Prokaryotes

J Cell Biol, 1992 Jul, 118(2), 481 - 90
Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane; Miao GH et al.; Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules . It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence . A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter . In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide . The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion . Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated . Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A . Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids . In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane . This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane . Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not . Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.

Eur J Biochem, 1992 Jul 1, 207(1), 1 - 11
The polyanion-binding domain of cytoplasmic Lys-tRNA synthetase from Saccharomyces cerevisiae is not essential for cell viability; Martinez R et al.; Cytoplasmic Lys-tRNA synthetase (LysRS) from Saccharomyces cerevisiae is a dimeric enzyme made up of identical subunits of 68 kDa . By limited proteolysis, this enzyme can be converted to a truncated dimer without loss of activity . Whereas the native enzyme strongly interacts with polyanionic carriers, the modified form displays reduced binding properties . KRS1 is the structural gene for yeast cytoplasmic LysRS . It encodes a polypeptide with an amino-terminal extension composed of about 60-70 amino acid residues, compared to its prokaryotic counterpart . This segment, containing 13 lysine residues, is removed upon proteolytic treatment of the native enzyme . The aim of the present study was to probe in vivo the significance of this amino-terminal extension . We have constructed derivatives of the KRS1 gene, encoding enzymes lacking 58 or 69 amino-terminal residues and, by site-directed mutagenesis, we have changed four or eight lysine residues from the amino-terminal segment of LysRS into glutamic acids . Engineered proteins were expressed in vivo after replacement of the wild-type KRS1 allele . The mutant enzymes displayed reduced specific activities (2-100-fold) . A series of carboxy-terminal deletions, encompassing 3, 10 or 15 amino acids, were introduced into the LysRS mutants with modified amino-terminal extensions . The removal of three residues led to a 2-7-fold increase in the specific activity of the mutant enzymes . This partial compensatory effect suggests that interactions between the two extreme regions of yeast LysRS are required for a proper conformation of the native enzyme . All KRS1 derivatives were able to sustain growth of yeast cells, although the mutant cell lines displaying a low LysRS activity grew more slowly . The expression, as single-copy genes, of mutant enzymes with a complete deletion of the amino-terminal extension or with four Lys----Glu mutations, that displayed specific activities close to that of the wild-type LysRS, had no discernable effect on cell growth . We conclude that the polycationic extensions of eukaryotic aminoacyl-tRNA synthetases are dispensable, in vivo, for aminoacylation activities . The results are discussed in relation to the triggering role in in situ compartmentalization of protein synthesis that has been ascribed to the polypeptide-chain extensions that characterize most, if not all, eukaryotic aminoacyl-tRNA synthetases.

EMBO J, 1992 Jul, 11(7), 2611 - 7
Activation of mammalian DNA methyltransferase by cleavage of a Zn binding regulatory domain; Bestor TH; Mammalian DNA (cytosine-5) methyltransferase contains a C-terminal domain that is closely related to bacterial cytosine-5 restriction methyltransferase . This methyltransferase domain is linked to a large N-terminal domain . It is shown here that the N-terminal domain contains a Zn binding site and that the N- and C-terminal domains can be separated by cleavage with trypsin or Staphylococcus aureus protease V8; the protease V8 cleavage site was determined by Edman degradation to lie 10 residues C-terminal of the run of alternating lysyl and glycyl residues which joins the two domains and six residues N-terminal of the first sequence motif conserved between the mammalian and bacterial cytosine methyltransferases . While the intact enzyme had little activity on unmethylated DNA substrates, cleavage between the domains caused a large stimulation of the initial velocity of methylation of unmethylated DNA without substantial change in the rate of methylation of hemimethylated DNA . These findings indicate that the N-terminal domain of DNA methyltransferase ensures the clonal propagation of methylation patterns through inhibition of the de novo activity of the C-terminal domain . Mammalian DNA methyltransferase is likely to have arisen via fusion of a prokaryotic-like restriction methyltransferase and an unrelated DNA binding protein . Stimulation of the de novo activity of DNA methyltransferase by proteolytic cleavage in vivo may contribute to the process of ectopic methylation observed in the DNA of aging animals, tumors and in lines of cultured cells.

J Bacteriol, 1992 Jul, 174(13), 4356 - 60
Tyrosine phosphorylation of a membrane protein from Pseudomonas solanacearum; Atkinson M et al.; We have investigated a tyrosine kinase activity from Pseudomonas solanacearum, an economically important plant pathogen . In vitro incubation of membrane fractions with {gamma-32P}ATP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an 85-kDa phosphoprotein . Phosphorylation of this protein on tyrosine residues was demonstrated by phosphoamino acid analysis of base hydrolysis products and by immunoanalysis of Western blots (immunoblots) with antiphosphotyrosine monoclonal antibody . In vitro incubation of membranes with ATP was not required for recognition by the antibody, indicating that the 85-kDa protein is phosphorylated in vivo . These results demonstrate that membranes from P . solanacearum exhibit a tyrosine kinase activity toward an endogenous membrane protein . This bacterium provides an opportunity to study the structure and function of a prokaryotic tyrosine kinase.

Arch Biochem Biophys, 1992 Jul, 296(1), 321 - 7
Purification and characterization of a catalase-peroxidase from the fungus Septoria tritici; Levy E et al.; Three classes of heme proteins, commonly designated hydroperoxidases, are involved in the metabolism of hydrogen peroxide: catalases, peroxidases, and catalase-peroxidases . While catalases and peroxidases are widely spread in animals, plants, and microorganisms, catalase-peroxidases were characterized only in prokaryotes . We report here, for the first time, on a catalase-peroxidase in a eukaryotic organism . The enzyme was purified from the fungal wheat pathogen Septoria tritici, and is one of three different hydroperoxidases synthesized by this organism . The S . tritici catalase-peroxidase, designated StCP, is similar to the enzymes previously isolated from the bacteria Rhodobacter capsulatus, Escherichia coli, and Klebsiella pneumoniae, although it is significantly more sensitive to denaturing conditions . In addition to its catalatic activity StCP catalyzes peroxidatic activity with o-dianisidine, diaminobenzidine, pyrogallol, NADH, and NADPH as electron donors . The enzyme is a tetramer with identical subunits of 61,000 Da molecular weight . StCP shows a typical high-spin ferric heme spectrum with a Soret band at 405 nm and a peak at 632 nm, and binding of cyanide causes a shift of the Soret band to 421 nm, the appearance of a peak at 537 nm, and abolition of the peak at 632 nm . Reduction with dithionite results in a decrease in the intensity of the Soret band and its shift to 436 nm, and in the appearance of a peak at 552 nm . The pH optimum is 6-6.5 and 5.4 for the catalatic and peroxidatic activities, respectively . Fifty percent of the apparent maximal activity is reached at 3.4 mM and 0.26 mM for the catalatic and peroxidatic activities, respectively . The enzyme is inactivated by ethanol/chloroform, and is inhibited by KCN and NaN3, but not by the typical catalase inhibitor 3-amino-1,2,4-triazole.

Comp Biochem Physiol B, 1992 Jul, 102(3), 437 - 44
Structure and possible function of heat-shock proteins in Falciparum malaria; Sharma YD; Like many prokaryotes and eukaryotes, the malaria parasite also synthesizes several stress proteins . Most widely studied stress proteins of this parasite are the heat-shock proteins (hsps) . Their discovery in malaria is a gift of recombinant DNA technology . Five hsp genes from Plasmodium falciparum have been identified which are located on different chromosomes . Thus the inheritance and expression of hsp genes are independent of each other . They share a large amount of sequence homology at N-terminus with the hsps of other organisms . Their gene regulatory sequences and other elements, important for gene expression, are yet to be determined . The biological role of these proteins in malaria is not fully understood but it is possible that they provide protection to the parasite from various stresses encountered in the host . In this process hsps probably bind to the toxic molecules as well as damaged proteins to flush them out of the parasite . Their involvement in the stage-specific parasite transformation to increase the infectivity and virulence, as observed in other parasites, remains to be determined . Malarial hsps are antigenic in humans . This antigenicity could be attributed to the non-homologous sequences in the C-terminus region . The potential of one of them (pfhsp 70I) for a future malaria vaccine and immunodiagnostics requires re-evaluation of the data.

Mol Gen Genet, 1992 Jul, 234(1), 147 - 54
New tools for mitochondrial genetics of Chlamydomonas reinhardtii: manganese mutagenesis and cytoduction; Bennoun P et al.; A novel and efficient genetic procedure is described for generating mitochondrial mutants of the green alga Chlamydomonas reinhardtii . The development of a mutagenesis procedure using manganese cations and the application of cytoduction techniques resulted in a combined approach for the generation and analysis of mitochondrial mutants . Although mitochondrial mutations are inherited in sexual crosses from the minus mating type parent, the cytoduction technique can be used to transfer mitochondrial mutations into recipient strains with different genetic backgrounds, irrespective of their mating type . Cytoduction allows the transfer of mitochondrial markers from diploid to haploid cells also, which is of great benefit since diploid cells do not germinate in C . reinhardtii . We report here the isolation and characterisation of eight mutants, which are resistant to the antibiotics myxothiazol and mucidin . The mutants all have point mutations in the mitochondrial gene for apocytochrome b . Using in vitro-amplified cytb gene fragments as probes for direct DNA sequencing, three different types of single base pair substitutions were revealed in all mutants tested . In particular, amino acid substitutions in the mutant apocytochrome b polypeptide have been identified at residues 129, 132 and 137, which have been implicated in forming part of an antibiotic-binding niche . The amino acid substitution at position 132 has not been so far described for mutant apocytochrome b in any other organism, prokaryotic or eukaryotic . The genetic approach presented here confirms C . reinhardtii as a model system that is unique among plant cells.

Int J Biochem, 1992 Jul, 24(7), 1125 - 33
Ribosomes from trichomonad protozoa have prokaryotic characteristics; Champney WS et al.; 1 . Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized . The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions . 2 . The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis . The ribosomes contained both 5.8S and 5S RNAs . 3 . The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC . Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T . gallinae, 40 in T . bactrachorum and 45 in the Tritrichomonas sp . The protein molecular mass range was 8.5-40 kDa . 4 . The HPLC analysis confirmed the protein number established by the gel methods . 5 . Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp . than between those of the more distantly related T . gallinae and T . bactrachorum.

Plant Cell, 1992 Jul, 4(7), 785 - 98
A novel light-regulated promoter is conserved in cereal and dicot chloroplasts; Christopher DA et al.; The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively . We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP) . It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants . To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined . Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals . Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied . An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity . The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco . The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon . We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region.

Protein Sci, 1992 Jul, 1(7), 925 - 34
Theory of chaperonin action: inertial model for enhancement of prokaryotic Rubisco assembly; Roy H et al.; We have performed a computational simulation of the aggregation and chaperonin-dependent reconstitution of dimeric prokaryotic ribulose bisphosphate carboxylase/oxygenase (Rubisco), based on the data of P . Goloubinoff et al . (1989, Nature 342, 884-889) and P . V . Viitanen et al . (1990, Biochemistry 29, 5665-5671) . The aggregation is simulated by a set of 12 differential equations representing the aggregation of the Rubisco folding intermediate, Rubisco-I, with itself and with aggregates of Rubisco-I, leading up to dodecamers . Four rate constants, applying to forward or reverse steps in the aggregation process, were included . Optimal values for these constants were determined using the ellipsoid algorithm as implemented by one of us (Ecker, J.G . & Kupferschmid, M., 1988, Introduction to Operations Research, Wiley, New York, pp . 315-322) . Intensive exploration of simpler aggregation models did not identify an alternative that could simulate the data as well as this one . The activity of the chaperonin in this system was simulated by using this aggregation model, combined with a model similar to that proposed by Goloubinoff et al . (1989) . The model assumes that the chaperonin can bind the folding intermediate rapidly, and that the chaperonin complex releases the Rubisco molecule slowly, permitting time for its spontaneous folding while interacting with the chaperonin . This is followed by self-association of the folded Rubisco monomer to yield the active dimeric Rubisco . A modification of the model that simulates temperature effects was also constructed . The most important results we obtained indicate that the chaperonin-dependent reconstitution of Rubisco can be simulated adequately without invoking any catalysis of folding by the chaperonin . In addition, the simulations predict values for the association rate constant of Rubisco-I with the chaperonin, and other variables, that are subject to experimental verification.

Trends Biochem Sci, 1992 Jul, 17(7), 262 - 6
Evolutionary conservation of the active site of soluble inorganic pyrophosphatase; Cooperman BS et al.; Soluble inorganic pyrophosphatases (PPases) are essential enzymes that are important for controlling the cellular levels of inorganic pyrophosphate (PPi) . Although prokaryotic and eukaryotic PPases differ substantially in amino acid sequence, recent evidence now demonstrates clearly that PPases throughout evolution show a remarkable level of conservation of both an extended active site structure, which has the character of a mini-mineral, and a catalytic mechanism . PPases require several (three or four) Mg2+ ions at the active site for activity and many of the 15-17 fully conserved active site residues are directly involved in the binding of metal ions . Each of the eight microscopic rate constants that has been evaluated for the PPases from both Escherichia coli and Saccharomyces cerevisiae is quite similar in magnitude for the two enzymes, supporting the notion of a conserved mechanism.

Genes Dev, 1992 Jul, 6(7), 1269 - 79
Trans and cis requirements for intron mobility in a prokaryotic system; Clyman J et al.; Intron mobility requires cleavage of an intronless allele by an intron-encoded endonuclease, followed by transfer of the intron into the cleaved recipient . The mobile phage introns provide an opportunity to identify accessory functions involved in the intron inheritance process . To test for trans and cis requirements of mobility in Escherichia coli, we have exploited the td intron of phage T4 in both phage T4 and lambda genetic backgrounds . Mobility depends on host or phage recombinase functions, RecA or UvsX, respectively . The process also requires a phage-encoded 5'----3' exonuclease activity and associated annealing function that can be provided by phage lambda . Finally, host-encoded 3'----5' exonuclease activities are also implicated in intron inheritance . We demonstrated further that restriction enzymes could substitute for the intron-encoded endonuclease, indicating that the endonuclease does not have an essential role in recombination . Neither the precise position nor the nature of the double-strand break was critical to intron transfer . These features provide insight into the recombination pathway and are factors impacting on the spread of introns throughout natural populations.

FEBS Lett, 1992 Jun 29, 305(2), 91 - 6
Fibronectin type III-like sequences and a new domain type in prokaryotic depolymerases with insoluble substrates; Hansen CK; Fibronectin type III-like sequences are present in many proteins from higher eukaryotes and are involved in protein-protein interactions, heparin binding and cell adhesion . A nine-member family of bacterial sequences is shown to be significantly homologous to the type III-like sequences . All the sequences are contained in secreted depolymerases acting on complex, energy-rich insoluble substrates, in which they apparently do not participate in catalysis or substrate-binding, their exact function remaining unclear . Furthermore, a new family of sequences, present in some cellulases, is presented.

Proc R Soc Lond B Biol Sci, 1992 Jun 22, 248(1323), 273 - 81
Amplification and rearrangement of a prokaryotic metallothionein locus smt in Synechococcus PCC 6301 selected for tolerance to cadmium; Gupta A et al.; Metal-tolerant cyanobacteria have been isolated from metal-polluted aquatic environments and also selected in culture, but no genes which confer metal tolerance have been described . To investigate the possibility that amplification of a prokaryotic metallothionein gene (smtA), or rearrangement of the smt locus, could be involved in the development of Cd tolerance in Synechococcus PCC 6301, Cd-tolerant lines were selected by stepwise adaptation of a Synechococcus culture . An increase in smtA gene copy number and the appearance of unique additional smtA restriction fragments (both larger and smaller) were detected in these tolerant lines (tolerant to 0.8 microM Cd, 1.3 microM Cd and 1.7 microM Cd) . Stepwise adaptation was repeated by using a culture of Synechococcus PCC 6301 inoculated from a single plated colony to obtain four new lines (tolerant to 1.4 microM Cd, 1.8 microM Cd, 2.6 microM Cd and 3.2 microM Cd) . Amplification of the smtA gene and development of unique smtA restriction fragments (larger and smaller) were once again detected in these tolerant lines . Amplification and rearrangement of the smt locus were only detected in the seven Cd-tolerant lines, with no evidence of amplification or rearrangement in the non-tolerant lines from which they were derived . As a control, another gene, psaE, was also monitored in these cell lines . There was no evidence of amplification or rearrangement of psaE in the non-tolerant or any of the Cd-tolerant lines.

J Mol Biol, 1992 Jun 20, 225(4), 933 - 8
From adenylate cyclase to guanylate cyclase . Mutational analysis of a change in substrate specificity; Beuve A et al.; Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination . A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity . In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity . This was due to a point mutation destroying most of the adenylate cyclase activity . A second selection step restored most of the original activity . This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 855 - 60
Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177; Chen HH et al.; A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109 . The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG) . The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol . In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system . Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s . These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity . Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.

FEBS Lett, 1992 Jun 15, 304(2-3), 119 - 23
Characterisation of a chloroplast-encoded secY homologue and atpH from a chromophytic alga . Evidence for a novel chloroplast genome organisation; Scaramuzzi CD et al.; secY is a prokaryotic gene that encodes the SecY protein, an integral membrane component of the prokaryotic protein translocation apparatus . A chloroplast-encoded secY homologue has been identified in the unicellular, chromophytic alga, Pavlova lutherii . The gene predicts a protein composed of ten membrane-spanning regions, that is approximately 25% homologous and 50% similar to bacterial and plastid SecY proteins . The secY gene from P . lutherii is independent of the ribosomal protein (rp) gene cluster to which it is closely linked in other organisms . In P . lutherii secY is located 5' to atpI and atpH . Since, in higher plants the atpIHFA gene cluster and the rp gene cluster are separated by approximately 50 kb, we conclude, this indicates a novel chloroplast gene arrangement in P . lutherii.

Gene, 1992 Jun 15, 115(1-2), 167 - 72
Regulation of secondary metabolism and cell differentiation in Streptomyces: A-factor as a microbial hormone and the AfsR protein as a component of a two-component regulatory system; Horinouchi S et al.; A-factor is a microbial hormone that functions as a key switch for secondary metabolite formation and morphogenesis in Streptomyces griseus . Genetic and biochemical studies on the A-factor-binding protein have implied that the binding protein present in the cytoplasm plays a role in repressing streptomycin (Sm) production and sporulation while the binding of A-factor to the binding protein releases this repression . The A-factor signal is transferred, probably via some additional regulatory proteins in the A-factor-regulatory cascade, to the strR gene, a regulator for Sm biosynthesis . A positive regulatory protein binds about 430-330 bp upstream from the transcription start point of the strR promoter and activates its transcription . The StrR product, in turn, activates the other Sm-biosynthesis genes . A global regulatory gene, afsR, of Streptomyces coelicolor A3(2) encodes a 993-amino acid protein that is phosphorylated by a specific phosphokinase, AfsK, encoded by the region just upstream from the afsR gene . Site-directed mutagenesis of afsR has revealed that phosphorylated AfsR globally stimulates transcription of antibiotic-production genes . It is most likely that AfsR and AfsK compose a two-component regulatory system . Although AfsR shows no significant homology with typical regulators of the two-component systems in other prokaryotes, such as OmpR and PhoB of Escherichia coli, it shows considerable homology with regulatory proteins in antibiotic biosynthetic gene clusters of Streptomyces spp., such as actII ORF4, dnrR1 ORF1 and redD ORF1.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1992 Jun 15, 206(3), 927 - 34
Structural and functional characterization of Escherichia coli peptidyl-prolyl cis-trans isomerases; Compton LA et al.; Peptidyl-prolyl cis-trans isomerases (PPIases), enzymes that catalyze the cis-trans isomerization of peptide bonds to which proline contributes the nitrogen, were purified from Escherichia coli . In this organism, at least two PPIases are present . Both the cationic (periplasmic) and anionic (cytoplasmic) PPIases are inhibited by cyclosporin A with a Ki of 25-50 microM, a concentration 1000-fold higher than that required for eukaryotic PPIases . Although isoelectric focusing indicates that the two enzymes differ in isoelectric point by at least 4.0 pH units, the specific activities of the enzymes toward the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-methyl-coumarylamide are equivalent . The activity of both enzymes for a series of substituted succinyl-Ala-Xaa-Pro-Phe-para-nitroanilide tetrapeptides suggests that the structure and function of the active site of the prokaryotic proteins is similar to that of eukaryotic cyclophilins . Both enzymes are capable of catalyzing the refolding of thermally denatured type III collagen . Antibodies against the periplasmic PPIase do not recognize the cytoplasmic enzyme, indicating significant differences in epitopes between the two forms . Circular dichroism spectroscopy indicates that the secondary structure of the cationic protein consists of 17% alpha-helix, 34% beta-sheet, 17% turns, 33% random coil and is very similar to human cytosolic PPIase.

FEMS Microbiol Lett, 1992 Jun 15, 72(3), 209 - 12
The effects of growth temperature on the methyl sterol and phospholipid fatty acid composition of Methylococcus capsulatus (Bath); Jahnke LL; Growth of Methylococcus capsulatus (Bath) at temperatures ranging from 30 to 50 degrees C resulted in changes to the whole cell lipid constituents . As temperature was lowered, the overall proportion of hexadecenoic acid (C16:1) increased, and the relative proportions of the delta 9, delta 10 and delta 11 C16:1 double bond positional isomers changed . Methyl sterol content also increased as the growth temperature was lowered . The highest amounts of methyl sterol were found in 30 degrees C cells and the lowest in 50 degrees C cells (sterol-phospholipid ratios of 0.077 and 0.013, respectively) . The data are consistent with a membrane modulating role for the sterol produced by this prokaryotic organism.

FEMS Microbiol Lett, 1992 Jun 15, 72(3), 243 - 7
'uidA-antibiotic-resistance cassettes for insertion mutagenesis, gene fusions and genetic constructions; Bardonnet N et al.; We have constructed a series of promoterprobe cassettes that provides powerful tools for insertion mutagenesis, transcription fusions and genetic constructions . These cassettes contain the Tn9 chloramphenicol (CmR) and the Tn903 kanamycin (KmR) resistance genes which are expressed in a large variety of microorganisms; these antibiotic-resistance markers were associated with the uidA promoterless gene . This beta-glucuronidase-encoding gene of Escherichia coli K-12 has been successfully used as reporter gene for various organisms including prokaryotes and eukaryotes . The resulting 'uidA-KmR and 'uidA-CmR cassettes (truncated at the ') can be excized with most of the commonly used restriction enzymes . Furthermore, they are borne by ApR or CmR plasmids which facilitate their utilization . These promoter-probe cassettes allow transcriptional signal localization and regulation studies.

Biochem J, 1992 Jun 15, 284 ( Pt 3), 861 - 7
Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein; van den Hombergh JP et al.; The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid . DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology . These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli . As a result, the DHQ synthase domain is overproduced in E . coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol . The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A . nidulans . The qutB gene of A . nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E . coli . The quinate dehydrogenase is not overproduced, but is active in E . coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E . coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.

Cell, 1992 Jun 12, 69(6), 1043 - 50
A cytoplasmic chaperonin that catalyzes beta-actin folding; Gao Y et al.; We have isolated a cytoplasmic chaperonin based on its ability to catalyze the folding of denatured beta-actin . The cytoplasmic chaperonin is organized as a multisubunit toroid and requires Mg2+ and ATP for activity . The folding reaction proceeds via the rapid ATP-independent formation of a binary complex, followed by a slower ATP-dependent release of the native product . Electron microscopic observations reveal a striking structural change that occurs upon addition of Mg2+ and ATP . The eukaryotic cytoplasm thus contains a chaperonin that is functionally analagous to its prokaryotic, mitochondrial, and chloroplastic counterparts.

Biochim Biophys Acta, 1992 Jun 11, 1107(1), 39 - 43
Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii; Nystrom S et al.; Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins . The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains . Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively . No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids . Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates . The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins . Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114 . This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.

Nucleic Acids Res, 1992 Jun 11, 20(11), 2667 - 71
An African swine fever virus gene with homology to DNA ligases; Hammond JM et al.; Sequence analysis of the SalI g region of the genome of a virulent isolate of ASFV (Malawi Lil 20/1) has revealed an open reading frame with the potential to encode a 48 kilodalton (kD) polypeptide which has significant homology with eukaryotic and prokaryotic DNA ligases . This ASFV encoded gene also contains the putative active site region of DNA ligases including the lysine residue which is necessary for enzyme-adenylate adduct formation, but lacks the C-terminal basic region conserved in other eukaryotic DNA ligases . A novel {32P}-labelled potential DNA ligase-adenylate adduct of M(r) 45 kD was observed upon incubation of ASFV infected cell cytoplasmic extracts with alpha-{32P}-ATP and subsequent analysis of products by SDS/PAGE . These data together suggest that ASFV encodes its own DNA ligase.

J Biol Chem, 1992 Jun 5, 267(16), 11553 - 8
The sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase; Birch-Machin MA et al.; The cDNA sequence of the flavoprotein subunit of bovine heart succinate dehydrogenase is reported . This is the first complete eukaryotic sequence of the flavoprotein subunit to be characterized, and it encodes a 665-amino acid protein that consists of a presequence and a 621-residue mature protein . The deduced bovine sequence shows homology to the corresponding peptides of prokaryotic succinate dehydrogenase and the related fumarate reductases; in particular, there is good overall homology (48%) to the flavoprotein subunit of Escherichia coli succinate dehydrogenase . The conserved sequences comprising the active site and those involved in FAD binding are also found in the bovine protein . The active site of the bovine polypeptide contains a cysteine that confers sensitivity of the enzyme to sulfhydryl reagents; this cysteine is only present in some sequences and thus provides a discriminatory biochemical marker . A putative flavoprotein subunit of human placental succinate dehydrogenase (partial sequence) that lacks this critical cysteine (Malcovati, M., Marchetti, T., Zanelli, T., and Tenchini, M . L . (1991) in Flavins and Flavoproteins 1990 (Curti, B., Ronchi, S., and Zanetti, G., eds) pp . 727-730, Walter de Gruyter & Co., Berlin) has only 16% homology to the bovine heart flavoprotein subunit . However, we show that the enzyme from human placenta is as sensitive to N-ethylmaleimide as that from bovine tissues . In addition, a transcript in human placenta and muscle hybridizes to the bovine heart flavoprotein cDNA and is the same size as that in bovine tissues.

Plant Mol Biol, 1992 Jun, 19(3), 355 - 65
Conserved relationship between psbH and petBD genes: presence of a shared upstream element in Prochlorothrix hollandica; Greer KL et al.; Prochlorophytes are an unusual group of prokaryotic oxygenic photoautotrophs that morphologically appear to bridge the gap between cyanobacteria and the chloroplasts of eukaryotic plants . Molecular data place this group evolutionarily within the cyanobacteria, but they have a photosynthetic apparatus that is very similar to that found in chloroplasts . We have sequenced from the prochlorophyte Prochlorothrix hollandica a set of genes (psbB, psbH, petB and petD) that has a conserved organization in chloroplast genomes that is different from the organization in cyanobacterial genomes . The four genes are linked as an operon in chloroplasts, but only petB and petD are closely linked and cotranscribed in cyanobacteria . Although the prochlorophyte gene arrangement resembles that of cyanobacteria, one feature suggests the coordinated regulation of the unlinked genes . A 93 bp region of absolute conservation occurs upstream of the psbH gene and the petBD operon, near the site of transcription initiation in each gene set . This conserved element may indicate an alternative to cotranscription for achieving co-regulation of the psbH and petBD genes in the prochlorophyte.

FEBS Lett, 1992 Jun 1, 303(2-3), 159 - 63
Cyanobacterial metallothionein gene expressed in Escherichia coli . Metal-binding properties of the expressed protein; Shi J et al.; The recently isolated Synechococcus gene smtA encodes the only characterised prokaryotic protein designated to be a metallothionein (MT) . To examine the metal-binding properties of its product the smtA gene was expressed in Escherichia coli as a carboxyterminal extension of glutathione-S-transferase . The pH of half dissociation of Zn, Cd and Cu ions from the expressed protein was determined to be 4.10, 3.50, 2.35, respectively, indicating a high affinity for these ions (in particular for Zn in comparison to mammalian MT) . E . coli expressing this gene showed enhanced (ca . 3-fold) accumulation of Zn.

Biochem J, 1992 Jun 1, 284 ( Pt 2), 469 - 76
Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons; Cheetham ME et al.; The bacterial heat-shock protein DnaJ has been implicated in protein folding and protein complex dissociation . The DnaJ protein interacts with the prokaryotic analogue of Hsp70, DnaK, and accelerates the rate of ATP hydrolysis by DnaK . Several yeast homologues of DnaJ, with different proposed subcellular localizations and functions, have recently been isolated and are the only eukaryotic forms of DnaJ so far described . We have isolated cDNAs corresponding to two alternatively spliced transcripts of a novel human gene, HSJ1, which show sequence similarity to the bacterial DnaJ protein and the yeast homologues . The cDNA clones were isolated from a human brain-frontal-cortex expression library screened with a polyclonal antiserum raised to paired-helical-filament (PHF) proteins isolated from extracts of the brains of patients suffering from Alzheimer's disease . The similarity between the predicted human protein sequences and the bacterial and yeast proteins is highest at the N-termini, this region also shows a limited similarity to viral T-antigens and is a possible common motif involved in the interaction with DnaK/Hsp70 . Northern-blot analysis has shown that human brain contains higher levels of mRNA for the DnaJ homologue than other tissues examined, and hybridization studies with riboprobes in situ show a restricted pattern of expression of the mRNA within the brain, with neuronal layers giving the strongest signal . These findings suggest that the DnaJ-DnaK (Hsp70) interaction is general to eukaryotes and, indeed, to higher organisms.

Eur J Biochem, 1992 Jun 1, 206(2), 503 - 10
Cloning and nucleotide sequence of the psrA gene of Wolinella succinogenes polysulphide reductase; Krafft T et al.; The polysulphide reductase (formerly sulphur reductase) of Wolinella succinogenes is a component of the phosphorylative electron transport system with polysulphide as the terminal acceptor . Using an antiserum raised against the major subunit (PsrA, 85 kDa) of the enzyme, the corresponding gene (psrA) was cloned from a lambda-gene bank . The N-terminal amino acid sequence of PsrA mapped within the psrA gene product, which also contained an apparent signal peptide . Downstream of the psrA gene two more open reading frames (psrB and psrC) were found . The three genes may form a transcriptional unit with the transcription start site in front of psrA . The three genes were present only once on the genome . PsrA is a hydrophilic protein homologous to the largest subunits of six prokaryotic molybdoenzymes . PsrB is predicted to be hydrophilic, to contain ferredoxin-like cysteine clusters and to be homologous to the smaller hydrophilic subunits of four molybdoenzymes . PsrC is predicted to be a hydrophobic protein that could possibly serve as the membrane anchor of the enzyme.

Proc Natl Acad Sci U S A, 1992 Jun 1, 89(11), 5188 - 91
Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages; Cianciotto NP et al.; Legionella pneumophila is an intracellular parasite of freshwater protozoa and human macrophages . Recent studies determined that the macrophage infectivity potentiator (Mip) surface protein, a prokaryotic homolog of the FK506-binding proteins, is required for optimal infection of macrophages . To determine whether Mip is also involved in L . pneumophila infection of protozoa, we examined the ability of a strain lacking Mip to parasitize Hartmannella amoebae and Tetrahymena ciliates . After 3 days of incubation, approximately 1000-fold fewer bacteria were recovered from protozoan cocultures infected with the Mip- strain than from those cocultures infected with an isogenic Mip+ strain . The mip mutant was, however, not impaired in its ability to bind to amoebae cell surfaces, indicating that Mip is involved in bacterial resistance to intracellular killing and/or intracellular multiplication . These data suggest that L . pneumophila employs similar genes and mechanisms to infect human cells and protozoa . Furthermore, they support the hypothesis that the ability of L . pneumophila to parasitize macrophages and hence to cause human disease is a consequence of its prior adaptation to intracellular growth within protozoa.

EMBO J, 1992 Jun, 11(6), 2219 - 28
The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent; Austin S et al.; The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme . Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent . We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites.

Res Microbiol, 1992 Jun, 143(5), 467 - 70
Two unrelated alkaliphilic Bacillus species possess identical deviations in sequence from those of other prokaryotes in regions of F0 proposed to be involved in proton translocation through the ATP synthase; Ivey DM et al.; The a and c subunits of two unrelated alkaliphilic Bacillus species contain unusual motifs in regions previously implicated by others in H(+)-coupled oxidative phosphorylation . The facultative alkaliphile B . firmus OF4 apparently does not contain genes encoding an alternative F0, supporting other evidence that a single species of proton-translocating F1F0-ATPase catalyses oxidative phosphorylation both at low and high pH . The unusual F0 sequence motifs may be part of the adaptation of the extreme alkaliphiles to growth at very high pH.

