Prokaryotes

Microbiol Mol Biol Rev, 1998 Sep, 62(3), 667 - 83
Chlorophyll fluorescence analysis of cyanobacterial photosynthesis and acclimation; Campbell D et al.; Cyanobacteria are ecologically important photosynthetic prokaryotes that also serve as popular model organisms for studies of photosynthesis and gene regulation . Both molecular and ecological studies of cyanobacteria benefit from real-time information on photosynthesis and acclimation . Monitoring in vivo chlorophyll fluorescence can provide noninvasive measures of photosynthetic physiology in a wide range of cyanobacteria and cyanolichens and requires only small samples . Cyanobacterial fluorescence patterns are distinct from those of plants, because of key structural and functional properties of cyanobacteria . These include significant fluorescence emission from the light-harvesting phycobiliproteins; large and rapid changes in fluorescence yield (state transitions) which depend on metabolic and environmental conditions; and flexible, overlapping respiratory and photosynthetic electron transport chains . The fluorescence parameters FV/FM, FV'/FM',qp,qN, NPQ, and phiPS II were originally developed to extract information from the fluorescence signals of higher plants . In this review, we consider how the special properties of cyanobacteria can be accommodated and used to extract biologically useful information from cyanobacterial in vivo chlorophyll fluorescence signals . We describe how the pattern of fluorescence yield versus light intensity can be used to predict the acclimated light level for a cyanobacterial population, giving information valuable for both laboratory and field studies of acclimation processes . The size of the change in fluorescence yield during dark-to-light transitions can provide information on respiration and the iron status of the cyanobacteria . Finally, fluorescence parameters can be used to estimate the electron transport rate at the acclimated growth light intensity.

Gene, 1998 Aug 31, 216(2), 255 - 65
Characterization of the enzymatic domains in the modular polyketide synthase involved in rifamycin B biosynthesis by Amycolatopsis mediterranei; Tang L et al.; Five clustered polyketide synthase (PKS) genes, rifA-rifE, involved in rifamycin (Rf) biosynthesis in Amycolatopsis mediterranei S699 have been cloned and sequenced (August, P.R . et al., 1998 . Chem . Biol . 5, 69-79) . The five multifunctional polypeptides constitute a type I modular PKS that contains ten modules, each responsible for a specific round of polyketide chain elongation . Sequence comparisons of the Rf PKS proteins with other prokaryotic modular PKSs elucidated the regions that have an important role in enzyme activity and specificity . The beta-ketoacyl:acyl carrier protein synthase (KS) domains show the highest degree of similarity between themselves (86-90%) and to other PKSs (78-85%) among all the constituent domains . Both malonyl-coenzyme A (MCoA) and methylmalonyl-coenzyme A (mMCoA) are substrates for chain elongation steps carried out by the Rf PKS . Since acyltransferase (AT) domains of modular PKSs can distinguish between these two substrates, comparison of the sequence of all ten AT domains of the Rf PKS with those found in the erythromycin (Er) (Donadio, S . and Katz, L., 1992 . Gene 111, 51-60) and rapamycin (Rp) (Haydock, S . et al., 1995 . FEBS Lett . 374, 246-248) PKSs revealed that the AT domains in module 2 of RifA and module 9 of RifE are specific for MCoA, whereas the other eight modules specify mMCoA . Dehydration of the beta-hydroxyacylthioester intermediates should occur during the reactions catalysed by module 4 of RifB and modules 9 and 10 of RifE, yet only the active site region of module 4 conforms closely to the dehydratase (DH) motifs in the Er and Rp PKSs . The DH domains of modules 9 and 10 diverge significantly from the consensus sequence defined by the Er and Rp PKSs, except for the active site His residues . Deletions in the DH active sites of module 1 in RifA and module 5 in RifB and in the N- and C-terminal regions of module 8 of RifD should inactivate these domains, and module 2 of RifA lacks a DH domain, all of which are consistent with the proposed biosynthesis of Rf . In contrast, module 6 of RifB and module 7 of RifC appear to contain intact DH domains even though DH activity is not apparently required in these modules . Module 2 of RifA lacks a beta-ketoacyl:acyl carrier protein reductase (KR) domain and the one in module 3 has an apparently inactive NADPH binding motif, similar to one found in the Er PKS, while the other eight KR domains of the Rf PKS should be functional . These observations are consistent with biosynthetic predictions . All the acyl carrier protein (ACP) domains, while clearly functional, nevertheless have active site signature sequences distinctive from those of the Er and Rp PKSs . Module 2 of RifA has only the core domains (KS, AT and ACP) . The starter unit ligase (SUL) and ACP domains present in the N-terminus of RifA direct the selection and loading of the starter unit, 3-amino-5-hydroxybenzoic acid (AHBA), onto the PKS . AHBA is made by the products of several other genes in the Rf cluster through a variant of the shikimate pathway (August, P.R . et al., inter alia) . RifF, produced by the gene immediately downstream of rifE, is thought to catalyse the intramolecular cyclization of the PKS product, thereby forming the ansamacrolide precursor of Rf B . 1998 Elsevier Science B.V.

Appl Environ Microbiol, 1998 Sep, 64(9), 3346 - 51
Growth rate regulation of rRNA content of a marine synechococcus (Cyanobacterium) strain
Binder BJ, Liu YC.
The relationship between growth rate and rRNA content in a marine Synechococcus strain was examined . A combination of flow cytometry and whole-cell hybridization with fluorescently labeled 16S rRNA-targeted oligonucleotide probes was used to measure the rRNA content of Synechococcus strain WH8101 cells grown at a range of light-limited growth rates . The sensitivity of this approach was sufficient for the analysis of rRNA even in very slowly growing Synechococcus cells (&mgr; = 0.15 day-1) . The relationship between growth rate and cellular rRNA content comprised three phases: (i) at low growth rates (< approximately 0.7 day-1), rRNA cell-1 remained approximately constant; (ii) at intermediate rates ( approximately 0 . 7 - 1.6 day-1), rRNA cell-1 increased proportionally with growth rate; and (iii) at the highest, light-saturated rates (> approximately 1.6 day-1), rRNA cell-1 dropped abruptly . Total cellular RNA (as measured with the nucleic acid stain SYBR Green II) was well correlated with the probe-based measure of rRNA and varied in a similar manner with growth rate . Mean cell volume and rRNA concentration (amount of rRNA per cubic micrometer) were related to growth rate in a manner similar to rRNA cell-1, although the overall magnitude of change in both cases was reduced . These patterns are hypothesized to reflect an approximately linear increase in ribosome efficiency with increasing growth rate, which is consistent with the prevailing prokaryotic model at low growth rates . Taken together, these results support the notion that measurements of cellular rRNA content might be useful for estimating in situ growth rates in natural Synechococcus populations.

Mol Biol Cell, 1998 Sep, 9(9), 2375 - 82
Evidence for the presence of 5S rRNA in mammalian mitochondria; Magalhaes PJ et al.; Mammalian mitochondrial ribosomes contain two prokaryotic-like rRNAs, 12S and 16S, both encoded by mitochondrial DNA . As opposed to cytosolic ribosomes, however, these ribosomes are not thought to contain 5S rRNA . For this reason, it has been unclear whether 5S rRNA, which can be detected in mitochondrial preparations, is an authentic organellar species imported from the cytosol or is merely a copurifying cytosol-derived contaminant . We now show that 5S rRNA is tightly associated with highly purified mitochondrial fractions of human and rat cells and that 5S rRNA transcripts derived from a synthetic gene transfected transiently into human cells are both expressed in vivo and present in highly purified mitochondria and mitoplasts . We conclude that 5S rRNA is imported into mammalian mitochondria, but its function there still remains to be clarified.

J Exp Zool, 1998 Sep-Oct 1, 282(1-2), 127 - 32
Structural analysis shows five glycohydrolase families diverged from a common ancestor; Robertus JD et al.; We have solved the X-ray structure of barley chitinase and bacterial chitosanase . Structural constraints predicted these would work by an inverting mechanism, which has been confirmed biochemically . The two enzymes were compared with lysozymes from goose (GEWL), phage (T4L), and hen (HEWL) . Although the proteins share no significant amino acid similarities, they are shown to have a structurally invariant core containing two helices and a three-stranded beta sheet that from the substrate binding and catalytic cleft . These enzymes represent a superfamily of hydrolases arising from the divergent evolution of an ancient protein . The glycohydrolase superfamily can be structurally divided into a bacterial family (chitosanase and T4L), and a eucaryotic family represented by chitinase, GEWL, and HEWL . Both families contain the ancestral core but differ at the amino and carboxy termini . The eucaryotes have a small N terminal domain, while the procaryotes have none . The C terminal domain of the eucaryotic family contains a single alpha-helix, while the prokaryotic domain has three antiparallel helices.

Bioessays, 1998 Jul, 20(7), 523 - 7
Tubulin and FtsZ structures: functional and therapeutic implications; Desai A et al.; The microtubule cytoskeleton has lagged nearly a decade behind the actin cytoskeleton with respect to structural information on the basic polymer subunit . This structural inferiority complex has finally been lifted by two recent papers describing the structures of the alpha beta tubulin dimer and FtsZ, a protein similar to tubulin that is essential for cell division in prokaryotes.

Trends Genet, 1998 Aug, 14(8), 307 - 11
You are what you eat: a gene transfer ratchet could account for bacterial genes in eukaryotic nuclear genomes; Doolittle WF; Recent phylogenetic analyses reveal that many eukaryotic nuclear genes whose prokaryotic ancestry can be pinned down are of bacterial origin . Among them are genes whose products function exclusively in cytosolic metabolism . The results are surprising: we had come to believe that the eukaryotic nuclear genome shares a most recent common ancestor with archaeal genomes, thus most of its gene should be 'archaeal' (loosely speaking) . Some genes of bacterial origin were expected as the result of transfer from mitochondria, of course, but these were thought to be relatively few, and limited to producing proteins reimported into mitochondria . Here, I suggest that the presence of many bacterial genes with many kinds of functions should not be a surprise . The operation of a gene transfer ratchet would inevitably result in the replacement of nuclear genes of early eukaryotes by genes from the bacteria taken by them as food.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10698 - 703
Replicational and transcriptional selection on codon usage in Borrelia burgdorferi; McInerney JO; With more than 10 fully sequenced, publicly available prokaryotic genomes, it is now becoming possible to gain useful insights into genome evolution . Before the genome era, many evolutionary processes were evaluated from limited data sets and evolutionary models were constructed on the basis of small amounts of evidence . In this paper, I show that genes on the Borrelia burgdorferi genome have two separate, distinct, and significantly different codon usages, depending on whether the gene is transcribed on the leading or lagging strand of replication . Asymmetrical replication is the major source of codon usage variation . Replicational selection is responsible for the higher number of genes on the leading strands, and transcriptional selection appears to be responsible for the enrichment of highly expressed genes on these strands . Replicational-transcriptional selection, therefore, has an influence on the codon usage of a gene . This is a new paradigm of codon selection in prokaryotes.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10419 - 24
Crystal structures of eukaryotic translation initiation factor 5A from Methanococcus jannaschii at 1.8 A resolution; Kim KK et al.; Eukaryotic translation initiation factor 5A (eIF-5A) is a ubiquitous protein found in all eukaryotic cells . The protein is closely associated with cell proliferation in the G1-S stage of the cell cycle . Recent findings show that the eIF-5A proteins are highly expressed in tumor cells and act as a cofactor of the Rev protein in HIV-1-infected cells . The mature eIF is the only protein known to have the unusual amino acid hypusine, a post-translationally modified lysine . The crystal structure of eIF-5A from Methanococcus jannaschii (MJ eIF-5A) has been determined at 1.9 A and 1.8 A resolution in two crystal forms by using the multiple isomorphous replacement method and the multiwavelength anomalous diffraction method for the first crystal form and the molecular replacement method for the second crystal form . The structure consists of two folding domains, one of which is similar to the oligonucleotide-binding domain found in the prokaryotic cold shock protein and the translation initiation factor IF1 despite the absence of any significant sequence similarities . The 12 highly conserved amino acid residues found among eIF-5As include the hypusine site and form a long protruding loop at one end of the elongated molecule.

Proc Natl Acad Sci U S A, 1998 Sep 1, 95(18), 10413 - 8
A novel DNA-binding motif in MarA: the first structure for an AraC family transcriptional activator; Rhee S et al.; A crystal structure for a member of the AraC prokaryotic transcriptional activator family, MarA, in complex with its cognate DNA-binding site is described . MarA consists of two similar subdomains, each containing a helix-turn-helix DNA-binding motif . The two recognition helices of the motifs are inserted into adjacent major groove segments on the same face of the DNA but are separated by only 27 A thereby bending the DNA by approximately 35 degrees . Extensive interactions between the recognition helices and the DNA major groove provide the sequence specificity.

EMBO J, 1998 Sep 1, 17(17), 5201 - 13
Copy number control of IncIalpha plasmid ColIb-P9 by competition between pseudoknot formation and antisense RNA binding at a specific RNA site; Asano K et al.; Replication of a low-copy-number IncIalpha plasmid ColIb-P9 depends on expression of the repZ gene encoding the replication initiator protein . repZ expression is negatively controlled by the small antisense Inc RNA, and requires formation of a pseudoknot in the RepZ mRNA consisting of stem-loop I, the Inc RNA target, and a downstream sequence complementary to the loop I . The loop I sequence comprises 5'-rUUGGCG-3', conserved in many prokaryotic antisense systems, and was proposed to be the important site of copy number control . Here we show that the level of repZ expression is rate-limiting for replication and thus copy number, by comparing the levels of repZ expression and copy number from different mutant ColIb-P9 derivatives defective in Inc RNA and pseudoknot formation . Kinetic analyses using in vitro transcribed RNAs indicate that Inc RNA binding and the pseudoknot formation are competitive at the level of initial base paring to loop I . This initial interaction is stimulated by the presence of the loop U residue in the 5'-rUUGGCG-3' motif . These results indicate that the competition between the two RNA-RNA interactions at the specific site is a novel regulatory mechanism for establishing the constant level of repZ expression and thus copy number.

J Biol Chem, 1998 Sep 4, 273(36), 22957 - 61
Coordination of Zn2+ (and Cd2+) by prokaryotic metallothionein . Involvement of his-imidazole; Daniels MJ et al.; In mammalian metallothionein Zn2+ is exclusively coordinated to Cys-thiolate to form clusters in which the metal is thermodynamically stable but also kinetically labile . By contrast, little is known about coordination to prokaryotic metallothionein, SmtA . 3 nmol of Zn2+ nmol-1 SmtA were displaced by 8 nmol of p-(hydroxymercuri)phenylsulfonate implicating eight of the nine Cys in the coordination of three metal ions . None of the Zn2+ associated with SmtA was accessible to 4-(2-pyridylazo)resorcinol prior to the addition of p-(hydroxymercuri)phenylsulfonate . An unusual feature of SmtA is the presence of three His residues, and we have investigated whether these contribute to metal coordination . Less Zn2+ was associated with purified SmtA(H40R/H49R/H55R), in which all three His residues were substituted with Arg, and approximately one equivalent of Zn2+ was immediately accessible to 4-(2-pyridylazo)resorcinol . Following incubation of SmtA with 111Cd, three 111Cd resonances were detected, two in a range expected for CdS4 and the third indicative of either CdNS3 or CdN2S2 coordination . Two-dimensional TOCSY 1H NMR and 111Cd-edited 1H NMR showed two His residues bound to 111Cd, confirming CdN2S2 coordination . The pH of half-dissociation of Zn2+ increased from 4.05 for SmtA to 5.37 for SmtA(H40R/H49R/H55R) . Equivalent values for single His mutants SmtA(H40R), SmtA(H49R), and SmtA(H55R) were 4.62, 4.48, and 3.81, respectively, revealing that conversion of His40 or His49 to Arg impairs Zn2+ binding at the CdN2S2 and CdS4 sites . Only approximately two equivalents of Zn2+ were associated with purified SmtA(H49R) . The appearance of a fourth 111Cd resonance at lower pH suggests that an alternative CdN2S2 site also exists.

Plant J, 1998 Jul, 15(2), 243 - 52
Differential regulation of the tomato ETR gene family throughout plant development; Lashbrook CC et al.; Ethylene perception in plants is co-ordinated by multiple hormone receptor candidates sharing sequence commonalties with prokaryotic environmental sensor proteins known as two-component regulators . Two tomato homologs of the Arabidopsis ethylene receptor ETR1 were cloned from a root cDNA library . Both cDNAs, termed LeETR1 and LeETR2, were highly homologous to ETR1, exhibiting approximately 90% deduced amino acid sequence similarity and 80% deduced amino acid sequence identity . LeETR1 and LeETR2 contained all the major structural elements of two-component regulators, including the response regulator motif absent in LeETR3, the gene encoding tomato NEVER RIPE (NR) . Using RNase protection analysis, the mRNAs of LeETR1, LeETR2 and NR were quantified in tissues engaged in key processes of the plant life cycle, including seed germination, shoot elongation, leaf and flower senescence, floral abscission, fruit set and fruit ripening . LeETR1 was expressed constitutively in all plant tissues examined . LeETR2 mRNA was expressed at low levels throughout the plant but was induced in imbibing tomato seeds prior to germination and was down-regulated in elongating seedlings and senescing leaf petioles . NR expression was developmentally regulated in floral ovaries and ripening fruit . Notably, hormonal regulation of NR was highly tissue-specific . Ethylene biosynthesis induced NR mRNA accumulation in ripening fruit but not in elongating seedlings or in senescing leaves or flowers . Furthermore, the abundance of mRNAs for all three LeETR genes remained uniform in multiple plant tissues experiencing marked changes in ethylene sensitivity, including the cell separation layer throughout tomato flower abscission.

J Bacteriol, 1998 Sep, 180(17), 4497 - 507
Molecular characterization and sequence of a methionine biosynthetic locus from Pseudomonas syringae; Andersen GL et al.; Two methionine biosynthetic genes in Pseudomonas syringae pv . syringae, metX and metW, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces . The metXW locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness . Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa . The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine O-acetyltransferases . This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus Leptospira, and several gram-positive bacterial genera . Both metX and metW were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine . MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes . Several classes of methionine auxotrophs, including metX and metW mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness . This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.

Genomics, 1998 Aug 1, 51(3), 459 - 62
Structural characterization and chromosomal localization of the gene encoding human biphenyl hydrolase-related protein (BPHL); Puente XS et al.; The gene encoding human biphenyl hydrolase-related protein (Bph-rp), a serine hydrolase with sequence similarity to prokaryotic enzymes involved in the degradation of polychlorinated biphenyls, has been cloned and its overall organization established . The gene, whose HGM-approved nomenclature is BPHL, spans more than 30 kb and is composed of eight exons and seven introns . The number and distribution of exons and introns differ from those reported for the genes encoding other serine hydrolases with sequence similarity to Bph-rp, indicating that these genes are distantly related . Nucleotide sequence analysis of the 5'-flanking region of BPHL revealed a high GC content, a ratio CpG/GpC close to unity, and the absence of consensus transcriptional sequences such as a TATA box or a CCAAT box . Chromosomal localization of BPHL revealed that it maps to chromosome 6p25, a unique location for all serine hydrolases mapped to date .

Genomics, 1998 Aug 1, 51(3), 332 - 9
Performance-guarantee gene predictions via spliced alignment; Mironov AA et al.; An important and still unsolved problem in gene prediction is designing an algorithm that not only predicts genes but estimates the quality of individual predictions as well . Since experimental biologists are interested mainly in the reliability of individual predictions (rather than in the average reliability of an algorithm) we attempted to develop a gene recognition algorithm that guarantees a certain quality of predictions . We demonstrate here that the similarity level with a related protein is a reliable quality estimator for the spliced alignment approach to gene recognition . We also study the average performance of the spliced alignment algorithm for different targets on a complete set of human genomic sequences with known relatives and demonstrate that the average performance of the method remains high even for very distant targets . Using plant, fungal, and prokaryotic target proteins for recognition of human genes leads to accurate predictions with 95, 93, and 91% correlation coefficient, respectively . For target proteins with similarity score above 60%, not only the average correlation coefficient is very high (97% and up) but also the quality of individual predictions is guaranteed to be at least 82% . It indicates that for this level of similarity the worst case performance of the spliced alignment algorithm is better than the average case performance of many statistical gene recognition methods .

Mol Microbiol, 1998 Jul, 29(2), 491 - 503
Assembly of the FtsZ ring at the central division site in the absence of the chromosome; Sun Q et al.; The FtsZ ring assembles between segregated daughter chromosomes in prokaryotic cells and is essential for cell division . To understand better how the FtsZ ring is influenced by chromosome positioning and structure in Escherichia coli, we investigated its localization in parC and mukB mutants that are defective for chromosome segregation . Cells of both mutants at non-permissive temperatures were either filamentous with unsegregated nucleoids or short and anucleate . In parC filaments, FtsZ rings tended to localize only to either side of the central unsegregated nucleoid and rarely to the cell midpoint; however, medial rings reappeared soon after switching back to the permissive temperature . Filamentous mukB cells were usually longer and lacked many potential rings . At temperatures permissive for mukB viability, medial FtsZ rings assembled despite the presence of apparently unsegregated nucleoids . However, a significant proportion of these FtsZ rings were mislocalized or structurally abnormal . The most surprising result of this study was revealed upon further examination of FtsZ ring positioning in anucleate cells generated by the parC and mukB mutants: many of these cells, despite having no chromosome, possessed FtsZ rings at their midpoints . This discovery strongly suggests that the chromosome itself is not required for the proper positioning and development of the medial division site.

Mol Microbiol, 1998 Jul, 29(2), 383 - 96
Molecular mechanisms of cytochrome c biogenesis: three distinct systems; Kranz R et al.; The past 10 years have heralded remarkable progress in the understanding of the biogenesis of c-type cytochromes . The hallmark of c-type cytochrome synthesis is the covalent ligation of haem vinyl groups to two cysteinyl residues of the apocytochrome (at a Cys-Xxx-Yyy-Cys-His signature motif) . From genetic, genomic and biochemical studies, it is clear that three distinct systems have evolved in nature to assemble this ancient protein . In this review, common principles of assembly for all systems and the molecular mechanisms predicted for each system are summarized . Prokaryotes, plant mitochondria and chloroplasts use either system I or II, which are each predicted to use dedicated mechanisms for haem delivery, apocytochrome ushering and thioreduction . Accessory proteins of systems I and II co-ordinate the positioning of these two substrates at the membrane surface for covalent ligation . The third system has evolved specifically in mitochondria of fungi, invertebrates and vertebrates . For system III, a pivotal role is played by an enzyme called cytochrome c haem lyase (CCHL) in the mitochondrial intermembrane space.

Mol Biochem Parasitol, 1998 Jul 1, 94(1), 13 - 25
Close linkage of three merozoite surface protein genes on chromosome 2 of Plasmodium falciparum; Marshall VM et al.; We have analysed a 10.5 kb region of chromosome 2 in Plasmodium falciparum that encompasses the coding region of four genes . Three genes are arranged in a head-to-tail orientation and encode the merozoite surface proteins MSP2 and MSP4 as well as a previously unreported sequence that encodes a polypeptide with the characteristics of a merozoite surface protein, now designated MSP5 . The fourth gene, asl, is arranged in a tail-to-tail orientation with msp2 and has homology with prokaryotic and eukaryotic genes encoding adenylosuccinate lyase (ASL), an enzyme involved in purine biosynthesis and salvage . The genes, arranged in the order msp4, msp5, msp2 and asl, are separated by intergenic distances of 1021, 1017 and 722 bp, respectively . msp4 and msp5 are clearly related genes, each being composed of 2 exons and encoding proteins of identical length . Both msp4 and msp5 encode proteins that contain hydrophobic signal sequences, apparent glycosylphosphatidylinositol (GPI) attachment signals and a single epidermal growth factor-like (EGF-like) domain at their carboxyl termini . Nevertheless, the remainder of their protein coding regions are quite dissimilar . It appears that one of these genes arose as a result of a relatively ancient gene duplication event and both genes have subsequently diverged considerably . This study shows that msp5 is transcribed in asexual stages and its encoded product is a 40 kDa protein that appears to be located on the merozoite surface as determined by immunofluorescence assays.

J Appl Microbiol, 1998 Jun, 84(6), 1035 - 42
Clarification of small volume microbial suspensions in an ultrasonic standing wave; Limaye MS et al.; The removal of Saccharomyces cerevisiae and Escherichia coli from 2.5 ml suspensions in ultrasonic standing wave formed at 1 or 3 MHz has been characterized . The standing wave was set up by a plane transducer and reflector mounted in the vertical plane . Cells in the ultrasonic field first concentrated in vertical planes at half wavelength separations . The ultrasound was then pulsed to allow clumps of concentrated cells to sediment in a controlled way during the short 'off' intervals . Yeast removal from suspension at a concentration of 3 x 10(9) ml-1 (14% volume v/v) was 99.5% in a total time of 4.5 min . Almost total (99.5%) clarification of prokaryote (E . coli) suspension was achieved here for the first time in a standing wave field . The clarification of a 1.3 x 10(11) ml-1 (16% v/v) E . coli suspension occurred over 11.5 min . The period decreased to 7 min in the presence of a polycationic flocculant, polyethyleneimine . The implications of the results for design of systems to further reduce clarification times are discussed . Removal efficiency for both S . cerevisiae and E . coli decreased with decrease in cell concentration . This concentration dependence is shown not to be simply a consequence of acoustic interaction between single cells . Flow cytometry of stained cells detected no loss of cell viability arising from the ultrasonic procedure.

Anal Biochem, 1998 Aug 1, 261(2), 183 - 90
A high-throughput fluorescence screen to monitor the specific binding of antagonists to RNA targets; Hamasaki K et al.; Since RNA molecules can form intricate three-dimensional structures, it should be possible to design specific, high-affinity antagonists directed against these structures . To begin to explore the validity of this possibility, high-throughput screening methods are required to assay for RNA antagonists . A fluorescence quenching technique is described here in a 96-well plate format which is capable of screening chemical diversity libraries . A pyrene-containing aminoglycoside analog is used to accurately monitor antagonist binding to a prokaryotic 16S rRNA A-site decoding region construct . This rRNA region comprises the natural target for aminoglycoside antibiotics . The fluorescence technique reported here should be generally adaptable to monitor the binding of structurally novel antagonists to any selected RNA target .

Gene, 1998 Jul 30, 215(2), 415 - 23
Site-specific recombination using an epitope tagged bacteriophage P1 Cre recombinase; Stricklett PK et al.; Since the original description of Cre mediated site-specific recombination in bacteriophage P1 (Sternberg, N., Hamilton, D., 1981 J . Mol . Biol., 150, 467-487), the Cre-lox system of recombination has been widely used to manipulate prokaryotic and eukaryotic genomes . Unfortunately, there are few means available to measure Cre protein expression in vivo . We have constructed an expression vector wherein the Cre protein is tagged at the carboxy terminus with an 11-amino-acid epitope to the herpes simplex virus (HSV) glycoprotein D coat protein (Isola, V.J., Eisenberg, R.J., Siebert, G.R., Heilman, C.J., Wilcox, W.C., Cohan, G.H., 1989 . J . Virol . 63, 2325-2334) . The epitope tag facilitates detection of Cre expression in vitro and in vivo using immunofluorescent labeling with a commercially available antibody . The epitope tag does not interfere with Cre recombinase activity or alter recombination efficiency between loxP sites . We have shown in mice that a transgene expressing our tagged Cre is capable of excising a loxP flanked sequence contributed by another transgenic mouse . In summary, we have developed an epitope-tagged Cre recombinase that is fully active and readily detectable.

J Biol Chem, 1998 Aug 28, 273(35), 22305 - 10
Oxygen-mediated inactivation of peptide deformylase; Rajagopalan PT et al.; Peptide deformylase catalyzes the removal of the N-formyl group from newly synthesized polypeptides in prokaryotes . Its essential character and unique presence in prokaryotes make it an attractive target for antibacterial chemotherapy . However, purification and characterization of the peptide deformylase have remained a major challenge because this enzyme is extraordinarily labile under a variety of conditions (t1/2 approximately 1 min at room temperature) . In this work, we show that this unusual instability is because of oxidation of the catalytic Fe2+ ion of the deformylase into catalytically inactive Fe3+ ion by atmospheric oxygen . Oxidation of Fe2+ is accompanied by the conversion of O2 into a yet unidentified reactive species, which covalently modifies the deformylase protein, most likely by oxidizing cysteine-90, a ligand residue of the Fe2+ ion, into a cysteine sulfonic acid . Enzymatic exclusion of O2 from the deformylase assays renders the deformylase highly stable under otherwise identical conditions . An improved, readily reproducible purification procedure has been developed that produces approximately 10 mg of pure, fully active Fe2+ deformylase from a liter of cells . In addition, active peptide deformylase can be reconstituted in vitro from the denatured deformylase.

J Biol Chem, 1998 Aug 28, 273(35), 22173 - 6
Biochemical characterization of the human arsenite-stimulated ATPase (hASNA-I); Kurdi-Haidar B et al.; Arsenic is a potent toxin and carcinogen . In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems . We have previously reported the cloning of hASNA-I, a human homologue of arsA encoding the ATPase component of the Escherichia coli arsenite transporter . Purified glutathione S-transferase (GST)-hASNA-I fusion protein was biochemically characterized, and its properties were compared with those of ArsA . The GST-hASNA-I exhibited a basal level of ATPase activity of 18.5 +/- 8 nmol/min/mg in the absence of arsenite . Arsenite produced a 1.6 +/- 0.1-fold stimulation of activity (p = 0 . 0044), which was related to an increase in Vmax; antimonite did not stimulate activity . Two lines of evidence suggest that an oligomer is the most likely native form of hASNA-I . First, lysates of human embryo kidney 293 cells overproducing recombinant hASNA-I produced a single monomeric 37-kDa band on SDS-polyacrylamide gel electrophoresis (PAGE) and two distinct species when analyzed using nondenaturing PAGE . Second, chemical cross-linking of the 63-kDa GST-hASNA-I resulted in the formation of dimeric and tetrameric protein forms . The results indicate that hASNA-I is a distinct human arsenite-stimulated ATPase belonging to the same superfamily of ATPases represented by the E . coli ArsA protein.

Cell Mol Life Sci, 1998 Jul, 54(7), 684 - 95
Ribonucleotide reductases and radical reactions; Fontecave M; Ribonucleotide reductases (RNRs) catalyse the reduction of ribonucleotides to deoxyribonucleotides . They play a pivotal role in the regulation of DNA synthesis and are targets for antiproliferative drugs . Ribonucleotide reductases are unique enzymes in that they all require a protein radical for activity . Class I nonheme iron RNRs (mammals, plants, Escherichia coli) use a tyrosyl/cysteinyl radical pair, class II adenosylcobalamin RNRs (prokaryotes, archaea) a cysteinyl radical, class III iron-sulphur RNRs (facultative anaerobes) a glycyl radical . Here we describe the reactivity of these radicals with respect to the natural ribonucleotide substrates as well as to a variety of enzyme inhibitors, radical scavengers, nitric oxide, superoxide radicals and substrate analogues.

Math Biosci, 1998 Aug 1, 151(2), 155 - 63
Mathematical relationships among DNA supercoiling, cation concentration, and temperature for prokaryotic transcription; Wang JY; DNA twist has been proposed to affect transcription from some promoters of Escherichia coli, but involvement of twist has been difficult to test because it cannot be measured in transcription reaction mixtures . However, changes in other factors affect both DNA twist and transcription . These parameters are expected to be related when maximum transcription initiation is considered . In the present work, mathematical relationships among supercoiling, cation concentration, and temperature are derived for prokaryotic transcription initiation . The relationships indicate that as DNA becomes more negatively supercoiled, maximal initiation occurs at a higher cation concentration and at a lower temperature . For example, when superhelical density becomes more negative by 0.0025, a 1.6-fold increase in potassium concentration is predicted to be required to maintain transcription initiation at its maximum rate . Experimental verification of the relationships should provide a useful test of the idea that transcription initiation is sensitive to DNA twist.

Proc Natl Acad Sci U S A, 1998 Aug 18, 95(17), 10003 - 8
The role of SOS and flap processing in microsatellite instability in Escherichia coli; Morel P et al.; Mutations affecting mismatch repair result in elevated frequencies of microsatellite length alteration in prokaryotes and eukaryotes . However, the finding that microsatellite instability is found often in cells with a functional mismatch repair system prompted a search for other factors of tract alteration . In the present report, we show that, in Escherichia coli, poly(AC/TG) tracts are destabilized by mutations that induce SOS . These observations may have implications for eukaryotic cells because recent results suggest the existence of a mammalian SOS response analogous to that in prokaryotes . In addition, a defect in the 5'-3' exonuclease domain of DNA polymerase I, homologous to the mammalian FEN1 and the yeast RAD27 nucleases, leads to a marked increase in repeat expansions characteristic of several genetic disorders . Finally, we found that the combination of a proofreading defect with mismatch repair deficiency results in extreme microsatellite instability.

EMBO J, 1998 Aug 17, 17(16), 4559 - 71
The solution structure of ribosomal protein S4 delta41 reveals two subdomains and a positively charged surface that may interact with RNA; Markus MA et al.; S4 is one of the first proteins to bind to 16S RNA during assembly of the prokaryotic ribosome . Residues 43-200 of S4 from Bacillus stearothermophilus (S4 Delta41) bind specifically to both 16S rRNA and to a pseudoknot within the alpha operon mRNA . As a first step toward understanding how S4 recognizes and organizes RNA, we have solved the structure of S4 Delta41 in solution by multidimensional heteronuclear nuclear magnetic resonance spectroscopy . The fold consists of two globular subdomains, one comprised of four helices and the other comprised of a five-stranded antiparallel beta-sheet and three helices . Although cross-linking studies suggest that residues between helices alpha2 and alpha3 are close to RNA, the concentration of positive charge along the crevice between the two subdomains suggests that this could be an RNA-binding site . In contrast to the L11 RNA-binding domain studied previously, S4 Delta41 shows no fast local motions, suggesting that it has less capacity for refolding to fit RNA . The independently determined crystal structure of S4 Delta41 shows similar features, although there is small rotation of the subdomains compared with the solution structure . The relative orientation of the subdomains in solution will be verified with further study.

