 1997 Oct 24, 273(2), 377 - 88
Role of upstream activation sequences and integration host factor in transcriptional activation by the constitutively active prokaryotic enhancer-binding protein PspF; Dworkin J et al.; PspF, the transcriptional activator of the pspA operon of Escherichia coli, which belongs to the enhancer binding protein (EBP) family of sigma54 activator proteins, is constitutively active in an in vitro transcription assay . PspF protein, together with RNA polymerase holoenzyme containing sigma54, is required for in vitro transcription from the pspA promoter . EBP proteins are typically subject to regulation either by post-translational modification or interaction of a specific ligand with an N-terminal regulatory domain . However, unlike other members of the EBP family, PspF lacks this domain . pspA is positively regulated by IHF in vitro, and this regulation is dependent on the topology of the DNA; a linear template is much more dependent on IHF than a supercoiled template . EBP binding to upstream activating sequences (UAS) in their target promoters is mediated by the C-terminal domain which contains a helix-turn-helix DNA-binding motif . A mutant PspF protein lacking the C-terminal DNA-binding domain is active in vitro, although at much higher concentrations than the wild-type protein . In vitro transcription from pspA templates missing one or both of the UAS sites is reduced relative to wild-type templates, but is still appreciable; however, IHF acts as a negative regulator of pspA transcription on these mutant templates . Thus, PspF bound to non-specific sequences upstream of the pspA promoter can activate pspA transcription, but this activation is inhibited by IHF . These data, taken together, support the model that a precise promoter geometry is necessary for IHF to positively regulate transcription and that IHF may act to prevent activation from inappropriately spaced upstream sites .

Adv Microb Physiol, 1998, 39, 291 - 338
Circadian and ultradian clock-controlled rhythms in unicellular microorganisms; Lloyd D; The time structure of a biological system is at least as intricate as its spatial structure . Whereas we have detailed information about the latter, our understanding of the former is still rudimentary . As techniques for monitoring intracellular processes continuously in single cells become more refined, it becomes increasingly evident that periodic behaviour abounds in all time domains . Circadian timekeeping dominates in natural environments . Here the free-running period is about 24 h . Circadian rhythms in eukaryotes and prokaryotes allow predictive matching of intracellular states with environmental changes during the daily cycles . Unicellular organisms provide excellent systems for the study of these phenomena, which pervade all higher life forms . Intracellular timekeeping is essential . The presence of a temperature-compensated oscillator provides such a timer . The coupled outputs (epigenetic oscillations) of this ultradian clock constitute a special class of ultradian rhythm . These are undamped and endogenously driven by a device which shows biochemical properties characteristic of transcriptional and translational elements . Energy-yielding processes, protein turnover, motility and the timing of the cell-division cycle processes are all controlled by the ultradian clock . Different periods characterize different species, and this indicates a genetic determinant . Periods range from 30 min to 4 h . Mechanisms of clock control are being elucidated; it is becoming evident that many different control circuits can provide these functions.

Mol Biotechnol, 1997 Aug, 8(1), 17 - 33
Antiviral ribozymes . New jobs for ancient molecules; Menke A et al.; Catalytic RNAs are a genetic property not only of some particular viroids or viruses, but also are more common naturally among eukaryotes and even prokaryotes than earlier expected . However, the major interest in ribozymes results from their potential for development of "tailor-made" cDNA constructions designed to be transcribed into catalytic RNAs that will recognize by hybridization and destroy by specific cleavage their cellular or viral RNA targets . The efficiency of an antiviral ribozyme is determined by both the accessibility and sequence conservation of the target region, as well as the design of the ribozyme: its type, size, and composition of flanking sequences; expression rates; and cellular compartment localization . Until now the most frequently selected viral target is the human immunodeficiency virus, where an up to a 10(4)-fold inhibition in its progeny production has been achieved . Although the first generation ribozymes focused on improvements in basic design and expression rates, more recently the efficiency of antiviral catalytic activity has been increased by employing polyribozymes and/or multitarget ribozymes, as well as special constructions to enhance the cellular co-compartmentation of the ribozyme with its viral RNA target.

FEBS Lett, 1997 Sep 22, 415(1), 75 - 80
A phage T4 site-specific endonuclease, SegE, is responsible for a non-reciprocal genetic exchange between T-even-related phages; Kadyrov FA et al.; The bacteriophage T4 segE gene encoding site-specific endonuclease lies between the hoc.1 and uvsW genes . The similar region of T-even-related phage RB30 lacks the segE gene . Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30 . The preferred inheritance of the segE gene depends on its own expression and is based on a non-reciprocal homologous recombination event providing the transfer of the gene from the segE-containing to the segE-lacking allele . The SegE endonuclease cleaves DNA in a site located at the 5' end of the uvsW gene in the RB30 genome . The T4 DNA is also cleaved by the enzyme, but less efficiently . The cleavage at the RB30 site appears to initiate the observed conversion, which is stimulated by DNA homology and accompanied by co-conversion of flanking markers . Our findings provide a novel example of endonuclease-dependent generation of genetic variation in prokaryotes.

DNA Cell Biol, 1997 Sep, 16(9), 1111 - 22
Characterization and expression of the mouse endonuclease G gene; Prats E et al.; Endonuclease G (Endo G) is a nuclease of prokaryotic lineage found in the mitochondria of vertebrates that has been suggested to play a role in mitochondrial DNA (mtDNA) replication . We have isolated and sequenced the entire mouse endo G gene, determined the limits of the mRNA, and mapped the promoter region . The coding sequence of the single copy gene is interrupted by two introns and analysis of the transcripts does not support a model by which more than one Endo G isoform could be produced by alternative splicing . We have also characterized a full-length human Endo G cDNA and comparison at the protein level of the human, bovine, and murine nucleases indicates a high degree of conservation except in the respective mitochondrial targeting signals . Endo G is ubiquitously expressed and the steady-state levels of its mRNA vary by a factor greater than seven between different tissues . The relationship between the mtDNA copy number and Endo G mRNA levels is not strictly proportional but tissues richer in mtDNA have higher amounts of the mRNA and vice versa.

Annu Rev Microbiol, 1997, 51, 593 - 628
Clues and consequences of DNA bending in transcription; Perez-Martin J et al.; This review attempts to substantiate the notion that nonlinear DNA structures allow prokaryotic cells to evolve complex signal integration devices that, to some extent, parallel the transduction cascades employed by higher organisms to control cell growth and differentiation . Regulatory cascades allow the possibility of inserting additional checks, either positive or negative, in every step of the process . In this context, the major consequence of DNA bending in transcription is that promoter geometry becomes a key regulatory element . By using DNA bending, bacteria afford multiple metabolic control levels simply through alteration of promoter architecture, so that positive signals favor an optimal constellation of protein-protein and protein-DNA contacts required for activation . Additional effects of regulated DNA bending in prokaryotic promoters include the amplification and translation of small physiological signals into major transcriptional responses and the control of promoter specificity for cognate regulators.

Annu Rev Microbiol, 1997, 51, 203 - 24
Against all odds: the survival strategies of Deinococcus radiodurans; Battista JR; Bacteria of the genus Deinococcus exhibit an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents-particularly the effects of ionizing radiation . These bacteria are the most DNA damage-tolerant organisms ever identified . Relatively little is known about the biochemical basis for this phenomenon; however, available evidence indicates that efficient repair of DNA damage is, in large part, responsible for the deinococci's radioresistance . Obviously, an explanation of the deinococci's DNA damage tolerance cannot be developed solely on the basis of the DNA repair strategies of more radiosensitive organisms . The deinococci's capacity to survive DNA damage suggests that (a) they employ repair mechanisms that are fundamentally different from other prokaryotes, or that (b) they have the ability to potentiate the effectiveness of the conventional complement of DNA repair proteins . An argument is made for the latter alternative.

Annu Rev Microbiol, 1997, 51, 179 - 202
Making and breaking disulfide bonds; Raina S et al.; It is now well established that protein folding requires the assistance of folding helpers in vivo . The formation or isomerization of disulfide bonds in proteins is a slow process requiring catalysis . In nascent polypeptide chains the cysteine residues are in the thiol form . The formation of the disulfide bonds usually occurs simultaneously with the folding of the polypeptide, which means in the endoplasmic reticulum of eukaryotes or in the periplasm of Gram-negative bacteria . In prokaryotes, the existence of redox proteins involved in the formation of disulfide bonds containing proteins has recently been revealed in the periplasm . The discovery of these redox proteins through various genetic approaches will be summarized, as well as the most recent insights regarding their biochemical and biological activities.

Proc Natl Acad Sci U S A, 1997 Oct 28, 94(22), 11786 - 91
Protein-protein interactions among the Aux/IAA proteins; Kim J et al.; The plant hormone indoleacetic acid (IAA) transcriptionally activates early genes in plants . The Aux/IAA family of early genes encodes proteins that are short-lived and nuclear-localized . They also contain a putative prokaryotic betaalphaalpha DNA binding motif whose formation requires protein dimerization . Here, we show that the pea PS-IAA4 and Arabidopsis IAA1 and IAA2 proteins perform homo- and heterotypic interactions in yeast using the two-hybrid system . Gel-filtration chromatography and chemical cross-linking experiments demonstrate that the PS-IAA4 and IAA1 proteins interact to form homodimers in vitro . Deletion analysis of PS-IAA4 indicates that the betaalphaalpha containing acidic C terminus of the protein is necessary for homotypic interactions in the yeast two-hybrid system . Screening an Arabidopsis lambda-ACT cDNA library using IAA1 as a bait reveals heterotypic interactions of IAA1 with known and newly discovered members of the Arabidopsis Aux/IAA gene family . The new member IAA24 has similarity to ARF1, a transcription factor that binds to an auxin response element . Combinatorial interactions among the various members of the Aux/IAA gene family may regulate a variety of late genes as well as serve as autoregulators of early auxin-regulated gene expression . These interactions provide a molecular basis for the developmental and tissue-specific manner of auxin action.

Eur J Biochem, 1997 Sep 15, 248(3), 748 - 61
Cloning and characterization of Manduca sexta and Plutella xylostella midgut aminopeptidase N enzymes related to Bacillus thuringiensis toxin-binding proteins; Denolf P et al.; We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, an insecticidal crystal protein {ICP} from Bacillus thuringiensis . Sequence information derived from this M . sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5 . Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M . sexta . On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin . Internal amino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides . From a nested PCR with M . sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes . This PCR fragment was used to screen cDNA libraries of larval midguts from M . sexta and P . xylostella . From the M . sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned . The ORF of the sequence encodes a 942-residue aminopeptidase N (M . sexta Apn2) containing two hydrophobic regions . The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins . Low-stringency hybridization of the P . xylostella midgut cDNA library with M . sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein . This aminopeptidase N (P . xylostella Apn1) displays 61% amino acid identity to M . sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition . Both M . sexta Apn2 and P . xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules . Treatment of Sf9 cells expressing the P . xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P . xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.

Eur J Biochem, 1997 Sep 15, 248(3), 700 - 6
Functional expression of a tobacco gene related to the serine hydrolase family -- esterase activity towards short-chain dinitrophenyl acylesters; Baudouin E et al.; We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens . We present and discuss here some structural and biochemical properties of the gene product . Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids . The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin . The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p-nitrophenylbutyrate, a general substrate for carboxylesterases . The enzyme was unable to hydrolyze lipids, and was active on short-chain acyl esters only . The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family . Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.

J Cell Sci, 1997 Sep, 110 ( Pt 18), 2271 - 80
The alternatively spliced domains EIIIB and EIIIA of human fibronectin affect cell adhesion and spreading; Hashimoto-Uoshima M et al.; Fibronectin has a complex pattern of alternative splicing at the pre-mRNA level leading to the expression of different isoforms . The alternatively spliced domains EIIIB and EIIIA are known to be prominently expressed during development and wound healing . While the other spliced domain (CS-segment) is known to promote cell adhesion in a cell type specific manner, the biological functions of the spliced domains EIIIB and EIIIA are not well understood . In the present study, we have prepared expression proteins of specific domains of human fibronectin using a prokaryotic expression system and used the purified fragments to test their ability to support adhesion and spreading of cultured cells . Fragments from type-III domains #7 to #12 were prepared in various combinations to include or exclude the spliced domains EIIIB and EIIIA . The results indicate that cultured NIL fibroblasts adhere to many of the fragments tested . However, the cell adhesion and spreading are enhanced, especially at lower concentrations, to fragments including the domain EIIIB . The inclusion of domain EIIIA led to a decrease in the adhesion of cells and those that adhered did not spread well . When tested in a centrifugal cell adhesion assay, fragments including domain EIIIB resisted the detaching forces and stayed adhered . Fragments that included domain EIIIA were unable to resist the detaching centrifugal forces to the same extent as the fragments that included domain EIIIB alone . These results suggest that the spliced domain EIIIB may be serving important biological functions in enhancing cell adhesion and spreading . This is likely to be mediated by conformational effects because domain EIIIB alone neither exhibited any adhesive activity nor competed in inhibiting adhesion to fragments #7-10.

Biosci Rep, 1997 Jun, 17(3), 303 - 17
Respiratory protection of nitrogenase activity in Azotobacter vinelandii--roles of the terminal oxidases; Poole RK et al.; Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes--nitrogenases--are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically . This review summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration . We review the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd gene expression . The roles of other oxidases and other mechanisms for limiting damage to nitrogenase are assessed.

Nature, 1997 Oct 2, 389(6650), 520 - 2
Protein memory through altered folding mediated by intramolecular chaperones; Shinde UP et al.; The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain . Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases . Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain . Subtilisin E folded by an intramolecular chaperone with an Ile(-48)-to-Val mutation acquires an 'altered' enzymatically active conformation that differs from wild-type subtilisin E . Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans . The two subtilisins also have different secondary structures, thermostability and substrate specificities . Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process . Such a phenomenon, which we term 'protein memory', may be important in investigations of protein folding.

Electrophoresis, 1997 Aug, 18(9), 1577 - 85
On simple repetitive DNA sequences and complex diseases; Epplen C et al.; Simple repetitive DNA sequences are abundantly interspersed in eukaryote genomes and therefore useful in genome research and genetic fingerprinting in plants, fungi and animals, including man . Recently, simple repeats were also identified in some prokaryotic genomes . Hence the same probes can be applied for multilocus DNA fingerprinting in medically relevant bacteria . Simple repeats including composite dinucleotide microsatellites are differentially represented in different compartments of eukaryote genomes . Expanded triplet blocks in and around certain genes may, for example, cause so-called trinucleotide diseases in man . As a consequence, simple repetitive sequences should also be characterized with respect to their influences on the DNA structure, gene expression, genomic (in)stability and their development on an evolutionary time scale . Here three examples of microsatellites in the human major histocompatibility complex (HLA) are investigated, a (GT)n microsatellite situated 2 kb 5' off the lymphotoxin alpha (LTA) gene, a (GAA)n block in the 5' part of the HLA-F gene and a composite (GT)n(GA)m stretch in the second intron of HLA-DRBl genes . Grossly differing mutation rates are evident in these elements as well as varying linkage disequilibria . The unfolding of these simple repeats in distant human populations is covered including Caucasians, Bushmen and South American Indians . Furthermore, implications of simple repeat neighboring genes are discussed for the multifactorial diseases multiple sclerosis (MS), rheumatoid arthritis (RA) and early onset pauciarticular arthritis (EOPA) . Polymorphisms of HLA-DRBl and T cell receptor beta variable (TCRBV) genes confer susceptibility for these autoimmune diseases as demonstrable by intronic simple repeat variability . Microsatellite polymorphisms within the TNF region reveal linkage disequilibria with HLA-DRBl and different promotor alleles of the TNFA gene . Disease associations with TNFA microsatellite alleles are, on the one hand, secondary to associations with HLA-DRBl genes (in MS) or they represent additional risk factors (in RA, EOPA) on the other hand . Evolutionary persistence, various structural conformations and the specific binding of nuclear proteins to several simple repeat sequences refute the preconceptions of biological insignificance for all of these ubiquitously interspersed elements.

Biochem Pharmacol, 1997 Sep 1, 54(5), 545 - 50
Interaction of the DNA topoisomerase II catalytic inhibitor meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane (ICRF-193), a bisdioxopiperazine derivative, with the conserved region(s) of eukaryotic but not prokaryotic enzyme; Sato M et al.; ICRF-193 {meso-2,3-bis(3,5-dioxopiperazine-1-yl)butane}, a bisdioxopiperazine compound, has been shown to be a catalytic inhibitor of DNA topoisomerase II by stabilizing the enzyme in the form of a closed "protein clamp," an intermediate form in the catalytic cycle (Roca et al., Proc Natl Acad Sci USA 91: 1781-1785, 1994) . In view of its usefulness as a probe in the functional analysis of the enzyme, we tried further to define the domain(s) of the enzyme interacting with the drug by examining its inhibitory activity on type II topoisomerases from various species of eukaryotes and prokaryotes . ICRF-193 inhibited the enzyme from yeast, fly, frog, plant, and mammals at IC50 values in the range of 1-13 microM . Experiments using fission yeast truncated mutant type II enzyme lacking both amino-terminal 74 amino acids and carboxy-terminal 265 amino acids revealed that ICRF-193 interacts with the 125 kDa "core" polypeptide of the enzyme . In contrast, prokaryotic type II enzymes, Escherichia coli DNA gyrase, topo IV, and phage T4 topo, were not affected by the drug . From these results, the domain(s) common to eukaryotic but not to prokaryotic type II enzymes interacting with ICRF-193 was speculated.

Nucleic Acids Res, 1997 Nov 1, 25(21), 4224 - 9
Prokaryotic 5'-3' exonucleases share a common core structure with gamma-delta resolvase; Artymiuk PJ et al.; The three dimensional crystal structure of T5 5'-3' exonuclease was compared with that of two other members of the 5'-3' exonuclease family: T4 ribonuclease H and the N-terminal domain of Thermus aquaticus DNA polymerase I . Though these structures were largely similar, some regions of these enzymes show evidence of significant molecular flexibility . Previous sequence analysis had suggested the existence of a helix-hairpin-helix motif in T5 exonuclease, but a distinct, though related structure is actually found to occur . The entire T5 exonuclease structure was then compared with all the structures in the complete Protein Data Bank and an unexpected similarity with gamma-delta (gamma delta) resolvase was observed . 5'-3' exonucleases and gamma delta resolvase are enzymes involved in carrying out quite different manipulations on nucleic acids . They appear to be unrelated at the primary sequence level, yet the fold of the entire catalytic domain of gamma delta resolvase is contained within that of the 5'-3'exonuclease . Different large-scale helical structures are used by both families to form DNA binding sites.