Immunol Lett, 1992 Jun, 33(1), 53 - 9
Rabbit single domain antibodies specific to protein C expressed in prokaryotes; Suter M et al.; VDJ genes were amplified by the polymerase chain reaction from mRNA isolated from peripheral blood B cells of rabbits immunized with protein C . The amplified genes were cloned into a lambda phage expression vector and packaged . A library of 6 x 10(5) recombinant phages was screened with labelled protein C and 30 positive clones were found . Three of them were plaque purified and the affinity of the single domain antibodies to the antigen determined to be 10(6)-10(7) l M-1 . The data indicate the feasibility of generating single domain antibody, specific to protein antigen, from rabbit.

FASEB J, 1992 Jun, 6(9), 2660 - 6
ATP-dependent bacterial transporters and cystic fibrosis: analogy between channels and transporters; Ames GF et al.; The traffic ATPases superfamily includes known transporters, both prokaryotic and eukaryotic, including the medically important proteins, P-glycoprotein, and the cystic fibrosis gene product (CFTR), which is known to be a Cl- channel . The structure and mechanism of action of the best-studied members of the superfamily, the periplasmic permeases, are described and related to that of CFTR and eukaryotic traffic ATPases in general . The contention is put forward that the distinction between the architecture and mechanisms of action of channels and transporters is blurred.

Immunol Invest, 1992 Jun, 21(3), 241 - 57
Immunoreactivity of recombinant carcinoembryonic antigen proteins expressed in Escherichia coli; Kuroki M et al.; Immunoreactivities of recombinant carcinoembryonic antigen (CEA) proteins expressed in Escherichia coli (E . coli) were analyzed in relation to the CEA domain structure {domains N, I (A1-B1), II (A2-B2), III (A3-B3) and M} . We reconstructed in a prokaryotic expression vector, pUCPL-cI, the cDNAs fro CEA-N, CEA-I, CEA-II, and CEA-III-M . The latter three were expressed as fusion products with bacterial beta-galactosidase . The recombinant proteins were solubilized by sonication in 1% sodium dodecyl sulfate (SDS) and purified by preparative SDS-polyacrylamide gel electrophoresis followed by electroelution . Their molecular weights judged from Western blotting coincided with those calculated from their cDNA sequences, respectively . By solid-phase enzyme immunoassays, the immunoreactivities of the purified recombinant proteins were tested with 21 distinct anti-CEA monoclonal antibodies (MAbs) which had been found to recognize the peptide epitopes of the CEA molecule and to be reactive with the recombinant CEA proteins expressed in Chinese hamster ovary (CHO) cells . Fourteen of the 21 MAbs reacted with the recombinant CEA proteins expressed in E . coli and confirmed the localization of the epitopes identified by using the recombinant CEA proteins expressed in CHO cells . The reactivities of 5 MAbs with the recombinant proteins expressed in E . coli were remarkably low when compared with those of the proteins expressed in CHO cells but also confirmed the localization of the epitopes identified with the recombinant CEA proteins expressed in CHO cells . The remaining 2 MAbs did not react with any recombinant protein expressed in E . coli . These results indicate that the fusion CEA-proteins expressed in E . coli are useful in the localization of the epitopes on the polypeptide chains when they reacted with the MAbs tested . However, one third of the epitopes of CEA peptides may be profoundly affected by the presence of disulfide bonds and/or sugar chains which do not seem to be formed well in E . coli.

J Virol, 1992 Jun, 66(6), 3918 - 24
Gene for the major antigenic structural protein (p100) of human herpesvirus 6; Neipel F et al.; A human herpesvirus 6 (HHV-6) structural protein of 100 kDa (p100) is the polypeptide most frequently and intensively reactive in immunoblotting analyses with human sera on HHV-6-infected cells or partially purified virions . The gene for p100 was identified by screening a bacteriophage lambda library with monospecific rabbit antisera . The gene codes for a polypeptide of 870 amino acids with a calculated molecular size of 97 kDa . Its amino-terminal third is weakly homologous to the immunogenic basic matrix phosphoprotein pp150 of human cytomegalovirus . Five fragments representing more than 93% of HHV-6 p100 were prokaryotically expressed . The antigenic epitopes of p100 were preliminary mapped by immunoblotting with human sera . They are located within the carboxy-terminal part which is neither homologous nor cross-reactive to pp150 of human cytomegalovirus . Availability of the gene for the immunodominant structural protein should provide tools for studies of pathogenesis by HHV-6.

Virology, 1992 Jun, 188(2), 938 - 47
A gene homologous to topoisomerase II in African swine fever virus; Garcia-Beato R et al.; A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases . The gene is located within EcoRI fragments P and H of the African swine fever virus genome . Sequencing of this region has revealed a long open reading frame, designated P1192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543 . Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA . The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29% . The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established . The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged.

J Biol Chem, 1992 May 15, 267(14), 9654 - 62
Guanine nucleotide-binding proteins in the intestinal parasite Giardia lamblia . Isolation of a gene encoding an approximately 20-kDa ADP-ribosylation factor; Murtagh JJ Jr et al.; Giardia lamblia is a protozoan intestinal parasite that has characteristics of both eukaryotes and prokaryotes . To determine whether genes for guanine nucleotide-binding proteins are present in Giardia, genomic DNA and cDNA libraries were screened by polymerase chain reaction and by hybridization with mixed oligonucleotide probes complementary to sequences encoding conserved GTP-binding domains . A gene with a high degree of sequence identity with mammalian ADP-ribosylation factors (ARFs), believed to be important in vesicular transport, was identified . The Giardia ARF gene had a 573-base open reading frame encoding 191 amino acids which are 63-70% identical with known mammalian and yeast ARFs . Sequence conservation among ARFs was greatest in putative GTP-binding domains . A single ARF mRNA species of approximately 750 bases was found in two different Giardia isolates . Primer extension and RNA sequencing of the Giardia ARF transcript revealed a short (6-base) 5'-untranslated region similar in size to those found in other Giardia transcripts . Giardia extracts contained ARF activity, as shown by stimulation of cholera toxin-catalyzed ADP-ribosylation and a Giardia ARF expressed in Escherichia coli as a fusion protein likewise exhibited biochemical activity . Its presence in Giardia is consistent with the view that ARF emerged before the divergence of this protozoan from other eukaryotes (approximately 1.5 billion years ago), and that an ARF-like protein may have been the ancestor of several other classes of signal-transducing guanine nucleotide-binding proteins, including the alpha subunits of the heterotrimeric G proteins.

Gene, 1992 May 15, 114(2), 239 - 43
A selectable bifunctional beta-galactosidase::phleomycin-resistance fusion protein as a potential marker for eukaryotic cells; Baron M et al.; The Sh ble gene, conferring phleomycin resistance (PhR), was fused in frame to both the 3' and 5' ends of the Escherichia coli lacZ gene . The bifunctionality of the resulting 130-kDa hybrid proteins was demonstrated in E . coli and in the fungus, Tolypocladium geodes . PhR transformants of both organisms could be selected for . All transformants from E . coli and most from T . geodes displayed beta Gal activity . In the fungal host, higher transformation frequencies and greater levels of beta Gal activity were observed in clones harboring the lacZ::Sh ble fusion, as compared to the Sh ble::lacZ configuration . This system appears to be a potentially useful tool for the direct selection of transformants, and the evaluation of gene expression and regulation in a wide variety of prokaryotic and eukaryotic hosts.

Eur J Biochem, 1992 May 15, 206(1), 69 - 77
Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp . PCC 6301; Marques S et al.; Ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp . PCC 6301 has been purified using, as main steps, ethanol fractionation in the presence of high ionic strength, ion-exchange chromatography and ferredoxin-Sepharose affinity chromatography . The overall process yielded an homogeneous enzyme with a specific activity of 30 U/mg protein, after a purification of 2800-fold with a recovery of 43% . The molecular mass of the native protein was 156 kDa, as calculated from its Stokes radius (rS, 4.32 nm) and sedimentation coefficient (S20,w, 8.46 S) . The size was also estimated by SDS/PAGE as 160 kDa, indicating that the native protein was a monomer . The enzyme exhibited absorption maxima at 279, 370 and 438 nm and a A279/A438 absorbance ratio of 11 . One molecule of FMN, but not FAD, was found/molecule native protein . The addition of dithionite resulted in the loss of the absorption peak at 438 nm, which was restored by the addition of 2-oxoglutarate, thus indicating that the prosthetic group is functional in catalysis . Classical hyperbolic kinetics with substrate inhibition was seen for 2-oxoglutarate . The Km values determined for glutamine and ferredoxin were 0.7 mM and 7 microM, respectively, and the apparent Km for 2-oxoglutarate was estimated to be 1.7 mM . Azaserine and 6-diazo-5-oxo-L-norleucine were potent inhibitors of the activity, while pyridoxal 5-phosphate, known to react with Lys residues, partially inactivated the enzyme . This ferredoxin-dependent glutamate synthase is, as far as we know, the first purified from prokaryotic organisms and resembles its counterpart from chloroplasts, suggesting that cyanobacterial glutamate synthase may have been the ancestor of ferredoxin-glutamate synthase in plants.

Proc Natl Acad Sci U S A, 1992 May 15, 89(10), 4437 - 41
A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942; Fukuzawa H et al.; To understand the CO2-concentrating mechanism in cyanobacteria, a genomic DNA fragment that complements a temperature-sensitive high-CO2 (5%)-requiring mutant of Synechococcus PCC7942 has been isolated . An open reading frame (ORF272) encoding a polypeptide of 272 amino acids (Mr, 30,184) was found within the genomic region located 20 kilobases downstream from the genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLS) . Insertion of a kanamycin-resistance gene cartridge within the ORF272 in wild-type cells led to a high-CO2-requiring phenotype . Strains carrying a gene disabled by insertional mutagenesis accumulated inorganic carbon in the cells, but they could not fix it efficiently, even though ribulose-1,5-bisphosphate carboxylase activity was comparable to that of the wild-type strain . Therefore, the ORF272 was designated as a gene icfA, which is essential to inorganic carbon fixation . Furthermore, the predicted icfA gene product shared significant sequence similarities with plant chloroplast carbonic anhydrases (CAs) from pea (22%) and spinach (22%) and also with the Escherichia coli cynT gene product (31%), which was recently identified to be E . coli CA . These results indicate that the putative CA encoded by icfA is essential to photosynthetic carbon dioxide fixation in cyanobacteria and that plant chloroplast CAs may have evolved from a common ancestor of the prokaryotic CAs, which are distinct from mammalian CAs and Chlamydomonas periplasmic CAs.

Cell, 1992 May 15, 69(4), 677 - 84
Signal peptides open protein-conducting channels in E . coli; Simon SM et al.; Plasma membrane vesicles and protoplasts of Escherichia coli were fused to planar lipid bilayers and studied with electrophysiological techniques . Large transmembrane aqueous channels were opened when 0.2 nM LamB signal peptide was added to the cytoplasmic side of the membrane . These aqueous pores are similar in conductance to those previously observed in mammalian endoplasmic reticulum when puromycin is used to release and thus unplug nascent translocating chains . Signal sequences have been previously shown to be necessary and sufficient for targeting proteins to cellular membranes . These results demonstrate that signal peptides are sufficient for opening the protein-conducting channels . We suggest that they are the physiological ligands that open protein-conducting channels at the initiation of protein translocation across prokaryotic plasma membrane and mammalian endoplasmic reticulum.

Nucleic Acids Res, 1992 May 11, 20(9), 2287 - 91
Isolation and characterization of the cDNA encoding human DNA methyltransferase; Yen RW et al.; We have cloned a series of overlapping cDNA clones encoding a 5194 bp transcript for human DNA methyltransferase (DNA MTase) . This sequence potentially codes for a protein of 1495 amino acids with a predicted molecular weight of 169 kDa . The human DNA MTase cDNA has eighty percent homology at the nucleotide level, and the predicted protein has seventy-four percent identity at the amino acid level, to the DNA MTase cDNA cloned from mouse cells . Like the murine DNA MTase, the amino terminal two-thirds of the human protein contains a cysteine-rich region suggestive of a metal-binding domain . The carboxy terminal one-third of the protein shows considerable similarity to prokaryotic (cytosine-5)-methyltransferases . The arrangement of multiple motifs conserved in the prokaryotic genes is preserved in the human DNA MTase, including the relative position of a proline-cysteine dipeptide thought to be an essential catalytic site in all (cytosine-5)-methyltransferases . A single 5.2 kb transcript was detected in all human tissues tested, with the highest levels of expression observed in RNA from placenta, brain, heart and lung . DNA MTase cDNA clones were used to screen a chromosome 19 genomic cosmid library . The DNA MTase-positive cosmids which are estimated to span a genomic distance of 93 kb have been localized to 19p13.2-p13.3 by fluorescence in situ hybridization . Isolation of the cDNA for human DNA MTase will allow further study of the regulation of DNA MTase expression, and of the role of this enzyme in establishing DNA methylation patterns in both normal and neoplastic cells.

FEBS Lett, 1992 May 4, 302(1), 1 - 4
Pseudouridine in the large-subunit (23 S-like) ribosomal RNA . The site of peptidyl transfer in the ribosome?
Lane BG, Ofengand J, Gray MW.
On evolutionary grounds, it has been advocated for more than 40 years that RNA generally, and more recently rRNA in particular, may participate, catalytically, in protein biosynthesis . A specific molecular mechanism has never been proposed . We suggest here that the N-1 position(s) in one or more of the approximately 4 pseudouridine (omega) residues in E . coli 23 S rRNA catalyzes transfer of the aminoacyl moiety from teh 3'-terminus of peptidyl tRNA in the P site to aminoacyl tRNA in the A site of the ribosome . Evidence that supports the proposal in the case of E . coli ribosomes, and relevant information pertaining to eukaryotic ribosomes, is summarized . Essential features of the evidence are that (i) the N-1 position in 1-acetylthymine (a direct analogue of 1-acetylpseudouridine) has an especially high potential for acyl-group transfer, comparable to that found for N-acetylimidazole (Spector, L.B . and Keller, E.B . (1958) J . Biol . Chem . 232, 185-192), (ii) most of the omega residues in prokaryotic 23 S rRNA are confined to the peptidyl transferase center in E . coli ribosomes, and (iii) Um-Gm-omega, the most densely modified sequence in eukaryotic 26 S rRNA, is universally conserved at a fixed site in the putative peptidyl transferase center of all eukaryotic ribosomes.

Mol Microbiol, 1992 May, 6(10), 1253 - 7
Membrane-associated GTPases in bacteria; March PE; Members of the GTPase superfamily are extremely important in regulating membrane signalling pathways in all cells . This review focuses on membrane-associated GTPases that have been described in prokaryotes . In bacteria, LepA and NodQ are very similar to protein synthesis elongation factors but apparently have membrane-related functions . The amino acid sequences of FtsY and Ffh are clearly related to eukaryotic factors involved in protein secretion . Obg and Era are not closely related to any GTPase subgroup according to amino acid sequence comparisons, but they are essential for viability . In spite of similarities to well-studied eukaryotic proteins the signalling pathways of these cellular regulators, with the exception of NodQ, have not yet been elucidated.

Bioessays, 1992 May, 14(5), 295 - 301
DNA precursor asymmetries, replication fidelity, and variable genome evolution; Mathews CK et al.; Balanced pools of deoxyribonucleoside triphosphates (dNTPs) are essential for DNA replication to occur with maximum fidelity . Conditions that create biased dNTP pools stimulate mutagenesis, as well as other phenomena, such as recombination or cell death . In this essay we consider the effective dNTP concentrations at replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication . We focus upon two questions . (1) In prokaryotic systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that do not equilibrate with the bulk dNTP pools in the cell . Since these pools cannot be analyzed directly, what indirect approaches can illuminate the nature of these replication-active pools? (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide . Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle . To what extent might these natural asymmetries contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome?

Cell, 1992 May 1, 69(3), 439 - 56
DMC1: a meiosis-specific yeast homolog of E . coli recA required for recombination, synaptonemal complex formation, and cell cycle progression; Bishop DK et al.; DMC1 is a new meiosis-specific yeast gene . Dmc1 protein is structurally similar to bacterial RecA proteins . dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase . dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related . dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation . dmc1 mutations confer prophase arrest . Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism . DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.

Genes Dev, 1992 May, 6(5), 825 - 36
Polar localization of a bacterial chemoreceptor; Alley MR et al.; The bacterial chemotaxis signal transducer MCP is an integral membrane receptor protein . The chemoreceptor is localized at the flagellum-bearing pole of Caulobacter crescentus swarmer cells . Amino-terminal sequences of the MCP target the protein to the membrane while the carboxy-terminal portion of the protein is responsible for polar localization . The C . crescentus and Escherichia coli MCPs have highly conserved carboxy-terminal domains, and when an E . coli MCP is expressed in C . crescentus, it is targeted to the swarmer cell progeny . These results suggest that subcellular localization of a prokaryotic protein involves interaction of specific regions of the protein with unique cell sites that contain either localized binding proteins or a specific secretory apparatus.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 4028 - 32
Enzymatic defect in "X-linked" sideroblastic anemia: molecular evidence for erythroid delta-aminolevulinate synthase deficiency; Cotter PD et al.; Recently, the human gene encoding erythroid-specific delta-aminolevulinate synthase was localized to the chromosomal region Xp21-Xq21, identifying this gene as the logical candidate for the enzymatic defect causing "X-linked" sideroblastic anemia . To investigate this hypothesis, the 11 exonic coding regions of the delta-aminolevulinate synthase gene were amplified and sequenced from a 30-year-old Chinese male with a pyridoxine-responsive form of X-linked sideroblastic anemia . A single T----A transition was found in codon 471 in a highly conserved region of exon 9, resulting in an Ile----Asn substitution . This mutation interrupted contiguous hydrophobic residues and was predicted to transform a region of beta-sheet structure to a random-coil structure . Prokaryotic expression of the normal and mutant cDNAs revealed that the mutant construct expressed low levels of enzymatic activity that required higher concentrations of pyridoxal 5'-phosphate to achieve maximal activation than did the normal enzyme . The amino acid substitution occurred in the exon containing the putative pyridoxal 5'-phosphate binding site and may account for the reduced ability of the cofactor to catalyze the formation of delta-aminolevulinic acid.

J Bacteriol, 1992 May, 174(9), 2929 - 34
Comparative physiology of two protozoan parasites, Leishmania donovani and Trypanosoma brucei, grown in chemostats; ter Kuile BH et al.; Cultures of the insect stage of the protozoan parasites Leishmania donovani and Trypanosoma brucei were grown in chemostats with glucose as the growth rate-limiting substrate . L . donovani has a maximum specific growth rate (mu max) of 1.96 day-1 and a Ks for glucose of 0.1 mM; the mu max of T . brucei is 1.06 day-1 and the Ks is 0.06 mM . At each steady state (specific growth rate, mu, equals D, the dilution rate), the following parameters were measured: external glucose concentration (Glcout), cell density, dry weight, protein, internal glucose concentration (Glcin), cellular ATP level, and hexokinase activity . L . donovani shows a relationship between mu and yield that allows an estimation of the maintenance requirement (ms) and the yield per mole of ATP (YATP) . Both the ms and the YATP are on the higher margin of the range found for prokaryotes grown on glucose in a complex medium . L . donovani maintains the Glcin at a constant level of about 50 mM as long as it is not energy depleted . T . brucei has a decreasing yield with increasing mu, suggesting that it oxidizes its substrate to a lesser extent at higher growth rates . Glucose is not concentrated internally but is taken up by facilitated diffusion, while phosphorylation by hexokinase is probably the rate-limiting step for glucose metabolism . The Ks is constant as long as glucose is the rate-limiting substrate . The results of this study demonstrate that L . donovani and T . brucei have widely different metabolic strategies for dealing with varying external conditions, which reflect the conditions they are likely to encounter in their respective insect hosts.

Infect Immun, 1992 May, 60(5), 1729 - 33
Consequences of microbial attachment: directing host cell functions with adhesins; Hoepelman AI et al.; We take the view that adherence is not just a static process of holding hands but rather elicits a response in the targeted cell . From this point of view, adherence is an active process with an outcome . This outcome or fate is predictable only when several parameters of the host cell-adhesin interaction are known: is the adhesin acting alone or in series with other products, is the receptor up- or down-regulated at the time of ligation, which domain of the receptor is bound, and finally, which intracellular response circuits are connected to the receptor in the cell type targeted? Variations in these parameters are the basis for the ability of the adhesins of pathogens to orchestrate outcomes as disparate as simple address recognition versus actin nucleation, cytokine induction, activation of plasmin, derangement of leukocyte migration, or deposition of antibody on host cell membranes . The recognition of the relatedness of some eukaryotic and prokaryotic adhesive domains and the shared use of existing eukaryotic cell-cell interaction systems between host and pathogen suggest that the cellular interactions of interest in eukaryotic cell biology can be revealed by taking clues from the pathogens, which have studied and adapted to them the longest.

J Virol, 1992 May, 66(5), 2934 - 42
Regulated expression of foreign genes in vaccinia virus under the control of bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor; Alexander WA et al.; The gene encoding bacteriophage T7 RNA polymerase (T7gene1) was placed under the control of regulatory elements from the Escherichia coli lac operon to construct an inducible vaccinia virus expression system consisting entirely of prokaryotic transcriptional machinery . Regulated expression of T7 RNA polymerase was necessary to construct a stable recombinant vaccinia virus harboring a T7 promoter; otherwise, uncontrolled expression led to interference with endogenous virus replication . To this end, the gene encoding the repressor protein of the lac operon was fused to a viral early/late promoter so that it was expressed constitutively, and the lac operator was interposed between a viral major late promoter and T7gene1 . Greater than 99% repression of T7 RNA polymerase, which was relieved approximately 80-fold in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), was obtained . An expression cassette containing a T7 promoter-controlled beta-galactosidase reporter gene was recombined into a different region of the viral genome containing T7gene1 . A stable, double recombinant virus was isolated and grown to a high titer . In the absence of inducer, beta-galactosidase expression was substantially repressed . Addition of increasing amounts of IPTG induced expression of beta-galactosidase to the point of suppression of viral replication . This hybrid vaccinia virus system (Vac/Op/T7) has potential applications for the efficient bioproduction of a wide variety of gene products.

J Neurochem, 1992 May, 58(5), 1782 - 9
Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line; Malherbe P et al.; Human catechol-O-methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies . When human membrane-bound catechol-O-methyltransferase (MB-COMT; amino acids 1-271) and the soluble catechol-O-methyltransferase COMT (S-COMT; delta membrane anchor hCOMT; amino acids 27-271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high-level synthesis of catalytically active enzymes was obtained . Insertion of the human MB-COMT-coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells . Subcellular fractionation of 293 cells transfected with pBC12/CMV-hCOMT showed hCOMT to be located predominantly in the membrane fraction . The catechol-O-methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37 degrees C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h) . Km values were 10 microM for MB-COMT and 108 microM for S-COMT, indicating that recombinant MB-COMT exhibits a higher affinity for catechol as the substrate than the soluble form . RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of approximately 1.4 kb . Its level in these various tissues was similar to those of COMT protein in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Antimicrob Agents Chemother, 1992 May, 36(5), 1119 - 24
The unstable tetracycline resistance gene of Streptomyces lividans 1326 encodes a putative protein with similarities to translational elongation factors and Tet(M) and Tet(O) proteins; Dittrich W et al.; Streptomyces lividans contains a genetically unstable tetracycline resistance determinant . Nucleotide sequencing revealed an open reading frame of 1,917 nucleotides . The transcriptional start site was mapped at about 110 bp upstream of the ATG codon . The proposed promoter contains an 8-bp perfect inverted repeat between the -10 and -35 regions . The deduced amino acid sequence showed several motifs which are commonly found in many GTP-binding proteins . On the basis of its amino acid sequence, the presumptive S . lividans 1326 protein belongs to the Tet(M)-Tet(O) group of tetracycline resistance proteins and shows significant similarity to translational elongation factors of prokaryotes and eukaryotes.

Drug Saf, 1992 May-Jun, 7(3), 214 - 22
Mutagenicity of quinolone antibacterials; Fort FL; The literature is summarised on the activity of quinolone antibacterial compounds in assays which are commonly used for risk assessment of new pharmaceuticals . These include assays for DNA damage, sister chromatid exchanges, chromosome aberrations and mutation induction . The general pattern of activity exhibited by these compounds is induction of DNA damage in both prokaryotic and eukaryotic cells, and induction of mutations in DNA repair-proficient bacteria and at the thymidine kinase locus in mammalian cells . They do not appear as a class to induce mutations at the hypoxanthine-guanine-phosphoribosyltransferase (HGPRT) or Na+,K(+)-ATPase loci or to cause chromosome aberrations . It is suggested that these actions may be the result of interference with eukaryotic topoisomerase and that this interference differs in some respects from the topoisomerase interference caused by certain antitumour compounds . The postulated mechanism of action has important implications for assessment of risk from consumption of quinolone antibacterials . The risk of adverse genotoxic events should vary directly with the concentration of drug reaching the intracellular enzyme target and the affinity of the drug for the target . Results of carcinogenicity studies conducted to date with the quinolone antibacterials suggest minimal risk from long term consumption of the newer, second-generation compounds.

Mol Microbiol, 1992 May, 6(10), 1375 - 83
Mip protein of Legionella pneumophila exhibits peptidyl-prolyl-cis/trans isomerase (PPlase) activity; Fischer G et al.; Legionella pneumophila is an intracellular parasite which is able to survive and multiply in human monocytes and alveolar macrophages . The Mip (macrophage infectivity potentiator) protein has been shown to be an essential virulence factor . A search of translated nucleic acid data bases has shown that the Mip protein from strain Wadsworth possesses regions homologous to those found in the FK506-binding proteins (FKBPs) of several different eukaryotic organisms . FKBPs are able to bind to the immunosuppressant macrolide FK506 and possess peptidyl-prolyl cis/trans isomerase (PPIase) activity . The gene coding for the Mip protein was cloned from the chromosome of L . pneumophila strain Philadelphia I and sequenced . It was synthesized in Escherichia coli K-12 and after purification it exhibited PPIase activity catalysing the slow cis/trans isomerization of prolyl peptide bonds in oligopeptides . Mip is inhibited by FK506 and fully resistant to cyclosporin A, as was also found for the recently characterized FKBP-type PPIases of eukaryotes . However, the N-terminal extension of Mip and/or the substitutions of the variable amino acids in the C-terminal FKBP core leads to variations, when compared with eukaryotic FKBPs, in substrate specificity with the oligopeptide substrates of type Suc-Ala-Xaa-Pro-Phe-4-nitroanilide . Nevertheless, the Legionella Mip factor represents a bacterial gene product which shares some characteristics normally found in eukaryotic proteins . In view of the activity of PPIases in protein-folding reactions, such prokaryotic FKBP analogues may represent a new class of bacterial pathogenicity factors.

Proc Natl Acad Sci U S A, 1992 May 1, 89(9), 3756 - 60
Phage display of ricin B chain and its single binding domains: system for screening galactose-binding mutants; Swimmer C et al.; We demonstrate that the B chain of ricin toxin preserves its lectin activity when expressed as a fusion protein on the surface of fd phage . Moreover, B chain, which folds into two topologically similar globular domains, can be dissected into amino-terminal and carboxyl-terminal domains to form single binding domains (SBDs) of B chain, each of which displays specificity for complex galactosides . The specific binding exhibited by the fusion protein of these SBDs was eliminated when amino acid substitutions Gly-46 in SBD1 or Gly-255 in SBD2 for native asparagine were introduced to alter key residues implicated in hydrogen bonding with substrate . These data demonstrate that it is possible to use a prokaryotic expression system to stably express and screen ricin B chain and its SBDs for sugar-binding mutants . Expression of ricin B chain on the surface of fd phage provides a method that can be used to efficiently select mutants with altered binding activities from a randomly generated library.

EMBO J, 1992 May, 11(5), 1785 - 96
Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo; Cardenas ME et al.; The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis . Phosphorylation has been shown to stimulate topoisomerase II activity in vitro . Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1 . Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II . Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant . The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II . This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.

J Bacteriol, 1992 May, 174(10), 3242 - 9
The lysP gene encodes the lysine-specific permease; Steffes C et al.; Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine . The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine . It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine) . Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA . A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport . This lysP mutation was mapped to about 46.5 min on the E . coli chromosome . The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene . The nucleotide sequence of the 2.7-kb BamHI fragment was determined . An open reading frame from nucleotides 522 to 1989 was observed . The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues . The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E . coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1) . Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels . These results demonstrate that the lysP gene encodes the lysine-specific permease.

J Bacteriol, 1992 May, 174(9), 3011 - 20
The lemA gene required for pathogenicity of Pseudomonas syringae pv . syringae on bean is a member of a family of two-component regulators; Hrabak EM et al.; The lemA gene of the plant pathogen Pseudomonas syringae pv . syringae is required for disease lesion formation on bean plants . Cosmid clones that complemented a lemA mutant in trans were isolated previously . The lemA gene was localized by subcloning and transposon mutagenesis . The lemA region and flanking DNA were sequenced, and an open reading frame of 2.7 kb was identified . The nucleotide and predicted amino acid sequences of the lemA gene showed sequence similarity to a family of prokaryotic two-component regulatory proteins . Unlike most of the previously described two-component systems, the lemA gene product contained homology to both components in one protein . Mutations introduced upstream and downstream of the lemA gene failed to locate a gene for a second protein component but identified the putative cysM gene of P . syringae pv . syringae . The cysM gene was located upstream of the lemA gene and was divergently transcribed . The lemA gene product was expressed at low levels in P . syringae pv . syringae and appeared to be positively auto-regulated.

J Biol Chem, 1992 Apr 25, 267(12), 8007 - 11
Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae; Chang YH et al.; A yeast gene for a methionine aminopeptidase, one of the central enzymes in protein synthesis, was cloned and sequenced . The DNA sequence encodes a precursor protein containing 387 amino acid residues . The mature protein, whose NH2-terminal sequence was confirmed by Edman degradation, consists of 377 amino acids . The function of the 10-residue sequence at the NH2 terminus, containing 1 serine and 6 threonine residues, remains to be established . In contrast to the structure of the prokaryotic enzyme, the yeast methionine aminopeptidase consists of two functional domains: a unique NH2-terminal domain containing two motifs resembling zinc fingers, which may allow the protein to interact with ribosomes, and a catalytic COOH-terminal domain resembling other prokaryotic methionine aminopeptidases . Furthermore, unlike the case for the prokaryotic gene, the deletion of the yeast MAP1 gene is not lethal, suggesting for the first time that alternative NH2-terminal processing pathway(s) exist for cleaving methionine from nascent polypeptide chains in eukaryotic cells.

Nature, 1992 Apr 23, 356(6371), 725 - 8
A eukaryotic DNA glycosylase/lyase recognizing ultraviolet light-induced pyrimidine dimers; Hamilton KK et al.; Cyclobutane pyrimidine dimers (CPDs) are the predominant product of photodamage in DNA after exposure of cells to ultraviolet light and are cytotoxic, mutagenic and carcinogenic in a variety of cellular and animal systems . In prokaryotes, enzymes and protein complexes have been characterized that remove or reverse CPDs in DNA . Micrococcus luteus and T4 phage-infected Escherichia coli contain a specific N-glycosylase/apurinic-apyrimidinic lyase that catalyses a two-step DNA incision process at sites of CPDs, thus initiating base excision repair of these lesions . It is well established that CPDs are recognized and removed from eukaryotic DNA by excision repair processes but very little information exists concerning the nature of the proteins involved in CPD recognition and DNA incision events . We report here that an enzyme functionally similar to the prokaryotic N-glycosylase/apurinic-apyrimidinic lyases exists in Saccharomyces cerevisiae . To our knowledge, this is the first time such an activity has been found in a eukaryote and is also the first example of an organism having both direct reversal and base excision repair pathways for the removal of CPDs from DNA.