Annu Rev Nutr, 1998, 18, 145 - 77
Newly discovered redox cofactors: possible nutritional, medical, and pharmacological relevance to higher animals; McIntire WS; Research spurred by the discovery of pyrroloquinoline quinone (PPQ) in 1979 led to the discovery of four additional oxidation-reduction (redox) cofactors, all of which result from transmogrification of amino acyl side chains in respective enzymes . These cofactors are (a) topa quinone in copper-containing amine oxidases, enzymes found in nearly all forms of life, including human; (b) lysyl topa quinone of the copper protein lysyl oxidase, an enzyme required for proper cross-linking of collagen and elastin; (c) tryptophan tryptophylquinone of alkylamine dehydrogenases from gram-negative soil bacteria; and (d) the copper-complexed cysteinyltyrosyl radical of fungal galactose oxidase . Originally, PQQ was thought to be a covalently bound cofactor in numerous enzymes from eukaryotes and prokaryotes . Today, PQQ is only found as a noncovalent cofactor in bacterial enzymes . The ubiquity of PQQ in the environment and its steady accessibility in the human diet has raised questions concerning its role as a vitamin, or an essential or helpful nutrient . The relevance to nutrition, medicine, and pharmacology of PQQ, topa quinone, lysyl topa quinone, tryptophan trytophylquinone, the galactose oxidase cofactor, and the enzymes harboring these cofactors are discussed in this review.

Biochem Biophys Res Commun, 1998 Aug 10, 249(1), 17 - 22
A mushroom fruiting body-inducing substance inhibits activities of replicative DNA polymerases; Mizushina Y et al.; We found and isolated two natural products in the extract from a basidiomycete, Ganoderma lucidum, as eukaryotic DNA polymerase inhibitors . The compounds were identified as cerebrosides, (4E,8E)-N-D-2'-hydroxypalmitoyl- 1-O-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and (4E,8E)-N-D-2'-hydroxystearoyl-1-O-beta-D-glucopyranos yl-9-methyl- 4,8-sphingadienine and were found to be identical to the mushroom fruiting body-inducing substances (FIS) reported . These cerebrosides selectively inhibited the activities of replicative DNA polymerases, especially the alpha-type, from phylogenetically broad eukaryotic species, whereas they hardly influenced the activities of DNA polymerase beta, prokaryotic DNA polymerases, terminal deoxynucleotidyl transferase, HIV reverse transcriptase, RNA polymerase, deoxyribonuclease I, and ATPase . The inhibition of another replicative polymerase, the delta-type, was moderate . The inhibitions of the replicative polymerases were dose-dependent, and the IC50 for animal or mushroom DNA polymerase alpha was achieved at approximately 12 micrograms/ml (16.2 microM) and for animal DNA polymerase delta at 57 micrograms/ml (77.2 microM) . FIS is possibly a DNA polymerase inhibitor specific to the replicative enzyme group, and the fruiting body formation may be required for the suppression of the DNA replication or the vegetative growth of the mycelium.

Nucleic Acids Res, 1998 Sep 1, 26(17), 4056 - 62
Distinct frequency-distributions of homopolymeric DNA tracts in different genomes; Dechering KJ et al.; The unusual base composition of the genome of the human malaria parasite Plasmodium falciparum prompted us to systematically investigate the occurrence of homopolymeric DNA tracts in the P . falciparum genome and, for comparison, in the genomes of Homo sapiens , Saccharomyces cerevisiae , Caenorhabditis elegans , Arabidopsis thaliana , Escherichia coli and Mycobacterium tuberculosis . Comparison of theobserved frequencies with the frequencies as expected for random DNA revealed that homopolymeric (dA:dT) tracts occur well above chance in the eukaryotic genome . In the majority of these genomes, (dA:dT) tract overrepresentation proved to be an exponential function of the tract length . (dG:dC) tract overrepresentation was absent or less pronounced in both prokaryotic and eukaryotic genomes . On the basis of our results, we propose that homopolymeric (dA:dT) tracts are expanded via replication slippage . This slippage-mediated expansion does not operate on tracts with lengths below a critical threshold of 7-10 bp.

Biotechnol Annu Rev, 1995, 1, 105 - 28
Prokaryotic promoters in biotechnology; Goldstein MA et al.; The basic properties of prokaryotic promoters and the promotor region are described with special emphasis on promoters that are found in Escherichia coli and Bacillus subtilis . Promoters recognized by major and minor forms of RNA polymerase holoenzymes are compared for their specificities and differences . Both natural and hybrid promoters that have been constructed for purposes of efficient and regulated transcription are discussed in terms of their utility . Since promoter regions contain sequences that are recognized not only by RNA polymerase but by positive and negative regulatory factors that regulate expression from promoters, the functions and properties of these promoter regions are also described . The current utility and the future prospects of the prokaryotic promoters in expressing heterologous genes for biotechnology purposes are discussed.

Mol Cell, 1998 Jul, 2(1), 23 - 32
Nucleotide-dependent prereplicative complex assembly by Cdc6p, a homolog of eukaryotic and prokaryotic clamp-loaders; Perkins G et al.; Expression of the Cdc6 protein (Cdc6p) is essential for formation of prereplicative complexes at budding yeast replication origins . Analysis of mutations in the conserved nucleoside triphosphate (NTP)-binding site of Cdc6p described here suggests that NTPs are required both for the productive interaction of Cdc6p with replication origins during G1 and the quantitative loading of the Mcm2-7 family of proteins onto chromatin . We show that Cdc6p exhibits significant sequence similarity to subunits of eukaryotic and prokaryotic clamp-loaders, which load ring-shaped DNA polymerase processivity factors onto DNA in an analogous reaction . Similarities in both sequence and mechanism suggest that Cdc6p and the clamp-loaders are members of a superfamily of nucleotide-dependent loading factors.

Mol Microbiol, 1998 Jul, 29(1), 13 - 8
The function of auxiliary operators; Muller-Hill B; Gene regulation by control of transcription has been analysed in great detail both in prokaryotes and in eukaryotes . The frequency of transcription may be decreased by repressors or increased by activators . A repressor may work by decreasing the concentration of RNA polymerase at a promoter capable of forming an open complex . An activator may work by increasing the concentration of RNA polymerase at a promoter capable of forming an open complex . For this purpose, a strategy is used over and over again . It is called increase in local concentration . How Escherichia coli uses this strategy efficiently is discussed.

Bioessays, 1998 Jun, 20(6), 505 - 10
A novel protein modification generating an aldehyde group in sulfatases: its role in catalysis and disease; von Figura K et al.; In multiple sulfatase deficiency, a rare human lysosomal storage disorder, all known sulfatases are synthesized as catalytically poorly active polypeptides . Analysis of the latter has shown that they lack a protein modification that was detected in all members of the sulfatase family . This novel protein modification generates a 2-amino-3-oxopropanoic acid (C alpha-formylglycine) residue by oxidation of the thiol group of a cysteine that is conserved among all eukaryotic sulfatases . The oxidation occurs in the endoplasmic reticulum at a stage when the nascent polypeptide is not yet folded . The aldehyde is part of the catalytic site and is likely to act as an aldehyde hydrate . One of the geminal hydroxyl groups accepts the sulfate during sulfate ester cleavage leading to the formation of a covalently sulfated enzyme intermediate . The other hydroxyl is required for the subsequent elimination of the sulfate and regeneration of the aldehyde group . In some prokaryotic members of the sulfatase gene family, the DNA sequence predicts a serine residue, and not a cysteine . Analysis of one of these prokaryotic sulfatases, however, revealed the presence of the C alpha-formylglycine indicating that the aldehyde group is essential for all members of the sulfatase family and that it can be generated from either cysteine or serine.

Biochim Biophys Acta, 1998 Jun 10, 1365(1-2), 71 - 8
The electron transport chain in anaerobically functioning eukaryotes; Tielens AG et al.; Many lower eukaryotes can survive anaerobic conditions via a fermentation pathway that involves the use of the reduction of endogenously produced fumarate as electron sink . This fumarate reduction is linked to electron transport in an especially adapted, anaerobically functioning electron-transport chain . An aerobic energy metabolism with Krebs cycle activity is accompanied by electron transfer from succinate to ubiquinone via complex II of the respiratory chain . On the other hand, in an anaerobic metabolism, where fumarate functions as terminal electron acceptor, electrons are transferred from rhodoquinone to fumarate, which is the reversed direction . Ubiquinone cannot replace rhodoquinone in the process of fumarate reduction in vivo, as ubiquinone can only accept electrons from complex II and cannot donate them to fumarate . Rhodoquinone, with its lower redox potential than ubiquinone, is capable of donating electrons to fumarate . Eukaryotic fumarate reductases were shown to interact with rhodoquinone (a benzoquinone), whereas most prokaryotic fumarate reductases interact with the naphtoquinones menaquinone and demethylmenaquinone . Fumarate reductase, the enzyme essential for the anaerobic functioning of many eukaryotes, is structurally very similar to succinate dehydrogenase, the Krebs cycle enzyme catalysing the reverse reaction . In prokaryotes these enzymes are differentially expressed depending on the external conditions . Evidence is now emerging that also in eukaryotes two different enzymes exist for succinate oxidation and fumarate reduction that are differentially expressed.

Biochemistry, 1998 Aug 4, 37(31), 10866 - 70
Rabbit beta-globin is extended beyond its UGA stop codon by multiple suppressions and translational reading gaps; Chittum HS et al.; Translational reading gaps occur when genetic information encoded in mRNA is not translated during the normal course of protein synthesis . This phenomenon has been observed thus far only in prokaryotes and is a mechanism for extending the reading frame by circumventing the normal stop codon . Reading frames of proteins may also be extended by suppression of the stop codon mediated by a suppressor tRNA . The rabbit beta-globin read-through protein, the only known, naturally occurring read-through protein in eukaryotes, was sequenced by ion trap mass spectrometry to determine how the reading frame is extended . Seven different proteolytic peptide fragments decoded by the same sequence that spans the UGA stop codon of rabbit beta-globin mRNA were detected . Three of these peptides contain translational reading gaps of one to three amino acids that correspond to the UGA stop codon site and/or one or two of the immediate downstream codons . To our knowledge, this is the first reported example of the occurrence of reading gaps in protein synthesis in eukaryotes . This event is unique in that it is associated with bypasses involving staggered lengths of untranslated information . Four of the seven peptides contain serine, tryptophan, cysteine, and arginine decoded by UGA and thus arise by suppression . Serine is donated by selenocysteine tRNA, and it, like the other tRNAs, has previously been shown to suppress UGA in vitro in mammals, but not in vivo.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9699 - 704
Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/ glyceraldehyde-3-phosphate dehydrogenase complex dissociation; Wedel N et al.; For higher plant chloroplasts, two key enzymes of the Calvin cycle, phosphoribulokinase (EC 2.7.1.19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), have recently been shown to be oligomerized onto the nonenzymatic peptide CP12 . Enzymatic activity depends on complex dissociation, mediated by NADPH . The discovery of genes for CP12 in mosses, green algae, and cyanobacteria, together with the analysis of equivalent multiprotein complexes of Chlamydomonas and Synechocystis suggests that light regulation of Calvin cycle activity via NADPH-mediated reversible phosphoribulokinase/CP12/GAPDH complex dissociation is conserved in all photosynthetic organisms, prokaryotes and eukaryotes . In vitro complex reconstitution assays with heterologously expressed Synechocystis wild-type and mutagenized CP12 demonstrate a conserved subunit composition, stoichiometry, and topology in this complex . Further finding of genes, coding for chimeric proteins, carrying CP12 or parts of it as genetic fusions, indicates that evolution has used the peptide loops of CP12 as universal modules to keep various enzymatic activities under the control of NADP(H) . These fusion events occurred at least twice in evolution . First was the fusion of the duplicated genes for CP12 and the ORF4 protein of Anabaena variabilis to the chimeric gene for the heterocyst-specific expressed ORF3 protein, most probably involved in N2 fixation . A second gene fusion, which led to the higher plant chloroplast-specific GAPDH subunit, GAPB, has taken place during the transition from water- to land plants.

Proc Natl Acad Sci U S A, 1998 Aug 4, 95(16), 9117 - 22
Structural homology between the Rap30 DNA-binding domain and linker histone H5: implications for preinitiation complex assembly; Groft CM et al.; The three-dimensional structure of the human Rap30 DNA-binding domain has been solved by multinuclear NMR spectroscopy . The structure of the globular domain is strikingly similar to that of linker histone H5 and its fold places Rap30 into the "winged" helix-turn-helix family of eukaryotic transcription factors . Although the domain interacts weakly with DNA, the binding surface was identified and shown to be consistent with the structure of the HNF-3/fork head-DNA complex . The architecture of the Rap30 DNA-binding domain has important implications for the function of Rap30 in the assembly of the preinitiation complex . In analogy to the function of linker histones in chromatin formation, the fold of the Rap30 DNA-binding domain suggests that its role in transcription initiation may be that of a condensation factor for preinitiation complex assembly . Functional similarity to linker histones may explain the dependence of Rap30 binding on the bent DNA environment induced by the TATA box-binding protein . Cryptic sequence identity and functional homology between the Rap30 DNA-binding domain and region 4 of Escherichia coli sigma70 may indicate that the sigma factors also possess a linker histone-like activity in the formation of a prokaryotic closed complex.

Curr Microbiol, 1998 Sep, 37(3), 214 - 6
Sequence analysis of a DNA fragment from buchnera aphidicola (Aphid endosymbiont) containing the genes dapD-htrA-ilvI-ilvH-ftsL-ftsI-murE
Thao ML, Baumann P.
Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum . One of the endosymbiont's functions is the synthesis of branched-chain amino acids . A 9.7-kilobase B . aphidicola chromosomal DNA fragment was cloned and sequenced and found to contain genes encoding acetohydroxy acid synthase (ilvIH), the first enzyme of the parallel pathway of isoleucine and valine biosynthesis . Previously we have detected ilvC and ilvD, encoding the two other enzymes of this pathway . In addition the DNA fragment contained genes for cell division (ftsL, ftsI), murein biosynthesis (murE), lysine biosynthesis (dapD) and a periplasmic protease (htrA) . In these properties B . aphidicola resembles free-living bacteria.

FEBS Lett, 1998 Jul 3, 430(3), 278 - 82
Interaction of the N-terminal domain of MukB with the bacterial tubulin homologue FtsZ; Lockhart A et al.; The MukB protein is involved in the process of chromosome partition in Escherichia coli and has a domain structure reminiscent of the eukaryotic motor proteins kinesin and myosin . This has led to the suggestion that MukB may function as a motor protein in vivo . In order to test this idea we have recombinantly expressed the N-terminal domain of MukB (residues 1-342) as a poly-His tagged fusion protein for biochemical characterisation . The purified protein (Muk342) is monomeric and has low basal Mg-ATPase (1.23 min(-1)) and Mg-GTPase (0.17 min(-1)) activities . Muk342 binds with high affinity to the prokaryotic tubulin homologue FtsZ and we have evidence that FtsZ can stimulate nucleotide turnover by Muk342 . These properties are consistent with MukB functioning as a motor protein using FtsZ as a track or anchor for generating force within E . coli.

Naturwissenschaften, 1998 Jun, 85(6), 278 - 82
Immunoelectron microscopic studies indicate the existence of a cell shape preserving cytoskeleton in prokaryotes; Mayer F et al.; Immunoelectron microscopic studies of prokaryotes were performed with anti-actin antibodies directed against the C terminus of actin . Studies on ultrathin sections revealed high proportions of the overall label close to the cell periphery in Escherichia coli, Ralstonia eutropha, Thermoanaerobacterium thermosulfurigenes, T . thermosaccharolyticum, and Methanococcus jannaschii . Substantial label also in the cytoplasm was observed in Bacillus sp., Methanococcus voltae, and Methanobacterium thermoautotrophicum . Only very minor amounts of label were found in the nucleoid region of the cells . Whole-mount immunogold studies, combined with negative staining, revealed the existence of an intracellular network of fibrils which could be labeled by anti-actin antibodies . This network is assumed to be located below the cytoplasmic membrane all around the cytoplasm . It appears to have properties that would allow its function as a cytoskeleton-like structure preserving cell shape.

Mutat Res, 1998 May 25, 400(1-2), 77 - 88
Effect of target gene CpG content on spontaneous mutation in299 transgenic mice; Skopek T et al.; Transgenic mutation assays utilizing bacterial target genes display a high frequency of spontaneous mutation at CpG sequences . This is believed to result from the fact that: (1) the prokaryotic genes currently being used as transgenic mutation targets have a high CpG content and (2) these sequences are methylated by mammalian cells to produce 5-methylcytosine (5MC), a known promutagenic base . To study the effect of CpG content on the frequency and type of spontaneous mutation, we have synthesized an analogue of the bacterial lacI target gene (mrkII) that contains a reduced number of CpG sequences . This gene was inserted into a lambda vector and used to construct transgenic mice that undergo vector rescue from genomic DNA upon in vitro packaging . Results on spontaneous mutation frequency and spectrum have been collected and compared to those observed at the lacI gene in Big Blue transgenic mice . Spontaneous mutations at the mrkII gene occurred at a frequency in the mid-10-5 range and were predominantly base pair substitutions, similar to results seen in Big Blue . However, mrkII mutations were distributed toward the carboxyl end of the gene instead of the bias toward the amino terminus seen in lacI . Unexpectedly, 23% of the spontaneous mrkII mutations were GC-->AT transitions at CpG sequences (compared to 32% in lacI), despite the reduction in CpG number from 95 in lacI to only 13 in mrkII . Nine of the CpG bases undergoing transition mutations in mrkII have not been recorded previously as spontaneous sites in Big Blue . Therefore, substantial reduction of the number of CpG sequences in the lacI transgene did not significantly reduce the rate of spontaneous mutation or alter the contribution of CpG-related events . This suggests that other factors are also operating to establish frequency and composition of spontaneous mutations in transgenic targets .

Nucleic Acids Res, 1998 Aug 15, 26(16), 3700 - 6
Genetic analysis of prokaryotic and eukaryotic DNA-binding proteins in Escherichia coli; Whipple FW; This report describes an Escherichia coli genetic system that permits bacterial genetic methods to be applied to the study of essentially any prokaryotic or eukaryotic site-specific DNA binding protein . It consists of two parts . The first part is a set of tools that facilitate construction of customized E.coli strains bearing single copy lacZYA reporters that are repressed by a specific target protein . The second part is a pair of regulatable protein expression vectors that permit in vivo production of the target protein at levels appropriate for genetic experiments . When expressed in a properly designed reporter strain, the target protein represses the lac genes, resulting in an E.coli phenotype that can be quantitatively measured or exploited in large scale genetic screens or selections . As a result, large plasmid-based libraries of protein genes or pools of mutagenized variants of a given gene may be examined in relatively simple genetic experiments . The strain construction technique is also useful for generating E.coli strains bearing reporters for other types of genetic systems, including repression-based and activation-based systems in which chimeric proteins are used to examine interactions between foreign protein domains.

Nucleic Acids Res, 1998 Aug 15, 26(16), 3619 - 25
Survey, analysis and genetic organization of genes encoding eukaryotic-like signaling proteins on a cyanobacterial genome; Zhang CC et al.; Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose . Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well . The sequence determination of the genome of the cyanobacterium Synechocystis sp . strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism . This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins . The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins . We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis . Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction . In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.

Genes Cells, 1998 May, 3(5), 265 - 78
How protein reads the stop codon and terminates translation; Nakamura Y et al.; Translation termination requires two codon-specific protein-release factors in prokaryotes and one factor in eukaryotes . The underlying mechanism for stop codon recognition, as well as the biological meaning of the conservation of one or two release factors in the evolutionary kingdoms, are not known . The recent discovery of release factor genes and the molecular mimicry between translational factors and tRNA provide us with clues to the mechanisms of how proteins read the stop codon and terminate translation, shedding some light on the evolutionary aspect of release factors.

Protein Sci, 1998 Jul, 7(7), 1626 - 31
The discoidin domain family revisited: new members from prokaryotes and a homology-based fold prediction; Baumgartner S et al.; Members of the discoidin (DS) domain family, which includes the C1 and C2 repeats of blood coagulation factors V and VIII, occur in a great variety of eukaryotic proteins, most of which have been implicated in cell-adhesion or developmental processes . So far, no three-dimensional structure of a known example of this extracellular module has been determined, limiting the usefulness of identifying a new sequence as member of this family . Here, we present results of a recent search of the protein sequence database for new DS domains using generalized profiles, a sensitive multiple alignment-based search technique . Several previously unrecognized DS domains could be identified by this method, including the first examples from prokaryotic species . More importantly, we present statistical, structural, and functional evidence that the D1 domain of galactose oxidase whose three-dimensional structure has been determined at 1.7 A resolution, is a distant member of this family . Taken together, these findings significantly expand the concept of the DS domain, by extending its taxonomic range and by implying a fold prediction for all its members . The proposed alignment with the galactose oxidase sequence makes it possible to construct homology-based three-dimensional models for the most interesting examples, as illustrated by an accompanying paper on the C1 and C2 domains of factor V.

FEBS Lett, 1998 Jul 10, 431(1), 39 - 44
Potential dual targeting of an Arabidopsis archaebacterial-like histidyl-tRNA synthetase to mitochondria and chloroplasts; Akashi K et al.; A cDNA clone encoding a histidyl-tRNA synthetase (HisRS) was characterized from Arabidopsis thaliana . The deduced amino acid sequence (AtHRS1) is surprisingly more similar to HisRSs from archaebacteria than those from eukaryotes and prokaryotes . AtHRS1 has an N-terminal extension with features characteristic of mitochondrial and chloroplast transit peptides . Transient expression assays in tobacco protoplasts clearly demonstrated efficient targeting of a fusion peptide consisting of the first 71 amino acids of AtHRS1 joined to jellyfish green fluorescent protein (GFP) to both mitochondria and chloroplasts . These observations suggest that the AtHisRS1 cDNA encodes both mitochondrial and chloroplast histidyl-tRNA synthetases.

Extremophiles, 1997 Aug, 1(3), 117 - 23
Molecular analyses of the sediment of the 11,000-m deep Mariana Trench; Kato C et al.; We have obtained sediment samples from the world's deepest sea-bottom, the Mariana Trench challenger point at a depth of 10,898 m, using the new unmanned submersible Kaiko . DNA was extracted from the sediment, and DNA fragments encoding several prokaryotic ribosomal RNA small-subunit sequences and pressure-regulated gene clusters, typically identified in deep-sea adapted bacteria, were amplified by the polymerase chain reaction . From the sequencing results, at least two kinds of bacterial 16S rRNAs closely related to those of the genus Pseudomonas and deep-sea adapted marine bacteria, and archaeal 16S rRNAs related to that of a planktonic marine archaeon were identified . The sequences of the amplified pressure-regulated clusters were more similar to those of deep-sea barophilic bacteria than those of barotolerant bacteria . These results suggest that deep-sea adapted barophilic bacteria, planktonic marine archaea, and some of the world's most widespread bacteria (the genus Pseudomonas) coexist on the world's deepest sea-bottom.

Mol Microbiol, 1998 Jun, 28(6), 1043 - 9
Hyperthermophiles and the problem of DNA instability; Grogan DW; Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication . The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face . The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form . Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation . Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo, and underlying mechanisms are beginning to be examined . Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair . However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.

Chem Biol Interact, 1998 Apr 24, 111-112, 41 - 50
Mechanistic imperative for the evolution of a metalloglutathione transferase of the vicinal oxygen chelate superfamily; Laughlin LT et al.; A number of glutathione (GSH) transferases are now known in prokaryotes and eukaryotes . The enzymes appear to be primarily involved in the metabolism of foreign compounds . At least six distinct classes of soluble GSH transferases have been identified in eukaryotes and named alpha, mu, pi, sigma, theta and kappa . Sequences and the known three-dimensional structures of the soluble enzymes suggest that these proteins share a common ancestry, though the precise details of their evolution remain obscure . A second distinct family of GSH transferases are the microsomal or membrane-bound enzymes that include leukotriene C4 synthase . A third family is represented by a bacterial GSH transferase (FosA) responsible for conferring resistance to the antibiotic fosfomycin, reported some years ago by Suarez and co-workers (Arca et al., Antimicrob . Agents Chemother . 34 (1990) 1552-1556) . The enzyme is quite specific for fosfomycin, which contains a very stable epoxide moiety . Evidence is presented that FosA is a metalloprotein related to iron- and manganese-dependent dioxygenases and to glyoxalase I . These enzymes are members of a previously unrecognized group of enzymes; the vicinal oxygen chelate superfamily . The mechanistic imperative driving the evolution of FosA and its relatives, which are enzymes catalyzing quite diverse chemical reactions, is proposed to be the electrophilic assistance provided by the metal through chelation of a substrate or intermediate.

Anticancer Res, 1998 May-Jun, 18(3B), 1967 - 71
Micronucleus analysis in peripheral blood lymphocytes from melanoma patients treated with dacarbazine; Miele M et al.; BACKGROUND: Dacarbazine is an antitumor drug used with considerable success in the chemotherapy of a number of human neoplasias, particularly advanced disseminated melanoma . Dacarbazine is mutagenic in prokaryotic and eukaryotic cells, but no effect in vivo have been evaluated . MATERIALS AND METHODS: Peripheral blood lymphocytes from patients with metastatic melanoma undergoing dacarbazine chemotherapy every 21 days for a total of 7 cycles, were analyzed for the presence of micronuclei with the CREST antikinetochore antibody technique . Cytogenetic analysis on blood samples collected just before and 2 hours after the therapy was carried out at 48, 72 and 96 hours following lymphocyte stimulation . RESULTS: A significant increase in micronucleus frequency was found at both 72 and 96 hours after therapy . For the only two patients analyzed after more than one cycle, a decrease in micronuclei was observed after the third and the fourth therapy . Moreover, the CREST antibody technique showed that the frequency of micronuclei containing whole chromosomes (CREST+) was significantly higher after therapy at 72 and 96 hours . As the frequency of micronuclei containing acentric chromosome fragments (CREST-) was not significantly increased after therapy, either at 72 or 96 hours after lymphocyte stimulation, we suppose that DTIC mainly acted as an aneugenic agent . CONCLUSIONS: The lack of a significant micronucleus increase at 48 hours could suggest that this culture time is too short for providing cultures with a sufficient large number of diving cells . In conclusion, our results have shown that dacarbazine induced chromosome loss in lymphocytes from patients treated with this drug.

FEMS Microbiol Lett, 1998 Jul 1, 164(1), 35 - 8
Co-migration of RAPD-PCR amplicons from Aeromonas hydrophila; Oakey HJ et al.; Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels . RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals . The analysis of such fingerprints is based upon the assumption that co-migration of amplicons does not occur and that any given band contains a single amplicon . This report shows that co-migration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila . The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.

Trends Microbiol, 1998 Jun, 6(6), 233 - 8
A green light for the bacterial cytoskeleton; Margolin W; Improved fluorescence techniques for visualizing proteins in whole bacterial cells have resulted in recent breakthroughs in our understanding of chromosome segregation and cytokinesis in prokaryotes . The dynamics and localization of some of these proteins reveal surprisingly cytoskeletal-like behavior.

Mutat Res, 1998 Jun 18, 402(1-2), 41 - 50
Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast; Kozmin SG et al.; Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms . In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog .

Arch Biochem Biophys, 1998 Jul 15, 355(2), 241 - 8
Decorin core protein fragment Leu155-Val260 interacts with TGF-beta but does not compete for decorin binding to type I collagen; Schonherr E et al.; It has been shown that small proteoglycans containing leucine-rich repeats in their core proteins can form complexes with TGF-beta . Decorin, a ubiquitously found molecule of the extracellular matrix, is the best-studied example . Therefore, binding domains on its core protein were investigated using recombinant decorin fragments generated as fusion proteins in prokaryotes . The peptide Leu155-Val260 immobilized by the polyhistidine tag on a nickel chelate column bound TGF-beta1 and -beta2 almost as effectively as the largest fragment (Asp45-Lys359) studied . Other peptides were less effective . For the two peptides Asp45-Lys359 and Leu155-Val260 dissociation constants in the nanomolar range for high-affinity binding sites were calculated in a solid-phase assay with immobilized TGF-beta2 . Peptide Asp45-Lys359 also contained a lower affinity binding site . Domains with lower affinity were also found in peptides Asp45-Leu155 and Arg63-Gly190 . Peptide Leu155-Val260 also formed complexes with TGF-beta in the liquid phase as determined by equilibrium gel filtration . Furthermore, F(ab') fragments of polyclonal antibodies against peptide Leu155-Val260 interfered with TGF-beta binding to peptide Asp45-Lys359 in a dose-dependent manner . Peptide Leu155-Val260, however, is only a weak competitor of the binding of wild-type decorin to reconstituted type I collagen fibrils . Therefore, independent binding sites of decorin for TGF-beta and type I collagen should exist . In support of this hypothesis saturable binding of TGF-beta1 and TGF-beta2 to collagen-bound native decorin could be demonstrated . The bound cytokine could be released in a biologically active form by collagenase treatment . Thus, decorin may play a biological role in storing this cytokine temporarily in the extracellular matrix and in thereby modulating an interaction of TGF-beta with its signaling receptors .

Arch Virol, 1997, 142(8), 1673 - 80
Banana bunchy top virus DNA-3 encodes the viral coat protein; Wanitchakorn R et al.; Banana bunchy top virus (BBTV) has a multicomponent genome consisting of at least six circular single-stranded DNAs each with a single large open reading frame (ORF) in the virion sense . A protein of approximately 20 kDa has been associated with purified virions and is assumed to be the viral coat protein . The N-terminus of this protein was sequenced and compared to the predicted amino acid sequence of the large ORF of BBTV DNA-1 to 6 . This comparison indicated that the ORF of BBTV DNA-3, which potentially encodes a protein of 19.3 kDa, was the coat protein gene of BBTV . The ORF sequence of BBTV DNA-3 was cloned into a prokaryotic expression vector, pMAL-c2, and the resulting maltose binding-BBTV coat protein fusion product was purified and used for the production of polyclonal antiserum in a rabbit . The resultant antiserum was able to detect the presence of BBTV in infected leaf tissue confirming that the large virion sense ORF of BBTV DNA-3 encodes the viral coat protein.

Nucleic Acids Res, 1998 Aug 1, 26(15), 3513 - 20
Microsporidian Encephalitozoon cuniculi, a unicellular eukaryote with an unusual chromosomal dispersion of ribosomal genes and a LSU rRNA reduced to the universal core; Peyretaillade E et al.; Microsporidia are eukaryotic parasites lacking mitochondria, the ribosomes of which present prokaryote-like features . In order to better understand the structural evolution of rRNA molecules in microsporidia, the 5S and rDNA genes were investigated in Encephalitozoon cuniculi . The genes are not in close proximity . Non-tandemly arranged rDNA units are on every one of the 11 chromosomes . Such a dispersion is also shown in two other Encephalitozoon species . Sequencing of the 5S rRNA coding region reveals a 120 nt long RNA which folds according to the eukaryotic consensus structural shape . In contrast, the LSU rRNA molecule is greatly reduced in length (2487 nt) . This dramatic shortening is essentially due to truncation of divergent domains, most of them being removed . Most variable stems of the conserved core are also deleted, reducing the LSU rRNA to only those structural features preserved in all living cells . This suggests that the E.cuniculi LSU rRNA performs only the basic mechanisms of translation . LSU rRNA phylogenetic analysis with the BASEML program favours a relatively recent origin of the fast evolving microsporidian lineage . Therefore, the prokaryote-like ribosomal features, such as the absence of ITS2, may be derived rather than primitive characters.

Proc Natl Acad Sci U S A, 1998 Jul 21, 95(15), 8550 - 5
Mechanism of DNA segregation in prokaryotes: replicon pairing by parC of plasmid R1; Jensen RB et al.; Prokaryotic chromosomes and plasmids encode partitioning systems that are required for DNA segregation at cell division . The systems are thought to be functionally analogous to eukaryotic centromeres and to play a general role in DNA segregation . The parA system of plasmid R1 encodes two proteins ParM and ParR, and a cis-acting centromere-like site denoted parC . The ParR protein binds to parC in vivo and in vitro . The ParM protein is an ATPase that interacts with ParR specifically bound to parC . Using electron microscopy, we show here that parC mediates efficient pairing of plasmid molecules . The pairing requires binding of ParR to parC and is stimulated by the ParM ATPase . The ParM mediated stimulation of plasmid pairing is dependent on ATP hydrolysis by ParM . Using a ligation kinetics assay, we find that ParR stimulates ligation of parC-containing DNA fragments . The rate-of-ligation was increased by wild type ParM protein but not by mutant ParM protein deficient in the ATPase activity . Thus, two independent assays show that parC mediates pairing of plasmid molecules in vitro . These results are consistent with the proposal that replicon pairing is part of the mechanism of DNA segregation in prokaryotes.

Mol Gen Genet, 1998 Jun, 258(5), 530 - 7
A gene coding for an RPS2 protein is present in the mitochondrial genome of several cereals, but not in dicotyledons; Vaitilingom M et al.; A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum) . The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid . Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus . The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences . This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene . Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.

Curr Opin Struct Biol, 1998 Jun, 8(3), 355 - 63
Beyond complete genomes: from sequence to structure and function; Koonin EV et al.; Computer analysis of complete prokaryotic genomes shows that microbial proteins are in general highly conserved--approximately 70% of them contain ancient conserved regions . This allows us to delineate families of orthologs across a wide phylogenetic range and, in many cases, predict protein functions with considerable precision . Sequence database searches using newly developed, sensitive algorithms result in the unification of such orthologous families into larger superfamilies sharing common sequence motifs . For many of these superfamilies, prediction of the structural fold and specific amino acid residues involved in enzymatic catalysis is possible . Taken together, sequence and structure comparisons provide a powerful methodology that can successfully complement traditional experimental approaches.

J Mol Biol, 1998 Jul 17, 280(3), 431 - 42
Crystal structures of native and recombinant yeast fumarase; Weaver T et al.; Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomyces cerevisiae have been completed to moderate resolution by two separate laboratories . The recombinant form was obtained by the construction of an expression plasmid for Escherichia coli . Despite a high level of amino acid sequence similarity, purification of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasible . The crystal structure of the native form, NY-fumarase, encompasses residues R22 through M484, while the recombinant form, RY-fumarase, consists of residues S27 through L485 . Both crystal structures lack the N-terminal translocation segment . Each subunit of the homo-tetrameric protein has three domains . The active site is formed by segments from each of three polypeptide chains . The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done in the absence of dicarboxylic acid and has an unoccupied active site . As a comparison, native fumarase was crystallized in the presence of the competitive inhibitor, meso-tartrate . Meso-tartrate occupies a position close to that of the bound citrate molecule found in the active site of the E . coli enzyme . This inhibitor participates in hydrogen bonding to an active-site water molecule . The independent determination of the two structures provides further evidence that an active-site water molecule may play an active role in the fumarase-catalyzed reaction .