Biochemistry, 1997 Oct 21, 36(42), 13054 - 9
Metal-dependent conformers of the periplasmic ferric ion binding protein; Nowalk AJ et al.; One of the better understood structural correlates of Fe3+ binding by the transferrins is the conformational shift demonstrated by both lobes . FbpA, a prokaryotic protein involved in periplasmic iron transport, has previously been shown to be structurally and functionally homologous to the transferrins . Similar to each individual lobe of the transferrins, it is hypothesized that FbpA exists in two distinct conformations depending on whether metal is bound . Evidence for these changes is provided by the differential susceptibility of FbpA to trypsin digestion . Binding of Fe3+ by FbpA significantly decreases the ability of trypsin to digest wild-type protein . Construction of a null binding mutant, Tyr195Ile, confirms that protein "locked" in the apo-conformation is similarly susceptible to trypsin . This mutant also marks the initial characterization of an FbpA molecule unable to bind iron, suggesting that the Tyr195 residue is directly involved in iron binding . Other FbpA mutants which do bind iron show moderate resistance to digestion which suggests that they remain in the holo-protein conformation when binding Fe3+ . The conformational states of FbpA may have important implications in protein-protein recognition during transport of Fe3+ between membranes, and may explain how these proteins function in the context of periplasm-to-cytosol Fe3+ transport.

J Bacteriol, 1997 Oct, 179(20), 6360 - 6
The presence of a dnaK (HSP70) multigene family in members of the orders Planctomycetales and Verrucomicrobiales; Ward-Rainey N et al.; Sequences of the dnaK gene, coding for the 70-kDa heat shock protein (HSP70), were determined for six members of the order Planctomycetales, including representatives of three genera, and for the only cultivated member of the order Verrucomicrobiales, Verrucomicrobium spinosum . A fragment of the dnaK gene was amplified from these strains by PCR with oligonucleotide primers targeting regions of the dnaK gene that are conserved at the amino acid level, and the resulting PCR products were cloned into a plasmid vector . Sequence analysis of the cloned dnaK fragments revealed the presence of two different types of dnaK sequence in one of the planctomycete strains, Planctomyces maris, and in V . spinosum . Only one type of dnaK sequence was found for each of the remaining strains . Phylogenetic analysis of the partial sequence data suggested that the majority of planctomycete strains, including one of the Planctomyces maris sequences, form a coherent phylogenetic group branching adjacent to other main lines of descent within the domain Bacteria, as has been shown previously by 16S rRNA sequence analysis . One of the two V . spinosum dnaK sequences also appears to constitute a separate lineage within the gram-negative bacteria . Each of the remaining sequences from P . maris and V . spinosum, together with the single sequence obtained from Planctomyces limnophilus, appeared to be unrelated to the other planctomycete sequences and to occupy a position distant from that of other gram-negative bacteria . The phylogenetic diversity of dnaK sequences exhibited by P . maris and V . spinosum was comparable to that found in Synechococcus sp . strain PCC7942 and Escherichia coli, the only other prokaryotes for which a dnaK multigene family has been demonstrated.

Nat Struct Biol, 1997 Oct, 4(10), 819 - 26
Solution structure of the DNA-binding domain and model for the complex of multifunctional hexameric arginine repressor with DNA; Sunnerhagen M et al.; The structure of the monomeric DNA-binding domain of the Escherichia coli arginine repressor, ArgR, determined by NMR spectroscopy, shows structural homology to the winged helix-turn-helix (wHTH) family, a motif found in a diverse class of proteins including both gene regulators and gene organizers from prokaryotes and eukaryotes . Biochemical data on DNA binding by intact ArgR are used as constraints to position the domain on its DNA target and to derive a model for the hexamer-DNA complex using the known structure of the L-arginine-binding domain . The structural independence of the wHTH fold may be important for multimeric DNA-binding proteins that contact extended DNA regions with imperfect match to consensus sequences, a feature of many wHTH-domain proteins.

Gene, 1997 Sep 15, 197(1-2), 353 - 60
Cloning and characterization of the gene encoding PspPI methyltransferase from the Antarctic psychrotroph Psychrobacter sp . strain TA137 . Predicted interactions with DNA and organization of the variable region; Rina M et al.; The gene (pspPIM) encoding the PspPI DNA methyltransferase (MTase) associated with the PspPI restriction-modification (R-M) system (5'-GGNCC-3') of Psychrobacter species TA137 has been cloned and expressed in E . coli, and its nucleotide (nt) sequence has been determined . The coding region was 1248 nt in length and capable of specifying a 46826-Da protein of 416 amino acids (aa) . The predicted sequence of the MTase protein displays ten sequence motifs characteristic of all prokaryotic m5C-MTases and shows the highest similarity to other MTases that methylate the GGNCC sequence, namely M . Eco47II and M . Sau96I . All three MTases methylate the internal cytosine within their recognition sequence . Sequence similarities between M . PspPI and its isospecific M . Eco47II and M . Sau96I as well as with four other m5C-MTases that methylate the related GGWCC sequence, namely M . SinI, M . HgiCII, M . HgiBI, M . HgiEI have been also found within the variable region of these proteins . On the basis of structural information from M . HhaI and M . HaeIII, several M . PspPI residues that are expected to interact with DNA can be predicted . Furthermore, an organization of the variable region of m5C-MTases into two segments exhibiting a pattern of conserved residues and a considerable degree of structural homologies is described.

Gene, 1997 Sep 15, 197(1-2), 337 - 41
An epitope tagged mammalian/prokaryotic expression vector with positive selection of cloned inserts; Schneider S et al.; A dual eukaryotic/prokaryotic expression vector has been developed which combines the features of positive selection for cloned inserts along with the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells as well as high level induced expression in E . coli cells harbouring T7 RNA polymerase . This vector, pZilch, has two MCSs flanking a mutant E . coli phenylalanyl-tRNA synthetase gene, pheS, which when expressed in combination with the phenylalanine analog p-CI-Phe, results in termination of host cell protein synthesis . Cloning of inserts using unique sites in the flanking MCS regions results in loss of the pZilch pheS allele and hence permits growth of colonies harbouring recombinants on p-Cl-Phe plates . Additional features of the vector include an optimal Kozak consensus sequence for high level eukaryotic cell expression and an efficient prokaryotic translation initiation site in frame and downstream from the eukaryotic initiation site . Recombinant proteins can be produced with an N-terminal FLAG epitope which can be removed via a specific protease cleavage site . Flanking T7 and SP6 RNA polymerase promoter sites permit in vitro transcription and translation of cloned inserts . A derivative of the vector has also been constructed enabling nuclear accumulation of the tagged proteins via an SV40 nuclear localisation signal upstream of the 5' MCS.

Gene, 1997 Sep 15, 197(1-2), 277 - 87
Molecular cloning and expression of a porcine chondrocyte nucleotide pyrophosphohydrolase; Masuda I et al.; The porcine 127-kDa nucleotide pyrophosphohydrolase (NTPPHase) had been previously purified from the conditioned culture media of porcine articular cartilage . Protein sequencing of an internal 61-kDa proteolytic fragment of NTPPHase (61-kDa NTPPHase) determined the 26 N-terminal amino acids . This sequence was used to amplify a DNA fragment, which was used as a probe to clone the gene encoding the 61-kDa NTPPHase from a porcine chondrocyte cDNA library . DNA sequence analysis showed the cDNA insert to be 2509 bp, corresponding to a predicted open reading frame (ORF) encoding 599 amino acids . The 26 N-terminal amino acids of the 61-kDa NTPPHase were located within the ORF immediately downstream of a putative protease recognition region, RRKRR . This is consistent with this cDNA insert representing an internal proteolytic fragment of the full length 127-kDa NTPPHase . BLAST and FASTA analysis confirmed that the deduced amino acid sequence of 61-kDa NTPPHase was unique and did not possess a high degree of homology to sequence in the non-redundant protein and nucleotide databases . Proteins that possess limited homology (< 17%) with the 61-kDa NTTPPHase include several prokaryotic and eukaryotic ATP pyrophosphate-lyases (adenylate cyclase) . Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61-kDa NTPPHase hybridized to a single 4.0-kb RNA transcript . This DNA probe also hybridized to a single species of human chondrocyte RNA . Expression of a 61-kDa protein was detected by coupled in-vitro transcription/translation . Western blot analysis of this in-vitro transcription/translation reaction detected a 61-kDa protein, using an antibody raised against the peptide sequence that was originally used to clone the 61-kDa NTPPHase . These data indicate the successful in-vitro cloning and expression of the porcine chondrocyte 61-kDa NTPPHase . Future studies that utilize the gene encoding the 61-kDa NTPPHase may allow the characterization of the role of NTPPHase in calcium pyrophosphate dihydrate (CPPD) crystal deposition disease.

Gene, 1997 Sep 15, 197(1-2), 9 - 17
Paralogous histidine biosynthetic genes: evolutionary analysis of the Saccharomyces cerevisiae HIS6 and HIS7 genes; Fani R et al.; The HIS6 gene from Saccharomyces cerevisiae strain YNN282 is able to complement both the S . cerevisiae his6 and the Escherichia coli hisA mutations . The cloning and the nucleotide sequence indicated that this gene encodes a putative phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxiamide isomerase (5' Pro-FAR isomerase, EC 5.3.1.16) of 261 amino acids, with a molecular weight of 29,554 . The HIS6 gene product shares a significant degree of sequence similarity with the prokaryotic HisA proteins and HisF proteins, and with the C-terminal domain of the S . cerevisiae HIS7 protein (homologous to HisF), indicating that the yeast HIS6 and HIS7 genes are paralogous . Moreover, the HIS6 gene is organized into two homologous modules half the size of the entire gene, typical of all the known prokaryotic hisA and hisF genes . The structure of the yeast HIS6 gene supports the two-step evolutionary model suggested by Fani et al . (J . Mol . Evol . 1994; 38: 489-495) to explain the present-day hisA and hisF genes . According to this idea, the hisF gene originated from the duplication of an ancestral hisA gene which, in turn, was the result of an earlier gene elongation event involving an ancestral module half the size of the extant gene . Results reported in this paper also suggest that these two successive paralogous gene duplications took probably place in the early steps of molecular evolution of the histidine pathway, well before the diversification of the three domains, and that this pathway was one of the metabolic activities of the last common ancestor . The molecular evolution of the yeast HIS6 and HIS7 genes is also discussed.

DNA Res, 1997 Jun 30, 4(3), 179 - 84
Prediction of translation initiation sites on the genome of Synechocystis sp . strain PCC6803 by Hidden Markov model; Hirosawa M et al.; We developed a computer program, GeneHackerTL, which predicts the most probable translation initiation site for a given nucleotide sequence . The program requires that information be extracted from the nucleotide sequence data surrounding the translation initiation sites according to the framework of the Hidden Markov Model . Since the translation initiation sites of 72 highly abundant proteins have already been assigned on the genome of Synechocystis sp . strain PCC6803 by amino-terminal analysis, we extracted necessary information for GeneHackerTL from the nucleotide sequence data . The prediction rate of the GeneHackerTL for these proteins was estimated to be 86.1% . We then used GeneHackerTL for prediction of the translation initiation sites of 24 other proteins, of which the initiation sites were not assigned experimentally, because of the lack of a potential initiation codon at the amino-terminal position . For 20 out of the 24 proteins, the initiation sites were predicted in the upstream of their amino-terminal positions . According to this assignment, the processed regions represent a typical feature of signal peptides . We could also predict multiple translation initiation sites for a particular gene for which at least two initiation sites were experimentally detected . This program would be effective for the prediction of translation initiation sites of other proteins, not only in this species but also in other prokaryotes as well.

Biochem J, 1997 Sep 15, 326 ( Pt 3), 897 - 902
Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments; Yang XL et al.; Copper is an essential trace element in prokaryotes and eukaryotes and is strictly regulated by biological mechanisms . Menkes and Wilson diseases are human disorders that arise from disruption of the normal process of copper export from the cytosol to the extracellular environment . Recently a gene for Wilson disease (WD)(also named the ATP7B gene) was cloned . This gene encodes a copper transporter of the P-type ATPase . We prepared monoclonal and polyclonal anti-(WD protein) antibodies and characterized the full-length WD protein as well as a shorter form that is produced by alternative splicing in the human brain . We found that the WD protein is localized mainly in the Golgi apparatus, whereas the shorter form is present in the cytosol . These results suggest that the alternative WD proteins act as key regulators of copper metabolism, perhaps by performing distinct roles in the intracellular transport and export of copper.

Proc Int Conf Intell Syst Mol Biol, 1997, 5, 15 - 24
Standardized representations of the literature: combining diverse sources of ribosomal data; Altman RB et al.; We are building a knowledge base (KB) of published structural data on the 30s ribosomal subunit in prokaryotes . Our KB is distinguished by a standardized representation of biological experiments and their results, in a reusable format . It can be accessed by computer programs that exploit the rich interconnections within the data . The KB is designed to support the construction of 3D models of the 30S subunit, as well as the analysis and extension of relevant functional and phylogenetic information . Most published information about the structure of the ubiquitous ribosome focuses on E . coli as a model system . At the same time, thousands of RNA sequences for the ribosome have been gathered and cataloged . The volume and complexity of these data can complicate attempts to separate structural data peculiar to E . coli from data of universal relevance . We have written an application that dynamically queries the KB and the Ribosome Database Project, a repository of ribosomal RNA sequences from other organisms, in order to assess the relevance of structural data to particular organisms . The application uses the RDP alignment to determine whether a set of data refer primarily to conserved, mismatched, or gapped positions . For a set of 16 representative articles evaluated over 211 sequences, 73% of observations have unambiguous translations from E . coli to the other organisms, 21% have somewhat ambiguous translations, and 6% have no translations . There is a wide variation in these numbers over different articles and organisms, confirming that some articles report structural information specific to E . coli while others report information that is quite general.

Biomed Environ Sci, 1997 Sep, 10(2-3), 182 - 9
RNA and protein requirements for eukaryotic selenoprotein synthesis; Berry MJ et al.; Selenium has been recognized as an essential nutrient in animals since the 1950s . Demonstration of the role of dietary selenium in protection from oxidative stress followed in the early 1970s, and was largely attributed to its presence as an integral part of cellular glutathione peroxidase . However, the functions of this enzyme did not explain many of the other effects of selenium deficiency . The identification of other mammalian selenoproteins during the last few years has provided new insights into the functions of this trace nutrient . The discovery that type 1 deiodinase (D1) is a selenoenzyme, in addition to unveiling an essential role for selenium in thyroid hormone action, has had more far-reaching implications . Studies of this protein opened the door for investigation of the requirements for eukaryotic selenoprotein synthesis, and the features that distinguish this pathway from the corresponding prokaryotic pathway . Selenium is present in a number of prokaryotic and eukaryotic proteins in the form of the unusual amino acid, selenocysteine . Incorporation of selenocysteine into these proteins requires a novel translation step in which UGA specifies selenocysteine insertion . Since UGA codons are typically recognized as translation stop signals, an intriguing question is raised: How does a cell recognize and distinguish a UGA selenocysteine codon from a UGA stop codon? In this review, we will focus on what is known about selenocysteine incorporation in eukaryotes, briefly summarizing initial studies and discussing a few recent advances in our understanding of this unique "recoding" process.

J Theor Biol, 1997 Oct 7, 188(3), 379 - 85
Nucleosomes: a solution to a crowded intracellular environment?
Minsky A, Ghirlando R, Reich Z.
The emergence of eukaryotes was accompanied by two major events that concern their genome and are of crucial significance when considered in terms of macromolecular crowding: (i) a substantial increase in the amount of DNA, and (ii) its confinement within a defined space . The resulting highly crowded environment would have strongly promoted DNA self-assembly processes, leading to extremely condensed and thermodynamically stable DNA aggregates . Such structural transitions have indeed been observed in vitro, as well as in virtually all cellular systems in which a nucleosomal assembly is absent . In this appear we raise the hypothesis that upon transition from prokaryotic systems to eukaryotes, the nucleosomes were rendered essential in order to negate extensive DNA condensation processes that would have resulted from excluded volume effects . By suppressing such processes, the nucleosomes act to maintain and regulate the conformational space of the DNA, thus enabling conformational flexibility and reversible structural modulations .

Immunol Cell Biol, 1997 Aug, 75(4), 364 - 9
DNA vaccines for bacterial infections; Strugnell RA et al.; DNA vaccines are an exciting development in vaccine technology which may have a special role in preventing viral infections and as 'theracines' for cancer . Their use in preventing bacterial infections has, by comparison, been less well documented . While it is unlikely that traditional, highly successful and cheap vaccines for diseases such as diphtheria will be replaced by DNA vaccines, naked DNA may be particularly appropriate for preventing bacterial infections where cytotoxic T cells confer protection, or where a Th1 type T cell response mediates resistance . For example, DNA vaccines containing different mycobacterial antigens have been shown to inhibit overt infections by Mycobacterium tuberculosis in rodent models . The use of DNA vaccines in bacterial infections may be complicated by fundamental differences between prokaryotic and eukaryotic genes and gene products, including mRNA stability, codon bias, secondary structures surrounding native start sequences and glycosylation . These problems can be solved by re-synthesis of bacterial genes to produce 'new' sequences which are more highly expressed by eukaryotic cells.

Biomed Environ Sci, 1997 Sep, 10(2-3), 177 - 81
Solution structure of SECIS, the mRNA element required for eukaryotic selenocysteine insertion--interaction studies with the SECIS-binding protein SBP; Walczak R et al.; Selenocysteine, a selenium-containing analog of cysteine, is found in the prokaryotic and eukaryotic kingdoms in active sites of enzymes involved in oxidation-reduction reactions . This aminoacid is cotranslationally incorporated at UGA codons which usually act as translation stop codons . In eukaryotes, decoding of selenocysteine necessitates the participation of the selenocysteine insertion sequence (SECIS), an element lying in the 3'-untranslated region of selenoprotein mRNAs . A detailed experimental study of the secondary structures of the SECIS elements of rat and human type 1 iodothyronine deiodinases and rat glutathione peroxidase was performed . Enzymatic and chemical structure probing led us to propose a secondary structure model, supported by sequence comparison of 23 SECIS mRNAs . The secondary structure model revealed the existence of a novel type of RNA motif composed of four consecutive non-Watson-Crick base-pairs . Using gel shift experiments, we identified in several mammalian cell type extracts the protein SBP, for SECIS-binding protein, that specifically recognizes the iodothyronine deiodinases and glutathione peroxidase SECIS elements . The structural model that we derived for the SECIS RNAs discloses RNA features possibly implicated in the binding of SBP and/or SECIS function.