J Mol Biol, 1992 Apr 20, 224(4), 1039 - 54
Mutational analysis of an inherently defective translation initiation site; Ivey-Hoyle M et al.; In a reverse of many studies of translational initiation sites, we have explored the basis for the inactivity of an apparently defective initiation site . Gene VII of the filamentous phage f1 has a translational start site with highly unusual functional properties and a sequence dissimilar to a prokaryotic ribosome binding site . The VII site shows no activity in assays of independent initiation, even in a deletion series designed to remove potentially interfering RNA secondary structure . Activity from the VII site is only observed if the site is coupled to a source of translation immediately upstream, but its efficiency is low at a one-nucleotide spacing from the stop codon of the upstream cistron and extremely sensitive to the distance between the stop codon and the gene VII AUG . These and other atypical characteristics of coupling distinguish the VII site from most coupled initiation sites . To identify the pattern of nucleotide substitutions that give the VII site the capacity for independent initiation, a series of designed and random point mutations were introduced in the sequence . Improving the Shine-Dalgarno complementarity from GG to GGAG or GGAGG made activity detectable, but at only low levels . Random substitutions, each increasing activity above background by a small increment, were found at 16 positions throughout the region of ribosome contact . These substitutions lengthened the Shine-Dalgarno complementarity or changed the G and C residues present in the wild-type site to A or T . Significant activity was not observed unless a strong Shine-Dalgarno sequence and a number of the up-mutations were present together . The nature and distribution of the substitutions and their agreement with the known preferences for nucleotides in initiation sites provide evidence that the VII site's major defect is its primary sequence overall . It appears to lack the specialized sequence required to bind free 30 S ribosomes, and thus depends on the translational coupling process to give it limited activity.

FEMS Microbiol Lett, 1992 Apr 15, 71(2), 175 - 80
Identification of putative multifunctional peptide synthetase genes using highly conserved oligonucleotide sequences derived from known synthetases; Borchert S et al.; Many peptide antibiotics in prokaryotes and lower eukaryotes are produced non-ribosomally by multi-enzyme complexes . Analysis of gene-derived amino acid sequences of some peptide synthetases of bacterial and fungal origins revealed a high degree of conservation (35-50% identity) . The genes encoding those peptide synthetases are clustered into large operons with repetitive domains (about 600 amino acids), in the case of synthetases activating more than one amino acid . We used two 35-mer oligonucleotides derived from two highly conserved regions of known peptide synthetases to identify the surfactin synthetase operon in Bacillus subtilis ATCC 21332, a strain not accessible to genetic manipulation . We show that the derived oligonucleotides can be used not only for the identification of unknown peptide synthetase genes by hybridization experiments but also in sequencing reactions as primers to identify internal domain sequences . Using this method, a 25.8-kb chromosomal DNA fragment bearing a part of the surfactin biosynthesis operon was cloned and partial sequences of two internal domains were obtained.

Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3536 - 40
RNA polymerase-associated transcription specificity factor encoded by vaccinia virus; Ahn BY et al.; Vaccinia virus encodes a multisubunit DNA-dependent RNA polymerase (EC 2.7.7.6) that is packaged in the infectious virus particle . This polymerase was found to contain a submolar polypeptide of approximately 85 kDa in addition to the core subunits, which consist of two larger and several smaller polypeptides . The polymerase containing the 85-kDa polypeptide was separated from the core polymerase by column chromatography . Although the core polymerase actively transcribed heterologous single-stranded DNA, only the form with the associated 85-kDa polypeptide could act in conjunction with an early stage-specific factor to transcribe double-stranded DNA containing a vaccinia virus early promoter . Peptide sequencing established that the RNA polymerase-associated 85-kDa protein was derived from the vaccinia virus H4L open reading frame, which encodes a 94-kDa polypeptide that we named RAP94 . RAP94 is not closely related to prokaryotic sigma 70 or eukaryotic RAP30 RNA polymerase-binding proteins, although there are short regions of sequence similarity . The specific association of RAP94 with viral RNA polymerase was corroborated with antibody raised to a recombinant fusion protein . Unlike the previously defined subunits of vaccinia virus RNA polymerase, RAP94 is synthesized exclusively late in infection, and synthesis could be prevented by a DNA replication inhibitor . The role of RAP94 in mediating specific transcription was demonstrated by using an extract from cells in which the H4L open reading frame had been transiently expressed.

Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3516 - 20
The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor; Liberek K et al.; The heat shock response and the heat shock proteins have been conserved across evolution . In Escherichia coli, the heat shock response is positively regulated by the sigma 32 transcriptional factor and negatively regulated by a subset of the heat shock proteins themselves . In an effort to understand the regulation of the heat shock response, we have purified the sigma 32 polypeptide to homogeneity . During the purification procedure, we found that a large fraction of the overexpressed sigma 32 polypeptide copurified with the universally conserved DnaK heat shock protein (the prokaryotic equivalent of the 70-kDa heat shock protein, HSP70) . Further experiments established that purified sigma 32 bound to DnaK and that this complex was disrupted in the presence of ATP . Consistent with the fact that dnaK756 mutant bacteria overexpress heat shock proteins at all temperatures, purified DnaK756 mutant protein did not appreciably bind to sigma 32.

J Biol Chem, 1992 Apr 15, 267(11), 7758 - 60
Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants; Habuka N et al.; Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity . In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP . When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP . Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect . In contrast, on the protein synthesis of E . coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect . These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E . coli ribosomes . These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity . When the R171K gene was expressed in E . coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected . This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.

Proc Natl Acad Sci U S A, 1992 Apr 15, 89(8), 3394 - 8
Identification of a mammalian 10-kDa heat shock protein, a mitochondrial chaperonin 10 homologue essential for assisted folding of trimeric ornithine transcarbamoylase in vitro; Hartman DJ et al.; We have identified a 10-kDa stress-inducible mitochondrial protein . The protein is synthesized at elevated rates in cultured rat hepatoma cells challenged with heat shock or amino acid analogues and, therefore, designated heat shock protein 10 (Hsp10) . Hsp10 was purified to homogeneity from rat liver and found to exhibit a native molecular mass of 65 kDa, as opposed to a monomeric molecular mass of 10,813.4 +/- 0.41 Da . The amino acid sequence of rat Hsp10 disclosed extensive sequence similarity with bacterial chaperonin (Cpn) 10 . Rat Hsp10 and Escherichia coli Cpn60 were used to reconstitute functional trimeric rat ornithine transcarbamoylase from a chemically denatured state with high efficiency . This process depended completely upon rat Hsp10 and was abolished in the presence of a nonhydrolyzable ATP analogue . We conclude that Hsp10 is a eukaryotic Cpn10 homologue and, therefore, together with Cpn60 essential for mitochondrial protein biogenesis . The Cpn-mediated protein-folding apparatus, thus, exhibits a high degree of conservation between prokaryotes and mitochondria of higher eukaryotes.

Nucleic Acids Res, 1992 Apr 11, 20(7), 1785 - 91
Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation; Fieck A et al.; Eukaryotic expression vectors designed to produce E . coli Lac repressor protein targeted to the nucleus of mammalian cells were constructed . These constructions carry the lac repressor gene (lacI) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a . When the NLS's were fused to the lacI gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified LacI protein . Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity . When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the LacI protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected . The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacI behind the nine base pair linker, AGC AGC CTG (ser-ser-leu) . This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction . The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-terminus of the protein . These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein . This modified lacI gene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.

Biochemistry, 1992 Apr 7, 31(13), 3507 - 13
Purification and characterization of delta helicase from fetal calf thymus; Li X et al.; A DNA helicase (delta helicase) which partially copurifies with DNA polymerase delta has been highly purified from fetal calf thymus . delta helicase differs in physical and enzymatic properties from other eukaryotic DNA helicases described thus far . The enzyme has an apparent mass of 57 kDa by gel filtration and is associated with polypeptides of 56 and 52 kDa by SDS-polyacrylamide gel electrophoresis . Photo-cross-linking of the purified enzyme with {alpha-32P}ATP resulted in labeling of a polypeptide of approximately 58 kDa, suggesting that the active site is present on the larger polypeptide . Unwinding of a partial duplex requires a nucleoside triphosphate which can be either ATP or dATP but not a nonhydrolyzable analogue of ATP . Other ribo- and deoxyribonucleoside triphosphates have little or no activity as cofactors . delta helicase also has DNA-dependent ATPase activity which has a relatively low Km for ATP (40 microM) . delta helicase binds to single-stranded DNA but has little or no affinity for double-stranded DNA or single-stranded RNA . Similar to replicative DNA helicases from prokaryotes and the herpes simplex virus type 1 helicase-primase, delta helicase translocates in the 5'-3' direction along the strand to which it is bound and preferentially unwinds DNA substrates with a forklike structure.

J Biol Chem, 1992 Apr 5, 267(10), 6801 - 6
Site-directed mutagenesis of a conserved domain in vaccinia virus thymidine kinase . Evidence for a potential role in magnesium binding; Black ME et al.; Alignment of prokaryotic and vertebrate type II thymidine kinases (TK) (EC 2.7.1.21), such as that encoded by vaccinia virus (VVTK), reveals three conserved regions: designated domains I, III, and VII . Domains I and III of VVTK contain residues which closely resemble segments A (ATP) and B (Mg2+), respectively, of a Mg.ATP binding descriptor proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J . (1982) EMBO J . 1, 945-951) . In support of this hypothesis, domain I of the VVTK enzyme has previously been identified as the ATP binding site (Black and Hruby, 1990b) . With regard to Mg2+ binding, several features of the VVTK domain III suggest that it may be responsible for this activity: 1) sequence similarity to a magnesium binding motif proposed previously (Walker, J.E., Saraster, M., Runswick, M-J., and Gay, N.J . (1982) EMBO J . 1, 945-951); 2) alignment of the predicted secondary structure of type II TK enzymes with other magnesium-binding enzymes such as adenylate kinase, EF-TU, and p21 reveals a conserved aspartic acid residue preceded by several hydrophobic residues with domain III; and 3) the conserved VVTK domain III aspartic acid residue (D82) aligns with D93 residue of adenylate kinase which is has been shown by NMR to participate in Mg2+ binding (Yan, H., and Tsai, M.-D., Biochemistry, in press) . To directly examine the potential contribution of the conserved domain III D82 residue of VVTK in magnesium binding, site-directed mutagenesis was performed on positions D82 and G84 to generate four mutants, N82, L82, I82, and V84 . Each mutant was analyzed for enzyme activity, divalent cation requirements, tetramer formation, and ATP binding ability . The results obtained were consistent with D82 playing a direct role in Mg2+ binding and suggest that while the aspartic acid does not appear to participate directly with ATP binding it may instead act to facilitate ATP hydrolysis by binding Mg2+ which aids to correctly position ATP for nucleophilic attack.

J Biol Chem, 1992 Apr 5, 267(10), 6455 - 8
Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product; Hartman J et al.; The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product . We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508) . Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP . Irreversible NBD-1 labeling by an ATP analog was demonstrated using {32P}8-azido-ATP . This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain . These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide . Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

New Biol, 1992 Apr, 4(4), 389 - 95
The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif; Doniger J et al.; We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins . Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived . This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins . Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units.

New Biol, 1992 Apr, 4(4), 290 - 8
The Y-box factors: a family of nucleic acid binding proteins conserved from Escherichia coli to man; Wolffe AP et al.; The Y-box factors interact specifically with both DNA and RNA . Biologically they have roles in both transcriptional and translational regulation . Conserved through evolution from prokaryotic to eukaryotic organisms they represent a new family of nucleic acid binding proteins.

Brain Res Mol Brain Res, 1992 Apr, 13(3), 179 - 87
Cu/Zn superoxide dismutase mRNA and enzyme activity, and susceptibility to lipid peroxidation, increases with aging in murine brains; de Haan JB et al.; To protect against reactive oxygen species, prokaryotic and eukaryotic cells have developed an antioxidant defence mechanism where O2- is converted to H2O2 by superoxide dismutase (Sod), and in a second step, H2O2 is converted to H2O by catalase (Cat) and/or glutathione peroxidase (Gpx) . If Sod levels are increased without a concomitant Gpx increase, then the intermediate H2O2 accumulates . This intermediate could undergo the Fenton's reaction, generating hydroxyl radicals which may lead to lipid peroxidation in cells . In this study, we investigate the expression of Sod1, Gpx1 and susceptibility to lipid peroxidation during the aging process in mouse brains . We demonstrate that the mRNA levels and enzyme activity of Sod1 are higher in brains from adult mice compared to neonatal mice . Furthermore, we show that a linear increase in Sod1 mRNA and enzyme activity occurs with aging (1-100 weeks) . On the contrary, we find that the mRNA and enzyme activity for Gpx1 does not increase with aging in mouse brains . In addition, our results demonstrate that the susceptibility of murine brains to lipid peroxidation increases with aging . The data in this study are consistent with the notion that reactive oxygen species may contribute to the aging process in mammalian brains . These results are discussed in relation to the normal aging process in mammals, and to the premature aging and mental retardation in Down syndrome.

Gene, 1992 Apr 1, 113(1), 55 - 65
Codon usage in the G+C-rich Streptomyces genome; Wright F et al.; The codon usage (CU) patterns of 64 genes from the Gram+ prokaryotic genus Streptomyces were analysed . Despite the extremely high overall G+C content of the Streptomyces genome (estimated at 0.74), individual genes varied in G+C content from 0.610 to 0.797, and had third codon position G+C contents (GC3s) that varied from 0.764 to 0.983 . The variation in GC3s explains a significant proportion of the variation in CU patterns . This is consistent with an evolutionary model of the Streptomyces genome where biased mutation pressure has led to a high average G+C content with random variation about the mean, although the variation observed is greater than that expected from a simple binomial model . The only gene in the sample that can be confidently predicted to be highly expressed, EF-Tu of Streptomyces coelicolor A3(2) (GC3s = 0.927), shows a preference for a third position C in several of the four codon families, and for CGY and GGY for Arg and Gly codons, respectively (Y = pyrimidine); similar CU patterns are found in highly expressed genes of the G+C-rich Micrococcus luteus genome . It thus appears that codon usage in Streptomyces is determined predominantly by mutation bias, with weak translational selection operating only in highly expressed genes . We discuss the possible consequences of the extreme codon bias of Streptomyces and consider how it may have evolved . A set of CU tables is provided for use with computer programs that locate protein-coding regions.

EMBO J, 1992 Apr, 11(4), 1487 - 92
RNA polymerase III catalysed transcription can be regulated in Saccharomyces cerevisiae by the bacterial tetracycline repressor-operator system; Dingermann T et al.; We have investigated whether the RNA polymerase III-driven transcription of eukaryotic tRNA genes can be regulated by the prokaryotic tetracycline operator-repressor system . The bacterial tet operator (tetO) was inserted at two different positions (-7 and -46) upstream of a tRNA(Glu) (amber) suppressor gene . Both constructs are transcribed in Saccharomyces cerevisiae and yield functional tRNAs as scored by suppression of an amber nonsense mutation in the met8-1 allele . Controlled expression of Tet repressor was achieved by fusing the bacterial tetR gene to the yeast gal1 promoter . This leads to expression of Tet repressor in yeast on galactose--but not on glucose--containing media . Regulation of the su-tRNA gene with the tetO fragment inserted at position -7 has been demonstrated . Under conditions which allow tetR expression, cells exhibit a met- phenotype . This methionine auxotrophy can be conditionally reverted to prototrophy by adding tetracycline . However, a su-tRNA gene with the tetO fragment inserted at position -46 cannot be repressed . Our results demonstrate clearly that the bacterial repressor protein binds to its operator in the yeast genome . Formation of this complex in the vicinity of the pol III transcription initiation site reduces the level of su-tRNA at least 50-fold as concluded from quantitative primer extension analyses . This indicates for the first time that class III gene expression can be regulated by a DNA binding protein with its target site in the 5'-flanking region and that a prokaryotic repressor can confer regulation of a suitably engineered tRNA gene.

Mol Cell Biol, 1992 Apr, 12(4), 1680 - 6
Fine-structure map of the human ribosomal protein gene RPS14; Diaz JJ et al.; We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J . Diaz, D . D . Rhoads, and D . J . Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14 . Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated . Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells . The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci . As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains . Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity . In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes . Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.

J Virol, 1992 Apr, 66(4), 2051 - 6
Activation of the human immunodeficiency virus type 1 long terminal repeat by vaccinia virus; Stellrecht KA et al.; A variety of DNA viruses are known to activate gene expression directed by the long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) . In light of the proposed use of recombinant vaccinia virus for HIV-1 vaccines, evaluation of the role of vaccinia virus in HIV-1 activation is warranted . To investigate whether vaccinia virus induces HIV LTR-directed gene expression, transient expression assays in Jurkat cells persistently infected with vaccinia virus (Jvac) using plasmid DNA containing the LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were performed . CAT activity in Jvac cells was always recorded, although the level appears to fluctuate independently of virus titers . Dual intracytoplasmic staining and fluorescence-activated cell sorter analysis showed that CAT activity was expressed in the infected cells . CAT expression was not due to plasmid replication, since plasmid DNA extracted from Jvac cells 48 h after transfection was restricted only by enzymes which recognize methylated sequences, indicating a prokaryotic source for the DNA . These findings suggest that a factor(s) present in vaccinia virus-infected cells is capable of activating the LTR of HIV-1.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2742 - 6
Sequence of Prochloron didemni atpBE and the inference of chloroplast origins; Lockhart PJ et al.; The prochlorophytes, oxygenic photosynthetic prokaryotes containing chlorophylls a and b, have been put forward as descended from the organisms that gave rise to chloroplasts of green plants and algae by endosymbiosis, although this has always been controversial . To assess the phylogenetic position of the prochlorophyte Prochloron didemni, we have cloned and sequenced its atpBE genes . Phylogenetic inference under a range of models gives moderate to strong support for a cyanobacterial grouping rather than a chloroplast one . Possible systematic errors in this and previous analyses of prochlorophyte sequences are discussed.

Curr Genet, 1992 Apr, 21(4-5), 409 - 16
The nucleotide sequence of the small subunit ribosomal RNA gene from Symbiodinium pilosum, a symbiotic dinoflagellate; Sadler LA et al.; The complete sequence of the small subunit ribosomal RNA (SSU rRNA) gene was determined for the symbiotic dinoflagellate Symbiodinium pilosum . This sequence was compared with sequences from two other dinoflagellates (Prorocentrum micans and Crypthecodinium cohnii), five Apicomplexa, five Ciliata, five other eukaryotes and one archaebacterium . The corresponding structurally conserved regions of the molecule were used to determine which portions of the sequences could be unambiguously aligned . Phylogenetic relationships were inferred from an analysis of distance matrices, where pair-wise distances were determined using a maximum likelihood model for transition and transversion ratios, and from maximum parsimony analysis, with bootstrap resampling . By either analytical approach, the dinoflagellates appear distantly related to prokaryotes, and are most closely related to two of the Apicomplexa, Sarcocystis muris and Theileria annulata . Among the dinoflagellates, C . cohnii was found to be more closely affiliated with the Apicomplexa than either P . micans or S . pilosum.

Hum Antibodies Hybridomas, 1992 Apr, 3(2), 81 - 5
Bacteriophage cloning and Escherichia coli expression of a human IgM Fab; Hay BN et al.; We have combined the molecular biology methods of the polymerase chain reaction and recombinant DNA cloning in bacteriophage lambda to express a human IgM Fab in Escherichia coli using genes derived from an Epstein-Barr virus transformed cell line . This method comprises three cDNA amplifications and a single cloning step, culminating in the stable overexpression of mammalian heterodimeric recombinant protein in a prokaryotic host.

Int J Syst Bacteriol, 1992 Apr, 42(2), 226 - 33
Phylogenetic relationships between the western aster yellows mycoplasmalike organism and other prokaryotes established by 16S rRNA gene sequence; Kuske CR et al.; Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene . Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced . The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli . Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes . We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes . Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.

Proc Natl Acad Sci U S A, 1992 Apr 1, 89(7), 2605 - 9
A uridine-rich sequence required for translation of prokaryotic mRNA; Zhang J et al.; Binding of 30S ribosomal subunits to mRNA during the initiation of prokaryotic translation is known to be influenced by the initiation codon and the Shine-Dalgarno sequence . Site-directed mutagenesis of rnd, the Escherichia coli gene encoding RNase D, has now shown that a U8 sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA . Alteration of two to five uridine residues within this sequence has no effect on mRNA levels but decreases RNase D protein and activity by as much as 95%, indicating that the U-rich sequence acts as an enhancer of translation . Moreover, mutant transcripts bind to 30S ribosomes in vitro with lower affinity than their wild-type counterparts, suggesting that the role of the U8 sequence is in the initial binding of ribosomes to the translation initiation region of the message . These data demonstrate that sequences other than those previously recognized can be essential for translation initiation.

Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Apr, 14(2), 79 - 84
{PCR amplification, cloning and expression of the gene coding for CD4 V1-V2 domains}; Li J; We describe the use of a modified technique of polymerase chain reaction (PCR) for facilitating cloning and expression of a cDNA fragment encoding CD4 V1-V2 domains . The modification includes introducing suitable signals to start primer and halt primer of the target gene as indicated in Fig . 1 . After verified by DNA sequencing, the amplified DNA fragment was cloned into prokaryotic expression vectors containing lambda PL promoter . The screened clones were induced to express the target fragment V1-V2 of CD4 gene and the expected gene product was estimated to be 8% or 10% of the total cellular proteins.

J Bacteriol, 1992 Apr, 174(7), 2338 - 43
Spectroscopic and genetic evidence for two heme-Cu-containing oxidases in Rhodobacter sphaeroides; Shapleigh JP et al.; It has recently become evident that many bacterial respiratory oxidases are members of a superfamily that is related to the eukaryotic cytochrome c oxidase . These oxidases catalyze the reduction of oxygen to water at a heme-copper binuclear center . Fourier transform infrared (FTIR) spectroscopy has been used to examine the heme-copper-containing respiratory oxidases of Rhodobacter sphaeroides Ga . This technique monitors the stretching frequency of CO bound at the oxygen binding site and can be used to characterize the oxidases in situ with membrane preparations . Oxidases that have a heme-copper binuclear center are recognizable by FTIR spectroscopy because the bound CO moves from the heme iron to the nearby copper upon photolysis at low temperature, where it exhibits a diagnostic spectrum . The FTIR spectra indicate that the binuclear center of the R . sphaeroides aa3-type cytochrome c oxidase is remarkably similar to that of the bovine mitochondrial oxidase . Upon deletion of the ctaD gene, encoding subunit I of the aa3-type oxidase, substantial cytochrome c oxidase remains in the membranes of aerobically grown R . sphaeroides . This correlates with a second wild-type R . sphaeroides is grown photosynthetically, the chromatophore membranes lack the aa3-type oxidase but have this second heme-copper oxidase . Subunit I of the heme-copper oxidase superfamily contains the binuclear center . Amino acid sequence alignments show that this subunit is structurally very highly conserved among both eukaryotic and prokaryotic species . The polymerase chain reaction was used to show that the chromosome of R . sphaeroides contains at least one other gene that is a homolog of ctaD, the gene encoding subunit I of the aa3-type cytochrome c oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature, 1992 Mar 26, 356(6367), 301 - 9
Oxygen activation and the conservation of energy in cell respiration; Babcock GT et al.; Many of the membrane-associated oxidases that catalyse respiratory reduction of O2 to water simultaneously couple this exergonic reaction to the translocation of protons across the inner mitochondrial membrane, or the cell membrane in prokaryotes, a process by which metabolic energy is conserved for subsequent synthesis of ATP . The molecular mechanism of O2 reduction and its linkage to H+ translocation are now emerging . The bimetallic haem iron-copper reaction centre in this family of enzymes is the critical structure for catalysis of both these processes.

J Biol Chem, 1992 Mar 25, 267(9), 5901 - 9
Examination of the functional roles of 5 highly conserved residues in the cytochrome b subunit of the bc1 complex of Rhodobacter sphaeroides; Yun CH et al.; The cytochrome b subunit of the bc1 complexes contains two cytochrome components (bL and bH) and is the locus of both a quinol-oxidizing site (Qo or Qz) and a quinone-reducing site (Qi or Qc) . Sequence alignments of this subunit from over 20 eukaryotic and prokaryotic species have revealed a remarkable degree of conservation, including approximately 20 totally conserved residues . In this paper, site-directed mutagenesis has been used to examine the structural or functional roles of 5 of these highly conserved residues, Gly48, Gln58, Ser102, Phe104, and Pro202, all predicted to be within transmembrane alpha-helical segments . The mutants were made in the bc1 complex of Rhodobacter sphaeroides, a photosynthetic bacterium . The ability to use spectroscopic, electrochemical, and flash-induced kinetic methods allows the mutants to be analyzed for influences both on cytochrome spectra and thermodynamic properties and on the kinetics of specific electron transfer reactions . The results show that none of the 5 residues is absolutely essential . Substitution of aspartate or valine for Gly48 results in the loss of photosynthetic growth . The G48V mutant assembles a bc1 complex, but with modified cytochromes bH and bL, and a dysfunctional quinone reductase (Qc) site; an alanine is tolerated at this position . Possibly, a small residue is important here for heme packing . Gln58 and Ser102 are the only highly conserved polar residues predicted to be within the transmembrane spans, apart from the histidines which are heme axial ligands . Neither Gln58 nor Ser102 is essential for assembly or function of the bc1 complex, although substitution of other amino acids in these positions does cause subtle, but measurable changes . Phe104 lies midway between the axial ligands to cytochromes bL and bH and can be modeled to project in the space separating the two hemes . Replacement of this highly conserved aromatic residue by isoleucine has no measurable influence on the rate of electron transfer through the cytochrome b chain containing the two hemes . Finally, Pro202 is a totally conserved proline which is in the middle of transmembrane helix D, in between the 2 histidines which provide ligands to the hemes . No major inhibition of electron transfer resulted from replacing this proline by a leucine, although subtle changes in spectra of the b cytochromes and their electrochemical properties were noted.

FEBS Lett, 1992 Mar 24, 299(1), 44 - 7
Prokaryotic calcium-binding protein of the calmodulin superfamily . Calcium binding to a Saccharopolyspora erythraea 20 kDa protein; Bylsma N et al.; The EF-hand calcium-binding protein from Saccharopolyspora erythraea has been shown, using 113Cd NMR, to possess three Cd(2+)-ion binding sites . This indicates that of the four EF-hand motifs in the molecule, one (probably site 2) is unable to bind Cd(2+)-ions . Data from the titration of the protein with Ca2+, in the presence of Quin2, were fitted to a curve calculated on the assumption that the protein contains three high affinity Ca2+ binding sites, two of which (pK1 = 8.0, pK2 = 9.0) are strongly cooperative, and one single site (pK3 = 7.5) . Preliminary 1H NMR experiments indicate marked structural changes upon Ca(2+)-binding.

Biochim Biophys Acta, 1992 Mar 23, 1105(1), 131 - 40
The enzymatic synthesis of membrane glucolipids in Acholeplasma laidlawii; Dahlqvist A et al.; In membranes of the prokaryote Acholeplasma laidlawii, the physiological regulation of the two major membrane lipids, monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), is governed by factors affecting the equilibria between lamellar and non-lamellar phases of the membrane lipids . The synthesis of the glucolipids is considered to be a two-step glucosylation: (i) DAG+UDP-Glc----MGlcDAG+UDP; and (ii) MGlcDAG+UDP-Glc----DGlcDAG+UPD . This was corroborated by in vivo pulse labelling experiments showing turnover of MGlcDAG but not DGlcDAG . The enzymatic synthesis of MGlcDAG was localized to fresh or freeze-dried membranes in vitro . Synthesis of DGlcDAG was minor in such membranes but of substantial magnitude in intact cells . Synthesis of MGlcDAG was stimulated by small amounts of SDS but completely inhibited upon solubilization of the membranes by a variety of detergents . The inhibitory effect of several UDP-Glc analogs on glucolipid synthesis demonstrated the importance of UDP-Glc as the sugar donor . Synthesis of both glucolipids was lost in freeze-dried plus lipid-extracted cells but restored when lipids were transferred back to the extracted cell membrane . By selectively adding specific lipids, a strong dependence on the acceptor lipid DAG, as well as the need for general matrix lipids for enzyme activity, was established . In addition, the anionic phosphatidylglycerol (PG), but not the other phospholipids, had a strong stimulatory effect . The presence of different phosphorylating agents stimulated the synthesis of DGlcDAG and partially inhibited that of MGlcDAG . This, together with the lipid dependency, may constitute mechanisms for the regulation of the enzyme activities in vivo.

Proc Natl Acad Sci U S A, 1992 Mar 15, 89(6), 2125 - 9
A developmentally regulated chlamydial gene with apparent homology to eukaryotic histone H1; Perara E et al.; We have developed a method for the isolation of genes whose expression is developmentally regulated from the murine strain of Chlamydia trachomatis . Here we describe the identification of two developmental stage-specific genes, one of which is predicted to encode a 26-kDa lysine- and alanine-rich protein that appears to be homologous to several eukaryotic histone H1 proteins . A substantial proportion of this homology relates to its distinctive amino acid composition . No sequence homology was observed between this protein and other bacterial "histone-like" chromosomal proteins, but homology does exist with two other recently described prokaryotic proteins . The protein is expressed late in chlamydial development, during the transition from reticulate bodies to elementary bodies . The basic nature of the protein predicts that it could bind DNA, and Southwestern blotting experiments confirm this finding . These properties are consistent with a role either in the regulation of late gene expression or in the compaction of the chlamydial genome.

J Biol Chem, 1992 Mar 15, 267(8), 5380 - 7
Molecular cloning, sequence analysis, and in vitro expression of flavanone 3 beta-hydroxylase from Petunia hybrida; Britsch L et al.; A cDNA encoding flavanone 3 beta-hydroxylase was isolated from petals of Petunia hybrida . The open reading frame of the nearly full length cDNA coded for a 369-amino acid polypeptide with a calculated Mr of 41,466 . The function of this nucleotide sequence was verified by comparison with amino acid sequence of the amino terminus and tryptic peptides from purified plant enzyme, by Northern blotting with RNA from wild type and mutant plants, and by prokaryotic expression yielding an enzymatically active hydroxylase . Computer-aided sequence analysis revealed high similarity (73.5%) to flavanone 3 beta-hydroxylase from barley . Genomic Southern blot analysis showed the presence of only one gene for flavanone 3 beta-hydroxylase in P . hybrida.

J Biol Chem, 1992 Mar 15, 267(8), 5036 - 9
A tomato gene expressed during fruit ripening encodes an enzyme of the carotenoid biosynthesis pathway; Bartley GE et al.; In the initial stages of carotenoid biosynthesis in plants the enzyme phytoene synthase converts two molecules of geranylgeranyl diphosphate into phytoene, the first carotenoid of the pathway . We show here that a tomato (Lycopersicon esculentum) cDNA for a gene (Psy1) expressed during fruit ripening directs the in vitro synthesis of a 47-kDa protein which, upon import into isolated chloroplasts, is processed to a mature 42-kDa form . The imported protein is largely associated with membranes, but it can be easily solubilized by dilution or by treatment at high pH . A plasmid construct containing prokaryotic promoter and ribosome-binding sequences fused to the Psy1 cDNA complements the carotenoidless phenotype of a Rhodobacter capsulatus crtB mutant . We conclude that Psy1 encodes phytoene synthase and that this enzyme is a peripheral plastid membrane protein.

J Biol Chem, 1992 Mar 15, 267(8), 5460 - 6
Overexpression of higher eukaryotic membrane proteins in bacteria . Novel insights obtained with the liver mitochondrial proton/phosphate symporter; Ferreira GC et al.; In order to better understand why higher eukaryotic membrane proteins, in contrast to soluble proteins, are not readily expressed in Escherichia coli, the gene encoding the liver mitochondrial phosphate transporter (H+/Pi symporter) (Ferreira, G . C., Pratt, R . D., and Pedersen, P . L . (1989) J . Biol . Chem . 264, 15628-15633), was subcloned into a plasmid (pFOG402) containing the alkaline phosphatase promoter and leader sequence . Although this system is highly efficient in overexpressing soluble mitochondrial proteins in E . coli, e.g . alpha and beta subunits of the liver ATP synthase, it fails to express the H+/Pi transporter . Expression is not obtained by truncation of the transporter gene from either the 3' or 5' end, by fusing the mature transporter gene to genes encoding either the alpha or beta ATP synthase subunits, or by using different expression plasmids . Significantly, the H+/Pi transporter is overexpressed in E . coli provided its cDNA is first truncated at the 3' end (carboxyl-terminal end) and fused to a cDNA fragment derived from the ATP synthase alpha subunit gene . In fact, progressive deletions from the 3' end of the transporter cDNA produce a ladder of increasingly overexpressed fusion proteins which account from the largest to the smallest for approximately 2.5-14% of the total bacterial cell protein . The minimal truncation necessary from the 3' end is 192 base pairs corresponding to 64 COOH-terminal amino acids . This corresponds to 20% of the transporter and involves removal of one of the six predicted membrane-spanning segments . In a variety of additional experiments designed to define the molecular basis for E . coli's inability to express the complete liver H+/Pi transporter, problems related to cell toxicity and transcription were ruled out . However, in vitro transcription-translation assays revealed that the complete transporter is readily expressed when eukaryotic, but not prokaryotic, ribosomes are present . Significantly, the fused transporter gene (i.e . Pi transporter cDNA truncated at the 3' end + ATP synthase alpha subunit cDNA) is expressed when prokaryotic ribosomes are present . These results support the view that the difficulty in expressing higher eukaryotic membrane proteins in bacteria may be related in some cases to a problem at the level of translation.