Biochemistry, 1998 Jul 14, 37(28), 10325 - 35
Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia; Mohsen AW et al.; Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric mitochondrial flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA . PCR of IVD genomic and complementary DNA was used to identify mutations occurring in patients with deficiencies in IVD activity . Western blotting, in vitro mitochondrial import, prokaryotic expression, and kinetic studies of IVD mutants were conducted to characterize the molecular defects caused by the amino acid replacements . Mutations leading to Arg21Pro, Asp40Asn, Ala282Val, Cys328Arg, Val342Ala, Arg363Cys, and Arg382Leu replacements were identified . Western blotting of fibroblast extracts and/or in vitro mitochondrial import experiments indicate that the seven precursor IVD mutant peptides, and a previously identified IVD Leu13Pro mutant, are synthesized and imported into mitochondria . While the IVD Leu13Pro, Arg21Pro, and Cys328Arg mutant peptides are rapidly degraded following mitochondrial import, the other mutant peptides exhibit greater mitochondrial stability, though less than the wild-type enzyme . Active IVD Ala282Val, Val342Ala, Arg363Cys, and Arg382Leu mutants were less stable than wild type when produced in Escherichia coli . The Km values of purified IVD Ala282Val, Val342Ala, and Arg382Leu mutants are 27.0, 2 . 8, and 6.9 microM isovaleryl-CoA, respectively, compared to 3.1 microM for the wild type, using the electron-transfer flavoprotein (ETF) fluorescence quenching assay . The catalytic efficiency per mole of FAD content of these three mutants is 4.8, 17.0, and 17.0 microM-1*min-1, respectively, compared to 170 microM-1*min-1 for wild type.

Mol Microbiol, 1998 Jun, 28(5), 1005 - 16
In vivo relations between pAMbeta1-encoded type I topoisomerase and plasmid replication; Bidnenko V et al.; A number of large extrachromosomal elements encode prokaryotic type I topoisomerases of unknown functions . Here, we analysed the topoisomerase Topbeta encoded by the Gram-positive broad-host-range plasmid pAMbeta1 . We show that this enzyme possesses the DNA relaxation activity of type I topoisomerases . Interestingly, it is active only on plasmids that use DNA polymerase I to initiate replication, such as pAMbeta1, and depends on the activity of this polymerase . This is the first example, to our knowledge, of prokaryotic type I topoisomerase that is specific for a given type of replicon . During pAMbeta1 replication in Bacillus subtilis cells, Topbeta promotes premature arrest of DNA polymerase I, approximately 190bp downstream of the replication initiation point . We propose that Topbeta acts on the early replication intermediates of pAMbeta1, which contain D-loops formed by DNA polymerase I-mediated strand displacement . The possible role of the resulting DNA Pol I arrest in plasmid replication is discussed.

Plant Physiol, 1998 Jul, 117(3), 997 - 1006
Molecular and biochemical characterization of cytosolic phosphoglucomutase in maize . Expression during development and in response to oxygen deprivation; Manjunath S et al.; Phosphoglucomutase (PGM) catalyzes the interconversion of glucose (Glc)-1- and Glc-6-phosphate in the synthesis and consumption of sucrose . We isolated two maize (Zea mays L.) cDNAs that encode PGM with 98.5% identity in their deduced amino acid sequence . Southern-blot analysis with genomic DNA from lines with different Pgm1 and Pgm2 genotypes suggested that the cDNAs encode the two known cytosolic PGM isozymes, PGM1 and PGM2 . The cytosolic PGMs of maize are distinct from a plastidic PGM of spinach (Spinacia oleracea) . The deduced amino acid sequences of the cytosolic PGMs contain the conserved phosphate-transfer catalytic center and the metal-ion-binding site of known prokaryotic and eukaryotic PGMs . PGM mRNA was detectable by RNA-blot analysis in all tissues and organs examined except silk . A reduction in PGM mRNA accumulation was detected in roots deprived of O2 for 24 h, along with reduced synthesis of a PGM identified as a 67-kD phosphoprotein on two-dimensional gels . Therefore, PGM is not one of the so-called "anaerobic polypeptides." Nevertheless, the specific activity of PGM was not significantly affected in roots deprived of O2 for 24 h . We propose that PGM is a stable protein and that existing levels are sufficient to maintain the flux of Glc-1-phosphate into glycolysis under O2 deprivation.

Plant Physiol, 1998 Jul, 117(3), 923 - 30
A new class of Arabidopsis mutants with reduced hexadecatrienoic acid fatty acid levels; Miquel M et al.; Chloroplast glycerolipids in a number of higher-plant species, including Arabidopsis thaliana, are synthesized by two distinct pathways termed the prokaryotic and eukaryotic pathways . The molecules of galactolipids produced by the prokaryotic pathway contain substantial amounts of hexadecatrienoic acid fatty acid . Here we describe a new class of mutants, designated gly1, with reduced levels of hexadecatrienoic acid . Lipid fatty acid profiles indicated that gly1 mutants exhibited a reduced carbon flux through the prokaryotic pathway that was compensated for by an increased carbon flux through the eukaryotic pathway . Genetic and biochemical approaches revealed that the gly1 phenotype could not be explained by a deficiency in the enzymes of the prokaryotic pathway . The flux of fatty acids into the prokaryotic pathway is sensitive to changes in glycerol-3-phosphate (G3P) availability, and the chloroplast G3P pool can be increased by exogenous application of glycerol to leaves . Exogenous glycerol treatment of gly1 plants allowed chemical complementation of the mutant phenotype . These results are consistent with a mutant lesion affecting the G3P supply within the chloroplast . The gly1 mutants may therefore help in determining the pathway for synthesis of chloroplast G3P.

Gene, 1998 May 28, 212(1), 87 - 94
Expression of Reticulomyxa filosa alpha- and beta-tubulins in Escherichia coli yields soluble and partially correctly folded material; Linder S et al.; Tubulins are highly conserved multidomain proteins that have to interact with eukaryotic chaperonins to gain their correct three-dimensional conformation . The prokaryotic chaperonin system of GroEL/ES is able to generate intermediate folding states but not natively folded tubulin . To create a system for studying these folding intermediates, tubulins from the giant amoeba Reticulomyxa filosa (alpha 2- and beta 2-tubulin) were expressed in Escherichia coli singly or in tandem . In all cases, soluble tubulin was generated in amounts of 5-10 mg/l culture . This is the first reported expression of soluble tubulin in bacterial cells . Of particular interest was the observation that upon coexpression with R . filosa beta 2-tubulin, proteolytic degradation of alpha 2-tubulin was reduced and more full-length product remained intact . This observation points to a specific interaction of alpha 2- and beta 2-tubulins in the E . coli cell . The sites of interaction are most probably the same that are responsible for the binding of native alpha 2- and beta 2-tubulin . The established expression system therefore seems well suited for further studies concerning the folding of tubulins.

Mol Cell, 1998 Mar, 1(4), 519 - 29
Regulated chromosomal DNA replication in the absence of a nucleus; Walter J et al.; Using Xenopus egg extracts, we have developed a completely soluble system for eukaryotic chromosomal DNA replication . In the absence of a nuclear envelope, a single, complete round of ORC-dependent DNA replication is catalyzed by cytosolic and nuclear extracts added sequentially to demembranated sperm chromatin or prokaryotic plasmid DNA . The absence of rereplication is explained by an activity present in the nucleus that prevents the binding of MCM to chromatin . Our results indicate that the role of the nuclear envelope in DNA replication is to concentrate activators and inhibitors of replication inside the nucleus . In addition, they provide direct evidence that metazoans use the same strategy as yeast to activate DNA replication and to restrict it to a single round per cell cycle.

J Biol Chem, 1998 Jul 17, 273(29), 18109 - 16
The repressor protein, Bm3R1, mediates an adaptive response to toxic fatty acids in Bacillus megaterium; Palmer CN et al.; Bm3R1 is a helix-turn-helix transcriptional repressor from Bacillus megaterium whose binding to DNA is inhibited by fatty acids and a wide range of compounds that modulate lipid metabolism . The inactivation of Bm3R1/DNA binding activity results in the activation of transcription of the operon encoding a fatty acid hydroxylase, cytochrome P450 102 . The metabolic role of this operon is unknown . It is possible that it is involved in the synthesis of modified fatty acids as part of normal cellular metabolism or may represent a protective mechanism by which B . megaterium detoxifies harmful foreign lipids . In this report we demonstrate that polyunsaturated fatty acids (PUFA) activate the transcription of the CYP102 operon . These PUFA are the most potent activators of the CYP102 operon observed to date, and we show that their effects are due to binding directly to Bm3R1 . In addition, cultures that have been treated with the CYP102 inducer, nafenopin, are protected against PUFA toxicity . Resistance to PUFA toxicity is also seen in a Bm3R1-deficient strain that constitutively expresses CYP102 . The resistant phenotype of this Bm3R1 mutant strain is reversed by specific chemical inactivation of CYP102 . These data demonstrate that Bm3R1 can act as a direct sensor of toxic fatty acids and, in addition, provide the first direct evidence of fatty acids binding to a prokaryotic transcription factor.

Eur J Biochem, 1998 Jun 1, 254(2), 356 - 62
Purification and characterization of an alcohol dehydrogenase from the Antarctic psychrophile Moraxella sp . TAE123; Tsigos I et al.; An NAD+-dependent alcohol dehydrogenase (ADH) of the Antarctic psychrophile Moraxella sp . TAE123 was purified to homogeneity with an overall yield of 16.7% and further characterized . The native enzyme had an apparent molecular mass of 240 kDa and consisted of four identical 52-kDa subunits . The pI of the enzyme was determined to be 5.5, while its optimum pH is 7.5 . The enzyme contained 1 zinc atom/subunit and exhibited a remarkable thermal lability . Moraxella sp . TAE123 ADH exhibited a wide range of substrate specificity similar to its mammalian counterparts and in contrast to other microbial ADHs . It oxidized mainly primary and secondary aliphatic alcohols . The highest reaction rate was observed when ethanol was used as substrate . A gradual decrease in rate was observed by increasing the length and branching of the carbon chain of the alcohol . This enzyme oxidized effectively large bulky alcohols, such as diphenylmethanol . Reduction of aldehydes and ketones was also observed . N-terminal amino acid sequence analysis of the enzyme did not reveal any similarity with the amino termini of all other ADHs, while an unexpected significant similarity was observed with the amino terminal sequence of four prokaryotic aldehyde dehydrogenases.

Eur J Biochem, 1998 Jun 1, 254(2), 325 - 32
A family of flavoproteins in the domains Archaea and Bacteria; Wasserfallen A et al.; A family of flavoproteins, called A-type flavoproteins, is described . It consists of 14 protein sequences of 385-597 amino acids in length, 7 from methanogens (domain: Archaea), 5 from phototrophic prokaryotes, one from Escherichia coli, and a partial sequence from the sulfate reducer Desulfovibrio gigas (domain: Bacteria) . No similar sequence could be found in the domain Eucarya . All sequences show significant similarity over a 385-400 amino acid portion overlapping a recognizable flavodoxin signature starting at positions 245-285 of the common core sequence . Cofactor analysis and, to some extent, analysis of the primary structure of six A-type flavoproteins, three of which are structurally characterized here, support the existence of four sub-families: (a) simple flavoproteins binding only FMN; (b) diflavin flavoproteins binding FMN and FAD; (c) a flavorubredoxin binding FMN and iron; (d) a hemoflavoprotein . The possible involvement of A-type flavoproteins in the metabolism of oxygen, as suggested for D . gigas hemoflavoprotein {Gomes, C . M., Silva, G., Oliveira, S., LeGall, J., Liu, M.-Y., Xavier, A . V., Rodrigues-Pousada, C . & Teixeira, M . (1997) J . Biol . Chem . 272, 22502-22508}, is discussed.

J Virol, 1998 Aug, 72(8), 6832 - 7
Localization of varicella-zoster virus gene 21 protein in virus-infected cells in culture; Mahalingam R et al.; Although four varicella-zoster virus (VZV) genes have been shown to be transcribed in latently infected human ganglia, their role in the development and maintenance of latency is unknown . To study these VZV transcripts, we decided first to localize their expression products in productively infected cells . We began with VZV gene 21, whose open reading frame (ORF) is 3,113 bp . We cloned the 5' and 3' ends and the predicted antigenic segments of the ORF as 1292-, 1280-, and 880-bp DNA fragments, respectively, into the prokaryotic expression vector pGEX-2T . The three VZV 21 ORFs were expressed as approximately 75-, 73-, and 59-kDa glutathione S-transferase fusion proteins in Escherichia coli . To prepare polyclonal antibodies that would recognize all potential epitopes on the VZV gene 21 protein, rabbits were inoculated with a mixture of the three fusion proteins, and antisera were obtained and affinity purified . Immunohistochemical and immunoelectron microscopic analyses using these antibodies revealed VZV ORF 21 protein in both the nucleus and cytoplasm of VZV-infected cells . When these antibodies were applied to purified VZV nucleocapsids, intense staining was seen in their central cores.

Science, 1998 Jul 10, 281(5374), 262 - 6
Structure of the Escherichia coli RNA polymerase alpha subunit amino-terminal domain; Zhang G et al.; The 2.5 angstrom resolution x-ray crystal structure of the Escherichia coli RNA polymerase (RNAP) alpha subunit amino-terminal domain (alphaNTD), which is necessary and sufficient to dimerize and assemble the other RNAP subunits into a transcriptionally active enzyme and contains all of the sequence elements conserved among eukaryotic alpha homologs, has been determined . The alphaNTD monomer comprises two distinct, flexibly linked domains, only one of which participates in the dimer interface . In the alphaNTD dimer, a pair of helices from one monomer interact with the cognate helices of the other to form an extensive hydrophobic core . All of the determinants for interactions with the other RNAP subunits lie on one face of the alphaNTD dimer . Sequence alignments, combined with secondary-structure predictions, support proposals that a heterodimer of the eukaryotic RNAP subunits related to Saccharomyces cerevisiae Rpb3 and Rpb11 plays the role of the alphaNTD dimer in prokaryotic RNAP.

Biochim Biophys Acta, 1998 Jul 9, 1398(3), 225 - 31
Structure and expression of an asparaginyl-tRNA synthetase gene located on chromosome IV of Arabidopsis thaliana and adjacent to a novel gene of 15 exons; Aubourg S et al.; The gene AtNS1 coding for an asparaginyl-tRNA synthetase and located on chromosome IV of Arabidopsis thaliana has been characterized . AtNS1 is the first asparaginyl-tRNA synthetase gene described in higher plants . The genomic environment of AtNS1 has been studied, as well as a partial cDNA of a second homologous asparaginyl-tRNA synthetase gene, AtNS2 . Both AtNS1 and AtNS2 exhibit the highest similarity with prokaryotic homologues . A large novel gene of 15 exons, named AtG2484-1, is located adjacent to AtNS1 . AtG2484-1 shows features rarely described in plants including large exons and one 3' non-coding exon . PCR and Northern analyses were carried out to obtain information about the expression of these genes in various A . thaliana tissues.

Structure, 1998 Jun 15, 6(6), 747 - 56
The structure of a methylated tetraloop in 16S ribosomal RNA; Rife JP et al.; BACKGROUND: Ribosomal RNAs contain many modified nucleotides . The functions of these nucleotides are poorly understood and few of them are strongly conserved . The final stem loop in 16S-like rRNAs is an exception in both regards . In both prokaryotes and eukaryotes, the tetranucleotide loop that caps the 3'-terminal stem contains two N6, N6-dimethyladenosine residues . The sequence and pattern of methylation are conserved within the loop, and there is evidence that these methylated nucleotides play an important role in subunit association and the initiation of protein synthesis . Because of the integral role that helix 45 plays in ribosome function, it is important to know what consequences these methylated nucleotides have on its structure . RESULTS: We have solved the solution structure of a 14-nucleotide analog of the terminal stem loop of bacterial 16S rRNA, which contains N2-methylguanosine as well as two N6,N6-dimethyladenosines . CONCLUSIONS: The methylation of the 16S rRNA stem loop completely alters its conformation, which would otherwise be a GNRA tetraloop . It is likely that the conformation of this loop is crucial for its function, having implications for its interaction with ribosomal subunits and its role in the initiation of protein synthesis.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8245 - 50
Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells; Delecluse HJ et al.; With current techniques, genetic alterations of herpesviruses are difficult to perform, mostly because of the large size of their genomes . To solve this problem, we have designed a system that allows the cloning of any gamma-herpesvirus in Escherichia coli onto an F factor-derived plasmid . Immortalized B cell lines were readily established with recombinant Epstein-Barr virus (EBV), demonstrating that the F factor-cloned EBV genome has all the characteristics of wild-type EBV . Because any genetic modification is possible in E . coli, this experimental approach opens the way to the genetic analysis of all EBV functions . Moreover, it is now feasible to generate attenuated EBV strains in vitro such that vaccine strains can be designed . Because we incorporated the genes for hygromycin resistance and green fluorescent protein onto the E . coli cloned EBV genome, the still open question of the EBV target cells other than B lymphocytes will be addressed.

Proc Natl Acad Sci U S A, 1998 Jul 7, 95(14), 8165 - 9
Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons; Ito K et al.; Prokaryotic translational release factors, RF1 and RF2, catalyze polypeptide release at UAG/UAA and UGA/UAA stop codons, respectively . In this study, we isolated a bacterial RF2 mutant (RF2*) containing an E167K substitution that restored the growth of a temperature-sensitive RF1 strain of Escherichia coli and the viability of a chromosomal RF1/RF2 double knockout . In both in vivo and in vitro polypeptide termination assays, RF2* catalyzed UAG/UAA termination, as does RF1, as well as UGA termination, showing that RF2* acquired omnipotent release activity . This result suggests that the E167K mutation abolished the putative third-base discriminator function of RF2 . These findings are interpreted as indicating that prokaryotic and eukaryotic release factors share the same anticodon moiety and that only one omnipotent release factor is sufficient for bacterial growth, similar to the eukaryotic single omnipotent factor.

Trends Biotechnol, 1998 Jun, 16(6), 272 - 7
Perfluorochemicals: their applications and benefits to cell culture; Lowe KC et al.; The properties of perfluorochemical liquids, particularly their high gas solubility, enables them to be exploited in cell biotechnology . They can facilitate respiratory-gas delivery to prokaryotic and eukaryotic cells in culture; in some systems, they can stimulate production of biomass, yields of commercially important cellular products and, for plant systems, expression of totipotency . The recoverability, and hence recycleability, of perfluorochemicals from aqueous systems makes their routine use a commercially feasible option . This article reviews the applications and beneficial effects of perfluorochemicals in cultured microbial, animal and plant cells, including both aerobic and anaerobic systems.

J Biol Chem, 1998 Jul 10, 273(28), 17879 - 85
Topoisomerase IV catalysis and the mechanism of quinolone action; Anderson VE et al.; Topoisomerase IV is a bacterial type II topoisomerase that is essential for proper chromosome segregation and is a target for quinolone-based antimicrobial agents . Despite the importance of this enzyme to the survival of prokaryotic cells and to the treatment of bacterial infections, relatively little is known about the details of its catalytic mechanism or the basis by which quinolones alter its enzymatic functions . Therefore, a series of experiments that analyzed individual steps of the topoisomerase IV catalytic cycle were undertaken to address these critical mechanistic issues . The following conclusions were drawn . First, equilibrium levels of DNA cleavage mediated by the bacterial enzyme were considerably (>10-fold) higher than those observed with its eukaryotic counterparts . To a large extent, this reflected decreased rates of DNA religation . Second, the preference of topoisomerase IV for catalyzing DNA decatenation over relaxation reflects increased rates of strand passage and enzyme recycling rather than a heightened recognition of intermolecular DNA helices . Third, quinolones stimulate topoisomerase IV-mediated DNA cleavage both by increasing rates of DNA scission and by inhibiting religation of cleaved DNA . Finally, quinolones inhibit the overall catalytic activity of topoisomerase IV primarily by interfering with enzyme-ATP interactions.

Int J Biol Macromol, 1998 May-Jun, 22(3-4), 151 - 62
Genealogy of the alpha-crystallin--small heat-shock protein superfamily; de Jong WW et al.; Sequences of 40 very diverse representatives of the alpha-crystallin-small heat-shock protein (alpha-Hsp) superfamily are compared . Their characteristic C-terminal 'alpha-crystallin domain' of 80-100 residues contains short consensus sequences that are highly conserved from prokaryotes to eukaryotes . There are, in addition, some positions that clearly distinguish animal from non-animal alpha-Hsps . The alpha-crystallin domain is predicted to consist of two hydrophobic beta-sheet motifs, separated by a hydrophilic region which is variable in length . Combination of a conserved alpha-crystallin domain with a variable N-terminal domain and C-terminal extension probably modulates the properties of the various alpha-Hsps as stress-protective and structural oligomeric proteins . Phylogeny reconstruction indicates that multiple alpha-Hsps were already present in the last common ancestor of pro- and eukaryotes . It is suggested that during eukaryote evolution, animal and non-animal alpha-Hsps originated from different ancestral gene copies . Repeated gene duplications gave rise to the multiple alpha-Hsps present in most organisms.

Mol Gen Genet, 1998 May, 258(4), 427 - 30
Genes encoding thymidylate synthases A and B in the genus Bacillus are members of two distinct families; Tam NH et al.; Bacillus subtilis strain 168 is known to possess two genes that encode thymidylate synthases, thyA and thyB . We have identified genes similar to the thyA and thyB genes in several Bacillus strains by Southern hybridization and by DNA amplification with sequence-specific primers . Analysis of thyA genes cloned from B . subtilis W23 strain 2A6, B . subtilis ATCC6633, B . amyloliquefaciens S18 and B . atrophaeus S223 reveals that they are very similar to the thyA genes from B . subtilis 168 and its phage phi3T, but differ considerably from the majority of known prokaryotic and eukaryotic thymidylate synthases.

Appl Environ Microbiol, 1998 Jul, 64(7), 2585 - 95
Seasonal and spatial variability of bacterial and archaeal assemblages in the coastal waters near Anvers Island, Antarctica; Murray AE et al.; A previous report of high levels of members of the domain Archaeal in Antarctic coastal waters prompted us to investigate the ecology of Antarctic planktonic prokaryotes . rRNA hybridization techniques and denaturing gradient gel electrophoresis (DGGE) analysis of the bacterial V3 region were used to study variation in Antarctic picoplankton assemblages . In Anvers Island nearshore waters during late winter to early spring, the amounts of archaeal rRNA ranged from 17.1 to 3.6% of the total picoplankton rRNA in 1996 and from 16.0 to 1.0% of the total rRNA in 1995 . Offshore in the Palmer Basin, the levels of archaeal rRNA throughout the water column were higher (average, 24% of the total rRNA) during the same period in 1996 . The archaeal rRNA levels in nearshore waters followed a highly seasonal pattern and markedly decreased during the austral summer at two stations . There was a significant negative correlation between archaeal rRNA levels and phytoplankton levels (as inferred from chlorophyll a concentrations) in nearshore surface waters during the early spring of 1995 and during an 8-month period in 1996 and 1997 . In situ hybridization experiments revealed that 5 to 14% of DAPI (4',6-diamidino-2-phenylindole)-stained cells were archaeal, corresponding to 0.9 x 10(4) to 2.7 x 10(4) archaeal cells per ml, in late winter 1996 samples . Analysis of bacterial ribosomal DNA fragments by DGGE revealed that the assemblage composition may reflect changes in water column stability, depth, or season . The data indicate that changes in Antarctic seasons are accompanied by significant shifts in the species composition of bacterioplankton assemblages and by large decrease in the relative proportion of archaeal rRNA in the nearshore water column.

J Immunol, 1998 Jul 1, 161(1), 90 - 6
H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro; Byers DE et al.; Most CTL responses to epitopes from influenza virus are restricted by MHC class Ia molecules . However, a synthetic peptide corresponding to residues 173 to 190 of influenza A/JAP/305/57 hemagglutinin (HA) can induce, in vitro, a CTL response to peptide presented by a mouse class Ib molecule encoded by a gene telomeric to H2-Q . Here, we identify the molecule as H2-M3 and show that the last five residues of HA173-190, MLIIW, is the minimal epitope for CTL recognition . Cells that express M3wt, from C57BL/6 or BALB/c mice, are sensitized by both MLIIW and the longer peptide HA173-190, whereas cells that express M3f, from A.CA or B10.M mice, are sensitized only by MLIIW; a single amino acid change at residue 31 (V-->M) of M3 accounts for this difference . Although M3-restricted CTLs preferably recognize N-formylated epitopes, i.e., those of mitochondrial or prokaryotic origin, our findings show that M3-restricted primary CTL responses can be generated in vitro against nonformylated epitopes.

Annu Rev Biophys Biomol Struct, 1998, 27, 199 - 224
Simulation of prokaryotic genetic circuits; McAdams HH et al.; Biochemical and genetic approaches have identified the molecular mechanisms of many genetic reactions, particularly in bacteria . Now a comparably detailed understanding is needed of how groupings of genes and related protein reactions interact to orchestrate cellular functions over the cell cycle, to implement preprogrammed cellular development, or to dynamically change a cell's processes and structures in response to environmental signals . Simulations using realistic, molecular-level models of genetic mechanisms and of signal transduction networks are needed to analyze dynamic behavior of multigene systems, to predict behavior of mutant circuits, and to identify the design principles applicable to design of genetic regulatory circuits . When the underlying design rules for regulatory circuits are understood, it will be far easier to recognize common circuit motifs, to identify functions of individual proteins in regulation, and to redesign circuits for altered functions.

J Biol Chem, 1998 Jul 3, 273(27), 16843 - 52
Recognition of a human arrest site is conserved between RNA polymerase II and prokaryotic RNA polymerases; Mote J Jr et al.; DNA sequences that arrest transcription by either eukaryotic RNA polymerase II or Escherichia coli RNA polymerase have been identified previously . Elongation factors SII and GreB are RNA polymerase-binding proteins that enable readthrough of arrest sites by these enzymes, respectively . This functional similarity has led to general models of elongation applicable to both eukaryotic and prokaryotic enzymes . Here we have transcribed with phage and bacterial RNA polymerases, a human DNA sequence previously defined as an arrest site for RNA polymerase II . The phage and bacterial enzymes both respond efficiently to the arrest signal in vitro at limiting levels of nucleoside triphosphates . The E . coli polymerase remains in a template-engaged complex for many hours, can be isolated, and is potentially active . The enzyme displays a relatively slow first-order loss of elongation competence as it dwells at the arrest site . Bacterial RNA polymerase arrested at the human site is reactivated by GreB in the same way that RNA polymerase II arrested at this site is stimulated by SII . Very efficient readthrough can be achieved by phage, bacterial, and eukaryotic RNA polymerases in the absence of elongation factors if 5-Br-UTP is substituted for UTP . These findings provide additional and direct evidence for functional similarity between prokaryotic and eukaryotic transcription elongation and readthrough mechanisms.

Photochem Photobiol, 1998 May, 67(5), 541 - 6
UV-DNA damage in mouse and human cells induces the expression of tumor necrosis factor alpha; Kibitel J et al.; Ultraviolet light induces the expression of tumor necrosis factor alpha (TNF alpha) in many mammalian cells . We have examined the signal for this induction in a human DNA repair-deficient cell line carrying a transgene composed of the murine TNF regulatory sequences fused to the chloramphenicol acetyltransferase (CAT) structural gene . When compared by fluence, UVC was a more efficient inducer of CAT than was UVB, but they were equivalent inducers when compared by the frequency of cyclobutyl pyrimidine dimers produced by each source . Further, treatment of UV-irradiated cells with the prokaryotic DNA repair enzyme T4 endonuclease V increased the level of repair of dimers and concomitantly reduced CAT gene expression . Membrane-bound TNF alpha expression was increased by UV and reduced by repair of dimers . Finally, in the TNFcat transgene system, DNA damage directly to the cell with the transgene was required as cocultivation of unirradiated TNFcat cells with UV-irradiated cells did not increase CAT activity . These results show that DNA damage is a signal for the induction of TNF alpha gene expression in mouse and human cells.

Mutagenesis, 1998 May, 13(3), 263 - 9
Expression of human cytochrome P450 1A2 in Escherichia coli: a system for biotransformation and genotoxicity studies of chemical carcinogens; Kranendonk M et al.; In this study we describe the development of strain BMX100, a new Escherichia coli K12 tester strain, derived from MX100, a strain which was constructed for detection of mutagens and for mechanistic studies of chemical carcinogens . We demonstrate here that strain BMX100 can be used for stable expression of human CYP1A2 or human CYP1A2 fused to rat liver NADPH cytochrome P450 reductase . Mutagenicity of precarcinogens known to be bioactivated by CYP1A2, namely 2-aminoanthracene (2-AA), aflatoxin B1 (AFB1) and 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), could be detected . The mutagenic activity of 2-AA using BMX100 expressing CYP1A2 alone and in combination with rat CYP reductase was respectively 10 and 20 times higher than in BMX100 with the standard metabolic activation system, rat liver S9 fraction . Furthermore, the mutagenicity of 2-AA could be nullified by alpha-naphthoflavone, a known inhibitor of CYP1A2 . IQ responded equally in BMX100 expressing the CYP1A2-reductase fusion protein as compared with usage of rat liver S9 fraction . Rat liver S9 fraction was much more potent in generating a mutagenic response to AFB1 in BMX100 than in the strain expressing human CYP1A2 alone or CYP1A2 fused to rat reductase . The results described in this study demonstrate that this new E.coli strain can function as a human CYP1A2-competent prokaryotic mutagenicity test system and they seem to characterize BMX100 as a strain of interest for studies to identify individual human CYPs involved in bioactivation and bioinactivation reactions of putative genotoxins.

J Mol Biol, 1998 Jun 26, 279(5), 1027 - 43
DNA microloops and microdomains: a general mechanism for transcription activation by torsional transmission; Travers A et al.; Prokaryotic transcriptional activation often involves the formation of DNA microloops upstream of the polymerase binding site . There is substantial evidence that these microloops function to bring activator and polymerase into close spatial proximity . However additional functions are suggested by the ability of certain activators, of which FIS is the best characterised example, to facilitate polymerase binding, promoter opening and polymerase escape . We review here the evidence for the concept that the topology of the microloop formed by such activators is tightly coupled to the structural transitions in DNA mediated by RNA polymerase . In this process, which we term torsional transmission, a major function of the activator is to act as a local topological homeostat . We argue that the same mechanism may also be employed in site-specific DNA inversion .

Wei Sheng Wu Xue Bao, 1996 Jun, 36(3), 173 - 80
Cloning and expression of full-length delta-endotoxin cryIA(c) gene in three kinds of prokaryotic systems using shuttle vector pHT3101; Yang Y et al.; Two fragments, 6.5kb and 4.3kb encoding 5' end and 3' end of delta-endotoxin cryIA(c) gene, respectively, were selected from the Bacillus thuringiensis kurstaki-HD-73 75kb plasmid gene pool using random primer labelling delta-endotoxin cryIA(b) gene probe . The full-length 3.92kb cryIA(c) gene including 5' end 152 bp promoter sequence and 3' end 198 bp terminater sequence was rebuilt after uncoding sequences were deleted . Three kinds of engineering strains harbouring the same recombinant plasmid pHTY1 were obtained after the cryIA(c) gene had been subcloned in shuttle vector pHT3101 and transformed to E . coli NM522, Bacillus subtilis AS1176 and Bacillus thuringiensis crystal-deficient 4D10(H3ab) . SDS-PAGE electrophoresis patterns reveal that the cryIA(c) gene expressed the same 133,300 protoxin proteins in all three host systems with the same molecular weight to the crystal protein from the Bacillus thuringiensis kurstaki HD-73 . Immuno-assays indicate that the expression proteins can react with antiserum of HD-73 paraspore crystal protein in the same pattern as the natural crystalprotein . Bioassays using crude expressed products from three host strains reveal that they all showed toxicities to second instar larvae of Plutella xylostella, and their LD50 were calculated to be 0.311 micrograms/cm2, 0.02 micrograms/cm2 and 0.017 micrograms/cm2, respectively.

Arch Microbiol, 1998 Jul, 170(1), 8 - 17
Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides; Paoli GC et al.; Photosynthetic prokaryotes that assimilate CO2 under anoxic conditions may also grow chemolithoautotrophically with O2 as the electron acceptor . Among the nonsulfur purple bacteria, two species (Rhodobacter capsulatus and Rhodopseudomonas acidophilus), exhibit aerobic chemolithoautotrophic growth with hydrogen as the electron donor . Although wild-type strains of Rhodobacter sphaeroides grow poorly, if at all, with hydrogen plus oxygen in the dark, we report here the isolation of a spontaneous mutant (strain HR-CAC) of Rba . sphaeroides strain HR that is fully capable of this mode of growth . Rba . sphaeroides and Rba . capsulatus fix CO2 via the reductive pentose phosphate pathway and synthesize two forms of ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO) . RubisCO levels in the aerobic-chemolithoautotrophic-positive strain of Rba . sphaeroides were similar to those in wild-type strains of Rba . sphaeroides and Rba . capsulatus during photoheterotrophic and photolithoautotrophic growth . Moreover, RubisCO levels of Rba . sphaeroides strain HR-CAC approximated levels obtained in Rba . capsulatus when the organisms were grown as aerobic chemolithoautotrophs . Either form I or form II RubisCO was able to support aerobic chemolithoautotrophic growth of Rba . capsulatus strain SB 1003 and Rba . sphaeroides strain HR-CAC at a variety of CO2 concentrations, although form II RubisCO began to lose the capacity to support aerobic CO2 fixation at high O2 to CO2 ratios . The latter property and other facets of the physiology of this system suggest that Rba . sphaeroides and Rba . capsulatus strains may be effectively employed for the biological selection of RubisCO molecules of altered substrate specificity.

J Exp Biol, 1998 Jul, 201 ( Pt 14), 2091 - 102
The electroneutral cation-chloride cotransporters; Mount DB et al.; Electroneutral cation-chloride cotransporters are widely expressed and perform a variety of physiological roles . A novel gene family of five members, encompassing a Na+-Cl- transporter, two Na+-K+-2Cl- transporters and two K+-Cl- cotransporters, encodes these membrane proteins; homologous genes have also been identified in a prokaryote and a number of lower eukaryotes . The cotransporter proteins share a common predicted membrane topology, with twelve putative transmembrane segments flanked by long hydrophilic N- and C-terminal cytoplasmic domains . The molecular identification of these transporters has had a significant impact on the study of their function, regulation and pathophysiology.

