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 sequ