J Microbiol Methods, 2003 Oct, 55(1), 73 - 81 Representational difference analysis: critical appraisal and method development for the identification of unique DNA sequences from prokaryotes; Allen NL et al.; Representational difference analysis (RDA) has great potential for preferential amplification of unique but uncharacterised DNA sequences present in one source such as a whole genome, but absent from a related genome or other complex population of sequences . While a few examples of its successful exploitation have been published, the method has not been well dissected and robust, detailed published protocols are lacking . Here we examine the method in detail, suggest improvements and provide a protocol that has yielded key unique sequences from a pathogenic bacterial genome. Res Microbiol, 2003 Sep, 154(7), 466 - 73 The microbiology of acidic mine waters; Johnson DB et al.; Acidic, metal-rich waters generated by the microbially accelerated dissolution of pyrite and other sulfide minerals, are frequently encountered in derelict mine sites, including many that have been long-abandoned . While these waters are major causes of environmental pollution and are toxic to the majority of prokaryotic and eukaryotic organisms, some life forms (mostly bacteria and archaea) thrive within them . "Acidophiles" comprise a surprisingly wide diversity (in terms of both physiology and phylogeny) of microorganisms . This article reviews current knowledge of the distribution and biodiversity of this group of extremophiles. Biophys Chem, 2003 Sep, 105(2-3), 361 - 70 Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA; Goulding CW et al.; The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets . With this background, the TB Structural Genomics Consortium has been formed . This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries . The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information . We summarize the efforts of the UCLA consortium members . Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination . Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes . Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms . In addition, some protein targets were chosen that are specific to M . tuberculosis, such as PE and PPE domains . Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials . Our efforts have yielded 13 crystals and eight structures . Seven structures are summarized here . Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M . tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase . We also report the structures of three proteins that are potentially essential to the survival of M . tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c . Our approach to the M . tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis . In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis. Biochim Biophys Acta, 2003 Sep 23, 1651(1-2), 163 - 71 Nuclear magnetic resonance structures of the zinc finger domain of human DNA polymerase-alpha; Evanics F et al.; The carboxy terminus of the human DNA polymerase-alpha contains a zinc finger motif . Three-dimensional structures of this motif containing 38 amino acid residues, W L I C E E P T C R N R T R H L P L Q F S R T G P L C P A C M K A T L Q P E, were determined by nuclear magnetic resonance (NMR) spectroscopy . The structures reveal an alpha-helix-like domain at the amino terminus, extending 13 residues from L2 through H15 with an interruption at the sixth residue . The helix region is followed by three turns (H15-L18, T23-L26 and L26-A29), all of which involve proline . The first turn appears to be type III, judging by the dihedral angles . The second and third turns appear to be atypical . A second, shorter helix is formed at the carboxy terminus extending from C30 through L35 . A fourth type III turn starting at L35 was also observed in the structure . Proline serves as the third residue of all the turns . Four cysteine residues, two located at the beginning of the helix at the N-terminus and two at the carboxy end, are coordinated to Zn(II), facilitating the formation of a loop . One of the cysteines at the carboxy terminus is part of the atypical turn, while the other is the part of the short helix . These structural features are consistent with the circular dichroism (CD) measurements which indicate the presence of 45% helix, 11% beta turns and 19% non-ordered secondary structures . The zinc finger motif described here is different from those observed for C(4), C(2)H(2), and C(2)HC modules reported in the literature . In particular, polymerase-alpha structures exhibit helix-turn-helix motif while most zinc finger proteins show anti-parallel sheet and helix . Several residues capable of binding DNA, T, R, N, and H are located in the helical region . These structural features imply that the zinc finger motif is most likely involved in binding DNA prior to replication, presumably through the helical region . These results are discussed in the context of other eukaryotic and prokaryotic DNA polymerases belonging to the polymerase B family. Biotechnol Adv, 2003 Sep, 21(6), 465 - 99 Ribosomally synthesized peptides with antimicrobial properties: biosynthesis, structure, function, and applications; Papagianni M; Ribosomally synthesized peptides with antimicrobial properties (antimicrobial peptides-AMPs) are produced by eukaryotes and prokaryotes and represent crucial components of their defense systems against microorganisms . Although they differ in structure, they are nearly all cationic and very often amphiphilic, which reflects the fact that many of them attack their target cells by permeabilizing the cell membrane . They can be roughly categorized into those that have a high content of a certain amino acid, most often proline, those that contain intramolecular disulfide bridges, and those with an amphiphilic region in their molecule if they assume an alpha-helical structure . Most of the known ribosomally synthesized peptides with antimicrobial functions have been identified and studied during the last 20 years . As a result of these studies, new knowledge has been acquired into biology and biochemistry . It has become evident that these peptides may be developed into useful antimicrobial additives and drugs . The use of two-peptide antimicrobial peptides as replacement for clinical antibiotics is promising, though their applications in preservation of foods (safe and effective for use in meat, vegetables, and dairy products), in veterinary medicine, and in dentistry are more immediate . This review focuses on the current status of some of the main types of ribosomally synthesized AMPs produced by eucaryotes and procaryotes and discusses the novel antimicrobial functions, new developments, e.g . heterologous production of bacteriocins by lactic acid bacteria, or construction of multibacteriocinogenic strains, novel applications related to these peptides, and future research paradigms. J Gen Virol, 2003 Oct, 84(Pt 10), 2647 - 59 In vivo transcription of the Epstein-Barr virus (EBV) BamHI-A region without associated in vivo BARF0 protein expression in multiple EBV-associated disorders; van Beek J et al.; The in vivo expression of the Epstein-Barr virus (EBV) BamHI-A rightward transcripts (BARTs) as well as the putative BART-encoded BARF0 and RK-BARF0 proteins in various EBV-associated malignancies was investigated . RT-PCRs specific for the different splice variants of the BARTs and both a nucleic acid sequence-based amplification assay and an RT-PCR specific for the BARF0 ORF were used . Abundant transcription of BARTs was found in EBV-associated Hodgkin's lymphomas, Burkitt's lymphomas (BL), T-cell non-Hodgkin's lymphomas, post-transplant lymphoproliferative disorders, AIDS-related lymphomas and gastric carcinomas . Using RNA in situ hybridization (RISH), BARTs were detected within the neoplastic cells of these malignancies . BARTs encoding RK-BARF0 were not detected . The BARTs detected were shown possibly to encode the RPMS1 and BARF0 proteins, based on their splicing . However, BARTs actually harbouring the BARF0 ORF were detected only in specimens containing a relatively large number of EBV-positive cells . New monoclonal antibodies against the BARF0 protein were generated that efficiently recognized prokaryotic and eukaryotic recombinant BARF0 . However, the BARF0 protein was not detected in clinical samples, nor in EBV-positive cell lines, even though these were positive for BARTs by RISH and/or BARF0 RNA in vitro analysis . Using immunoblot analysis, no antibodies against baculovirus-expressed BARF0 protein were detected in the sera of nasopharyngeal carcinoma patients, BL patients and Hodgkin's disease patients, patients with chronic EBV infection, infectious mononucleosis patients or EBV-positive healthy donors . Thus, BARTs containing the BARF0 ORF are expressed in vivo but the BARF0 protein cannot be detected and may be expressed only marginally . It is concluded that the BARF0 protein is unlikely to play a role in vivo in EBV-positive malignancies. Int J Antimicrob Agents, 2003 Sep, 22(3), 347 - 51 Since phenothiazines alter antibiotic susceptibility of microorganisms by inhibiting efflux pumps, are these agents useful for evaluating similar pumps in phenothiazine-sensitive parasites? Gracio MA, Gracio AJ, Viveiros M, Amaral L. Phenothiazines have activity against Schistosoma mansoni, Trypanosoma brucei, Trypanasoma gambiensi, Molinema dessetae, Leishmania spp., Plasmodium falciparum and free-living protozoa . These organisms and other parasitic infections are prevalent in HIV-infected humans . These infections are becoming more frequently resistant to commonly employed antibiotics, and due to the absence of economic motivation, new and effective compounds against these infections are not anticipated in the near future . Resistance of prokaryotes and eukaryotes to antibiotics is now known to be also due to the presence of efflux pumps that extrude the antibiotic prior to the agent reaching its target . Because phenothiazines are known to inhibit some efflux pumps and therefore alter the susceptibility of the organism to an antibiotic to which it is resistant, and also because of the sensitivity of the above parasites to phenothiazines, efflux pumps may play a role in emerging antibiotic resistance of these organisms . Furthermore, their prevalence is known to be greatest in areas that have high rates of HIV infection; therefore, it would be necessary that these agents should receive close scrutiny . This review concerns the attributes afforded by phenothiazines related to their effective activity against a wide range of parasites . Because these agents are inexpensive and many are no longer protected by patent, they may be exploited as anti-parasitic agents in the poorer areas of the world. Int J Antimicrob Agents, 2003 Sep, 22(3), 188 - 99 ABC-transporters: implications on drug resistance from microorganisms to human cancers; Lage H; Resistance to chemotherapy is a common clinical problem in patients with infectious diseases as well as in patients with cancer . During treatment of infections or malignant tumors, the drug targets of prokaryotic or eukaryotic microorganisms and neoplastic cells are often found to be refractory to a variety of drugs that have different structures