Gene, 1992 Mar 15, 112(2), 257 - 60
Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay; Sorensen MS et al.; Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells . We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene . The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Nippon Rinsho, 1992 Mar, 50(3), 444 - 9
{Possible roles of infections and heat shock proteins in rheumatoid arthritis}; Minota S; Heat shock protein, one of the most conserved proteins from prokaryotes to man, was shown to be strongly immunogenic in bacterial and helminthic infections . Evidences have accumulated suggesting that mycobacterial hsp65 plays a crucial role in the development of adjuvant arthritis induced in rats . By analogy, the pathogenetic roles of hsp were investigated in rheumatoid arthritis in man . The evidences obtained so far are highly suggestive but still circumstantial . Hsp's strong immunogenicity and high conservation, which seem to be mutually exclusive, make this molecule very mysterious . In this regard, Coutinho's new network theory or Cohen and Young's theory of immunological homunculus nicely reconciles these aspects.

Microbiol Rev, 1992 Mar, 56(1), 123 - 36
DNA looping; Matthews KS; DNA-looping mechanisms are part of networks that regulate all aspects of DNA metabolism, including transcription, replication, and recombination . DNA looping is involved in regulation of transcriptional initiation in prokaryotic operons, including ara, gal, lac, and deo, and in phage systems . Similarly, in eukaryotic organisms, the effects of enhancers appear to be mediated at least in part by loop formation, and examples of DNA looping by hormone receptor proteins and developmental regulatory proteins have been found . In addition, instances of looped structures have been found in replication and in recombination in both prokaryotes and eukaryotes . DNA loop formation may have different functions in different cellular contexts; in some cases, the loop itself is requisite for regulation, while in others the increase in the effective local concentration of protein may account for the effects observed . The ability of DNA to form loops is affected by the distance between binding sites; by the DNA sequence, which determines deformability and bendability; and by the presence of other proteins that exert an influence on the conformation of a particular sequence . Alteration of the stability of DNA loops and/or protein-DNA binding by extra- or intracellular signals provides responsivity to changing metabolic or environmental conditions . The fundamental property of site-specific protein binding to DNA can be combined with protein-protein and protein-ligand interaction to generate a broad range of physiological states.

Gene, 1992 Mar 1, 112(1), 139 - 44
Evidence favouring the hypothesis of a conserved 3'-5' exonuclease active site in DNA-dependent DNA polymerases; Blanco L et al.; The complete amino acid (aa) alignment of the N-terminal domain of 33 DNA-dependent DNA polymerases encompassing the putative segments Exo I, Exo II and Exo III, proposed by Bernad et al . {Cell 59 (1989) 219-228} to form a conserved 3'-5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases, allowed us to identify and/or correct some of the most conserved segments (Exo I, II and III) in certain DNA polymerases . In particular, the aa region of T4 DNA polymerase and other eukaryotic (viral and cellular) DNA polymerases previously proposed as Exo I segment 1, did not align with the Exo I segment of Escherichia coli DNA polymerase I (PolI)-like and protein-primed DNA polymerases; instead, a new conserved region of aa similarity was identified in T4 DNA polymerase and eukaryotic (viral and cellular) DNA polymerases as their corresponding Exo I segment . Therefore, according to our alignment, the recently reported T4 DNA polymerase site-directed mutants, D189A and E191A {Reha-Krantz et al., Proc . Natl . Acad . Sci . USA 88 (1991) 2417-2421}, do not correspond to what we now consider the critical Exo I motif of PolI . As discussed in this communication, the functional importance of conserved segments Exo I, Exo II and Exo III is supported by site-directed mutagenesis in PolI, and in phi 29, T7 and delta(Sc) DNA polymerases . Furthermore, genetically selected T4 DNA polymerase mutator mutants form two main clusters, centered in the conserved segment Exo III and in the newly identified Exo I segment.

Gene, 1992 Mar 1, 112(1), 133 - 7
Are there highly conserved DNA polymerase 3'----5' exonuclease motifs?
Reha-Krantz LJ.
It was proposed by Bernad et al . {Cell 59 (1989) 219-228} and Blanco et al . {Gene 100 (1991) 27-38} that the 3'----5' exonuclease (Exo) domain of Escherichia coli DNA polymerase I (PolI) is structurally and functionally conserved among prokaryotic and eukaryotic DNA polymerases . The basis for this claim is the presence of three short peptide sequences in many DNA polymerases that resemble PolI sequences that have been shown by x-ray crystallographic and genetic engineering studies to be metal ion binding sites that are essential for PolI 3'----5' Exo activity {Derbyshire et al., Science 240 (1988) 199-201} . This claim is made even though there is little amino acid (aa) sequence similarity between PolI and many eukaryotic and viral DNA polymerases and in spite of significant differences in the amount of 3'----5' Exo activity in the DNA polymerases compared . For at least one DNA polymerase, bacteriophage T4 DNA polymerase, one of the proposed conserved Exo sequences does not appear to be important for 3'----5' Exo activity . This T4 DNA polymerase result provides a reminder that caution must be used when weak aa sequence similarities are used to predict protein structure and function.

Trends Biochem Sci, 1992 Mar, 17(3), 110 - 3
Fructose-bisphosphate aldolases: an evolutionary history; Marsh JJ et al.; Two mechanistically distinct forms of fructose-bisphosphate aldolase are known to exist . It has been assumed that the Class II (metallo) aldolases are evolutionary more primitive than their Class I (Schiff-base) analogs since the latter had only been found in eukaryotes . With the identification of prokaryotic Class I aldolases, we present here an alternative scheme of aldolase evolution . This scheme proposes that both aldolase classes are evolutionarily ancient and rationalizes the observed highly variable expression of both enzyme types in contemporary file forms.

Bioessays, 1992 Mar, 14(3), 145 - 50
The homeodomain: a new face for the helix-turn-helix?
Treisman J, Harris E, Wilson D, Desplan C.
The discovery of conserved protein domains found in many Drosophila and mammalian developmental gene products suggests that fundamental developmental processes are conserved throughout evolution . Our understanding of development has been enhanced by the discovery of the widespread role of the homeodomain (HD) . The action of HD-containing proteins as transcriptional regulators is mediated through a helix-turn-helix motif which confers sequence specific DNA binding . Unexpectedly, the well conserved structural homology between the HD and the prokaryotic helix-turn-helix proteins contrasts with their divergent types of physical interaction with DNA . A C-terminal extension of the HD recognition helix has assumed the role that the N-terminus of the prokaryotic helix plays for specification of DNA binding preference . However, the HD appears also capable of recognizing DNA in an alternative way and its specificity in vivo may be modified by regions outside the helix-turn-helix motif . We propose that this intrinsic complexity of the HD, as well as its frequent association with other DNA binding domains, explains the functional specificity achieved by genes encoding highly related HDs.

Plant Mol Biol, 1992 Mar, 18(5), 873 - 85
cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock; Prasad TK et al.; Mitochondria contain a nuclear-encoded heat shock protein, HSP60, which functions as a chaperonin in the post-translational assembly of multimeric proteins encoded by both nuclear and mitochondrial genes . We have isolated and sequenced full-length complementary DNAs coding for this mitochondrial chaperonin in Arabidopsis thaliana and Zea mays . Southern-blot analysis indicates the presence of a single hsp60 gene in the genome of A . thaliana . There is a high degree of homology at the predicted amino acid levels (43 to 60%) between plant HSP60s and their homologues in prokaryotes and other eukaryotes which indicates that these proteins must have similar evolutionarily conserved functions in all organisms . Northern- and western-blot analyses indicate that the expression of the hsp60 gene is developmentally regulated during seed germination . It is also heat-inducible . Developmental regulation of the (beta-subunit of F1-ATPase, an enzyme complex that is involved in the cyanide-sensitive mitochondrial electron transport system, indicates that imbibed embryos undergo rapid mitochondrial biogenesis through the early stages of germination . Based on the functional role of HSP60 in macromolecular assembly, these data collectively suggest that the presence of higher levels of HSP60 is necessary during active mitochondrial biogenesis, when the need for this protein is greatest in assisting the rapid assembly of the oligomeric protein structures.

Mol Biol (Mosk), 1992 Mar-Apr, 26(2), 244 - 63
{Enzymatic methylation of regulatory elements in controlling the activity of genes from various groups of organisms}; Mazin AL; About 1800 sequences of gene promoters, enhancers and other types of regulatory elements (REG) have been statistically analysed for investigation of a role for enzymatic DNA methylation in prokaryotes, yeasts, plants, invertebrates, animal viruses, vertebrates and human . The frequencies and localizations of CG and CNG methylated sites and also the number of CG-->TG+CA transitions in different series of REGs have been studied . It was showed that the pro- and eukaryotic REGs with the exception of yeast and drosophila ones have higher CpG-suppression values than the main genome in the same species . About 40% of all the point substitutions in pro- and eukaryotic REGs were found in the CG and CNG methylated sites, that are "hot spots" for C-->T transitions . More than 30% of all analysed REGs have neither sites CG nor CNG and so they are not capable of methylation in vivo . The methylated sites have not been localized in any specific regions of promoters and other types of REGs nor in the flanking sequences of the same genes . Only part of the homological REG's sequences have CG and CNG methylated sites . Therefore the methylation of cytosine residues in any REGs may be not an obligatory condition for normal regulation of the REG activity in cells . Two main REG's families of different length were unexpectedly found in the study . The length of the first one is 9-12 n . and the second is 17-20 n . The families are about 60-80% of other REGs . The essential deficiency of cytosine residues and also triplets of CGG, CCG, CTG and CAG has been showed in the "sense" chain of the REGs . The chain has some abundance of TTG, CCA and CAA triplets . The REG's chains have a strong asymmetry in purine and pyrimidine contents and also in duplets TG and CA frequencies . It may be the result of different reparation effectivity of G-T pairs produced by 5-meC residues deamination in DNA complementary chains . Therefore cytosine methylation in REGs may strongly destabilize the structure, accelerate its divergence in evolution, and disturb the REGs binding with protein factors regulating activity of the genes . The results showed that a function of DNA enzymatic methylation may be hardly realized through the modification of gene regulatory elements.

Vet Microbiol, 1992 Mar, 30(4), 387 - 94
Characterization of the glycoprotein D gene products of equine herpesvirus 1 using a prokaryotic cell expression vector; Love DN et al.; The gene encoding equine herpesvirus 1 (equine abortion virus; EHV-1) glycoprotein D was engineered into the prokaryotic vector pEX, and expressed as a beta-galactosidase fusion product, which was recognized by pooled equine sera and anti-EHV-1 rabbit sera . Antibodies raised against the EHV-1 gD fusion product identified strong bands in infected cells at 66 and 68 K and at 138 K in purified virus, thus characterizing the several forms of this major envelope glycoprotein which is an important candidate for inclusion in subunit vaccines.

Biochemistry, 1992 Feb 25, 31(7), 1904 - 8
RNA polymerases react differently at d(ApG) and d(GpG) adducts in DNA modified by cis-diamminedichloroplatinum(II); Corda Y et al.; Two duplexes (20-mers) were constructed containing either a single cis-{Pt(NH3)2{d(GpG)}} or cis-{Pt(NH3)2{d(ApG)}} intrastrand cross-link, the major DNA adducts of the antitumor drug cis-diamminedichloroplatinum(II) . These synthetic duplexes were multimerized and the resultant polymers used as templates in single-step addition reactions of condensation of a single nucleoside triphosphate substrate to a dinucleotide primer (abortive elongation reaction) catalyzed by prokaryotic or eukaryotic RNA polymerases . Primer-substrate combinations were selected so as to direct trinucleotide product formation within the platinated bases of the templates . Transcription experiments established that cis-DDP-DNA adducts formed at d(ApG) or d(GpG) sites are not an absolute block to formation of a single phosphodiester bond by either Escherichia coli RNA polymerase or wheat germ RNA polymerase II . Furthermore, the kinetic data indicate that single-step addition reactions are much more impeded at the platinated d(GpG) than at the platinated d(ApG) site and that the mechanisms of inhibition of RNA polymerase activity are different at the two platinated sites . In particular, binding affinity between E . coli RNA polymerase and the d(GpG)-containing platinated template is lowered, as the apparent Km of enzyme for the platinated polymer is increased by a factor of 4-5 . In contrast, binding affinity between the RNA polymerase and the d(ApG)-containing template is not affected by modification of the d(ApG) site by cis-diamminedichloroplatinum(II) . Similar experiments were carried out with synthetic templates containing the adducts at the d(GpG) sites, in which one of the two platinated dG residues is paired with a dT residue.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1992 Feb 25, 267(6), 3764 - 70
Characterization of the hemolysin transporter, HlyB, using an epitope insertion; Juranka P et al.; The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P . F., Zastawny, R . L., and Ling, V . (1989) FASEB J . 3, 2583-2592) . Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance . In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function . This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.

J Theor Biol, 1992 Feb 21, 154(4), 519 - 39
Towards the molecular bases of polymerase dynamics; Chela-Flores J; One aspect of the strong relationship that is known to exist between the processes of DNA replication and transcription is manifest in the coupling of the rates of movement of the replication fork (rf) and RNA polymerase (rt) . We address two issues concerning the largely unexplored area of polymerase dynamics: (i) The validity of an approximate kinematic formula linking rf and rt suggested by experiments in which transcription is inhibited in some prokaryotes with the antibiotic streptolydigin, and (ii) What are the molecular bases of the kinematic formula? An analysis of the available data suggests possible molecular bases for polymerase dynamics . In particular, we are led to a hypothesis: In active chromatin rt may depend on the length (lambda t) of the transcript of the primary messenger RNA (pre-mRNA) . This new effect is subject to experimental verification . We discuss possible experiments that may be performed in order to test this prediction.

FEBS Lett, 1992 Feb 17, 298(1), 93 - 6
A secY homologue is found in the plastid genome of Cryptomonas phi; Douglas SE; An open reading frame with significant similarity to the secY gene of Escherichia coli has been found within a ribosomal protein operon on the plastid genome of the chlorophyll c-containing alga Cryptomonas phi . The gene encodes a protein of 420 amino acids (molecular weight 46,906 daltons) and contains ten potential membrane-spanning domains, as in the E . coli homologue . This report of a secY homologue in a plastid genome provides preliminary evidence that a prokaryotic-like protein export system may be operating in plastids.

Proc Natl Acad Sci U S A, 1992 Feb 15, 89(4), 1358 - 62
Over- and under-representation of short oligonucleotides in DNA sequences; Burge C et al.; Strand-symmetric relative abundance functionals for di-, tri-, and tetranucleotides are introduced and applied to sequences encompassing a broad phylogenetic range to discern tendencies and anomalies in the occurrences of these short oligonucleotides within and between genomic sequences . For dinucleotides, TA is almost universally under-represented, with the exception of vertebrate mitochondrial genomes, and CG is strongly under-represented in vertebrates and in mitochondrial genomes . The traditional methylation/deamination/mutation hypothesis for the rarity of CG does not adequately account for the observed deficiencies in certain sequences, notably the mitochondrial genomes, yeast, and Neurospora crassa, which lack the standard CpG methylase . Homodinucleotides (AA.TT, CC.GG) and larger homooligonucleotides are over-represented in many organisms, perhaps due to polymerase slippage events . For trinucleotides, GCA.TGC tends to be under-represented in phage, human viral, and eukaryotic sequences, and CTA.TAG is strongly under-represented in many prokaryotic, eukaryotic, and viral sequences . The CCA.TGG triplet is ubiquitously over-represented in human viral and eukaryotic sequences . Among the tetranucleotides, several four-base-pair palindromes tend to be under-represented in phage sequences, probably as a means of restriction avoidance . The tetranucleotide CTAG is observed to be rare in virtually all bacterial genomes and some phage genomes . Explanations for these over- and under-representations in terms of DNA/RNA structures and regulatory mechanisms are considered.

Experientia, 1992 Feb 15, 48(2), 118 - 29
Proteolysis in protein import and export: signal peptide processing in eu- and prokaryotes; Muller M; Numerous proteins in pro- and eukaryotes must cross cellular membranes in order to reach their site of function . Many of these proteins carry signal sequences that are removed by specific signal peptidases during, or shortly after, membrane transport . Signal peptidases have been identified in the rough endoplasmic reticulum, the matrix and inner membrane of mitochondria, the stroma and thylakoid membrane of chloroplasts, the bacterial plasma membrane and the thylakoid membrane of cyanobacteria . The composition of these peptidases varies between one and several subunits . No site-specific inhibitors are known for the majority of these enzymes . Accordingly, signal peptidases recognize structural motifs rather than linear amino acid sequences . Such motifs have become evident by employing extensive site-directed mutagenesis to investigate the anatomy of signal sequences . Analysis of the reaction specificities and the primary sequences of several signal peptidases suggests that the enzymes of the endoplasmic reticulum, the inner mitochondrial membrane and the thylakoid membrane of chloroplasts all have evolved from bacterial progenitors.

Cancer Res, 1992 Feb 15, 52(4), 983 - 9
Control of Ha-ras-mediated mammalian cell transformation by Escherichia coli regulatory elements; Liu HS et al.; Inducible eukaryotic promoters, particularly those responsive to glucocorticoids or heavy metals, have been extensively used to study the consequences of induction of a target gene in mammalian cells . An alternative approach, intended to improve the selectivity of gene induction and to minimize perturbation of chromatin structure, is to utilize elements from prokaryotic regulatory systems that are unlikely to be shared by mammalian cells . We and others previously have shown that the lac repressor can function in mammalian cells and repress expression of a reporter gene controlled by a eukaryotic promoter containing a lac operator sequence . The reporter gene can be specifically activated by administration of the lactose analogue isopropyl beta-D-thiogalactoside . The target genes tested so far encode the biochemical and histochemical markers, chloramphenicol acetyltransferase and beta-galactosidase . As a model system to establish whether or not the lactose regulatory system can also be used to effectively modulate a cellular phenotype, NIH 3T3 cells were made transgenic for a constitutively expressed lacI gene, encoding lac repressor, and an activated human Ha-ras gene directed by a simian virus 40 promoter within which a lac operator sequence had been embedded . In the absence of inducer, cells were phenotypically untransformed . Consequent to isopropyl beta-D-thiogalactoside administration, four biological end points characteristic of a transformed phenotype were observed . Consistent with transformation, the cells assumed an altered morphology; they displayed a reduced density inhibition of growth; they acquired the capacity to grow in soft agar; and they were released from a G0 block following serum deprivation . The data demonstrate that regulation of gene expression in mammalian cells by the lactose regulatory system affords a sensitive means for modulating cellular phenotype.

Gene, 1992 Feb 15, 111(2), 175 - 81
Isolation and characterization of genes encoding chaperonin 60 beta from Arabidopsis thaliana; Zabaleta E et al.; Chaperonins (Cpn) are implicated in the folding and assembly of multimeric proteins in plastids and mitochondria of eukaryotes and in prokaryotes . Plastid Cpn is composed of two different polypeptides termed Cpn60 alpha and Cpn60 beta . We have isolated cDNA and genomic clones encoding Cpn60 beta from Arabidopsis thaliana . The steady-state level of the cpn60 beta mRNAs is higher in etiolated leaves and sucrose-treated plants as compared to control leaves . The A . thaliana cpn60 beta gene family consists of at least three different coding units . It was confirmed that Cpn beta-encoding genes have a high level of conservation among plants.

FEBS Lett, 1992 Feb 10, 297(3), 250 - 2
Mechanism and site of action of a ribosome-inactivating protein type 1 from Dianthus barbatus which inactivates Escherichia coli ribosomes; Prestle J et al.; A single chain ribosome-inactivating protein with RNA N-glycosidase activity, here named Dianthin 29, was isolated from leaves of Dianthus barbatus L . Incubation of intact Escherichia coli ribosomes with Dianthin 29 and subsequent aniline treatment of the isolated rRNA releases a rRNA fragment of 243 nucleotides from 23 S rRNA . Nucleotide sequence studies showed that the site of N-glycosidic bond cleavage is at A-2660 within the universally conserved sequence 5'-AGUACGAGAGGA-3' near the 3'-end of 23/28 S rRNAs . To our knowledge, Dianthin 29 is the first ribosome-inactivating protein which is shown to inactivate intact prokaryotic ribosomes in the same manner as eukaryotic ribosomes.

FEBS Lett, 1992 Feb 10, 297(3), 209 - 11
'boxA'-like sequence between the 16 S/23 S spacer in rRNA operon of mycoplasmas; Harasawa R et al.; We have found that a boxA-like sequence is conserved in the 16 S and 23 S rRNA intergenic spacer regions of mycoplasmas, and that it always locates on loop regions of the hypothetical secondary stem-loop structures . A nucleotide sequence similar to the '-10' box of prokaryotic promoters was identified at upstream sites of the boxA-like sequence in the 16 S/23 S spacer regions . These structures may represent an internal promoter between the 16 S and 23 S rRNA genes in mycoplasmas.

J Biol Chem, 1992 Feb 5, 267(4), 2214 - 21
Bacterial expression, purification, and functional mapping of the amyloid beta/A4 protein precursor; Roch JM et al.; The secreted form of Alzheimer amyloid beta/A4 protein precursor (APP) has been shown to be involved in cell growth regulation (Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Oltersdorf, T., and Schenk, D.B . (1989) Cell 58, 615-622) . Using a strong prokaryotic expression system, we expressed, in Escherichia coli, peptide fragments covering different regions of the secreted form of APP-695 . The longest of these fragments (KB75, 572 amino acids from Val-20 to Ile-591), which contained neither the Kunitz-type protease inhibitor (KPI) domain nor the amyloid beta/A4-protein domain, was purified and shown to be biologically active in terms of growth regulation . Two other APP fragments (KB48, 316 amino acids from Val-20 to Met-335; and RB17, 150 amino acids from Thr-296 to Pro-445), overlapping by only 40 amino acids at a close site C-terminal to the KPI insertion site, were also active . Furthermore, a chemically synthesized 40-residue peptide corresponding to this region of overlap also stimulated the growth of A-1 fibroblasts . These results establish the presence of growth-promoting activity in the secreted form of APP-695 and suggest that the site of this activity of APP-695 lies within a 40-amino acid domain next to the KPI insertion site.

FEBS Lett, 1992 Feb 3, 297(1-2), 196 - 200
Functional and immunological relationships between metyrapone reductase from mouse liver microsomes and 3 alpha-hydroxysteroid dehydrogenase from Pseudomonas testosteroni; Maser E et al.; 3 Alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) from Pseudomonas testosteroni was shown to reduce the xenobiotic carbonyl compound metyrapone (MPON) . Reversely, MPON reductase purified from mouse liver microsomes and previously characterized as aldehyde reductase, was competitively inhibited by 3 alpha-HSD steroid substrates . For MPON reduction both enzymes can use either NADH or NADPH as co-substrate . Immunoblot analysis after native and SDS gel electrophoresis of 3 alpha-HSD gave a specific crossreaction with the antibodies against the microsomal mouse liver MPON reductase pointing to structural homologies between these enzymes . In conclusion, there seem to exist structural as well as functional relationships between a mammalian liver aldehyde reductase and prokaryotic 3 alpha-HSD . Moreover, based on the molecular weights and the co-substrate specificities microsomal mouse liver MPON reductase and Pseudomonas 3 alpha-HSD seem to be members of the short-chain alcohol dehydrogenase family.

J Bacteriol, 1992 Feb, 174(4), 1314 - 23
Role of the two-component leader sequence and mature amino acid sequences in extracellular export of endoglucanase EGL from Pseudomonas solanacearum; Huang JZ et al.; The egl gene of Pseudomonas solanacearum encodes a 43-kDa extracellular endoglucanase (mEGL) involved in wilt disease caused by this phytopathogen . Egl is initially translated with a 45-residue, two-part leader sequence . The first 19 residues are apparently removed by signal peptidase II during export of Egl across the inner membrane (IM); the remaining residues of the leader sequence (modified with palmitate) are removed during export across the outer membrane (OM) . Localization of Egl-PhoA fusion proteins showed that the first 26 residues of the Egl leader sequence are required and sufficient to direct lipid modification, processing, and export of Egl or PhoA across the IM but not the OM . Fusions of the complete 45-residue leader sequence or of the leader and increasing portions of mEgl sequences to PhoA did not cause its export across the OM . In-frame deletion of portions of mEGL-coding sequences blocked export of the truncated polypeptides across the OM without affecting export across the IM . These results indicate that the first part of the leader sequence functions independently to direct export of Egl across the IM while the second part and sequences and structures in mEGL are involved in export across the OM . Computer analysis of the mEgl amino acid sequence obtained from its nucleotide sequence identified a region of mEGL similar in amino acid sequence to regions in other prokaryotic endoglucanases.

J Bacteriol, 1992 Feb, 174(3), 778 - 84
Cloning and DNA sequence of a mycoplasmal recA gene; Dybvig K et al.; Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria . In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii . DNA sequence data indicate extensive homology between the A . laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis . The recA sequences from three A . laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A . laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein . By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype . When carried on a multicopy plasmid, the wild-type A . laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.

Indian J Biochem Biophys, 1992 Feb, 29(1), 9 - 12
Relationship of single-stranded DNA-binding proteins of Ehrlich ascites tumour to cell growth phase and DNA replications; Koterov AN et al.; The possible involvement of SSB-proteins in DNA replication in Ehrlich ascites tumour (EAT) has been investigated . A direct relation (the computer-generated correlation coefficient was 0.9) between the SSB-proteins content in chromatin and intensity of the replicative synthesis of DNA in various preparation of EAT in vivo and in vitro is observed . Addition of exogenous SSB-proteins to the permeable EAT cells has been found to increase the replicative synthesis . Although eukaryotic SSB-proteins are not complete analogs of the prokaryotic SSB-proteins, they evidently participate in DNA replication in eukaryotic cells and possibly are intracellular regulators of proliferation.

J Med Virol, 1992 Feb, 36(2), 142 - 6
Prevalence of antibodies to recombinant virion infectivity factor in the sera of prospectively studied patients with HIV-1 infection; Schwander S et al.; Two hundred twenty-four sera were collected from 34 HIV-1 infected patients during an observation period of up to 4.5 years (109 patient years of observation) . The sera were tested for the presence of antibodies against the HIV-1 virion infectivity factor (vif) protein . Thirty sera from 6 HIV-1 seronegative individuals served as negative controls . The sera were immunoblotted against a recombinant, prokaryotically expressed vif protein . The prevalence of anti-vif antibodies increased significantly with progression of the disease from 18% to 81% (P less than 0.0001) which suggests a possible role of vif in HIV-1 replication and pathogenicity.

J Lipid Res, 1992 Feb, 33(2), 167 - 78
Structure and evolution of the lipase superfamily; Hide WA et al.; The lipase superfamily includes three vertebrate and three invertebrate (dipteran) proteins that show significant amino acid sequence similarity to one another . The vertebrate proteins are lipoprotein lipase (LPL), hepatic lipase (HL), and pancreatic lipase (PL) . The dipteran proteins are Drosophila yolk proteins 1, 2, and 3 . We review the relationships among these proteins that have been established according to gene structural relatedness and introduce our findings on the phylogenetic relationships, distance relationships, and evolutionary history of the lipase gene superfamily . Drosophila yolk proteins contain a 104 amino acid residue segment that is conserved with respect to the lipases . We have used the yolk proteins as an outgroup to root a phylogeny of the lipase family . Our phylogenetic reconstruction suggests that ancestral PL diverged earlier than HL and LPL, which share a more recent root . Human and bovine LPL are shown to be more closely related to murine LPL than to guinea pig LPL . A comparison of the distance (a measure of the number of substitutions between sequences) between mammalian and avian LPL reveals that guinea pig LPL has the largest distance from the other mammals . Human, rodent, and rabbit HL show marked divergence from one another, although they have similar relative rates of amino acid substitution when compared to human LPL as an outgroup . Human and porcine PL are not as divergent as human and rat HL, suggesting that PL is more conserved than HL . However, canine PL demonstrates an unusually rapid rate of substitution with respect to the other pancreatic lipases . The lipases share several structurally conserved features . One highly conserved sequence (Gly-Xaa-Ser-Xaa-Gly) contains the active site serine . This feature, which agrees with that found in serine esterases and proteases, is found within the entire spectrum of lipases, including the evolutionarily unrelated prokaryotic lipases . We review the location and possible activity of putative lipid binding domains . We have constructed a conservation index (CI) to display conserved structural features within the lipase gene family, a CI of 1.0 signifying perfect conservation . We have found a correlation between a high CI and the position of conserved functional structures . The putative lipid-binding domains of LPL and HL, the disulfide-bridging cysteine residues, catalytic residues, and N-linked glycosylation sites of LPL, HL, and PL all lie within regions having a CI of 0.8 or higher . A number of amino acid substitutions have been identified in familial hyperchylomicronemia which result in loss of LPL function.(ABSTRACT TRUNCATED AT 400 WORDS)

J Mol Evol, 1992 Feb, 34(2), 126 - 9
Analyzing the mosaic structure of genes; Smith JM; Some genes in prokaryotes consist of a mosaic of regions derived from different ancestors by horizontal gene transfer . A method is described for demonstrating the statistical significance of such mosaic structure and for locating the crossover points separating different regions.

J Clin Microbiol, 1992 Feb, 30(2), 305 - 11
Prokaryotic expression of a VP1 polypeptide antigen for diagnosis by a human parvovirus B19 antibody enzyme immunoassay; Soderlund M et al.; To produce parvovirus B19 antigen for diagnostic purposes, partially overlapping segments covering the genes encoding the viral structural proteins VP1 and VP2 were cloned into expression vectors . The constructs were induced in Escherichia coli, resulting in the expression of beta-galactosidase fusion proteins . In immunoblotting experiments with sera from patients with erythema infectiosum, immunoglobulin G (IgG) and IgM antibodies bound to a single polypeptide of 235 amino acids at the N terminus of VP1 . The DNA fragment encoding this polypeptide was amplified by the polymerase chain reaction and cloned into an expression vector . The viral capsid antigen expressed in E . coli was purified by preparative agarose gel electrophoresis and used in IgG and IgM solid-phase enzyme immunoassays . Comparison with reference gamma- and mu-capture radioimmunoassays using whole virus antigen showed that these antibody tests are suitable for the serodiagnosis of human infections caused by parvovirus B19.

EMBO J, 1992 Feb, 11(2), 699 - 704
Identification and characterization of yeast mutants and the gene for a cruciform cutting endonuclease; Kleff S et al.; An assay was developed that detected DNA cruciform cutting endonuclease activity in crude extracts of Saccharomyces cerevisiae . A collection of temperature-sensitive strains was screened using this assay, and a mutant lacking the activity was found . The mutation leading to the enzymatic defect was mapped to the left arm of chromosome XI within 3 cM of the centromere . Cloning of the gene for this endonuclease was achieved by chromosome walking from the nearby PUT3 locus . The gene, called CCE1 (cruciform cutting endonuclease), was sequenced and found to have an open reading frame encoding a 41 kDa protein . The amino acid sequence of this eukaryotic endonuclease shows homology neither to its prokaryotic counterparts nor to other proteins in available databases . A cce1 null mutant has no obvious growth defect, and despite the ability of the CCE1 enzyme to cleave Holliday junction analogs, the mutant shows no defect in meiotic or mitotic recombination . A second cruciform cutting activity was detected in extracts from a cce1 null mutant, indicating that yeast has at least two such enzymes . The only phenotype observed for cce1 mutants is a higher than normal frequency of appearance of petite cells, suggesting that the CCE1 protein is important for the maintenance of mitochondrial DNA.

Plant Mol Biol, 1992 Feb, 18(3), 467 - 76
Heat shock Hsp70 protein is chloroplast-encoded in the chromophytic alga Pavlova lutherii; Scaramuzzi CD et al.; Heat shock proteins are ubiquitous and highly conserved . Recently they have become implicated in the import of proteins into organelles . All the heat shock genes characterized to date, however, are known or assumed to be encoded in the nuclear genome even if the corresponding protein can be localised in the mitochondrion or chloroplast . In contrast, we identify here an hsp70 gene in the unicellular chromophytic alga Pavlova lutherii which is located on the chloroplast genome . Localisation of this gene to the chloroplast chromosome is confirmed by Southern blot analysis and pulse-field gel electrophoresis which also reveals that the length of the P . lutherii chloroplast chromosome is 115 kb . We compare the predicted protein of this hsp70 gene with that of maize and of the analogous proteins in the prokaryotic organisms Escherichia coli and Synechocystis PCC6803 . The greatest identity is found with the cyanobacterium Synechocystis PCC6803.