Biochem Mol Biol Int, 1997 Sep, 43(1), 161 - 71
Transport of glutathione S-conjugates in Escherichia coli; Kaluzna A et al.; There are no reports concerning glutathione-S-conjugates transport in prokaryotic cells, although many studies have been performed in eukaryotic systems . This study demonstrates that glutathione S-conjugates (2,4-dinitrophenyl-S-glutathione and bimane-S-glutathione) are transported also out of Escherichia coli K12 cells . This transport is inhibited by vanadate, fluoride and other inhibitors of glutathione S-conjugate transport in mammalian cells suggesting a similarity of this process to that known in eukaryotes.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10961 - 6
Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis; Bardarov S et al.; Transposon mutagenesis provides a direct selection for mutants and is an extremely powerful technique to analyze genetic functions in a variety of prokaryotes . Transposon mutagenesis of Mycobacterium tuberculosis has been limited in part because of the inefficiency of the delivery systems . This report describes the development of conditionally replicating shuttle phasmids from the mycobacteriophages D29 and TM4 that enable efficient delivery of transposons into both fast- and slow-growing mycobacteria . These shuttle phasmids consist of an Escherichia coli cosmid vector containing either a mini-Tn10(kan) or Tn5367 inserted into a nonessential region of the phage genome . Thermosensitive mutations were created in the mycobacteriophage genome that allow replication at 30 degrees C but not at 37 degrees C (TM4) or 38.5 degrees C (D29) . Infection of mycobacteria at the nonpermissive temperature results in highly efficient transposon delivery to the entire population of mycobacterial cells . Transposition of mini-Tn10(kan) occurred in a site-specific fashion in M . smegmatis whereas Tn5367 transposed apparently randomly in M . phlei, Bacille Calmette-Guerin (BCG), and M . tuberculosis . Sequence analysis of the M . tuberculosis and BCG chromosomal regions adjacent to Tn5367 insertions, in combination with M . tuberculosis genomic sequence and physical map data, indicates that the transpositions have occurred randomly in diverse genes in every quadrant of the genome . Using this system, it has been readily possible to generate libraries containing thousands of independent mutants of M . phlei, BCG, and M . tuberculosis.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 11096 - 101
Link between light and fatty acid synthesis: thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase; Sasaki Y et al.; Fatty acid synthesis in chloroplasts is regulated by light . The synthesis of malonyl-CoA, which is catalyzed by acetyl-CoA carboxylase (ACCase) and is the first committed step, is modulated by light/dark . Plants have ACCase in plastids and the cytosol . To determine the possible involvement of a redox cascade in light/dark modulation of ACCase, the effect of DTT, a known reductant of S-S bonds, was examined in vitro for the partially purified ACCase from pea plant . Only the plastidic ACCase was activated by DTT . This enzyme was activated in vitro more efficiently by reduced thioredoxin, which is a transducer of redox potential during illumination, than by DTT alone . Chloroplast thioredoxin-f activated the enzyme more efficiently than thioredoxin-m . The ACCase also was activated by thioredoxin reduced enzymatically with NADPH and NADP-thioredoxin reductase . These findings suggest that the reduction of ACCase is needed for activation of the enzyme, and a redox potential generated by photosynthesis is involved in its activation through thioredoxin as for enzymes of the reductive pentose phosphate cycle . The catalytic activity of ACCase was maximum at pH 8 and 2-5 mM Mg2+, indicating that light-produced changes in stromal pH and Mg2+ concentration modulate ACCase activity . These results suggest that light directly modulates a regulatory site of plastidic prokaryotic form of ACCase via a signal transduction pathway of a redox cascade and indirectly modulates its catalytic activity via stromal pH and Mg2+ concentration . A redox cascade is likely to link between light and fatty acid synthesis, resulting in coordination of fatty acid synthesis with photosynthesis.

Proc Natl Acad Sci U S A, 1997 Sep 30, 94(20), 10967 - 72
Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance; Yeh CH et al.; A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress . It is known that E . coli does not normally produce class I LMM HSPs . We compared the survivability of E . coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)-Oshsp16.9 fusion protein (pGST-FL cells) with the control E . coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl beta-D-thiogalactopyranoside induction . The pGST-FL cells demonstrated thermotolerance at 47.5 degrees C, a treatment that was lethal to the pGST cells . When the cell lysates from these two E . coli transformants were heated at 55 degrees C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells . Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion) . The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5 degrees C . This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.

J Biol Chem, 1997 Oct 3, 272(40), 25275 - 82
DNA translocation across planar bilayers containing Bacillus subtilis ion channels; Szabo I et al.; The mechanisms by which genetic material crosses prokaryotic membranes are incompletely understood . We have developed a new methodology to study the translocation of genetic material via pores in a reconstituted system, using techniques from electrophysiology and molecular biology . We report here that planar bilayer membranes become permeable to double-stranded DNA (kilobase range) if Bacillus subtilis membrane vesicles containing high conductance channels have been fused into them . The translocation is an electrophoretic process, since it does not occur if a transmembrane electrical field opposing the movement of DNA, a polyanion, is applied . It is not an aspecific permeation through the phospholipid bilayer, since it does not take place if no proteins have been incorporated into the membrane . The transport is also not due simply to the presence of polypeptides in the membrane, since it does not occur if the latter contains gramicidin A or a eukaryotic, multi-protein vesicle fraction exhibiting 30-picosiemens anion-selective channel activity . The presence of DNA alters the behavior of the bacterial channels, indicating that it interacts with the pores and may travel through their lumen . These results support the idea that DNA translocation may take place through proteic pores and suggest that some of the high conductance bacterial channels observed in electrophysiological experiments may be constituents of the DNA translocating machinery in these organisms.

EMBO J, 1997 Sep 1, 16(17), 5455 - 63
Ca2+-mediated GTP-dependent dynamic assembly of bacterial cell division protein FtsZ into asters and polymer networks in vitro; Yu XC et al.; FtsZ, a tubulin-like GTPase that forms a dynamic ring marking the division plane of prokaryotic cells, is essential for cytokinesis . It is not known what triggers FtsZ ring assembly . In this work, we use a FtsZ-green fluorescent protein (Gfp) chimera to assay FtsZ assembly over time by using fluorescence microscopy . We show that FtsZ polymers can assemble dynamically in solution in a GTP-dependent manner . Initially, FtsZ nucleation centers grow into aster-like structures that dramatically resemble microtubule organizing centers . As assembly proceeds further, protofilament bundles emanating from different asters interconnect, mimicking the closure of the FtsZ ring in vivo . Surprisingly, millimolar levels of Ca2+ promote FtsZ dynamic assembly . FtsZ can undergo repeated GTP-dependent assembly and disassembly in solution by sequential addition and removal of Ca2+ . In addition, GTP binding and hydrolysis by FtsZ are regulated by Ca2+ concentration . Although the concentration of Ca2+ required for FtsZ assembly in vitro is high, its clear and specific effect on FtsZ dynamics suggests the possibility that Ca2+ may have a role in regulating FtsZ ring assembly in the cell.

Biochem Pharmacol, 1997 Sep 15, 54(6), 639 - 47
A new nomenclature for the aldo-keto reductase superfamily; Jez JM et al.; The aldo-keto reductases (AKRs) represent a growing oxidoreductase superfamily . Forty proteins have been identified and characterized as AKRs, and an additional fourteen genes may encode proteins related to the superfamily . Found in eukaryotes and prokaryotes, the AKRs metabolize a wide range of substrates, including aliphatic aldehydes, monosaccharides, steroids, prostaglandins, and xenobiotics . This broad substrate specificity has caused problems in naming these proteins . Enzymes capable of these reactions have been referred to as aldehyde reductase (ALR1), aldose reductase (ALR2), and carbonyl reductase (ALR3); however, ALR3 is not a member of the AKR superfamily . Also, some AKRs have multiple names based upon substrate specificity . For example, human 3alpha-hydroxysteroid dehydrogenase (3apha-HSD) type I is also known as dihydrodiol dehydrogenase 4 and chlordecone reductase . To address these issues, we propose a new nomenclature system for the AKR superfamily based on amino acid sequence identities . Cluster analysis of the AKRs shows seven distinct families at the 40% amino acid identity level . The largest family (AKR1) contains the aldose reductases, aldehyde reductases, and HSDs . Other families include the prokaryotic AKRs, the plant chalcone reductases, the Shaker channels, and the ethoxyquin-inducible aflatoxin B1 aldehyde reductase . At the level of 60% amino acid identity, subfamilies are discernible . For example, the AKR1 family includes five subfamilies: (A) aldehyde reductases (mammalian); (B) aldose reductases; (C) HSDs; (D) delta4-3-ketosteroid-5beta-reductases; and (E) aldehyde reductases (plant) . This cluster analysis forms the basis for our nomenclature system . Recommendations for naming an aldo-keto reductase include the root symbol "AKR," an Arabic number designating the family, a letter indicating the subfamily when multiple subfamilies exist, and an Arabic numeral representing the unique protein sequence . For example, human aldehyde reductase would be assigned as AKR1A1 . Our nomenclature is both systematic and expandable, thereby allowing assignment of consistent designations for newly identified members of the superfamily.

Structure, 1997 Aug 15, 5(8), 1017 - 32
The structure of the cofactor-binding fragment of the LysR family member, CysB: a familiar fold with a surprising subunit arrangement; Tyrrell R et al.; BACKGROUND: CysB is a tetrameric protein of identical subunits (M(r) = 36,000) which controls the expression of genes associated with the biosynthesis of cysteine in bacteria . CysB is both an activator and a repressor of transcription whose activity is responsive to the inducer N-acetylserine; thiosulphate and sulphide act as anti-inducers . CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over approximately 280 residues including a putative helix-turn-helix DNA-binding motif at their N terminus . The aims of the present study were to explore further the complex molecular biology and curious ligand binding properties of CysB and to provide structural insights into the LysR family of proteins . RESULTS: The crystal structure of a dimeric chymotryptic fragment of Klebsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refined against data extending to 1.8 A resolution . The protein comprises two alpha/beta domains (I and II) connected by two short segments of polypeptide . The two domains enclose a cavity lined by polar sidechains, including those of two residues whose mutation is associated with constitutive expression of the cysteine regulon . A sulphate anion and a number of well ordered water molecules have been modelled into discrete electron-density peaks within this cavity . In the dimer, strands beta B from domain I and strands beta G from domain II come together so that a pair of antiparallel symmetry-related 11-stranded twisted beta-pleated sheets is formed . CONCLUSIONS: The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins . A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins . In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain . This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.

Prog Nucleic Acid Res Mol Biol, 1998, 58, 239 - 61
Molecular and structural features of the proton-coupled oligopeptide transporter superfamily; Fei YJ et al.; Work in the area of molecular biology of transport proteins has unveiled the presence of a distinct peptide transporter superfamily whose members extend from the prokaryotic to the eukaryotic kingdom . There are two subgroups within this superfamily, one subgroup harnessing the energy necessary for active transport from a transmembrane H+ gradient and the other subgroup relying directly on ATP hydrolysis . In addition to the use of different driving forces, the two subgroups are also distinguishable with regard to molecular structure and operational mechanism . This review is intended to analyze critically the molecular nature of the members of the H+ gradient-dependent peptide transporter subgroup, with emphasis on the cloning strategies utilized in the isolation of the individual transporter cDNAs or genes; on the structural patterns, motifs, and conserved amino acid residues common to constituent members of the subgroup; and on the characteristic topological features of the individual members.

Prog Nucleic Acid Res Mol Biol, 1998, 58, 127 - 64
Lactose repressor protein: functional properties and structure; Matthews KS et al.; The lactose repressor protein (LacI), the prototype for genetic regulatory proteins, controls expression of lactose metabolic genes by binding to its cognate operator sequences in E . coli DNA . Inducer binding elicits a conformational change that diminishes affinity for operator sequences with no effect on nonspecific binding . The release of operator is followed by synthesis of mRNA encoding the enzymes for lactose utilization . Genetic, chemical and physical studies provided detailed insight into the function of this protein prior to the recent completion of X-ray crystallographic structures . The structural information can now be correlated with the phenotypic data for numerous mutants . These structures also provide the opportunity for physical and chemical studies on mutants designed to examine various aspects of lac repressor structure and function . In addition to providing insight into protein structure-function correlations, LacI has been utilized in a wide variety of applications both in prokaryotic gene expression and in eukaryotic gene regulation and studies of mutagenesis.

Microbiology, 1997 Sep, 143 ( Pt 9), 2891 - 902
Towards the phylogeny of APS reductases and sirohaem sulfite reductases in sulfate-reducing and sulfur-oxidizing prokaryotes; Hipp WM et al.; The genes for adenosine-5'-phosphosulfate (APS) reductase, aprBA, and sirohaem sulfite reductase, dsrAB, from the sulfur-oxidizing phototrophic bacterium Chromatium vinosum strain D (DSMZ 180(T)) were cloned and sequenced . Statistically significant sequence similarities and similar physicochemical properties suggest that the aprBA and dsrAB gene products from Chr . vinosum are true homologues of their counterparts from the sulfate-reducing chemotrophic archaeon Archaeoglobus fulgidus and the sulfate-reducing chemotrophic bacterium Desulfovibrio vulgaris . Evidence for the proposed duplication of a common ancestor of the dsrAB genes is provided . Phylogenetic analyses revealed a greater evolutionary distance between the enzymes from Chr . vinosum and D . vulgaris than between those from A . fulgidus and D . vulgaris . The data reported in this study are most consistent with the concept of common ancestral protogenotic genes both for dissimilatory sirohaem sulfite reductases and for APS reductases . The aprA gene was demonstrated to be a suitable DNA probe for the identification of apr genes from organisms of different phylogenetic positions . PCR primers and conditions for the amplification of apr homologous regions are described.

EMBO J, 1997 Aug 15, 16(16), 4880 - 6
Co-translational protein targeting catalyzed by the Escherichia coli signal recognition particle and its receptor; Powers T et al.; The Ffh-4.5S ribonucleoprotein particle (RNP) and FtsY from Escherichia coli are homologous to essential components of the mammalian signal recognition particle (SRP) and SRP receptor, respectively . The ability of these E . coli components to function in a bona fide co-translational targeting pathway remains unclear . Here we demonstrate that the Ffh-4.5S RNP and FtsY can efficiently replace their mammalian counterparts in targeting nascent secretory proteins to microsomal membranes in vitro . Targeting in the heterologous system requires a hydrophobic signal sequence, utilizes GTP and, moreover, occurs co-translationally . Unlike mammalian SRP, however, the Ffh-4.5S RNP is unable to arrest translational elongation, which results in a narrow time window for the ribosome nascent chain to interact productively with the membrane-bound translocation machinery . The highly negatively charged N-terminal domain of FtsY, which is a conserved feature among prokaryotic SRP receptor homologs, is important for translocation and acts to localize the protein to the membrane . Our data illustrate the extreme functional conservation between prokaryotic and eukaryotic SRP and SRP receptors and suggest that the basic mechanism of co-translational protein targeting is conserved between bacteria and mammals.

Can J Microbiol, 1997 Aug, 43(8), 734 - 43
Cloning, functional expression, and complementation analysis of an inorganic pyrophosphatase from Bartonella bacilliformis; Mitchell SJ et al.; We have cloned the inorganic pyrophosphatase gene (ppa) from the facultative intracellular pathogen Bartonella bacilliformis and characterized its encoded product . The 531-bp gene is located approximately 1 kb downstream of, and in opposite orientation to, the invasion-associated locus (ialAB) of B . bacilliformis . The predicted protein encoded by ppa is 177 amino acid residues, which is in agreement with in vitro and in vivo synthesis of a protein with an apparent molecular mass of 22-23 kDa . The predicted B . bacilliformis pyrophosphatase (PPase) sequence is 53% identical and 85% similar to the E . coli PPase (EC 3.6.1.1), and contains all 12 of the amino acid residues implicated in the catalytic active site . The isolated B . bacilliformis PPase exhibits an activity of 51 +/- 2 mumol PO4 released/(mg protein.min) at 28 degrees C and pH 8, and is sensitive to inhibition by Ca2+ . In keeping with other prokaryotic PPases, B . bacilliformis PPase activity occurs from pH 6 to 10 (optimal pH = 8) and demonstrates high thermostability in the presence of Mg2+ (highest activity at 55 degrees C, relative activity = 80 +/- 3% at pH 8) . The cloned B . bacilliformis ppa is able to genetically complement a ppa- mutant strain of E . coli.

Free Radic Biol Med, 1997, 23(6), 851 - 8
Oxidative damage to nucleic acids photosensitized by titanium dioxide; Wamer WG et al.; The semiconductor TiO2 is known to have photobiological activity in prokaryotic and eukaryotic cells . Applications of this photobiological activity have been suggested including sterilization of waste water and phototherapy of malignant cells . Here, several model and cellular systems were used to study the mechanism of photocatalysis by TiO2 . Treatment of TiO2 (anatase, 0.45 microns), suspended in water containing a spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), with UV radiation (320 nm) resulted in an electron spin resonance (ESR) signal characteristic of the hydroxyl radical . Irradiation of solutions containing calf thymus DNA and TiO2 with UVA (320-400 nm) radiation resulted in hydroxylation of guanine bases . The degree of hydroxylation was dependent on both UVA fluence and amount of TiO2 in suspension . Human skin fibroblasts, preincubated 18 h with 10 micrograms/cm2 TiO2 and then UVA-irradiated (0-58 KJ/m2), showed dose dependent photocytoxicity . RNA, isolated from similarly treated fibroblasts, contained significant levels of photooxidation, measured as hydroxylation of guanine bases . However, no oxidative damage was detectable in cellular DNA . These results suggest that nucleic acids are a potential target for photooxidative damage sensitized by TiO2, and support the view that TiO2 photocatalyzes free radical formation.