and functions . This phenomenon has been termed multidrug resistance (MDR) . The mechanisms leading to MDR are frequently caused by trans-membrane xenobiotic transport molecules belonging to the superfamily of ATP-binding cassette (ABC) transporters . There is an urgent need to understand the structure-function relationships of these efflux pumps that underlie their transport mechanism and drug selectivity . This knowledge may allow the rational design of new drugs that can inhibit or circumvent the activity of these MDR transport molecules . Furthermore, the development of such chemosensitizing agents would help us learn more about the physiological functions and substrates of these pump proteins . This review will discuss the current state of knowledge of the functional and structural similarities among ABC-transporters in prokaryotic and eukaryotic cells and their impact on MDR. Int J Antimicrob Agents, 2003 Sep, 22(3), 177 - 87 Protein-mediated transbilayer movement of lipids in eukaryotes and prokaryotes: the relevance of ABC transporters; Tannert A et al.; Lipid distribution across cellular membranes is regulated by specific membrane proteins controlling transbilayer movement of lipids . Flippases facilitate flip-flop of lipids and allow them to equilibrate between the two membrane leaflets independent of ATP . Distinct P-Type-ATPases transport specific lipids unidirectionally across the membrane at the expense of ATP . A group of ATP-dependent lipid transporters, the ATP-binding cassette (ABC) transporter family, was identified in studies originally related to multidrug resistance (MDR) in cancer cells . Meanwhile, lipid transport activity has been shown for full and half size ABC proteins in eukaryotic and prokaryotic cells . This activity may not only modify the organisation of lipids in membranes, but could also be of significant consequence for cell homeostasis . The various types of lipid movement mediating proteins and their cellular localisation in eukaryotes and prokaryotes are reviewed. Int J Parasitol, 2003 Sep 30, 33(11), 1245 - 58 Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative; Hotez PJ et al.; Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering . An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics . Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly . Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness . These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm . Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages . The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems . However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens . A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres . In some cases, antigen-specific IgE responses are required to mediate protection . Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics. PLoS Biol . 2003 Oct;1(1):E19 . Epub 2003 Sep 15. From gene trees to organismal phylogeny in prokaryotes: the case of the gamma-Proteobacteria; Lerat E et al.; The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes . However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies . The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the gamma-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals . We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the gamma-Proteobacteria . We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses . Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event . The concatenated sequences of the concordant families yield a fully resolved phylogeny . This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages . Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT . This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization. Proc Natl Acad Sci U S A, 2003 Sep 30, 100(20), 11684 - 9 Epub 2003 Sep 15. Borrelia oxidative stress response regulator, BosR: a distinctive Zn-dependent transcriptional activator; Boylan JA et al.; The ability of a pathogen to cause infection depends on successful colonization of the host, which, in turn, requires adaptation to various challenges presented by that host . For example, host immune cells use a variety of mechanisms to control infection by bacterial pathogens, including the production of bactericidal reactive oxygen species . Prokaryotic and eukaryotic cells have developed ways of protecting themselves against this oxidative damage; for instance, Borrelia burgdorferi alters the expression of oxidative-stress-related proteins, such as a Dps/Dpr homolog NapA (BB0690), in response to increasing levels of oxygen and reactive oxygen species . These stress-related genes appear to be regulated by a putative metal-dependent DNA-binding protein (BB0647) that has 50.7% similarity to the peroxide-specific stress response repressor of Bacillus subtilis, PerR . We overexpressed and purified this protein from Escherichia coli and designated it Borrelia oxidative stress regulator, BosR . BosR bound to a 50-nt region 180 bp upstream of the napA transcriptional start site and required DTT and Zn2+ for optimal binding . Unlike the Bacillus subtilis PerR repressor, BosR did not require Fe2+ and Mn2+ for binding, and oxidizing agents, such as t-butyl peroxide, enhanced, not eliminated, BosR binding to the napA promoter region . Surprisingly, transcriptional fusion analysis indicated that BosR exerted a positive regulatory effect on napA that is inducible with t-butyl peroxide . On the basis of these data, we propose that, despite the similarity to PerR, BosR functions primarily as a transcriptional activator, not a repressor of oxidative stress response, in B . burgdorferi. BMC Genomics . 2003 Sep 15;4(1):37. An improved probability mapping approach to assess genome mosaicism; Zhaxybayeva O et al.; BACKGROUND: Maximum likelihood and posterior probability mapping are useful visualization techniques that are used to ascertain the mosaic nature of prokaryotic genomes . However, posterior probabilities, especially when calculated for four-taxon cases, tend to overestimate the support for tree topologies . Furthermore, because of poor taxon sampling four-taxon analyses suffer from sensitivity to the long branch attraction artifact . Here we extend the probability mapping approach by improving taxon sampling of the analyzed datasets, and by using bootstrap support values, a more conservative tool to assess reliability . RESULTS: Quartets of orthologous proteins were complemented with homologs from selected reference genomes . The mapping of bootstrap support values from these extended datasets gives results similar to the original maximum likelihood and posterior probability mapping . The more conservative nature of the plotted support values allows to focus further analyses on those protein families that strongly disagree with the majority or plurality of genes present in the analyzed genomes . CONCLUSION: Posterior probability is a non-conservative measure for support, and posterior probability mapping only provides a quick estimation of phylogenetic information content of four genomes . This approach can be utilized as a pre-screen to select genes that might have been horizontally transferred . Better taxon sampling combined with subtree analyses prevents the inconsistencies associated with four-taxon analyses, but retains the power of visual representation . Nevertheless, a case-by-case inspection of individual multi-taxon phylogenies remains necessary to differentiate unrecognized paralogy and shared phylogenetic reconstruction artifacts from horizontal gene transfer events. Cell Res, 2003 Aug, 13(4), 285 - 94 Novel SLA class I alleles of Chinese pig strains and their significance in xenotransplantation; Chen FX et al.; To lay background for studying rejection mechanisms in xenotransplantation and developing the strategies for intervention, class I genes of swine leukocyte antigens (SLA) of three Chinese pig strains Bm, Gz and Yn were cloned and sequenced . The cDNA of the class I loci P1 and P14 were amplified by RT-PCR and subjected to insert into sequencing vectors . All six allelic sequences we examined, each two for one Chinese strain, are not identical to those reported, which allows these novel sequences receiving their accession numbers AY102467-AY102472 from GenBank . This study further reveals that the homologies of MHC class I genes in their primary structures and the deduced amino acids between Chinese pigs (SLA) and human (HLA-A*0201) are better than those between pigs and mice (H-2Db/H-2Kb) . The comparison also indicates that the amino acid residues critical for recognition by human KIRs are altered in the swine class I molecules . The amino acids responsible for binding human CD8 coreceptor are largely conserved although there are two critical residues substituted . A functional test indicated that the human T cells specific for the prokaryotically expressed SLA P1 protein could respond quite well in vitro to the class I-positive swine chondrocytes and PBMCs in presence of human APCs . This implies that, due to the substitution of two critical residues, the inaccessibility of human CD8 coreceptor to swine class I molecule might be contributable to the indirect pathway that the human T cells have to use for recognizing the SLA class I xenogeneic antigens. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2003 Aug, 25(4), 451 - 6 {Immuno-therapeutic study of anti-idiotype minibody (single chain Fv-CH3) on ovarian carcinoma bearing mice}; Li Y et al.; OBJECTIVE: An anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer . METHODS: Using gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region . The fusion protein named minibody was induced with IPTG in E . coli and analyzed with Western blot and inhibition ELISA tests respectively . Twenty human-PBL-SCID mice bearing i.p . Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times . Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3) . The latent period of ascites growth and the mean survival time were observed respectively . CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry . RESULTS: SDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3) . The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot . Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen . Ab3 could be detected in the sera of immunized mice with minibody by ELISA test . Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks . The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination . The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively . CONCLUSIONS: These results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function . Antigenicity is increased without adjuvants and partial humanization is realized . Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo . When ascites formation was delayed or prevented and the survival was prolonged in minibody group . We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails. Mol Biol Cell, 2003 Sep, 14(9), 3848 - 56 Epub 2003 Jun 13. The role of the 3' untranslated region in mRNA sorting to the vicinity of mitochondria is conserved from yeast to human cells; Sylvestre J et al.; We recently demonstrated, using yeast DNA microarrays, that mRNAs of polysomes that coisolate with mitochondria code for a subset of mitochondrial proteins . The majority of these mRNAs encode proteins of prokaryotic origin . Herein, we show that a similar association occurs between polysomes and mitochondria in human cells . To determine whether mRNA transport machinery is conserved from yeast to human cells, we examined the subcellular localization of human OXA1 mRNA in yeast . Oxa1p is a key component in the biogenesis of mitochondrial inner membrane and is conserved from bacteria to eukaryotic organelles . The expression of human OXA1 cDNA partially restores the respiratory capacity of yeast oxa1- cells . In this study, we demonstrate that 1) OXA1 mRNAs are remarkably enriched in mitochondrion-bound polysomes purified from yeast and human cells; 2) the presence of the human OXA1 3' untranslated region (UTR) is required for the function of the human Oxa1p inside yeast mitochondria; and 3) the accurate sorting of the human OXA1 mRNA to the vicinity of yeast mitochondria is due to the recognition by yeast proteins of the human 3' UTR . Therefore, it seems that the recognition mechanism of OXA1 3' UTR is conserved throughout evolution and is necessary for Oxa1p function. J Mol Biol, 2003 Sep 26, 332(4), 809 - 19 Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA; Christensen SK et al.; Prokaryotic chromosomes encode toxin-antitoxin loci, often in multiple copies . In most cases, the function of these genes is not known . The chpA (mazEF) locus of Escherichia coli has been described as a cell killing module that induces bacterial apoptosis during nutritional stress . However, we found recently that ChpAK (MazF) does not confer cell killing but rather, induces a bacteriostatic condition from which the cells could be resuscitated . Results presented here yield a mechanistic explanation for the detrimental effect on cell growth exerted by ChpAK and the homologous ChpBK protein of E.coli . We show that both proteins inhibit translation by inducing cleavage of translated mRNAs . Consistently, the inhibitory effect of the proteins was counteracted by tmRNA . Amino acid starvation induced strong transcription of chpA that depended on Lon protease but not on ppGpp . Simultaneously, ChpAK cleaved tmRNA in its coding region . Thus, ChpAK and ChpBK inhibit translation by a mechanism very similar to that of E.coli RelE . On the basis of these results, we propose a model that integrates TA loci into general prokaryotic stress physiology. J Immunol Methods, 2003 Aug, 279(1-2), 149 - 61 Llama-derived phage display antibodies in the dissection of the human disease oculopharyngeal muscular dystrophy; van Koningsbruggen S et al.; Functional analysis of the estimated 30,000 genes of the human genome requires fast and reliable high-throughput methods to study spatio-temporal protein dynamics . To explore the suitability of heavy-chain antibodies (HCAbs) for studying mechanisms underlying human disease, we used oculopharyngeal muscular dystrophy (OPMD) as a paradigm for the expanding group of protein aggregation disorders that is characterized by subcellular dislocalization and aggregation of mutant protein . OPMD is caused by a moderate alanine expansion in the poly-A binding protein nuclear 1 (PABPN1) and is associated with intranuclear PABPN1 deposition exclusively in muscle . An experimental approach was designed in which the primary sequence of the PABPN1 gene was employed for generating a prokaryotic expression construct that permitted its expression in the host Escherichia coli . The purified product was used for immunization of a llama as well as for the selection of an antigen-specific antibody fragment from the derived phage display library . This single-domain antibody was able to recognize the native gene product in mammalian cell lines and in human muscle tissue by immunocytochemical, immunohistochemical and immunoblot analysis . Our results suggest that phage display derived heavy-chain antibodies can be used in proteomics to study the localization and function of hypothetical gene products, relevant to human disease. J Biol Chem, 2003 Nov 28, 278(48), 48413 - 21 Epub 2003 Sep 10. Identification of a stomatin orthologue in vacuoles induced in human erythrocytes by malaria parasites . A role for microbial raft proteins in apicomplexan vacuole biogenesis; Hiller NL et al.; When the human malaria parasite Plasmodium falciparum infects erythrocytes, proteins associated with host-derived detergent-resistant membrane (DRM) rafts are selectively recruited into the newly formed vacuole, but parasite proteins that contribute to raft-based vacuole development are unknown . In mammalian cells, DRM-associated integral membrane proteins such as caveolin-1 and flotillin-1 that form oligomers have been linked to the formation of DRM-based invaginations called caveolae . Here we show that the P . falciparum genome does not encode caveolins or flotillins but does contain an orthologue of human band 7 stomatin, a protein known to oligomerize, associate with non-caveolar DRMs and is distantly related to flotillins . Stomatins are members of a large protein family conserved in evolution and P . falciparum (Pf) stomatin appears to be a prokaryotic-like molecule . Evidence is presented that it associates with DRMs and may oligomerize, suggesting that these features are conserved in the stomatin family . Further, Pfstomatin is an integral membrane protein concentrated at the apical end of extracellular parasites, where it co-localizes with invasion-associated rhoptry organelles . A resident rhoptry protein, RhopH2 also resides in DRMs . This provides the first evidence that rhoptries of an apicomplexan parasite contain DRM rafts . Further, when the parasite invades erythrocytes, rhoptry Pfstomatin and RhopH2 are inserted into the newly formed vacuole . Thus, like caveolin-1 and flotillin-1, a stomatin may also associate with non-clathrin coated, DRM-enriched vacuoles . We propose a new model of invasion and vacuole formation involving DRM-based interactions of both host and parasite molecules. Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 55 - 8 {Structural and functional reorganization of staphylococci and air-bearing system following experimental intratracheal introduction of pathogen}; Kozlova AN et al.; Morphological changes in the cells of the mucous membrane of the tracheo-bronchial system and in staphylococci under the conditions of intratracheal infection were studied . Different forms of interaction between the host cell and staphylococci, reflecting the organ specific properties and a wide range of compensation and adaptation reactions of eukaryotic and prokaryotic cells were revealed with the use of electron microscopy. Microbiol Mol Biol Rev, 2003 Sep, 67(3), 303 - 42, table of contents Ancient origin of the tryptophan operon and the dynamics of evolutionary change; Xie G et al.; The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes . A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways . These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level . These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution . Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux . Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced . Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion . The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria . The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea . Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence . The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting features that can be distinguished . As additional genomes are thoroughly analyzed, an increasingly refined resolution of the sequential evolutionary steps is clearly possible . These comparisons suggest that present-day trp operons that possess finely tuned regulatory features are under strong positive selection and are able to resist the disruptive evolutionary events that may be experienced by simpler, poorly regulated operons. BMC Genomics . 2003 Sep 10;4(1):36. Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E . coli strain MG1655 and B . subtilis strain 168 genomes, prepared without passaging through E . coli; Haney SA et al.; BACKGROUND: Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable . However, the expression of genes or gene fragments that are capable of interacting with E . coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library . RESULTS: Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E . coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries . This includes the rapid loss of a fragment of the E . coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA . Expression of this clone causes slower growth in E . coli . This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed . We have demonstrated in this report that the construction of libraries for the E . coli and B . subtilis genomes without passaging through E . coli is practical, but the number of transformants is less than for libraries cloned using E . coli as a host . Analysis of several clones in the libraries that are strongly growth inhibitory in E . coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport . CONCLUSION: Expression of Y2H clones capable of interacting with E . coli and yeast targets are rapidly lost, causing a loss of complexity . The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E . coli, or yeast, including many genes that represent potential antibacterial targets . While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes. Biomed Environ Sci, 2003 Jun, 16(2), 149 - 56 Purification and immunity analysis of recombinant 6His-HPT protein expressed in E . coli; Yang LC et al.; OBJECTIVE: To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from E . coli and to prepare the polyclonal antibody (pAbs) against it . METHODS: HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222 . Then the constructed recombinant plasmid pBV222-HPT was transferred into E . coli DH5alpha for HPT expression . The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression . E . coli cells were lysed by sonication and detergent dissolution . After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition . The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit . The titer and specificity of antisera were detected by ELISA and Western blot respecitively . RESULTS: Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct . The amount of recombinant HPT expressed in E . coli accounted for 30% of total cellular proteins . From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95% . The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity . Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein) . CONCLUSION: HPT protein can be expressed and purified from E . coli by a relatively simple method, which has high immunity activity. Protein Expr Purif, 2003 Sep, 31(1), 99 - 107 Purification of recombinant plasminogen activator inhibitor-1 in the active conformation by refolding from inclusion bodies; Lee HJ et al.; Plasminogen activator inhibitor-1 (PAI-1) acts as the major inhibitor of fibrinolysis by inhibiting tissue-type and urokinase-type plasminogen activators . Although it shares a common tertiary structure with other serine protease inhibitors, PAI-1 is unique in its conformational lability, which allows conversion of the active form to the latent conformation under physiological conditions . Therefore, recombinant PAI-1 expressed in eukaryotic or prokaryotic cells almost always contains its inactive, latent form, with very low specific activity . In this study, we developed a simple and efficient method for purifying the active form of recombinant PAI-1 rather than the latent conformation from PAI-1 overexpressing Escherichia coli cells . The overall level of expression and the amount of PAI-1 found in inclusion bodies were found to increase with culture temperature and with time after induction . Refolding of unfolded PAI-1 from inclusion bodies and ion-exchange column chromatography were sufficient to purify PAI-1 . The purified protein yielded a single, 43kDa protein band upon SDS-polyacrylamide gel electrophoresis, and it efficiently inhibited tissue-type and urokinase-type plasminogen activators similar to PAI-1 from natural sources . Activity measurements showed that PAI-1 purified from inclusion bodies exhibited a specific activity near the theoretical maximum, unlike PAI-1 prepared from cytosolic fractions . Conformational analysis by urea gel electrophoresis also indicated that the PAI-1 protein purified from inclusion bodies was indeed in its active conformation. Nippon Rinsho, 2003 Aug, 61(8), 1317 - 22 {Heat shock protein 105 in multiple sclerosis}; Minohara M; Heat shock proteins are known to be immunodominant antigens of bacteria . They are strongly conserved proteins present in all eukaryotic and prokaryotic cellular organisms . HSP105/HSP110 family is expressed most abundantly in the brain . Here, we demonstrate the immune responses to HSP105 in multiple sclerosis(MS) and experimental autoimmune encephalomyelitis(EAE) . We found that (a) the frequency of positive IgG anti-hHSP105 antibody increased in MS patients, (b) hHSP105 expression was found to be enhanced in MS lesions, (c) in the ELISPOT assay, significantly lower the IL-10 response to hHSP105 in MS patients than healthy controls, (d) mice vaccinated with HSP105-DNA developed an exacerbated form of EAE . Our data indicated that immune responses to HSP105 may play a regulatory role in inflammation during MS and EAE. Microb Drug Resist, 2003 Fall, 9(3), 249 - 55 Promoter strength of folic acid synthesis genes affects sulfa drug resistance in Saccharomyces cerevisiae; Iliades P et al.; The enzyme dihydropteroate synthase (DHPS) is an important target for sulfa drugs in both prokaryotic and eukaryotic microbes . However, the understanding of DHPS function and the action of antifolates in eukaryotes has been limited due to technical difficulties and the complexity of DHPS being a part of a bifunctional or trifunctional protein that comprises the upstream enzymes involved in folic acid synthesis (FAS) . Here, yeast strains have been constructed to study the effects of FOL1 expression on growth and sulfa drug resistance . A DHPS knockout yeast strain was complemented by yeast vectors expressing the FOL1 gene under the control of promoters of different strengths . An inverse relationship was observed between the growth rate of the strains and FOL1 expression levels . The use of stronger promoters to drive FOL1 expression led to increased sulfamethoxazole resistance when para-aminobenzoic acid (pABA) levels were elevated . However, high FOL1 expression levels resulted in increased susceptibility to sulfamethoxazole in pABA free media . These data suggest that up-regulation of FOL1 expression can lead to sulfa drug resistance in Saccharomyces cerevisiae. Appl Environ Microbiol, 2003 Sep, 69(9), 5656 - 63 Novel eukaryotes from the permanently anoxic Cariaco Basin (Caribbean Sea); Stoeck T et al.; Present knowledge of microbial diversity is decidedly incomplete (S . J . Giovannoni and M . S . Rappe, p . 47-84, in D . Kirchman, ed., Microbial Ecology of the Oceans, 2000; E . Stackebrandt and T . M . Embley, p . 57-75, in R . R . Colwell and D . J . Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) . Protistan phylogenies are particularly deficient and undoubtedly exclude clades of principal ecological and evolutionary importance (S . L . Baldauf, Science 300:1703-1706, 2003) . The rRNA approach has been extraordinarily successful in expanding the global prokaryotic record (S . J . Giovannoni and M . S . Rappe, p . 47-84, in D . Kirchman, ed., Microbial Ecology of the Oceans, 2000; E . Stackebrandt and T . M . Embley, p . 57-75, in R . R . Colwell and D . J . Grimes, ed., Nonculturable Microorganisms in the Environment, 2000) but has rarely been used in protistan discovery . Here we report the first application of the 18S rRNA approach to a permanently anoxic environment, the Cariaco Basin off the Venezuelan coast . On the basis of rRNA sequences, we uncovered a substantial number of novel protistan lineages . These included new clades of the highest taxonomic level unrelated to any known eukaryote as well as deep branches within established protistan groups . Three novel lineages branch at the base of the eukaryotic evolutionary tree preceding, contemporary with, or immediately following the earliest eukaryotic branches . These newly discovered protists may retain traits reminiscent of an early eukaryotic ancestor(s). Appl Environ Microbiol, 2003 Sep, 69(9), 5192 - 7 Spatial distribution of Rhodopseudomonas palustris ecotypes on a local scale; Bent SJ et al.; The number, spatial distribution, and significance of genetically distinguishable ecotypes of prokaryotes in the environment are poorly understood . Oda et al . (Y . Oda, B . Star, L . A . Huisman, J . C . Gottschal, and L . J . Forney, Appl . Environ . Microbiol . 69:xxx-xxx, 2003) have shown that Rhodopseudomonas palustris ecotypes were lognormally distributed along a 10-m transect and that multiple strains of the species could coexist in 0.5-g sediment samples . To extend these observations, we investigated the clonal diversity of R . palustris in 0.5-g samples taken from the corners and center of a 1-m square . A total of 35 or 36 clones were recovered by direct plating from each sample and were characterized by BOX A1R repetitive element-PCR genomic DNA fingerprinting . Isolates with fingerprint images that were >/=80% similar to each other were defined as the same genotype . Among the 178 isolates studied, 32 genotypes were identified, and each genotype contained between 1 and 40 isolates . These clusters were consistent with minor variations found in 16S rRNA gene sequences . The Shannon indices of the genotypic diversity within each location ranged from 1.08 (5 genotypes) to 2.18 (13 genotypes) . Comparison of the rank abundance of genotypes found in pairs of locations showed that strains from three locations were similar to each other, with Morisita-Horn similarity coefficients ranging from 0.59 to 0.71 . All comparisons involving the remaining two locations resulted in coefficients between 0 and 0.12 . From these results we inferred that the patterns of ecotype diversity at the sampling site are patchy at a 1-m scale and postulated that factors such as mixing, competitive interactions, and microhabitat variability are likely to be responsible for the maintenance of the similarities between some locations and the differences between others. Nucleic Acids Res, 2003 Sep 15, 31(18), 5389 - 98 The Drosophila melanogaster BTB proteins bric à brac bind DNA through a composite DNA binding domain containing a pipsqueak and an AT-Hook motif; Lours C et al.; The bric a brac (bab) locus is composed of two paralogous genes, bab1 and bab2, in Drosophila melanogaster . Bab1 and Bab2 are nuclear proteins that contain a broad complex, tramtrack, bric a brac/poxviruses and zinc-finger (BTB/POZ) domain . Many BTB/POZ proteins are transcriptional regulators of which the majority contain C(2)H(2) zinc-finger motifs . There is no detectable zinc-finger motif in either Bab protein . However, they share the Bab conserved domain (BabCD) that is highly conserved between Bab1 and Bab2, and the Bab proteins of several other species, e.g . Anopheles gambiae, Apis mellifera and Drosophila virilis . Here we show that Bab2 binds to several discrete sites on polytene chromosomes including the bab locus, and that the BabCD of both Bab1 and Bab2 binds in vitro to the cis-regulatory regions of bab1 and bab2 . Our results indicate that the BabCD binds to A/T-rich regions and that its optimum binding sites contain TA or TAA repeats . The BabCD is a composite DNA binding domain with a psq motif and an AT-Hook motif; both motifs are required for DNA binding activity . Structural similarities suggest that the BabCD may bind to DNA in a similar manner as some prokaryotic recombinases. J Biol Chem, 2003 Nov 14, 278(46), 46052 - 63 Epub 2003 Sep 03. Functional importance of polar and charged amino acid residues in transmembrane helix 14 of multidrug resistance protein 1 (MRP1/ABCC1): identification of an aspartate residue critical for conversion from a high to low affinity substrate binding state; Zhang DW et al.; Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions . We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance . We have now assessed the functional importance of all polar and charged amino acids in this TM helix . Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein . Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16) . In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity . In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected . We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2 . Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs. Genome Res, 2003 Sep, 13(9), 2178 - 89 OrthoMCL: identification of ortholog groups for eukaryotic genomes; Li L et al.; The identification of orthologous groups is useful for genome annotation, studies on gene/protein evolution, comparative genomics, and the identification of taxonomically restricted sequences . Methods successfully exploited for prokaryotic genome analysis have proved difficult to apply to eukaryotes, however, as larger genomes may contain multiple paralogous genes, and sequence information is often incomplete . OrthoMCL provides a scalable method for constructing orthologous groups across multiple eukaryotic taxa, using a Markov Cluster algorithm to group (putative) orthologs and paralogs . This method performs similarly to the INPARANOID algorithm when applied to two genomes, but can be extended to cluster orthologs from multiple species . OrthoMCL clusters are coherent with groups identified by EGO, but improved recognition of "recent" paralogs permits overlapping EGO groups representing the same gene to be merged . Comparison with previously assigned EC annotations suggests a high degree of reliability, implying utility for automated eukaryotic genome annotation . OrthoMCL has been applied to the proteome data set from seven publicly available genomes (human, fly, worm, yeast, Arabidopsis, the malaria parasite Plasmodium falciparum, and Escherichia coli) . A Web interface allows queries based on individual genes or user-defined phylogenetic patterns . Analysis of clusters incorporating P . falciparum genes identifies numerous enzymes that were incompletely annotated in first-pass annotation of the parasite genome. Proc R Soc Lond B Biol Sci, 2003 Aug 7, 270 Suppl 1, S69 - 72 Evidence for the presence of a cellulase gene in the last common ancestor of bilaterian animals; Lo N et al.; Until recently, the textbook view of cellulose hydrolysis in animals was that gut-resident symbiotic organisms such as bacteria or unicellular eukaryotes are responsible for the cellulases produced . This view has been challenged by the characterization and sequencing of endogenous cellulase genes from some invertebrate animals, including plant-parasitic nematodes, arthropods and a mollusc . Most of these genes are completely unrelated in terms of sequence, and their evolutionary origins remain unclear . In the case of plant-parasitic nematodes, it has been suggested that their ancestor obtained a cellulase gene via horizontal gene transfer from a prokaryote, and similar suggestions have been made about a cellulase gene recently discovered in a sea squirt . To improve understanding about the evolution of animal cellulases, we searched for all known types of these enzymes in GenBank, and performed phylogenetic comparisons . Low phylogenetic resolution was found among most of the sequences examined, however, positional identity in the introns of cellulase genes from a termite, a sea squirt and an abalone provided compelling evidence that a similar gene was present in the last common ancestor of protostomes and deuterostomes . In a different enzyme family, cellulases from beetles and plant-parasitic nematodes were found to cluster together . This result questions the idea of lateral gene transfer into the ancestors of the latter, although statistical tests did not allow this possibility to be ruled out . Overall, our results suggest that at least one family of endogenous cellulases may be more widespread in animals than previously thought. Genome Biol . 