Blood, 1992 Feb 1, 79(3), 559 - 62
Localization of a PlA1 epitope to the amino terminal 66 residues of platelet glycoprotein IIIa; Bowditch RD et al.; A platelet glycoprotein (GP) IIIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector lambda gt22 and screened with purified anti-PlA1 antibodies for clones expressing a PlA1 epitope . Five independent clones were isolated and characterized by nucleotide sequencing . The smallest anti-PlA1 reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa . Substitution of leucine33 (PlA1) with a proline33 (PlA2) by in vitro mutagenesis resulted in the loss of anti-PlA1 reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies . These data indicate that a PlA1 alloantigenic epitope is located within a small, unglycosylated fragment of GPIIIa containing the polymorphism responsible for the PIA phenotype . Furthermore, these results prove that small recombinant mimics of a PlA1 epitope may be synthesized and used for detection of these alloantibodies.

Protein Sci, 1992 Feb, 1(2), 191 - 200
Stein and Moore Award address . Reconstructing history with amino acid sequences; Doolittle RF; The main goal of the protein evolutionist is the reconstruction of past events leading to the structures of contemporary proteins . The common strategy is to align amino acid sequences and make inferences about matters of common ancestry . The rate of change of amino acid sequence varies greatly from protein to protein, and this naturally affects how far back a given protein's ancestry can be traced . Happily, the rate of change of many proteins is slow enough that very ancient events can be inferred . Many mainstream metabolic enzymes, for example, are 40-50% identical in prokaryotes and eukaryotes, groups that diverged from a common ancestor more than 1.5 billion years ago . Moreover, some eukaryotic proteins like actin and tubulin change so slowly that they are seldom less than 60% identical, no matter from what source they are drawn . As it happens, prokaryotic counterparts for many eukaryotic cytoskeletal proteins are unknown . A recent exception involves the finding that a heat shock protein cognate is a relative of actin . The gene duplication that gave rise to these two proteins must have been an ancient event . The more recent invention of other proteins whose distribution is restricted to one or the other of the major kingdoms may be easier to trace . Among the factors that can confound the reconstruction of events, however, are occasional horizontal gene transfers and exon shuffling . The latter has led to a number of mosaic proteins, many of which contain various combinations of a relatively small set of modules like the epidermal growth factor domain.

J Biol Chem, 1992 Jan 25, 267(3), 1623 - 32
A model for the aminoacyl-tRNA binding site of eukaryotic elongation factor 1 alpha; Kinzy TG et al.; Eukaryotic elongation factor 1 alpha (EF-1 alpha) binds all the aminoacyl-tRNAs except the initiator tRNA in a GTP-dependent manner . While the GTP binding site is delineated by the three GTP binding consensus elements, less is known about the aminoacyl-tRNA binding sites . In order to better understand this site, we have initiated cross-linking and protease mapping studies of the EF-1 alpha-GTP-aminoacyl-tRNA complex . Two different chemical cross-linking reagents, trans-diaminedichloroplatinum(II) and diepoxybutane, were used to cross-link four different aminoacyl-tRNA species to EF-1 alpha . A series of peptides were obtained, located predominantly in domains II and III . The ability of aminoacyl-tRNA to protect protease digestion sites was also monitored, and domain II was found to be protected from digestion by aminoacyl-tRNA . Last, an aminoacyl-tRNA analog with a reactive group on the aminoacyl side chain, N epsilon-bromoacetyl-Lys-tRNA, was cross-linked to EF-1 alpha . This reagent cross-liked to histidine 296 in a GTP-dependent manner and thus localizes the aminoacyl group adjacent to domain II . A model is developed for aminoacyl-tRNA binding to EF-1 alpha based on its similarity to the prokaryotic factor EF-Tu, for which an x-ray crystal structure is available.

J Biol Chem, 1992 Jan 25, 267(3), 1502 - 9
Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis . Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase; Miquel M et al.; The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation . Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants . These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R . (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids . Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids . Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids . This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.

J Biol Chem, 1992 Jan 25, 267(3), 2073 - 9
Purification and initial characterization of peptidyl-tRNA hydrolase from rabbit reticulocytes; Gross M et al.; We have identified an activity in rabbit reticulocyte lysate as peptidyl-tRNA hydrolase, based upon its ability to hydrolyze native reticulocyte peptidyl-tRNA, isolated from polyribosomes, and N-acylaminoacyl-tRNA, and its inability to hydrolyze aminoacyl-tRNA, precisely the same substrate specificity previously reported for peptidyl-tRNA hydrolase from bacteria or yeast . The physiological role of the reticulocyte enzyme may be to hydrolyze and recycle peptidyl-tRNA that has dissociated prematurely from elongating ribosomes, as suggested for the bacterial and yeast enzymes, since reticulocyte peptidyl-tRNA hydrolase is completely incapable of hydrolyzing peptidyl-tRNA that is still bound to polyribosomes . We have purified reticulocyte peptidyl-tRNA hydrolase over 5,000-fold from the postribosomal supernatant with a yield of 14% . The purified product shows a 72-kDa band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis that has co-purified with enzyme activity and comprises about 90% of the total stained protein, strongly suggesting that the 72-kDa protein is the enzyme . Sucrose density gradient analysis indicates an apparent molecular mass for the native enzyme of 65 kDa, implying that it is a single polypeptide chain . The enzyme is almost completely inactive in the absence of a divalent cation: Mg2+ (1-2 mM) promotes activity best, Mn2+ is partly effective, and Ca2+ and spermidine are ineffective . The hydrolase shows a Km of 0.60 microM and Vmax of 7.1 nmol/min/mg with reticulocyte peptidyl-tRNA, a Km of 60 nM and Vmax of 14 nmol/min/mg with Escherichia coli fMet-tRNA(fMet), and a Km of 100 nM and Vmax of 2.2 nmol/min/mg with yeast N-acetyl-Phe-tRNA(Phe) . The enzyme has a pH optimum of 7.0-7.25, it is inactivated by heat (60 degrees C for 5 min), and its activity is almost completely inhibited by pretreatment with N-ethylmaleimide or incubation with 20 mM phosphate . The fact that the enzyme hydrolyzes E . coli but not yeast or reticulocyte fMet-tRNA(fMet) may be explained, at least in part, by structural similarities between prokaryotic tRNA(fMet) and eukaryotic elongator tRNA that are not shared by eukaryotic tRNA(fMet).

Nature, 1992 Jan 16, 355(6357), 267 - 70
Multiple evolutionary origins of prochlorophytes within the cyanobacterial radiation; Urbach E et al.; The taxonomic group Prochlorales (Lewin 1977) Burger-Wiersma, Stal and Mur 1989 was established to accommodate a set of prokaryotic oxygenic phototrophs which, like plant, green algal and euglenoid chloroplasts, contain chlorophyll b instead of phycobiliproteins . Prochlorophytes were originally proposed (with concomitant scepticism) to be a monophyletic group sharing a common ancestry with these 'green' chloroplasts . Results from molecular sequence phylogenies, however, have suggested that Prochlorothrix hollandica is not on a lineage that leads to plastids . Our results from 16S ribosomal RNA sequence comparisons, which include new sequences from the marine picoplankter Prochlorococcus marinus and the Lissoclinum patella symbiont Prochloron sp., indicate that prochlorophytes are polyphyletic within the cyanobacterial radiation, and suggest that none of the known species is specifically related to chloroplasts . This implies that the three prochlorophytes and the green chloroplast ancestor acquired chlorophyll b and its associated structural proteins in convergent evolutionary events . We report further that the 16S rRNA gene sequence from Prochlorococcus is very similar to those of open ocean Synechococcus strains (marine cluster A), and to a family of 16S rRNA genes shotgun-cloned from plankton in the north Atlantic and Pacific Oceans.

Nature, 1992 Jan 16, 355(6357), 265 - 7
Multiple evolutionary origins of prochlorophytes, the chlorophyll b-containing prokaryotes; Palenik B et al.; Prochlorophytes are prokaryotes that carry out oxygenic photosynthesis using chlorophylls a and b, but lack phycobiliproteins as light-harvesting pigments . These characteristics distinguish them from cyanobacteria, which contain phycobiliproteins, but no chlorophyll b . Three prochlorophyte genera have been described: Prochloron, Prochlorothrix and Prochlorococcus . The prochlorophytes share their pigment characteristics with green plant and euglenoid chloroplasts, which has led to a debate on whether these chloroplasts may have arisen from an endosymbiotic prochlorophyte rather than a cyanobacterium . Molecular sequence data, including those presented here based on a fragment of the rpoC1 gene encoding a subunit of DNA-dependent RNA polymerase, indicate that the known prochlorophyte lineages do not include the direct ancestor of chloroplasts . We also show that the prochlorophytes are a highly diverged polyphyletic group . Thus the use of chlorophyll b as a light-harvesting pigment has developed independently several times in evolution . Similar conclusions have been reached in parallel studies using 16S ribosomal RNA sequences.

J Biol Chem, 1992 Jan 15, 267(2), 819 - 24
The orotidine-5'-monophosphate decarboxylase gene of Myxococcus xanthus . Comparison to the OMP decarboxylase gene family; Kimsey HH et al.; The nucleotide sequence of the Myxococcus xanthus orotidine-5'-monophosphate decarboxylase (OMP DCase) gene was determined . The derived protein sequence is not closely related to other prokaryotic OMP DCase sequences; nor is it closely related to any eukaryotic OMP DCase sequences . Progressive multiple alignment of the M . xanthus OMP DCase protein sequence with 19 other OMP DCase sequences revealed four conserved regions present in all 20 sequences . Ten entirely conserved residues were found in these four regions and one region contains a tight cluster of 5 conserved residues, certain of which may be catalytically active residues . A second open reading frame was found upstream of uraA and oriented in the same direction as uraA . A stretch of 21 consecutive pyrimidine (C or T) residues were found in the intercistronic region between the potential ribosome-binding site of uraA and the UGA stop codon of the upstream open reading frame . RNA directly upstream of the pyrimidine run, including the UGA stop codon of the upstream open reading frame, could be folded into a stable hairpin structure resembling Rho-independent terminators of Escherichia coli . Expression of the uraA gene may be regulated by an intercistronic transcription termination mechanism.

Eur J Biochem, 1992 Jan 15, 203(1-2), 99 - 105
Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp . PCC 6803; Muro-Pastor MI et al.; NADP-dependent isocitrate dehydrogenase activity has been screened in several cyanobacteria grown on different nitrogen sources; in all the strains tested isocitrate dehydrogenase activity levels were similar in cells grown either on ammonium or nitrate . The enzyme from the unicellular cyanobacterium Synechocystis sp . PCC 6803 has been purified to electrophoretic homogeneity by a procedure that includes Reactive-Red-120-agarose affinity chromatography and phenyl-Sepharose chromatography as main steps . The enzyme was purified about 600-fold, with a yield of 38% and a specific activity of 15.7 U/mg protein . The native enzyme (108 kDa) is composed of two identical subunits with an apparent molecular mass of 57 kDa . Synechocystis isocitrate dehydrogenase was absolutely specific for NADP as electron acceptor . Apparent Km values were 125, 59 and 12 microM for Mg2+, D,L-isocitrate and NADP, respectively, using Mg2+ as divalent cation and 4, 5.7 and 6 microM for Mn2+, D,L-isocitrate and NADP, respectively, using Mn2+ as a cofactor . The enzyme was inhibited non-competitively by ADP (Ki, 6.4 mM) and 2-oxoglutarate, (Ki, 6 mM) with respect to isocitrate and in a competitive manner by NADPH (Ki, 0.6 mM) . The circular-dichroism spectrum showed a protein with a secondary structure consisting of about 30% alpha-helix and 36% beta-pleated sheet . The enzyme is an acidic protein with an isoelectric point of 4.4 and analysis of the NH2-terminal sequence revealed 45% identity with the same region of Escherichia coli isocitrate dehydrogenase . The aforementioned data indicate that NADP isocitrate dehydrogenase from Synechocystis resembles isocitrate dehydrogenase from prokaryotes and shows similar molecular and structural properties to the well-known E . coli enzyme.

Cell, 1992 Jan 10, 68(1), 119 - 31
Prokaryotic-like cis elements in the cap-independent internal initiation of translation on picornavirus RNA; Pilipenko EV et al.; Initiation of translation on picornavirus RNAs is accomplished through internal binding of ribosomes to a complex cis-acting element . Here we show that efficient function of this element involves two appropriately spaced smaller elements: UUUCC and an AUG . This conclusion emerged from analysis of the genome structures of spontaneous revertants of mutant polioviruses with extended insertions between the UUUCC and AUG motifs . It was confirmed by the results obtained with specially designed constructs . A similarity to the prokaryotic translation initiation mechanism, which involves the Shine-Dalgarno sequence, is emphasized, but in the picornavirus system the position of the UUUCC must be strictly fixed relative to upstream cis-acting elements, and the AUG may not necessarily serve as an initiation codon.

Gene, 1992 Jan 2, 110(1), 81 - 8
A universal compositional correlation among codon positions; D'Onofrio G et al.; We have investigated the compositional distributions of third codon positions of genes from the 16 prokaryotes and seven eukaryotes for which the largest numbers of coding sequences are available in data banks . In prokaryotes, both narrow and broad distributions were found . In eukaryotes, distributions were very broad (except for Saccharomyces cerevisiae) and remarkably different for different genomes . In low-GC genomes, third codon positions were lower in GC than first + second codon positions and trailed towards high GC; the opposite situation was found for high-GC genomes . In all genomes, first codon positions were higher in GC than second codon positions . We then investigated the compositional correlations between third and first + second codon positions in prokaryotic genomes (the 16 mentioned above plus 87 additional ones) and in genome compartments of eukaryotes . A general, common relationship was found, which also holds within the same (heterogeneous) genomes . This universal correlation is due to the fact that the relative effects of compositional constraints on different codon positions are the same, on the average, whatever the genome under consideration.

Plasmid, 1992 Jan, 27(1), 72 - 9
Germanium and silver resistance, accumulation, and toxicity in microorganisms; Slawson RM et al.; Germanium is an inert metal with no known biological function in prokaryotic or eukaryotic organisms . Its toxicity is low compared to that of silver . Germanium is accumulated in certain bacterial strains by either energy-independent passive binding or an energy-dependent mechanism . Little is known about the molecular aspects of silver resistance, toxicity, and accumulation in bacterial strains . This is surprising because silver has been used as an antimicrobial agent in the medical field for centuries . It is likely that silver ions are excluded (resulting in decreased silver accumulation) from certain bacterial strains or immobilized intracellularly to prevent toxic effects from being exerted . These mechanisms of silver resistance have not been fully elucidated . This review examines the toxicity and accumulation of germanium and silver in selected microbial species . In addition, resistance mechanisms to these biologically nonessential metals is discussed, with more emphasis placed on silver-resistant bacteria due to the knowledge available.

EMBO J, 1992 Jan, 11(1), 233 - 40
Recombination of constant and variable modules alters DNA sequence recognition by type IC restriction-modification enzymes; Gubler M et al.; EcoR124 and EcoDXXI are allelic type I restriction-modification (R-M) systems whose specificity genes consist of common structural elements: two variable regions are separated by a constant, homologous region containing a number of repetitive sequence elements . In vitro recombination of variable and constant elements has led to fully active, hybrid R-M systems exhibiting new and predictable target site specificities . Methylation of synthetic DNA sequences with purified, hybrid modification methylases was used to confirm the proposed recognition sequences . The results clearly demonstrate the correlation between protein domains and target site specificity . Our data suggest that a bacterial population may switch the recognition sequences of its type I R-M system by single recombination events and thus is able to maintain a prokaryotic analogue of the immune system of variable specificity.

DNA Cell Biol, 1992 Jan-Feb, 11(1), 71 - 82
Isolation and characterization of eft-1, an elongation factor 2-like gene on chromosome III of Caenorhabditis elegans; Ofulue EN et al.; A gene (eft-1) encoding an elongation factor 2-like protein was isolated from a region adjacent to the polyubiquitin gene, ubq-1, of Caenorhabditis elegans . Sequence analysis of genomic and cDNA clones revealed that the deduced amino acid sequence of the protein (EFT-1) is 38% identical to that of mammalian and Drosophila elongation factor 2 (EF-2) . The entire eft-1 gene is approximately 3.8 kb in length and contains 5 exons separated by short introns of 46-75 bp . The 2,547-bp open reading frame predicts a protein of 849 amino acid residues (calculated Mr, 96,151) . Conserved sequences shared among a variety of GTP-binding proteins including EF-2 are found in the amino-terminal third of EFT-1 . The carboxy-terminal half contains regions with 40-57% similarity (including conservative changes) with segments characteristic of EF-2 and its prokaryotic homolog, EF-G . However, the histidyl residue target for ADP-ribosylation of EF-2 by diphtheria toxin is replaced by tyrosine in EFT-1 . Southern and Northern blot analyses indicate that eft-1 is a single-copy gene that is expressed at all stages of nematode development . Amplification of fragments encoding highly conserved regions of EF-2 using the polymerase chain reaction led to the isolation of a fragment encoding the modifiable histidyl residue and which likely represents part of the C . elegans EF-2 gene (eft-2) . This suggests that EFT-1 is not the C . elegans homolog of EF-2, but a closely related protein.

Plant Mol Biol, 1992 Jan, 18(2), 275 - 85
An aberrant plastid ribosomal RNA gene cluster in the root parasite Conopholis americana; Wimpee CF et al.; The plastid ribisomal RNA (rRNA) operon of the achlorophyllous root parasite Conopholis americana was completely sequenced . Full-length rRNA genes are retained in the gene cluster, but significant divergence has occurred in the 16S, 23S and 5S genes . Both the 16S-23S intergenic spacer and the 4.5S-5S intergenic spacer have suffered substantial deletions, including the two tRNA genes typically found in prokaryotic and plastid 16S-23S spacers.

Proc Natl Acad Sci U S A, 1992 Jan 1, 89(1), 118 - 22
Pertussis toxin has eukaryotic-like carbohydrate recognition domains; Saukkonen K et al.; Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin . The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits . Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer . Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3 . Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides . Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity . Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.

Arch Microbiol, 1992, 157(4), 336 - 42
Construction of insertion mutants of Synechocystis sp . PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex; Osiewacz HD; The gene encoding subunit IV of the cytochrome b6/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp . PCC 6803 . The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa . The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostoc sp . PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g . Chlorella prototecoides, Nicotiana tabacum) . Transformation of Synechocystis sp . PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3'-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants . The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies . Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp . PCC 6803.

Genetika, 1992 Jan, 28(1), 28 - 37
{Principles of prokaryotic genome organization}; Sukhodolets VV; The peculiarities of bacterial chromosome organization are discussed, based mainly on the data on Escherichia coli . Highly important for bacterial genome organization is its division into two approx . equal half-genomes undergoing periodically "exchanges" of some kind displayed as continuous inversions including the oriC region of replication initiation . It is believed that short oligonucleotides are comprised in either of half-genomes . The former are predominantly oriented as direct repeats, which ensures the possibility of formation of tandem duplications consisting of identical genes--under conditions when selection for enhancing functions of corresponding genes takes place . Multiple tandem duplications capable of excision of plasmatic gene copies seem to initiate horizontal gene transfer in bacteria . Tandem gene duplications are probably being formed in the process of bacterial genetic recombination as well, when, as a result of non-equal crossing over, gene alleles derived from different strains are united into a tandem.

Comput Biol Med, 1992 Jan-Mar, 22(1-2), 97 - 112
DNA spatial considerations in the arrangement of G/C and A/T blocks; Nussinov R; Intensive computations have been carried out on eukaryotic and prokaryotic DNA sequences present in the GenBank database . These calculations were aimed at studying particular sequence patterns . Previously we have observed a trend in the relative positioning of DNA oligomers with respect to each other . Specifically, we have shown that there is a preference for A/T stretches to be inserted inside G/C blocks, partitioning the latter . This arrangement is preferred over having a longer G/C block with an A/T stretch next to it . That is (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2(A/T)m . Previously we have attributed this preferred pattern to nucleosome packaging of the DNA . Since the average length of the DNA involved in a single nucleosome formation is 200 bp, oligomers were scored in the analysis only if two identical ones occurred within that distance . Since, in addition, the total length of the oligomers studied is n + m + 2 less than or equal to 7, the counts of the longer oligomers were quite low . Here we have repeated the analysis on the newer, larger database, with that restriction removed . Our new analysis confirms and strengthens the older findings . Moreover, we have carried out DNA structural analysis of these trends, focusing on the twist and roll angular parameters . We have obtained these values from detailed structural computations on the Cray super computer . Some correlations between these values, DNA flexibility and the sequence trends are observed . We suggest that the positioning of the (A/T)m sequence stretch between the two (G/C) blocks affords greater flexibility in the spatial positioning of the G/C sequence elements.

Mol Carcinog, 1992, 5(2), 161 - 9
Isolation and partial characterization of murine O6-alkylguanine-DNA-alkyltransferase: comparative sequence and structural properties; Santibanez-Koref M et al.; A cDNA encoding murine O6-alkylguanine-DNA-alkyltransferase (ATase) has been sequenced after isolation from total liver RNA by the polymerase chain reaction using oligonucleotide primers derived from the rat ATase cDNA sequence . Functionally active murine ATase protein has been expressed in Escherichia coli at high levels (about 2% of total protein) and purified to apparent homogeneity (molecular mass 26 kDa) . In liquid hybridization experiments, anti-human ATase polyclonal antibodies inhibited human but not rat or mouse ATase, whereas anti-rat polyclonal antibodies inhibited rat and mouse but not human ATase . Both antibodies detected all mammalian ATases tested by western analysis so far . These results indicate some common epitopes and at least one unique human epitope . We compared the amino-acid sequence of the murine ATase with those of other mammalian and bacterial ATases . The proteins of this family all have a large domain (approximately 70 amino acids) of highly conserved residues flanking the sequence PCHRV, which contains the alkyl-accepting cysteine residue of the active site . No evidence was found in the sequences for helix-turn-helix, leucine-zipper, or zinc-finger motifs for DNA recognition and binding . Nuclear localization signals (basic-residue-rich regions) could not be uniquely identified in the mammalian members of the family . Outside of the conserved PCHRV region, there were major differences between prokaryotic and eukaryotic proteins at the primary structure level: there was a series of proline-rich motifs, but these also varied between sequences.

Mol Microbiol, 1992 Jan, 6(2), 189 - 95
The relationship between disulphide bond formation, processing and secretion of lipo-beta-lactamase in yeast; Shani O et al.; The hybrid prokaryotic lipo-beta-lactamase mature and precursor proteins spontaneously form an intramolecular disulphide bond when oxidized in vitro . When expressed in Saccharomyces cerevisiae (in vivo) the lipo-beta-lactamase precursor is in a reduced form whereas the majority of the mature protein is oxidized . The results indicate that in yeast, the lipo-beta-lactamase precursor is first processed (the signal peptide is removed) and then oxidized to form a disulphide bond in the mature protein . Reduced-mature lipo-beta-lactamase was found to reach the yeast periplasm and the process depends on endoplasmic reticulum (ER) entry even though the protein is not oxidized . This result is remarkable since in eukaryotes, disulphide bond formation occurs in the ER . Oxidized mature lipo-beta-lactamase can also be released from the sphaeroplast into the yeast periplasm . Mutant lipo-beta-lactamase genes in which cysteine residue 131 was changed to either tyrosine or threonine, were efficiently processed and secreted in yeast, which is consistent with the finding that reduced-mature non-mutant lipo-beta-lactamase can be secreted . We discuss the possibility that the folding mechanism of lipo-beta-lactamase in vitro may be fundamentally different from the process in the eukaryotic system of S . cerevisiae.

Physiol Chem Phys Med NMR, 1992, 24(2), 97 - 107
Small acidic peptides from wheat germ chromatin . I . Isolation and biochemical characterization; Mancinelli L et al.; RNA synthesis in cell and cell-free systems is inhibited by a family of acidic, low molecular weight chromatin peptides (CPs) . These peptides were extracted from deproteinized DNA of prokaryotic and eukaryotic cells, but the low yield of purified material by this procedure hinders efforts aimed at understanding their action mechanism in gene regulation . In this report we describe two purification methods of CPs from an easily available source, wheat germ . A comparison is made between the method starting from deproteinized DNA and the method from purified chromatin . The biological effects (inhibition of L1210 cell growth and DNA in vitro transcription) of CPs from wheat germ together with their chemical characteristics (molecular weight, amino acid composition and presence of phosphoserine) show strong homology with those of CPs from other sources . These results suggest a possible role of these chromatin peptides in controlling gene expression.

Mol Biol (Mosk), 1992 Jan-Feb, 26(1), 25 - 58
{Single-stranded DNA from viruses, prokaryotes and eukaryotes}; Popov LS; The review presents the results of investigations of single-stranded DNAs of viruses, bacteria and cells of higher organisms . Methods of revealing, isolating and analysis of these DNAs are presented . A large variety of single-stranded DNA containing genomes of plant and animal viruses was revealed . Attention is drawn to the integration and replication of viral genomes . Results of SV40 integration during the first two days after infection of Chinese hamster cells are shown . Results of studying multi-copy single-stranded DNA in bacterial cells were analysed . In separate sections, the replication of plasmid single-stranded DNA was studied as well as the problem of plasmid stability in cells . Advances in bacteria transformation studies are stated . Data of single-stranded DNA investigation in cells of higher organisms are mainly presented on the example of early embryos . Data on the analysis of gene hypersensitivity to nuclease S1 are given . A table of proteins destabilizing and unweaving single-stranded DNA and a classification table of proteins bound with single-stranded DNA according to their functional significance are presented . It is stated that the problem of single-stranded DNA significance in cells remains open, although some results have been achieved.

Annu Rev Biochem, 1992, 61, 199 - 223
DNA looping; Schleif R; DNA looping is widely used in nature . It is well documented in the regulation of prokaryotic and eukaryotic gene expression, DNA replication, and site-specific DNA recombination . Undoubtedly looping also functions in other protein-DNA transactions such as repair and chromosome segregation . While the underlying physical chemistry of DNA looping is common to all systems, the precise biochemical details of looping and the utilization of looping by different systems varies widely . Looping appears to have been chosen by nature in such a wide variety of contexts because it solves problems both of binding and of geometry . The cooperativity inherent in binding a protein to multiple sites on DNA facilitates high occupancy of DNA sites by low concentrations of proteins . DNA looping permits a sizeable number of DNA-binding proteins to interact with one of their number, for example RNA polymerase . Finally, DNA looping may simplify evolution by not requiring a precise spacing between a protein's binding site and a second site on the DNA.

DNA Seq, 1992, 3(3), 177 - 9
The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals; Seraphin B; By comparing the sequence of a putative translation product from a Saccharomyces cerevisiae split gene with several data-bases, I have uncovered a new protein family . Members of this family are found in prokaryotes as well as in lower and higher eukaryotes . The function of these proteins is unknown but they share a characteristic histidine triad that may be involved in zinc binding . This group of protein has been named the HIT protein family.

Tsitologiia, 1992, 34(5), 3 - 33
{Eukaryotes devoid of the most important cellular organelles (flagella, Golgi apparatus, mitochondria) and the main task of organellology}; Seravin LN; Comparative evidence on the lack of three important organelles (flagella, Golgi-complex, mitochondria) in cells and organisms at the cellular level of organization has been summarized for all the four eukaryotic kingdoms--Protista, Fungi, Plantae and Animalia (Metazoa) . It is established that in the course of evolution these organelles may undergo the total reduction . There is no cellular organelle to be regarded as universal, indispensable . There are only three main obligatory cell components--the plasmalemma, nucleus and cytoplasm (with applied cytoskeleton, cytomembranes and ribosomes) . The reduction of flagella (cilia) is occurring in different taxa independent of the transition of protists from the flagellate type of locomotion to the amoeboid, gliding of metabolizing ones, and in the number of metazoan cells . The members of Protista and Fungi, which line in microaerobic or anaerobic conditions, nearly inevitably lose their mitochondria . The tendency to lose Golgi-complex is demonstrated in protists with parasitic mode of life, especially in combination with anaerobiosis . There is so far no satisfied morphological criterium that could say with certainty whether the lacking of flagella, Golgi complex or mitochondria in the low eukaryotes may be primary or secondary (as the result of reduction) . Data on the composition, structure and RNA nucleotide sequences cannot be either the straight evidence . A comparative analysis of these data shows that the ribosomes of the primary eukaryotes were, presumably, of a prokaryotic type . Their eukaryotization was carried out for a long time during the evolution of the low eukaryotes (Protista and Fungi), probably, independently in different phylogenetic lines . It is unknown at what steps and in what main phylogenetic lines the three above mentioned organelles may have appeared . It is proposed to single out a special division of cytology--organellology (organoidology)--as an individual science whose main purpose may be investigation of the origination, evolution and disappearance of organelles.

Annu Rev Microbiol, 1992, 46, 429 - 59
Positive regulation in the gram-positive bacterium: Bacillus subtilis; Klier A et al.; Temporally and environmentally regulated gene expression in prokaryotes occurs primarily at the level of transcription initiation . Two main modes of regulation have been described, including either the binding of a repressor that blocks transcription or the interaction of a positive regulator with the transcription complex, leading to transcription initiation . Several classes can be distinguished among positive regulators according to their mechanisms of action . This review describes the different types of positive regulators identified in Bacillus subtilis, a gram-positive bacterium . These include accessory regulatory polypeptides, classical positive regulators that bind to target sites located just upstream from the promoter, ambiactive regulators that can act both positively and negatively, antiterminators, two-component signal transduction systems, and positive regulators associated with specific secondary sigma factors.

Curr Top Microbiol Immunol, 1992, 181, 169 - 88
Lipopolysaccharide receptors and signal transduction pathways in mononuclear phagocytes; Chen TY et al.; There is little question but that bacterial lipopolysaccharides (LPS) remain one of the most potent stimuli which can affect macrophage activation . Although the precise biochemical mechanisms responsible for this remain to be fully defined, there is now evidence accumulating from a number of laboratories that functional receptors for these bacterial products do exist and may contribute to the initial triggering event . Unfortunately, there is currently no consensus as to which of the candidate receptors identified to date serves as the primary binding target for LPS, and it is possible that the difference in macrophage cell types, LPS probes, and detection systems will all influence the nature of the binding . At the present time, therefore, macromolecules of 96-kDa, 95-kDa (adhesion beta chain), 80-kDa, 65-kDa, and 55-kDa may be considered as possible LPS targets . With the exception of the 96-kDa protein identified by Hampton and his co-workers, there exists some experimental evidence for a functional role for each of the molecules so far identified . It is apparent that the molecular cloning and sequencing and subsequent biochemical characterization of these LPS receptors will be required to determine unequivocally their role in LPS-mediated triggering events . Such information will be invaluable in sorting out the relevant biochemical second signals involved in macrophage activation . Although much new information has recently been accumulated on potential signaling pathways for LPS, the definitive events remain far from unequivocally established . In view of the obvious importance of LPS-macrophage interactions in the overall capacity of the mammalian host to respond appropriately to the potentially hostile prokaryotic environment, a precise delineation of LPS-mediated macrophage activation is critical to our understanding of this important inflammatory mediator cell.

Curr Top Microbiol Immunol, 1992, 176, 195 - 211
Tracing the origin of retroviruses; Doolittle RF et al.; Reverse transcriptase sequences, which are fundamental to retrovirus existence, are widely distributed in the living world . Phylogenies based on their sequences set vertebrate retroviruses apart as relatively modern creations . Their nearest evolutionary relatives are a large group of transposable elements that have all the standard retrovirus equipment except spliced envelope proteins . The distribution of these elements suggests a long-standing presence predating the radiation of plants, fungi, and animals . There is another large group of elements, LINEs, that also contain recognizable reverse transcriptase sequences and which likely diverged even earlier, as evidenced by their presence in trypanosomes and other protists . They lack tRNA priming sites--which they could have lost--but they do exhibit characteristic eukaryotic polyadenylation . These elements are problematic in that the sequences are so degenerate in most instances that it is not possible to identify the accessory enzymes or structural proteins with any confidence, leaving major gaps in our reconstruction of events . Even with these gaps, however, the historical beginnings of retroviruses can be traced back to events coincident with the prokaryotic invasion of primitive eukaryotes.