Biol Chem, 1997 Aug, 378(8), 769 - 77
Drug efflux proteins in multidrug resistant bacteria; van Veen HW et al.; Bacteria contain an array of transport proteins in their cytoplasmic membrane . Many of these proteins play an important role in conferring resistance to toxic compounds . The multidrug efflux systems encountered in prokaryotic cells are very similar to those observed in eukaryotic cells . Therefore, a study of the factors which determine the substrate specificity and energy coupling to drug translocation in bacteria has significance for the general field of multidrug resistance . Three issues will be dealt with in this review . First, an overview of the various classes of prokaryotic multidrug transporters will be presented . Second, the current understanding of the regulation of bacterial multidrug resistance will be summarized . Third, the present knowledge of the molecular mechanisms involved in drug transport processes will be discussed.

Planta, 1997 Sep, 203(1), 111 - 20
Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L.) by exogenously supplied carbohydrates in excised leaf discs; Duwenig E et al.; A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated . Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes . Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low affinity to glycogen . Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity . Plastidic phosphorylase was transcribed in flowers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds . This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds . Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation . The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates . Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined . A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species . In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates . These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.

Biochem Biophys Res Commun, 1997 Aug 28, 237(3), 673 - 7
Characterization of a putative receptor for intestinal trefoil factor in rat small intestine: identification by in situ binding and ligand blotting; Tan XD et al.; Intestinal trefoil factor (ITF), a small peptide secreted by intestinal goblet cells, maintains mucosal integrity and promotes epithelial wound healing . Although ITF has been cloned, the detailed mechanism by which ITF interacts with intestinal epithelium remains elusive . In the present study, we expressed mature rat ITF (rITF) with pXa (a prokaryotic expression vector) in Escherichia coli to generate a biotinylated rITF fusion protein (bTag-ITF) . By using bTag-ITF probe, we identified ITF binding cells in the crypts of the small intestine and mucous cells of the region of gastric glands . Using a ligand blotting technique, we further characterized a 50 kDa glycosylated protein from the membrane fraction of the small intestine, which bound to bTag-ITF . Our data suggest that this 50 kDa membrane glycoprotein is a putative receptor for ITF in the gastrointestinal tract.

Mol Phylogenet Evol, 1997 Oct, 8(2), 167 - 76
Evolution of the tryptophan biosynthetic pathway in Buchnera (aphid endosymbionts): studies of plasmid-associated trpEG within the genus Uroleucon; Rouhbakhsh D et al.; Aphids obtain tryptophan from prokaryotic endosymbionts assigned to the genus Buchnera . The rate-limiting enzyme in tryptophan biosynthes, anthranilate synthase, is encoded by the genes trpE and trpG . In Buchnera within the family Aphididae, trpEG is plasmid-borne, apparently as an adaptation to overproduce tryptophan for hosts . To explore the evolution of these plasmids, sequences for trpEG, the upstream region containing the plasmid origin of replication, and chromosomal trpB were obtained for Buchnera of three species in the aphid genus Uroleucon and analyzed together with sequences for six other aphid species . Phylogenies based on trpB and trpEG agree with each other and with previous views of aphid phylogeny . Synonymous substitutions are about twice as high for plasmid-borne genes as for chromosomal genes in the same lineages, suggesting higher mutation rates for genes on plasmids . Nonsynonymous rates for trpEG are accelerated within Buchnera of Uroleucon, indicating a change in selection intensity within this genus . Accelerated evolution within Uroleucon also seems to characterize the upstream region containing the putative origin of replication .

Biochemistry, 1997 Sep 23, 36(38), 11298 - 303
Subunit II of the cytochrome bo3 ubiquinol oxidase from Escherichia coli is a lipoprotein; Ma J et al.; The purified Escherichia coli cytochrome bo3 ubiquinol oxidase contains four subunits that are each integral components of the cytoplasmic membrane . The molecular weight of each of the subunits has been determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI) . The observed molecular weight of subunit II (CyoA) is considerably less than the calculated value from the deduced amino acid sequence, indicating possible posttranslational processing . The similarity of a portion of the sequence near the N-terminus of CyoA with the sequences of known prokaryotic membrane-bound lipoproteins suggested that CyoA is proteolytically processed to generate an N-terminus at Cys25, and that Cys25 is covalently modified by the addition of lipids . This would be consistent with the observed molecular mass, and was confirmed by demonstrating the incorporation of radioactive palmitic acid into subunit II of the cytochrome bo3 oxidase . Site-directed mutagenesis replacing Cys25 by alanine prevents the processing, generating a precursor form of CyoA with a higher molecular mass . The C25A mutant of CyoA still assembles as an active quinol oxidase capable of supporting growth of the cells by aerobic respiration . Hence, this unusual processing of a cytoplasmic membrane protein, which is already anchored to the membrane by two transmembrane helices, is not essential for either assembly or function.

Biochim Biophys Acta, 1997 Aug 21, 1358(1), 53 - 60
Characterization of prokaryotic recombinant Aspergillus ribotoxin alpha-sarcin; Sylvester ID et al.; The Aspergillus ribonuclease alpha-sarcin is toxic to intact mammalian cells but the mechanism by which it enters the cells to reach its ribosomal RNA substrate is unclear . Here we have compared the cytotoxicity of alpha-sarcin to that of ricin, another catalytic toxin that targets the same rRNA sequence but whose mechanism of cell entry is better understood . Intact ricin binds to cell surface components and enters the cells by receptor-mediated endocytosis, whereas the catalytic polypeptide of ricin (the A chain or RTA) which, like alpha-sarcin, is unable to bind to surface components directly and enters cells by fluid phase uptake . Recombinant alpha-sarcin was produced in Escherichia coli and purified to homogeneity . The protein was soluble, stable and its ability to inhibit in vitro protein synthesis was indistinguishable from that of native alpha-sarcin . Further, recombinant alpha-sarcin had the same in vitro protein synthesis inhibition activity as ricin A chain . The cytotoxicity of alpha-sarcin and ricin A chain to HeLa cells was also the same . The cytotoxicity of alpha-sarcin was due to its RNAase activity rather than to specific membrane effects at the cell surface, since a mutant containing a single substitution at a putative key catalytic residue had reduced ribonuclease activity and an equivalent reduction in cytotoxicity . One interpretation of the data is that a-sarcin enters mammalian cells in the same way as free ricin A chain.

Antonie Van Leeuwenhoek, 1997 Jul, 72(1), 49 - 61
Protein phylogenies and signature sequences: evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes; Gupta RS; The evolutionary relationships within prokaryotes and between prokaryotes and eukaryotes is examined based on protein sequence data . Phylogenies and common signature sequences in some of the most conserved proteins point to a close evolutionary relationship between Archaebacteria and Gram-positive bacteria . The monophyletic nature and distinctness of the Archaebacterial domain is not supported by many of the phylogenies . Within Gram-negative bacteria, cyanobacteria are indicated as the deepest branching lineage, and a clade consisting of Archaebacteria, Gram-positive bacteria and cyanobacteria is supported by signature sequences in many proteins . However, the division within the prokaryotic species, viz . Archaebacteria<-->Gram-positive bacteria-->Cyanobacteria-->other groups of Gram-negative bacteria, is indicated to be not very rigid but, instead is an evolutionary continuum . It is expected that certain species will be found which represent intermediates in the above transitions . By contrast to the evolutionary relationships within prokaryotes, the eukaryotic species, which are structurally very different, appear to have originated by a very different mechanism . Protein phylogenies and signature sequences provide evidence that the eukaryotic nuclear genome is a chimera which has received major contributions from both an Archaebacterium and a Gram-negative bacterium . To explain these observations, it is suggested that the ancestral eukaryotic cell arose by a symbiotic fusion event between the above parents and that this fusion event led to the origin of both nucleus and endoplasmic reticulum . The monophyletic nature of all extant eukaryotic species further suggests that a 'successful primary fusion' between the prokaryotic species that gave rise to the ancestral eukaryotic cell took place only once in the history of this planet.

Antonie Van Leeuwenhoek, 1997 Jul, 72(1), 39 - 48
Non-cultivable microorganisms from symbiotic associations of insects and other hosts; Baumann P et al.; Many symbiotic associations involve microorganisms which cannot be cultivated on laboratory media . These organisms remained little known until the recent advent of methods of recombinant DNA analysis and molecular phylogenetics . Applications of these methods to endosymbionts have resulted in substantial new insights concerning the genetics and evolution of these organisms . This communication provides a listing of recently studied associations involving non-cultivable symbionts . The associations involve a diverse set of host taxa and a wide range of effects, both favorable and deleterious, on host biology . Among beneficial endosymbionts, a variety of nutritional interactions have been documented . One type of association has been demonstrated for a number of animal hosts, namely endosymbioses that result from a single infection of an ancestral host by a prokaryote . In these associations, endosymbionts are transmitted maternally and are not exchanged between host lineages, resulting in a long-term pattern of codiversification of hosts and endosymbionts . The association between aphids and non-cultivable prokaryotic endosymbionts is a well studied example of such a symbiosis.

Antonie Van Leeuwenhoek, 1997 Jul, 72(1), 3 - 19
Prokaryotic diversity and the importance of culturing; Palleroni NJ; Modern approaches based on the use of molecular techniques presumed to circumvent the need for culturing prokaryotes, fail to provide sufficient and reliable information for estimation of prokaryote diversity . Many properties that make these organisms important members of the living world are amenable to observation only through the study of living cultures . Since current culture techniques do not always satisfy the need of providing a balanced picture of the microflora composition, future developments in the study of bacterial diversity should include improvements in the culture methods to approach as closely as possible the conditions of natural habitats . Molecular methods of microflora analysis have an important role as guides for the isolation and characterization of new prokaryotic taxa . Although the species concept is central to biodiversity studies, it is extremely difficult to propose a definition applicable without constraints to all groups of living organisms . However, in prokaryote systematics much improvement has been achieved by comprehensive descriptions that include not only molecular data, but also the relevant aspects of the biology of the organisms under study (polyphasic approach).

J Biol Chem, 1997 Sep 19, 272(38), 23592 - 6
Molecular cloning and expression of fatty acid alpha-hydroxylase from Sphingomonas paucimobilis; Matsunaga I et al.; Fatty acid alpha-hydroxylase (FAAH) catalyzes the initial reaction in alpha-oxidation of fatty acid to produce 2-hydroxy fatty acid . FAAH activity has been detected in a wide range of organisms from prokaryotes to eukaryotes . Here, we describe cloning of the FAAH gene from Sphingomonas paucimobilis, a sphingolipid- and 2-hydroxymyristic acid-rich bacterium . The isolated gene encoded 415 amino acids . A homology search revealed that amino acid sequences highly conserved in cytochrome P450 (P450) were present in FAAH . Although the heme-binding cysteine was recognizable at position 361, the consensus in the heme-binding region was modified by an insertion . Overall, FAAH has no significant identity to the known P450s . CO difference spectrum of recombinant FAAH showed the characteristic one of P450, except this peak was at 445 nm . These results suggest bacterial FAAH is a novel member of the P450 superfamily.

J Bacteriol, 1997 Sep, 179(18), 5728 - 35
A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus synthesis; Haase J et al.; TraF, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4 (IncP), which is located at the periplasmic side of the cytoplasmic membrane, encodes a specific protease . The traF gene products of IncP and Ti plasmids show extensive similarities to prokaryotic and eukaryotic signal peptidases . Mutational analysis of RP4 TraF revealed that the mechanism of the proteolytic cleavage reaction resembles that of signal and LexA-like peptidases . Among the RP4 transfer functions, the product of the Tra2 gene, trbC, was identified as a target for the TraF protease activity . TrbC is homologous to VirB2 of Ti plasmids and thought to encode the RP4 prepilin . The maturation of TrbC involves three processing reactions: (i) the removal of the N-terminal signal peptide by Escherichia coli signal peptidase I (Lep), (ii) a proteolytic cleavage at the C terminus by an as yet unidentified host cell enzyme, and (iii) C-terminal processing by TraF . The third reaction of the maturation process is critical for conjugative transfer, pilus synthesis, and the propagation of the donor-specific bacteriophage PRD1 . Thus, cleavage of TrbC by TraF appears to be one of the initial steps in a cascade of processes involved in export of the RP4 pilus subunit and pilus assembly mediated by the RP4 mating pair formation function.

Proc Natl Acad Sci U S A, 1997 Sep 16, 94(19), 10227 - 32
Compositional differences within and between eukaryotic genomes; Karlin S et al.; Eukaryotic genome similarity relationships are inferred using sequence information derived from large aggregates of genomic sequences . Comparisons within and between species sample sequences are based on the profile of dinucleotide relative abundance values (The profile is rho*XY = f*XY/f*Xf*Y for all XY, where f*X denotes the frequency of the nucleotide X and f*XY denotes the frequency of the dinucleotide XY, both computed from the sequence concatenated with its inverted complement) . Previous studies with respect to prokaryotes and this study document that profiles of different DNA sequence samples (sample size >/=50 kb) from the same organism are generally much more similar to each other than they are to profiles from other organisms, and that closely related organisms generally have more similar profiles than do distantly related organisms . On this basis we refer to the collection (rho*XY) as the genome signature . This paper identifies rho*XY extremes and compares genome signature differences for a diverse range of eukaryotic species . Interpretations on the mechanisms maintaining these profile differences center on genome-wide replication, repair, DNA structures, and context-dependent mutational biases . It is also observed that mitochondrial genome signature differences between species parallel the corresponding nuclear genome signature differences despite large differences between corresponding mitochondrial and nuclear signatures . The genome signature differences also have implications for contrasts between rodents and other mammals, and between monocot and dicot plants, as well as providing evidence for similarities among fungi and the diversity of protists.

RNA, 1997 Sep, 3(9), 1016 - 27
Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity; Nikolcheva T et al.; The effect of genetic context on splicing of group I introns is not well understood at present . The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA . Mutations that block splicing in E . coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes . The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit . Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients . This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix . Association of the intron with 50S subunits correlates with slow cell growth . The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.

J Gen Virol, 1997 Sep, 78 ( Pt 9), 2365 - 77
Baculoviruses contain a gene for the large subunit of ribonucleotide reductase; van Strien EA et al.; In the genomes of two baculoviruses, Spodoptera exigua and S . littoralis multicapsid nucleopolyhedroviruses (SeMNPV and SpliMNPV, respectively), an open reading frame (ORF) encoding the large subunit of ribonucleotide reductase (RR1) was identified . The predicted amino acid sequences of SeMNPV and SpliMNPV RR1 showed high homology to RR1 proteins from eukaryotes (ca . 70% and 80% similarity, respectively) . The amino acid residues thought to be involved in catalytic function were conserved in the baculoviral RR1 ORFs . The RR1 ORFs in SeMNPV and SpliMNPV were located in different genomic positions . In SeMNPV, the RR1 ORF was located upstream of the polyhedrin gene, in an anti-genomic orientation . In SpliMNPV, the RR1 ORF preceded the p74 gene . By searching databanks, sequences homologous to the N terminus of RR1 were also detected upstream of the polyhedrin gene of three other baculoviruses, Mamestra brassicae multicapsid NPV, Panolis flammea multicapsid NPV and Orgyia pseudotsugata single nucleocapsid NPV . The baculovirus type species, Autographica californica multicapsid NPV, however, does not encode RR . A 2.7 kb transcript could be detected throughout infection with SeMNPV, classifying SeMNPV rr1 as an early gene . Primer extension analysis revealed several early and late start sites . None of the major start sites showed similarity to previously characterized baculoviral transcriptional start motifs . Phylogenetic analysis of prokaryotic, eukaryotic and viral RR1 proteins suggested that SeMNPV and SpliMNPV acquired the gene for RR1 from a eukaryotic source, but independently from each other.

Genet Res, 1997 Jun, 69(3), 183 - 95
Role of transposable elements in age-related genomic instability; Nikitin AG et al.; Genetic instability is associated with aging in many species . One of the initiating factors for genetic instability is the movement of transposable elements (TEs), which occur in all prokaryotic and eukaryotic organisms . The hypothesis that TEs could be involved in the aging process is discussed and the correlation between aging and activity of TEs is analysed in a variety of biological systems.

Biochemistry, 1997 Sep 16, 36(37), 11125 - 30
Mutations induced by 2-hydroxyadenine on a shuttle vector during leading and lagging strand syntheses in mammalian cells; Kamiya H et al.; An oxidatively damaged base, 2-hydroxyadenine (2-OH-Ade), was incorporated into a predetermined site of one of the strands inverted question mark(+)- or (-)-strand inverted question mark of the double-stranded shuttle vector, pSVK3, and the modified DNAs were transfected into simian COS-7 cells . The nucleotide sequences in which the modified base was incorporated were 5'-GTCGA*C and 5'-CTTA*AG (A* represents 2-OH-Ade) . The former is the recognition site for the restriction enzyme SalI, and the latter is that for AflII . The DNAs replicated in the cells were recovered and were transfected again into Escherichia coli . The DNAs recovered from the COS-7 cells transfected with a plasmid containing 2-OH-Ade at either site of the (+)-strand (a template strand for lagging strand synthesis) formed colonies about 50%-70% as frequently as the unmodified DNA . This indicated that the base weakly blocked DNA replication during lagging strand synthesis . On the other hand, the base in the (-)-strand did not appear to affect the efficiency of leading strand synthesis in COS-7 cells . The mutation frequencies of 2-OH-Ade in COS-7 cells were 0.6%-0.1%, depending on the sequence and the strand location . Although the mutation spectra of 2-OH-Ade also differed with sequences and strands, the base elicited substitution and deletion mutations in mammalian cells, as in E . coli . These results indicate that 2-OH-Ade is mutagenic in eukaryotic cells as well as in prokaryotic cells.

Arch Microbiol, 1997 Sep, 168(3), 169 - 75
Glycoproteins in prokaryotes; Moens S et al.; Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins . A literature survey revealed the different types of glycoproteins . They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes . Only in a few cases is structural information available . Many different structures have been observed that display much more variation than that observed in eukaryotes . A few studies have given evidence for the function of the prokaryotic glycoprotein glycans . Also from the biosynthetic point of view, information is rather scarce . Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins . However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed.