2003;4(9):R59 . Epub 2003 Aug 29. Inference of protein function and protein linkages in Mycobacterium tuberculosis based on prokaryotic genome organization: a combined computational approach; Strong M et al.; The genome of Mycobacterium tuberculosis was analyzed using recently developed computational approaches to infer protein function and protein linkages . We evaluated and employed a method to infer genes likely to belong to the same operon, as judged by the nucleotide distance between genes in the same genomic orientation, and combined this method with those of the Rosetta Stone, Phylogenetic Profile and conserved Gene Neighbor computational methods for the inference of protein function. Genome Biol . 2003;4(9):R55 . Epub 2003 Aug 29. Evolution of mosaic operons by horizontal gene transfer and gene displacement in situ; Omelchenko MV et al.; BACKGROUND: Shuffling and disruption of operons and horizontal gene transfer are major contributions to the new, dynamic view of prokaryotic evolution . Under the 'selfish operon' hypothesis, operons are viewed as mobile genetic entities that are constantly disseminated via horizontal gene transfer, although their retention could be favored by the advantage of coregulation of functionally linked genes . Here we apply comparative genomics and phylogenetic analysis to examine horizontal transfer of entire operons versus displacement of individual genes within operons by horizontally acquired orthologs and independent assembly of the same or similar operons from genes with different phylogenetic affinities . RESULTS: Since a substantial number of operons have been identified experimentally in only a few model bacteria, evolutionarily conserved gene strings were analyzed as surrogates of operons . The phylogenetic affinities within these predicted operons were assessed first by sequence similarity analysis and then by phylogenetic analysis, including statistical tests of tree topology . Numerous cases of apparent horizontal transfer of entire operons were detected . However, it was shown that apparent horizontal transfer of individual genes or arrays of genes within operons is not uncommon either and results in xenologous gene displacement in situ, that is, displacement of an ancestral gene by a horizontally transferred ortholog from a taxonomically distant organism without change of the local gene organization . On rarer occasions, operons might have evolved via independent assembly, in part from horizontally acquired genes . CONCLUSIONS: The discovery of in situ gene displacement shows that combination of rampant horizontal gene transfer with selection for preservation of operon structure provides for events in prokaryotic evolution that, a priori, seem improbable . These findings also emphasize that not all aspects of operon evolution are selfish, with operon integrity maintained by purifying selection at the organism level. FEMS Microbiol Lett, 2003 Aug 29, 225(2), 241 - 7 The gene encoding pyolysin, the pore-forming toxin of Arcanobacterium pyogenes, resides within a genomic islet flanked by essential genes; Rudnick ST et al.; The plo gene, encoding the Arcanobacterium pyogenes cholesterol-dependent cytolysin, pyolysin (PLO), was localized to a 2.7-kb genomic islet of reduced %G+C content and alternate codon usage frequency . This islet, conserved among isolates from diverse hosts and geographical locations, separated the housekeeping genes smc and ftsY, which are found adjacent in many prokaryotes . The ftsY and ffh genes, located downstream of the plo islet, encode components of the signal recognition particle . Mutational analysis suggested that these genes were essential for viability in A . pyogenes . The A . pyogenes ffh gene was unable to complement a conditional ffh mutant of Escherichia coli and its overexpression was toxic in E . coli . Mutagenesis of the islet-encoded orf121 did not affect plo expression, indicating that it may not be involved directly in the regulation of plo expression . Regardless, the presence of the plo gene as part of a genomic islet inserted between genes essential for normal growth may provide selective pressure for the retention of this important virulence factor. Mol Microbiol, 2003 Sep, 49(6), 1523 - 36 Essential role of DivIVA in polar growth and morphogenesis in Streptomyces coelicolor A3(2); Flardh K; Streptomycetes grow by cell wall extension at hyphal tips . The molecular basis for such polar growth in prokaryotes is largely unknown . It is reported here that DivIVASC, the Streptomyces coelicolor homologue of the Bacillus subtilis protein DivIVA, is essential and directly involved in hyphal tip growth and morphogenesis . A DivIVASC-EGFP hybrid was distinctively localized to hyphal tips and lateral branches . Reduction of divIVASC expression to about 10% of the normal level produced a phenotype strikingly similar to that of many tip growth mutants in fungi, including irregular curly hyphae and apical branching . Overexpression of the gene dramatically perturbed determination of cell shape at the growing tips . Furthermore, staining of nascent peptidoglycan with a fluorescent vancomycin conjugate revealed that induction of overexpression in normal hyphae disturbed tip growth, and gave rise to several new sites of cell wall assembly, effectively causing hyperbranching . The results show that DivIVASC is a novel bacterial morphogene, and it is localized at or very close to the apical sites of peptidoglycan assembly in Streptomyces hyphae. Mol Microbiol, 2003 Sep, 49(6), 1493 - 508 PknB kinase activity is regulated by phosphorylation in two Thr residues and dephosphorylation by PstP, the cognate phospho-Ser/Thr phosphatase, in Mycobacterium tuberculosis; Boitel B et al.; Bacterial genomics revealed the widespread presence of eukaryotic-like protein kinases and phosphatases in prokaryotes, but little is known on their biochemical properties, regulation mechanisms and physiological roles . Here we focus on the catalytic domains of two trans-membrane enzymes, the Ser/Thr protein kinase PknB and the protein phosphatase PstP from Mycobacterium tuberculosis . PstP was found to specifically dephosphorylate model phospho-Ser/Thr substrates in a Mn2+-dependent manner . Autophosphorylated PknB was shown to be a substrate for Pstp and its kinase activity was affected by PstP-mediated dephosphorylation . Two threonine residues in the PknB activation loop, found to be mostly disordered in the crystal structure of this kinase, namely Thr171 and Thr173, were identified as the target for PknB autophosphorylation and PstP dephosphorylation . Replacement of these threonine residues by alanine significantly decreased the kinase activity, confirming their direct regulatory role . These results indicate that, as for eukaryotic homologues, phosphorylation of the activation loop provides a regulation mechanism of mycobacterial kinases and strongly suggest that PknB and PstP could work as a functional pair in vivo to control mycobacterial cell growth. EMBO Rep, 2003 Sep, 4(9), 850 - 4 A genomic overview of pyridoxal-phosphate-dependent enzymes; Percudani R et al.; Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts . Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families . In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism . Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships . Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities . In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes . This observation presumably applies to many other classes of protein-encoding genes. Nat Rev Immunol, 2003 Sep, 3(9), 733 - 44 Immune regulation by helminth parasites: cellular and molecular mechanisms; Maizels RM et al.; Immunology was founded by studying the body's response to infectious microorganisms, and yet microbial prokaryotes only tell half the story of the immune system . Eukaryotic pathogens--protozoa, helminths, fungi and ectoparasites--have all been powerful selective forces for immune evolution . Often, as with lethal protozoal parasites, the focus has been on acute infections and the inflammatory responses they evoke . Long-lived parasites such as the helminths, however, are more remarkable for their ability to downregulate host immunity, protecting themselves from elimination and minimizing severe pathology in the host. Microbiology, 2003 Sep, 149(Pt 9), 2571 - 84 Spontaneous zygogenesis in Escherichia coli, a form of true sexuality in prokaryotes; Gratia JP et al.; A new type of mating, differing from classic conjugation and previously observed in a certain strain of Escherichia coli K-12, has also been found in strains derived from ordinary F- cells of E . coli K-12 exposed to an exogenous factor originating in an E . coli clinical isolate . Immunofluorescence and electron microscopy after single and double labelling of DNA were used to produce evidence in favour of a novel mating mechanism by cell contact at the poles of the bacterial rod . These findings are supported by genetic analyses indicating complete genetic mixing . Unstable complementing diploids were formed, which throw off phenotypically haploid cells, of which some showed a parental phenotype and some were true genetic recombinants . Recombination was observed even when one parent was a UV-inactivated F- RecA- strain . The name 'spontaneous zygogenesis' (Z-mating, for short) is proposed for this kind of mating. Mol Biol Evol, 2004 Jan, 21(1), 86 - 9 Epub 2003 Aug 29. Quartet mapping and the extent of lateral transfer in bacterial genomes; Daubin V et al.; Several recent analyses have used quartet-based methods to assess the