J Anim Sci, 1992 Jan, 70(1), 289 - 95
Use of DNA probes to monitor nutritional effects on ruminal prokaryotes and Fibrobacter succinogenes S85; Briesacher SL et al.; We used DNA probes to study dietary effects on the prokaryotic population in the rumen . Procedures used to isolate and quantify prokaryotic 16S ribosomal RNA (rRNA) from the rumen using universal and species-specific DNA probes were evaluated . In this experiment, three ruminally fistulated steers were fed orchard-grass hay, and ruminal digesta were collected at 0, 3, and 9 h after offering hay (0800) . Samples of ruminal digesta were taken from the interior portion of the digesta mat and from the fluid below the mat in the dorsal rumen . Freezing (-65 degrees C) and blending samples both increased (P less than .07) the yield of 16S rRNA from ruminal digesta . Extraction of prokaryotic rRNA was greater (P less than .04) when phenol buffered with sodium acetate was used than when it was buffered with hydroxymethyl-amino-methane . Prokaryotic 16S rRNA concentration of the fluid phase was similar (P greater than .10) at 0, 3, and 9 h after offering hay . Prokaryotic 16S rRNA concentration of the mat phase increased up to the 9 h after feeding . The proportion of Fibrobacter succinogenes remained constant in both digesta phases at all times measured . From these data we concluded that DNA probes can be used to monitor bacterial population shifts in the rumen.

Clin Exp Immunol, 1992 Jan, 87(1), 80 - 6
Use of recombinant epitopes to study the heterogeneous nature of the autoantibodies against thyroid peroxidase in autoimmune thyroid disease; Zanelli E et al.; Microsomal antigen is often recognized by the sera from patients with autoimmune thyroid disease (AITD) . Human thyroid peroxidase (hTPO) is the main component of this antigen . In a previous study, we expressed hTPO cDNA as fusion proteins in prokaryotic vector; we thereby defined seven antigenic peptides by using two rabbit polyclonal anti-hTPO antibodies . In the present study we used the seven epitopes and three widened peptides to define the reactivity pattern of 61 sera from patients with AITD . Thirty-eight of them reacted against at least one of the seven hTPO-restricted epitopes; 14 were negative against the seven determinants but recognized one or two of the extended peptides . Thus, the antibody response against hTPO appeared to be highly heterogeneous in AITD patient sera . Moreover, we demonstrated that the immunodetection of the hTPO on Western blotting with deoxycholate solubilized microsomes can be perfectly correlated with the recognition of one of the epitopes in the region 554-735.

Genes Dev, 1992 Jan, 6(1), 135 - 48
A 5'-terminal stem-loop structure can stabilize mRNA in Escherichia coli; Emory SA et al.; The 5'-untranslated region of the long-lived Escherichia coli ompA transcript functions as an mRNA stabilizer capable of prolonging the lifetime in E . coli of a number of heterologous messages to which it is fused . To elucidate the structural basis of differential mRNA stability in bacteria, the domains of the ompA 5'-untranslated region that allow it to protect mRNA from degradation have been identified by mutational analysis . The presence of a stem-loop no more than 2-4 nucleotides from the extreme 5' terminus of this RNA segment is crucial to its stabilizing influence, whereas the sequence of the stem-loop is relatively unimportant . The potential to form a hairpin very close to the 5' end is a feature common to a number of stable prokaryotic messages . Moreover, the lifetime of a normally labile message (bla mRNA) can be prolonged in E . coli by adding a simple hairpin structure at its 5' terminus . Accelerated degradation of ompA mRNA in the absence of a 5'-terminal stem-loop appears to start downstream of the 5' end . We propose that E . coli messages beginning with a single-stranded RNA segment of significant length are preferentially targeted by a degradative ribonuclease that interacts with the mRNA 5' terminus before cleaving internally at one or more distal sites.

J Bacteriol, 1992 Jan, 174(2), 530 - 40
Identification of elements involved in transcriptional regulation of the Escherichia coli cad operon by external pH; Watson N et al.; Expression of the lysine decarboxylase gene (cadA) of Escherichia coli is induced upon external acidification . To dissect the molecular mechanisms responsible for this regulation, we analyzed a 4.2-kbp region upstream from cadA . DNA sequencing revealed two long open reading frames upstream of and on the same strand as cadA . One of these, cadB, is 444 codons long and is situated immediately upstream of cadA . Transcriptional fusions between fragments upstream of cadA and lacZ, Northern (RNA) hybridization, primer extension, and site-directed mutagenesis experiments defined a promoter, Pcad, upstream of cadB that was responsible for pH-regulated expression of cadA . Upstream of Pcad is an open reading frame, cadC, consisting of 512 codons . The predicted amino terminal region of the cadC gene product (CadC) resembles the carboxy-terminal domain of prokaryotic transcriptional activators involved in environmental sensing . Tn10 insertions within or immediately upstream of cadC abolished Pcad activity, suggesting that cadC encodes a positive transcription factor . Expression of plasmid-borne cadC in the Tn10 mutants restored Pcad activity, while introduction of a plasmid expressing truncated CadC resulted in the inability to complement . The presence of Pcad on a multicopy plasmid was found to lower expression arising from chromosomal Pcad, suggesting that a positive-acting factor is limiting . Our data suggests that cadA, cadB, and the acid-inducible Pcad comprise, at least in part, the cad operon which is under control of the cadC product.

Arch Med Res, 1992, 23(2), 23 - 5
P-glycoprotein genes of Entamoeba histolytica; Descoteaux S et al.; Six different P-glycoprotein gene segments were identified from an emetine-resistant E . histolytica mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene . The open reading frames of two completely sequenced genes EhPgp1 and EhPgp2 were 1,302 and 1,310 amino acids long, respectively, and showed a 67% positional identity with each other and 41 and 40% positional identities, respectively, with human mdr1 gene . Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins . A phylogenetic tree showed that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins . In addition, there were two P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site.

Bull Soc Pathol Exot, 1992, 85(5 Pt 2), 457 - 9
Role of associated bacteria in growth and toxicity of cultured benthic dinoflagellates; Gonzalez I et al.; Clonal cultures of the toxic benthic dinoflagellate Ostreopsis lenticularis isolated from the coastal waters of southwest of Puerto Rico show peak toxicities during the stationary phase of growth, correlated with significant increases in bacteria directly associated with these cells . The specific toxicity (MU/mg) of dinoflagellate extracts in control cultures increased 340% during the static phase of culture growth, while those cultures treated with antibiotics that inhibit prokaryote protein synthesis showed no significant increase in toxicity during this phase of culture growth . There was a significant decrease in the diversity of dinoflagellate associated bacterial strains in antibiotic treated cultures . These data indicate that associated bacteria play a role in toxin production by dinoflagellate-bacteria consortia when grown in laboratory culture.

Arch Med Res, 1992, 23(2), 27 - 9
Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica; Bruchhaus I et al.; A recombinant iron-containing superoxide dismutase (recFeSOD) of Entamoeba histolytica was produced in a prokaryotic expression system . Purified recFeSOD was found to be enzymatically active as determined by (i) inhibition of ferri-cytochrome c reduction, (ii) dismutation of superoxide anions generated by human neutrophils and (iii) inhibition of nitroblue tetrazolium reduction . The enzymatic properties of recFeSOD were similar to those of the native protein in trophozoite extracts . In an ELISA using recFeSOD as antigen, 96% of sera from patients having invasive amebiasis were reactive whereas none of the healthy controls or of patients suffering from malaria, bacterial or viral infections were reactive . Only sera of Toxoplasma-, Leishmania- or Trypanosoma-infected individuals exhibited partial cross-reactivity to recFeSOD.

Drugs Exp Clin Res, 1992, 18(7), 275 - 82
Synthesis and antitumour activities of quinolone antineoplastic agents; Chu DT et al.; DNA topoisomerases, found in all prokaryotic and eukaryotic cells, play a key role in controlling the topological state of DNA . Quinolone antibacterial agents have been shown to be inhibitors of DNA gyrase, a bacterial topoisomerase II enzyme . The eukaryotic topoisomerase II is the target of various cytotoxic agents such as adriamycin and etoposide . Recently, several quinolones having C-8 fluoro and C-8 chloro substituents have been found to have cytotoxic activities and to interact with mammalian topoisomerase II . In searching for an antitumour agent of the quinolone class, we identified several quinolones having excellent in vitro cytotoxic activity . A-74932 also possesses good activity in vivo against both systemic tumour and subcutaneously implanted murine solid tumours as well as human tumour xenografts . The chemical synthesis as well as biological properties of A-74932 are described.

Nucleic Acids Symp Ser, 1992, (27), 195 - 6
Inhibitory effect of oligoribonucleotide phosphorodithioates against the 3'-exonuclease activity; Sakatsume O et al.; Oligoribonucleotides consisted of modified nucleotides at 3'-turmini were chemically synthesized on the solid phase method . Moreover, these analogues were investigated the stability in a prokaryotic cell-free translation system and the resistance against a few kind of nucleases containing 3'-exonuclease activity.

Folia Microbiol (Praha), 1992, 37(5), 323 - 9
Biological function of DNA methylation; Hubacek J; Structural and functional properties of prokaryotic DNA methyltransferases are summarized . The different aspects of the role of DNA methylation which influences DNA-protein interaction in restriction and modification of DNA and in mismatch repair, DNA replication and gene expression are discussed.

Genetica, 1992, 86(1-3), 47 - 53
Nonautonomous transposable elements in prokaryotes and eukaryotes; Hartl DL et al.; Defective (nonautonomous) copies of transposable elements are relatively common in the genomes of eukaryotes but less common in the genomes of prokaryotes . With regard to transposable elements that exist exclusively in the form of DNA (nonretroviral transposable elements), nonautonomous elements may play a role in the regulation of transposition . In prokaryotes, plasmid-mediated horizontal transmission probably imposes a selection against nonautonomous elements, since nonautonomous elements are incapable of mobilizing themselves . The lower relative frequency of nonautonomous elements in prokaryotes may also reflect the coupling of transcription and translation, which may bias toward the cis activation of transposition . The cis bias we suggest need not be absolute in order to militate against the long-term maintenance of prokaryotic elements unable to transpose on their own . Furthermore, any cis bias in transposition would also decrease the opportunity for trans repression of transposition by nonautonomous elements.

Genetica, 1992, 86(1-3), 269 - 74
Evolutionary dynamics of transposable elements in prokaryotes and eukaryotes; Hickey DA; This paper summarizes some recent theories about the evolution of transposable genetic elements in outbreeding, sexual eukaryotic organisms . The evolutionary possibilities available to self-replicating transposable elements are shown to vary depending on the reproductive biology of the host genome . This effect can be used to explain, in part, the differences in abundance of transposable elements between prokaryotes and eukaryotes . It is argued that the pattern of sexual outbreeding seen in mammals and plants is especially favorable to the spread of transposons . Moreover, because transposon spread is facilitated by zygote formation, the evolutionary origin of sexual conjugation may have been due to selection on transposon-encoded genes . Finally, evidence is also presented that introns could have originated as transposable genetic elements.

Genetica, 1992, 86(1-3), 215 - 46
Genome canalization: the coevolution of transposable and interspersed repetitive elements with single copy DNA; von Sternberg RM et al.; Transposable and interspersed repetitive elements (TIREs) are ubiquitous features of both prokaryotic and eukaryotic genomes . However, controversy has arisen as to whether these sequences represent useless 'selfish' DNA elements, with no cellular function, as opposed to useful genetic units . In this review, we selected two insect species, the Dipteran Drosophila and the Lepidopteran Bombyx mori (the silkmoth), in an attempt to resolve this debate . These two species were selected on the basis of the special interest that our laboratory has had over the years in Bombyx with its well known molecular and developmental biology, and the wealth of genetic data that exist for Drosophila . In addition, these two species represent contrasting repetitive element types and patterns of distribution . On one hand, Bombyx exhibits the short interspersion pattern in which Alu-like TIREs predominate while Drosophila possesses the long interspersion pattern in which retroviral-like TIREs are prevalent . In Bombyx, the main TIRE family is Bm-1 while the Drosophila group contains predominantly copia-like elements, non-LTR retroposons, bacterial-type retroposons and fold-back transposable elements sequences . Our analysis of the information revealed highly non-random patterns of both TIRE biology and evolution, more indicative of these sequences acting as genomic symbionts under cellular regulation rather than useless or selfish junk DNA . In addition, we extended our analysis of potential TIRE functionality to what is known from other eukaryotic systems . From this study, it became apparent that these DNA elements may have originated as innocuous or selfish sequences and then adopted functions . The mechanism for this conversion from non-functionality to specific roles is a process of coevolution between the repetitive element and other cellular DNA often times in close physical proximity . The resulting interdependence between repetitive elements and other cellular sequences restrict the number of evolutionarily successful mutational changes for a given function or cistron . This mutual limitation is what we call genome canalization . Well documented examples are discussed to support this hypothesis and a mechanistic model is presented for how such genomic canalization can occur . Also proposed are empirical studies which would support or invalidate aspects of this hypothesis.

Yi Chuan Xue Bao, 1992, 19(6), 558 - 68
{The correlation between the maturation of rbcL transcripts and its flanking sequence in a prokaryotic system}; Xu Y et al.; There is a 15bp large reverse repeated sequence proceeded by a 7bp small one in the 3'flanking region of rbcL of Nicotiana tabacum . A 383 bp of XbaI fragment containing these tandemly repeats was inserted into the plasmid p lambda S delta, at the position between the lambda p and the cat gene . Then these two repeats were separated and deleted systematically to obtain various deletions . The deletion pRT65, pRT74 and pRT83 was sequenced to determine the deleted base pairs exactly . S1 mapping analysis was adopted to investigate the transcripts of these deletions in E . coli JM83 . The results showed us that the stability of mature 306bp mRNA relied on the large repeat and a short sequence downstream . The small one was not efficient . The regulation level of the rbcL termination was also investigated . The XbaI-EcoRI fragments from pRT65, pRT74 and pRT83 were transferred into pSP-TT* at the position between the spinach promoter and threonine terminator to construct pRT65, pRT74 and pRT83 respectively . The results from S1 mapping analysis showed that the E . coli RNA polymerase read through the 3' flanking region of rboL gene and terminated at the stop site of threonine terminator . These results suggested that mature rbcL mRNA might be the product precisely processed from a precursor mRNA and the 3' flanking sequence might be the signal for precursor mRNA to be processed to the correct position.

Ciba Found Symp, 1992, 171, 113 - 24; discussion 124-8
Genes for the biosynthesis of beta-lactam compounds in microorganisms; Turner G; Rapid progress has recently been made in the characterization of genes and gene clusters involved in the biosynthesis of beta-lactam antibiotics such as penicillins, cephalosporins and cephamycins . The biosynthetic pathways are found in a wide range of microorganisms, including fungi, actinomycetes and Gram-negative bacteria . Comparisons of gene sequences (particularly the genes encoding isopenicillin N synthetase) and gene organization in these different microorganisms have led to proposals about the evolution of this group of pathways, and how they might have been transferred from prokaryotes to eukaryotes . The isolation and characterization of the genes encoding ACV (tripeptide) synthetase, the first step in the beta-lactam biosynthetic pathway, have revealed the presence of three partly repeated domains, most likely responsible for the recognition, adenylation and activation of the three amino acid precursors of the penams and cephems . This has confirmed their classification as peptide synthetases, distantly related to enzymes responsible for the synthesis of peptide antibiotics in Bacillus brevis and other bacteria and fungi.

Biosystems, 1992, 28(1-3), 75 - 90
Gene phylogenies and the endosymbiotic origin of plastids; Morden CW et al.; The endosymbiotic origin of chloroplasts from cyanobacteria has long been suspected and has been confirmed in recent years by many lines of evidence . Debate now is centered on whether plastids are derived from a single endosymbiotic event or from multiple events involving several photosynthetic prokaryotes and/or eukaryotes . Phylogenetic analysis was undertaken using the inferred amino acid sequences from the genes psbA, rbcL, rbcS, tufA and atpB and a published analysis (Douglas and Turner, 1991) of nucleotide sequences of small subunit (SSU) rRNA to examine the relationships among purple bacteria, cyanobacteria and the plastids of non-green algae (including rhodophytes, chromophytes, a cryptophyte and a glaucophyte), green algae, euglenoids and land plants . Relationships within and among groups are generally consistent among all the trees; for example, prochlorophytes cluster with cyanobacteria (and not with green plastids) in each of the trees and rhodophytes are ancestral to or the sister group of the chromophyte algae . One notable exception is that Euglenophytes are associated with the green plastid lineage in psbA, rbcL, rbcS and tufA trees and with the non-green plastid lineage in SSU rRNA trees . Analysis of psbA, tufA, atpB and SSU rRNA sequences suggests that only a single bacterial endosympbiotic event occurred leading to plastids in the various algal and plant lineages . In contrast, analysis of rbcL and rbcS sequences strongly suggests that plastids are polyphyletic in origin, with plastids being derived independently from both purple bacteria and cyanobacteria . A hypothesis consistent with these discordant trees is that a single bacterial endosymbiotic event occurred leading to all plastids, followed by the lateral transfer of the rbcLS operon from a purple bacterium to a rhodophyte.

Biosystems, 1992, 28(1-3), 57 - 68
Eukaryote-eukaryote endosymbioses: insights from studies of a cryptomonad alga; Douglas SE; It has been proposed that those plants which contain photosynthetic plastids surrounded by more than two membranes have arisen through secondary endosymbiotic events . Molecular evidence confirms this proposal, but the nature of the endosymbiont(s) and the number of endosymbioses remain unresolved . Whether plastids arose from one type of prokaryotic ancestor or multiple types is the subject of some controversy . In order to try to resolve this question, the plastid gene content and arrangement has been studied from a cryptomonad alga . Most of the gene clusters common to photosynthetic prokaryotes and plastids are preserved and seventeen genes which are not found on the plastid genomes of land plants have been found . Together with previously published phylogenetic analyses of plastid genes, the present data support the notion that the type of prokaryote involved in the initial endosymbiosis was from within the cyanobacterial assemblage and that an early divergence giving rise to the green plant lineage and the rhodophyte lineage resulted in the differences in plastid gene content and sequence between these two groups . Multiple secondary endosymbiotic events involving a eukaryotic (probably rhodophytic alga) and different hosts are hypothesized to have occurred subsequently, giving rise to the chromophyte, cryptophyte and euglenophyte lineages.

Nucleic Acids Symp Ser, 1992, (27), 183 - 4
Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification; Suiko M et al.; In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form . For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation . TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2 . TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63 . Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.

Folia Biol (Praha), 1992, 38(5), 277 - 83
Expression of chimeric proteins encoded by the fused BLV reverse transcriptase: beta-galactosidase in Escherichia coli; Supek F et al.; The gene for bovine leukemia virus (BLV) reverse transcriptase was cloned in prokaryotic expression vector pUC8-2 . After fusion of Escherichia coli lacZ gene to different parts of reverse transcriptase we detected expression of new proteins with molecular weights corresponding to the size of the hybrid genes . A coding region most probably responsible for about a hundred-fold decrease in expression of long fusion proteins has been identified . A few possible causes of this phenomenon were tested.

Cell, 1991 Dec 20, 67(6), 1241 - 50
Interaction of TFIID in the minor groove of the TATA element; Lee DK et al.; TFIID binding in the minor groove of DNA at the TATA element was demonstrated by methylation interference and hydroxyl radical footprinting assays, and by binding studies with thymine analog substituted oligonucleotides . These results provide an explanation for TFIID-dependent DNA bending at the TATA element . TFIID binding shows phosphate contacts with the same residues that were found to be essential for TFIID interactions by methylation and thymine-specific modification interference assays . Based on previous studies implicating residues conserved between the direct repeats in DNA binding, as well as models of prokaryotic DNA binding proteins, these results also suggest a model in which the direct repeats of TFIID form two basic antiparallel beta ribbon arms that could contact DNA through the minor groove.

J Biol Chem, 1991 Dec 15, 266(35), 23921 - 6
Mapping of trypsin cleavage and antibody-binding sites and delineation of a dispensable domain in the beta subunit of Escherichia coli RNA polymerase; Borukhov S et al.; We have mapped principal sites in the Escherichia coli RNA polymerase molecule that are exposed to attack by trypsin under limited proteolysis conditions . The 1342-amino acid-long beta subunit is alternatively cleaved at Arg903 or Lys909 . The cleavage occurs adjacent to a dispensable domain (residues 940-1040) that is absent in the homologous RNA polymerase subunits from chloroplasts, eukaryotes, and archaebacteria . In E . coli, this region can be disrupted with genetic deletions and insertions without the loss of RNA polymerase function . Insertion of 127 amino acids into this region introduces a new highly labile site for trypsin proteolysis . The dispensable domain carries the epitope for monoclonal antibody PYN-6 (near residue 1000), which can be used for anchoring the catalytically active enzyme on a solid support . We also report the identification of a secondary trypsin cleavage at Arg81 of the beta' subunit within a putative zinc-binding domain that is conserved in prokaryotes and chloroplasts.

J Biol Chem, 1991 Dec 15, 266(35), 23698 - 705
A residue substitution in phosphoribulokinase of Synechocystis PCC 6803 renders the mutant light-sensitive; Su X et al.; We have isolated a light-sensitive mutant (BRLS) of the photosynthetic cyanobacterium Synechocystis 6803 (S . 6803) that does not survive exposure to bright light: 70% of BRLS cells die upon exposure to light of greater than 3,000 lux for 2 h . A complementing DNA fragment from wild-type cells and the corresponding DNA from the BRLS cells have been cloned and sequenced . An open reading frame is found to encode phosphoribulokinase, a key enzyme in the enzyme system for photosynthetic carbon reduction (ES-PCR) . The deduced peptide sequence of this enzyme is highly homologous to eukaryotic phosphoribulokinases but is not similar to known prokaryotic phosphoribulokinases . The mutation responsible for the phenotype of BRLS is a single nucleotide change that results in substitution of phenylalanine for Ser-222 in the phosphoribulokinase . The catalytic activity and the apparent affinity for ATP of the mutated kinase are about one-tenth and one-seventh those of the wild-type kinase, respectively . Furthermore, the mutated kinase is selectively degraded in BRLS cells in bright light . Degradation of the mutated kinase and cell death in bright light can be suppressed by inhibiting photosynthetic electron flow (PS-EF) with 3-(3,4-dichlorophenyl)-1,1-dimethylurea . The data indicate that PS-EF is not impeded by an impaired ES-PCR although the ES-PCR activity is controlled by the rate of PS-EF . Continued PS-EF in the absence of the normal substrates for carbon reduction appears to result in damage to cellular components essential for life or in the generation of lethal components.

New Biol, 1991 Dec, 3(12), 1137 - 47
A multiplicity of potential carbon catabolite repression mechanisms in prokaryotic and eukaryotic microorganisms; Saier MH Jr; The discovery of cyclic AMP (cAMP) and its receptor protein in Escherichia coli and the convincing demonstration that these molecules mediate catabolite repression of the synthesis of carbohydrate catabolic enzymes led to the widespread belief that the phenomenon of catabolite repression in bacteria was understood . It is now recognized that cAMP-independent catabolite repression mechanisms are operative in both prokaryotic and eukaryotic microorganisms . New evidence has led to the identification of a diversity of cAMP-independent regulatory mechanisms that may mediate catabolite repression in bacteria . These mechanisms utilize (i) novel transcription factors, (ii) starvation-induced RNA polymerase sigma factors, and (iii) three evolutionarily distinct protein phosphorylating enzyme systems . Although these mechanisms are not fully understood, it is suggested that they exert their effects at the transcriptional level and that phosphorylation and allosteric control by regulatory proteins are involved in these processes.

Nucleic Acids Res, 1991 Dec 11, 19(23), 6379 - 82
Identity determinants of E . coli tryptophan tRNA; Himeno H et al.; The first base pair of the acceptor stem A1-U72 and the discriminator base G73, as well as the anticodon nucleotides, characterize the tryptophan tRNA in E . coli . To determine the contribution of these nucleotides to the tryptophan acceptor activity, various transcripts of E . coli tryptophan tRNA mutants were constructed . Substitutions of the discriminator base G73, which is conserved within prokaryotic tryptophan tRNAs, impaired aminoacylation with tryptophan . Substitutions of other purine-pyrimidine pairs for A1-U72 revealed that only U72 weakly contributed to recognition by tryptophanyl-tRNA synthetase . The E . coli aspartic acid tRNA transcript introducing the tryptophan anticodon CCA showed almost the same tryptophan charging activity as the tryptophan tRNA transcript possessing a G1-C72 base pair . Only a low activity was detected in the mutant tryptophan tRNA transcript possessing a set of G1-C72 and A73, which is observed in eukaryotic tryptophan tRNAs . These results indicate that the anticodon and G73 are major identity determinants of tryptophan tRNA in E . coli, whereas the A1-U72 base pair is only a weak recognition element.

J Biol Chem, 1991 Dec 5, 266(34), 23103 - 11
Bombinin-like peptides with antimicrobial activity from skin secretions of the Asian toad, Bombina orientalis; Gibson BW et al.; The structures and hemolytic and bactericidal activities of three bombinin-like peptides, or BLP-1-3, from the skin of Bombina orientalis are described . The peptides were isolated from the skin of B . orientalis and sequenced by tandem mass spectrometry and are amphipathic, cationic peptides of 25-27 amino acids in length . The sequence of the most abundant member (BLP-1) is: Gly-Ile-Gly-Ala-Ser-Ile-Leu-Ser-Ala-Gly-Lys-Ser-Ala-Leu-Lys-Gly-Leu- Ala-Lys-Gly-Leu-Ala-Glu-His-Phe-Ala-Asn-NH2 . All three peptides were found to share considerable, but not complete, homology with bombinin, an antimicrobial, hemolytic peptide first isolated by Michl and Csordas (Csordas, A., and Michl, A . (1970) Monatsh . Chem . 101, 182-189) from the skin of Bombina variegata . The BLPs have been assayed for antibiotic and hemolytic activity and found to be more potent than magainin 2 (a related antimicrobial peptide from Xenopus laevis) in their ability to kill bacteria . However, no significant hemolytic activity was found for these peptides which suggests a selectivity for prokaryotic over eukaryotic membranes . The molecular basis for antibacterial activity is presumed to be due to their predicted amphipathic alpha-helical structures which is supported by circular dichroism measurements that found significant helical content (63-69% alpha-helix) in 40% trifluoroethanol . Last, a cDNA library was constructed from the skin of B . orientalis and screened with an oligonucleotide probe complementary to the COOH terminus of BLP-1 . Several clones were isolated and sequenced that encode BLP-1 and BLP-3, as well as an additional peptide (BLP-4) that differs by two amino acid substitutions from BLP-3.

J Biol Chem, 1991 Dec 5, 266(34), 22800 - 2
Ribosome-independent GTPase activity of translation initiation factor IF2 and of its G-domain; Severini M et al.; In the absence of ribosomes, Bacillus stearothermophilus translation initiation factor IF2 (Mr = 82 kDa) and its GTP-binding domain (i.e . the G-domain, Mr = 41 kDa) promote barely detectable hydrolysis of GTP . Upon addition of some aliphatic alcohols, however, the rate of nucleotide cleavage is substantially increased with both IF2 and G-domain, the highest stimulation being observed with 20% (v/v) ethanol . Under these conditions, the rates of ribosome-independent GTP hydrolysis with both IF2 and G-domain are approximately 30-fold lower than the corresponding rates obtained in the presence of ribosomes, while the Km for GTP is approximately the same in all cases . These results indicate that, as with the other two prokaryotic G proteins involved in translation (i.e . elongation factors EF-Tu and EF-G), also in the case of IF2, the GTPase catalytic center resides in the factor and, more specifically, in its G-domain.

J Biol Chem, 1991 Dec 5, 266(34), 22826 - 31
Histidine tRNA guanylyltransferase from Saccharomyces cerevisiae . I . Purification and physical properties; Pande S et al.; Compared to other tRNAs all known histidine tRNAs have the unique feature of possessing an additional nucleotide at their 5' end . It is usually a guanosine residue but not in bacteriophage T5 tRNA which carries an additional uridine . The additional nucleotide is not encoded in eukaryotic histidine tRNA genes but is added in a post-transcriptional modification reaction (Cooley, L., Appel, B., and Soll, D . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 6475-6479) by histidine tRNA guanylyltransferase (tRNAHis guanylyltransferase) . Here we report the purification of this enzyme from Saccharomyces cerevisiae and the determination of some of its physical properties . Six different steps including Polymin P precipitation, chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, ATP-agarose, and gel filtration on Superose 12 were employed for the purification of the guanylyltransferase from an S-100 extract . A Stokes radius of 46.5 +/- 0.5 A and a sedimentation coefficient (S20,w) of 7.8 +/- 0.2 were determined by gel filtration and rate zonal sedimentation, respectively . A relative molecular weight (Mr) of approximately 120,000 was calculated for the purified native enzyme . The Mr of the denatured protein is approximately 58,000 as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . These results indicate a homodimeric (alpha 2) structure for the enzyme . Among all acceptor RNAs in unfractionated tRNA only tRNAHis is a substrate for the purified guanylyltransferase . The reaction requires ATP, a guanosine substrate, and a divalent metal ion . Treatment of guanylyltransferase with 5,5'-dithiobis(2-nitrobenzoic)-acid abolishes activity; this suggests the importance of sulfhydryl groups for enzymatic activity . The enzyme shows discrimination among different histidine tRNA species; tRNA from plant and prokaryotes are better substrates than mammalian and insect tRNAs.

Plant Mol Biol, 1991 Dec, 17(6), 1241 - 3
The full precursor of the 33 kDa oxygen-evolving complex protein of wheat is exported by Escherichia coli and processed to the mature size; Meadows JW et al.; A full-length cDNA encoding the precursor of the lumenal 33 kDa oxygen-evolving complex protein from wheat was inserted into a prokaryotic expression vector . Cell-free transcription-translation of this construct generates a precursor protein of the correct size . However, when expressed in Escherichia coli, the protein is quantitatively exported into the periplasm and processed to the mature size . The results indicate that the thylakoid transfer sequence of this precursor can function as an internal E . coli export signal.

Nucleic Acids Res, 1991 Dec, 19(25), 7131 - 7
Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice; Lettmann C et al.; In cultured mammalian cells, foreign DNA can be integrated into the host genome . Foreign DNA is frequently de novo methylated in specific patterns with successive cell generations . The sequence-specific methylation of promoter sequences in integrated foreign DNA is associated with the long-term inactivation of eukaryotic genes . We have now extended these experiments to studies on transgenic mice . As in previous work, a construct (pAd2E2AL-CAT) has been used which consists of the late E2A promoter of adenovirus type 2 (Ad2) DNA fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) . This construct has been integrated in the non-methylated in the 5'-CCGG-3' premethylated form in the genomes of transgenic mice . DNA from various organs was analyzed by HpaII/MspI cleavage to assess the state of methylation in 5'-CCGG-3' sequences . The results demonstrate that the transgenic construct is in general stable . Non-methylated constructs have remained partly non-methylated for four generations or can become de novo methylated at all or most 5'-CCGG-3' sequences in the founder animal . Preimposed patterns of 5'-CCGG-3' methylation have been preserved for up to four generations beyond the founder animal . In the testes of two different founder animals and two F1 males, the transgenic DNA has become demethylated by an unknown mechanism . In all other organs, the transgenic DNA preserves the preimposed 5'-CCGG-3' methylation pattern . In the experiments performed so far we have not observed differences in the transmission of methylation patterns depending on whether the transgene has been maternally or paternally inherited . The 5'-CCGG-3' premethylated transgene does not catalyze CAT activity in several organs, except in one example of the testes of an animal in which the transgenic construct has become demethylated . In contrast, when the nonmethylated construct has been integrated and remained largely non-methylated, CAT activity has been detected in extracts from some of the organs.

Biosci Rep, 1991 Dec, 11(6), 445 - 74; discussion 474-5
Chemiosmotic systems in medicine; Garland PB; The concept of chemiosmotic systems arises from the pioneering work of Peter Mitchell on two fronts . One is concerned with the mechanisms by which molecules are transported across membranes which are generally barriers to such transport . These mechanisms are inevitably molecular, and are now yielding their secrets to a combination of structural protein chemistry and molecular biology . The other front is more physiological, and explores the functional relationships between metabolism and transport . Nevertheless, the two fronts form a continuum of mutually related structure and function . Chemiosmotic systems provide a hierarchy of complexity, starting from say a uniporter reconstituted in a chemically defined bilayer, and proceeding to greater complexity in mitochondria, chloroplasts, eukaryotic and prokaryotic cell membranes, and multicellular systems . Their relationship to medicine is profound, because they provide many opportunities for therapeutic intervention . In this paper I present an overview of chemiosmotic systems at different levels of complexity, both molecular and biological, of their involvements in pathology, and of possible pharmacological treatment or prevention of disease.