J Biomed Mater Res, 1997 Sep 5, 36(3), 284 - 8
Glutaraldehyde-containing dentin bonding agents are mutagens in mammalian cells in vitro; Schweikl H et al.; The mutagenic potential of glutaraldehyde-containing dentin bonding agents was shown in previous studies using a bacterial gene mutation assay, the Ames test . However, current strategies of genotoxicity testing and regulatory requirements for the biological evaluation of medical devices recommend a battery of tests that indicate induced mutations in prokaryotic and eukaryotic cells . Accordingly, the mutagenicity of three glutaraldehyde-containing bonding agents (Syntac adhesive, Prisma Universal Bond 3 adhesive, and Gluma 3) was investigated using a quantitative mammalian cell gene mutation assay (V79/HPRT test) in the present investigation . The materials were extracted in dimethyl sulfoxide (0.1 g/2 mL) for 24 h and original extracts were then serially diluted in cell culture medium before exposure to V79 cells . Cytotoxic and mutagenic effects were observed with identical concentrations of extracts of the different test materials . There was a moderate decrease of the number of surviving cells immediately after the end of exposure . Mutagenicity at the hprt locus in V79 cells was found with all materials tested, and the increases in the absolute numbers of mutants were dose dependent . The mutant frequencies were about 15- (Syntac adhesive and Gluma 3) to 20-fold (Prisma UB3 adhesive) higher than solvent control values . Since other substances than glutaraldehyde may be responsible for the mutagenic effects in mammalian cells in this study, work is currently in progress to identify the individual mutagenic compounds of dentin adhesives and related composite materials.

Proc Natl Acad Sci U S A, 1997 Sep 2, 94(18), 9881 - 6
Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase; Hartzell PL; The complex prokaryote, Myxococcus xanthus, undergoes a program of multicellular development when starved for nutrients, culminating in sporulation . M . xanthus makes MglA, a 22-kDa, soluble protein that is required for both multicellular development and gliding motility . MglA is similar in sequence to the Saccharomyces cerevisiae SAR1 protein, a member of the Ras/Rab/Rho superfamily of small eukaryotic GTPases . The SAR1 gene, when integrated into the M . xanthus genome, complements the sporulation defect of a DeltamglA strain . A forward, second-site mutation on the M . xanthus chromosome, rpm, in combination with SAR1, restores fruiting body morphogenesis and gliding motility to a DeltamglA strain . The result that the rpm mutation suppresses the substitution of SAR1 for mglA suggests that Sar1p interacts with other M . xanthus proteins to control the motility-dependent aggregation of cells during development.

Mol Biol Evol, 1997 Sep, 14(9), 951 - 8
Phylogenetic relationships among prokaryotic and eukaryotic catalases; Klotz MG et al.; Seventy-four catalase protein sequences, including 29 bacterial, 8 fungal, 7 animal, and 30 plant sequences, were compiled, and 70 were used for phylogenetic reconstruction . The core of the resulting tree revealed unique, separate groups of plant and animal catalases, two groups of fungal catalases, and three groups of bacterial catalases . The only overlap of kingdoms occurred within one branch and involved fungal and bacterial large-subunit enzymes . The other fungal branch was closely linked to the group of animal enzymes . Group I bacterial catalases were more closely related to the plant enzymes and contained such diverse taxa as the Gram-positive Listeria seeligeri, Deinocococcus radiodurans, and gamma-proteobacteria . Group III bacterial sequences were more closely related to fungal and animal sequences and included enzymes from a broad range of bacteria including high- and low-GC Gram positives, proteobacteria, and a bacteroides species . Group II was composed of large-subunit catalases from diverse sources including Gram positives (low-GC Bacilli and high-GC Mycobacteria), proteobacteria, and species of the filamentous fungus Aspergillus . These data can be interpreted in terms of two gene duplication events that produced a minimum of three catalase gene family members that subsequently evolved in response to environmental demands . Horizontal gene transfer may have been responsible for the group II mixture of bacterial and fungal large-subunit catalases.

Mol Biol Evol, 1997 Sep, 14(9), 914 - 25
Mutation accumulation in nuclear, organelle, and prokaryotic transfer RNA genes; Lynch M; A comparative analysis of the transfer RNA genes in the genomes of the major kingdoms of eukaryotes and prokaryotes leads to the general conclusion that the rate of evolution of organelle tRNA genes is typically equal to of greater than that of their nuclear counterparts . Situations where this is not the case, most notably in vascular plants, are attributable to an elevated mutation rate in the nuclear genome . Through a comparison of rates of mutation with rates of nucleotide substitution, it is shown that there is a reduction in the efficiency of selection on new mutations in organelle genes . Numerous lines of evidence, including observed reductions in stem duplex stability and changes in loop sizes, suggest that the excess changes observed in the organelle genes are mildly deleterious . Uniparental inheritance of organelles causes a reduction in the efficiency of selection through the joint effects of an increase in linkage disequilibrium and a decrease in effective population size . These results provide molecular support for the idea that asexually propagating genomes are subject to long-term, gradual fitness loss and raise questions about the role of organelle mutations in the long-term survival of major phylogenetic lineages.

Mol Cell Biol, 1997 Sep, 17(9), 5559 - 70
Palindrome resolution and recombination in the mammalian germ line; Akgun E et al.; Genetic instability is promoted by unusual sequence arrangements and DNA structures . Hairpin DNA structures can form from palindromes and from triplet repeats, and they are also intermediates in V(D)J recombination . We have measured the genetic stability of a large palindrome which has the potential to form a one-stranded hairpin or a two-stranded cruciform structure and have analyzed recombinants at the molecular level . A palindrome of 15.3 kb introduced as a transgene was found to be transmitted at a normal Mendelian ratio in mice, in striking contrast to the profound instability of large palindromes in prokaryotic systems . In a significant number of progeny mice, however, the palindromic transgene is rearranged; between 15 and 56% of progeny contain rearrangements . Rearrangements within the palindromic repeat occur both by illegitimate and homologous, reciprocal recombination . Gene conversion within the transgene locus, as quantitated by a novel sperm fluorescence assay, is also elevated . Illegitimate events often take the form of an asymmetric deletion that eliminates the central symmetry of the palindrome . Such asymmetric transgene deletions, including those that maintain one complete half of the palindromic repeat, are stabilized so that they cannot undergo further illegitimate rearrangements, and they also exhibit reduced levels of gene conversion . By contrast, transgene rearrangements that maintain the central symmetry continue to be unstable . Based on the observed events, we propose that one mechanism promoting the instability of the palindrome may involve breaks generated at the hairpin structure by a hairpin-nicking activity, as previously detected in somatic cells . Because mammalian cells are capable of efficiently repairing chromosome breaks through nonhomologous processes, the resealing of such breaks introduces a stabilizing asymmetry at the center of the palindrome . We propose that the ability of mammalian cells to eliminate the perfect symmetry in a palindromic sequence may be an important DNA repair pathway, with implications regarding the metabolism of palindromic repeats, the mutability of quasipalindromic triplet repeats, and the early steps in gene amplification events.

Nucleic Acids Res, 1997 Sep 1, 25(17), 3465 - 70
Specific polyadenylation and purification of total messenger RNA from Escherichia coli; Amara RR et al.; Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages . We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese . The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb . We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA . Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes . Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species . Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis . Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Neurochem Int, 1997 Sep, 31(3), 379 - 92
Regulation of gene expression by natural antisense RNA transcripts; Knee R et al.; The use of synthetic antisense oligonucleotides as specific inhibitors of gene expression exploits the susceptibility of mRNA to functional blockade at several levels, including mRNA processing, transport, translation and degradation . It is becoming increasingly apparent that the actions of these synthetic oligomers are analogous to those of endogenous RNA molecules involved in the regulation of gene expression in both prokaryotes and eukaryotes . A growing number of eukaryotic genes are now thought to be regulated at least in part by natural antisense RNA transcribed from the presumptive non-coding DNA strand . This possibility is supported by the presence of a complex system of double-stranded (ds) RNA-specific proteins and dsRNA-induced signal transduction pathways in eukaryotic cells . The presence of functional open reading frames in a number of recognized natural antisense RNA transcripts indicates that, in addition to regulating gene function at the RNA level, the antisense strand of many genes may code for as yet unidentified proteins . In the present study we review the current literature on the role(s) played by natural antisense RNA in eukaryotic cells, with an emphasis on genes for which clear evidence of regulation, or potential regulation by natural antisense RNA is available.

Biochemistry, 1997 Aug 26, 36(34), 10538 - 44
3-Phosphohistidine cannot replace phosphotyrosine in high-affinity binding to phosphotyrosine binding or Src homology 2 domains; Senderowicz L et al.; Posttranslational phosphorylation of proteins is an important event in many cellular processes . Phosphorylated tyrosine residues can serve as association sites for other proteins in signal transduction cascades of tyrosine kinase receptors . Formation of phosphohistidine residues in proteins has been found in eukaryotic and prokaryotic organisms . Furthermore, it has been suggested that phosphohistidine might substitute for phosphotyrosine in conferring high-affinity binding to proteins involved in signal transduction . We have analyzed the ability of 3-phosphohistidine to associate with the known phosphotyrosine-specific phosphotyrosine binding and src homology 2 protein domains . From our binding studies using synthetic peptides, we conclude that 3-phosphohistidine cannot replace phosphotyrosine in conferring high-affinity binding to the phosphotyrosine binding domain of shc or the src homology 2 domain of phospholipase C-gamma1.

Biochemistry, 1997 Aug 19, 36(33), 10335 - 42
Tetrameric stoichiometry of a prokaryotic K+ channel; Heginbotham L et al.; Genes with sequences reminiscent of neuronal K+ channels have recently been identified in prokaryotes . These putative K+ channels appear to be integral membrane proteins, with multiple transmembrane sequences identified by hydrophobicity analysis and a sequence strikingly similar to the pore-lining "P-region" motif found in all known eukaryotic K+ channels . This study examines the oligomeric state and stability in detergent micelles of SliK, a K+ channel homologue from Streptomyces lividans . A synthetic gene for SliK was expressed at high levels in Escherichia coli, and the protein was purified . The predominant form of the protein runs in SDS-PAGE gels as an oligomer of the 19-kDa polypeptide, but harsh treatments such as heat or high pH convert this slowly-migrating material into monomeric form . A "mass-tagging" strategy developed to examine subunit stoichiometry shows that SliK is a homotetramer in SDS and dodecyl maltoside micelles . The tetrameric structure can be disrupted by P-region mutations known to prevent the functional expression of neuronal K+ channels . The tetramer is remarkably stable, showing no conversion to the monomeric form after 14 days at room temperature . Although SliK-mediated cation flux activity was not observed, the tetrameric behavior of the protein argues that SliK may provide a system for a direct attack on the structure of a K+ channel P-region sequence.

Nucleic Acids Res, 1997 Aug 15, 25(16), 3332 - 8
Functional analysis of point mutations in human flap endonuclease-1 active site; Shen B et al.; Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases . Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue . In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity . Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160) . We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme . Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively) . The other mutants (D158A, D179A and D233A) have no detectable binding activity . We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme . These two metal coordinated active sites are distinguishable but interrelated . One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding . These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.

Biochemistry, 1997 Aug 12, 36(32), 9780 - 90
Comparative EPR and redox studies of three prokaryotic enzymes of the xanthine oxidase family: quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase; Canne C et al.; For three prokaryotic enzymes of the xanthine oxidase family, namely quinoline 2-oxidoreductase, quinaldine 4-oxidase, and isoquinoline 1-oxidoreductase, the electron transfer centers were investigated by electron paramagnetic resonance . The enzymes are containing a molybdenum-molybdopterin cytosine dinucleotide cofactor, two distinct {2Fe-2S} clusters and, apart from isoquinoline 1-oxidoreductase, a flavin adenine dinucleotide . The latter cofactor yields two different organic radical signals in quinoline 2-oxidoreductase and quinaldine 4-oxidase, typical for the neutral and anionic form, respectively . A "rapid" Mo(V) species is present in all enzymes with small differences in magnetic parameters . From spectra simulation of 95Mo-substituted quinoline 2-oxidoreductase, a deviation of 25 degrees between the maximal g and 95Mo-hyperfine tensor component was derived . The very rapid Mo(V) species was detected in small amounts upon reduction with substrates in quinoline 2-oxidoreductase and quinaldine 4-oxidase, but showed a different kinetic behavior with considerable EPR intensities in isoquinoline 1-oxidoreductase . The FeSI and FeSII centers produced different signals in all three enzymes and, in case of isoquinoline 1-oxidoreductase, revealed a dipolar interaction, from which a maximum distance of 15 A between FeSI and FeSII was estimated . The midpoint potentials of the FeS centers were surprisingly different and determined for FeSI/FeSII with -155/-195 mV in quinoline 2-oxidoreductase, -250/-70 mV in quinaldine 4-oxidase, and +65/+10 mV in isoquinoline 1-oxidoreductase . The slopes of the fitting curves for the Nernst equation are indicative for nonideal behavior . Only in quinoline 2-oxidoreductase, an averaged midpoint potential of the molybdenum redox pairs of about -390 mV could be determined . Both of the other enzymes did not produce Mo(V) signals in redox titration experiments, probably because of direct reduction of Mo(VI) to Mo(IV) in the presence of dithionite.

J Biol Chem, 1997 Aug 8, 272(32), 20299 - 305
Human lysophosphatidic acid acyltransferase . cDNA cloning, expression, and localization to chromosome 9q34.3; Eberhardt C et al.; Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate (LPA)) is a phospholipid with diverse biological activities . The mediator serves as an intermediate in membrane phospholipid metabolism but is also produced in acute settings by activated platelets . LPA is converted to phosphatidic acid, itself a lipid mediator, by an LPA acyltransferase (LPAAT) . A human expressed sequence tag was identified by homology with a coconut LPAAT and used to isolate a full-length human cDNA from a heart muscle library . The predicted amino acid sequence bears 33% identity with a Caenorhabditis elegans LPAAT homologue and 23-28% identity with plant and prokaryotic LPAATs . Recombinant protein produced in COS 7 cells exhibited LPAAT activity with a preference for LPA as the acceptor phosphoglycerol and arachidonyl coenzyme A as the acyl donor . Northern blotting demonstrated that the mRNA is expressed in most human tissues including a panel of brain subregions; expression is highest in liver and pancreas and lowest in placenta . The human LPAAT gene is contained on six exons that map to chromosome 9, region q34.3.

J Gen Virol, 1997 Aug, 78 ( Pt 8), 1867 - 73
Monoclonal antibodies to the ORF5 product of porcine reproductive and respiratory syndrome virus define linear neutralizing determinants; Pirzadeh B et al.; Complementary DNA encoding the ORF5 gene of a Quebec reference isolate (IAF-Klop) of porcine reproductive and respiratory syndrome virus (PRRSV) was cloned into the prokaryotic expression vectors pGEX-4T and pET21a to produce ORF5-glutathione S-transferase and ORF5-polyhistidine fusion proteins . Five hybridoma cell lines producing monoclonal antibodies (MAbs) to the 25 kDa viral envelope glycoprotein (GP5) were obtained from BALB/c mice immunized with the affinity chromatography-purified GST-ORF5 fusion protein . The polypeptide specificity of these anti-PRRSV MAbs, belonging to the IgG1 isotype, was confirmed by Western immunoblotting assays with recombinant and native viral proteins, and by radioimmunoprecipitation using {35S}methionine-labelled concentrated extracellular virus . All these MAbs showed virus-neutralizing (VN) activity, with VN titres ranging from 1:32 to 1:128 . Two MAbs (IAF-1B8 and IAF-8A8) reacted with similar titres with the modified live attenuated vaccine strain ATCC VR-2332, but all five failed to react to the prototype European strain, the Lelystad virus, in VN and indirect immunofluorescence tests . The results obtained suggest that these five anti-PRRSV MAbs are directed to serotype-specific linear neutralizing epitopes which are not affected by the absence of carbohydrate residues.

Biotechnol Appl Biochem, 1997 Aug, 26 ( Pt 1), 15 - 7
Expression in Escherichia coli and purification of human thrombopoietin; Li S et al.; Human thrombopoietin (TPO) has been successfully overexpressed in Escherichia coli, with an expression level of about 12% of total cellular protein . The full-length TPO gene was subcloned into the prokaryotic expression vector pKK233-2 under the control of the inducible tac promoter . The recombinant protein was produced mainly in the form of inclusion body . By efficient renaturation and one-step purification, the recombinant protein was purified to homogeneity . The specific activity and yield of recombinant TPO can reach 2 x 10(4) units/mg and 2 mg/g of wet E . coli cells respectively.

J Bacteriol, 1997 Aug, 179(16), 5188 - 94
Protection of DNA during oxidative stress by the nonspecific DNA-binding protein Dps; Martinez A et al.; Reactive oxygen species can damage most cellular components, but DNA appears to be the most sensitive target of these agents . Here we present the first evidence of DNA protection against the toxic and mutagenic effects of oxidative damage in metabolically active cells: direct protection of DNA by Dps, an inducible nonspecific DNA-binding protein from Escherichia coli . We demonstrate that in a recA-deficient strain, expression of Dps from an inducible promoter prior to hydrogen peroxide challenge increases survival and reduces the number of chromosomal single-strand breaks . dps mutants exhibit increased levels of the G x C-->T x A mutations characteristic of oxidative damage after treatment with hydrogen peroxide . In addition, expression of Dps from the inducible plasmid reduces the frequency of spontaneous G x C-->T x A and A x T-->T x A mutations and can partially suppress the mutator phenotype of mutM (fpg) and mutY alleles . In a purified in vitro system, Dps reduces the number of DNA single-strand breaks and Fpg-sensitive sites introduced by hydrogen peroxide treatment, indicating that the protection observed in vivo is a direct effect of DNA binding by Dps . The widespread conservation of Dps homologs among prokaryotes suggests that this may be a general strategy for coping with oxidative stress.

J Bacteriol, 1997 Aug, 179(16), 5111 - 7
Induction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp . strain PCC 7942; Porankiewicz J et al.; The heat shock protein ClpB is essential for acquired thermotolerance in cyanobacteria and eukaryotes and belongs to a diverse group of polypeptides which function as molecular chaperones . In this study we show that ClpB is also strongly induced during moderate cold stress in the unicellular cyanobacterium Synechococcus sp . strain PCC 7942 . A fivefold increase in ClpB (92 kDa) content occurred when cells were acclimated to 25 degrees C over 24 h after being shifted from the optimal growth temperature of 37 degrees C . A corresponding increase occurred for the smaller ClpB' (78 kDa), which arises from a second translational start within the clpB gene of prokaryotes . Shifts to more extreme cold (i.e., 20 and 15 degrees C) progressively decreased the level of ClpB induction, presumably due to retardation of protein synthesis within this relatively cold-sensitive strain . Inactivation of clpB in Synechococcus sp . increased the extent of inhibition of photosynthesis upon the shift to 25 degrees C and markedly reduced the mutant's ability to acclimate to the new temperature regime, with a threefold drop in growth rate . Furthermore, around 30% fewer delta clpB cells survived the shift to 25 degrees C after 24 h compared to the wild type, and more of the mutant cells were also arrested during cell division at 25 degrees C, remaining attached after septum formation . Development of a cold thermotolerance assay based on cell survival clearly demonstrated that wild-type cells could acquire substantial resistance to the nonpermissive temperature of 15 degrees C by being pre-exposed to 25 degrees C . The same level of cold thermotolerance, however, occurred in the delta clpB strain, indicating ClpB induction is not necessary for this form of thermal resistance in Synechococcus spp . Overall, our results demonstrate that the induction of ClpB contributes significantly to the acclimation process of cyanobacteria to permissive low temperatures.