congruence among phylogenies derived for large sets of genes from prokaryotic genomes . The principal conclusion from these studies is that lateral gene transfer (LGT) has blurred prokaryotic phylogenies to such a degree that the darwinian scheme of treelike evolution might be abandoned in favor of a net or web . Here, we focus on one of these methods, quartet mapping, and show that its application can lead to overestimation of the extent of inferred LGT in prokaryotes, particularly when applied to distantly related taxa. Curr Opin Struct Biol, 2003 Aug, 13(4), 432 - 42 Structure and mechanism in prokaryotic mechanosensitive channels; Perozo E et al.; Mechanosensitive channels function as electromechanical switches with the capability to sense the physical state of lipid bilayers . The X-ray crystal structures of MscL and MscS offer a unique opportunity to identify the types of protein motions associated with the opening and closing of these structurally unrelated channels, while providing the framework to address a mechanism of tension sensing that is defined by channel-lipid interactions . Recent functional, structural and dynamic data offer fresh insights into the molecular basis of gating for these membrane proteins. Beijing Da Xue Xue Bao, 2003 Aug, 35(4), 426 - 8 Cloning and expression of xeroderma pigmentosum complementation group A cDNA and the identification of its recombinant protein; Yang Y et al.; OBJECTIVE: To clone and express xeroderma pigmentosum, complementation group A (XPA) cDNA and to identify its recombinant protein . METHODS: Coding region of XPA cDNA was amplified from human tonsil cDNA by reverse transcription and nested PCR . The PCR product was cloned into pBluescript vector, confirmed by DNA sequencing, cloned in frame into a prokaryotic expression vector pTrcHis C and expressed in E . coli . TOP10 . The recombinant fusion protein was identified by immunoblotting . RESULTS: The entire coding region of XPA cDNA was cloned and expressed . The fusion XPA protein was identified by anti-XPA monoclonal antibody on western blot . CONCLUSION: Cloning of human XPA cDNA and successful expression of recombinant XPA protein will be useful for the construction of a viral gene transfer vector for the gene therapy of XPA. Beijing Da Xue Xue Bao, 2003 Aug, 35(4), 360 - 3 {Purification and stability studies of prokaryotic PDCD5 protein}; Wang L et al.; OBJECTIVE: To set up an effective and simple purification method to obtain highly purified prokaryotic protein of PDCD5 and study its stability . METHODS: Recombinant PDCD5 protein expressed in E . coli . was accumulated as an inclusion body . After washing, the inclusion body was denatured, renatured, digested with thrombine and then purified by two steps of chromatography . The purity of the products was analyzed by capillary electrophoresis and the stability was identified by SDS-PAGE . RESULTS: Capillary electrophoresis showed that the purity of protein was 100%, and molecular weight was 15,800 with pI 5.9 . Further bioactivity assay indicated that the purified PDCD5 could enhance the apoptosis of HL-60 cells withdrawing cytokine, which was in a dose-dependent manner . Stability analysis showed that the PDCD5 protein was sensitive to temperature and easy to degrade at 4 degrees C and 25 degrees C . However, it was relatively stable at -20 degrees C or lyophilized . CONCLUSION: Highly purified and stable recombinant PDCD5 protein was obtained, which lays a foundation for the functional study and application investigation of PDCD5. J Mol Biol, 2003 Sep 5, 332(1), 13 - 21 The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors; Oefner C et al.; Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides . There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses . Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme . Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents . Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution . The protein has a typical "pita-bread" fold as observed for the other MetAP structures . The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions . The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is. Acta Cient Venez, 2002, 53(4), 297 - 306 {Mitochondria and apoptosis}; Arvelo F; It is now accepted that mitochondria are endosymbionts, originated in aerobic bacteria which were integrated by the ancestor of eukaryotic cells . A part of the apoptotic machinery could exist in unicellular eukaryotic and some controlling apoptosis components might be present in prokaryotes . It is therefore possible that the mechanism originally involved in the maintenance of the symbiosis between the bacterial ancestor of the mitochondria and the host cell precursor of eukaryotes, provided the basis for the actual mechanism controlling cell survival . Metazoans would have improved this possibility by connecting to the mitochondria as principal effector of cellular death to the pathways of signal transduction . A variety of events appoint to the mitochondria as principal effector of the apoptosis . This including the release caspase activators (cytochrome c), changes in electron transport, loss of mitochondrial transmembrane potential, altered cellular oxidation-reduction, and participation of pro and antiapoptotic Bcl-2 proteins . The different signals that converge on mitochondria for activation or inhibition of these events, delineate several pathways in the physiology of the cellular death. Mol Immunol, 2003 Sep, 40(5), 279 - 86 Native and recombinant interleukin-2, two functionally distinct molecules; Denis MC et al.; Recombinant IL-2 (rIL-2) produced in prokaryotes, is widely used in lieu of native IL-2, which is secreted by T cells, to assess the functional profile of this cytokine . We provide evidence that a naturally occurring species of post-translationally modified IL-2 (moIL-2) exhibits enhanced bioactivity compared to rIL-2, as tested at the biochemical and functional level . We show that moIL-2 has high binding affinity for the IL-2 receptor (IL-2R), induces the immediate expression of the IL-2R alpha chain and rapidly activates downstream signaling molecules . Finally, in contrast to rIL-2, moIL-2 can promote the antigen-independent proliferation of resting lymphocytes . Collectively, our data demonstrate that native moIL-2 is functionally distinct from rIL-2, suggesting the existence of diverse IL-2 variants which may be critical for the shaping of the immune response. Plant J, 2003 Jan, 33(1), 97 - 106 Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases; Carmi N et al.; Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage . These sugars are initially metabolised by alpha-galactosidases (alpha-gal) . We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species . The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families . In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity . Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step . This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate. Cell Mol Life Sci, 2003 Jul, 60(7), 1477 - 88 Thiamine triphosphate and thiamine triphosphatase activities: from bacteria to mammals; Makarchikov AF et al.; In most organisms, the main form of thiamine is the coenzyme thiamine diphosphate . Thiamine triphosphate (ThTP) is also found in low amounts in most vertebrate tissues and can phosphorylate certain proteins . Here we show that ThTP exists not only in vertebrates but is present in bacteria, fungi, plants and invertebrates . Unexpectedly, we found that in Escherichia coli as well as in Arabidopsis thaliana, ThTP was synthesized only under particular circumstances such as hypoxia (E . coli) or withering (A . thaliana) . In mammalian tissues, ThTP concentrations are regulated by a specific thiamine triphosphatase that we have recently characterized . This enzyme was found only in mammals . In other organisms, ThTP can be hydrolyzed by unspecific phosphohydrolases . The occurrence of ThTP from prokaryotes to mammals suggests that it may have a basic role in cell metabolism or cell signaling . A decreased content may contribute to the symptoms observed during thiamine deficiency. Cell Mol Life Sci, 2003 Jul, 60(7), 1333 - 41 The free-radical hypothesis of aging goes prokaryotic; Nystrom T; With respect to oxidative damage and its targets, growth-arrested bacterial cells show some of the same signs of senescence as aging insects, worms and mammals . In addition, the fact that the life span of growth-arrested Escherichia coli cells is greatly extended by limiting oxygen availability suggests that free radicals may be one causal factor behind bacterial senescence . Recent analysis reveals a novel culprit in this oxidation, namely the production of aberrant proteins, which are especially susceptible to oxidative attack . This route of oxidation appears to elude the classical oxidative defense proteins . In addition, the failure of growth-arrested cells to fully combat oxidative damage may be linked to a trade-off between proliferation activities and stress management . Even during stasis, E . coli cells maintain a basal transcription of reproduction-related genes, and resources are thus partly diverted from maintenance and stress defences to activities relating to proliferation . Thus, some aspects of bacterial senescence may lend support to contemporary theories of aging, including the free radical, antagonistic pleiotropy, and disposable soma theories. Mol Biol (Mosk), 2003 Jul-Aug, 37(4), 674 - 87 {Identification of the horizontal gene transfer based on phylogenetic data}; V'iugin VV et al.; We suggest a new procedure to search for the genes with horizontal transfer events in their evolutionary history . The search is based on analysis of topology difference between the phylogenetic trees of gene (protein) groups and the corresponding phylogenetic species trees . Numeric values are introduced to measure the discrepancy between the trees . This approach was applied to analyze 40 prokaryotic genomes classified into 132 classes of orthologs . This resulted in a list of the candidate genes for which the hypothesis of horizontal transfer in evolution looks true. Immunology, 2003 Sep, 110(1), 1 - 9 Facets of heat shock protein 70 show immunotherapeutic potential; Todryk SM et al.; Amongst the families of intracellular molecules that chaperone and assist with the trafficking of other proteins, notably during conditions of cellular stress, heat shock protein (hsp) 70 is one of the most studied . Although its name suggests that expression is exclusively induced during cellular hyperthermia, members of the hsp70 family of proteins can be constitutively expressed and/or induced by a range of other cellular insults . The ubiquitous presence of hsp70 in eukaryotic and prokaryotic cells, combined with its high degree of sequence homology and intrinsic immunogenicity, have prompted the suggestion that inappropriate immune reactivity to hsp70 might lead to pro-inflammatory responses and the development of autoimmune disease . Indeed, hsp70 has been shown to be a potent activator of innate immunity and aberrant expression of hsp70 in certain organs promotes immunopathology . However, studies also suggest that hsp70 might have immunotherapeutic potential, as hsp70 purified from malignant and virally infected