Curr Microbiol, 1998 Jul, 37(1), 58 - 9
The endosymbiont (Buchnera) of the aphid Diuraphis noxia contains all the genes of the tryptophan biosynthetic pathway; Baumann L et al.; Previously it has been shown that the prokaryotic endosymbiont (Buchnera) of the aphid Diuraphis noxia contains a plasmid consisting of one copy of the gene for anthranilate synthase (trpEG) and seven trpEG pseudogenes . In the present communication we show that this endosymbiont contains the remaining genes of the tryptophan biosynthetic pathway {trpDC(F)BA} which appear to be functional in that they code for the complete enzyme proteins and are not pseudogenes . As in the case of Buchnera from other endosymbionts, these genes appear to be organized as one transcription unit and are located on the endosymbiont chromosome.

Biotechnology (N Y), 1995 Apr, 13(4), 383 - 8
Phosphoric acid entrapment leads to apparent protein heterogeneity; Fountoulakis M et al.; Recombinant proteins produced in prokaryotes or eukaryotes show certain types of heterogeneity due to post-translational modifications . Some preparations of a soluble interferon gamma receptor, produced in Escherichia coli, appeared as a double band with slightly different mobilities in non-reducing sodium dodecylsulfate and native polyacrylamide gels . Ion spray mass spectrometry showed that the two forms had a mass difference of one to three multiples of 97 +/- 2 D . Gas chromatography-mass spectrometry analysis revealed the presence of phosphoric acid in the hydrolysate and in the intact protein . The more slowly migrating protein species had trapped molecules of phosphoric acid during the protein extraction . Most of the trapped phosphoric acid was loosely associated with the protein . One to three molecules were tightly, but non-covalently linked per receptor molecule . Phosphoric acid entrapment did not affect biological activity and most likely did not affect protein conformation . The species carrying phosphoric acid showed higher solubility . Trapping of phosphoric acid by proteins may be a general phenomenon and the results reported here thus useful in the characterization of other recombinant proteins.

J Biol Chem, 1998 Jun 19, 273(25), 15794 - 803
Ultraviolet radiation triggers the ribotoxic stress response in mammalian cells; Iordanov MS et al.; The ribotoxic stress response, which is conserved between prokaryotes and eukaryotes, is a cellular reaction to cytotoxic interference with the function of the 3'-end of the large (23 S/28 S) ribosomal RNA . The 3'-end of the large rRNA is directly involved in the three sequential steps of translational elongation: the aminoacyl-tRNA binding, the peptidyl transfer, and the ribosomal translocation . In mammalian cells, the ribotoxic stress response involves activation of the stress-activated protein kinase/c-Jun NH2-terminal kinase and the p38 mitogen-activated protein kinase and transcriptional induction of immediate early genes such as c-fos and c-jun . Active ribosomes are essential mediators of the ribotoxic stress response . We demonstrate here that the transcriptional response of mammalian cells to ultraviolet radiation (UV response) displays the characteristics of a ribotoxic stress response, inasmuch as (i) the activation of stress kinases and gene expression in response to UV requires the presence of active ribosomes at the moment of irradiation; (ii) UV irradiation inhibits protein synthesis; and (iii) irradiation of cells with UV causes specific damage to the 3'-end of the 28 S rRNA . In contrast, the activation of the stress kinases by hyperosmolarity, by the DNA-cross-linking agent diepoxybutane, or by growth factors and cytokines does not depend on the presence of active ribosomes . Our results identify UV as a potential ribotoxic stressor and support the notion that some of the cellular signaling cascades in response to UV might be generated in the ribosome, possibly triggered by damage to rRNA.

Bioinformatics, 1998, 14(4), 367 - 71
Frame: detection of genomic sequencing errors; Brown NP et al.; MOTIVATION: The underlying error rate for genomic sequencing sometimes results in the introduction of artificial frameshifts and in-frame stop codons into putative protein encoding genes . Severe errors are then introduced into the inferred transcripts through mis-translation or premature termination . RESULTS: We describe a system for screening segments of DNA for frameshift and in-frame stop errors in coding regions . The method is based on homology matching using blastx to compare all six reading frames of the query nucleotide sequence against selected protein sequence databases . Fragments of protein matching neighbouring regions of the query DNA are united and extended laterally to define candidate open reading frames, within which, frameshifts and stops are identified . Suitable targets include prokaryotic or other intron-free genomic sequence and complementary DNAs . As an example of its use, we report here two frameshifted ORFs that deviate from the original TIGR sequence annotations for the recently released Helicobacter pylori genome . AVAILABILITY: The tool is accessible via the URL CONTACT: brown@ebi.ac.uk.

J Biol Chem, 1998 Jun 26, 273(26), 15961 - 70
Purification, characterization, and cloning of a cytosolic aspartyl aminopeptidase; Wilk S et al.; An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4 . 11.7) was purified to near homogeneity from rabbit brain cytosol . Its properties were similar to an enzyme described previously (Kelly, J . A., Neidle, E . L., and Neidle, A . (1983) J . Neurochem . 40, 1727-1734) . Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates . Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5) . The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis . The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags . Human and mouse clones described as "similar to a yeast vacuolar aminopeptidase" and containing full-length cDNAs were identified and sequenced . The human cDNA was expressed in Escherichia coli . The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases . Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates . Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.

Science, 1998 Jun 12, 280(5370), 1757 - 60
Promotion of met-tRNAiMet binding to ribosomes by yIF2, a bacterial IF2 homolog in yeast; Choi SK et al.; Delivery of the initiator methionine transfer RNA (Met-tRNAiMet) to the ribosome is a key step in the initiation of protein synthesis . Previous results have indicated that this step is catalyzed by the structurally dissimilar translation factors in prokaryotes and eukaryotes-initiation factor 2 (IF2) and eukaryotic initiation factor 2 (eIF2), respectively . A bacterial IF2 homolog has been identified in both eukaryotes and archaea . By using a combination of molecular genetic and biochemical studies, the Saccharomyces cerevisiae IF2 homolog is shown to function in general translation initiation by promoting Met-tRNAiMet binding to ribosomes . Thus, the mechanism of protein synthesis in eukaryotes and prokaryotes is more similar than was previously realized.

J Mol Biol, 1998 May 22, 278(5), 903 - 14
Translational activation by an NtrC enhancer-binding protein; Cullen PJ et al.; The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-binding protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor . The nifR3-ntrB-ntrC operon in R . capsulatus codes for the nitrogen-sensing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes . Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported . The nifR3-ntrB-ntrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and ntrC mutants under nitrogen-limited or nitrogen-sufficient conditions . LacZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain constant, production of NifR3 is at least ten to 40-fold reduced in NtrC- strains . NifR3 is increased at least fivefold upon nitrogen limitation whereas NtrC production is constitutive . Surprisingly, the purified NtrC protein binds cooperatively to the nifR3 promoter region in vitro at two sets of tandem binding sites centered at +1 and -81 nucleotides relative to the transcriptional start site . Deletion analysis demonstrates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo , but are not necessary for nifR3 transcription . These experiments indicate that NtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA . This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA and anfA) in R . capsulatus which are transcriptionally activated by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site .

J Mol Biol, 1998 May 8, 278(3), 599 - 608
Protein identification with N and C-terminal sequence tags in proteome projects; Wilkins MR et al.; Genome sequences are available for increasing numbers of organisms . The proteomes (protein complement expressed by the genome) of many such organisms are being studied with two-dimensional (2D) gel electrophoresis . Here we have investigated the application of short N-terminal and C-terminal sequence tags to the identification of proteins separated on 2D gels . The theoretical N and C termini of 15, 519 proteins, representing all SWISS-PROT entries for the organisms Mycoplasma genitalium, Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and human, were analysed . Sequence tags were found to be surprisingly specific, with N-terminal tags of four amino acid residues found to be unique for between 43% and 83% of proteins, and C-terminal tags of four amino acid residues unique for between 74% and 97% of proteins, depending on the species studied . Sequence tags of five amino acid residues were found to be even more specific . To utilise this specificity of sequence tags for protein identification, we created a world-wide web-accessible protein identification program, TagIdent , which matches sequence tags of up to six amino acid residues as well as estimated protein pI and mass against proteins in the SWISS-PROT database . We demonstrate the utility of this identification approach with sequence tags generated from 91 different E . coli proteins purified by 2D gel electrophoresis . Fifty-one proteins were unambiguously identified by virtue of their sequence tags and estimated pI and mass, and a further 11 proteins identified when sequence tags were combined with protein amino acid composition data . We conlcude that the TagIdent identification approach is best suited to the identification of proteins from prokaryotes whose complete genome sequences are available . The approach is less well suited to proteins from eukaryotes, as many eukaryotic proteins are not amenable to sequencing via Edman degradation, and tag protein identification cannot be unambiguous unless an organism's complete sequence is available .

J Biol Chem, 1998 May 15, 273(20), 12407 - 14
Structural and functional properties of complement-activating protein M161Ag, a Mycoplasma fermentans gene product that induces cytokine production by human monocytes; Matsumoto M et al.; Human malignant cells are targeted by homologous complement C3b if they express M161Ag, a 43-kDa protein with C3-activating property . cDNA of M161Ag cloned from human leukemia cell lines predicted M161Ag as a novel secretory protein comprised of 428 amino acids including 5 amino acids encoded by TGA codons (Matsumoto M., Takeda, J., Inoue, N., Hara, T., Hatanaka, M., Takahashi, K., Nagasawa, S., Akedo, H., and Seya, T . (1997) Nat . Med . 3, 1266-1270), although the origin of this gene was obscure . Here we clarified this point through genomic and biochemical analysis: 1) 5'-UT and genomic sequences represented the prokaryote promoter and ribosomal binding site; 2) the TGA codons in M161Ag cDNA were translated not into selenocysteines but into tryptophans; 3) M161Ag anchored onto the membrane secondary to its N-terminal palmitoylation like prokaryote lipoproteins; 4) genomic and cDNA clones of M161Ag were highly homologous to Mycoplasma fermentans gene encoding P48, a monocytic differentiation/activation factor, recently released in the data base, although the resultant proteins were different in the amino acid sequences . Additionally, purified soluble M161Ag efficiently provoked IL-1beta, tumor necrosis factor alpha, and IL-6 like P48, and further IL-10 and IL-12 in human peripheral blood monocytes . Thus, M161Ag originates from M . fermentans, and latently infected M . fermentans allows human cells to produce M161Ag . The liberated protein serves as a potent modulator of innate and cellular immune responses via its complement-activating and cytokine-producing activities.

Nucleic Acids Res, 1998 May 1, 26(9), 2230 - 6
Using neural networks for prediction of the subcellular location of proteins; Reinhardt A et al.; Neural networks have been trained to predict the subcellular location of proteins in prokaryotic or eukaryotic cells from their amino acid composition . For three possible subcellular locations in prokaryotic organisms a prediction accuracy of 81% can be achieved . Assigning a reliability index, 33% of the predictions can be made with an accuracy of 91% . For eukaryotic proteins (excluding plant sequences) an overall prediction accuracy of 66% for four locations was achieved, with 33% of the sequences being predicted with an accuracy of 82% or better . With the subcellular location restricting a protein's possible function, this method should be a useful tool for the systematic analysis of genome data and is available via a server on the world wide web.

Bioessays, 1998 Mar, 20(3), 256 - 63
My favorite cell: Giardia; Upcroft J et al.; The gut protozoan parasite, Giardia duodenalis, is the best characterized example of the most ancient eukaryotes, which are anaerobic and appear to be primitively amitochondrial . Apart from its obvious medical importance, Giardia is fascinating in its own right . Its prokaryotic-like anaerobic metabolism renders it selectively sensitive to some bacterial drugs, especially the nitroimidazoles, which are activated to form toxic radicals . Other features, including an enzyme that reduces oxygen directly to water, cysteine as the keeper of redox balance, a plasmid, and toxin-like genes are also distinctly prokaryotic-like . But, unlike prokaryotes, Giardia has a sophisticated, highly developed cytoskeleton, bounded nuclei, linear chromosomes capped with telomeric repeats, and telomere positional regulation of gene expression.

J Mol Evol, 1998 Jun, 46(6), 703 - 15
The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits; Hilario E et al.; Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed . The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported . Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote . The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes . Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus . The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex . Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase . Their possible implication in the evolution of V/F/A-ATPases is discussed.

Nucleic Acids Res, 1998 Jul 1, 26(13), 3188 - 93
Gene length and codon usage bias in Drosophila melanogaster, Saccharomyces cerevisiae and Escherichia coli; Moriyama EN et al.; The relationship between gene length and synonymous codon usage bias was investigated in Drosophila melanogaster, Escherichia coli and Saccharomyces cerevisiae . Simulation studies indicate that the correlations observed in the three organisms are unlikely to be due to sampling errors or any potential bias in the methods used to measure codon usage bias . The correlation was significantly positive in E.coli genes, whereas negative correlations were obtained for D . melanogaster and S.cerevisiae genes . When only ribosomal protein genes were used, whose expression levels are assumed to be similar, E.coli and S.cerevisiae showed significantly positive correlations . For the two eukaryotes, the distribution of effective number of codons was different in short genes (300-500 bp) compared with longer genes; this was not observed in E.coli . Both positive and negative correlations can be explained by translational selection . Energetically costly longer genes have higher codon usage bias to maximize translational efficiency . Selection may also be acting to reduce the size of highly expressed proteins, and the effect is particularly pronounced in eukaryotes . The different relationships between codon usage bias and gene length observed in prokaryotes and eukaryotes may be the consequence of these different types of selection.

Biol Chem, 1998 Apr-May, 379(4-5), 553 - 7
Chromosomal inactivation of Bacillus subtilis exfusants: a prokaryotic model of epigenetic regulation; Grandjean V et al.; Epigenetic mechanisms are not exclusively reserved to eukaryotic organisms . They are also observed in prokaryotes . As described first by Hotchkiss and Gabor, protoplast fusion between strains of Bacillus subtilis produces heterodiploid cells . Heterodiploidy is associated with the inactivation of one of the chromosomes . To study the physical structure of the fusion product and the molecular mechanisms of inactivation, we constructed heterodiploid clones containing two chromosomes labeled by a NotI restriction fragment length polymorphism . In the progeny, we identified haploid recombinant clones that contain a chromosome carrying large regions of inactivated DNA . Studies of both recombinants of the latter kind and heterodiploid cells indicated that chromosomal inactivation was not determined by alteration of the inactivated nucleotide sequence, but was probably due to a modification in the structure of the bacterial chromatin.

Plant J, 1998 May, 14(3), 345 - 51
Targeting and modification of prokaryotic cell-cell junctions by tobacco mosaic virus cell-to-cell movement protein; Heinlein M et al.; The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants . Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp . strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated . It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa . It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related . The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.

FEMS Microbiol Lett, 1998 May 15, 162(2), 257 - 64
Dissimilatory ATP sulfurylase from the hyperthermophilic sulfate reducer Archaeoglobus fulgidus belongs to the group of homo-oligomeric ATP sulfurylases; Sperling D et al.; In the hyperthermophilic sulfate reducer Archaeoglobus fulgidus DSM 4304T, two open reading frames (sat and ORF2) are located upstream of the aprBA genes encoding adenosine-5'-phosphosulfate (APS) reductase . sat-ORF2-aprBA probably form a transcriptional unit, since sat is preceded by putative promoter sequences and termination signals are found downstream of aprA . While the 117-residue ORF2 product does not show significant similarity to known proteins, the 456-residue, 52.78-kDa, sat-encoded polypeptide exhibits similarity to the homo-oligomeric adenosine triphosphate (ATP) sulfurylases from sulfur-oxidizing bacteria and from sulfate-assimilating bacteria and eukaryotes . Functional overexpression of sat in Escherichia coli proved that the encoded protein acts as an ATP sulfurylase . The recombinant protein was purified to homogeneity and found to be a homo-dimer . Comparison of sulfate and thiosulfate grown A . fulgidus revealed that ATP sulfurylase and APS reductase are constitutive enzymes . Distance matrix analyses allowed insights into the evolution of prokaryotic ATP sulfurylases.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6728 - 32
Two-domain reconstitution of a functional protein histidine kinase; Park H et al.; In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses . Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals . In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function . Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A {EnvZ(C) . (223-289); 67 residues} and subdomain B {EnvZ(C).(290-450); 161 residues} . Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ . Subdomain B, an alpha/beta-protein, exists as a monomer . When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP . Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR . The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6711 - 5
Thermostable archaeal O6-alkylguanine-DNA alkyltransferases; Skorvaga M et al.; Archaea represent some of the most ancient organisms on earth, and they have relatively uncharacterized DNA repair processes . We now show, using an in vitro assay, that extracts of two Crenarchaeota (Sulfolobus acidocaldarius and Pyrobaculum islandicum) and two Euryarchaeota (Pyrococcus furiosus and Thermococcus litoralis) contain the DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) . The ATase activities found in the archaea were extremely thermostable, with half-lives at 80 degreesC ranging from 0.5 hr (S . acidocaldarius) to 13 hr (T . litoralis) . The temperature optima of the four proteins ranged from approximately 75 to approximately 100 degreesC, although activity was seen at 37 degreesC, the temperature optimum of the Escherichia coli and human ATases . In all cases, preincubaton of extracts with a short oligonucleotide containing a single O6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkylphosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea . The ATase from Pyrobaculum islandicum had an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described . In higher organisms, ATase is responsible for the repair of toxic and mutagenic O6-alkylguanine lesions in alkylated DNA . The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generate appropriate substrates in DNA may be an early event in evolution.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6687 - 92
The RIIbeta regulatory subunit of protein kinase A binds to cAMP response element: an alternative cAMP signaling pathway; Srivastava RK et al.; cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation . In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity . In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons . The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation . Herein we report that RIIbeta subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE . In contrast to CREB/ATF, the binding of RIIbeta to a CRE was enhanced by cAMP, and in addition, RIIbeta exhibited transcriptional activity as a Gal4-RIIbeta fusion protein . These experiments identify RIIbeta as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 6578 - 83
Prokaryotes: the unseen majority; Whitman WB et al.; The number of prokaryotes and the total amount of their cellular carbon on earth are estimated to be 4-6 x 10(30) cells and 350-550 Pg of C (1 Pg = 10(15) g), respectively . Thus, the total amount of prokaryotic carbon is 60-100% of the estimated total carbon in plants, and inclusion of prokaryotic carbon in global models will almost double estimates of the amount of carbon stored in living organisms . In addition, the earth's prokaryotes contain 85-130 Pg of N and 9-14 Pg of P, or about 10-fold more of these nutrients than do plants, and represent the largest pool of these nutrients in living organisms . Most of the earth's prokaryotes occur in the open ocean, in soil, and in oceanic and terrestrial subsurfaces, where the numbers of cells are 1.2 x 10(29), 2.6 x 10(29), 3.5 x 10(30), and 0 . 25-2.5 x 10(30), respectively . The numbers of heterotrophic prokaryotes in the upper 200 m of the open ocean, the ocean below 200 m, and soil are consistent with average turnover times of 6-25 days, 0.8 yr, and 2.5 yr, respectively . Although subject to a great deal of uncertainty, the estimate for the average turnover time of prokaryotes in the subsurface is on the order of 1-2 x 10(3) yr . The cellular production rate for all prokaryotes on earth is estimated at 1.7 x 10(30) cells/yr and is highest in the open ocean . The large population size and rapid growth of prokaryotes provides an enormous capacity for genetic diversity.

J Biol Chem, 1998 Jun 12, 273(24), 14869 - 76
The Aspergillus nidulans cnxF gene and its involvement in molybdopterin biosynthesis . Molecular characterization and analysis of in vivo generated mutants; Appleyard MV et al.; The product of the Aspergillus nidulans cnxF gene was found by biochemical analysis of cnxF mutants to be involved in the conversion of precursor Z to molybdopterin . Mutants cnxF1242 and cnxF8 accumulate precursor Z, while the level of molybdopterin is undetectable . The DNA sequence of the cnxF gene was determined, and the inferred protein of 560 amino acids was found to contain a central region (residues around 157 to 396) similar in sequence to the prokaryotic proteins MoeB, which is thought to encode molybdopterin synthase sulfurylase, ThiF, required for thiamine biosynthesis, and HesA, involved in heterocyst formation, as well as eukaryotic ubiquitin-activating protein E1 . Based on these similarities, a possible mechanism of action is discussed . Sequence comparisons indicate the presence of one and possibly two nucleotide binding motifs, Gly-X-Gly-X-X-Gly, as well as two metal binding Cys-X-X-Cys motifs in this central region of the CnxF protein . Seven in vivo generated A . nidulans cnxF mutants were found to have amino acid substitutions of conserved residues within this central region of similarity to molybdopterin synthase sulfurylase, indicating that these seven amino acids are essential and that this domain is crucial for function . Of these seven, the cnxF1285 mutation results in the replacement of Gly-178, the last glycine residue of the N-proximal Gly-X-Gly-X-X-Gly motif, indicating that this motif is essential . Mutation of the conserved Arg-208, also probably involved in nucleotide binding, leads to a loss-of-function phenotype in cnxF200 . Alteration of Cys-263, the only conserved Cys residue (apart from the metal binding motifs), in cnxF472 suggests this residue as a candidate for thioester formation between molybdopterin synthase and the sulfurylase . Substitution of Gly-160 in two independently isolated mutants, cnxF21 and cnxF24, results in temperature-sensitive phenotypes and indicates that this residue is important in protein conformation . The C-terminal CnxF stretch (residues 397-560) shows substantial sequence conservation to a yeast hypothetical protein, Yhr1, such conservation between species suggesting that this region has function . Not inconsistent with this proposition is the observation that mutant cnxF8 results from loss of the 34 C-terminal residues of CnxF . There is no obvious similarity of the CnxF C-terminal region with other proteins of known function . Two cnxF transcripts are found in low abundance and similar levels were observed in nitrate- or ammonium-grown cells.

J Gen Physiol, 1998 Jun, 111(6), 741 - 9
Functional reconstitution of a prokaryotic K+ channel; Heginbotham L et al.; SliK, a K+ channel encoded by the Streptomyces KcsA gene, was expressed, purified, and reconstituted in liposomes . A concentrative 86Rb+ flux assay was used to assess the ion transport properties of SliK . SliK-mediated ionic flux shows strong selectivity for K+ over Na+ and is inhibited by micromolar concentrations of Ba2+, mirroring the basic permeation characteristic of eukaryotic K+ channels studied by electrophysiological methods . 86Rb+ uptake kinetics and equilibrium measurements also demonstrate that the purified protein is fully active.

Biochemistry, 1998 May 5, 37(18), 6327 - 35
Deletion of a highly motional residue affects formation of the Michaelis complex for Escherichia coli dihydrofolate reductase; Miller GP et al.; Analysis of the dihydrofolate reductase (DHFR) complex with folate by two-dimensional heteronuclear (1H-15N) nuclear magnetic relaxation revealed that isolated residues exhibit diverse backbone fluctuations on the nanosecond to picosecond time scale {Epstein, D . M., Benkovic, S . J., and Wright, P . E . (1995) Biochemistry 34, 11037-11048} . These dynamical features may be significant in forming the Michaelis complex . Of these residues, glycine 121 displays large-amplitude backbone motions on the nanosecond time scale . This amino acid, strictly conserved for prokaryotic DHFRs, is located at the center of the betaF-betaG loop . To investigate the catalytic importance of this residue, we report the effects of Gly121 deletion and glycine insertion into the modified betaF-betaG loop . Relative to wild type, deletion of Gly121 dramatically decreases the rate of hydride transfer 550-fold and the strength of cofactor binding 20-fold for NADPH and 7-fold for NADP+ . Furthermore, DeltaG121 DHFR requires conformational changes dependent on the initial binary complex to attain the Michaelis complex poised for hydride transfer . Surprisingly, the insertion mutants displayed a significant decrease in both substrate and cofactor binding . The introduction of glycine into the modified betaF-betaG loop, however, generally eliminated conformational changes required by DeltaG121 DHFR to attain the Michaelis complex . Taken together, these results suggest that the catalytic role for the betaF-betaG loop includes formation of liganded complexes and proper orientation of substrate and cofactor . Through a transient interaction with the Met20 loop, alterations of the betaF-betaG loop can orchestrate proximal and distal effects on binding and catalysis that implicate a variety of enzyme conformations participating in the catalytic cycle.

J Biol Chem, 1998 May 8, 273(19), 11826 - 38
Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence; Asano K et al.; The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids . In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA . In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I . In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top . The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner . On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction . Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA . This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K . (1998) J . Biol . Chem . 273, 11815-11825) is formed specifically with structure I . A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed.

J Biol Chem, 1998 May 8, 273(19), 11405 - 8
A signal peptide that directs non-Sec transport in bacteria also directs efficient and exclusive transport on the thylakoid Delta pH pathway; Mori H et al.; Signal peptides that specifically direct precursor proteins to the thylakoid Delta pH pathway possess an N domain RR motif . Signal peptides that direct transport of bacterial proteins across a non-Sec export pathway possess an N domain RRXFLK consensus motif . Recent genetic studies suggest an evolutionary link between these two protein translocation pathways . To further explore this relationship, we examined the thylakoid targeting capability of the signal peptide for Escherichia coli hydrogenase 1 small subunit (HyaA) by linking it to plastocyanin and assaying the chimeric protein in an in vitro thylakoid transport assay . The chimeric precursor was transported across thylakoids with high efficiency . Transport was characteristic of the Delta pH but not the Sec pathway, i.e . it was eliminated by ionophores that dissipate the DeltapH but occurred in the absence of stromal extract or ATP . This result was confirmed by competition with chemical quantities of a Delta pH pathway precursor . This indicates that the HyaA signal peptide has the necessary elements for efficient and exclusive targeting to the Delta pH pathway and further supports the notion that the alternate targeting pathways in prokaryotes and plant thylakoids are analogous.

Biotechniques, 1998 Apr, 24(4), 624 - 8, 630-2
Lac/Tet dual-inducible system functions in mammalian cell lines; Liu HS et al.; The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels . In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell . The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms) . The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell . The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis . The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed . Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes . In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.

Bull Acad Natl Med, 1998, 182(1), 49 - 57
{Malignant progression and resistance of cancer cells: an inducible survival program similar to the SOS system of unicellular organisms induced by environmental assaults}; Israel L; The hypothesis discussed in this paper states that defence and survival strategies of cancer cells against therapeutic approaches are similar in their mechanisms and homologous in several genes to the SOS program known in bacteria and induced by several assaults . The almost ineluctable malignant progression and its acceleration in response to various therapies should then be considered as an inducible system inherited from prokaryotes and repressed in multicellular organisms through the anti-oncogene system . The later, weakened in case of some inherited mutations yields even in sporadic cases to repeated assaults and to the decrease with time of internal defences, including antioxidant mechanisms . This theory which presents in a new perspective the biological status of cancer in the frame of Darwinian evolution and hence the strategies able to control its progression, leads itself to some predictions and testable assertions: absence of any anti-oncogene homologues in unicellular organisms, built-in weaknesses in anti-oncogenes, existence of a common repressor and a common derepressor mechanism for several distinct genes involved in cancer and in response to an environmental assault, and finally a possible transfer of drug resistance genes in malignant cells as it is the case for bacteria submitted to stress conditions.

J Bacteriol, 1998 Jun, 180(12), 3260 - 4
Characterization of the glnK-amtB operon of Azotobacter vinelandii; Meletzus D et al.; To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized . Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product . A gene designated amtB was found downstream of and was contranscribed with glnK as in E . coli . The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote . glnK and amtB comprise an operon . Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A . vinelandii . amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport {14C}methylammonium.

J Biol Chem, 1998 Jun 5, 273(23), 14435 - 41
Domain mapping of human apurinic/apyrimidinic endonuclease . Structural and functional evidence for a disordered amino terminus and a tight globular carboxyl domain; Strauss PR et al.; We recently described the pre-steady state enzymatic binding kinetics of apurinic/apyrimidinic endonuclease (AP endo) . In this report we describe the domain structure of the enzyme in solution determined by mild protease digestion in the presence and absence of substrate, product, and an efficient competitive inhibitor (HDP) . AP endo is a 35.5-kDa protein with a high degree of homology to its prokaryotic counterpart, exonuclease III (Exo III), except for the amino terminus, which is lacking in the prokaryotic enzyme . The entire conserved region plus an additional 20 residues unique to the eukaryotic enzyme was inaccessible to trypsin and V8 protease, indicating that it forms a tight globular structure . In contrast, the amino-terminal 35 residues were readily accessible to all the proteases investigated, leading us to conclude that they associate poorly with the rest of the structure and constitute a highly fluid region . When AP endo was boiled with SDS and cooled prior to the addition of V8 protease, several acidic residues within the globular domain became protease-accessible, indicating rapid renaturation except along the nuclease fold with restoration of globular conformation for the carboxyl two-thirds of the molecule . Of all the proteases tested, only chymotrypsin was able to cleave internal to the globular portion without prior denaturation . Although AP endo cleaved with chymotrypsin retained full enzymatic activity, the activity was lost when the digested peptides were recovered after denaturation by heat and/or boiling in SDS, precipitation, and renaturation or when fragments were recovered from an SDS gel and renatured . Thus, the protein is probably held together strongly by noncovalent interactions that maintain enzymatic function after protease nicking . The three major chymotrypsin cleavage sites, Tyr-144, Leu-179, and Leu-205, became strikingly less accessible to protease digestion in the presence of abasic site-containing DNA . Since the three residues form a spherical triangle on the surface of the molecule on one side of the nuclease fold, there must be multiple means by which DNA containing an abasic site associates with the enzyme . The most likely explanation is that substrate and product, both of which were present during proteolysis, bind differently to the enzyme . Finally, the two cysteine residues thought to be involved in the redox reaction of AP endo with Jun protein were entirely inaccessible to proteolysis even after prolonged exposure of AP endo to reducing agents . Consequently, if AP endo plays a role in the physiological function of Jun, it must undergo major conformational changes in the process . Alternatively, the two cysteines could maintain an appropriate conformation such that other residues participate directly in the redox activity.

Glycobiology, 1998 Jun, 8(6), 625 - 32
Conserved sequences in enzymes of the UDP-GlcNAc/MurNAc family are essential in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase; Dal Nogare AR et al.; The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols . A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family . To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) . Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT . While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity . Further, the C and D scramble mutants had an unexpected sorting defect . Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E) . For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected . These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable . Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.

Microbiol Mol Biol Rev, 1998 Jun, 62(2), 275 - 93
Short-sequence DNA repeats in prokaryotic genomes; van Belkum A et al.; Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes . These loci harbor short or long stretches of repeated nucleotide sequence motifs . DNA sequence motifs in a single locus can be identical and/or heterogeneous . SSRs are encountered in many different branches of the prokaryote kingdom . They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins . SSRs enable genetic and consequently phenotypic flexibility . SSRs function at various levels of gene expression regulation . Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies . These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10(-4) per bacterial cell division and allowing high-frequency genetic switching . Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure . SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness . Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria . The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.

Nucleic Acids Res, 1998 May 15, 26(10), 2380 - 4
Step-wise DNA relaxation and decatenation by NaeI-43K; Jo K et al.; Nae I protein was originally isolated for its restriction endonuclease properties . Nae I was later discovered to either relax or cleave supercoiled DNA, depending upon whether Nae I position 43 contains a lysine (43K) or leucine (43L) respectively . Nae I-43K DNA relaxation activity appears to be the product of coupling separate endonuclease and ligase domains within the same polypeptide . Whereas Nae I relaxes supercoiled DNA like a topoisomerase, even forming a transient covalent intermediate with the substrate DNA, Nae I shows no obvious sequence similarity to the topoisomerases . To further characterize the topoisomerase activity of Nae I, we report here that Nae I-43K changes the linking number of a single negatively supercoiled topoisomer of pBR322 by units of one and therefore is a type I topoisomerase . Positively supercoiled pBR322 was resistant to Nae I-43K . At low salt concentration Nae I-43K was processive; non-saturating amounts of enzyme relaxed a fraction of the DNA . At high salt concentration the same non-saturating amounts of Nae I-43K partially relaxed all the DNA in a step-wise fashion to give a Gaussian distribution of topoisomers, demonstrating a switch from a processive to a distributive mode of action . Nae I-43K decatenated kinetoplast DNA containing nicked circles, implying that Nae I-43K can cleave opposite a nick . The products of the reaction are decatenated nicked circles under both processive and distributive conditions . The behavior of Nae I-43K is consistent with that of a prokaryotic type I topoisomerase.

J Bacteriol, 1998 Jun, 180(11), 2883 - 8
Posttranscriptional modifications in 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfataricus; Noon KR et al.; Posttranscriptional modification is common to many types of RNA, but the majority of information concerning structure and function of modification is derived principally from tRNA . By contrast, less is known about modification in rRNA in spite of accumulating evidence for its direct participation in translation . The structural identities and approximate molar levels of modifications have been established for 16S and 23S rRNAs of the archaeal hyperthermophile Sulfolobus solfactaricus by using combined chromatography-mass spectrometry-based methods . Modification levels are exceptionally high for prokaryotic organisms, with approximately 38 modified sites in 16S rRNA and 50 in 23S rRNA for cells cultured at 75 degrees C, compared with 11 and 23 sites, respectively, in Escherichia coli . We structurally characterized 10 different modified nucleosides in 16S rRNA, 64% (24 residues) of which are methylated at O-2' of ribose, and 8 modified species in 23S rRNA, 86% (43 residues) of which are ribose methylated, a form of modification shown in earlier studies to enhance stability of the polynucleotide chain . From cultures grown at progressively higher temperatures, 60, 75, and 83 degrees C, a slight trend toward increased ribose methylation levels was observed, with greatest net changes over the 23 degrees C range shown for 2'-O-methyladenosine in 16S rRNA (21% increase) and for 2'-O-methylcytidine (24%) and 2'-O-methylguanosine (22%) in 23S rRNA . These findings are discussed in terms of the potential role of modification in stabilization of rRNA in the thermal environment.

J Bacteriol, 1998 May, 180(10), 2652 - 9
(Methyl)ammonium transport in the nitrogen-fixing bacterium Azospirillum brasilense; Van Dommelen A et al.; An ammonium transporter of Azospirillum brasilense was characterized . In contrast to most previously reported putative prokaryotic NH4+ transporter genes, A . brasilense amtB is not part of an operon with glnB or glnZ which, in A . brasilense, encode nitrogen regulatory proteins PII and PZ, respectively . Sequence analysis predicts the presence of 12 transmembrane domains in the deduced AmtB protein and classifies AmtB as an integral membrane protein . Nitrogen regulates the transcription of the amtB gene in A . brasilense by the Ntr system . amtB is the first gene identified in A . brasilense whose expression is regulated by NtrC . The observation that ammonium uptake is still possible in mutants lacking the AmtB protein suggests the presence of a second NH4+ transport mechanism . Growth of amtB mutants at low ammonium concentrations is reduced compared to that of the wild type . This suggests that AmtB has a role in scavenging ammonium at low concentrations.