Pharmacol Ther, 1991 Dec, 52(3), 331 - 63
Angelicins, angular analogs of psoralens: chemistry, photochemical, photobiological and phototherapeutic properties; Bordin F et al.; Angelicin and some of its derivatives are naturally occuring compounds which show interesting photobiological properties . In this review various aspects of angelicin and its derivatives have been reported . The natural occurrence and the chemical synthesis both of naturally occurring and synthetic angelicins have been reviewed . Photochemical and photophysical properties of angelicins have been considered with particular reference to the capacity to generate active forms of oxygen, photoreactions with nucleic acids, proteins and unsaturated fatty acids . Photobiological effects have been considered: skin phototoxicity, antiproliferative effects, genotoxicity, ability to induce hemolysis in erythrocytes, inactivation of prokaryotic and eukaryotic microorganism and of viruses . The ability of some angelicins to induce photocarcinogenesis has been reviewed as well as in the activity as photochemotherapeutic agents.

J Biomol Struct Dyn, 1991 Dec, 9(3), 537 - 52
Eukaryotic DNAs in solution contain characteristic components of tertiary structure; Reinert KE et al.; For natural eukaryotic DNA in solution, we suggest the existence of secondary-helix components superponed to parts of the DNA double helices . In a previous report we found, for calf thymus DNA in solution and of different mean molar mass Mr, an electrostatically driven rise of the hydrodynamically operative contour length of the double helix . This result was derived from Mr-dependent systematic deviations from the almost but not exactly linear plots of intrinsic viscosity {eta} as a function of 1/cs1/2 (cs = Na+ concentration) accurately determined by a titration technique {K.G . and K.E.R., Nucl . Acids Res . 8, 2807 (1980)} . In order to discriminate between DNA elongation contributions caused by secondary or by tertiary structure effects, respective measurements have now been extended to different temperatures for two eukaryotic and two prokaryotic DNA species . The slope of the curves obtained for the (apparent) gradual elongation effect as a function of temperature is negative for the eukaryotic DNAs investigated and is smaller and positive for the prokaryotic species, thus revealing different underlying main elongation mechanisms . We propose that, for the eukaryotic DNA samples, an electrostatically driven partial abolition of tertiary structure components is responsible for the prevailing part of the DNA elongation effect measured . (A helix elongation of this type may be the result of an abolition of an apparent helix shortening as realized in a very high degree on formation of nucleosome chains or in a less degree by DNA molecules with a respective evolutionarily fitted tertiary structure) . For the smaller effects of prokaryotic DNA species something like a base breathing seems to dominate . Recent literature results support such an interpretation.

J Cell Sci, 1991 Dec, 100 (Pt 4), 687 - 91
How relevant is the Escherichia coli UvrABC model for excision repair in eukaryotes?
Hoeijmakers JH.
Knowledge about the DNA excision repair system is increasing rapidly . A detailed model for this process in Escherichia coli has emerged in which a lesion in the DNA is first recognized by the UvrA2B helicase complex . Subsequently, UvrC mediates incision on both sites of the DNA injury . Finally, the concerted action of helicase II (UvrD), polymerase and ligase takes care of removal of the damage-containing oligonucleotide, DNA resynthesis and sealing of the residual nick . In the eukaryotes, yeast and mammals a total of 10 excision repair genes have been analysed thus far . However, little is still known about the molecular mechanism of this repair reaction . Amino acid sequence comparison suggests that at least three DNA helicases operate in eukaryotic nucleotide excision . In addition, a striking sequence conservation is noted between human and yeast repair proteins . But no eukaryotic homologs of the UvrABC proteins have been identified . In this Commentary the parallels and differences between the prokaryotic and eukaryotic excision repair pathways are weighed in an attempt to assess the relevance of the E . coli model for the eukaryotic system.

Curr Opin Cell Biol, 1991 Dec, 3(6), 1013 - 8
Stability and degradation of mRNA; Higgins CF; Differential mRNA stability plays an important role in the regulation of gene expression . Several recent advances have helped to define the general pathways by which mRNA is degraded in prokaryotic cells, although many details remain to be elucidated . Much less is known about the pathways of degradation in eukaryotic cells, but recent studies on specific systems have highlighted both differences from and similarities to prokaryotic pathways.

Biochimie, 1991 Dec, 73(12), 1557 - 66
Structural and functional domains of E coli initiation factor IF2; Laalami S et al.; Initiation of translation in prokaryotes requires the participation of at least three soluble proteins: the initiation factors IF1, IF2 and IF3 . Initiation factor 2, which is one of the largest proteins involved in translation (97.3 kDa) has been shown to stimulate in vitro the binding of fMet-tRNA(fMet) to the 30S ribosomal subunit . After formation of 70S translation initiation complex, IF2 is believed to participate in GTP hydrolysis, thereby promoting its own release . Here we review evidence which indicates the functional importance of the different structural domains of IF2, emphasizing new information obtained by in vivo experiments.

Microbiol Rev, 1991 Dec, 55(4), 675 - 83
Recombination-dependent concatemeric plasmid replication; Viret JF et al.; The replication of covalently closed circular supercoiled (form I) DNA in prokaryotes is generally controlled at the initiation level by a rate-limiting effector . Once initiated, replication proceeds via one of two possible modes (theta or sigma replication) which do not rely on functions involved in DNA repair and general recombination . Recently, a novel plasmid replication mode, leading to the accumulation of linear multigenome-length plasmid concatemers in both gram-positive and gram-negative bacteria, has been described . Unlike form I DNA replication, an intermediate recombination step is most probably involved in the initiation of concatemeric plasmid DNA replication . On the basis of structural and functional studies, we infer that recombination-dependent plasmid replication shares important features with phage late replication modes and, in several aspects, parallels the synthesis of plasmid concatemers in phage-infected cells . The characterization of the concatemeric plasmid replication mode has allowed new insights into the mechanisms of DNA replication and recombination in prokaryotes.

Mol Biochem Parasitol, 1991 Dec, 49(2), 289 - 96
The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases; Fox BA et al.; We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum . Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources . Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P . falciparum DNA polymerase delta . P . falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels . The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active.

Gene, 1991 Dec 1, 108(1), 47 - 53
Identification and characterization of the ribosomal RNA-encoding genes in Clavibacter xyli subsp . cynodontis; Sathyamoorthy M et al.; Clavibacter xyli subsp . cynodontis (Cxc) is a xylem-inhabiting bacterial endophyte of Bermudagrass . This organism is classified with Gram-positive, high G + C content, coryneform-actinomycete bacteria . Southern-blot analysis showed that Cxc contains only one copy of the ribosomal RNA-encoding genes (rRNA) . A clone containing the rRNA genes was isolated from a genomic library of Cxc DNA cloned in the lambda EMBL3 vector . The gene cluster was partially sequenced, revealing the gene order 5'-16S-23S-5S-3', similar to that found in other prokaryotes . Low-resolution S1 mapping suggested multiple transcription start points of the rRNA operon.

EMBO J, 1991 Dec, 10(13), 4267 - 77
A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase; Cerini C et al.; In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa . We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex . We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase . It is composed of three major domains, two of them specifying distinct synthetase activities . The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively . The central domain is made of six 46 amino acid repeats . In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes . The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells . This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle . The involvement of the internal repeats in mediating complex assembly is discussed.

Clin Exp Immunol, 1991 Dec, 86(3), 478 - 82
Anti-human thyroid peroxidase and anti-human thyroglobulin antibodies present no cross-reactivity on recombinant peptides; Henry M et al.; Thyroglobulin (Tg) and thyroid peroxidase (TPO) are two antigens largely recognized by the sera from patients with autoimmune thyroid disease (AITD) . Recently, the complete mapping of both antigens was established with rabbit polyclonal antibodies by the use of recombinant proteins expressed in prokaryotic vector . Several investigators have argued for the existence of a cross-reactivity of some hetero- and autologous antibodies versus these two proteins . In the present study, using rabbit polyclonal antibody, mouse polyclonal antibody and autoimmune antibody (aAb), we observed no common epitope on human Tg (hTg) and human TPO (hTPO).

Exp Cell Res, 1991 Dec, 197(2), 229 - 33
Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells; Izumi M et al.; Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes . The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection . The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells . The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells . Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S . Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene . Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.

Nucleic Acids Res, 1991 Dec, 19(25), 7193 - 9
Visualization of novel simian virus 40 DNA recombination intermediates induced by ultraviolet light irradiation; Hsu MT; Electron microscopic technique was used to examine the structures of SV40 DNA recombination intermediates induced by ultraviolet irradiation as an approach for understanding recombination mechanisms in animal cells . Putative recombination intermediate with the characteristic Holliday junction was observed in both SV40 and CV-1 monkey kidney cell DNA . These results suggest that Holliday recombination intermediate is a common intermediate in eukaryotic as well as prokaryotic recombination pathways . In UV irradiated cells, putative SV40 DNA recombination intermediates with multiple recombining partners were observed . In addition, UV irradiation induced two types of novel joint molecules of SV40 DNA . The first type contains replication intermediates as one of the joint molecules with the putative recombination junction located in the newly replicated DNA arms . The second type of novel joint molecules is represented by of the 'dumbbell' structures with two circular SV40 DNA linked by a linear DNA of varying lengths . The structures of these novel recombination intermediates suggest a strand-invasion mechanism for UV-induced DNA recombination.

Nucleic Acids Res, 1991 Nov 25, 19(22), 6215 - 20
Cloning, sequencing and overexpression of the gene for prokaryotic factor EF-P involved in peptide bond synthesis; Aoki H et al.; A soluble protein EF-P (elongation factor P) from Escherichia coli has been purified and shown to stimulate efficient translation and peptide-bond synthesis on native or reconstituted 70S ribosomes in vitro . Based on the partial amino acid sequence of EF-P, 18- and 24-nucleotide DNA probes were synthesized and used to screen lambda phage clones from the Kohara Gene Bank . The entire EF-P gene was detected on lambda clone #650 which contains sequences from the 94 minute region of the E.coli genome . Two DNA fragments, 3.0 and 0.78 kilobases in length encompassing the gene, were isolated and cloned into pUC18 and pUC19 . Partially purified extracts from cells transformed with these plasmids overrepresented a protein which co-migrates with EF-P upon SDS polyacrylamide gel electrophoresis, and also exhibited increased EF-P mediated peptide-bond synthetic activity . Based on DNA sequence analysis of this gene, the EF-P protein consists of 187 amino acids with a calculated molecular weight of 20,447 . The sequence and chromosomal location of EF-P establishes it as a unique gene product.

Nucleic Acids Res, 1991 Nov 25, 19(22), 6183 - 90
The sequence specificity domain of cytosine-C5 methylases; Klimasauskas S et al.; Prokaryotic DNA{cytosine-C5}methyltransferases (m5C-methylases) share a common architectural arrangement of ten conserved sequence motifs . A series of eleven hybrids have been constructed between the HpaII (recognition sequence: Cm5CGG) and HhaI (recognition sequence: Gm5CGC) DNA-methylases . The hybrids were over-expressed in E.coli and their in vivo methylation phenotypes investigated . Six were inactive by our assay while five of them retained partial methylation activity and full specificity . In all five cases the specificity matched that of the parent methylase which contributed the so-called variable region, located between conserved motifs VIII and IX . This was the only sequence held in common between the active hybrids and for the first time provides unequivocal evidence that the specificity determinants of the mono-specific m5C-methylases are located within the variable region . Correlation of the hybrid methylase structure with the efficiency of methylation suggests that conserved motif IX may interact with the variable region whereas motif X most probably interacts with the N-terminal half of the molecule.

Science, 1991 Nov 22, 254(5035), 1205 - 7
Functional importance of sequence in the stem-loop of a transcription terminator; Cheng SW et al.; Intrinsic transcription terminators of prokaryotes are distinguished by a common RNA motif: a stem-loop structure high in guanine and cytosine content, followed by multiple uridine residues . Models explaining intrinsic terminators postulate that the stem-loop sequence is necessary only to form structure . In the tR2 terminator of coliphage lambda, single-nucleotide changes reducing potential RNA stem stability eliminated tR2 activity, and a compensatory change that restored the stem structure restored terminator activity . However, multiple changes in the stem sequence that should have either maintained or increased stability reduced terminator activity . These results suggest that the ability of the stem-loop structure to signal transcription termination depends on sequence specificity and secondary structure.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 9999 - 10003
The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex; Flores O et al.; We found that transcription factor IIF mediates the association of RNA polymerase II with promoter sequences containing transcription factors IID, IIB, and IIA (DAB complex) . The resulting DNA-protein complex contained RNA polymerase II and the two subunits of transcription factor IIF (RAP 30 and RAP 74) . Cloned human RAP 30 was sufficient for the recruitment of RNA polymerase II to the DAB complex . This ability of RAP 30 to recruit RNA polymerase to a promoter is also a characteristic of sigma factors in prokaryotes.

Proc Natl Acad Sci U S A, 1991 Nov 15, 88(22), 10104 - 8
Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific; Shuman S; Specialized type I topoisomerases catalyze DNA strand transfer during site-specific recombination in prokaryotes and fungi . As a rule, the site specificity of these systems is determined by the DNA binding and cleavage preference of the topoisomerase per se . The Mr 32,000 topoisomerase I encoded by vaccinia virus (a member of the eukaryotic family of "general" type I enzymes) is also selective in its interaction with DNA; binding and cleavage occur in vitro at a pentameric motif 5'-(C or T)CCTT in duplex DNA . Expression of vaccinia virus DNA topoisomerase I in a lambda lysogen of Escherichia coli promotes int-independent excisive recombination of the prophage . To address whether the topoisomerase directly catalyzes DNA strand transfer in vivo, the recombination junctions of plaque-purified progeny phage were cloned and sequenced . In five of six distinct excision events examined, a topoisomerase cleavage sequence is present in one strand of the DNA duplex of both recombining partners . Recombination entails no duplication, insertion, or deletion of nucleotides at the crossover points, consistent with excision via conservative strand exchange at sites of topoisomerase cleavage . Three of these five recombination events are distinguished by the presence of direct repeats at the parental half-sites that extend beyond the pentameric cleavage motif, suggesting that sequence homology may facilitate excision . The data are consistent with a model in which vaccinia topoisomerase catalyzes reciprocal strand transfer, leading to the formation of a nonmigrating Holliday junction, the resolution of which can lead to excisive recombination.

Nucleic Acids Res, 1991 Nov 11, 19(21), 5923 - 7
Eukaryotic DNA does not form nucleosomes as readily as some prokaryotic DNA; Getts RC et al.; Nucleosomal-length DNA was prepared from the genomic DNA of various prokaryotic and eukaryotic organisms by limited nuclease digestion after reconstitution with core histones . The DNAs ranged in base composition from 26.5% to 72% guanosine-plus-cytosine (%GC) . The nucleosomal-length DNAs were then used in a competitive reconstitution assay in order to quantitatively determine their relative abilities to form nucleosomes . The results of the assay indicate a linear dependence of the free energy of nucleosome formation on base composition and, surprisingly, show that several prokaryotic DNAs form nucleosomes as well as or better than eukaryotic DNAs.

J Theor Biol, 1991 Nov 7, 153(1), 111 - 35
Trends in the 5' vs . 3' flanks of oligonucleotides in eukaryotic and prokaryotic genomes: the asymmetric roles played by cytosine and guanine; Nussinov R et al.; Studies of sequence context preferences of oligonucleotides composed of (G/C)n and (A/T)m blocks (n + m = 3,4,5) unravel strong patterns . Comparisons of the 5' and 3' nearest neighbor doublets flanking these oligomers reveal the preference of (G/C)2 to be positioned immediately next to the (A/T)m block, enclosing it by (G/C) nucleotides rather than extending the (G/C)n block . That is, for a (G/C)n(A/T)m oligomer and a (G/C)2 doublet, (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2 (A/T)m . Similarly for an (A/T)m(G/C)n oligomer, (G/C)2(A/T)m(G/C)n greater than (A/T)m(G/C)n + 2 . In an analogous manner, (A/T)2 flanking doublets prefer enclosing the (G/C)n blocks, although these patterns are weaker . Here we show a strong, direct relationship between the magnitude of the trends and the presence of Cs in the (G/C)n block in the (G/C)n(A/T)m oligomer, and the presence of Gs in the complementary (A/T)m(G/C)n oligomers . The trends are stronger in eukaryotic than in prokaryotic sequences . They are stronger for longer (G/C)n and shorter (A/T)m blocks . We suggest that the preference for (A/T)m to be enclosed by (G/C) rather than be flanked by them on only one side is related to DNA structure and DNA-protein interaction . Sequences of the (G/C)(A/T)(G/C) type may have more homogeneous minor groove geometry . In particular, the strong G vs . C asymmetry in the trends may be related to pyrimidine-purine junctions, possibly to CG sequences.

FEBS Lett, 1991 Nov 4, 292(1-2), 76 - 8
Immuno-chemical non-cross-reactivity between eukaryotic and prokaryotic seryl-tRNA synthetases; Sidorik LL et al.; Monospecific polyclonal antibodies (pAbs) against highly purified bovine seryl-tRNA synthetase (SerRS, EC 6.1.1.1) were prepared and their specificity tested . The interactions of pAbs with SerRS from different organisms were investigated by protein immunoblotting and ELISA methods . pAbs inhibit eukaryotic SerRS aminoacylating activity and exert no effect on SerRS activity from prokaryotes . It is proposed that prokaryotic and eukaryotic SerRS evolve from different ancestor genes.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9751 - 4
A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli; Andersson H et al.; Signal sequences serve to target proteins to the secretory pathway in both prokaryotic and eukaryotic cells . However, although necessary, the presence of a signal sequence is not always sufficient to ensure efficient membrane translocation . One feature of the nascent chain that adversely affects secretion, at least in Escherichia coli, is the presence of positively charged amino acids immediately downstream of the signal sequence . We have exploited this sensitivity to positively charged residues to demonstrate the presence of a sharply delimited "export initiation domain" that comprises the signal sequence and its approximately 30 downstream residues . A string of six consecutive lysines completely blocks translocation when placed inside this domain but not when placed only a few residues further away.

Proc Natl Acad Sci U S A, 1991 Nov 1, 88(21), 9553 - 7
Sequence of general transcription factor TFIIB and relationships to other initiation factors; Malik S et al.; Transcription factor TFIIB is a ubiquitous factor required for transcription initiation by RNA polymerase II . Previous studies have suggested that TFIIB serves as a bridge between the "TATA"-binding factor (TFIID) and RNA polymerase II during preinitiation complex assembly and, more recently, that TFIIB can be a target of acidic activators . We have purified TFIIB to homogeneity, shown that activity resides in a 33-kDa polypeptide, and obtained cDNAs encoding functional TFIIB . TFIIB contains a region with amino acid sequence similarity to a highly conserved region of prokaryotic sigma factors . This is consistent with analogous functions for these factors in promoter recognition by RNA polymerases and with similar findings for TFIID, TFIIE, and TFIIF/RAP30 . Like TFIID, TFIIB contains both a large imperfect repeat that could contribute an element of symmetry to the folded protein and clusters of basic residues that could interact with acidic activator domains . These findings argue for a common origin of TFIIB, TFIID, and other general transcription factors and for the evolutionary segregation of complementary functions.

Mol Gen Genet, 1991 Nov, 230(1-2), 120 - 8
Cloning and sequence analysis of genes involved in erythromycin biosynthesis in Saccharopolyspora erythraea: sequence similarities between EryG and a family of S-adenosylmethionine-dependent methyltransferases; Haydock SF et al.; The gene cluster (ery) responsible for production of the macrolide antibiotic erythromycin by Saccharopolyspora erythraea is also known to contain ermE, the gene conferring resistance to the antibiotic . The nucleotide sequence has been determined of a 4.5 kb portion of the biosynthetic gene cluster, from a region lying between 3.7 kb and 8.2 kb 3' of ermE . This has revealed the presence of four complete open reading frames, including the previously known ery gene eryG, which catalyses the last step in the biosynthetic pathway . Comparison of the amino acid sequence of EryG with the sequence of other S-adenosylmethionine (SAM)-dependent methyltransferases has revealed that one of the sequence motifs previously suggested to be part of the SAM-binding site is present not only in EryG but also in many other recently sequenced SAM-dependent methyltransferases . Previous genetic studies have shown that this region also contains gene(s) involved in hydroxylation of the intermediate 6-deoxyerythronolide B . One of the three other open reading frames (eryF) in fact shows very high sequence similarity to known cytochrome P450 hydroxylases . An adjacent gene (ORF5) shows a strikingly high degree of similarity to prokaryotic and eukaryotic acyltransferases and thioesterases.

Infect Immun, 1991 Nov, 59(11), 4117 - 24
Identification of Mycobacterium leprae antigens from a cosmid library: characterization of a 15-kilodalton antigen that is recognized by both the humoral and cellular immune systems in leprosy patients; Sela S et al.; Screening of the Mycobacterium leprae cosmid library with pooled sera from lepromatous leprosy (LL) patients by a colony immunoblot technique resulted in the identification of about 100 colonies that produced immunologically reactive proteins . Twenty-four of these clones were purified, analyzed, and found to comprise two groups according to the reactivity of the recombinant proteins with LL sera and to the DNA restriction patterns of the recombinant plasmids and cosmids . Proteins specified by clones from group I reacted strongly with LL patients' sera on a Western blot (immunoblot), demonstrating a 15-kDa protein band designated A15 . The A15 antigen also reacted with pooled sera from patients with tuberculoid leprosy from the United States and Brazil . Clones from group II did not show any reactive protein band on a Western blot, when reacted with patients' sera . DNAs from cosmids of group II all contain a 10-kb PstI fragment that hybridized to the unique repetitive M . leprae DNA . Sequence analysis of a 1.2-kb fragment containing the entire coding sequence of A15 revealed three open reading frames (ORFs), only one of which (ORF II) contains sufficient genetic information to encode for A15 . Part of the A15 gene was found to exist also in a group of lambda gt11:M . leprae clones previously isolated in our laboratory by immunological screening with LL patients' sera . One of the lambda gt11 clones (L8) expresses a beta-galactosidase fusion protein with 89 amino acids from the C terminus of A15 . An important result was that the fusion protein was clearly recognized by T cells from leprosy patients . Interestingly, Mycobacterium tuberculosis-stimulated T cells from M . leprae nonresponder (LL as well as borderline tuberculoid) patients were able to respond to the isolated recombinant M . leprae antigen, indicating that nonresponsiveness to M . leprae antigens can be reversible . The sequence of the M . leprae DNA fused to the beta-galactosidase gene of lambda gt11 clone L8 was identical to that of a lambda gt11:M . leprae clone isolated recently that expresses an immunologically reactive fusion protein (S . Laal, Y . D . Sharma, H . K . Prasad, A . Murtaza, S . Singh, S . Tangri, R . Misra, and I . Nath, Proc . Natl . Acad . Sci . USA 88:1054-1058, 1991) . Besides the complete sequence of the A15 gene, sequencing data of two flanking ORFs are presented . Downstream from ORF II (A15), ORF III has a high degree of similarity to the genes for tomato ATP-dependent proteases that are members of a larger class of highly conserved proteases ubiquitous among prokaryotes and eukaryotes.(ABSTRACT TRUNCATED AT 400 WORDS)

EMBO J, 1991 Nov, 10(11), 3379 - 86
Anatomy of the parp gene promoter of Trypanosoma brucei; Sherman DR et al.; While growing in the tsetse fly, Trypanosoma brucei expresses a major surface glycoprotein, the procyclic acidic repetitive protein (PARP) . The parp genes are transcribed by an alpha-amanitin-resistant RNA polymerase . We have determined the sequence requirements for parp promoter activity . Studies of RNA produced from input DNA in transiently transfected trypanosomes indicate that the RNA is correctly processed by trans-splicing and polyadenylation . Deletion analyses show that 330 bp are sufficient for full promoter and splicing activity and that the promoter structure is complex, involving at least three elements whose mutual spacing is important . Mutagenesis pin-pointed two sequences vital for promoter activity; neither bears any resemblance to known prokaryotic or eukaryotic promoter elements.

Curr Genet, 1991 Nov, 20(5), 427 - 30
Plastid genomes of the Rhodophyta and Chromophyta constitute a distinct lineage which differs from that of the Chlorophyta and have a composite phylogenetic origin, perhaps like that of the Euglenophyta; Markowicz Y et al.; A phylogenetic tree has been constructed from comparisons of entire 16S rRNA gene sequences from different prokaryotes and from several algal plastids . According to this study, and to previous work on the ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) large and small subunit genes, we postulate that: (1) rhodophyte and chromophyte plastid genomes have a common, composite phylogenetic origin which implies at least two different ancestors, a cyanobacterial and a beta-proteobacterial ancestor; (2) chlorophyte (green algae and land plants) plastids have a cyanobacterial ancestor which probably differs from that of rhodophyte and chromophyte plastids, and in any case constitute a different lineage; (3) euglenophyte plastid genomes also seem to have a composite phylogenetic origin which involves two different lineages.

Neurosci Behav Physiol, 1991 Nov-Dec, 21(6), 559 - 68
Pathways of the evolution of hormonal signal realization systems; Pertseva MN; The problem of the structural-functional organization, and of the origin and evolution of the chemosignal systems which realize the effect of hormones and hormone-like substances in the higher eukaryotes-lower eukaryotes-prokaryotes series, is reviewed on the basis of an analysis of published information and our own data . The notion that the systems of the conduction and transduction of chemical signals are related to universal and evolutionarily ancient structures is formulated . The roots of these systems take their origin in the single-celled eukaryotes, while individual of their functional units take their origin even in the prokaryotes . The hypothesis is advanced that the progressive evolution of chemosignal systems has traveled common pathways, and has consisted in the linking up and the functional combination of originally general and conservative units in the direction of the ever-increasing specialization of these systems and augmentation of their efficiency.

Biol Chem Hoppe Seyler, 1991 Nov, 372(11), 971 - 4
Nucleotide-activated oligosaccharides are intermediates of the cell wall polysaccharide of Methanosarcina barkeri; Hartmann E et al.; The cell wall of Methanosarcina barkeri consists of a heteropolysaccharide (methanochondroitin), which resembles the eukaryotic chondroitin . From cell extracts of Methanosarcina barkeri four uridine diphosphate and one undecaprenyl pyrophosphate-activated intermediate(s) of the methanochondroitin were isolated . In contrast to the known biosynthetic pathways of polysaccharides from other prokaryotes and eukaryotes, nucleotide activated oligosaccharide precursors are involved in the case of the methanochondroitin . Usually, oligosaccharides are synthesized at the lipid stage.

Trends Biochem Sci, 1991 Nov, 16(11), 397 - 402
Prokaryotic transcriptional enhancers and enhancer-binding proteins; Kustu S et al.; A number of prokaryotic enhancer-binding proteins activate transcription by specialized forms of RNA polymerase . The enhancer-binding proteins catalyse isomerization of the initial complex formed between RNA polymerase and a promoter from the closed to the open state . To do so, one class of enhancer-binding proteins contacts its cognate polymerase by DNA loop formation but the other, which is represented by a single member, does not . Despite this difference, both classes of enhancer-binding proteins must hydrolyse ATP to catalyse open complex formation.

Trends Biochem Sci, 1991 Nov, 16(11), 394 - 7
Competing promoters in prokaryotic transcription; Goodrich JA et al.; Two (or more) bacterial promoters are often found in close proximity, and may compete for the binding of RNA polymerase . These competing promoters have interesting characteristics in vitro and analysis of the competition should be valuable to kinetic studies of more complex transcription systems.

Mol Biochem Parasitol, 1991 Nov, 49(1), 61 - 71
Pathogenic and nonpathogenic Entamoeba histolytica: identification and molecular cloning of an iron-containing superoxide dismutase; Tannich E et al.; Superoxide dismutase (SOD) activity was determined in the cell lysate of the axenically cultured Entamoeba histolytica isolate HM-1:IMSS . Under anaerobic culture conditions, 18.7 (+/- 4.9) units SOD activity (mg protein)-1 were found . By inhibition studies the activity was attributed to an iron-containing type of SOD (FeSOD) . Using degenerate oligonucleotide primers derived from regions highly conserved in prokaryotic FeSOD sequences, a genomic DNA fragment was amplified by the polymerase chain reaction . The fragment was used to isolate FeSOD specific cDNA clones from a pathogenic and a nonpathogenic E . histolytica isolate . A comparison of the 2 sequences revealed 5% nucleotide differences resulting in a single amino acid exchange . The primary structure showed the characteristics of an iron-containing type of SOD with a homology of approximately 55% with other FeSOD sequences . The enzyme was found to be encoded by single copy genes in both the pathogenic and the nonpathogenic E . histolytica, but restriction fragment lengths differed between the 2 groups . In 5 isolates studied, no correlation was found between pathogenic behavior of the amebae and the expression of FeSOD-related mRNA.

J Biochem (Tokyo), 1991 Nov, 110(5), 780 - 4
Identification of amino acid residues modified by two ATP analogs in Bacillus subtilis glutamine synthetase; Tanaka E et al.; Bacillus subtilis glutamine synthetase was modified by two ATP analogs, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP), each one containing either Mg2+ or Mn2+ . The FSBA labeled peptide was monitored by measuring the characteristic absorbance of the 4-carboxybenzenesulfonyl (CBS) part at 243 nm . The 8-N3ATP photolabeled peptide could also be monitored by measuring its absorption at 310 nm . A single CBS-labeled tryptic peptide was obtained, spanning residues 89-91 from the N-terminal of the subunit polypeptide chain, and sequence analysis by Edman degradation revealed that CBS-arginine was at position 91 . The amino acids photolabeled by 8-N3ATP at the ATP-binding site in B . subtilis GS were His-186, His-187, and Trp-424 . These results suggested that these four amino acids constitute an ATP-binding active site located at the interface between two subunits . The region surrounding Trp-424, which varies among different prokaryotic enzymes, was considered to be involved in a catalytic or regulatory role in B . subtilis GS . Since the same amino acids were labeled when B . subtilis GS was modified with FSBA or 8-N3ATP in the presence of Mn2+ or Mg2+, no conformational difference between B . subtilis GS binding Mn(2+)-ATP and that binding Mg(2+)-ATP was detected by affinity labeling with ATP analogs.

Mol Biol Evol, 1991 Nov, 8(6), 835 - 56
Evolution of retroposons by acquisition or deletion of retrovirus-like genes; McClure MA; The retroid family consists of all genetic elements that encode a potential reverse transcriptase (RT) . Members of this family include a diversity of eukaryotic genetic elements (viruses, transposable elements, organelle introns, and plasmids) and the retrons of prokaryotes . Some retroid elements have, in addition to the RT gene, other genes in common with the retroviruses . On the basis of RT sequence similarity, the retroposon group is defined as the eukaryotic long interspersed nuclear elements, the transposable elements of (1) Drosophila melanogaster (I and F factors), (2) Trypanosoma brucei (ingi element), (3) Zea mays (Cin4), (4) Bombyx mori (R2Bm), and members of the group II introns and plasmids of yeast mitochondria . The data presented here elucidate the extent of the relationships between the retroposons and other retroid-family members . Protein-sequence alignment data demonstrate that subsets of the retroposons contain different assortments of retroviral-like genes . Sequence similarities can be detected between the capsid, protease, ribonuclease H, and integrase proteins of retroviruses and several retroposon sequences . The relationships among the retroposon capsid-like sequences are congruent with the RT sequence phylogeny . In contrast, the similarity between ribonuclease H sequences varies in different subbranches of the retroposon lineage . These data suggest that xenologous recombination (i.e., the replacement of a homologous resident gene by a homologous foreign gene) and/or independent gene assortment have played a role in the evolution of the retroposons.

J Biol Chem, 1991 Oct 25, 266(30), 20007 - 10
Primary structure of rat ribosomal protein S2 . A ribosomal protein with arginine-glycine tandem repeats and RGGF motifs that are associated with nucleolar localization and binding to ribonucleic acids; Suzuki K et al.; The amino acid sequence of the rat 40 S ribosomal subunit protein S2 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed from the amino acid sequence of a cyanogen bromide peptide obtained from the protein . Ribosomal protein S2 has 293 amino acids and has a molecular weight of 31,211 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 23-28 copies of the S2 gene . The mRNA for the protein is about 1,000 nucleotides in length . The highly conserved repetitive mammalian gene family designated LLRep3, but not identified before, encodes ribosomal protein S2 . Rat S2 is related to Saccharomyces cerevisiae S4, Methanococcus vannielii S5, Escherichia coli S5, and other members of the prokaryotic S5 family . S . cerevisiae S4 and E . coli S5 are involved in the binding of aminoacyl-tRNA to ribosomes and in conditioning the fidelity of translation; it is plausible to assume that rat S2 serves similar functions . The NH2-terminal region of S2 is rich in arginine-glycine repeats including eight that occur in tandem and has two consecutive copies of the motif RGGF; these sequences have been associated with nucleolar localization and binding to RNA.