cells can transfer and deliver antigenic peptides to antigen-presenting cells to elicit peptide-specific immunity and, in contrast to its reported pro-inflammatory effects, the administration of recombinant hsp70 can attenuate experimental autoimmune disease . This review focuses on the immunoregulatory capacity of hsp70 and its potential therapeutic value. Mol Microbiol, 2003 Sep, 49(5), 1157 - 65 Chromosomal replicases as asymmetric dimers: studies of subunit arrangement and functional consequences; McHenry CS; Studies of the DNA polymerase III holoenzyme of Escherichia coli support a model in which both the leading and lagging strand polymerases are held together in a complex with the replicative helicase and priming activities, allowing two identical alpha catalytic subunits to assume different functions on the two strands of the replication fork . Creation of distinct functions for each of the two polymerases within the holoenzyme depends on the asymmetric character of the entire complex . The asymmetry of the holoenzyme is created by the DnaX complex, a heptamer that includes tau and gamma products of the dnaX gene . tau and gamma perform unique functions in the DnaX complex, and the interaction between alpha and tau appears to dictate the catalytic subunit's role in the replicative reaction . This review considers the properties of the DnaX complex including both tau and gamma, with the goal of understanding the properties of the replicase and its function in vivo . Recent studies in eukaryotic and other prokaryotic systems suggest that an asymmetric dimeric replicase may be universal . The leading and lagging strand polymerases may be distinct in some systems . For example, Pol e and Pol delta may function as distinct leading and lagging strand polymerases in eukaryotes, and PolC and DnaE may function as distinct leading and lagging strand polymerases in low GC content Gram-positive bacteria. Arch Microbiol, 2003 Sep, 180(3), 176 - 84 Epub 2003 Jul 09. Repressed multidrug resistance genes in Streptomyces lividans; Lee LF et al.; Multidrug resistance (MDR) systems are ubiquitously present in prokaryotes and eukaryotes and defend both types of organisms against toxic compounds in the environment . Four families of MDR systems have been described, each family removing a broad spectrum of compounds by a specific membrane-bound active efflux pump . In the present study, at least four MDR systems were identified genetically in the soil bacterium Streptomyces lividans . The resistance genes of three of these systems were cloned and sequenced . Two of them are accompanied by a repressor gene . These MDR gene sequences are found in most other Streptomyces species investigated . Unlike the constitutively expressed MDR genes in Escherichia coli and other gram-negative bacteria, all of the Streptomyces genes were repressed under laboratory conditions, and resistance arose by mutations in the repressor genes. FEBS Lett, 2003 Aug 28, 550(1-3), 46 - 50 Transposition and targeting of the prokaryotic mobile element IS30 in zebrafish; Szabo M et al.; We provide evidence that a prokaryotic insertion sequence (IS) element is active in a vertebrate system . The transposase of Escherichia coli element IS30 catalyzes both excision and integration in extrachromosomal DNA in zebrafish embryos . The transposase has a pronounced target preference, which is shown to be modified by fusing the enzyme to unrelated DNA binding proteins . Joining the transposase to the cI repressor of phage lambda causes transposition primarily into the vicinity of the lambda operator in E . coli, and linking to the DNA binding domain of Gli1 also directs the recombination activity of transposase near to the Gli1 binding site in zebrafish . Our results demonstrate the possibility of fusion transposases to acquire novel target specificity in both prokaryotes and eukaryotes. Proc Natl Acad Sci U S A, 2003 Sep 2, 100(18), 10477 - 82 Epub 2003 Aug 19. Chaperone-mediated in vitro assembly of Polyomavirus capsids; Chromy LR et al.; The polyomavirus coat protein viral protein 1 (VP1) has the intrinsic ability to self-assemble in vitro into polymorphic capsid-like structures on addition of calcium . In contrast, polyomavirus assembly in vivo is rigorously controlled, such that virions of uniform size are formed only in the cell nucleus . During viral infection, the 72 kDa cellular chaperone heat shock cognate protein (hsc70) binds VP1 posttranslation and colocalizes with VP1 to the nucleus, thereby suggesting a role for approximately 70-kDa heat shock protein (hsp70) family chaperones in regulating the quality and location of capsid assembly . We found that, after expression of recombinant VP1 in Escherichia coli, the prokaryotic hsp70 chaperone DnaK copurified with the VP1 C-terminal domain that links pentamers in an assembled capsid . When stably bound to VP1, DnaK inhibited in vitro assembly induced by calcium . However, in the presence of ATP, the hsp70 chaperone system comprised of DnaK, DnaJ, and GrpE assembled VP1 into uniform capsids without requiring calcium . Chaperone-mediated assembly was similarly catalyzed by the eukaryotic hsc70 protein, in combination with the J-domain function of the simian virus 40 large T-antigen protein . Thus, polyomavirus capsid assembly can be recapitulated with high-fidelity in vitro using either prokaryotic or eukaryotic hsp70 chaperone systems, thereby supporting a role for cellular chaperones in the in vivo regulation of virion assembly. Hum Mol Genet, 2003 Oct 15, 12(20), 2693 - 702 Epub 2003 Aug 19. Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency; Antonicka H et al.; Deficiencies in the activity of cytochrome c oxidase (COX) are an important cause of autosomal recessive respiratory chain disorders . Patients with isolated COX deficiency are clinically and genetically heterogeneous, and mutations in several different assembly factors have been found to cause specific clinical phenotypes . Two of the most common clinical presentations, Leigh Syndrome and hypertrophic cardiomyopathy, have so far only been associated with mutations in SURF1 or SCO2 and COX15, respectively . Here we show that expression of COX10 from a retroviral vector complements the COX deficiency in a patient with anemia and Leigh Syndrome, and in a patient with anemia, sensorineural deafness and fatal infantile hypertrophic cardiomyopathy . A partial rescue was also obtained following microcell-mediated transfer of mouse chromosomes into patient fibroblasts . COX10 functions in the first step of the mitochondrial heme A biosynthetic pathway, catalyzing the conversion of protoheme (heme B) to heme O via the farnesylation of a vinyl group at position C2 . Heme A content was reduced in mitochondria from patient muscle and fibroblasts in proportion to the reduction in COX enzyme activity and the amount of fully assembled enzyme . Mutation analysis of COX10 identified four different missense alleles, predicting amino acid substitutions at evolutionarily conserved residues . A topological model places these residues in regions of the protein shown to have important catalytic functions by mutation analysis of a prokaryotic ortholog . Mutations in COX10 have previously been reported in a single family with tubulopathy and leukodystrophy . This study shows that mutations in this gene can cause nearly the full range of clinical phenotypes associated with early onset isolated COX deficiency. Acta Crystallogr D Biol Crystallogr, 2003 Sep, 59(Pt 9), 1676 - 8 Epub 2003 Aug 19. Structure and oligomeric state of the mammalian tumour-associated antigen UK114; Deriu D et al.; The tumour-associated antigen UK114, isolated from goat liver, belongs to the YER057c/YIL051c/YjgF protein family, which has members in both the prokaryotes and eukaryotes . The crystal structure of a mammalian representative, goat UK114, was determined, revealing a trimeric arrangement in the crystal . It was confirmed by ultracentrifugation that UK114 is a trimer in solution . These results are in agreement with the published structures of homologues from unicellular organisms, but contrast with those reported for the rat homologue of UK114, for which a dimeric quaternary structure was proposed. Biochem J, 2003 Oct 15, 375(Pt 2), 231 - 46 The unique features of glycolytic pathways in Archaea; Verhees CH et al.; An early divergence in evolution has resulted in two prokaryotic domains, the Bacteria and the Archaea . Whereas the central metabolic routes of bacteria and eukaryotes are generally well-conserved, variant pathways have developed in Archaea involving several novel enzymes with a distinct control . A spectacular example of convergent evolution concerns the glucose-degrading pathways of saccharolytic archaea . The identification, characterization and comparison of the glycolytic enzymes of a variety of phylogenetic lineages have revealed a mosaic of canonical and novel enzymes in the archaeal variants of the Embden-Meyerhof and the Entner-Doudoroff pathways . By means of integrating results from biochemical and genetic studies with recently obtained comparative and functional genomics data, the structure and function of the archaeal glycolytic routes, the participating enzymes and their regulation are re-evaluated. World J Gastroenterol, 2003 Aug, 9(8), 1756 - 61 Construction and characterization of bivalent vaccine candidate expressing HspA and M(r)18,000 OMP from Helicobacter pylori; Jiang Z et al.; AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with M(r)18,000 and heat shock protein A (HspA) from Helicobacter pylori (H . pylori) in E . coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H . pylori infection . METHODS: The target gene of HspA was amplified from H . pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn I, BamH I simultaneously . The recombinant vector was used to sequence, and then together with pET32a (+)/Omp(18), digested by restrictive endonuclease enzyme Hind III and BamH I simultaneously . pET32a(+)/HspA and Omp(18) were recovered from 1 % agarose gel by gel kit, and ligated with T(4) ligase by BamH I digested viscidity end . The recombinant plasmid of pET32a(+)/HspA/Omp(18) was transformed and expressed in E . coli BL21 (DE3) under induction of IPTG . After purification, its antigenicity of the fusion protein was detected by Western blot . RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues . Compared with GenBank reported by Tomb et al, there were 1.3 % and 1.4 % differences in obtained H . pylori nucleotide sequence and amino acid residues, respectively . SDS-PAGE analysis showed that relative molecule mass (M(r)) of the expressed product was M(r) 51,000, M(r) of protein expressed by pET32a (+) was about M(r) 20,000, and soluble expression product accounted for 18.96 % of total bacterial protein . After purification with Ni(+2)-NTA agarose resins, the purification of recombinant fusion protein was about 95 % . Western blot showed that recombinant fusion protein could be recognized by the patients' serum infected with H . pylori and anti-Omp(18) monoclone, suggesting that this protein had good antigenicity . CONCLUSION: The gene coding for H . pylori M(r)18,000 OMP and HspA was cloned and expressed successfully . The results obtained lay the foundation for development