Exp Cell Res, 1997 Aug 1, 234(2), 205 - 16
Cloning and some novel characteristics of mitochondrial Hsp70 from Chinese hamster cells; Singh B et al.; The cDNA for Chinese hamster mitochondrial Hsp70 (mHsp70) was cloned and sequenced using a polymerase chain reaction probe based on conserved regions in the Hsp70 family of proteins . The encoded protein consists of 679 amino acids which includes a N-terminal mitochondrial targeting sequence of 46 amino acids . The mHsp70 protein contains several sequence signatures that are characteristics of prokaryotic and eukaryotic organellar Hsp70 homologs . In a phylogenetic tree based on Hsp70 sequences, it branches with the gram-negative proteobacteria, supporting the endosymbiotic origin of mitochondria from this group of prokaryotes . The mHsp70 cDNA was transcribed and translated in vitro and its import into isolated rat heart mitochondria was examined . The precursor mHsp70 was converted into a mature form of lower molecular mass (approximately 71 kDa) which became resistant to trypsin digestion . The import of mHsp70 into mitochondria was not observed in the presence of an uncoupler of energy metabolism or when the N-terminal presequence was lacking . The cDNA for mHsp70 was expressed in Escherichia coli and a polyclonal antibody to the purified recombinant protein was raised . The antibody shows no cross-reactivity to recombinant cytosolic Hsp70 protein and in 2-D gel blots it reacted specifically with the mHsp70 protein only . In immunofluorescence experiments, the antibody predominantly labeled mitochondria, and the observed labeling pattern was identical to that seen with a monoclonal antibody to the mitochondrial Hsp60 chaperonin . The affinity-purified antibody to mHsp70 was also employed to examine the subcellular distribution of the protein by cryoelectron microscopy and the immunogold-labeling technique . In these experiments, in addition to mitochondria, labeling with mitochondrial Hsp70 antibody was also observed on the plasma membrane and in unidentified cytoplasmic vesicles and granules . These studies raise the possibility that similar to the Hsp60 chaperonin and a number of other mitochondrial proteins, mHsp70 may have an extramitochondrial role.

Nat Biotechnol, 1997 Aug, 15(8), 751 - 3
Natural antisense RNA/target RNA interactions: possible models for antisense oligonucleotide drug design; Delihas N et al.; Current antisense oligonucleotides designed for drug therapy rely on Watson-Crick base pairing for the specificity of interactions between antisense and target molecules . However, thermodynamically stable duplexes containing non-Watson-Crick pairs have been formed with synthetic oligonucleotides . There are also numerous examples of non-canonical base pairs that participate in stable intra- and inter-molecular RNA/RNA pairing in prokaryotic and eukaryotic cells . Several natural antisense RNA/target RNA duplexes contain looped-out and bulged positions as well as non-canonical pairs as exemplified by formation of the Escherichia coli antisense micF RNA/ompF mRNA duplex . Secondary structures and the phylogenetic conservation of nucleotide sequences are well characterized in this system . Natural antisense/ target interactions may serve as models for determining possible and optimal antisense/target interactions in oligonucleotide drug design.

Mol Biol Evol, 1997 Aug, 14(8), 861 - 6
The age of the common ancestor of eukaryotes and prokaryotes: statistical inferences; Gu X; In this paper, a simple distance measure was used to estimate the age (T) of the common ancestor of eukaryotes and prokaryotes which takes the rate variation among sites and the pattern of amino acid substitutions into account . Our new estimate of T based on Doolittle et al.'s data is about 2.5 billion years ago (Ga), with 95% confidence interval from 2.1 to 2.9 Ga . This result indicates (1) that Doolittle et al.'s estimate (approximately 2.0 Ga) seems too recent, and (2) that the traditional view about the divergence time between eukaryotes and prokaryotes (T0 = 3.5 Ga) can be rejected at the 0.1% significance level.

Appl Environ Microbiol, 1997 Aug, 63(8), 3314 - 7
Glutamate dehydrogenase activity profiles for type strains of ruminal Prevotella spp; Wen Z et al.; The glutamate dehydrogenase (GDH) activities for the type strains of Prevotella ruminicola (strain 23), Prevotella brevis (strain GA33), and Prevotella bryantii (strain B(1)4) were assessed by a combination of enzyme assays and analysis of migration patterns of GDH proteins following nondenaturing polyacrylamide gel electrophoresis . Unlike results with most other prokaryotes, but similar to results with other members of the family Bacteroidaceae, NADPH-utilizing specific activity was greatest in all species following ammonia-limited growth . Similar also to previous findings with P . bryantii, the NAD(P)H-utilizing GDH activity of P . ruminicola can be attributed to a single protein . However, P . brevis produces an additional GDH protein(s) in response to growth with peptides . These results conclusively demonstrate that all type strains of the ruminal Prevotella sp . grouping possess GDH activity.

Appl Environ Microbiol, 1997 Aug, 63(8), 3003 - 9
Polyhydroxyalkanoate production in Rhodobacter capsulatus: genes, mutants, expression, and physiology; Kranz RG et al.; Like many other prokaryotes, the photosynthetic bacterium Rhodobacter capsulatus produces high levels of polyhydroxyalkanoates (PHAs) when a suitable carbon source is available . The three genes that are traditionally considered to be necessary in the PHA biosynthetic pathway, phaA (beta-ketothiolase), phaB (acetoacetylcoenzyme A reductase), and phaC (PHA synthase), were cloned from Rhodobacter capsulatus . In R . capsulatus, the phaAB genes are not linked to the phaC gene . Translational beta-galactosidase fusions to phaA and phaC were constructed and recombined into the chromosome . Both phaC and phaA were constitutively expressed regardless of whether PHA production was induced, suggesting that control is posttranslational at the enzymatic level . Consistent with this conclusion, it was shown that the R . capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis . The doubling times of R . capsulatus transcriptional nitrogen-sensing circuits were not involved in PHA synthesis . The doubling times of R . capsulatus grown on numerous carbon sources were determined, indicating that this bacterium grows on C2 to C12 fatty acids . Grown on acetone, caproate, or heptanoate, wild-type R . capsulatus produced high levels of PHAs . Although a phaC deletion strain was unable to synthesize PHAs on any carbon source, phaA and phaAB deletion strains were able to produce PHAs, indicating that alternative routes for the synthesis of substrates for the synthase are present . The nutritional versatility and bioenergetic versatility of R . capsulatus, coupled with its ability to produce large amounts of PHAs and its genetic tractability, make it an attractive model for the study of PHA production.

Orig Life Evol Biosph, 1997 Aug, 27(4), 405 - 12
Evolutionary consideration on 5-aminolevulinate synthase in nature; Oh-hama T; 5-Aminolevulinic acid (ALA), a universal precursor of tetrapyrrole compounds can be synthesized by two pathways: the C5 (glutamate) pathway and ALA synthase . From the phylogenetic distribution it is shown that distribution of ALA synthase is restricted to the alpha subclass of purple bacteria in prokaryotes, and further distributed to mitochondria of eukaryotes . The monophyletic origin of bacterial and eukaryotic ALA synthase is shown by sequence analysis of the enzyme . Evolution of ALA synthase in the alpha subclass of purple bacteria is discussed in relation to the energy-generating and biosynthetic devices in subclasses of this bacteria.

J Clin Invest, 1997 Aug 1, 100(3), 514 - 21
Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen; Chiang TM et al.; A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor . Computer search revealed that this cDNA represents the coding sequence of a unique protein . Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity . In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m) . The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography . The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner . The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner . Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of {14C}serotonin-labeled platelets to type I collagen in a dose-dependent manner . The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

J Infect Dis, 1997 Aug, 176(2), 533 - 6
Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay; Nash KA et al.; An adaption of an RNA/RNA duplex, base pair-mismatch assay is capable of detecting rifampin resistance in Mycobacterium tuberculosis . The specificity and sensitivity of the mismatch assay in detecting rifampin resistance were 100% and 96%, respectively, when tested against 46 rifampin-resistant and rifampin-susceptible strains of M . tuberculosis . By use of a range of mycobacterial and nonmycobacterial prokaryote pathogens, the mismatch assay was shown to be specific for M . tuberculosis and Mycobacterium bovis . The assay is cost-effective compared with DNA sequencing and other molecular methods and is simple to perform and interpret . Furthermore, the assay can return a result within 24 h after receipt of an isolated organism and potentially can be used directly with smear-positive specimens.

J Biol Chem, 1997 Aug 1, 272(31), 19277 - 81
Effect of metal on 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis in the Hansenula polymorpha copper amine oxidase; Cai D et al.; Previous studies of wild-type and mutant forms of a recombinant copper amine oxidase from Hansenula polymorpha, expressed in Saccharomyces cerevisiae, have indicated a self-processing mechanism for 2,4,5-trihydroxyphenylalanine (topa) quinone biogenesis involving the active site copper (Cai, D., and Klinman, J . P . (1994) J . Biol . Chem . 269, 32039-32042) . In contrast to prokaryotic copper amine oxidases, however, it has not been possible to initiate topa quinone formation by the addition of exogenous copper to precursor H . polymorpha amine oxidase lacking copper . Metal analysis of copper-depleted wild-type enzyme reveals 0.2-0.3 mol copper, together with 0.6 mol zinc . Despite changes in the zinc and copper levels in growth media, the level of zinc in purified enzyme remains fairly constant . Further, we have been unable to displace protein-bound zinc by exogenously added copper . The H . polymorpha amine oxidase gene was subsequently expressed in Escherichia coli and found to be almost completely free of copper and zinc . In vitro reconstitution of this apoprotein confirms that zinc binds to H . polymorpha amine oxidase and prevents reconstitution with copper . By contrast, addition of copper first to apoprotein leads to formation of topa quinone and stable activity in the presence of added zinc . These findings indicate efficient binding of either zinc or copper to a site that undergoes little or no exchange . The data confirm that topa quinone biogenesis in the H . polymorpha system is catalyzed by copper and occurs in the absence of added factors . We conclude that the mechanisms of cofactor biogenesis in pro- and eukaryotic systems are likely to be similar or identical . The results described herein imply different pathways for the in vivo assembly of heterologously expressed amine oxidases in S . cerevisiae and E . coli.

Science, 1997 Aug 1, 277(5326), 690 - 3
Simplification of DNA topology below equilibrium values by type II topoisomerases; Rybenkov VV et al.; Type II DNA topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another . The steady-state fraction of knotted or catenated DNA molecules produced by prokaryotic and eukaryotic type II topoisomerases was found to be as much as 80 times lower than at thermodynamic equilibrium . These enzymes also yielded a tighter distribution of linking number topoisomers than at equilibrium . Thus, topoisomerases do not merely catalyze passage of randomly juxtaposed DNA segments but control a global property of DNA, its topology . The results imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation.

J Virol, 1997 Aug, 71(8), 5758 - 63
The 85-kilodalton phosphoprotein (pp85) of human herpesvirus 7 is encoded by open reading frame U14 and localizes to a tegument substructure in virion particles; Stefan A et al.; A family of antigenically related proteins present in cells infected with human herpesvirus 7 (HHV-7), designated phosphoprotein 85 (pp85), comprises a complex of proteins, of which the 85-kDa species is phosphorylated . pp85 is a major determinant of human response to HHV-7 infection (L . Foa-Tomasi, E . Avitabile, L . Ke, and G . Campadelli-Fiume, J . Gen . Virol . 75:2719-2727, 1994; L . Foa-Tomasi, M . P . Fiorilli, E . Avitabile, and G . Campadelli-Fiume, J . Gen . Virol . 77:511-518, 1996; J . B . Black et al., Clin . Diagn . Lab . Immunol . 3:79-83, 1996) . By immunoscreening of a cDNA library from HHV-7-infected cells with monoclonal antibody (MAb) 5E1, directed to the proteins of the pp85 complex, we mapped the gene encoding pp85 to the U14 open reading frame of the HHV-7 genome . A prokaryotically expressed fusion protein containing the U14 open reading frame reacted with MAb 5E1 in an immunoblot assay . A functional role for pp85 was defined by immunoelectron microscopy studies . Immunogold labeling of cryosections of HHV-7-infected cord blood mononuclear cells at high resolution localized the reactivity of MAb 5E1 to the outer surface of the virion tegument . This finding demonstrates that pp85, the product of the U14 gene, is a component of the HHV-7 tegument and suggests that the HHV-7 tegument is not a homogeneous structure but rather is composed of substructures, including an outermost layer containing pp85 . The present findings, together with previously reported properties of MAb 5E1, including its ability to react with formalin-fixed paraffin-embedded samples, make this antibody a specific tool useful for etiopathogenetic studies of HHV-7 infection in humans and provide the basis for further development of pp85 into a specific recombinant diagnostic reagent.

Curr Microbiol, 1997 Aug, 35(2), 84 - 9
The (F1F0) ATP synthase of Buchnera aphidicola (endosymbiont of aphids): genetic analysis of the putative ATP operon; Clark MA et al.; Buchnera aphidicola is an intracellular, non-cultivable prokaryotic symbiont of the aphid Schizaphis graminum . A 6.8-kilobase fragment from B . aphidicola was cloned and sequenced and was found to contain genes encoding for proteins of the ATP synthase . The order of the genes, atpBEFHAGDC, is identical to that found inEscherichia coli and many other prokaryotes . This genetic organization is different from that observed in organelles such as mitochondria and chloroplasts, in which the genes are partitioned between the organellar and nuclear genomes . One difference between B . aphidicola and E . coli was the absence of atpI, a gene of unknown function, which in E . coli precedes atpB . As is the case of many other prokaryotes, atpBEFHAGDC appears to constitute a single transcription unit . The detection in B . aphidicola of the genes encoding the ATP synthase as well as past observations indicating that this organism is capable of respiration are consistent with the utilization byB . aphidicola of a proton gradient for the generation of ATP.

Gene, 1997 Jul 31, 194(2), 297 - 9
A novel gene encoding a ferredoxin reductase-like protein expressed in the neuroectoderm in Xenopus neurula; Hatada S et al.; In an attempt to elucidate the molecular mechanisms of early neural development in Xenopus laevis, we identified, using a differential display method, several genes that are induced after Concanavalin A treatment in the animal caps prepared from stage 9 blastula . One such gene was found to encode a possible type IIIa membrane protein of 66.2 kDa sharing similarities with several prokaryotic and eukaryotic redox enzymes, hence the putative product was named Nfrl, neurula-specific ferredoxin reductase-like protein . Northern blot analysis confirmed that the expression of the Nfrl gene is up-regulated around the neurula stage, and is much lower in embryos of earlier stages and in adult tissues . The temporally limited expression of this gene implies neurula- and early larva-specific redox reactions of certain substrates, the nature of which remains to be elucidated.

Gene, 1997 Jul 31, 194(2), 215 - 25
Three different genes encode NM23/nucleoside diphosphate kinases in Xenopus laevis; Ouatas T et al.; Nucleoside diphosphate kinases (NDPKs) catalyse the phosphorylation of nucleoside diphosphates . In mammals, the functional enzyme is a hexamer composed of different amounts of two homologous acidic (A) and basic (B) subunits encoded by separate genes . In prokaryotes and invertebrate eukaryotes, only one cytoplasmic enzyme has been isolated . Other genes encoding chloroplastic and mitochondrial forms as well as related proteins have been cloned . Here, we show that in Xenopus laevis, as in mammals, the cytoplasmic NDPK is encoded by several homologous genes . With Xenopus laevis being a pseudotetraploid species, each monomer is encoded by two genes . The amino acid sequences are very similar, and all the differences concern amino acids located at the outer surface of the hexameric enzyme . The Xenopus genes share 82-87% identity with their human counterparts . Interestingly, in vitro, the Xenopus X1 enzyme binds to a specific nuclease hypersensitive element (NHE) of the human c-myc promoter, as does its human counterpart . X1 also binds to a single-stranded (CT)(n) dinucleotide repeat . The NHE is present in the coding strand of a pyrimidine-rich region of the 3' non-coding sequence of the Xenopus NDPK genes . We propose that NDPK is indeed able to bind to its own mRNA and prevent polyadenylation at the normal position . This could provide an autoregulatory translation mechanism . A phylogenetic tree of the vertebrate NDPK sequences supports the idea that in amphibians, as in mammals, gene duplication has resulted in functional diversification.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 8249 - 54
Cloning of a polycistronic cDNA from tomato encoding gamma-glutamyl kinase and gamma-glutamyl phosphate reductase; Garcia-Rios M et al.; We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR) . This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons . The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR . DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato . We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon . In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR . Antibodies against the 44-kDa GPR purified from E . coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots . These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping . The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.

Proc Natl Acad Sci U S A, 1997 Jul 22, 94(15), 7698 - 703
Origin of genes; Gilbert W et al.; We discuss two tests of the hypothesis that the first genes were assembled from exons . The hypothesis of exon shuffling in the progenote predicts that intron phases will be correlated so that exons will be an integer number of codons and predicts that the exons will be correlated with compact regions of polypeptide chain . These predictions have been tested on ancient conserved proteins (proteins without introns in prokaryotes but with introns in eukaryotes) and hold with high statistical significance . We conclude that introns are correlated with compact features of proteins 15-, 22-, or 30-amino acid residues long, as was predicted by "The Exon Theory of Genes."

Biochem Biophys Res Commun, 1997 Jul 18, 236(2), 510 - 6
The eukaryotic translation initiation factor 5, eIF-5, a protein from Zea mays, containing a zinc-finger structure, binds nucleic acids in a zinc-dependent manner; Lopez Ribera I et al.; A maize cDNA encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from an 8-day-old seedling cDNA library . The 1975 bp cDNA encodes a protein of 451 amino acids, with a predicted molecular weight of 49.04 kDa, and hybridizes to a single sequence in the maize genome . The deduced sequence contains motifs characteristic of proteins belonging to the GPTase superfamily, a zinc finger well conserved in all the protein sequences for eIF-5 reported so far, and a fragment also present in prokaryotic and chloroplast L11 ribosomal protein . Polymer-binding assays have been used to assess the predicted RNA binding property of the protein and to characterize its function . It is shown that the eIF-5-encoded protein binds to single-stranded DNA and to polyuridylic acid and that the binding is dependent on the presence of Zn2+ ions . These results suggest that the zinc-finger structure is involved in the binding of the eIF-5 protein to RNA.