Genes Dev, 1998 May 1, 12(9), 1254 - 9
Characterization of a prokaryotic SMC protein involved in chromosome partitioning; Britton RA et al.; smc of Bacillus subtilis encodes a homolog of eukaryotic SMC proteins involved in chromosome condensation, pairing, and partitioning . A null mutation in B . subtilis smc caused a temperature-sensitive-lethal phenotype in rich medium . Under permissive conditions, the mutant had abnormal nucleoids, approximately 10% of the cells were anucleate, and assembly of foci of the chromosome partitioning protein Spo0J was altered . In combination with a null mutation in spo0J, the smc mutation caused a synthetic phenotype; cell growth was slower and approximately 25% of the cells were anucleate . Our results demonstrate that the B . subtilis Smc protein, like its eukaryotic counterpart, plays an important role in chromosome structure and partitioning.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5401 - 6
Association of the Arabidopsis CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors; Clark KL et al.; In Arabidopsis thaliana, signal transduction of the hormone ethylene involves at least two receptors, ETR1 and ERS, both of which are members of the two-component histidine protein kinase family that is prevalent in prokaryotes . The pathway also contains a negative regulator of ethylene responses, CTR1, which closely resembles members of the Raf protein kinase family . CTR1 is thought to act at or downstream of ETR1 and ERS based on double mutant analysis; however, the signaling mechanisms leading from ethylene perception to the regulation of CTR1 are unknown . By using the yeast two-hybrid assay, we detected a specific interaction between the CTR1 amino-terminal domain and the predicted histidine kinase domain of ETR1 and ERS . We subsequently verified these interactions by using an in vitro protein association assay(s) . In addition, we determined that the amino-terminal domain of CTR1 can associate with the predicted receiver domain of ETR1 in vitro . Based on deletion analysis, the portion of CTR1 that interacts with ETR1 roughly aligns with the regulatory region of Raf kinases . These physical associations support the genetic evidence that CTR1 acts in the pathway of ETR1 and ERS and suggest that these interactions could be involved in the regulation of CTR1 activity.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 5003 - 8
Transmembrane heme delivery systems; Goldman BS et al.; Heme proteins play pivotal roles in a wealth of biological processes . Despite this, the molecular mechanisms by which heme traverses bilayer membranes for use in biosynthetic reactions are unknown . The biosynthesis of c-type cytochromes requires that heme is transported to the bacterial periplasm or mitochondrial intermembrane space where it is covalently ligated to two reduced cysteinyl residues of the apocytochrome . Results herein suggest that a family of integral membrane proteins in prokaryotes, protozoans, and plants act as transmembrane heme delivery systems for the biogenesis of c-type cytochromes . The complete topology of a representative from each of the three subfamilies was experimentally determined . Key histidinyl residues and a conserved tryptophan-rich region (designated the WWD domain) are positioned at the site of cytochrome c assembly for all three subfamilies . These histidinyl residues were shown to be essential for function in one of the subfamilies, an ABC transporter encoded by helABCD . We believe that a directed heme delivery pathway is vital for the synthesis of cytochromes c, whereby heme iron is protected from oxidation via ligation to histidinyl residues within the delivery proteins.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4900 - 5
Escherichia coli rho factor induces release of yeast RNA polymerase II but not polymerase I or III; Lang WH et al.; Purified RNA polymerase II (pol II) from the yeast Saccharomyces cerevisiae pauses without releasing at many locations during in vitro transcription . Pausing can be induced by intrinsic DNA sequence as well as by specific DNA bound proteins such as the RNA pol I termination factor, Reb1p, or lac repressor . Addition of rho termination factor from E . coli induces RNA pol II to release at all of these pause sites . Rho-induced release of pol II requires both a rho binding site in the transcript upstream of the pause sites as well as hydrolysis of ATP . In contrast, rho factor has no effect on either pausing or release by RNA pol I or III . When combined with previous observations, these results suggest that RNA pol II may terminate by a mechanism closely related to the rho-dependent mechanism of prokaryotes . In contrast, pol I and III appear to utilize a mechanism more related to the rho-independent terminators of prokaryotes.

Proc Natl Acad Sci U S A, 1998 Apr 28, 95(9), 4853 - 8
A primitive pathway of porphyrin biosynthesis and enzymology in Desulfovibrio vulgaris; Ishida T et al.; Culture of Desulfovibrio vulgaris in a medium supplemented with 5-aminolevulinic acid and L-methionine-methyl-d3 resulted in the formation of porphyrins (sirohydrochlorin, coproporphyrin III, and protoporphyrin IX) in which the methyl groups at the C-2 and C-7 positions were deuterated . A previously unknown hexacarboxylic acid was also isolated, and its structure was determined to be 12, 18-didecarboxysirohydrochlorin by mass spectrometry and 1H NMR . These results indicate a primitive pathway of heme biosynthesis in D . vulgaris consisting of the following enzymatic steps: (i) methylation of the C-2 and C-7 positions of uroporphyrinogen III to form precorrin-2 (dihydrosirohydrochlorin); (ii) decarboxylation of acetate groups at the C-12 and C-18 positions of precorrin-2 to form 12,18-didecarboxyprecorrin-2; (iii) elimination of acetate groups of the C-2 and C-7 positions of 12,18-didecarboxyprecorrin-2 to form coproporphyrinogen III; and (iv) conversion of coproporphyrinogen III to protoporphyrin IX via protoporphyrinogen IX . We isolated the following three enzymatic activities involved in steps i-iii from the soluble fraction of the cells by anion-exchange chromatography: S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase, precorrin-2 12,18-acetate decarboxylase, and 12, 18-didecarboxyprecorrin-2 2,7-decarboxymethylase; all enzymic products were converted into autooxidized methyl esters and analyzed by thin-layer chromatography, UV-visible (UV-VIS) absorption, and mass spectrometry . The enzymatic reactions in D . vulgaris shed new light on porphyrin biosynthesis at an early stage in the evolution of prokaryotes.

Mol Gen Genet, 1998 Apr, 258(1-2), 26 - 33
Cloning and characterisation of the pknD gene encoding an eukaryotic-type protein kinase in the cyanobacterium Anabaena sp . PCC7120; Zhang CC et al.; Protein phosphorylation catalysed by protein kinases is an important mechanism for signal transduction in both prokaryotes and eukaryotes . A novel gene, pknD, encoding a protein similar to eukaryotic-type protein kinases, was cloned from Anabaena sp . PCC7120 . The N-terminal region of PknD is 60% identical to that of PknA, another putative Ser/Thr kinase from the same strain . Both PknA and PknD have C-terminal regions that are rich in Pro and Thr residues . Expression of pknD was undetectable by RNA/DNA hybridisation and was thus examined by RT-PCR . The pknD transcript was detected in filaments cultured in the presence of either nitrate or ammonium as a source of combined nitrogen, and also in filaments transferred from nitrate-sufficient to N2-fixing conditions . pknD mutants were created, and their growth characteristics under different nitrogen regimes and their capacity for heterocyst development were investigated . The growth rates of the mutants were similar to those of the wild-type strain in the presence of either nitrate or ammonium, but were only 20% that of the wild type under N2-fixing conditions . The rate of nitrogenase activity is normal in pknD mutant under aerobic conditions . Under nitrogen-fixing conditions, the inactivation of pknD led to enhanced modification of the PII protein compared to the weak phosphorylation of PII observed in the wild-type strain . This high level of PII phosphorylation in the pknD mutant is reminiscent of the situation in nitrogen-starved Synechococcus PCC7942 cells . PknD might be involved in regulating nitrogen metabolism or nitrogen trafficking from heterocysts to vegetative cells.

Bioorg Khim, 1998 Mar, 24(3), 171 - 4
{Characteristics of N-terminal 60-kDa fragment of elongation factor 2}; Korotkov KV et al.; The N-terminal 60-kDa-fragment of elongation factor 2 from rat liver (EF-2) was obtained by the limited proteolysis of native EF-2 with elastase . This fragment consists of 506 N-terminal amino acid residues of EF-2 . The conformational properties of both this fragment and EF-2 in solution were studied by circular dichroism and fluorescent spectroscopy . The contents of secondary structure components in the fragment and in the factor that were deduced from CD measurements agreed well with values predicted from their primary structures . Both proteins were resistant to denaturation with < or = 3 M urea and exhibited cooperative denaturation transitions . Temperature melting also proceeded cooperatively for the fragment and EF-2 . Structural properties of the N-terminal 60-kDa-fragment are discussed in comparison with the biochemical characteristics and 3D structure of prokaryotic elongation factor EF-G.

Microbiology, 1998 May, 144 ( Pt 5), 1281 - 90
Structural elements of the Streptomyces oriC region and their interactions with the DnaA protein; Jakimowicz D et al.; Streptomycetes differ from other prokaryotic organisms in their mycelial life cycle and in possessing a large, linear, GC-rich chromosome . To deduce structural features of the Streptomyces origin of chromosomal replication, the oriC sequences of three Streptomyces species (S . antibioticus, S . chrysomallus and S . lividans) were compared . In Streptomyces, the oriC region contains 19 DnaA boxes whose location, orientation and spacing are conserved . The consensus sequence of the DnaA box identified within Streptomyces oriC is (T/C)(T/C)(G/A/C)TCCACA (preferred bases underlined) . The interactions of DnaA with DNA fragments containing single, two or three DnaA boxes were studied using surface plasmon resonance . The dissociation constant (KD) for specific binding of individual DnaA boxes varied between 12 and 78 nM . Streptomyces oriC does not contain the three AT-rich 13-mer direct repeats present in the 5' part of the Escherichia coli oriC region . However, short AT-rich sequences are distributed among the DnaA boxes of Streptomyces oriC . Repeated attempts to unwind Streptomyces oriC have been unsuccessful . It remains to be elucidated whether DnaA interacts with putative accessory proteins which help in unwinding Streptomyces oriC.

Microbiology, 1998 May, 144 ( Pt 5), 1257 - 62
An antigenic protein gene of a phytoplasma associated with sweet potato witches' broom; Yu YL et al.; A gene encoding the major antigenic protein of phytoplasma associated with sweet potato witches' broom (SPWB) was cloned and analysed by screening the genomic library of SPWB phytoplasma with monoclonal antibodies for SPWB phytoplasma . The entire predicted structural gene encoded an antigenic protein composed of 172 amino acids with a computed molecular mass of 19.15 kDa and a pl value of 9.78 . The -10 region of the promoter and the terminator region of the gene were identified and found to be similar to those of prokaryotes . The hydropathy profile of the deduced amino acid sequence consisted of two distinct regions, a strongly hydrophobic N-terminus and a highly hydrophilic C-terminus . This major antigenic protein was also present in phytoplasma associated with peanut witches' broom (PNWB) and the two showed homology based on the results of Western blot analysis, Southern hybridization, Northern hybridization, primer extension analysis and PCR . The homologous genes of the antigenic protein of SPWB phytoplasma and PNWB phytoplasma were not found in other phytoplasmas tested.

Int J Biochem Cell Biol, 1998 Feb, 30(2), 169 - 72
The aquaporins . A family of water channel proteins; Connolly DL et al.; The recent discovery of aquaporins, a family of highly conserved water channel proteins, which are expressed in both eukaryotes and prokaryotes, has provoked a re-evaluation of the physiology of water transport in all organisms . So far, seven distinct aquaporins have been characterised in mammals, but highly homologous family members have also been found in amphibians, insects, plants and bacteria . These transmembrane proteins serve to facilitate water transport down osmotic gradients with low activation energy . Alterations in channel expression, cellular targeting and perhaps channel permeability regulate membrane water transport . Naturally occurring and experimentally produced mutations in aquaporins cause a variety of perturbations of water homeostasis . Manipulation of aquaporin expression may have a therapeutic role in several disease processes including cardiac failure and the ascites associated with liver disease.

Appl Environ Microbiol, 1998 Jun, 64(6), 2220 - 8
Hemoglobin biosynthesis in Vitreoscilla stercoraria DW: cloning, expression, and characterization of a new homolog of a bacterial globin gene; Joshi M et al.; In the strictly aerobic, gram-negative bacterium Vitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb) . The recently determined crystal structure of Vitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs . In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla, V . stercoraria DW . Several silent changes were observed within the coding region of the V . stercoraria vgb gene . Apart from that, V . stercoraria Hb exhibited interesting differences between the A and E helices . Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation of V . stercoraria Hb displays a slower autooxidation rate . The differences between Vitreoscilla Hb and V . stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within the V . stercoraria Hb fall in the region where the A and E helices contact each other . Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V . stercoraria Hb can be correlated to its slower autooxidation rate . In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscilla strain C1, production of Hb in V . stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V . stercoraria vgb promoter region . Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species of Vitreoscilla . The relatively slower autooxidation rate of V . stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V . stercoraria DW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

FEBS Lett, 1998 May 8, 427(2), 259 - 62
Biochemical and phylogenetic analyses of methionyl-tRNA synthetase isolated from a pathogenic microorganism, Mycobacterium tuberculosis; Kim S et al.; Mycobacterium tuberculosis methionyl-tRNA synthetase (MetRS) has been cloned and characterized . The protein contains class I signature sequences but lacks the Zn2+ binding motif and the C-terminal dimerization appendix that are found in MetRSs from several organisms including E . coli MetRS . Consistent with these features, the enzyme behaved as a monomer in a gel filtration chromatography and did not contain the bound Zn2+ . Nonetheless, it was active to the tRNAMet of E . coli as determined by in vivo genetic complementation and in vitro reaction . Phylogenetic analysis separated the M . tuberculosis and E . coli MetRSs into prokaryote and eukaryote-archaea group, respectively . This result is consistent with the taxonomic locations of the organism but is an interesting contrast to the case of its paralogous protein, isoleucyl-tRNA synthetase, and suggests that the two enzymes evolved in separate idiosyncratic pathways.

Arch Microbiol, 1998 May, 169(5), 404 - 10
Genetic evidence for a role of thioesterase domains, integrated in or associated with peptide synthetases, in non-ribosomal peptide biosynthesis in Bacillus subtilis; Schneider A et al.; Next to almost all prokaryotic operons encoding peptide synthetases, which are involved in the nonribosomal synthesis of peptide antibiotics, distinct genes have been detected that encode proteins with strong sequence similarity to type II fatty acid thioesterases of vertebrate origin . Furthermore, sequence analysis of bacterial and fungal peptide synthetases has revealed a region at the C-terminal end of modules that are responsible for adding the last amino acid to the peptide antibiotics; that region also exhibits significant similarities to thioesterases . In order to investigate the function of these putative thioesterases in non-ribosomal peptide synthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis, srfA fragments encoding the thioesterase domain of the surfactin synthetase 3 and the thioesterase-like protein SrfA-TE were deleted . This led to a 97 and 84% reduction of the in vivo surfactin production, respectively . In the double mutant, however, no surfaction production was detectable . These findings demonstrate for the first time that the C-terminal thioesterase domains and the SrfA-TE protein are directly involved in nonribosomal peptide biosynthesis.

Protein Sci, 1998 May, 7(5), 1208 - 13
The role of tyrosine 121 in cofactor binding of 5-aminolevulinate synthase; Tan D et al.; 5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthesis in nonplant eukaryotes and some prokaryotes . It functions as a homodimer and requires pyridoxal 5'-phosphate as an essential cofactor . Tyr-121 is a conserved residue in all known sequences of 5-aminolevulinate synthases . Further, it corresponds to Tyr-70 of Escherichia coli aspartate aminotransferase, which has been shown to interact with the cofactor and prevent the dissociation of the cofactor from the enzyme . To test whether Tyr-121 is involved in cofactor binding in murine erythroid 5-aminolevulinate synthase, Tyr-121 of murine erythroid 5-aminolevulinate synthase was substituted by Phe and His using site-directed mutagenesis . The Y121F mutant retained 36% of the wild-type activity and the Km value for substrate glycine increased 34-fold, while the activity of the Y121H mutant decreased to 5% of the wild-type activity and the Km value for glycine increased fivefold . The pKa1 values in the pH-activity profiles of the wild-type and mutant enzymes were 6.41, 6.54, and 6.65 for wild-type, Y121F, and Y121H, respectively . The UV-visible and CD spectra of Y121F and Y121H mutants were similar to those of the wild-type with the exception of an absorption maximum shift (420 --> 395 nm) for the Y121F mutant in the visible spectrum region, suggesting that the cofactor binds the Y121F mutant enzyme in a more unrestrained manner . Y121F and Y121H mutant enzymes also exhibited lower affinity than the wild-type for the cofactor, reflected in the Kd values for pyridoxal 5'-phosphate (26.5, 6.75, and 1.78 microM for Y121F, Y121H, and the wild-type, respectively) . Further, Y121F and Y121H proved less thermostable than the wild type . Taken together, these findings indicate that Tyr-121 plays a critical role in cofactor binding of murine erythroid 5-aminolevulinate synthase.

Mol Gen Genet, 1998 Apr, 257(6), 686 - 92
Deletion of the Saccharomyces cerevisiae gene RAD30 encoding an Escherichia coli DinB homolog confers UV radiation sensitivity and altered mutability; Roush AA et al.; The dinB gene of Escherichia coli is an SOS-inducible gene of unknown function . Its mode of regulation and the amino acid sequence similarity of the predicted DinB protein to the UmuC protein of E . coli both suggest a role in cellular responses to DNA damage and probably in error-prone repair . Proteins with sequence similarity to DinB have been predicted from genes cloned from various prokaryotic and eukaryotic organisms, including Caenorhabditis elegans . Here we present the phenotypic characterization of a haploid Saccharomyces cerevisiae strain deleted for the ORF YDR419W, encoding a yeast DinB homolog . The deletion mutant is viable but is moderately sensitive to killing following exposure to ultraviolet (UV) radiation . Hence, we have named the gene RAD30 . Steady-state levels of RAD30 transcripts are increased following UV irradiation . UV-induced locus-specific reversion of an ochre allele (arg4-17) is reduced in the rad30 deletion mutant . However, enhanced mutability was observed following treatment with the alkylating agent methylmethanesulfonate (MMS) . Spontaneous mutability was also slightly increased . We conclude that RAD30 encodes an accessory function involved in DNA repair and mutagenesis . We speculate that the relatively weak phenotype and the opposite effects on mutability as a function of the type of DNA damage involved may derive from a functional redundancy of yeast proteins which facilitate replicative bypass of non-coding DNA lesions.

J Biochem (Tokyo), 1998 Jun, 123(6), 1000 - 9
Sequence-function relationships of prokaryotic and eukaryotic galactosyltransferases; Breton C et al.; Galactosyltransferases are enzymes which transfer galactose from UDP-Gal to various acceptors with either retention of the anomeric configuration to form alpha1,2-, alpha1,3-, alpha1,4-, and alpha1, 6-linkages, or inversion of the anomeric configuration to form beta1, 3-, beta1,4-, and beta1-ceramide linkages . During the last few years, several (c)DNA sequences coding for galactosyltransferases became available . We have retrieved these sequences and conducted sequence similarity studies . On the basis of both the nature of the reaction catalyzed and the protein sequence identity, these enzymes can be classified into twelve groups . Using a sensitive graphics method for protein comparison, conserved structural features were found in some of the galactosyltransferase groups, and other classes of glycosyltransferases, resulting in the definition of five families . The lengths and locations of the conserved regions as well as the invariant residues are described for each family . In addition, the DxD motif that may be important for substrate recognition and/or catalysis is demonstrated to occur in all families but one.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6239 - 44
Genomic evidence for two functionally distinct gene classes; Rivera MC et al.; Analyses of complete genomes indicate that a massive prokaryotic gene transfer (or transfers) preceded the formation of the eukaryotic cell . In comparisons of the entire set of Methanococcus jannaschii genes with their orthologs from Escherichia coli, Synechocystis 6803, and the yeast Saccharomyces cerevisiae, it is shown that prokaryotic genomes consist of two different groups of genes . The deeper, diverging informational lineage codes for genes which function in translation, transcription, and replication, and also includes GTPases, vacuolar ATPase homologs, and most tRNA synthetases . The more recently diverging operational lineage codes for amino acid synthesis, the biosynthesis of cofactors, the cell envelope, energy metabolism, intermediary metabolism, fatty acid and phospholipid biosynthesis, nucleotide biosynthesis, and regulatory functions . In eukaryotes, the informational genes are most closely related to those of Methanococcus, whereas the majority of operational genes are most closely related to those of Escherichia, but some are closest to Methanococcus or to Synechocystis.

Proc Natl Acad Sci U S A, 1998 May 26, 95(11), 6067 - 72
Translation inhibitors stabilize Escherichia coli mRNAs independently of ribosome protection; Lopez PJ et al.; Translation inhibitors such as chloramphenicol in prokaryotes or cycloheximide in eukaryotes stabilize many or most cellular mRNAs . In Escherichia coli, this stabilization is ascribed generally to the shielding of mRNAs by stalled ribosomes . To evaluate this interpretation, we examine here how inhibitors affect the stabilities of two untranslated RNAs, i.e., an engineered lacZ mRNA lacking a ribosome binding site, and a small regulatory RNA, RNAI . Whether they block elongation or initiation, all translation inhibitors tested stabilized these RNAs, indicating that stabilization does not necessarily reflect changes in packing or activity of translating ribosomes . Moreover, both the initial RNase E-dependent cleavage of RNAI and lacZ mRNA and the subsequent attack of RNAI by polynucleotide phosphorylase and poly(A)-polymerase were slowed . Among various possible mechanisms for this stabilization, we discuss in particular a passive model . When translation is blocked, rRNA synthesis is known to increase severalfold and rRNA becomes unstable . Meanwhile, the pools of RNase E and polynucleotide phosphorylase, which, in growing cells, are limited because these RNases autoregulate their own synthesis, cannot expand . The processing/degradation of newly synthesized rRNA would then titrate these RNases, causing bulk mRNA stabilization.

J Virol, 1998 Jun, 72(6), 5279 - 84
Cloning and expression of a human T-lymphotropic virus type 1 protein with reverse transcriptase activity; Owen SM et al.; Unlike most other characterized retroviruses, there is little published information on the biochemical properties of human T-lymphotropic virus type 1 (HTLV-1) reverse transcriptase (RT) . Specifically, no reports of a cloned functional RT enzyme have been published . Since the RT enzyme is an essential component of the virus, our objective was to clone, express, and purify a functional RT enzyme from HTLV-1 . Our approach was to clone and express a protein of approximately 60 to 65 kDa that we hypothesized would correspond to the RT region encoded by the pol reading frame . The predicted region encoding the RT enzyme comprised nucleotides 2617 to 4312 of the HTLV-1 MT-2 isolate . A putative RT gene was obtained by PCR and was ligated into various prokaryotic expression vectors . A novel cloning approach allowed us to generate a stable clone in the prokaryotic expression vector pGEX-4T-1 and produce a recombinant protein of approximately 60 to 65 kDa . The partially purified protein displays RT activity in both amplification RT (AMP-RT) assays and traditional RT assays . This is the first report of a cloned protein from HTLV-1 which displays RT activity and is the first step in the characterization of HTLV-1 RT.

J Biol Chem, 1998 Apr 24, 273(17), 10302 - 7
Reconstitution of a protein disulfide catalytic system; Bader M et al.; Disulfide bonds are important for the structure and stability of many proteins . In prokaryotes their formation is catalyzed by the Dsb proteins . The DsbA protein acts as a direct donor of disulfides to newly synthesized periplasmic proteins . Genetic evidence suggests that a second protein called DsbB acts to specifically reoxidize DsbA . Here we demonstrate the direct reoxidation of DsbA by DsbB . We have developed a fluorescence assay that allows us to directly follow the reoxidation of DsbA . We show that membranes containing catalytic amounts of DsbB can rapidly reoxidize DsbA to completion . The reaction strongly depends on the presence of oxygen, implying that oxygen serves as the final electron acceptor for this disulfide bond formation reaction . Membranes from a dsbB null mutant display no DsbA reoxidation activity . The ability of DsbB to reoxidize DsbA fits Michaelis-Menten behavior with DsbA acting as a high affinity substrate for DsbB with a Km = 10 microM . The in vitro reconstitution described here is the first biochemical analysis of DsbB and allows us to study the major pathway of disulfide bond formation in Escherichia coli.

J Biol Chem, 1998 Apr 24, 273(17), 10216 - 22
Inhibition of assembly of bacterial cell division protein FtsZ by the hydrophobic dye 5,5'-bis-(8-anilino-1-naphthalenesulfonate); Yu XC et al.; To gain further insight into the structural relatedness of tubulin and FtsZ, the tubulin-like prokaryotic cell division protein, we tested the effect of tubulin assembly inhibitors on FtsZ assembly . Common tubulin inhibitors, such as colchicine, colcemid, benomyl, and vinblastine, had no effect on Ca2+-promoted GTP-dependent assembly of FtsZ into polymers . However, the hydrophobic probe 5, 5'-bis-(8-anilino-1-naphthalenesulfonate) (bis-ANS) inhibited FtsZ assembly . The potential mechanisms for inhibition are discussed . Titrations of FtsZ with bis-ANS indicated that FtsZ has one high affinity binding site and multiple low affinity binding sites . ANS (8-anilino-1-naphthalenesulfonate), a hydrophobic probe similar to bis-ANS, had no inhibitory effect on FtsZ assembly . Because tubulin assembly has also been shown to be inhibited by bis-ANS but not by ANS, it supports the idea that FtsZ and tubulin share similar conformational properties . Ca2+, which promotes GTP-dependent FtsZ assembly, stimulated binding of bis-ANS or ANS to FtsZ, suggesting that Ca2+ binding induces changes in the hydrophobic conformation of the protein . Interestingly, depletion of bound Ca2+ with EGTA further enhanced bis-ANS fluorescence . These findings suggest that both binding and dissociation of Ca2+ are capable of inducing FtsZ conformational changes, and these changes could promote the GTP-dependent assembly of FtsZ.

J Biol Chem, 1998 Apr 24, 273(17), 10091 - 4
ATP binding induces large conformational changes in the apical and equatorial domains of the eukaryotic chaperonin containing TCP-1 complex; Llorca O et al.; The chaperonin-containing TCP-1 complex (CCT) is a heteromeric particle composed of eight different subunits arranged in two back-to-back 8-fold pseudo-symmetric rings . The structural and functional implications of nucleotide binding to the CCT complex was addressed by electron microscopy and image processing . Whereas ADP binding to CCT does not reveal major conformational differences when compared with nucleotide-free CCT, ATP binding induces large conformational changes in the apical and equatorial domains, shifting the latter domains up to 40 degrees (with respect to the inter-ring plane) compared with 10 degrees for nucleotide-free CCT or ADP-CCT . This equatorial ATP-induced shift has no counterpart in GroEL, its prokaryotic homologue, which suggests differences in the folding mechanism for CCT.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1749 - 54
The joining of blunt DNA ends to 3'-protruding single strands in Escherichia coli; King J et al.; In eukaryotic and prokaryotic organisms DNA double-strand breaks with non-complementary ends can be joined by mechanisms of illegitimate recombination . We examined the joining of 3'-protruding single strand (PSS) ends, which do not have recessed 3' hydroxyls that can allow for fill-in DNA synthesis, to blunt ends . End-joining was examined by electro-transforming Escherichia coli strains with linearized plasmid DNA, sequencing the resulting junctions, and determining the transformation frequencies . Three different E.coli strains were examined: MC1061, which has no known recombination or DNA repair defects, HB101 (rec A-) and SURE (recB- recJ-) . No striking differences were found in either the spectrum of products observed or the efficiency of end-joining between these strains . As in vertebrate systems, the majority of the products were overlaps between directly repeated DNA sequences . 3'-PSS are frequently preserved in vertebrate systems, but they were not preserved in our experiments unless the transforming DNA was pretreated with a DNA polymerase.

Nucleic Acids Res, 1998 Apr 1, 26(7), 1668 - 74
Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA; Bhaduri T et al.; We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes . Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA . The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases . More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases . The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT . Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

Gene, 1998 May 28, 212(1), 57 - 65
Polyadenylation of oop RNA in the regulation of bacteriophage lambda development; Wrobel B et al.; We have shown that Escherichia coli pcnB mutants are lysogenized by bacteriophage lambda with lower efficiency as compared to the pcnB+ strains . Our genetic analysis revealed that expression of the lambda cII gene is decreased in the pcnB mutants . However, using various lacZ fusions we demonstrated that neither activities of pL and pR promoters nor transcription termination at tR1 were significantly impaired in the pcnB- host . On the other hand, we found that oop RNA, an antisense RNA for cII expression, is involved in this regulation . Primer protection experiments revealed that oop RNA was polyadenylated and that this polyadenylation was impaired in the pcnB mutant . We found that the oop RNA was more abundant in the pcnB mutant than in the pcnB+ strain . Furthermore, we showed that activity of the pO promoter was not stimulated in the pcnB mutant . Such findings indicated that degradation of oop RNA in the pcnB strain was slower because of inefficient polyadenylation, which could lead to more effective inhibition of cII expression by the antisense oop RNA, resulting in less efficient lysogenization of the host . The oop RNA was found previously to play a role in phage lambda development only under conditions of overproduction of this transcript . Here we demonstrate for the first time, the physiological function of oop RNA in lambda development, confirming that this short transcript plays an important role in the negative regulation of cII gene expression during lambda infection . Moreover, polyadenylation of oop RNA is one of very few known examples of specific RNA polyadenylation by PAP I in prokaryotic cells and its role in gene expression regulation.

Immunol Rev, 1998 Apr, 162, 143 - 51
Amino acid insertions and deletions contribute to diversify the human Ig repertoire; Wilson P et al.; The sequence analysis of Ig variable region genes transcribed within different B-cell subpopulations from human tonsil led us to identify a rare DNA sequence modification event consisting of bp insertions and/or deletions (I/D) . Although these events were previously reported, they had never been formally associated with the somatic hypermutation process . I/D events share with more conventional somatic hypermutation events their localization within hypervariable regions and, most particularly, within DNA motifs known to be mutational hot spots . Repetitive DNA tracts or DNA elements capable of forming DNA loop intermediates seem to be the preferred substrate for I/D to occur . These characteristics suggest a model for somatic hypermutation reminiscent of the "polymerase slippage" model involved in replication and repair mutations in prokaryotes, yeast, and mammals.

Immunol Rev, 1998 Apr, 162, 67 - 76
Recombination-based mechanisms for somatic hypermutation; Kong Q et al.; We review some experiments designed to test recombination-based mechanisms for somatic hypermutation in mice, particularly mechanisms involving templated mutation or gene conversion . As recombination and repair functions are highly conserved among prokaryotes and eukaryotes, pathways of mutation in microorganisms may prove relevant to the mechanism of somatic hypermutation . Escherichia coli initiates a recombination-based pathway of mutation in response to environmental stimuli, and this "adaptive" pathway of mutation has striking similarities with somatic hypermutation, as does a process of mutagenic repair that occurs at double-strand breaks in Saccharomyces cerevisiae . We present a model for recombination-based hypermutation of the immunoglobulin loci which could result in either templated or non-templated mutation.

Gene, 1998 Apr 28, 211(1), 1 - 9
Do natural antisense transcripts make sense in eukaryotes?
Vanhee-Brossollet C, Vaquero C.
The existence of naturally occurring antisense RNAs has been illustrated, in eukaryotes, by an increasing number of reports . The following review presents the major findings in this field, with a special focus on the regulation of gene expression exerted by endogenous complementary transcripts . A large variety of eukaryotic organisms, contains antisense transcripts . Moreover, the great diversity of genetic loci encoding overlapping sense and antisense RNAs suggests that such transcripts may be involved in numerous biological functions, such as control of development, adaptative response . viral infection . The regulation of gene expression by endogenous antisense RNAs seems of general importance in eukaryotes as already established in prokaryotes: it is likely to be involved in the control of various biological functions and to play a role in the development of pathological situations . Several experimental evidences for coupled, balanced or unbalanced expression of sense and antisense RNAs suggest that antisense transcripts may govern the expression of their sense counterparts . Furthermore, documented examples indicate that this control may be exerted at many levels of gene expression (transcription, maturation, transport, stability and translation) . This review also addresses the underlying molecular mechanisms of antisense regulation and presents the current mechanistic hypotheses.

Curr Biol, 1998 May 7, 8(10), R338 - 41
Cytoskeletal proteins: the evolution of cell division; Faguy DM et al.; The prokaryotic cell division protein FtsZ and eukaryotic tubulin have been shown to have very similar structures and are most likely homologs . The evolutionary transition from FtsZ to tubulin could provide a window into the transition from prokaryotic cells to eukaryotic cells.

J Cell Sci, 1998 Jun, 111 ( Pt 12), 1623 - 34
Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility; Palin AH et al.; Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions . They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells . Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance . Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified . In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility . Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon . FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands . Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome . We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus . Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly {A}.{T}-rich 156 bp region (86.5%) almost adjacent to the integration site . Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the {A}.{T}-rich sequences . This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments . Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.

Glycobiology, 1998 Jan, 8(1), 87 - 94
Conserved structural features in eukaryotic and prokaryotic fucosyltransferases; Breton C et al.; Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages . Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed . A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria . On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-fucosyltransferases, (2) the alpha-3-fucosyltransferases, (3) the mammalian alpha-6-fucosyltransferases, and (4) the bacterial alpha-6-fucosyltransferases . Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily . In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-fucosyltransferase . The alpha-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases . All these observations strongly suggest that the fucosyltransferases share some common structural and catalytic features.

Hum Mutat, 1998, 11(5), 354 - 9
Molecular basis of phenylketonuria in Venezuela: presence of two novel null mutations; De Lucca M et al.; This report describes the mutational spectrum and linked haplotypes of the phenylalanine hydroxylase gene in Venezuela . In this study, we have detected European mutations such as IVS10nt-11, R243Q, and R408W on the same haplotype background (6.7, 1.8, and 2.3, respectively) as in Europe . In this sample, we have found two novel mutations: S349L detected in two homozygous siblings on the background of haplotype 6.7, and a small deletion, P314fsdelC, that results in a frameshift and a premature stop codon detected on the background of haplotype 4.3 . The definite demonstration that mutation S349L results in a nonfunctional protein was shown by expression analysis in prokaryotic and eukaryotic systems . This mutation results in an unstable phenylalanine hydroxylase (PAH) protein completely devoid of enzymatic activity well correlated with the severe form of the disease exhibited by the homozygous patients.