J Biol Chem, 1991 Oct 15, 266(29), 19154 - 7
Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small; Wise CA et al.; The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA . We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs . DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene . Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich . The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5253 - 61
Species-specific patterns of DNA bending and sequence; VanWye JD et al.; Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence . The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes . The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA . However, base composition was not an accurate predictor for DNA bending in eukaryotes . Sequences from C . elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL . Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics . Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms . The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA . This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively . These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.

Nucleic Acids Res, 1991 Oct 11, 19(19), 5169 - 72
Initiation of translation at an AUA codon for an archaebacterial protein gene expressed in E.coli; Kopke AK et al.; Overexpression of the Sulfolobus solfataricus L12 ribosomal protein gene in E.coli cells yielded two products of different size . If the E.coli cells carrying the overexpression plasmid were induced in the early stage of bacterial growth, the smaller of the two products was almost exclusively produced . However, induction in a late stage of bacterial growth yielded the larger product in significant excess . The larger protein was identified as the translation product of the entire SsoL12 gene, while the smaller product was a N-terminally shortened version of the L12 protein (sh-SsoL12), starting with a N-terminal methionine at position 22 of the coded protein and continuing with the predicted protein sequence . Position 22 is an isoleucine in the complete SsoL12 protein sequence, coded by an AUA codon . A subclone (SsoL12**) of the SsoL12 gene containing overexpression plasmid, lacking the regular AUG start codon and the putative Shine Dalgarno sequence, was constructed to determine if E.coli ribosomes could initiate at this AUA codon . During overexpression the SsoL12** construct yielded exclusively the sh-SsoL12 product in significant amounts . An AUA start codon has never been found before in a natural message . However, experiments utilizing site directed mutagenesis to generate AUA start codons showed that this codon can be functional for initiation in prokaryotes and eukaryotes . The findings presented in this paper show that AUA acts as an initiation codon in a natural message expressed in a heterologous organism.

Biochemistry, 1991 Oct 8, 30(40), 9642 - 8
Biochemical and kinetic characteristics of the interaction of the antitumor antibiotic sparsomycin with prokaryotic and eukaryotic ribosomes; Lazaro E et al.; Using 125I-labeled phenol-alanine sparsomycin, an analogue of sparsomycin having higher biological activity than the unmodified antibiotic, we studied the requirements and the characteristics of its interaction with the ribosome . The drug does not bind to either isolated ribosomal subunits or reconstituted whole ribosomes . For sparsomycin binding to 70S and 80S ribosomes, the occupation of the peptidyltransferase P-site by an N-blocked aminoacyl-tRNA is a definitive requirement . The sparsomycin analogue binds to bacterial and yeast ribosomes with Ka values of around 10(6) M-1 and 0.6 x 10(6) M-1, respectively, but its affinity is probably affected by the character of the peptidyl-tRNA bound to the P-site . Chloramphenicol, lincomycin, and 16-atom ring macrolides compete with sparsomycin for binding to bacterial ribosomes, but streptogramins and 14-atom ring macrolides do not . Considering the reported low affinity of puromycin for bacterial ribosomes, this antibiotic is also a surprisingly good competitor of sparsomycin binding to these particles . In the case of yeast ribosomes, blasticidin is a relatively good competitor of sparsomycin interaction, but anisomycin, trichodermin, and narciclasin are not . As expected, puromycin is a poor competitor of the binding in this case . The results from competition studies carried out with different sparsomycin analogues reveal, in some cases, a discrepancy between the drug ribosomal affinity and its biological effects . This suggests that some intermediate step, perhaps a ribosomal conformational change, is required for the inhibition to take place.

Biochim Biophys Acta, 1991 Oct 8, 1090(2), 195 - 203
Immunoanalysis of ultraviolet radiation induced DNA damage and repair within specific gene segments of plasmid DNA; Wani AA et al.; The region-specific heterogeneity of repairing DNA damage has been established in several biological systems . A flexible and sensitive approach, based upon DNA damage specific antibodies, is described to monitor the repair of specific lesions within discrete genomic segments . Membrane transblotted DNA restriction fragments are immunoanalyzed for the initial formation and repair of 254 nm radiation induced pyrimidine dimers . Sensitivity of dimer immunodetection increases proportional to fragment concentration and size . Antibody binding was detectable in a 0.5 kb fragment obtained from approx . 100 ng of restriction digested phage lambda DNA irradiated with 50 J m-2 . Dimers within larger fragments (greater than 5 kb) could be detected at ultraviolet doses as low as 1 to 2 J m-2 . To determine the occurrence of preferential repair in prokaryotic cells, damage was assessed in DNA sequences established in various Escherichia coli strains . In vivo repair of 8.9 kb vector and 6.4 and 3.2 kb gene inserts occurred with an approximate t1/2 of 45 min in UvrABC excision repair-proficient strains . Antibody binding sites were retained by DNA within repair-deficient strains . Compared to UvrABC, the repair of DNA fragments mediated by T4 endonuclease V was rapid and complete within 30 min of cellular irradiation . The efficient repair in DenV+ strain is attributable to a highly processive repair enzyme rather than to selective repair of actively replicating target genes . The results demonstrate the exceptional ability of antibodies specific for altered biomolecular lesions to map damage and repair in gene segments episomally established within cells.

J Biol Chem, 1991 Oct 5, 266(28), 18714 - 9
Structure-function analysis of the histidine permease and comparison with cystic fibrosis mutations; Shyamala V et al.; Traffic ATPases constitute a superfamily of transporters that include prokaryotic permeases and medically important eukaryotic proteins, such as the multidrug resistance P-glycoprotein and the cystic fibrosis gene product . We present a structure-function analysis of a member of this superfamily, the prokaryotic histidine permease, using mutations generated both in vitro and in vivo, and assaying several biochemical functions . The analysis supports a previously predicted structural model and allows the assignment of specific functions to several predicted structural features . Mutations in the secondary structure features which form the nucleotide-binding pocket in general cause the loss of ATP binding activity . Mutations in the helical domain retain ATP binding activity . Several mutations have been identified which may affect the signaling mechanism between ATP hydrolysis and membrane translocation . We relate our findings to those emerging from the recent biochemical and genetic analyses of cystic fibrosis mutations.

J Biol Chem, 1991 Oct 5, 266(28), 18427 - 30
Heavy metal resistance: a new role for P-glycoproteins in Leishmania; Callahan HL et al.; P-glycoproteins are responsible for multidrug resistance in tumor cell lines and are thought to have a physiologic role in exporting cellular metabolites . We now report that a P-glycoprotein gene in the H region of the trypanosomatid protozoan Leishmania confers resistance to heavy metals when present in multiple copies . The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance . Leishmania expression vectors were used to introduce multiple copies of segments of the Leishmania major H region into wild-type L . major promastigotes . Only constructs bearing a segment of L . major DNA containing the P-glycoprotein lmpgpA conferred arsenite resistance . Deletional analysis of the arsenite-resistant construct mapped resistance to the lmpgpA protein coding region . Lines expressing lmpgpA showed resistance to arsenite and trivalent antimonials, but not to pentavalent antimonials, zinc, cadmium, or the typical multidrug-resistant P-glycoprotein substrates vinblastine and puromycin . Transfection of the Leishmania tarentolae P-glycoprotein homologue ltpgpA resulted in a similar resistance profile . Thus, these pgpAs represent a functionally distinct group of P-glycoproteins which exhibit a substrate specificity similar to prokaryotic heavy metal pumps . Additionally, several arguments suggest that pgpAs may play a role in the susceptibility of Leishmania to clinically utilized antimonials.

Science, 1991 Oct 4, 254(5028), 109 - 11
A bacterial system for investigating transport effects of cystic fibrosis--associated mutations; Gibson AL et al.; LIV-I, a high-affinity system that transports neutral, branched-chain amino acids into Escherichia coli, has two components, LivG and LivF, that are homologous to the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) . CF-associated mutations of human CFTR were introduced into corresponding regions of LivG, and their effects on leucine transport could be grouped into three classes . Mutations were found that (i) abolished LIV-I--directed transport, (ii) retained about a quarter of wild-type activity at the Michaelis-Menten constant (KM), and (iii) had minimal activity at the KM . A mutation equivalent to a benign polymorphism had no effect on transport . The correlation of these mutational phenotypes in LivG and CFTR suggests that the LIV-I prokaryotic transporter is functionally similar to the CF protein and that this similarity can be exploited to clarify the properties of the nucleotide-binding fold in this superfamily of proteins.

DNA Cell Biol, 1991 Oct, 10(8), 603 - 11
Molecular cloning and characterization of the Caenorhabditis elegans elongation factor 2 gene (eft-2); Ofulue EN et al.; A Caenorhabditis elegans lambda ZAP cDNA library was screened using a fragment amplified from highly conserved regions of the mammalian and Drosophila elongation factor 2 (EF-2) . Two types of cDNA clones were obtained, corresponding to two mRNA species with 3'-untranslated regions of 60 and 115 nucleotides, both encoding identical polypeptides . Sequence analysis of these clones and comparisons with hamster and Drosophila EF-2 sequences suggests that they encode C . elegans EF-2 . Clone pCef6A, encoding the entire C . elegans EF-2 mRNA sequence including 45 nucleotides of 5'-untranslated region, contains a 2,556-bp open reading frame which predicts a polypeptide of 852 amino acid residues (Mr 94,564) . The deduced amino acid sequence is greater than 80% identical to that of mammalian and Drosophila EF-2 . Conserved sequence segments shared among a variety of GTP-binding proteins are found in the amino-terminal region . The carboxy-terminal half contains segments unique to EF-2 and its prokaryotic homolog, EF-G, as well as the histidyl residue which is ADP-ribosylated by diphtheria toxin . The C . elegans protein contains a 12-amino-acid insertion between positions 90 and 100, and a 13-amino-acid deletion between positions 237 and 260, relative to hamster EF-2 . Partial sequencing of a genomic clone encoding the entire C . elegans EF-2 gene (named eft-2) has so far revealed two introns of 48 and 44 bp following codons Gln-191 and Gln-250, respectively . Southern and Northern blot analyses indicate that eft-2 is a single-copy gene and encodes a 3-kb mRNA species which is present throughout nematode development.

Mol Gen Genet, 1991 Oct, 229(2), 229 - 37
Analysis of a ribosomal RNA methylase gene from Streptomyces tenebrarius which confers resistance to gentamicin; Holmes DJ et al.; Resistance to the aminoglycoside gentamicin in the nebramycin producer, Streptomyces tenebrarius, occurs at the level of the ribosome . A resistance determinant isolated from this actinomycete was previously shown to encode a methylase enzyme which modifies residue G-1405 of 16S ribosomal RNA . This gene (kgmB) has been sequenced and expressed in Escherichia coli using lacZ transcriptional signals since, like many other actinomycete genes, kgmB is not expressed in E . coli from its own promoter . The 5' end of the kgmB transcript has been mapped revealing a single promoter which does not obviously conform to the prokaryotic consensus.

J Exp Med, 1991 Oct 1, 174(4), 941 - 4
Specialized functions of major histocompatibility complex class I molecules . II . Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin; Shawar SM et al.; The physiological functions of the mouse telomeric major histocompatibility complex (MHC) class I molecules, including Hmt, are unknown . Hmt presents a polymorphic, N-formylated peptide encoded by the mitochondrial gene ND1 forming the cell surface maternally transmitted antigen (Mta) . Because the N-formyl moiety is required for Hmt binding, we proposed that Hmt may function generally in presentation of N-formylated antigens . This hypothesis was validated by a competitive binding assay, demonstrating that synthetic N-formyl peptides from other mitochondrial genes also bound Hmt . Bacteria similarly initiate protein synthesis with N-formylmethionine; indeed, we established that Hmt can also present prokaryotic peptides in an N-formyl-dependent manner . These results indicate biochemical specialization of this MHC-peptide interaction and suggest a unique role for Hmt in prokaryotic host defenses.

FASEB J, 1991 Oct, 5(13), 2833 - 42
RNA polymerase: regulation of transcript elongation and termination; Kerppola TK et al.; Expanded interest in studying the mechanisms of elongation and termination during transcription has come as a result of several recent findings that highlight the importance of the regulation of these processes in human health . Several cellular proto-oncogenes contain regulated blocks to elongation (1), and the human immunodeficiency viruses also control gene expression in part by regulating the efficiency of elongation in response to the trans-activating protein, TAT (2) . This review considers these recent findings and compares potential mechanisms of regulation used by prokaryotic and eukaryotic RNA polymerases during elongation and termination . In all these systems, many of the detailed mechanisms of transcription elongation and termination are still to be defined; however, we have tried to group examples that may share some common regulatory elements into simplified categories.

Curr Opin Genet Dev, 1991 Oct, 1(3), 336 - 41
Genetic analysis of cyanobacterial development; Wolk CP; Filamentous cyanobacteria, the only prokaryotes that form linear patterns of differentiated cells, can be genetically manipulated by the conjugative transfer of plasmids from Escherichia coli . It has become possible to determine the cellular localization of genetic transcription, including transcription of developmentally critical genes before morphological differentiation takes place, by using luciferase as a reporter . These techniques are facilitating developmental analysis.

J Cell Biol, 1991 Oct, 115(1), 267 - 77
Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin; Kennedy SP et al.; Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin . Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known . We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins . Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments . Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin . A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs . These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin . The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat . It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity . We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure . Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

Antonie Van Leeuwenhoek, 1991 Oct-Nov, 60(3-4), 313 - 23
Determination of the maximum product yield from glucoamylase-producing Aspergillus niger grown in the recycling fermentor; van Verseveld HW et al.; Aspergillus niger has been grown in glucose- and maltose-limited recycling cultures to determine the maximum growth yield, the maximum product yield for glucoamylase production, and the maintenance requirements at very slow specific growth rates . Using the linear equation for substrate utilization, and using the experimental data from both recycling experiments, both the maximum growth yield, Yxsm, and the maximum product yield, Ypsm, could be determined . The values estimated were 157 g biomass per mol maltose for Yxsm and 100 g protein per mol maltose for Ypsm . Expressed on a C1-basis these values are 0.52 and 0.36 C-mole per C-mol for respectively Yxsm and Ypsm . The found value for Ypsm is half the value found for alkaline serine protease production in Bacillus licheniformis, and it can be concluded that formation of extracellular protein is more energy consuming in filamentous fungi than in prokaryotic organisms . Maintenance requirements are no significant factor during growth of Aspergillus niger, and reported maintenance requirements are most probably due to differentiation.

Biochimie, 1991 Oct, 73(10), 1261 - 8
The remote control of transcription, DNA looping and DNA compaction; Amouyal M; mRNA synthesis can be controlled at some distance from the start of transcription in eukaryotes and prokaryotes . It is generally assumed that the distal elements of the transcriptional machinery directly interact with the proximal elements, forcing the DNA to bend in a loop . DNA loop formation and transcription can be affected by the distance between the sites, their helical positioning, their orientation, their concentration (responsible for a cis- or a trans-effect of the DNA sequences), and DNA compaction in chromatin . The corresponding in vitro and in vivo situations have been critically examined for a number of systems, mostly prokaryotic.

Hum Antibodies Hybridomas, 1991 Oct, 2(4), 172 - 89
Recombinant antibodies; Larrick JW et al.; Many fundamental advances in our understanding of the structure and function of eukaryotic genes were derived from the study of antibody genes . Examples include mRNA splicing and rearrangement to generate antibody diversity . The capacity to immortalize an individual B cell using cell fusion permitted the generation of monoclonal antibodies . Monoclonal antibodies have had wide application in many fields of the life sciences and beyond . Recent advances permitting manipulation of antibody genes using recombinant DNA techniques offer many advantages over conventional somatic cell hybridization techniques . Rodent monoclonals can be "humanized" and antibody isotype readily changed . Grafting of the complementarity determining regions from rodent to human framework regions demonstrated the importance of these hypervariable portions of the immunoglobulin to the integrity of the antibody combining site . Recombinant monoclonal antibodies (rMAb) or fragments thereof have been successfully produced in both prokaryotic and eukaryotic hosts at levels equal to those produced by hybridomas . Successful efforts to express rMAbs in plants and other large capacity systems suggest that rMAbs can be produced inexpensively . Use of antibody catalysis and antibodies mimicking various receptors or ligands have numerous applications . Technology developed to immortalize the heavy and light chain repertoire permits the generation in vitro of recombinatorial libraries of antibodies . The capacity to artificially generate high-affinity antibodies in vitro using the methods of recombinant DNA technology has enormous pharmaceutical and industrial potential.

Comput Appl Biosci, 1991 Oct, 7(4), 495 - 500
A new method for finding long consensus patterns in nucleic acid sequences; Taylor P et al.; We describe a fast computer algorithm for identifying consensus patterns in DNA sequences . The method requires no prior assumptions about the consensus pattern other than its length . In particular no previous knowledge of the frequency or spacing of consensus patterns is required . However, a priori information about the shape of the consensus pattern, or invariability of individual positions, or the overall conservation level, can be utilized to enhance the selectivity and sensitivity of search . As the number of all possible consensus words increases very rapidly with length, comprehensive searches have usually been restricted to a maximum of 10-12 nucleotides, even when large mainframes are used . Our algorithm enables searching for consensus patterns of this order on current mid-range and powerful microcomputers . Searches may be conducted on single, long sequences or a set of possibly aligned shorter sequences . We give examples of identified consensus patterns in both prokaryotic and eukaryotic DNA sequences, along with some typical program timings.

Can J Physiol Pharmacol, 1991 Oct, 69(10), 1486 - 92
cis-dominance of rat atrial natriuretic factor gene regulatory sequences in transgenic mice; Seidman CE et al.; We have produced transgenic mice that express the prokaryotic marker protein chloramphenicol acetyltransferase under the control of regulatory sequences derived from the rat atrial natriuretic factor gene . The transgene, which contains 2.4 kilobases of the rat atrial natriuretic factor gene regulatory region, was found to direct 4000-fold more chloramphenicol acetyltransferase expression in adult atria than in ventricles . Low-level activity was also detected in the hypothalamus, demonstrating that these sequences contain the signals necessary for cardiac and central nervous system expression of the hormone atrial natriuretic factor . Developmental analyses showed early, high-level transgene expression in fetal atrial and ventricular tissues but marked reduction of ventricular transgene expression following birth . Further, the developmental expression patterns of the endogenous murine atrial natriuretic factor gene and rat transgene were found to be quite distinct . Although both the rat and mouse atrial natriuretic factor genes are activated early in embryogenesis, perinatal ventricular expression appears to differ in these two rodent species . The transgene is expressed in a pattern analogous to the neonatal rat rather than the endogenous murine gene . These studies demonstrate that the cis-acting signals required for correct tissue specificity and developmental regulation of the rat atrial natriuretic factor gene are encoded in this 2.4-kilobase fragment and that these sequences act in a dominant fashion.

Tijdschr Kindergeneeskd, 1991 Oct, 59(5), 178 - 82
{Heat-shock proteins and arthritis}; Rijkers GT et al.; Heat-shock proteins are a category of proteins which are synthesized under stressful conditions (such as increased temperatures) both by prokaryotic and eukaryotic cells . Heat-shock proteins are a major target of the immune response and thus can be considered dominant antigens . Under physiological circumstances the response to heat-shock proteins is considered to play a role in overall defence against bacterial infections . An aberrant immune response against heat-shock proteins may lead to autoimmunity; adjuvant arthritis in Lewis rats . Current evidence also points towards a role of T cell immunity against heat-shock proteins in the etiology and pathogenesis of human autoimmune diseases such as juvenile chronic arthritis.

J Biol Chem, 1991 Sep 25, 266(27), 18363 - 9
Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves; Nour JM et al.; One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes . In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide . The structural organization of these enzymes in plants had not previously been examined . We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it . The protein was a dimer with a subunit molecular weight of Mr = 67,000 . The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively . The enzyme required both monovalent and divalent cations for maximum activity . The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column . On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted . In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves . These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional . Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.

J Biol Chem, 1991 Sep 25, 266(27), 17804 - 8
Heterologous cooperativity in Escherichia coli . The CytR repressor both contacts DNA and the cAMP receptor protein when binding to the deoP2 promoter; Pedersen H et al.; Promoters in Escherichia coli that are negatively regulated by the CytR repressor are also activated by the cAMP receptor protein (CRP) complexed to cAMP; as a characteristic, these promoters encode two binding sites for the cAMP.CRP complex . Biochemical and genetic studies have shown that CytR relies on interactions with the cAMP.CRP complex in order to bind promoter DNA and repress transcription . Here we have purified CytR to near homogeneity and addressed the question of how it interacts with the deoP2 promoter . Gel retardation and DNase I footprinting analyses show that CytR is a true sequence-specific DNA-binding protein that binds to the sequence between the two CRP sites in deoP2 with a relatively low affinity . In the presence of the cAMP.CRP complex the two protein species bind cooperatively to deoP2, forming a complex in which CytR occupies the sequence between the two DNA bound cAMP.CRP complexes . Furthermore, the inducer (cytidine) does not affect independent DNA binding of CytR, rather the CytR/cAMP.CRP cooperativity is perturbed . These results indicate that CytR binding to deoP2 relies on both repressor-DNA interactions and protein-protein interactions to cAMP.CRP . This combinatorial repression mechanism, in which an activator functions as an adaptor for a repressor that is not capable of blocking transcription on its own, is unprecedented in prokaryotes; it is, however, reminiscent of repression mechanisms found in eukaryotes.

J Mol Biol, 1991 Sep 20, 221(2), 431 - 40
Mutations that confer de novo activity upon a maintenance methyltransferase; Kelleher JE et al.; DNA methyltransferases are not only sequence specific in their action, but they also differentiate between the alternative methylation states of a target site . Some methyltransferases are equally active on either unmethylated or hemimethylated DNA and consequently function as de novo methyltransferases . Others are specific for hemimethylated target sequences, consistent with the postulated role of a maintenance methyltransferase in perpetuating a pattern of DNA modification . The molecular basis for the difference between de novo and maintenance methyltransferase activity is unknown, yet fundamental to cellular activities that are affected by different methylation states of the genome . The methyltransferase activity of the type I restriction and modification system, EcoK, is the only known prokaryotic methyltransferase shown to be specific for hemimethylated target sequences . We have isolated mutants of Escherichia coli K-12 which are able to modify unmethylated target sequences efficiently in a manner indicative of de novo methyltransferase activity . Consistent with this change in specificity, some mutations shift the balance between DNA restriction and modification as if both activities now compete at unmethylated targets . Two genes encode the methyltransferase and all the mutations are loosely clustered within one of them.

Cell, 1991 Sep 20, 66(6), 1269 - 78
Poly(A) polymerase and a dissociable polyadenylation stimulatory factor encoded by vaccinia virus; Gershon PD et al.; mRNA made in eukaryotic cells typically has a 3' poly(A) tail that is added posttranscriptionally . To investigate mechanisms by which 3' poly(A) is formed, we identified the genes for the two vaccina virus-encoded polypeptides, VP55 and VP39 . Primer-dependent polyadenylation activity was associated exclusively with purified VP55-VP39 heterodimer, which, although stable to column chromatography and glycerol gradient sedimentation, was readily dissociated by antibody to an N-terminal peptide of VP55 . Poly(A) polymerase activity was associated with immunopurified VP55, but not with immunopurified or chromatographically purified VP39 . VP39 was, however, required for the formation of long poly(A) molecules, in conjunction with either purified VP55 or low concentrations of the heterodimer, and was shown to bind free poly(A) . Thus, a catalytic polypeptide and a dissociable poly(A)-binding stimulatory factor each contribute to poly(A) tail formation . No prokaryotic or eukaryotic homologs of either polypeptide were detected in sequence data bases, consistent with the absence of previously reported poly(A) polymerase genes from any source.

J Biol Chem, 1991 Sep 15, 266(26), 17158 - 64
Expression and characterization of a recombinant yeast isoleucyl-tRNA synthetase; Racher KI et al.; We describe the heterologous expression of a recombinant Saccharomyces cerevisiae isoleucyl-tRNA synthetase (IRS) gene in Escherichia coli, as well as the purification and characterization of the recombinant gene product . High level expression of the yeast isoleucyl-tRNA synthetase gene was facilitated by site-specific mutagenesis . The putative ribosome-binding site of the yeast IRS gene was made to be the consensus of many highly expressed genes of E . coli . Mutagenesis simultaneously created a unique BclI restriction site such that the gene coding region could be conveniently subcloned as a "cassette." The variant gene was cloned into the expression vector pKK223-3 (Brosius, J., and Holy, A . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 6929-6933) thereby creating the plasmid pKR4 in which yeast IRS expression is under the control of the isopropyl-thio-beta-galactopyranoside (IPTG)-inducible tac promoter . Recombinant yeast IRS, on the order of 10 mg/liter of cell culture, was purified from pKR4-infected and IPTG-induced E . coli strain TG2 . Yeast IRS was purified to homogeneity by a combination of anion-exchange and hydroxyapatite gel chromatography . Inhibition of yeast IRS activity by the antibiotic pseudomonic acid A was tested . The yeast IRS enzyme was found to be 10(4) times less sensitive to inhibition by pseudomonic acid A (Ki = 1.5 x 10(-5) M) than the E . coli enzyme . E . coli strain TG2 infected with pKR4, and induced with IPTG, had a plating efficiency of 100% at inhibitor concentrations in excess of 25 micrograms/ml . At the same concentration of pseudomonic acid A, E . coli strain TG2 infected with pKK223-3 had a plating efficiency less than 1% . The ability of yeast IRS to rescue E . coli from pseudomonic acid A suggests that the eukaryotic synthetase has full activity in its prokaryotic host and has specificity for E . coli tRNA(ile).

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 8232 - 6
Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor; Selby CP et al.; In eukaryotic and prokaryotic cells, activity transcribed genes and, in some instances, the template strand of these genes have been found to be repaired 2-10 times more rapidly than nontranscribed genes or the coding strand of transcribed genes . We demonstrate here gene- and template strand-specific repair synthesis in vitro by using an Escherichia coli cell-free extract and a plasmid carrying a gene with the strong tac promoter . Strand-specific repair of UV, 4'-hydroxymethyl1-4,5',8-trimethylpsoralen, and cis-dicholorodiammine platinum(II) damage was dependent upon transcription and a functional nucleotide excision repair system and was stimulated by 6% (wt/vol) polyethylene glycol . A defined system consisting of the transcription and repair proteins in highly purified form did not perform strand-specific repair; however, active fractions of extract conferred strand specificity to the defined system . Transcription-repair coupling activity was partially purified from extract by successive DEAE-agarose and gel filtration chromatography . The coupling factor is heat-labile, with an estimated Mr of 100,000.

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 8121 - 5
Evolution and relatedness in two aminoacyl-tRNA synthetase families; Nagel GM et al.; Sequence segments of about 140 amino acids in length, each containing a selected consensus region, were used in alignments of the aminoacyl-tRNA synthetases with the aim of discerning their evolutionary relationships . In all cases tested, enzymes specific for the same amino acid from a variety of organisms grouped together, reinforcing the supposition that the aminoacyl-tRNA synthetases are very ancient enzymes that evolved to include the full complement of 20 amino acids long before the divergence leading to prokaryotes and eukaryotes . The enzymes are divided into two mutually exclusive groups that appear to have evolved from independent roots . Group I, for which two sequence segments were analyzed, contains the enzymes specific for glutamic acid, glutamine, tryptophan, tyrosine, valine, leucine, isoleucine, methionine, and arginine . Group II enzymes include those activating threonine, proline, serine, lysine, aspartic acid, asparagine, histidine, alanine, glycine, and phenylalanine . Both groups contain a spectrum of amino acid types, suggesting the possibility that each could have once supported an independent system for protein synthesis . Within each group, enzymes specific for chemically similar amino acids tend to cluster together, indicating that a major theme of synthetase evolution involved the adaptation of binding sites to accommodate related amino acids with subsequent specialization to a single amino acid . In a few cases, however, synthetases activating dissimilar amino acids are grouped together.

J Biol Chem, 1991 Sep 15, 266(26), 17060 - 6
Aminolevulinic acid dehydratase in pea (Pisum sativum L.) . Identification of an unusual metal-binding domain in the plant enzyme; Boese QF et al.; Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells . Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs . A set of overlapping cDNAs was characterized that encode the pea enzyme . The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases . The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases . Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate . Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots . Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 7958 - 62
Spectra of spontaneous and mutagen-induced mutations in the lacI gene in transgenic mice; Kohler SW et al.; Transgenic mice with a lambda shuttle vector containing a lacI target gene were generated for use as a short-term, in vivo mutagenesis assay . The gene is recovered from the treated mice by exposing mouse genomic DNA to in vitro packaging extracts and plating the rescued phage on agar plates containing 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) . Phage with mutations in the lacI gene form blue plaques, whereas phage with a nonmutated lacI form colorless plaques . Spontaneous background mutant rates using this system range from 0.6 x 10(-5) to 1.7 x 10(-5), depending upon tissue analyzed, with germ cells exhibiting less than one-third the background rate of somatic tissue . Treatment of the mice with N-ethyl-N-nitrosourea (EtNU), benzo{a}pyrene (B{a}P), or cyclophosphamide caused an induction of mutations over background . Recovery of the lacI target for sequence analysis was performed by genetic excision of a plasmid from the phage using partial filamentous phage origins . The predominant mutations identified from untreated and treated populations were base substitutions . Although it has been shown by others that 70% of all spontaneous mutations within the lacI gene, when replicated in Escherichia coli, occur at a hot spot located at bases 620-632, only 1 of 21 spontaneous mutations has been identified in this region in the transgenic mouse system . In addition, 5 of 9 spontaneous transitions analyzed occur at CpG dinucleotides, whereas no transition mutations were identified at the prokaryotic deamination hot spots occurring at dcm sites (CCA/TGG) within the lacI gene . For EtNU, approximately equal amounts of transitions and transversions were observed, contrasting with B{a}P-induced mutations, in which only transversions were obtained . In addition, B{a}P mutagenesis showed a predominance of mutations (81%) involving cytosines and/or guanines, consistent with its known mode of action . The discovery of a spontaneous mutation spectrum different from that of bacterial assays, coupled with the concordance of EtNU and B{a}P base mutations with the known mechanisms of activity for these mutagens, suggests that this transgenic system will be useful as a short-term, in vivo system for mutagen assessment and analysis of mechanisms leading to mutations.

J Ind Microbiol, 1991 Sep, 8(2), 121 - 5
The use of particle concentration fluorescence immunoassay technology for the analysis of rDNA products; Del Tito BJ Jr; The electrophoretic and immunological techniques typically used to detect potentially useful biopharmaceutical proteins are sensitive with detection limits in the nanogram range . However, quantitation of a recombinant protein can be cumbersome, and involve large numbers of samples throughout process optimization schemes . Although electrophoretic methods (i.e., SDS-PAGE and Western blots) now avail themselves to quantitation by densitometry, these techniques are time consuming because of the lack of appropriate automated systems . Biological activity assays, when available, often require relatively pure material and are not suitable for analyzing and quantitating impure or semi-purified samples, typical of the fermentation milieu . The optimization of several rDNA-derived protein systems from both prokaryotic and eukaryotic hosts has been completed using PCFIA, a rapid, sensitive system with high throughput . The development of Particle Concentration Fluorescence Immunoassay (CFIA) procedures for several of these rDNA-derived proteins of interest as potential biopharmaceuticals (e.g., alpha-1-antitrypsin, tPA, soluble CD4, and a malaria vaccine candidate) are discussed.

Nucleic Acids Res, 1991 Sep 11, 19(17), 4647 - 53
Analysis of inducers of the E.coli lac repressor system in mammalian cells and whole animals; Wyborski DL et al.; Although the inducible prokaryotic lac repressor system has been successfully adapted for control of gene expression in mammalian cells, little information is available on the pharmacokinetics of beta-galactoside inducers in mammalian cells for optimizing this system . These studies directly measure the cell uptake and clearance in cultured cells and animal tissue cells of lac inducers . In these cells, the beta-galactosides, isopropyl beta-D-thiogalactoside (IPTG) and methyl beta-D-thiogalactoside (MTG), are rapidly taken up, exceeding extracellular levels in less than 2 hours . Greater than 5% of this inducer is found in the nuclear fraction, slightly exceeding the cytoplasmic concentration . Although similar in uptake, IPTG is cleared from the cultured cells significantly faster than MTG . In the mouse, the half-life of both inducers in the blood ranges from 15-30 minutes . HPLC analysis of tissue extracts from inducer-injected mice indicates that the inducer is metabolically stable and functionally able to bind to lac repressor . These results should permit improvement in the adaptation of the lac repressor system to mammalian cells and aid in the development of an adaptable system for gene control in transgenic animals.

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