J Mol Biol, 1997 Jul 18, 270(3), 360 - 73
Expression of a coronavirus ribosomal frameshift signal in Escherichia coli: influence of tRNA anticodon modification on frameshifting; Brierley I et al.; Eukaryotic ribosomal frameshift signals generally contain two elements, a heptanucleotide slippery sequence (XXXYYYN) and an RNA secondary structure, often an RNA pseudoknot, located downstream . Frameshifting takes place at the slippery sequence by simultaneous slippage of two ribosome-bound tRNAs . All of the tRNAs that are predicted to decode frameshift sites in the ribosomal A-site (XXXYYYN) possess a hypermodified base in the anticodon-loop and it is conceivable that these modifications play a role in the frameshift process . To test this, we expressed slippery sequence variants of the coronavirus IBV frameshift signal in strains of Escherichia coli unable to modify fully either tRNA(Lys) or tRNA(Asn) . At the slippery sequences UUUAAAC and UUUAAAU (underlined codon decoded by tRNA(Asn), anticodon 5' QUU 3'), frameshifting was very inefficient (2 to 3%) and in strains deficient in the biosynthesis of Q base, was increased (AAU) or decreased (AAC) only two-fold . In E . coli, therefore, hypomodification of tRNA(Asn) had little effect on frameshifting . The situation with the efficient slippery sequences UUUAAAA (15%) and UUUAAAG (40%) (underlined codon decoded by tRNA(Lys), anticodon 5' mnm5s2UUU 3') was more complex, since the wobble base of tRNA(Lys) is modified at two positions . Of four available mutants, only trmE (s2UUU) had a marked influence on frameshifting, increasing the efficiency of the process at the slippery sequence UUUAAAA . No effect on frameshifting was seen in trmC1 (cmnm5s2UUU) or trmC2 (nm5s2UUU) strains and only a very small reduction (at UUUAAAG) was observed in an asuE (mnm5UUU) strain . The slipperiness of tRNA(Lys), therefore, cannot be ascribed to a single modification site on the base . However, the data support a role for the amino group of the mnm5 substitution in shaping the anticodon structure . Whether these conclusions can be extended to eukaryotic translation systems is uncertain . Although E . coli ribosomes changed frame at the IBV signal (UUUAAAG) with an efficiency similar to that measured in reticulocyte lysates (40%), there were important qualitative differences . Frameshifting of prokaryotic ribosomes was pseudoknot-independent (although secondary structure dependent) and appeared to require slippage of only a single tRNA.

FEBS Lett, 1997 Jul 14, 411(2-3), 313 - 6
A common core for binding single-stranded DNA: structural comparison of the single-stranded DNA-binding proteins (SSB) from E . coli and human mitochondria; Webster G et al.; The crystal structure of the DNA-binding domain of E . coli SSB (EcoSSB) has been determined to a resolution of 2.5 A . This is the first reported structure of a prokaryotic SSB . The structure of the DNA-binding domain of the E . coli protein is compared to that of the human mitochondrial SSB (HsmtSSB) . In spite of the relatively low sequence identity between them, the two proteins display a high degree of structural similarity . EcoSSB crystallises with two dimers in the asymmetric unit, unlike HsmtSSB which contains only a dimer . This is probably a consequence of the different polypeptide chain lengths in the EcoSSB heterotetramer . Crucial differences in the dimer-dimer interface of EcoSSB may account for the inability of EcoSSB and HsmtSSB to form cross-species heterotetramers, in contrast to many bacterial SSBs.

Biol Chem, 1997 Jul, 378(7), 599 - 607
The role of DNA conformation in transcriptional initiation and activation in Escherichia coli; Geiselmann J; Transcription activation in prokaryotes relies on a multitude of molecular mechanisms . Many of them use DNA bending or other deformations of the DNA to control the rate of transcription initiation . All steps of initiation involve a particular transconformation reaction of the DNA and can be controlled individually by activators . This review discusses the thermodynamic and kinetic basis of transcription initiation and illustrates the strategies used by activators . Particular emphasis is given to mechanisms that necessitate DNA-bending by the activator.

Appl Microbiol Biotechnol, 1997 Jul, 48(1), 66 - 72
Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli; Hsih MH et al.; Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitary glands . However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB) . We studied the solubilization of IB and the renaturation of rGH to help facilitate the production of a large amount of biologically active rGH . A 100-ml sample of rGH-producing E . coli produced 73.43 +/- 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl beta-o-thiogalactopyranoside . Interestingly, if the bacteria were induced by 0.1 mM beta-lactose, 95.3 +/- 3.43 mg of IB was obtained . The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM L-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 degrees C in a volume ratio of 3 to 500 . At least 20% of the denaturated rGH in IB was renatured . Juvenile black sea bream injected with 0.05 microgram/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period . This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system.

Trop Med Int Health, 1997 Jul, 2(7), 691 - 4
pOVEX vector: prokaryotic expression and purification of onchocerciasis vaccine candidate antigens as fusion proteins with the 24 kD Onchocerca volvulus glutathione S-transferase; Liebau E et al.; An expression vector, pOVEX, has been designed and constructed, combining the advantages of the expression vectors pGEX-3X and pJC2o . The pOVEX vector produces a fusion protein with the 24 kD Onchocerca volvulus glutathione S-transferase (OvGST2) which is easy to purify in one step from bacterial extracts under non-denaturing conditions using glutathione-sepharose chromatography . High yields of fusion protein were produced from this T7 RNA polymerase-dependent expression vector, which were then cleaved by digestion with the factor Xa protease to separate the OVGST2 polypeptide from the expressed protein of interest . This vector will be particularly useful to O . volvulus investigators for the production of O . volvulus antigens for the analyses of host humoral and cellular responses to these proteins and for immunization studies.

Glycobiology, 1997 Jul, 7(5), 697 - 701
Purification and characterization of the Escherichia coli K1 neuB gene product N-acetylneuraminic acid synthetase; Vann WF et al.; Escherichia coli K1 produces a capsular polysaccharide of alpha(2-8) poly-N-acetylneuraminic acid . This polysaccharide is an essential virulence factor of these neuropathogenic bacteria . The genes necessary for the synthesis of neuNAc were localized to a plasmid containing the neuBAC genes of the K1 gene cluster . Cells harboring the neuB+ allele in an aldolase (nanA-) negative background produce neuNAc in vivo . Enzymatic synthesis of neuNAc could be demonstrated in extracts of cells harboring an expression plasmid (pNEUB) containing the neuB gene alone . NeuNAc synthetase was purified to homogeneity from extracts of cells harboring pNEUB . The molecular weight of the purified enzyme is 40 kDa, similar to that predicted by the nucleotide sequence of the neuB gene . The amino terminal sequence of the purified protein matches that predicted by the nucleotide sequence of the neuB gene . NeuNAc synthetase catalyzes the formation of neuNAc as indicated by its coupling to the CMP-neuNAc synthetase reaction . The enzyme condenses manNAc and PEP with the release of phosphate . The E . coli neuNAc synthetase is specific for manNAc and PEP, unlike rat liver enzyme that utilizes N-acetylmannosamine-6-phosphate to form neuNAc-9-PO4 . This represents the first report of a purification of a sialic acid synthetase from either a eukaryotic or prokaryotic source to homogeneity . These experiments clearly demonstrate an aldolase-independent sialic acid synthetase activity in E . coli K1.

Chem Res Toxicol, 1997 Jul, 10(7), 713 - 32
What structural features determine repair enzyme specificity and mechanism in chemically modified DNA?
Singer B, Hang B.
A crucial question in repair is how do enzymes recognize substrates . In surveying the relevant literature, it becomes evident that there are no rules which can be clearly applied . At this time it appears that uracil glycosylase is the only repair enzyme for which all the known substrates can be rationalized on the basis of chemical structure . When surveying the multiplicity of substrates for m3A-DNA glycosylase, it is difficult, on the basis of present knowledge, to explain why 1,N6-etheno-A (epsilon A) is as good a substrate, if not better, than m3A for which the enzyme is named . There is no apparent unifying chemical structure which is required for recognition . It should also be noted that many studies of the mechanism of m3A-DNA glycosylase only utilized-N-3- and N-7-alkylpurines . On this basis, an electron-deficient purine, and later pyrimidine, was considered to be the recognition signal . Since epsilon A and Hx do not fall in this class, this is one illustration of why exploring new substrates becomes important in elucidating enzyme mechanisms . Ubiquitous enzymes, such as 5'-AP endonucleases, are present in both prokaryotes and eukaryotes . The primary function is the same, i.e., repair of an AP site which occurs through natural processes or from the action of DNA glycosylases . There are, however, completely unrelated substrates such as the exocyclic adducts pBQ-dC and pBQ-dG . pBQ-dC is repaired by both the human HAP1 and E . coli Exo III and Endo IV, while pBQ-dG is only repaired by the E . coli enzymes . Yet, when repair of these adducts occurs, it is by the same unusual pathway which differs from the usual base excision repair mechanism . This finding may ultimately not be as unusual as it now seems . The understanding of substrate recognition by repair enzymes, which can have different repair pathways, is complex . For example, three exocyclic derivatives which each have either the same modification (1,N4-epsilon dA and 3,N4-epsilon dC) or the same base with different modifying groups (3,N4-epsilon dC and 3,N4-pBQ-dC) are repaired by three separate enzymes and two mechanism (Figure 9) . Investigators have also reported that two separate enzymes and pathways can be found for simple adducts such as m6G and O4T . It is not clear why, for these adducts, both MGMT and excision repair can be utilized . This could be visualized as a "backup" system and may be more common than now known . We cannot think like an enzyme or vice versa . In the absence of enough necessary information, we can only be descriptive . What information is necessary for further understanding? (1) More detailed structural studies of adducts in defined oligonucleotides would be useful . (2) New substrates should be explored . For example, is the mechanism for PBQ-dC (and pBQ-dG) repair unique? This involves guesswork and intuition . (3) For the adducts mentioned in this Perspective and others, understanding enzyme/substrate recognition will be facilitated by cocrystallography and site-directed mutagenesis . (4) Genetic approaches, such as knockouts or targeted mutations in repair genes, should be expanded in order to focus on the physiological role of a specific enzyme . Above all: structure, structure, structure! Enzymologists, organic chemists, physical chemiste, X-ray crystallographers, and others must combine forces if the fundamental problems addressed here are to be understood.

Trends Genet, 1997 Jul, 13(7), 281 - 5
Origin of genes encoding multi-enzymatic proteins in eukaryotes; Davidson JN et al.; In several biosynthetic pathways of eukaryotes, multiple steps are catalyzed by enzymes physically linked as domains of multi-enzymatic proteins . The same steps in prokaryotes are frequently carried out by mono-enzymatic proteins . If genes encoding mono-enzymatic proteins are the precursors to those genes encoding multi-enzymatic proteins, how these genes fused remains an open question . However, the recent discovery of a cleavage-polyadenylation signal within an intron of the GART gene provides clues to this process and might also have more general implications for the origin of genes that contain alternative RNA processing reactions at their 5' or 3' ends.

Clin Infect Dis, 1997 Jul, 25 Suppl 1, S48 - 51
The antimicrobial agent melittin exhibits powerful in vitro inhibitory effects on the Lyme disease spirochete; Lubke LL et al.; Borrelia burgdorferi has demonstrated a capacity to resist the in vitro effects of powerful eukaryotic and prokaryotic metabolic inhibitors . However, treatment of laboratory cultures on Barbour-Stoenner-Kelly medium with melittin, a 26-amino acid peptide contained in honeybee venom, showed immediate and profound inhibitory effects when they were monitored by dark-field microscopy, field emission scanning electron microscopy, and optical density measurements . Furthermore, at melittin concentrations as low as 100 microg/mL, virtually all spirochete motility ceased within seconds of inhibitor addition . Ultrastructural examination of these spirochetes by scanning electron microscopy revealed obvious alterations in the surface envelope of the spirochetes . The extraordinary sensitivity of B . burgdorferi to mellitin may provide both a research reagent useful in the study of selective permeability in microorganisms and important clues to the development of effective new drugs against lyme disease.

Protein Sci, 1997 Jul, 6(7), 1405 - 11
Synthesis and characterization of histidine-phosphorylated peptides; Medzihradszky KF et al.; Posttranslational phosphorylation of proteins is an important event in many cellular processes . Whereas phosphoesters of serine, threonine, and tyrosine have been studied extensively, only limited information is available for other amino acids modified by a phosphate group . The formation of phosphohistidine residues in proteins was discovered originally in prokaryotic organisms, but also has been found recently in eukaryotic cells . We describe methods for the synthesis and analysis of phosphohistidine-containing peptides, a prerequisite for the investigation of the role of this posttranslational modification in cellular processes.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 65 - 73
Improved methods for in situ enzymatic amplification and detection of low copy number genes in bacteria; Jacobs D et al.; We present alternative and improved protocols for in situ analysis of single copy genes in prokaryotes . Primed in situ amplification (PRINS) and cycle PRINS were used to detect, via the incorporation of a fluorescein labelled nucleotide, the presence of specific genes carried on both high and low copy number plasmids in individual cells of Escherichia coli and a marine bacterium, SW5 . The optimised protocols described enabled a significant reduction in non-specific signals whilst maintaining high fluorescent activity via labelled nucleotide incorporation . In addition, nucleic acids were amplified linearly and were retained within the permeabilised microbial cells . These methods provide considerable advances in sensitivity, specificity and reliability compared to current protocols for bacterial in situ nucleic acid amplification.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 1 - 10
Secreted enzymes of Aeromonas; Pemberton JM et al.; A hallmark characteristic of species of Aeromonas is their ability to secrete a wide variety of enzymes associated with pathogenicity and environmental adaptability . Among the most intensively studied are beta-lactamases, lipases, hemolytic enterotoxins, proteases, chitinases, nucleases and amylases . Multiple copies of genes encoding each type of enzyme provide additional biological diversity . Except for the chitinases, these multiple copies show little evolutionary relatedness at the DNA level and only limited similarity at the protein level . Indeed a number of the genes, such as nuclease H of A . hydrophila, have no similarity to known prokaryotic or eukaryotic sequences . The challenge is to determine how these genes evolved, where they originated and why Aeromonas possesses them in such abundance and variety.

Clin Microbiol Rev, 1997 Jul, 10(3), 369 - 400
Yeast killer systems; Magliani W et al.; The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists . The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications . The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections . In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed.

Protein Expr Purif, 1997 Jul, 10(2), 256 - 62
High-level expression of human thymidylate synthase; Pedersen-Lane J et al.; A method is presented for expressing human thymidylate synthase (TS) to the extent of 25-30% of the protein in Escherichia coli . By this procedure, 200-400 mg of pure enzyme can be obtained from a 2-liter culture of cells . The key to the level of expression appears to be related to the conversion of purine bases in the third, fourth, and fifth codons of the TS cDNA to thymine, without altering the encoded protein product . Conversion of the penultimate proline to a leucine did not diminish expression, but while the isolated native enzyme contained only proline on its amino-terminal end, the mutated enzyme was found to contain methionine on its amino terminus . By contrast, the expression of the unmodified TS cDNA represented only about 0.1-0.2% of the total cellular protein . Unlike recombinant rat and human TSs, the respective enzymes purified to homogeneity from eukaryotic cells were blocked at the amino ends and possessed 2- to 4-fold lower specific activities . To determine at what level the impairment of expression occurred, an in vitro transcription, translation system was employed and the results showed that while transcription was unaffected, the translation of native TS mRNA was reduced by at least 20-fold relative to modified TS mRNA using a rabbit reticulocyte translation system . Thus, it appears that at least for the TS gene, expression is greatly influenced by the GC content of the 5' coding region of the gene in both prokaryote and eukaryote systems.

Proteins, 1997 Jul, 28(3), 375 - 9
A potential processing enzyme in prokaryotes: oligopeptidase B, a new type of serine peptidase; Polgar L; Basic amino acid pairs in polypeptides represent important markers for processing enzymes to produce biologically active products . Such enzymes related to the serine peptidase subtilisin have recently been identified in eukaryotes . Herein is described and kinetically characterized a new type of processing enzyme, oligopeptidase B, which is encountered in the prokaryote Escherichia coli, and belongs to the prolyl oligopeptidase family of serine peptidase . The enzyme hydrolyzes the peptides at the carboxy end of dibasic sites by two orders of magnitude faster with respect to monobasic substrates . The kcat/K(m) is extremely high, 63 microM-1 s-1, for the substrate benzyloxycarbonyl-L-arginyl-L-arginyl-7-(4-methylcoumaryl)amide . The bell-shaped pH dependence of the rate constant is perturbed by some ionizing group(s) . This effect is abolished at 1 M NaCl . In addition, high ionic strength inhibits the reaction considerably by increasing K(m), which is indicative of an electrostatic interaction between the arginyl residues and the enzymatic carboxy groups . In distinction from that found with most serine endopeptidases, kinetic deuterium isotope measurements with oligopeptidase B indicate that the rate-limiting step of the reaction is a physical step rather than a chemical one characterized by general acid/base catalysis . The present result will contribute to our understanding of the processing phenomena in prokaryotes, as well as in higher organisms.

J Neurosurg, 1997 Jul, 87(1), 89 - 95
Regulated expression of the diphtheria toxin A gene in human glioma cells using prokaryotic transcriptional control elements; Paulus W et al.; Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of diphtheria toxin A (DTA) fragment expression in human glioma cell lines using two transcriptional control systems derived from Escherichia coli: the tetracycline (Tet) system and the lactose (Lac) system . The Tet system includes a tetracycline-controlled transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and tetracycline as an allosteric inhibitor . The Lac system includes the lac repressor (lacR), a lacR-regulated Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-beta-D-galactoside (IPTG) as an allosteric inducer . Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG glioma cells along with the responsive DTA plasmid . Cell killing was monitored by the ability of the toxin to abolish protein synthesis and was quantitated using a luciferase reporter gene . In the Tet system, tumor cell killing could be regulated by tetracycline up to 120-fold . In contrast, only a twofold IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state . Replacement of the RSV-LTR promoter with the heavy metal-inducible mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system . In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.