J Allergy Clin Immunol, 1998 May, 101(5), 691 - 8
Superior biologic activity of the recombinant bee venom allergen hyaluronidase expressed in baculovirus-infected insect cells as compared with Escherichia coli; Soldatova LN et al.; BACKGROUND: Hyaluronidase (Hya) is one of several allergens in honeybee venom . Its cDNA sequence was recently described . OBJECTIVE: We sought to express recombinant Hya in prokaryotic and eukaryotic systems and to compare it with natural (n)Hya for biologic activity . METHODS: In Escherichia coli Hya was produced as inclusion body 6 x His-fusion protein . In baculovirus-infected insect cells expression was obtained by cotransfection of linearized Bac-N-Blue DNA and pMelBac transfer vector into Spodoptera frugiperda cells . RESULTS: Enzymatic activity of Hya from the baculovirus system was equal to nHya, and that of the enzyme expressed in E . coli was only 20% to 30% of nHya . In vitro IgE binding was similar in nHya and the enzyme from baculovirus but markedly lower in Hya expressed in E . coli . CONCLUSIONS: Biologic activity of Hya expressed in baculovirus-infected insect cells was comparable with that of the natural enzyme, indicating a native-like conformation of the recombinant protein . In contrast, the enzyme expressed in E . coli as an inclusion-body protein and reconstituted in vitro reached only 20% to 30% of the activity of nHya.

FEBS Lett, 1998 Apr 17, 426(2), 221 - 4
Increased cellular resistance to oxidative stress by expression of cyanobacterium catalase-peroxidase in animal cells; Ishikawa T et al.; To exploit prokaryotic antioxidant enzymes for protection of animal cells from oxidative damage, we expressed catalase-peroxidase of cyanobacterium Synechococcus PCC 7942 in 104C1 cells . The gene for this enzyme was inserted into the mammalian expression vector pRc/CMV . The stable transfectants obtained had higher specific activities of catalase and as a result became more resistant to H2O2 or paraquat than the parental cells . Subcellular fractionation and immunoblot analysis revealed that the expressed catalase-peroxidase was confined to the cytosol; this localization may be the basis for the effective protection of the transfectants from the oxidative cell damage.

Int J Biochem Cell Biol, 1998 Jan, 30(1), 1 - 5
Pyruvate carboxylase; Wallace JC et al.; Pyruvate carboxylase {EC 6.4.1.1} is a member of the family of biotin-dependent carboxylases and is found widely among eukaryotic tissues and in many prokaryotic species . It catalyses the ATP-dependent carboxylation of pyruvate to form oxaloacetate which may be utilised in the synthesis of glucose, fat, some amino acids or their derivatives and several neurotransmitters . Diabetes and hyperthyroidism increase the level of expression of pyruvate carboxylase in the long term, while its activity in the short term is controlled by the intramitochondrial concentrations of acetyl-CoA and pyruvate . Many details of this enzyme's regulation are yet to be described in molecular terms . However, progress towards this goal and towards understanding the relationship of pyruvate carboxylase structure to its catalytic reaction mechanism, has been enormously enhanced recently by the cloning and sequencing of genes and cDNAs encoding the approximately 130 kDa subunit of this homotetramer . Defects in the expression or biotinylation of pyruvate carboxylase in humans almost invariably results in early death or at best a severely debilitating psychomotor retardation, clearly reflecting the vital role it plays in intermediary metabolism in many tissues including the brain.

Biochemistry, 1998 Apr 28, 37(17), 5981 - 7
Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogues of 2, 3-oxidosqualene; Zheng YF et al.; Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evaluated as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius . In these analogues, sulfur replaces carbons at C-6, C-10, C-14, C-18, or C-19 of OS . Each analogue was a submicromolar inhibitor of SHC with IC50 values ranging from 60 to 570 nM . Enzyme inhibition kinetic analysis was performed using homogeneous recombinant A . acidocaldarius SHC . While analogues 9 (S-14, Ki = 109 nM, kinact = 0.058 min-1) and 11 (S-19, Ki = 83 nM, kinact = 0.054 min-1) were time-dependent inhibitors of SHC, analogues 7 (S-6, Ki = 127 nM) and 8 (S-10, Ki = 971 nM) showed no time dependency with SHC . Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (Ki = 31 nM, kinact = 0.071 min-1) . Kinetic analysis for the five analogues with purified rat liver OSLC was conducted to compare the vertebrate and prokaryotic enzymes . Affinity labeling experiments, using either {17-3H}10 or {22-3H}10 with crude and with pure recombinant SHC, clearly showed specific labeling . A single major radioactive band at 72 kDa on SDS-PAGE indicated that irreversible covalent modification of SHC had occurred . These results suggest that the presence of sulfur at C-18 of OS can interrupt the cyclization and that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.

Biochim Biophys Acta, 1998 Mar 30, 1391(2), 223 - 32
Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids; Tippelt A et al.; We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC) . This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis . Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3 . The deduced amino acid sequence of the M . capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs) . Like other triterpene cyclases, the M . capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain . The recombinant M . capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation . The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS .

Cryobiology, 1998 Mar, 36(2), 75 - 83
Cold stress responses in mesophilic bacteria; Panoff JM et al.; The diversity of the prokaryotes that have been studied, combined with the many different effects of low temperature, has led to an extensive literature concerning cold stress responses in mesophilic bacteria . The aim of this review is to discuss the effects of cold on the behavior of bacteria . The following three responses will be described: (i) biochemical modifications consisting first of membrane fatty acid desaturation and second of the synthesis of cold stress proteins, (ii) physiological responses of the cells to permit growth at low temperatures above 0 degrees C and cryotolerance at lower temperatures, and (iii) control of the cold shock response at a transcriptional and/or translational level . This paper reviews knowledge, most of which has been acquired in the last 10 years, in the field of cold stress responses . It is hoped that these data will help to focus attention on the metabolic responses associated with environmental disturbance .

Prog Nucleic Acid Res Mol Biol, 1998, 60, 267 - 315
Molecular genetics of succinate:quinone oxidoreductase in eukaryotes; Scheffler IE; Succinate:quinone oxidoreductase is a membrane-associated complex in mitochondria, often referred to as complex II, based on the fractionation scheme developed by Y . Hatefi and colleagues . It consists of four peptides, two of which are integral membrane proteins (15 and 12-13 kDa, respectively) and two others that are peripheral membrane proteins, i.e., a flavoprotein (Fp, 70 kDa) and an iron-protein (Ip, 27 kDa) . The mature, functional complex contains a cytochrome in association with the membrane proteins, a flavin linked covalently to the largest peptide, and three iron-sulfur clusters in the 27-kDa subunit . The present review touches only briefly on the biochemical and biophysical properties of this complex . Instead, the focus is on the molecular-genetic studies that have become possible since the first genes from eukaryotes were cloned in 1989 . The evolutionary conservation of the amino acid sequence of both the Fp and the Ip peptides has facilitated the cloning of these genes from a large variety of eukaryotic organisms by PCR-based methods . The review addresses questions related to the regulation of the expression of these genes, with an emphasis on mammals and yeast, for which most of the information is available . Four different genes have to be co-ordinately regulated . Transcriptional as well as posttranscriptional regulatory mechanisms have been observed in diverse organisms . Intriguing observations have been made in studies of this enzyme during the life cycle of organisms existing alternately under aerobic and anaerobic conditions . Naturally occurring or induced mutations in these genes have shed light on several questions related to the assembly of this complex, and on the relationship between structure and function . Four different peptides are imported into the mitochondria . They have to be modified, folded, and assembled . The stage is set for the exploration of highly specific changes introduced by site-directed mutagenesis . Until recently the genes were believed to be exclusively nuclear in all eukaryotes, but exceptions have since been found . This finding has relevance in the discussion of the evolution of mitochondria from prokaryotes . A highly conserved set of genes is found in prokaryotes, and some informative comparisons on gene organization and expression in prokaryotes and eukaryotes have been included.

Prog Nucleic Acid Res Mol Biol, 1998, 60, 47 - 78
Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling; Negrutskii BS et al.; This review offers a comprehensive analysis of eukaryotic translation elongation factor 1 (eEF-1 alpha) in comparison with its bacterial counterpart EF-Tu . Altogether, the data presented indicate some variances in the elongation process in prokaryotes and eukaryotes . The differences may be attributed to translational channeling and compartmentalization of protein synthesis in higher eukaryotic cells . The functional importance of the EF-1 multisubunit complex and expression of its subunits under miscellaneous cellular conditions are reviewed . A number of novel functions of EF-1 alpha, which may contribute to the coordinate regulation of multiple cellular processes including growth, division, and transformation, are characterized.

J Biochem (Tokyo), 1998 Apr, 123(4), 555 - 63
His-Asp phosphotransfer signal transduction; Mizuno T; In general, protein phosphorylation is one of the most widely used mechanisms for regulating biological processes, including intracellular signal transduction . In eukaryotes, the cascades of protein phosphorylation and dephosphorylation events involving a number of protein tyrosine or serine/threonine kinases have been well studied . In contrast, recent intensive studies revealed that bacteria have devised a quite different phosphotransfer signaling mechanism for eliciting a variety of adaptive responses to their environment . Such a bacterial signal transduction mechanism was originally referred to as a "two-component regulatory system." The mode of molecular communication between a "sensor kinase" and its cognate phospho-accepting "response regulator" is principally based on histidine-to-aspartate (His-Asp) phosphotransfer . In Escherichia coli, for example, at least 30 different sensor-regulator pairs operate in a wide variety of adaptive responses . This particular signal transduction mechanism was once thought to be restricted to prokaryotes . However, many instances have recently been uncovered in diverse eukaryotic species . Furthermore, recent studies suggested that the molecular mechanism underlying the bacterial signal transduction is not simple as, and, in fact, is more sophisticated than thought previously . The new concept should be referred to as the "multi-step His-Asp phosphotransfer signaling mechanism."

Electrophoresis, 1998 Apr, 19(4), 613 - 6
Differential display approach to quantitation of environmental stimuli on bacterial gene expression; Fislage R; The differential display of the mRNA technique for eukaryotes is fruitful in identifying genes with altered transcription rates caused by exogenous or endogenous stimuli . Prokaryotic analogues of the method using arbitrary oligonucleotides may reach a complete statistical genome coverage . Thus a genome-wide mass screening for transcriptionally regulated sequences will be possible . However, the primer sets have to be optimized for a given species to result in maximum band yields . Hence the construction of primers requires the calculation of oligonucleotide frequency distributions from known coding regions to choose sequences with frequent occurrence in the bacterial genome . After completion of many whole genome sequencing projects, differentially regulated cDNA sequences are readily identified by sequence comparisons.

Biochimie, 1998 Jan, 80(1), 43 - 8
Post-translational modification of proteins by reversible phosphorylation in prokaryotes; Cozzone AJ; Microorganisms have developed three different systems for catalyzing protein phosphorylation and using this reversible modificaiton to regulate their cellular activities . The first 'classical' system utilizes nucleoside-triphosphates as phosphoryl donors and leads to the modification of protein substrates at serine/threonine or tyrosine residues . The second system, called 'two-component system', requires first a sensor kinase which autophosphorylates at a histidine residue at the expense of adenosine-triphosphate, then a response regulator which is modified in turn at an aspartate residue and thereafter induces a metabolic change within the cell . The third system, called 'PTS system', makes use of phosphoenol pyruvate to generate a phosphoryl group which is passed down a chain of several proteins and finally transferred to a sugar . There is increasing evidence that, contrary to an early concept, these systems and the corresponding enzymes (protein kinases and phosphoprotein phosphatases) share a number of structural and functional similarities with the phosphorylation-dephosphorylation machineries found in eukaryotes . Therefore one can expect that microorganisms will serve, once again, as a basic model for exploring and understanding a key regulatory mechanism, reversible protein phosphorylation, which concerns all organisms.

Eur J Pediatr, 1998 Apr, 157 Suppl 2, S54 - 9
Methylenetetrahydrofolate reductase and methionine synthase: biochemistry and molecular biology; Matthews RG et al.; Methylenetetrahydrofolate reductase and cobalamin-dependent methionine synthase catalyze the penultimate and ultimate steps in the biosynthesis of methionine in prokaryotes, and are required for the regeneration of the methyl group of methionine in mammals . Defects in either of these enzymes can lead to hyperhomocysteinemia . The sequences of the human methylenetetrahydrofolate reductase and methionine synthase are now known, and show clear homology with their bacterial analogues . Mutations in both enzymes that are known to occur in humans and to be associated with hyperhomocysteinemia affect residues that are conserved in the bacterial enzymes . Structure/function studies on the bacterial proteins, summarized in this review, are therefore relevant to the function of the human enzymes; in particular studies on the effects of bacterial mutations analogous to those causing hyperhomocysteinemia in human may shed light on the defects associated with these mutations.

Nucleic Acids Symp Ser, 1997, (37), 195 - 6
Mitochondrial methionyl-tRNA transformylase from bovine liver; Takeuchi N et al.; Substrate specificities of mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) toward tRNA substrates were characterized in vitro . The MTFmt is able to formylate E . coli initiator methionyl-tRNA (Met-tRNA(fMet)) as efficiently as mammalian mitochondrial methionyl-tRNA . Furthermore, E . coli elongator methionyl-tRNA (Met-tRNA(mMet)) also serves as a substrate for mt MTF, whereas E . coli MTF rigorously excludes E . coli Met-tRNA(mMet) from formylation reaction . Thus, mammal mt MTF is suggested to have recognition mechanism different from E . coli MTF . To pursue the relationship between protein structure and unexpected substrate specificity of mammalian MTFmt, the nucleotide sequence of MTFmt gene was determined and its amino acids sequence was compared to other MTFs of prokaryotic origin.

Biochemistry, 1998 Apr 14, 37(15), 5074 - 85
The crystal structure of phosphoribulokinase from Rhodobacter sphaeroides reveals a fold similar to that of adenylate kinase; Harrison DH et al.; The essential photosynthetic enzyme phosphoribulokinase (PRK) is responsible for the conversion of ribulose 5-phosphate (Ru5P) to ribulose 1,5-bisphosphate, the substrate for the CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) . We have determined the structure of the octameric bacterial form of PRK to a resolution of 2.5 A . The protein is folded into a seven-member mixed beta-sheet surrounded by alpha-helices, giving the overall appearance of the nucleotide monophosphate family of kinases . Homology with the nucleotide monophosphate kinases suggests a number of amino acid residues that are likely to be important in catalysis and suggests the roles of some amino acid residues that have been mutated prior to the determination of the structure . Further, sequence identity across eukaryotic and prokaryotic species and a calculation of the buried surface area suggests the identity within the octamer of a dimer conserved throughout evolution . The width of the groove leading to the active site is consistent with an oriented molecule of thioredoxin controlling the oxidation state of two cysteines that regulate activity in the eukaryotic enzymes . Although neither Asp 42 nor Asp 169 can be definitively assigned as the catalytic base, the crystal structure suggests the location of a ribulose 5-phosphate binding site and suggests a role for several of the conserved basic residues.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4458 - 62
Deleterious mutations destabilize ribosomal RNA in endosymbiotic bacteria; Lambert JD et al.; In populations that are small and asexual, mutations with slight negative effects on fitness will drift to fixation more often than in large or sexual populations in which they will be eliminated by selection . If such mutations occur in substantial numbers, the combined effects of long-term asexuality and small population size may result in substantial accumulation of mildly deleterious substitutions . Prokaryotic endosymbionts of animals that are transmitted maternally for very long periods are effectively asexual and experience smaller effective population size than their free-living relatives . The contrast between such endosymbionts and related free-living bacteria allows us to test whether a population structure imposing frequent bottlenecks and asexuality does lead to an accumulation of slightly deleterious substitutions . Here we show that several independently derived insect endosymbionts, each with a long history of maternal transmission, have accumulated destabilizing base substitutions in the highly conserved 16S rRNA . Stabilities of Domain I of this subunit are 15-25% lower in endosymbionts than in closely related free-living bacteria . By mapping destabilizing substitutions onto a reconstructed phylogeny, we show that decreased ribosomal stability has evolved separately in each endosymbiont lineage . Our phylogenetic approach allows us to demonstrate statistical significance for this pattern: becoming endosymbiotic predictably results in decreased stability of rRNA secondary structure.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4241 - 6
DnaA-stimulated transcriptional activation of orilambda: Escherichia coli RNA polymerase beta subunit as a transcriptional activator contact site; Szalewska-Palasz A et al.; We present evidence that Escherichia coli RNA polymerase beta subunit may be a transcriptional activator contact site . Stimulation of the activity of the pR promoter by DnaA protein is necessary for replication of plasmids derived from bacteriophage lambda . We found that DnaA activates the pR promoter in vitro . Particular mutations in the rpoB gene were able to suppress negative effects that certain dnaA mutations had on the replication of lambda plasmids; this suppression was allele-specific . When a potential DnaA-binding sequence located several base pairs downstream of the pR promoter was scrambled by in vitro mutagenesis, the pR promoter was no longer activated by DnaA both in vivo and in vitro . Therefore, we conclude that DnaA may contact the beta subunit of RNA polymerase during activation of the pR promoter . A new classification of prokaryotic transcriptional activators is proposed.

Biochem J, 1998 Apr 1, 331 ( Pt 1), 201 - 9
Purification and characterization of high- and low-molecular-mass isoforms of phosphoenolpyruvate carboxylase from Chlamydomonas reinhardtii . Kinetic, structural and immunological evidence that the green algal enzyme is distinct from the prokaryotic and higher plant enzymes; Rivoal J et al.; Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme in the supply of carbon skeletons for the assimilation of nitrogen by green algae . Two PEPC isoforms with respective native molecular masses of 400 (PEPC1) and 650 (PEPC2) kDa have been purified from Chlamydomonas reinhardtii CW-15 cc1883 (Chlorophyceae) . SDS/PAGE, immunoblot and CNBr peptide-mapping analyses indicate the presence of the same 100 kDa PEPC catalytic subunit in both isoforms . PEPC1 is a homotetramer, whereas PEPC2 seems to be a complex between the PEPC catalytic subunit and other immunologically unrelated polypeptides of 50-70 kDa . Kinetic analyses indicate that these PEPC isoforms are (1) differentially regulated by pH, (2) activated by glutamine and dihydroxyacetone phosphate and (3) inhibited by glutamate, aspartate, 2-oxoglutarate and malate . These results are consistent with the current model for the regulation of anaplerotic carbon fixation in green algae, and demonstrate that green algal PEPCs are uniquely regulated by glutamine . Several techniques were used to assess the structural relationships between C . reinhardtii PEPC and the higher plant or prokaryotic enzyme . Immunoblot studies using anti-(green algal or higher plant PEPC) IgGs suggested that green algal (C . reinhardtii, Selenastrum minutum), higher plant (maize, banana fruit, tobacco) and prokaryotic (Synechococcus leopoliensis, Escherichia coli) PEPCs have little or no immunological relatedness . Moreover, the N-terminal amino acid sequence of the C . reinhardtii PEPC subunit did not have significant similarity to the highly conserved corresponding region in enzymes from higher plants, and CNBr cleavage patterns of green algal PEPCs were distinct from those of higher plant and cyanobacterial PEPCs . These results point to significant evolutionary divergence between green algal, higher plant and prokaryotic PEPCs.

Oral Microbiol Immunol, 1998 Apr, 13(2), 113 - 9
Putative heat shock protein 70 gene from Actinobacillus actinomycetemcomitans: molecular cloning and sequence analysis of its gene; Minami J et al.; We have cloned and sequenced two overlapping fragments of chromosomal DNA from Actinobacillus actinomycetemcomitans . The nucleotide sequence contained two open reading frames . The deduced amino acid sequences of the two open reading frames showed significant homology with the heat shock proteins hsp70 and hsp40 of other organisms respectively . The upstream open reading frame consisted of 1902 bp, corresponding to 634-amino-acid residues . The CAA codon for glutamines was frequently seen in hsp70, i.e., in 30 of 32 glutamines (93.8%) . The spacing region between the two open reading frames was unusually long compared with other prokaryotic organisms . A number of unique and distinguishing features of the sequences in the hsp70 family were verified, and it was found that a particular spacing sequence between the hsp70 and hsp40 gene loci can be used to identify A . actinomycetemcomitans from the periodontal pocket.

Biochim Biophys Acta, 1998 Mar 6, 1391(1), 52 - 66
Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus; Iwasaki Y et al.; Two phosphatidylinositol-specific phospholipase C (PI-PLC) genes from Streptomyces antibioticus were cloned by a shotgun method using Streptomyces lividans TK24 as a host . The genes of the two PI-PLCs (named as PLC1 and PLC2) were adjoined and opposite in the direction of transcription/translation . Both of them were confirmed to be expressed in S . antibioticus . The two enzymes were different in the following properties . (i) PLC2 had considerable sequence similarity to other bacterial PI-PLCs, while PLC1 had a short stretch that was similar to PI-PLCs of eukaryotes rather than the other bacterial enzymes . (ii) PLC1 was Ca2+-dependent, whereas PLC2 was not . (iii) PLC1 generated myo-inositol-1-phosphate and myo-inositol-1:2-cyclic phosphate simultaneously from PI, but PLC2 showed sequential formation of them . (iv) PLC2 has GPI-anchor-degrading activity while PLC1 does not have . Both enzymes did not hydrolyze phosphatidylcholine, phosphatidylinositol-4-monophosphate and phosphatidylinositol-4,5-bisphosphate . Both PLC1 and PLC2 contained two histidine residues that might be catalytic residues . PLC1 has residues that possibly form a Ca2+-binding site . Then it was suggested that both PLC1 and PLC2 act according to the catalytic mechanism using the two histidine residues as proposed in both eukaryotic and prokaryotic enzymes, but that PLC1 has a more 'eukaryotic' mechanism in which Ca2+ participates than that of the Ca2+-independent bacterial enzymes . Thus, we propose that PLC2 is a conventional 'bacteria-type' enzyme, while PLC1 is more closely related to the eukaryotic enzymes rather than the bacterial enzymes .

J Biotechnol, 1998 Feb 5, 60(1-2), 119 - 29
Direct sequencing of bacterial artificial chromosomes (BACs) and prokaryotic genomes by biotin-capture PCR; Sterky F et al.; Determination of unknown DNA sequences adjacent to known segments is an important task in genome-related research . We have applied the methodology of biotin-capture PCR for direct sequencing of bacterial artificial chromosomes (BACs) and bacterial genomes . The strategy involves extension of a biotinylated primer from a known locus into unknown regions of the template to yield single-stranded DNA, which is immobilised onto paramagnetic beads . An arbitrary primer initiates extension from the unknown region and back towards the known locus . The arbitrary primer contains a universal primer 'handle', which is utilised for subsequent amplification . The PCR products are then directly sequenced by solid-phase or cycle sequencing . The fact that BACs or bacterial chromosomes can be sequenced without prior purification or subcloning might be useful in numerous applications, such as gap-filling, sequencing of regulatory regions upstream known genes and determination of intron/exon-boundaries.

Nutr Rev, 1998 Feb, 56(2 Pt 2), s38 - 46; discussion s54-75
Leptin, leptin receptors, and the control of body weight; Friedman JM; The assimilation, storage, and disposition of nutrient energy constitute a complex homeostatic system central to the survival of both prokaryotic and eukaryotic organisms . In vertebrates, and especially among land dwelling mammalian species, the ability to store large quantities of energy-dense fuel in the form of adipose tissue triglyceride permits survival during prolonged periods of food deprivation . In order to maintain such fuel stores during times of dietary scarcity or surfeit, some balance between energy intake and expenditure must be achieved . Lesions of the hypothalamus alter body weight suggesting that this brain region regulates nutritional state . These and other studies led to the hypothesis that body weight was regulated by a feedback loop in which peripheral signals reported nutritional information to an integratory center in the brain . However, the identity of these nutrition signals proved elusive.

Anal Biochem, 1998 Apr 10, 258(1), 48 - 52
Enzymatic synthesis of {3H}Cytidine 5'-diphospho-1, 2-diacyl-sn-glycerol; Zhao M et al.; Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diacylglycerol; CDP-DG) is an important intermediate in the biosynthesis of the major glycerophosphate-based phospholipids of prokaryotes and eukaryotes . This compound is expensive to purchase and inefficient to prepare chemically . Radiolabeled CDP-diacylglycerol is unavailable commercially . We describe a simple and inexpensive method to synthesize {3H}CDP-DG enzymatically . The three-step enzymatic procedure includes phosphorylation of {3H}glycerol to sn-{3H}glycerol 3-phosphate (G3P) by glycerokinase,acylation of {3H}G3P to {3H}phosphatidic acid (PA) by G3P acyltransferase, and conversion of {3H}PA and CTP to {3H}CDP-DG by CDP-DG synthase . This procedure is considerably less labor intensive and less expensive than is chemical synthesis, and the yield is at least 30% .

Biol Chem, 1998 Mar, 379(3), 367 - 71
Homo-dimeric recombinant dihydrofolate reductase from Thermotoga maritima shows extreme intrinsic stability; Dams T et al.; Dihydrofolate reductase (DHFR) from the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli . Sequence determination of the reported dyrA gene was repeated, and a corrected version deposited in the nucleotide sequence databank (accession number Y11021) . Ultracentrifugational analysis and gel permeation chromatography prove that the enzyme forms a stable homodimer . The enzyme exhibits long-term stability at physiological temperature (80 degrees C) and in the presence of high denaturant concentrations (half-time in 6 M guanidinium chloride: 24h) . Alignments of DHFRS from different species, as well as comparative modeling based on the homology to the crystal structures of the enzyme from prokaryotes and eukaryotes, were used to generate a model of the three-dimensional structure . The apoenzyme was crystallized and a data set was collected to a resolution of about 2 A.

FEBS Lett, 1998 Apr 3, 425(3), 407 - 10
The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum; Gilberger TW et al.; The thioredoxin system is one of the major thiol reducing systems of the cell . Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme . We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base {Gilberger, T.-W., Walter, R.D . and Muller, S., J . Biol . Chem . 272 (1997) 29584-29589} . In addition to the active site thiols Cys88 and Cys93 the P . falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540 . To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (C-terminal deletion of the last nine amino acids) were constructed . All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate) . These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

Virus Genes, 1998, 16(1), 7 - 11
Molecular evolution of viruses--Past and Present, Part 2--An introduction; Becker Y; The evolution of viruses is reviewed within the perspective of the concepts on the evolution of the lipid membrane bound vesicular structures in the prebiotic soup through the ideas on evolution of cells during the RNA World and the transition into the DNA World . The ancient Archeae bacteria and their retrons that carry the bacterial reverse transcriptase gene and their unique protein splicing capability provide an indication of the evolutionary path for retroviruses and, independently, for RNA and DNA viruses of the prokaryotic Archeae bacteria and the eukaryotic yeast and fungi.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 115 - 23
Cloning and expression of an iron-containing superoxide dismutase in the parasitic protist, Trichomonas vaginalis; Viscogliosi E et al.; A superoxide dismutase (SOD) gene of the parasitic protist Trichomonas vaginalis was cloned, sequenced, expressed in Escherichia coli, and its gene product characterized . It is an iron-containing dimeric protein with a monomeric mass of 22,067 Da . Southern blots analyses suggested the presence of seven iron-containing (FeSOD) gene copies . Hydrophobic cluster analysis revealed some peculiarities in the 2D structure of the FeSOD from T . vaginalis and a strong structural conservation between prokaryotic and eukaryotic FeSODs . Phylogenetic reconstruction of the SOD sequences confirmed the dichotomy between FeSODs and manganese-containing SODs . FeSODs of protists appeared to group together with homologous proteobacterial enzymes suggesting a possible origin of eukaryotic FeSODs through an endosymbiotic event.

J Bacteriol, 1998 Apr, 180(8), 1995 - 2004
Identification of the omega4400 regulatory region, a developmental promoter of Myxococcus xanthus; Brandner JP et al.; Omega4400 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter . Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac omega4400 . The DNA upstream of the omega4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified . From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac omega4400 may encode a protein with an ATP- or GTP-binding site . Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by beta-galactosidase production in cells containing Tn5 lac omega4400 . The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of -101 bp is sufficient for C-signal-dependent, developmental activation of this promoter . A deletion to -76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein . The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M . xanthus sigB or sigC gene did not affect Tn5 lac omega4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M . xanthus promoter transcribed by a known form of RNA polymerase . However, the omega4400 promoter does contain the sequence 5'-CATCCCT-3' centered at -49 and the C-signal-dependent omega4403 promoter also contains this sequence at the same position . Moreover, the two promoters match at five of six positions in the -10 regions, suggesting that these promoters may share one or more transcription factors . These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.

Adv Parasitol, 1998, 40, 351 - 95
Microsporidiosis: molecular and diagnostic aspects; Weiss LM et al.; The term 'microsporidia' is a nontaxonomic designation which is used to refer to a group of intracellular parasites belonging to the phylum Microspora . These eukaryotic obligate intracellular protozoans have been described infecting every major animal group, especially insects, fish and mammals . They are important agricultural parasites in commercially important insects, fish, laboratory rodents, rabbits, fur-bearing animals, and primates . There is now an increasing recognition of microsporidia as important opportunistic pathogens in persons infected with the human immunodeficiency virus (HIV) . Microsporidia possess ribosomes with features resembling prokaryotes . Phylogenetic analysis of the rRNA sequence from several of the microsporidia suggests that these organisms were early branches in the eukaryotic evolutionary line . The data on these molecular phylogenetic relationships are reviewed in this paper . Inroads have recently been made into the molecular biology of these organisms and these data are also presented . Diagnosis of microsporidia infection from stool examination is possible and has replaced biopsy as the initial diagnostic procedure in many laboratories . These staining techniques can be difficult, however, due to the small size of the spores . The specific identification of microsporidian species has classically depended on ultrastructural examination . With the cloning of the rRNA genes from the human pathogenic microsporidia it has been possible to apply polymerase chain reaction (PCR) techniques for the diagnosis of microsporidial infection at the species level . Both staining and PCR techniques for the diagnosis of microsporidia are reviewed.

Mol Biol Evol, 1998 Apr, 15(4), 456 - 62
A phylogenetic approach to the identification of phosphoglucomutase genes; Whitehouse DB et al.; The expanding molecular database provides unparalleled opportunities for characterizing genes and for studying groups of related genes . We use sequences drawn from the database to construct an evolutionary framework for examining the important glycolytic enzyme phosphoglucomutase (PGM) . Phosphoglucomutase plays a pivotal role in the synthesis and utilization of glycogen and is present in all organisms . In humans, there are three well-described isozymes, PGMI, PGM2, and PGM3 . PGM1 was cloned 5 years ago; however, repeated attempts using both immunological approaches and molecular probes designed from PGM1 have failed to isolate either PGM2 or PGM3 . Using a phylogenetic strategy, we first identified 47 highly divergent prokaryotic and eukaryotic PGM-like sequences from the database . Although overall amino acid identity often fell below 20%, the relative order, position, and sequence of three structural motifs, the active site and the magnesium--and sugar-binding sites, were conserved in all 47 sequences . The phylogenetic history of these sequences was complex and marked by duplications and translocations; two instances of transkingdom horizontal gene transfer were identified . Nonetheless, the sequences fell within six well-defined evolutionary lineages, three of which contained only prokaryotes . Of the two prokaryotic/eukaryotic lineages, one contained bacterial, yeast, slimemold, invertebrate, and vertebrate homologs to human PGM1 and the second contained likely homologs to human PGM2 . Indeed, an amino acid sequence, derived from a partial human cDNA, that fell within the second cross-kingdom lineage bears several characteristics expected for PGM2 . A third lineage may contain homologs to human PGM3 . On a general level, our phylogenetic-based approach shows promise for the further utilization of the extensive molecular database.

Science, 1998 Apr 3, 280(5360), 106 - 9
Structural conservation in prokaryotic and eukaryotic potassium channels; MacKinnon R et al.; Toxins from scorpion venom interact with potassium channels . Resin-attached, mutant K+ channels from Streptomyces lividans were used to screen venom from Leiurus quinquestriatus hebraeus, and the toxins that interacted with the channel were rapidly identified by mass spectrometry . One of the toxins, agitoxin2, was further studied by mutagenesis and radioligand binding . The results show that a prokaryotic K+ channel has the same pore structure as eukaryotic K+ channels . This structural conservation, through application of techniques presented here, offers a new approach for K+ channel pharmacology.

Curr Microbiol, 1998 Mar, 36(3), 158 - 63
Sequence analysis of a 34.7-kb DNA segment from the genome of Buchnera aphidicola (endosymbiont of aphids) containing groEL, dnaA, the atp operon, gidA, and rho; Clark MA et al.; Buchnera aphidicola is a prokaryotic endosymbiont of the aphid Schizaphis graminum . From past and present nucleotide sequence analyses of the B . aphidicola genome, we have assembled a 34 . 7-kilobase (kb) DNA segment . This segment contains genes coding for 32 open reading frames (ORFs), which corresponded to 89.9% of the DNA . All of these ORFs could be identified with homologous regions of the Escherichia coli genome . The order of the genes with established functions was groELS-trmE-rnpA-rpmH-dnaA-dnaN-gyrB-atpCDGAH FEB-gidA-fdx-hscA- hscB-nifS-ilvDC-rep-trxA-rho . The order of genes in small DNA fragments was conserved in both B . aphidicola and E . coli . Most of these fragments were in approximately the same region of the E . coli genome . The latter organism, however, contained many additional inserted genes within and between the fragments . The results of the B . aphidicola genome analyses indicate that the endosymbiont has many properties of free-living bacteria.