J Clin Invest, 1997 Jul 1, 100(1), 68 - 73
CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract; Schwartz DA et al.; Since unmethylated CpG motifs are more frequent in DNA from bacteria than vertebrates, and the unmethylated CpG motif has recently been reported to have stimulatory effects on lymphocytes, we speculated that bacterial DNA may induce inflammation in the lower respiratory tract through its content of unmethylated CpG motifs . To determine the role of bacterial DNA in lower airway inflammation, we intratracheally instilled prokaryotic and eukaryotic DNA in C3H/HeBFEJ mice and performed whole lung lavage 4 h after the exposure . Heat denatured, single stranded Escherichia coli genomic DNA (0.06 ng endotoxin/microg DNA) was compared to heat denatured, single stranded calf thymus DNA (0.007 endotoxin/microg DNA) . 10 microg of bacterial DNA, in comparison to 10 microg of calf thymus DNA, resulted in a fourfold increase in the concentration of cells (P = 0.0002), a fivefold increase in the concentration of neutrophils (P = 0.0002), a 50-fold increase in the concentration of TNF-alpha (P = 0.001), and a fourfold increase in the concentration of both IL-6 (P = 0.0003) and macrophage inflammatory protein-2 (P = 0.0001) in the lavage fluid . Importantly, instillation of 0.60 ng of E . coli LPS resulted in a negligible inflammatory response . To test whether the stimulatory effects of bacterial DNA are due to its unmethylated CpG dinucleotides, we methylated the bacterial DNA and also prepared 20 base pair oligonucleotides with and without CpG motifs . In comparison to instillation of untreated bacterial DNA, methylation of the bacterial DNA resulted in a significant reduction in the concentration of cells and cytokines in the lower respiratory tract . Moreover, oligonucleotides containing embedded unmethylated CpG motifs resulted in inflammation in the lower respiratory tract that was indistinguishable from that observed with untreated bacterial DNA . In contrast, oligonucleotides without the embedded CpG motifs or with embedded but methylated CpG motifs resulted in significantly less inflammation in the lower respiratory tract . The possible relevance of these data to human disease was shown by extracting and analyzing DNA in sputum from patients with cystic fibrosis (CF) . Approximately 0.1 to 1% of this sputum DNA was bacterial . Intratracheal instillation of highly purified CF sputum DNA caused acute inflammation similar to that induced by bacterial DNA . These findings suggest that bacterial DNA, and unmethylated CpG motifs in particular, may play an important pathogenic role in inflammatory lung disease.

Mol Cell Biol, 1997 Jul, 17(7), 3687 - 93
A circadian enhancer mediates PER-dependent mRNA cycling in Drosophila melanogaster; Hao H et al.; Genes expressed under circadian-clock control are found in organisms ranging from prokaryotes to humans . In Drosophila melanogaster, the period (per) gene, which is required for clock function, is transcribed in a circadian manner . We have identified a circadian transcriptional enhancer within a 69-bp DNA fragment upstream of the per gene . This enhancer drives high-amplitude mRNA cycling under light-dark-cycling or constant-dark conditions, and this activity is per protein (PER) dependent . An E-box sequence within this 69-bp fragment is necessary for high-level expression, but not for rhythmic expression, indicating that PER mediates circadian transcription through other sequences in this fragment.

Pflugers Arch, 1997 Jul, 434(3), 323 - 31
Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia; Spindler B et al.; The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase) . In order to contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved . Epithelia were treated for 1 h with 10(-6 )M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied . Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase alpha1 and beta1 subunit mRNAs were slightly increased (1.2- to 1.4-fold) . In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR) . After 1 h of aldosterone treatment, approximately 0.5% of these appeared to be regulated . Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned and for two of them cDNAs containing entire coding sequences were isolated by library screening . ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2 . The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D . The K1/2 values were similar or slightly below those reported for stimulation of Na transport . These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.

Curr Microbiol, 1997 Jul, 35(1), 28 - 31
Iron and salicylate induction of cytochrome P450BM-1 in Bacillus megaterium; Shaw GC et al.; The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report . Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression . Substantial induction of chloramphenicol acetyltransferase (CAT) activity by iron and salicylate in B . megaterium cells bearing a P450BM-1 promoter-cat transcriptional fusion vector suggests that the induction of P450BM-1 by iron and salicylate occurs at the transcriptional level . Unexpectedly, in contrast to the bm1P1-dependent induction of P450BM-1 by pentobarbital, disruption of bm1P1 gene did not affect induction of P450BM-1 by iron and salicylate . This result suggests that the induction of P450BM-1 by iron and salicylate occurs by a bm1P1-independent mechanism . To our knowledge, this is the first example of an iron-regulated cytochrome P450 gene in prokaryotes.

Comp Biochem Physiol A Physiol, 1997 Jul, 117(3), 291 - 9
Mitogen-activated protein kinase cascades and the signaling of hyperosmotic stress to immediate early genes; Cohen DM; Among prokaryotes and lower eukaryotes, the threat of exposure to hyperosmotic stress is ubiquitous . Among higher eukaryotes, in contrast, only specific tissues are routinely exposed to marked hypertonicity . The mammalian renal medulla, the prototypical example, is continually subjected to an elevated solute concentration as a consequence of the renal concentrating mechanism . Until recently, the investigative focus has concerned the effects of diverse solutes on the regulation of genes essential for the adaptive accumulation of osmotically active organic solutes . Recent and sweeping developments elucidating the molecular mechanisms underlying stress signaling to the nucleus have focused interest on earlier events in the response to hyperosmotic stress . Such events include the transcriptional activation and post-translational modification of transcriptional activating proteins, a large subset of which represent the protein products of so-called immediate early genes . This review highlights developments in the understanding of stress signaling in general and hypertonic stress signaling in particular in both yeast and higher eukaryotic models . The relationship between hyperosmotic stress signaling and the transcription and activation of immediate-early gene transcription factors is explored.

FEBS Lett, 1997 Jun 30, 410(2-3), 433 - 6
Light-harvesting in Acaryochloris marina--spectroscopic characterization of a chlorophyll d-dominated photosynthetic antenna system; Schiller H et al.; Oxygenic photosynthesis of the prokaryote Acaryochloris marina involves chlorophyll d (Chl d) as the major pigment {Miyashita et al . (1996) Nature 383, 402} . Four spectral forms of Chl d (peak wavelengths: 694, 714, 726 and 740 nm) are resolvable by low-temperature absorption spectroscopy on intact cells . Based on fluorescence spectra (at 290 K and 77 K) and on analysis of fluorescence induction curves we conclude: (1) excitation energy is efficiently transferred between the various spectral forms of Chl d and the PS II reaction center; (2) Chl d serves as a light-harvesting pigment for both, Photosystem II (PS II) and PS I; (3) excitation energy transfer between PS II units occurs.

FEBS Lett, 1997 Jun 30, 410(2-3), 428 - 32
Isolation and characterization of biliprotein aggregates from Acaryochloris marina, a Prochloron-like prokaryote containing mainly chlorophyll d; Marquardt J et al.; Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment . In the electron microscope the biliprotein aggregates appear as rod-shaped structures of 26.0 x 11.3 nm, composed of four ring-shaped subunits 5.8 nm thick and 11.7 nm in diameter . Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin-type pigment, with very efficient energy transfer from the phycocyanin- to allophycocyanin-type constituent . The chromophore-binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa . They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.

FEBS Lett, 1997 Jun 30, 410(2-3), 303 - 8
Identification of a biological inactive complex form of pokeweed antiviral protein; Desvoyes B et al.; Pokeweed antiviral protein (PAP) inactivates both eukaryotic and prokaryotic ribosomes via a specific depurination of rRNA . The sensitivity of pokeweed ribosomes to PAP implies the existence of a mechanism to protect the plant . Using monoclonal antibodies specific to PAP, a protein complex (PAPi) which contained PAP was identified in leaf extract . In this complex, the enzymatic activity of the toxin was strongly inhibited . This protein complex had a pI lower than that of PAP and was separated from free PAP by a preparative native gel electrophoresis . PAPi had an apparent molecular mass of 57 kDa and was dissociated by heating for 5 min at 80 degrees C or by treatment by alkaline or acidic pH or by 7 M urea . The other components involved in the complex remain unknown.

Biochem Biophys Res Commun, 1997 Jun 27, 235(3), 631 - 5
Reconstitution and characterization of a divergent plastocyanin from the photosynthetic prokaryote, Prochlorothrix hollandica, expressed in Escherichia coli; Babu CR et al.; Plastocyanin (PC) is a copper protein that serves as a mobile electron carrier between cytochrome f and Photosystem I in the light reactions of photosynthesis . Despite large variability in amino acid sequences and isoelectric points, PCs from cyanobacterial and chloroplast sources reveal considerable similarities with respect to their secondary and tertiary structures . In this paper, we have expressed in Escherichia coli a PC from the prokaryote Prochlorothrix hollandica, and efficiently reconstituted the protein with copper under conditions yielding the characteristics of a native holoPC, as judged by redox titration (Eo' = +376 mV), near and far UV circular dichroism, and electron paramagnetic resonance (EPR) spectroscopy . By comparison of amino acid sequences, P . hollandica PC is the most divergent homolog identified to date, and analysis of this reconstituted preparation may reveal new insights as to the structural requirements for electron transport between the PC copper center and neighboring reaction partners.

Biochem Biophys Res Commun, 1997 Jun 27, 235(3), 545 - 52
Purification and characterization of a homodimeric catalase-peroxidase from the cyanobacterium Anacystis nidulans; Obinger C et al.; Cytosolic extracts of the cyanobacterium Anacystis nidulans exhibit both catalase and o-dianisidine peroxidase activity . Native polyacrylamide gel electrophoresis demonstrates one distinct enzyme, which has been purified to essential homogeneity and found to be composed of two identical subunits of equal size (80.5 kDa) . The isoelectric point is at pH 4.7 . It is a very efficient catalase with a broad pH optimum between 6.5 and 7.5 and a Km for H2O2 of 4.3 mM, a calculated turnover number of 7200 s(-1), and an overall-rate constant of 3.5 x 10(6) M(-1) s(-1) . The behaviour of this protoheme-enzyme is typical of the class of prokaryotic catalase-peroxidases, which is sensitive to cyanide (Ki = 27.2 microM) and insensitive to the eukaryotic catalase inhibitor 3-amino-1,2,4-triazole . The enzyme accepts electrons from o-dianisidine, but not from ascorbate, glutathione, and NADH . With hydrogen peroxide in steady-state conditions the enzyme is mainly in the ferric state indicating that Compound I is much faster reduced by H2O2 than it is formed . The native enzyme is in the high-spin state, which is transformed to low-spin upon addition of cyanide . With peroxoacetic acid Compound I is formed at a rate of 5.9 x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C with about 50% hypochromicity, a Soret-maximum at 405 nm and isosbestic points at 354 and 427 nm.

Proc Natl Acad Sci U S A, 1997 Jun 24, 94(13), 6718 - 23
Activation of RNA polymerase II by topologically linked DNA-tracking proteins; Ouhammouch M et al.; Almost all proteins mediating transcriptional activation from promoter-distal sites attach themselves, directly or indirectly, to specific DNA sequence elements . Nevertheless, a single instance of activation by a prokaryotic topologically linked DNA-tracking protein has also been demonstrated . The scope of the latter class of transcriptional activators is broadened in this work . Heterologous fusion proteins linking the transcriptional activation domain of herpes simplex virus VP16 protein to the sliding clamp protein beta of the Escherichia coli DNA polymerase III holoenzyme are shown to function as topologically DNA-linked activators of yeast and Drosophila RNA polymerase II . The beta:VP16 fusion proteins must be loaded onto DNA by the clamp-loading E . coli gamma complex to be transcriptionally active, but they do not occupy fixed sites on the DNA . The DNA-loading sites of these activators have all the properties of enhancers: they can be inverted and their locations relative to the transcriptional start site are freely adjustable.

Biochim Biophys Acta, 1997 Jun 23, 1346(3), 237 - 46
Effect of growth temperature on the biosynthesis of eukaryotic lipid molecular species by the cyanobacterium Spirulina platensis; Quoc KP et al.; The incorporation of linoleic acid added at mmolar concentrations to the culture medium of the photosynthetic prokaryote Spirulina platensis results in the synthesis of membrane glycerolipids with a eukaryotic distribution of fatty acid chain length on the glycerol backbone (Pham Quoc et al., Biochim . Biophys . Acta {1993} 1168, 94-99) . This distribution contrasts with the usual prokaryotic one found in lipids of cyanobacteria . A subsequent desaturation of the exogenously supplied fatty acid resulted in a large increase of gamma-linolenic acid . In order to estimate the capacities of S . platensis for bioconversion of fatty acids in lipid classes, the effects of different temperatures of growth were studied in linoleic acid-supplemented cultures . The lipid composition was affected by growth temperature, the synthesis of SQDG was stimulated at low temperature . The molecular species of each lipid were isolated and analyzed . Whatever the temperature of growth, the biosynthesis of eukaryotic C18/C18 lipid molecular species was observed in all lipid classes . Furthermore, the proportion of eukaryotic lipids increased at low temperature (24 degrees C) . The desaturation of C18 fatty acids at C1 and C2 positions of the glycerol moiety occurred and was further stimulated when the growth temperature was lowered . The resulting proportion of gamma-linolenic acid increased significantly in cultures supplemented with linoleate at low temperatures . Finally a pathway for the synthesis of eukaryotic lipids and the desaturation of fatty acids esterified to the acyl lipids of linoleate-supplemented S . platensis can be suggested.

Nucleic Acids Res, 1997 Jun 15, 25(12), 2303 - 10
V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element; Roch FA et al.; The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates . We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates . These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination . Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination . We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not . Also, prokaryotic sequences markedly suppress V(D)J recombination . This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context . The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation . The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.

Nucleic Acids Res, 1997 Jun 15, 25(12), 2537 - 8
Glutathione S-transferase fusion proteins as an affinity reagent for rapid isolation of specific sequence directly from genomic DNA; Majka J et al.; We describe a DNA binding assay for isolation of specific sequence(s) recognized by protein of interest directly from genomic or cosmid DNA . In our assay, the protein is fused to the glutathione-S-transferase and bound to glutathione-Sepharose beads . Then the immobilized fusion protein can be used to search for DNA fragment(s) that interact specifically with the protein of interest . As an example of such an approach, we identified and cloned a few prokaryotic oriC regions using the initiator DnaA protein fused to the glutathione-S-transferase.

J Biol Chem, 1997 Jun 13, 272(24), 15329 - 38
RNA polymerase switches between inactivated and activated states By translocating back and forth along the DNA and the RNA; Komissarova N et al.; Important regulatory events in both prokaryotic and eukaryotic transcription are currently explained in terms of an inchworming model of elongation . In this model, RNA extension is carried out by a mobile catalytic center that, at certain DNA sites, advances within stationary RNA polymerase . This idea emerged from the observation that footprints of individual elongation complexes, halted in vitro at consecutive DNA positions, can remain fixed on the template for several contiguous nucleotide additions . Here, we examine in detail the structural transitions that occur immediately after the enzyme stops at sites where discontinuous advancement of RNA polymerase is observed . We demonstrate that halting at such special sites does not "freeze" RNA polymerase at one location but induces it to leave its initial position and to slide backward along the DNA and the RNA without degrading the transcript . The resulting loss of contact between the RNA 3'-hydroxyl and the enzyme's catalytic center leads to temporary loss of the catalytic activity . This process is equilibrated with enzyme return to the original location, so that RNA polymerase is envisaged as an oscillating object switching between catalytically active and inactive states . The retreated isoform constitutes a principal intermediate in factor-induced endonucleolytic RNA cleavage . These oscillations of RNA polymerase can explain its apparent discontinuous advancement, which had been interpreted as indicating flexibility within the enzyme.

Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6164 - 9
Expression of a gene encoding a tRNA synthetase-like protein is enhanced in tumorigenic human myeloid leukemia cells and is cell cycle stage- and differentiation-dependent; Sen S et al.; We cloned a tumorigenic phenotype-associated cDNA encoding a tRNA synthetase-like protein from an acute-phase human myeloid leukemia cell line . The cDNA was isolated by reiterative subtraction of cDNAs synthesized from tumor-generating parental leukemia cells versus those from a nontumorigenic variant of the same cells . The selected cDNA encodes a protein that is a close homolog of one subunit of prokaryote and yeast phenylalanyl-tRNA synthetase (PheRS) . The expressed protein reacts specificially with polyclonal antibodies raised against mammalian phenylalanyl-tRNA synthetase . Expression of the gene (designated CML33) was directly confirmed by Northern blot hybridization to be substantially enhanced in the tumorigenic cells compared with the nontumorigenic variant . In addition, expression of CML33 in myeloid leukemia cells was sensitive to the stage of the cell cycle and to induction of differentiation . Although the relationship between these observations and the tumorigenic state of the human myeloid leukemia cell line used in these studies is unknown, to our knowledge, this is the first demonstration in mammalian cells of tumor-selective and cell cycle stage- and differentiation-dependent expression of a member of the tRNA synthetase gene family.

Proc Natl Acad Sci U S A, 1997 Jun 10, 94(12), 6025 - 9
The NG domain of the prokaryotic signal recognition particle receptor, FtsY, is fully functional when fused to an unrelated integral membrane polypeptide; Zelazny A et al.; Recent studies have revealed that Escherichia coli possesses an essential targeting system for integral membrane proteins, similar to the mammalian signal recognition particle (SRP) machinery . One essential protein in this system is FtsY, a homologue of the alpha-subunit of the mammalian SRP-receptor (SR-alpha) . However, E . coli does not possess a close homologue of the integral membrane protein SR-beta, which anchors SR-alpha to the membrane . Moreover, although FtsY can be found as a peripheral membrane protein, the majority is found soluble in the cytoplasm . In this study, we obtained genetic and biochemical evidence that FtsY must be targeted to the membrane for proper function . We demonstrate that the essential membrane targeting activity of FtsY is mediated by a 198-residue-long acidic N-terminal domain . This domain can be functionally replaced by unrelated integral membrane polypeptides, thus avoiding the need for specific FtsY membrane targeting factors . Therefore, the N terminus of FtsY constitutes an independent domain, which is required only for the targeting of the C-terminal NG domain of FtsY to the membrane.

FEBS Lett, 1997 Jun 9, 409(2), 307 - 11
Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S . cerevisiae; Ohba M; In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E . coli . To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S . cerevisiae . The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations . Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon . Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

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