Genes Dev, 1998 Mar 15, 12(6), 894 - 900
A protein-induced DNA bend increases the specificity of a prokaryotic enhancer-binding protein; Dworkin J et al.; Control of transcription in prokaryotes often involves direct contact of regulatory proteins with RNA polymerase from binding sites located adjacent to the target promoter . Alternatively, in the case of genes transcribed by Escherichia coli RNA polymerase holoenzyme containing the alternate sigma factor sigma54, regulatory proteins bound at more distally located enhancer sites can activate transcription via DNA looping by taking advantage of the increasing flexibility of DNA over longer distances . While this second mechanism offers a greater possible flexibility in the location of these binding sites, it is not clear how the specificity offered by the proximity of the regulatory protein and the polymerase intrinsic to the first mechanism is maintained . Here we demonstrate that integration host factor (IHF), a protein that induces a sharp bend in DNA, acts both to inhibit DNA-looping-dependent transcriptional activation by an inappropriate enhancer-binding protein and to facilitate similar activation by an appropriate enhancer-binding protein . These opposite effects have the consequence of increasing the specificity of activation of a promoter that is susceptible to regulation by proteins bound to a distal site.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2891 - 5
Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor Tu; Georgiou T et al.; Phage exclusion is a form of programmed cell death in prokaryotes in which death is triggered by infection with phage, a seemingly altruistic response that limits multiplication of the phage and its spread through the population . One of the best-characterized examples of phage exclusion is the exclusion of T-even phages such as T4 by the e14-encoded Lit protein in many Escherichia coli K-12 strains . In this exclusion system, transcription and translation of a short region of the major head coat protein gene late in phage infection activates proteolysis of translation elongation factor Tu (EF-Tu), blocking translation and multiplication of the phage . The cleavage occurs between Gly-59 and Ile-60 in the nucleotide-binding domain . In the present work, we show that a 29-residue synthetic peptide spanning the activating region of the major head coat protein can activate the cleavage of GDP-bound EF-Tu in a purified system containing only purified EF-Tu and purified Lit protein . Lit behaves as a bona fide enzyme in this system, cleaving EF-Tu to completion when present at substoichiometric amounts . Two mutant peptides with amino acid changes that reduce the activation of cleavage of EF-Tu in vivo were also greatly reduced in their ability to activate EF-Tu cleavage in vitro but were still able to activate cleavage at a high concentration . Elongation factor G, which has the same sequence at the cleavage site and a nucleotide-binding domain similar to EF-Tu, was not cleaved by this system, and neither was heat-inactivated EF-Tu, suggesting that the structural context of the cleavage site may be important for specificity . This system apparently represents an activation mechanism for proteolysis that targets one of nature's most evolutionarily conserved proteins for site-specific cleavage.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2885 - 90
Inhibition of FtsZ polymerization by SulA, an inhibitor of septation in Escherichia coli; Mukherjee A et al.; The bacterial cell division protein FtsZ assembles into the cytokinetic Z ring that directs cytokinesis in prokaryotes . In Escherichia coli the formation of the Z ring is prevented by induction of the cell division inhibitor SulA (SfiA), a component of the SOS response . Here we show that a MalE-SulA fusion that retains this inhibitory function in vivo inhibits the GTPase activity and polymerization of FtsZ in vitro . MalE-SulA10, which does not block Z ring formation in vivo, is unable to inhibit the GTPase activity and polymerization in vitro . Furthermore, FtsZ114, which is refractory to SulA in vivo, is not inhibited by MalE-SulA . These results indicate that SulA blocks Z ring formation by blocking FtsZ polymerization.

Proc Natl Acad Sci U S A, 1998 Mar 17, 95(6), 2818 - 23
Seeking an ancient enzyme in Methanococcus jannaschii using ORF, a program based on predicted secondary structure comparisons; Aurora R et al.; We have developed a simple procedure to identify protein homologs in genomic databases . The program, called ORF, is based on comparisons of predicted secondary structure . Protein structure is far better conserved than amino acid sequence, and structure-based methods have been effective in exploiting this fact to find homologs, even among proteins with scant sequence identity . ORF is a secondary structure-based method that operates solely on predictions from sequence and requires no experimentally determined information about the structure . The approach is illustrated by an example: Thymidylate synthase, a highly conserved enzyme essential to thymidine biosynthesis in both prokaryotes and eukaryotes, is thought to be used by Archaea, but a corresponding gene has yet to be identified . Here, a candidate thymidylate synthase is identified as a previously unassigned open reading frame from the genome of Methanococcus jannaschii, viz., MJ0757 . Using primary structure information alone, the optimally aligned sequence identity between MJ0757 and Escherichia coli thymidylate synthase is 7%, well below the threshold of sensitivity for detection by sequence-based methods.

FEBS Lett, 1998 Mar 20, 425(1), 151 - 6
Identification of an ABC transporter gene that exhibits mRNA level overexpression in fluoroquinolone-resistant Mycobacterium smegmatis; Banerjee SK et al.; We describe here the PCR amplification of a DNA fragment (mtp1) from Mycobacterium smegmatis using primers derived from consensus sequences of the ABC family of transporters . The fragment encodes amino acid sequences that exhibited significant homology with different ABC transporters . Amino acid sequence alignment of the full length gene with other transporters identified the ABC protein as the B-subunit of the phosphate specific transporter . Strikingly, a M . smegmatis colony which exhibited a high level of ciprofloxacin resistance showed mRNA level overexpression of mtp1 . Thus this is the first report in any prokaryote indicating differential expression of an ABC transporter in a fluoroquinolone resistant colony.

FEBS Lett, 1998 Mar 20, 425(1), 87 - 90
Proteasome function is dispensable under normal but not under heat shock conditions in Thermoplasma acidophilum; Ruepp A et al.; Hitherto the biology of proteolysis in prokaryotes, particularly in archaea, is only poorly understood . We have used the tri-peptide vinyl sulfone inhibitor carboxybenzyl-leucyl-leucyl-leucine vinyl sulfone (Z-L3VS) to study the in vivo function of proteasomes in Thermoplasma acidophilum . Z-L3VS is a potent inhibitor of the Thermoplasma proteasome and is capable of modifying 75 to 80% of the proteasomal beta-subunits in cell cultures . Inhibition of proteasomes has only marginal effects under normal growth conditions . Under heat shock conditions, however, the effects of proteasome inhibition are much more severe, to the extent of complete cell growth arrest . These data suggest that other proteolytic systems may exist that can compensate for the loss of proteasome function in T . acidophilum.

FEBS Lett, 1998 Mar 6, 424(1-2), 1 - 5
Comparative molecular analysis of Na+/H+ exchangers: a unified model for Na+/H+ antiport?
Dibrov P, Fliegel L.
Despite 30 years of study on Na+/H+ exchange, the molecular mechanisms of antiport remain obscure . Most challenging, the identity of amino acids involved in binding transported cations is still unknown . We review data examining the identity of residues that are involved in cation binding and translocation of prokaryotic and eukaryotic Na+/H+ antiporters . Several polar residues specifically distributed within or immediately adjacent to membrane spanning regions are implicated as being important . These key amino acids are conserved in prokaryotes and in some lower eukaryotic forms of the Na+/ H+ antiporter, despite their being dispersed throughout the protein and despite an overall low similarity in the linear sequence of these Na+/H+ antiporters . We suggest that this conservation of isolated residues (together with distances between them) reflects a general physicochemical mechanism of cation binding by exchangers . The binding could be based on coordination of the substrate cation by a crown ether-like cluster of polar atomic groups amino acids, as has been hypothesized by Boyer . Traditional screening for the extended, highly conserved linear protein sequences might not be applicable when searching for functional domains of ion transporters . Three-dimensional constellations of polar residues (3D-motifs) may be evolutionary conserved rather than linear primary sequence.

Biochem Biophys Res Commun, 1998 Mar 27, 244(3), 908 - 11
Conserved enzyme-substrate electrostatic attraction in prokaryotic Cu,Zn superoxide dismutases; Folcarelli S et al.; The catalytic activity of wild type Escherichia coli Cu,Zn superoxide dismutases and of two mutants in which two lysine residues conserved in most bacterial Cu,Zn superoxide dismutases have been replaced by serine was investigated by pulse radiolysis and Brownian dynamics simulations . Experimental and computational data show that neutralization of Lys60 strongly reduces the catalytic activity of the enzyme (approximately 50%), indicating that this residue has a primary role in the electrostatic attraction of the substrate towards the catalytic copper . Neutralization of Lys63 does not significantly influence the catalytic rate constant . The results suggest that prokaryotic Cu,Zn superoxide dismutases have evolved an electrostatic mechanism to facilitate the enzyme-substrate encounter that is functionally equivalent to that already found in the eukaryotic enzymes.

FASEB J, 1998 Apr, 12(6), 421 - 31
Binding to four-way junction DNA: a common property of architectural proteins?
Zlatanova J, van Holde K.
Proteins that can be shown to strongly bind in vitro to the four-way (Holliday) junction DNA include not only the obvious candidates such as enzymes involved in recombination, but also a remarkably diverse group of seemingly unrelated proteins . These include the HMG1 box proteins, members of the HMGI-Y family, winged helix proteins (including linker histones), the SWI/SNF complex, and some totally unrelated prokaryotic proteins . What these proteins seem to share is a propensity to bind to bent DNA, to bend DNA upon binding, and/or to preferentially interact with DNA crossings . Thus, they appear to be, in the main, architectural proteins, although some (like the SWI/SNF complex) have very specific functional roles as well . Perhaps because they bind to or promote the formation of particular DNA structures, the four-way junction binding proteins are frequently interchangeable in cellular function . Furthermore, since a given kind of structure can be recognized by many different protein motifs, it is not surprising that apparently unrelated proteins can fall into such a single functional class.

Insect Mol Biol, 1998 May, 7(2), 101 - 5
Analysis of Wolbachia protein synthesis in Drosophila in vivo; Sasaki T et al.; Intracellular Wolbachia infections are extremely common in arthropods and exert profound control over the reproductive biology of the host . However, very little is known about the underlying molecular mechanisms which mediate these interactions with the host . We examined protein synthesis by Wolbachia in a Drosophila host in vivo by selective metabolic labelling of prokaryotic proteins and subsequent analysis by 1D and 2D gel electrophoresis . Using this method we could identify the major proteins synthesized by Wolbachia in ovaries and testes of flies . Of these proteins the most abundant was of low molecular weight and showed size variation between Wolbachia strains which correlated with the reproductive phenotype they generated in flies . Using the gel systems we employed it was not possible to identify any proteins of Wolbachia origin in the mature sperm cells of infected flies.

Microbiology, 1998 Mar, 144 ( Pt 3), 801 - 5
A 28 kbp segment from the spoVM region of the Bacillus subtilis 168 genome; Foulger D et al.; The sequence of a 28 kbp segment of DNA surrounding the spoVM gene of Bacillus subtilis 168 (lying at approximately 145 degrees on the standard genetic map) has been determined . The region contains 27 ORFs, a number of which have predicted products significantly similar to proteins in sequence databases, particularly to proteins involved in macromolecular synthesis of nucleic acids, proteins and phospholipids . A pair of closely linked genes encode a likely serine protein phosphatase and a serine protein kinase, respectively . Such proteins play important regulatory roles in eukaryotic cells but are rare in prokaryotes.

Curr Microbiol, 1998 Apr, 36(4), 238 - 40
Endosymbionts (Buchnera) from the aphids Schizaphis graminum and Diuraphis noxia have different copy numbers of the plasmid containing the leucine biosynthetic genes; Thao ML et al.; The prokaryotic endosymbiont (Buchnera) of the aphid Schizaphis graminum contains 24 copies of a plasmid that has genes encoding enzymes of the leucine biosynthetic pathway while the endosymbiont of the related aphid Diuraphis noxia has only one copy of this plasmid . These results, in conjunction with similar results for the trpEG-containing plasmids, suggest that D . noxia has a reduced demand for endosymbiont-derived essential amino acids.

Genes Dev, 1998 Mar 1, 12(5), 745 - 54
Conversion of the omega subunit of Escherichia coli RNA polymerase into a transcriptional activator or an activation target; Dove SL et al.; Evidence obtained in both eukaryotes and prokaryotes indicates that arbitrary contacts between DNA-bound proteins and components of the transcriptional machinery can activate transcription . Here we demonstrate that the Escherichia coli omega protein, which copurifies with RNA polymerase, can function as a transcriptional activator when linked covalently to a DNA-binding protein . We show further that omega can function as an activation target when this covalent linkage is replaced by a pair of interacting polypeptides fused to the DNA-binding protein and to omega, respectively . Our findings imply that the omega protein is associated with RNA polymerase holoenzyme in vivo, and provide support for the hypothesis that contact between a DNA-bound protein and any component of E . coli RNA polymerase can activate transcription.

J Biol Chem, 1998 Mar 6, 273(10), 5443 - 6
Identification and molecular cloning of a human selenocysteine insertion sequence-binding protein . A bifunctional role for DNA-binding protein B; Shen Q et al.; Prokaryotic and eukaryotic cells incorporate the unusual amino acid selenocysteine at a UGA codon, which conventionally serves as a termination signal . Translation of eukaryotic selenoprotein mRNA requires a nucleotide selenocysteine insertion sequence in the 3'-untranslated region . We report the molecular cloning of the binding protein that recognizes the selenocysteine insertion sequence element in human cellular glutathione peroxidase gene (GPX1) transcripts and its identification as DNA-binding protein B, a member of the EFIA/dbpB/YB-1 family . The predicted amino acid sequence contains four arginine-rich RNA-binding motifs, and one segment shows strong homology to the human immunodeficiency virus Tat domain . Recombinant DNA-binding protein B binds the selenocysteine insertion sequence elements from the GPX1 and type I iodothyronine 5'-deiodinase genes in RNA electrophoretic mobility shift assays and competes with endogenous GPX1 selenocysteine insertion sequence binding activity in COS-1 cytosol extracts . Addition of antibody to DNA-binding protein B to COS-1 electromobility shift assays produces a slowly migrating "supershift" band . The molecular cloning and identification of DNA-binding protein B as the first eukaryotic selenocysteine insertion sequence-binding protein opens the way to the elucidation of the entire complex necessary for the alternative reading of the genetic code that permits translation of selenoproteins.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2691 - 6
Response regulators implicated in His-to-Asp phosphotransfer signaling in Arabidopsis; Imamura A et al.; The His to Asp phosphotransfer signal transduction mechanism involves three common signaling domains: the transmitter (or His-kinase), the receiver, and the histidine-containing phototransfer (HPt) domain . Typically, a sensor kinase has a His-kinase domain and a response regulator has a receiver domain containing a phosphoaccepting aspartate, whereas a histidine-containing phototransfer domain serves as a mediator of the histidine-to-aspartate phosphotransfer . This signal transduction mechanism was thought to be restricted to prokaryotes . However, many examples have been discovered in diverse eukaryotic species including higher plants . In Arabidopsis, three sensor kinases have been characterized, namely, ETR1, ERS, and CKI1, which were suggested to be involved in ethylene- and cytokinin-dependent signal transduction pathways, respectively . To date, no response regulator has been discovered in higher plants . We identify five distinct Arabidopsis response regulator genes, each encoding a protein containing a receiver-like domain . In vivo and in vitro evidence that ARRs can function as phosphoaccepting response regulators was obtained by employing the Escherichia coli His-Asp phosphotransfer signaling system.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2250 - 5
Oxa1p, an essential component of the N-tail protein export machinery in mitochondria; Hell K et al.; A number of nuclear encoded inner membrane proteins of mitochondria span the membrane in such a manner that their N termini are located in the intermembrane space . Many of these proteins attain this membrane orientation by undergoing an export step from the matrix across the inner membrane . This export process, which resembles bacterial N-tail export from energetic and topogenic signal requirements, is facilitated by Oxa1p, a protein that has homologues throughout prokaryotes and eukaryotes . Oxa1p, as we have previously shown, is required to export the N and C termini of the mitochondrially encoded pCoxII to the intermembrane space . We demonstrate here that imported nuclear encoded proteins physically interact with Oxa1p and depend on Oxa1p for efficient export of their N termini to the intermembrane space . Furthermore, Oxa1p interacts with nascent polypeptide chains synthesized in mitochondria, including the fully synthesized pCoxII and CoxIII species . Thus, Oxa1p represents a component of a general export machinery of the mitochondrial inner membrane.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2238 - 43
A poly(A) binding protein functions in the chloroplast as a message-specific translation factor; Yohn CB et al.; High-affinity binding of a set of proteins with specificity for the 5' untranslated region (UTR) of the Chlamydomonas reinhardtii chloroplast psbA mRNA correlates with light-regulated translational activation of this message . We have isolated a cDNA encoding the main psbA RNA binding protein, RB47, and identified this protein as a member of the poly(A) binding protein family . Poly(A) binding proteins are a family of eukaryotic, cytoplasmic proteins thought to bind poly(A) tails of mRNAs and play a role in translational regulation . In vitro translation of RNA transcribed from the RB47 cDNA produces a precursor protein that is efficiently transported into the chloroplast and processed to the mature 47-kDa protein . RB47 expressed and purified from Escherichia coli binds to the psbA 5' UTR with similar specificity and affinity as RB47 isolated from C . reinhardtii chloroplasts . The identification of a normally cytoplasmic translation factor in the chloroplast suggests that the prokaryotic-like chloroplast translation machinery utilizes a eukaryotic-like initiation factor to regulate the translation of a key chloroplast mRNA . These data also suggest that poly(A) binding proteins may play a wider role in translation regulation than previously appreciated.

Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2192 - 7
A dynamic model for PC4 coactivator function in RNA polymerase II transcription; Malik S et al.; Human positive cofactor (PC4) acts as a general coactivator for activator-dependent transcription by RNA polymerase II . Here we show that PC4 coactivator function, in contrast to basal (activator-independent) transcription, is dependent both on TATA binding protein (TBP)-associated factors (TAFs) in TFIID and on TFIIH . Surprisingly, PC4 strongly represses transcription initiation by minimal preinitiation complexes in the absence of TAFs and TFIIH, while simultaneously promoting the formation of these complexes . Furthermore, TFIIH and TAFII250, the largest subunit of TFIID, can both phosphorylate PC4 . These results provide evidence for an inactive, PC4-induced intermediate in preinitiation complex assembly and point to TFIIH and TAF requirements for its progression into a functional preinitiation complex . Thus PC4 coactivator activity is realized in a stepwise series of events reminiscent of prokaryotic activation pathways involving conversion of inactive RNA polymerase-promoter complexes to an initiation-competent state.

Biochem J, 1998 Feb 15, 330 ( Pt 1), 453 - 60
The plasma membrane of Leishmania donovani promastigotes is the main target for CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide; Diaz-Achirica P et al.; Reports on the lethal activity of animal antibiotic peptides have largely focused on bacterial rather than eukaryotic targets . In these, involvement of internal organelles as well as mechanisms different from those of prokaryotic cells have been described . CA(1-8)M(1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity . Using Leishmania donovani promastigotes as a model system we have studied the mechanism of action of CA(1-8)M(1-18), its two parental peptides and two analogues . At micromolar concentration CA(1-8)M(1-18) induces a fast permeability to H+/OH-, collapse of membrane potential and morphological damage to the plasma membrane . Effects on other organelles are related to the loss of internal homeostasis of the parasite rather than to a direct effect of the peptide . Despite the fast kinetics of the process, the parasite is able to deactivate in part the effect of the peptide, as shown by the higher activity of the d-enantiomer of CA(1-8)M(1-18) . Electrostatic interaction between the peptide and the promastigote membrane, the first event in the lethal sequence, is inhibited by polyanionic polysaccharides, including its own lipophosphoglycan . Thus, in common with bacteria, the action of CA(1-8)M(1-18) on Leishmania promastigotes has the same plasma membrane as target, but is unique in that different peptides show patterns of activity that resemble those observed on eukaryotic cells.

Proteins, 1998 Mar 1, 30(4), 424 - 34
Oligopeptidase B: cloning and probing stability under nonequilibrium conditions; Polgar L et al.; Oligopeptidase B is a member of a new serine peptidase family, unrelated to the trypsin and subtilisin families . It is a potential processing enzyme of prokaryotes, being very specific for the basic amino acid pairs of polypeptides . An understanding of the kinetics of the enzyme requires the examination of its conformational stability under a variety of conditions . To this end, the enzyme was cloned from Escherichia coli HB101 by the PCR method, expressed with high yield in E . coli XL1-Blue, and purified essentially in two chromatographic steps . The denatured enzyme failed to refold, which precluded the calculation of free energy of stability, deltaG0 . Therefore, the unfolding rates were measured to probe the stability against urea, pH, and heat . Denaturation processes were monitored by intrinsic fluorescence, circular dichroism, and activity measurements . A static method, intrinsic fluorescence vs . pH, was indicative of significant changes in the tertiary structure of the enzyme pH < 6 and pH > 8.5 . The more sensitive dynamic methods, unfolding rates in urea and inactivation rates at high temperature, revealed increased flexibility in the protein structure between pH 6 and pH 7, where the static method did not show significant changes . Inactivation of the enzyme in the acidic pH range correlated with the results obtained with the static rather than with the dynamic method . Acid denaturation at pH 3 was markedly retarded by 1 M NaCl . Against heat inactivation the enzyme was also considerably protected in the presence of salt, and the higher enthalpy and entropy of activation suggested the importance of hydration in the stabilization . The kinetics of unfolding followed single-exponential decay under strongly denaturing conditions (high urea concentration or high temperature), but deviated from the apparently two-state mechanism at low urea concentrations and at slightly acidic pH . The results indicate that under harsher denaturing conditions there is a single rate-limiting step in unfolding, whereas under milder conditions partly unfolded intermediates are populated.

Biophys J, 1998 Feb, 74(2 Pt 1), 1031 - 42
Electron tomography of ice-embedded prokaryotic cells; Grimm R et al.; Whole cells of archaea were embedded in vitreous ice by plunge freezing and investigated by automated energy-filtered electron tomography at 120 kV . The embedded cells were between 300 and 750 nm thick, and their structures were reconstructed to a resolution of 20-40 nm from tilt series comprising 50-140 images . The dose was kept within tolerable limits . A resolution of 20 nm allowed visualization of the individual stalks of the S-layer of Pyrobaculum aerophilum cells, which had undergone partial lysis, in three dimensions . The attainable resolution for low-dose electron tomography under different experimental conditions was theoretically investigated in terms of the specimen thickness . To obtain 2-nm resolution at 120 kV (300 kV), the specimen must not be thicker than 100 nm (150 nm) . For a resolution of 10 nm, the maximum thickness is 450 nm (700 nm) . An accelerating voltage of 300 kV is advantageous, mainly for specimens thicker than 100 nm . Experimental investigations so far have resulted in a resolution that is worse by a factor of 2-5 as compared to theory.

Cytogenet Cell Genet, 1997, 79(1-2), 132 - 8
Cytochrome b in human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the components in liver mitochondria and chromosome assignment of the genes for the large (SDHC) and small (SDHD) subunits to 1q21 and 11q23; Hirawake H et al.; Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms . In this study, the amino acid sequences of the large (cybL) and small (cybS) subunits of cytochrome b in human liver complex II were deduced from cDNAs isolated by homology probing with mixed primers for the polymerase chain reaction . The mature cybL and cybS contain 140 and 103 amino acids, respectively, and show little similarity to the amino acid sequences of the subunits from other species in contrast to the highly conserved features of the flavoprotein (Fp) subunit and iron-sulfur protein (Ip) subunit . From hydrophobicity analysis, both cybL and cybS appear to have three transmembrane segments, indicating their role as membrane-anchors for the enzyme complex . Histidine residues, which are possible heme axial ligands in cytochrome b of complex II, were found in the second transmembrane segment of each subunit . The genes for cybL (SDHC) and cybS (SDHD) were mapped to chromosome 1q21 and 11q23, respectively by fluorescent in situ hybridization (FISH).

Baillieres Clin Endocrinol Metab, 1997 Oct, 11(3), 451 - 79
Understanding the thyrotropin receptor function-structure relationship; Sanders J et al.; The thyrotropin (TSH) receptor (TSHR) is a key protein in the control of thyroid function and a major thyroid autoantigen . Recently, molecular cloning of the receptor has been carried out and we now review the impact of this work on our understanding of the physiology and pathophysiology of the TSHR . Analysis of recombinant TSHR proteins expressed in prokaryotic and eukaryotic systems has indicated that post-translational processing is important for the formation of active receptors . Studies of TSHR glycosylation have shown that a 'mature' form of the receptor containing mainly complex-type sugar residues is principally involved in TSH and TSHR autoantibody (TRAb) binding . In addition, the processing of the TSHR peptide chain into two subunits observed with native TSHR has been confirmed using recombinant TSHR . However, despite considerable efforts in many laboratories, the binding site(s) for TSH and TRAb on the TSHR have not been well characterized as yet and lessons learned from the discovery of naturally occurring amino acid mutations of the TSHR confirm the complexity of the hormone and autoantibody binding sites . Future progress in producing large amounts of pure TSHR as well as monoclonal TRAbs, followed by crystallographic analysis of TSHR-TSH complexes and TSHR-TRAb complexes, should be helpful in providing a better insight into the relationship between TSHR structure and function.

Bull Math Biol, 1998 Jan, 60(1), 163 - 94
An evolutionary analytical model of a complementary circular code simulating the protein coding genes, the 5' and 3' regions; Arques DG et al.; The self-complementary subset T0 = X0 {symbol: see text} inverted question markAAA, TTT inverted question mark with X0 = inverted question markAAC, AAT, ACC, ATC, ATT, CAG, CTC, CTG, GAA, GAC, GAG, GAT, GCC, GGC, GGT, GTA, GTC, GTT, TAC, TTC inverted question mark of 22 trinucleotides has a preferential occurrence in the frame 0 (reading frame established by the ATG start trinucleotide) of protein (coding) genes of both prokaryotes and eukaryotes . The subsets T1 = X1 {symbol: see text} inverted question markCCC inverted question mark and T2 = X2 {symbol: see text} inverted question markGGG inverted question mark of 21 trinucleotides have a preferential occurrence in the shifted frames 1 and 2 respectively (frame 0 shifted by one and two nucleotides respectively in the 5'-3' direction) . T1 and T2 are complementary to each other . The subset T0 contains the subset X0 which has the rarity property (6 x 10(-8) to be a complementary maximal circular code with two permutated maximal circular codes X1 and X2 in the frames 1 and 2 respectively . X0 is called a C3 code . A quantitative study of these three subsets T0, T1, T2 in the three frames 0, 1, 2 of protein genes, and the 5' and 3' regions of eukaryotes, shows that their occurrence frequencies are constant functions of the trinucleotide positions in the sequences . The frequencies of T0, T1, T2 in the frame 0 of protein genes are 49, 28.5 and 22.5% respectively . In contrast, the frequencies of T0, T1, T2 in the 5' and 3' regions of eukaryotes, are independent of the frame . Indeed, the frequency of T0 in the three frames of 5' (respectively 3') regions is equal to 35.5% (respectively 38%) and is greater than the frequencies T1 and T2, both equal to 32.25% (respectively 31%) in the three frames . Several frequency asymmetries unexpectedly observed (e.g . the frequency difference between T1 and T2 in the frame 0), are related to a new property of the subset T0 involving substitutions . An evolutionary analytical model at three parameters (p, q, t) based on an independent mixing of the 22 codons (trinucleotides in frame 0) of T0 with equiprobability (1/22) followed by t approximately 4 substitutions per codon according to the proportions p approximately 0.1, q approximately 0.1 and r = 1 - p - q approximately 0.8 in the three codon sites respectively, retrieves the frequencies of T0, T1, T2 observed in the three frames of protein genes and explains these asymmetries . Furthermore, the same model (0.1, 0.1, t) after t approximately 22 substitutions per codon, retrieves the statistical properties observed in the three frames of the 5' and 3' regions . The complex behaviour of these analytical curves is totally unexpected and a priori difficult to imagine.

Mol Cell Biol, 1998 Apr, 18(4), 1935 - 45
Analysis of the interaction of the novel RNA polymerase II (pol II) subunit hsRPB4 with its partner hsRPB7 and with pol II; Khazak V et al.; Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival . In S . cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions . Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus . Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes . In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4- phenotypes in yeast . However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II . hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions . Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins . This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.

Biotechniques, 1998 Mar, 24(3), 478 - 82
Fusion of green fluorescent protein with the Zeocin-resistance marker allows visual screening and drug selection of transfected eukaryotic cells; Bennett RP et al.; Green fluorescent protein (GFP) and the Zeocin-resistance gene Sh ble (ZeoR) were fused together to generate a bifunctional protein for the identification and selection of transfected mammalian cells . Expression of this hybrid selectable marker, GFP-ZeoR, was visually detected and conferred Zeocin resistance in prokaryotes and eukaryotes . This selectable marker provides a way to determine transient transfection efficiencies in tissue culture cells using fluorescence microscopy . Expression of the GFP-ZeoR was also used to identify and select stable mammalian cell lines expressing a heterologous gene . Selection was efficient and GFP fluorescence provides an excellent, noninvasive technique to monitor the success of Zeocin selection during the development of the stable cell lines . This hybrid resistance gene combines the functional properties of the Zeocin-resistance marker and GFP and should be useful for combined selection and fluorescence in a variety of organisms.

Plant Mol Biol, 1998 Mar, 36(5), 709 - 16
The 38 kDa chlorophyll a/b protein of the prokaryote Prochlorothrix hollandica is encoded by a divergent pcb gene; van der Staay GW et al.; The chlorophyll (Chl) a/b proteins of the photosynthetic prokaryotes appear to have evolved by gene duplication and divergence of the core Chl a antenna family, which also includes CP43 and CP47 and the iron-stress induced Chl a-binding IsiA proteins . We show here that Prochlorothrix hollandica has a cluster of three pcb (prochlorophyte chlorophyll b) genes which are co-transcribed . The major antenna polypeptides of 32 and 38 kDa are encoded by pcbA and pcbC respectively . The pcbC gene is significantly divergent from the other two and may have originated by a gene duplication independent of the one that led to isiA and the other prochlorophyte pcb genes . The distant relatedness of the three prochlorophyte genera implies that not only the ability to make Chl b and use it for light-harvesting arose independently in the three lineages, but also that the pcb genes may have arisen as the result of independent gene duplications in each lineage.

Biochemistry (Mosc), 1998 Jan, 63(1), 31 - 7
Effect of lysophosphatidylcholine on transmembrane signal transduction; Prokazova NV et al.; Lysophosphatidylcholine (LPC), 1-acyl-sn-glycero-3-phosphocholine, is well known as an intermediate of metabolism of phosphatidylcholine (PC), the main phospholipid component in all eukaryotic and many prokaryotic cells . LPC is produced as a result of PC hydrolysis by several isoforms of phospholipase A2 (PLA2) and in the reaction mediated by lecithin-cholesterol acyltransferase that transfers the fatty acid residue from PC to cholesterol . LPC is classified as a second messengers that is produced by activation of cytosolic hormone-activated PLA2 . It was shown that LPC inhibits transmembrane signaling via receptors, which in their active form are linked to G-proteins . There is a viewpoint that LPC abolishes formation of the complex between the receptor and G-protein . The effect of LPC on protein kinase C (PKC) activation is considered in this review . It was shown that low (less than 20 microM) and high (more than 30 microM) concentrations of LPC activated and inhibited PKC, respectively . The mechanism of LPC-induced activation of PKC still remains unclear . However, the studies of the effect of LPC on signal transduction through the PKC-mediated pathway showed that LPC probably plays an auxiliary role . It was suggested that LPC may prolong the effect of the direct activators of PKC (such as 1,2-diacylglycerol or phorbol esters) . The physiological role of the elevation of LPC level in tissues is associated with its ability to enhance or even evoke cell proliferation, stimulate adhesion and differentiation of lymphoid cells, have mitogenic effect on macrophages, activate human T-lymphocytes, initiate monocyte chemotaxis, decrease myocardial sensitivity to cholinergic stimulation, impair contractility of arterial smooth muscle, and modulate aggregation of platelets.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1282 - 7
The post-incision steps of the DNA base excision repair pathway in Escherichia coli: studies with a closed circular DNA substrate containing a single U:G base pair; Sandigursky M et al.; The DNA base excision repair pathway is responsible for removal of oxidative and endogenous DNA base damage in both prokaryotes and eukaryotes . This pathway involves formation of an apurinic/apyrimidinic (AP) site in the DNA, which is further processed to restore the integrity of the DNA . In Escherichia coli it has been suggested that the major mode of repair involves replacement of a single nucleotide at the AP site, based on repair synthesis studies using oligonucleotide substrates containing a unique uracil base . The mechanism of the post-incision steps of the bacterial base excision repair pathway was examined using a DNA plasmid substrate containing a single U:G base pair . Repair synthesis carried out by repair-proficient ung, recJ and xon E.coli cell extracts was analyzed by restriction endonuclease cleavage of the DNA containing the uracil lesion . It was found that replacement of the uracil base was always accompanied by replacement of several nucleotides ( approximately 15) 3' of the uracil and this process was absolutely dependent on initial removal of the uracil base by the action of uracil-DNA glycosylase . In contrast to findings with oligonucleotide substrates, replacement of just a single nucleotide at the lesion site was not detected . These results suggest that repair patch length may be substrate dependent and a re-evaluation of the post-incision steps of base excision repair is suggested.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1214 - 22
Isolation, characterization and baculovirus-mediated expression of the cDNA encoding cytosine DNA methyltransferase from Pisum sativum; Pradhan S et al.; A series of overlapping clones complementary to the Arabidopsis cytosine-5 DNA methyltransferase (C-5 MTase) has been isolated from pea cDNA libraries . The assembled nucleic acid sequence contains an open reading frame of 4761 bp encoding a protein of 1554 amino acids . Like other eukaryotic C-5 MTases, the inferred protein has a presumed regulatory N-terminal region linked to a catalytic C-terminal domain, which has eight of the ten conserved motifs found in prokaryotic C-5 MTases . The pea C-5 MTase has 65% identity at the nucleotide level and 61% identity at the protein level, with the Arabidopsis C-5 MTase . The catalytic domain of the pea enzyme shares 78% identity with Arabidopsis and approximately 52% identity with murine and human C-5 MTases, including the relative position of the proline-cysteine dipeptides of the catalytic centre . Using the conserved region of the cDNA as a probe, we have identified a transcript of 5 kb . Southern blot analysis of pea genomic DNA with the above probe indicates the presence of a single gene . Using poly(A)+ RNA from different developmental stages and different tissues, we have observed that expression is confined mostly to the rapidly dividing tissues of the plant . Expression of this assembled cDNA in a baculovirus system gives a protein of approximately 174 kDa . The expressed protein can be cross-linked, in an AdoMet-dependent manner, to duplex oligonucleotide substrates containing FdC in place of target cytosines in either CG or CAG/CTG sequences.

Nucleic Acids Res, 1998 Mar 1, 26(5), 1185 - 90
Site-selective inhibition of plastid RNA editing by heat shock and antibiotics: a role for plastid translation in RNA editing; Karcher D et al.; RNA editing in higher plant plastids changes single cytidine residues to uridine through an unknown mechanism . In order to investigate the relation of editing to physiological processes and to other steps in plastid gene expression, we have tested the sensitivity of chloroplast RNA editing to heat shock and antibiotics . We show that heat shock conditions as well as treatment of plants with prokaryotic translational inhibitors can inhibit plastid RNA editing . Surprisingly, this inhibitory effect is confined to a limited number of plastid editing sites suggesting that some site-specific factor(s) but none of the general components of the plastid RNA editing machinery are compromised . Contrary to previous expectations, our results provide evidence for a role of plastid translation in RNA editing.

Orig Life Evol Biosph, 1998 Apr, 28(2), 215 - 25
First steps in eukaryogenesis: physical phenomena in the origin and evolution of chromosome structure; Chela-Flores J; Our present understanding of the origin and evolution of chromosomes differs considerably from current understanding of the origin and evolution of the cell itself . Chromosome origins have been less prominent in research, as the emphasis has not shifted so far appreciably from the phenomenon of primeval nucleic acid encapsulation to that of the origin of gene organization, expression, and regulation . In this work we discuss some reasons why preliminary steps in this direction are being taken . We have been led to examine properties that have contributed to raise the ancestral prokaryotic programmes to a level where we can appreciate in eukaryotes a clear departure from earlier themes in the evolution of the cell from the last common ancestor . We shift our point of view from evolution of cell morphology to the point of view of the genes . In particular, we focus attention on possible physical bases for the way transmission of information has evolved in eukaryotes, namely, the inactivation of whole chromosomes . The special case of the inactivation of the X chromosome in mammals is discussed, paying particular attention to the physical process of the spread of X inactivation in monotremes (platypus and echidna) . When experimental data is unavailable some theoretical analysis is possible based on the idea that in certain cases collective phenomena in genetics, rather than chemical detail, are better correlates of complex chemical processes.

Eur J Biochem, 1998 Feb 15, 252(1), 79 - 89
A gene encoding a vicilin-like protein is specifically expressed in fern spores . Evolutionary pathway of seed storage globulins; Shutov AD et al.; The isolation and characterisation of a cDNA coding for a vicilin-like protein of the fern Matteuccia struthiopteris is described . The corresponding gene is specifically expressed during late stages of spore development . Extensive sequence comparisons suggest that the fern protein can be considered as a molecular missing link between single-domain germin/spherulin-like proteins and two-domain seed storage globulins of gymnosperms and angiosperms . Further, evidence is provided for the existence of a superfamily of structurally related, functionally different proteins which includes storage globulins of the vicilin and legumin families, a membrane-associated sucrose-binding protein of soybean, a Forssman antigen-binding lectin of velvet bean, the precursor of the vacuolar membrane bound proteins MP27/MP32 of pumpkin, the embryogenesis-specific protein Gea8 of carrot, the fern-spore-specific protein described here as well as the functionally diverse family of germins/germin-like proteins and the spherulins of myxomycetes . We propose that seed storage globulins of spermatophytes evolved from desiccation-related single-domain proteins of prokaryotes via a duplicated two-domain ancestor that is best represented by the extant fern spore-specific vicilin-like protein.

Biochimie, 1997 Dec, 79(12), 731 - 40
Cysteinyl-tRNA synthetase from Saccharomyces cerevisiae . Purification, characterization and assignment to the genomic sequence YNL247w; Motorin Y et al.; Cysteinyl-tRNA synthetase (CRS) from Saccharomyces cerevisiae was purified 2300-fold with a yield of 33%, to a high specific activity (kcat4.3 s-1 at 25 degrees C for the aminoacylation of yeast tRNACys) . SDS-PAGE revealed a single polypeptide corresponding to a molecular mass of 86 kDa . Polyclonal antibodies to the purified protein inactivated CRS activity and detected only one polypeptide of 86 kDa in a yeast extract subjected to SDS-PAGE followed by immunoblotting . In contrast to bacterial CRS which is a monomer of about 50 kDa, the native yeast enzyme behaved as a dimer, as assessed by gel filtration and cross-linking . Its subunit molecular mass is in good agreement with the value of 87.5 kDa calculated for the protein encoded by the yeast genomic sequence YNL247w . The latter was previously tentatively assigned to CRS, based on limited sequence similarities to the corresponding enzyme from other sources . Determination of the amino acid sequence of internal polypeptides derived from the purified yeast enzyme confirmed this assignment . Alignment of the primary sequences of prokaryotic and yeast CRS reveals that the larger size of the latter is accounted for mostly by several insertions within the sequence.

DNA Seq, 1997, 8(1-2), 17 - 29
Gene identification and classification in the Synechocystis genomic sequence by recursive gene mark analysis; Hirosawa M et al.; The GeneMark method has proven to be an efficient gene-finding tool for the analysis of prokaryotic genomic sequence data . We have developed a procedure of deriving and utilizing several GeneMark models in order to get better gene-detection performance . Upon applying this procedure to the 1.0 Mb contiguous DNA sequence of Synechocystis sp . strain PCC6803, we were able to cluster predicted genes into distinct classes and to produce the class-specific GeneMark models reflecting statistical characteristics of each gene class . One gene class apparently includes genes of exogenous origin . Using class-specific models reduces the gene under prediction error rate down to 1.7% in comparison with 8.1% reported in the previous study when only one GeneMark model was used.

Biochemistry, 1998 Mar 10, 37(10), 3509 - 17
Aspartate-279 in aminolevulinate synthase affects enzyme catalysis through enhancing the function of the pyridoxal 5'-phosphate cofactor; Gong J et al.; 5-Aminolevulinate synthase (ALAS) catalyzes the first step in the heme biosynthetic pathway in nonplant eukaryotes and some prokaryotes, which is the condensation of glycine with succinyl-coenzyme A to yield coenzyme A, carbon dioxide, and 5-aminolevulinate . ALAS requires pyridoxal 5'-phosphate as an essential cofactor and functions as a homodimer . D279 in murine erythroid enzyme was found to be conserved in all aminolevulinate synthases and appeared to be homologous to D222 in aspartate aminotransferase, where the side chain of the residue stabilizes the protonated form of the cofactor ring nitrogen, thus enhancing the electron sink function of the cofactor during enzyme catalysis . D279A mutation in ALAS resulted in no detectable enzymatic activity under standard assay conditions, and the conservative D279E mutation reduced the catalytic efficiency for succinyl-CoA 30-fold . The D279A mutation resulted in a 19-fold increase in the dissociation constant for binding of the pyridoxal 5'-phosphate cofactor . UV-visible and CD spectroscopic analyses indicated that the D279A mutant binds the cofactor in a different mode at the active site . In contrast to the wild-type and D279E mutant, the D279A mutant failed to catalyze the formation of a quinonoid intermediate upon binding of 5-aminolevulinate . Importantly, this partial reaction could be rescued in D279A by reconstitution of the mutant with the cofactor analogue N-methyl-PLP . The steady-state kinetic isotope effect when deuteroglycine was substituted for glycine was small for the wild-type enzyme (kH/kD = 1.2 +/- 0.1), but a strong isotope effect was observed with the D279E mutant (kH/kD = 7.7 +/- 0.3) . pH titration of the external aldimine formed with ALA indicated the D279E mutation increased the apparent pKa for quinonoid formation from 8.10 to 8.25 . The results are consistent with the proposal that D279 plays a crucial role in aminolevulinate synthase catalysis by enhancing the electron sink function of the cofactor.

Hum Genet, 1998 Feb, 102(2), 197 - 202
A germline mutation abolishing the original stop codon of the human adenine phosphoribosyltransferase (APRT) gene leads to complete loss of the enzyme protein; Taniguchi A et al.; Adenine phosphoribosyltransferase (APRT) is a purine metabolic enzyme and a homozygous deficiency in this enzyme causes 2,8-dihydroxyadenine urolithiasis . Various germline abnormalities have been described, but we report here a unique type of germline mutation in a homozygous individual (SY) who had excreted 2,8-dihydroxyadenine crystals . In SY, TCA was substituted for the physiological stop codon TGA . This base substitution generates a new HinfI restriction site, and, using the polymerase chain reaction and subsequent digestion by this enzyme, it was confirmed that SY is homozygous for the base substitution . This base change is unique in that it generates an open reading frame that extends to the poly(A) addition site . The amount of mRNA in transformed B cells from SY was approximately a quarter of that in control subjects and no APRT proteins were detected . In eukaryotes, unlike in prokaryotes, no rescue systems for defective polypeptide termination caused by a missing stop codon have been found . Therefore, the outcome of the defect of SY is unclear from present knowledge about termination of polypeptide synthesis . Investigations into the mechanisms of the absence of protein in the cells of SY may lead to a better understanding of the physiological and nonphysiological termination of polypeptide synthesis in eukaryotic cells.

Protein Sci, 1998 Feb, 7(2), 512 - 5
A LexA mutant repressor with a relaxed inter-domain linker; Oertel-Buchheit P et al.; The LexA protein is part of a large family of prokaryotic transcriptional repressors that contain an amino-terminal DNA binding domain and a carboxy-terminal dimerization domain . These domains are separated by a linker or hinge region, which is generally considered to be rather flexible and unconstrained . So far, no structure of any of the full-length repressors is available . Here we show that a mutant LexA repressor harboring several point mutations in the hinge region gets sensitive to trypsin and Glu-C cleavage over a segment of at least 20 amino acids, whereas the LexA wild-type hinge region is resistant to these proteases . These data are not compatible with the hypothesis of an fully flexible and/or unstructured inter-domain linker and suggest that the LexA hinge region is, in fact, constrained by contacts with the carboxy-terminal domain and/or a fairly stable local structure of the linker region.

Semin Liver Dis, 1998, 18(1), 77 - 84
Molecular genetics of congenital erythropoietic porphyria; Desnick RJ et al.; Congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis, results from the markedly deficient activity of the cytosolic enzyme, uroporphyrinogen III synthase (URO-synthase) . The accumulation of the nonphysiological and pathogenic porphyrin isomers, uroporphyrin I and coproporphyrin I, leads to the clinical manifestations of CEP . Disease severity in unrelated patients is markedly heterogeneous, ranging from fetal demise or severe transfusion dependency throughout life to milder adult cases with only cutaneous photosensitivity . To date, 18 mutations causing CEP have been described in the URO-synthase gene, including single base substitutions, insertions and deletions, and splicing defects . Most mutations have been identified in one or a few unrelated families with the exception of C73R, L4F, and T228M which occurred in about 33%, 8%, and 7% of the mutant alleles studied, respectively . Prokaryotic expression of the mutant URO-synthase alleles identified those with significant residual activity, thereby permitting genotype/phenotype predictions for severe to milder phenotypes of this clinically heterogeneous disease . As successful bone marrow transplantation in severely affected patients has proven curative, current efforts are underway to develop hematopoietic stem cell gene therapy for CEP.

J Biol Chem, 1998 Feb 27, 273(9), 4835 - 7
Arylsulfatase from Klebsiella pneumoniae carries a formylglycine generated from a serine; Miech C et al.; Eukaryotic sulfatases share an unusual posttranslational protein modification, which converts a cysteine into alpha-formylglycine . The alpha-formylglycine is essential for the catalytic activity . Klebsiella pneumoniae expresses an inducible arylsulfatase for which the DNA predicts a serine at the position occupied by the alpha-formylglycine residue in eukaryotic sulfatases . Structural analysis showed that the majority of the arylsulfatase polypeptides from K . pneumoniae carries the alpha-formylglycine, whereas the remaining arylsulfatase polypeptides contain the predicted serine residue . This demonstrates the evolutionary conservation between prokaryotes and eukaryotes of this novel protein modification that so far has been found only in sulfatases . alpha-Formylglycine in Klebsiella is generated from a serine and not from a cysteine as in eukaryotes.

J Biol Chem, 1998 Feb 13, 273(7), 3980 - 5
The nuclear RPL4 gene encodes a chloroplast protein that co-purifies with the T7-like transcription complex as well as plastid ribosomes; Trifa Y et al.; We have cloned and sequenced the cDNA and the gene coding for plastid ribosomal protein L4 (RPL4) from two higher plant species, spinach and Arabidopsis thaliana . Ribosomal protein L4 is one of the ribosomal proteins for which extraribosomal functions in transcriptional regulation has been demonstrated in prokaryotes . Sequence comparison of the two plant cDNAs and genes shows that the RPL4 gene has acquired a remarkable 3' extension during evolutionary transfer to the nuclear genome . This extension harbors an intron and codes for a glutamic and aspartic acid-rich amino acid sequence that resembles highly acidic C-terminal tails of some transcription factors . Co-purification of ribosomal protein L4 with plastid RNA polymerase and transcription factor CDF2 using different purification protocols as well as the surprising amino acid sequence of the L4 protein make it a likely candidate to play a role in plastid transcriptional regulation.

J Biol Chem, 1998 Feb 13, 273(7), 3871 - 7
Regulation of the activity of chloroplast translational initiation factor 3 by NH2- and COOH-terminal extensions; Yu NJ et al.; The mature form of the chloroplast translational initiation factor 3 (IF3chl) from Euglena gracilis consists of an internal region homologous to prokaryotic IF3 flanked by long NH2- and COOH-terminal extensions . Sequences in these extensions reduce the activity of the homology domain in promoting initiation complex formation with chloroplast mRNAs and 30 S ribosomal subunits . A series of deletions of the NH2- and COOH-terminal extensions of IF3chl were constructed and tested for their effects on the activity of the homology domain . About half of the inhibitory effect arises from sequences within 9 residues of the junction between the NH2-terminal extension and the homology domain . The remaining inhibitory effect is the result of sequences in the COOH-terminal extension . The equilibrium constant governing the binding of the homology domain of IF3chl to 30 S subunits is estimated to be 1.3 x 10(7) M-1 . Sequences close to the junction of the NH2-terminal extension and the homology domain reduce this binding constant about 10-fold . Sequences in the COOH-terminal extension have a similar negative effect . The negative effects of these two regions are cumulative, resulting in a 100-fold reduction of the binding constant . The 9 residues at the NH2-terminal extension effectively prevent the proofreading activity of IF3chl . The entire COOH-terminal extension reduces the proofreading ability by about half . These results are discussed in terms of the proposed three-dimensional structure of the homology domain of IF3chl.

Curr Biol, 1998 Jan 1, 8(1), R28 - 31
Cell cycle: the bacterial approach to coordination; Levin PA et al.; Despite the power of bacterial genetics, the prokaryotic cell cycle has remained poorly understood . But recent work with three different bacterial species has shed light on how chromosomes and plasmids are oriented and partitioned during the cell cycle, and on mechanisms regulating the initiation of DNA replication.

Mol Microbiol, 1998 Feb, 27(4), 853 - 69
Xanthine dehydrogenase from the phototrophic purple bacterium Rhodobacter capsulatus is more similar to its eukaryotic counterparts than to prokaryotic molybdenum enzymes; Leimkuhler S et al.; Fourteen Rhodobacter capsulatus mutants unable to grow with xanthine as sole nitrogen source were isolated by random Tn5 mutagenesis . Five of these Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments hybridizing to oligonucleotides synthesized according to conserved amino acid sequences of eukaryotic xanthine dehydrogenases . DNA sequence analysis of this region revealed two open reading frames, designated xdhA and xdhB, encoding xanthine dehydrogenase . The deduced amino acid sequence of XDHA contains binding sites for two {2Fe-2S} clusters and FAD, whereas XDHB is predicted to contain the molybdopterin cofactor . In contrast to R . capsulatus, these three cofactor binding sites reside within a single polypeptide chain in eukaryotic xanthine dehydrogenases . The amino acid sequence of xanthine dehydrogenase from R . capsulatus showed a higher degree of similarity to eukaryotic xanthine dehydrogenases than to the xanthine dehydrogenase-related aldehyde oxidoreductase from Desulphovibrio gigas . The expression of an xdhA-lacZ fusion was induced when hypoxanthine or xanthine was added as sole nitrogen source . Mutations in nifR1 (ntrC) and nifR4 (rpoN, encoding sigma54) had no influence on xdh gene expression . A putative activator sensing the availability of substrate seems to respond to xanthine but not to hypoxanthine . The transcriptional start site of xdhA was mapped by primer extension analysis . Comparison with known promoter elements revealed no significant homology . Xanthine dehydrogenase from R . capsulatus was purified to homogeneity . The enzyme consists of two subunits with molecular masses of 85 kDa and 50 kDa respectively . N-terminal amino acid sequencing of both subunits confirmed the predicted start codons . The molecular mass of the native enzyme was determined to be 275 kDa, indicating an alpha2beta2-subunit structure . Analysis of the molybdenum cofactor of xanthine dehydrogenase from R . capsulatus revealed that it contains the molybdopterin cofactor and not a molybdopterin dinucleotide derivative.

Biochem Pharmacol, 1998 Feb 15, 55(4), 537 - 41
Studies on inhibitors of mammalian DNA polymerase alpha and beta: sulfolipids from a pteridophyte, Athyrium niponicum; Mizushina Y et al.; Three sulfolipid compounds, 1, 2, and 3, have been isolated from a higher plant, a pteridophyte, Athyrium niponicum, as potent inhibitors of the activities of calf DNA polymerase alpha and rat DNA polymerase beta . The inhibition by the sulfolipids was concentration dependent, and almost complete inhibition of DNA polymerase alpha and DNA polymerase beta was achieved at 6 and 8 microg/mL, respectively . The compounds did not influence the activities of calf thymus terminal deoxynucleotidyl transferase, prokaryotic DNA polymerases such as the Klenow fragment of DNA polymerase I, T4 DNA polymerase and Taq polymerase, the DNA metabolic enzyme DNase I, and even a DNA polymerase from a higher plant, cauliflower . Similarly, the compounds did not inhibit the activity of the human immunodeficiency virus type 1 reverse transcriptase . The kinetic studies of the compounds showed that DNA polymerase alpha was inhibited non-competitively with respect to the DNA template and substrate, whereas DNA polymerase beta was inhibited competitively with both the DNA template and substrate . The binding to DNA polymerase beta could be stopped with non-ionic detergent, but the binding to DNA polymerase alpha could not.

FEMS Microbiol Lett, 1997 Nov 15, 156(2), 287 - 91
The cell wall-less rickettsia Eperythrozoon wenyonii is a Mycoplasma; Neimark H et al.; The 16S rRNA gene of Eperythrozoon (Haemobartonella) wenyonii, a wall-less hemotrophic prokaryote currently classified as a rickettsia, was sequenced to determine the relationship of this organism to other wall-less prokaryotes . Comparison to the GenBank data base showed that this hemotrophic organism is a Mycoplasma (family Mollicutes) . Phylogenetic analysis of 16S rRNA genes indicated that this and other recently sequenced 16S rRNA genes of hemotrophic bacteria form a new, separate branch which shares a node in common with the pneumoniae group of mycoplasmas . This result will require that Eperythrozoon wenyonii be reclassified as a Mycoplasma . A main point of this study is that this and related hemotrophic bacteria represent an entirely new group of pathogens among the mycoplasmas.

J Mol Biol, 1998 Feb 20, 276(2), 379 - 89
Distinct functions of N and C-terminal domains of GreA, an Escherichia coli transcript cleavage factor; Koulich D et al.; The prokaryotic transcription factors GreA and GreB are involved in the regulation of transcript elongation by RNA polymerase (RNAP) . Their known activities include suppression of transcription arrest, enhancement of transcription fidelity, and facilitation of the transition from abortive initiation to productive elongation . Presumably, Gre proteins exert their functions by altering the conformation of the enzyme in ternary elongation complexes (TEC) and inducing the cleavage of nascent RNA . GreA and GreB have a similar structural organization and consist of two domains: a C-terminal globular and an extended N-terminal coiled-coil domain . To investigate the functional roles of Gre domains, we expressed separately the N and C-terminal domains of GreA (NTD and CTD, respectively) and characterized their activities with in vitro assays . We demonstrate that the NTD possesses the residual transcript cleavage activity of the wild-type GreA . The CTD does not display any nucleolytic activity; however, it substantially increases the cleavage activity of the NTD . In contrast to NTD, the CTD competes with GreA and GreB for binding to RNAP and inhibits their transcript cleavage and antiarrest activities . Both domains individually and together inhibit transcription elongation . From these results we conclude that the NTD is responsible for the GreA induction of nucleolytic activity while the CTD determines the binding of GreA to RNAP . Both domains are required for full functional activity of GreA.

Curr Biol, 1998 Mar 12, 8(6), R215 - 7
Chloroplast biogenesis: mixing the prokaryotic and the eukaryotic?
Heins L, Soll J.
Chloroplast biogenesis requires the translocation of proteins across the outer and inner envelopes . The membrane components of this transport machinery completely differ from those of other organelles, but recently homologues of some of the components have been detected in prokaryotes.

Parasitology, 1998 Feb, 116 ( Pt 2), 157 - 64
Trypanosoma brucei mitochondrial ribonucleoprotein complexes which contain 12S and 9S ribosomal RNAs; Shu HH et al.; Antibiotics have been widely used to identify ribosomal activity in Trypanosoma brucei mitochondria . The validity of some of the results has been questioned because the permeability of the trypanosome cell membrane for some antibiotics was not adequately addressed . Here we describe translation inhibition experiments with digitonin-permeabilized trypanosomes to exclude diffusion barriers through the cell membrane . Using this system we were able to confirm, next to the eukaryotic and thus cycloheximide-sensitive translation system, the existence of a prokaryotic-type translational activity being cycloheximide resistant, chloramphenicol sensitive and streptomycin dependent . We interpret this observation analogous to what has been found for other eukarya as the independent protein synthesis activity of the mitochondrial organelle . We further examined the putative translational apparatus by using isokinetic density-gradient analysis of mitochondrial extracts . The 2 mitochondrially encoded rRNAs, the 9S and 12S rRNAs, were found to co-fractionate in a single RNP complex, approximately 80S in size . This complex disassembled at reduced MgCl2 concentrations into 2 unusually small complexes of 17.5S, containing the 9S rRNA, and 20S containing the 12S rRNA . A preliminary stoichiometry determination suggested a multicopy assembly of these putative subunits in a 2:3 ratio (20S:17.5S).

Bioessays, 1998 Jan, 20(1), 49 - 57
Cold shock and adaptation; Thieringer HA et al.; Adaptation to environmental stresses, such as temperature fluctuation, is essential for the survival of all living organisms . Cellular responses in both prokaryotes and eukaryotes to high temperature include the synthesis of a set of highly conserved proteins known as the heat shock proteins . In contrast to the heat shock response, adaptation to low temperatures has not been as extensively studied . However, a family of cold-inducible proteins is evident in prokaryotes . In addition, most organisms have developed adaptive mechanisms that alter both membrane fluidity and the protein translation machinery at low temperature . This review addresses the different adaptive mechanisms used by a variety of organisms with a focus on the molecular mechanisms of cold adaptation that have recently been identified during the cold shock response in Escherichia coli.

J Biochem (Tokyo), 1998 Jan, 123(1), 157 - 61
Expression of a synthetic gene for initiation factor 4E-binding protein 1 in Escherichia coli and its interaction with eIF-4E and eIF-4E x m7GTP complex; Nishi N et al.; An artificial gene coding for the human initiation factor (eIF) 4E-binding protein 1 (4E-BP1) was chemically synthesized and cloned . Although the expression of the 4E-BP1 gene alone has not yet been accomplished, the gene was expressed in Escherichia coli {BL21(DE3)} as a fusion gene with the glutathione-S-transferase (GST) gene using a prokaryotic gene fusion vector (pGEX-4T-2), which contains a gene sequence coding the cleavage site for a specific protease, alpha-thrombin . The fusion gene product was purified to homogeneity by glutathione Sepharose-4B affinity column chromatography . It was shown by m7GTP- and glutathione-affinity chromatography that the binding ability of 4E-BP1 to eIF-4E is nearly the same as that to the eIF-4E x m7GTP complex, implying different binding sites of eIF-4E and its nonallosteric obligation for 4E-BP1 and mRNA cap structure . In contrast with the binding of eIF-4E to the mRNA cap structure, where some functional amino acids play an important role in the binding, the binding to 4E-BP1 was suggested to occur via multiple nonspecific interactions.

J Biochem (Tokyo), 1998 Jan, 123(1), 16 - 23
Metalloid resistance mechanisms in prokaryotes; Xu C et al.; Resistance to antibiotics and other chemotherapeutic agents is becoming a wide spread health issue . The biochemical mechanisms of resistance vary, but active efflux of the toxic agents is one of the most common . Bacterial resistances to metals provide good model systems for transport-related resistances . One of the best understood metal resistance systems is the product of the ars operon, which provides resistance to arsenicals and antimonials . As a reflection of the ubiquity of arsenic in the environment, ars operons are found in all species of bacteria, carried in chromosomes, plasmids, and transposons . This review focuses on the biochemistry of the proteins of the ars operon of R-factor R773 . The system is novel in several respects . First, it is regulated at the transcriptional and allosteric levels, and regulation is effected through cysteine thiol interaction with As(III) or Sb(III) . Thus soft metal-thiol chemistry provides a high affinity digital switch to turn the regulated protein on with rapidity . The transport system that provides resistance, on the other hand, uses oxyanions of arsenic or antimony as substrates . This nonmetal chemistry allows for low affinity interactions of the membrane transporter with substrate, conductive with translocation and release of substrate on the outside of the cell membrane . Second, the transporter is uniquely capable of coupling to either electrochemical energy as a secondary carrier protein or the chemical energy of ATP when binding of a catalytic subunit converts it into an anion-translocating ATPase.

Int J Parasitol, 1998 Jan, 28(1), 11 - 20
Evolution of the protists and protistan parasites from the perspective of molecular systematics; Sogin ML et al.; Unlike prokaryotes, the Protista are rich in morphological and ultrastructure information . Their amazing phenotypic diversity permits assignment of many protists to cohesive phyletic assemblages but sometimes blurs relationships between major lineages . With the advent of molecular techniques, it became possible to test evolutionary hypotheses that were originally formulated according to shared phenotypic traits . More than any other gene family, studies of rRNAs changed our understanding of protist evolution . Stramenopiles (oomycetes, chrysophytes, phaeophytes, synurophytes, diatoms, xanthophytes, bicosoecids, slime nets) and alveolates (dinoflagellates, apicomplexans, ciliates) are two novel, complex evolutionary assemblages which diverged nearly simultaneously with animals, fungi, plants, rhodophytes, haptophytes and a myriad of independent amoeboid lineages . Their separation may have occurred one billion years ago and collectively these lineages make up the "crown" of the eukaryotic tree . Deeper branches in the eukaryotic tree show 16S-like rRNA sequence variation that is much greater than that observed within the Archaea and the Bacteria . A progression of independent protist branches, some as ancient as the divergence between the two prokaryotic domains, preceded the sudden radiation of "crown" groups . Trichomonads, diplomonads and Microsporidia are basal to all other eukaryotes included in rRNA studies . Together with pelobionts, oxymonads, retortamonads and hypermastigids, these amitochondriate taxa comprise the Archaezoa . This skeletal phylogeny suggested that early branching eukaryotes lacked mitochondria, peroxisomes and typical stacked Golgi dictyosomes . However, recent studies of heat shock proteins indicate that the first eukaryotes may have had mitochondria . When evaluated in terms of evolution of ultrastructure, lifestyles and other phenotypic traits, the rRNA phylogenies provide the most consistent of molecular trees . They permit identification of the phylogenetic affinity of many parasitic groups as well as a means to integrate molecular and cell biological information from diverse eukaryotes . We must place greater emphasis upon improved phylogenetic inference techniques and investigations of genomic diversity in protists.

Curr Opin Biotechnol, 1998 Feb, 9(1), 90 - 6
Macromolecular matchmaking: advances in two-hybrid and related technologies; Frederickson RM; The success of the original yeast two-hybrid system has stimulated the development of a number of 'hybrid technologies' in yeast (and now prokaryotes and mammals) to widen the scope of the protein-protein interactions that can be analyzed, and to enable comparable studies of the interactions of proteins with DNA, RNA or small chemical ligands . In addition, the application of the two-hybrid system to entire genomes is being used to create protein linkage maps which catalog the network of interactions of an organism's complete proteome.

Biochem Mol Biol Int, 1998 Jan, 44(1), 41 - 9
Prokaryotic iron superoxide dismutase replaces cytosolic copper, zinc superoxide dismutase in protecting yeast cells against oxidative stress; Agius DR et al.; The iron superoxide dismutase (FeSOD) gene of Escherichia coli was cloned in Saccharomyces cerevisiae cells deficient in copper,zinc superoxide dismutase (Cu,ZnSOD) . FeSOD replaced Cu,ZnSOD in protecting the yeast cells against oxidative stress . In the recombinant strains the FeSOD gene, which was under the transcriptional control of the yeast phosphoglycerate kinase gene promoter, was functionally expressed at two different levels on episomal and centromeric plasmids . Despite suppression of methionine and lysine auxotrophy, the higher level of FeSOD activity was more beneficial to growth of the mutant yeast cells only when these were exposed to higher levels of oxidative stress induced by paraquat or 100% oxygen . In the presence of paraquat, there was a novel stimulation of FeSOD activity . This was associated with a marked increase in catalase activity, and a decrease in glutathione reductase activity.

J Biol Chem, 1998 Feb 20, 273(8), 4470 - 7
Cloning and expression of a prokaryotic enzyme, arginine deiminase, from a primitive eukaryote Giardia intestinalis; Knodler LA et al.; Arginine deiminase (EC 3.5.3.6) catalyzes the irreversible catabolism of arginine to citrulline in the arginine dihydrolase pathway . This pathway has been regarded as restricted to prokaryotic organisms but is an important source of energy to the primitive protozoan Giardia intestinalis . In this paper we report the cloning and expression of the arginine deiminase gene from this parasite . Degenerate oligonucleotides based on amino acid sequences of tryptic peptides from the purified protein were used to amplify a portion of the arginine deiminase gene . This was then used as a probe to screen HindIII and PstI "mini" libraries to obtain two overlapping clones that contained the arginine deiminase gene . The open reading frame encoded 581 amino acids including all of the tryptic peptides that were sequenced and corresponded to a molecular mass of 67 kDa . Northern blot analysis identified a single 1.8-kilobase transcript in both trophozoites and encysting cells . Arginine deiminase was successfully expressed in Escherichia coli and purified to homogeneity . The recombinant protein was found to have characteristics comparable with those of the native enzyme.

Proc Natl Acad Sci U S A, 1998 Feb 17, 95(4), 1478 - 83
Intermolecular cleavage by UmuD-like mutagenesis proteins; McDonald JP et al.; The activity of a number of proteins is regulated by self-processing reactions . Elegant examples are the cleavage of the prokaryotic LexA and lambdaCI transcriptional repressors and the UmuD-like mutagenesis proteins . Various studies support the hypothesis that LexA and lambdaCI cleavage reactions are predominantly intramolecular in nature . The recently described crystal structure of the Escherichia coli UmuD' protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 A away from the catalytic active site . We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and lambdaCI, occurs via an intermolecular rather than intramolecular reaction . To test this hypothesis, we introduced into E . coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins . Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature . Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E . coli strains . Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD' protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD') can also act as a classical enzyme.

Curr Biol, 1998 Jan 15, 8(2), R59 - 61
Mismatch repair: origin of species?
Sniegowski P.
Mismatch repair reverses replication errors and inhibits recombination between diverged sequences . This has been suggested to be important in speciation, especially in prokaryotes, but theoretical analysis indicates that genetic divergence in bacterial populations is not constrained by naturally occurring recombination levels.

Arch Biochem Biophys, 1998 Mar 1, 351(1), 82 - 8
The low expression level of pokeweed antiviral protein (PAP) gene in Escherichia coli by the inducible lac promoter is due to inefficient transcription and translation and not to the toxicity of the PAP; Xu J et al.; Pokeweed (Phytolacca americana) antiviral protein (PAP) is a glycosidase which inactivates both eukaryotic and prokaryotic ribosomes . Due to this activity the wild-type PAP gene encoding mature protein has not so far been expressed in Escherichia coli . In spite of the ribosome impairing activity of the pre-PAP (containing two signal peptides at both termini) on bacterial ribosomes in vitro, the full-length PAP gene has been expressed successfully, although at a low level in E . coli under an inducible lac promoter . In this study we show that the full-length PAP gene, but not the PAP gene devoid of the N-terminal signal peptide codons, can be expressed constitutively in E . coli cells to produce a much higher yield as compared with the inducible expression . The full-length PAP is biologically active and it accumulates as inclusion bodies in bacterial cytoplasm . RNA analysis together with protein measurements show that the PAP gene is poorly transcribed and the PAP mRNA is poorly translated when a lac operator sequence is placed in front of the Shine/Dalgarno (SD) sequence . Nucleotide folding analysis of the 5' untranslated mRNA revealed that the SD sequence in the presence of a lac operator is involved in a stable secondary structure, whereas it is more relaxed in the mRNA transcribed from the constitutive vector . These results provide evidence that the low expression level of full-length PAP gene is due to inefficient transcription and translation but not to the toxicity of the expressed PAP.

J Biochem (Tokyo), 1997 Dec, 122(6), 1122 - 8
Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: II . Evolutionary analysis of protein and gene structures; Yoshida Y et al.; Phylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily . The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution . This may be strong evidence supporting the prokaryotic origin of P450 . Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s . This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures . CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family . One CYP51 processed a pseudogene was found in rat genome . Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence . This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.

Microbiology, 1998 Feb, 144 ( Pt 2), 529 - 41
A dissimilatory sirohaem-sulfite-reductase-type protein from the hyperthermophilic archaeon Pyrobaculum islandicum; Molitor M et al.; A sulfite-reductase-type protein was purified from the hyperthermophilic crenarchaeote Pyrobaculum islandicum grown chemoorganoheterotrophically with thiosulfate as terminal electron acceptor . In common with dissimilatory sulfite reductases the protein has an alpha 2 beta 2 structure and contains high-spin sirohaem, non-haem iron and acid-labile sulfide . The oxidized protein exhibits absorption maxima at 280, 392, 578 and 710 nm with shoulders at 430 and 610 nm . The isoelectric point of pH 8.4 sets the protein apart from all dissimilatory sulfite reductases characterized thus far . The genes for the alpha- and beta-subunits (dsrA and dsrB) are contiguous in the order dsrAdsrB and most probably comprise an operon with the directly following dsrG and dsrC genes . dsrG and dsrC encode products which are homologous to eukaryotic glutathione S-transferases and the proposed gamma-subunit of Desulfovibrio vulgaris sulfite reductase, respectively . dsrA and dsrB encode 44.2 kDa and 41.2 kDa peptides which show significant similarity to the two homologous subunits DsrA and DsrB of dissimilatory sulfite reductases . Phylogenetic analyses indicate a common protogenotic origin of the P . islandicum protein and the dissimilatory sulfite reductases from sulfate-reducing and sulfide-oxidizing prokaryotes . However, the protein from P . islandicum and the sulfite reductases from sulfate-reducers and from sulfur-oxidizers most probably evolved into three independent lineages prior to divergence of archaea and bacteria.

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