of H . pylori protein vaccine and a quick diagnostic kit. World J Gastroenterol, 2003 Aug, 9(8), 1719 - 24 Isolation of a novel member of small G protein superfamily and its expression in colon cancer; Yan W et al.; AIM: APMCF1 is a novel human gene whose transcripts are up-regulated in apoptotic MCF-7 cells . In order to learn more about this gene's function in other tumors, we cloned its full length cDNA and prepared its polyclonal antibody to investigate its expression in colon cancers with immunohistochemistry . METHODS: With the method of 5' rapid amplification of cDNA end (RACE) and EST assembled in GenBank, we extended the length of APMCF1 at 5' end . Then the sequence encoding the APMCF1 protein was amplified by RT-PCR from the total RNA of apoptotic MCF-7 cells and cloned into the prokaryotic expression vector pGEX-KG to construct recombinant expression vector pGEX-APMCF1 . The GST-APMCF1 fusion protein was expressed in E . coli and used to immunize rabbits to get the rabbit anti-APMCF1 serum . The specificity of polyclonal anti-APMCF1 antibody was determined by Western blot . Then we investigated the expression of Apmcf1 in colon cancers and normal colonic mucosa with immunohistochemistry . RESULTS: A cDNA fragment with a length of 1 745 bp was obtained . APMCF1 was mapped to chromosome 3q22.2 and spanned at least 14.8 kb of genomic DNA with seven exons and six introns contained . Bioinformatic analysis showed the protein encoded by APMCF1 contained a small GTP-binding protein (G proteins) domain and was homologous to mouse signal recognition particle receptor beta(SRbeta) . A coding region covering 816 bp was cloned and polyclonal anti-APMCF1 antibody was prepared successfully . The immunohistochemistry study showed that APMCF1 had a strong expression in colon cancer . CONCLUSION: APMCF1 may be the gene coding human signal recognition particle receptor beta and belongs to the small-G protein superfamily . Its strong expression pattern in colon cancer suggests it may play a role in colon cancer development. J Gen Virol, 2003 Sep, 84(Pt 9), 2531 - 44 Phylogenetic analysis and possible function of bro-like genes, a multigene family widespread among large double-stranded DNA viruses of invertebrates and bacteria; Bideshi DK et al.; Baculovirus repeated open reading frame (bro) genes and their relatives constitute a multigene family, typically with multiple copies per genome, known to occur among certain insect dsDNA viruses and bacteriophages . Little is known about the evolutionary history and function of the proteins encoded by these genes . Here we have shown that bro and bro-like (bro-l) genes occur among viruses of two additional invertebrate viral families, Ascoviridae and Iridoviridae, and in prokaryotic class II transposons . Analysis of over 100 sequences showed that the N-terminal region, consisting of two subdomains, is the most conserved region and contains a DNA-binding motif that has been characterized previously . Phylogenetic analysis indicated that these proteins are distributed among eight groups, Groups 1-7 consisting of invertebrate virus proteins and Group 8 of proteins in bacteriophages and bacterial transposons . No bro genes were identified in databases of invertebrate or vertebrate genomes, vertebrate viruses and transposons, nor in prokaryotic genomes, except in prophages or transposons of the latter . The phylogenetic relationship between bro genes suggests that they have resulted from recombination of viral genomes that allowed the duplication and loss of genes, but also the acquisition of genes by horizontal transfer over evolutionary time . In addition, the maintenance and diversity of bro-l genes in different types of invertebrate dsDNA viruses, but not in vertebrate viruses, suggests that these proteins play an important role in invertebrate virus biology . Experiments with the unique orf2 bro gene of Autographa californica multicapsid nucleopolyhedrovirus showed that it is not required for replication, but may enhance replication during the occlusion phase of reproduction. Wei Sheng Yan Jiu, 2003 May, 32(3), 236 - 9 {Human liver glutathione-S-transferase M1 gene cloning and temperature-dependent expression in E . coli}; Chen P et al.; In order to study the temperature-dependent expression of human liver glutathione-S-transferase M1 gene in prokaryotic cells, the recombinant of prokaryotic expression vector pBV220 with human glutathione-S-transferase M1 gene was constructed . The recombined plasmid pBV220-hGSTM1 was verified with PCR, restriction analysis and sequencing . It's expression was induced with temperature-dependent in 42 degrees C and the expressed non-fusion protein in HD5 alpha, with molecular weight of about 28 kDa, was about 28.3% of the total cell protein determined by SDS-PAGE . The sequence of human liver glutathione-S-transferase M1 gene was verified to be correctly recombined with pBV220 compared with the same sequence in Gene Bank, and code 619 C-->A, the amino acid changed from Pro to Thr was observed . The recombined plasmid pBV220-hGSTM1 may be applicable in toxicological and pharmacogenetical studies. Antibiot Khimioter, 2003, 48(3), 11 - 6 {A-Factor as a selective agent for isolation of the soil Gram-negative bacterium strain--the producent of an antibacterial antibiotic}; Gruzina VD et al.; It was shown the stimulating role of A-factor on soil prokaryotes growth . Soil sample culturing on agar medium, containing A-factor, resulted in the colony forming units (CFU) increasing in comparison with culturing on the medium without this regulator . Gram-negative bacteria were the reason of CFU increasing; previously the effect of A-factor on bacteria of this group was not shown . Gram-negative rod bacterial strain No . 35 was isolated and shown that CFU number was approximately twice increased at A-factor concentration in agar medium 2 and 7 mcg/ml; high A-factor concentration up to 28 mcg/ml was not effective . Isolated strain No . 35 is the producer of antibiotic, effective against gram-positive bacteria. Vet Res, 2003 Jul-Aug, 34(4), 361 - 77 Health hazards for terrestrial vertebrates from toxic cyanobacteria in surface water ecosystems; Briand JF et al.; Toxigenic cyanobacteria are photosynthetic prokaryotes that are most often recognized in marine and freshwater systems, such as lakes, ponds, rivers, and estuaries . When environmental conditions (such as light, nutrients, water column stability, etc.) are suitable for their growth, cyanobacteria may proliferate and form toxic blooms in the upper, sunlit layers . The biology and ecology of cyanobacteria have been extensively studied throughout the world during the last two decades, but we still know little about the factors and processes involved in regulating toxin production for many cyanobacterial species . In this minireview, we discuss these microorganisms, and more especially the toxins they produce, as a potential and important health risk for wild and domestic animals. Biochemistry, 2003 Aug 19, 42(32), 9804 - 12 Interaction of amoebapores and NK-lysin with symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers; Gutsmann T et al.; Amoebapores from protozoan parasite Entamoeba histolytica and NK-lysin of porcine cytotoxic lymphocytes belong to the same family of saposin-like proteins . In addition to the structural similarity, amoebapores and NK-lysin are both highly effective against prokaryotic and eukaryotic target cells in that they permeabilize the target cell membranes . Here, we have investigated in detail the protein/lipid interaction for the three isoforms of amoebapore and NK-lysin . Results obtained from electrical measurements on planar bilayer membranes, including reconstitution models of the lipid matrix of the outer membrane of Escherichia coli and phospholipid membranes, fluorescence energy transfer spectroscopy with liposomes, and monolayer measurements on a Langmuir trough, provided information on lipid preferences, pH dependences, and membrane interaction mechanisms . The three amoebapores led to the formation of transient pores with similar characteristics in conductance, sublevels, and lifetime for the different isoforms . The conductance of the pores was dependent on the polarity of the applied clamp voltage, and the distribution of the sublevels was affected by the value of the clamp voltage . The size of the pores and distribution of conductance sublevels differed between symmetric phospholipid and asymmetric lipopolysaccharide/phospholipid bilayers . Notably, NK-lysin caused the formation of well-defined pores, which were lipid- and voltage-dependent, and their characteristics differed from those induced by amoebapores; e.g., the protein concentration necessary to induce pore formation was 20 times higher . The biophysical data give important information on the mode of action of these small effector proteins, which may further lead to a better understanding of peptide-membrane interactions in general. Sichuan Da Xue Xue Bao Yi Xue Ban, 2003 Jul, 34(3), 390 - 4 {Construction of a site-directed mutant of FALL-39 and antibacterial activity of its E . coli-based product}; Yang Y et al.; OBJECTIVE: Constructing a point-directed mutant of FALL-39 and prokaryotic expressive vector, and determining its antibacterial activity . METHODS: A two-step polymerase chain reaction (PCR) was used for the site-directed mutagenesis . Two sets of primers (P1, P2, P3, P4) were designed according to FALL-39 gene sequence and mismatch was introduced into P2 for a substitution of AAG for AAT at position 32 . Mutagenesis was performed in a two-step PCR, the amplified fragments from the second PCR, which contain the mutation site, were subcloned into the vector PGEX lambda 1T and verified by sequencing analysis . Antibacterial activities of the E . coli-based product against E . coli and P . aeruginosa were detected by using minimal inhibitory concentration (MIC), minimal effective concentration (MEC) and minimal bactericidal concentration (MBC) . RESULTS: A mutant of FALL-39, whose AAT at position 32 is substituted by AAG, was obtained by the two-step PCR . The antibacterial activities of the E . coli-based product of the recombinant FALL-39-Lys32 against E . coli ML-35p and P . aeruginosa ATCC27853 were much stronger than those of FALL-39 . MIC and MBC of the FALL-39 against E . coli were 18.75 micrograms/ml and 37.50 micrograms/ml, whereas those of FALL-39-Lys32, 10.94 micrograms/ml and 21.88 micrograms/ml respectively . CONCLUSION: A point-directed FALL-39-Lys32 mutant was obtained by using two-step PCR and its antibacterial activity was increased, suggesting that increased cationic FALL-39 mutant might enhance its antibacterial activity. Proteins, 2003 Sep 1, 52(4), 585 - 97 Survey for g-proteins in the prokaryotic genomes: prediction of functional roles based on classification; Pandit SB et al.; The members of the family of G-proteins are characterized by their ability to bind and hydrolyze guanosine triphosphate (GTP) to guanosine diphosphate (GDP) . Despite a common biochemical function of GTP hydrolysis shared among the members of the family of G-proteins, they are associated with diverse biological roles . The current work describes the identification and detailed analysis of the putative G-proteins encoded in the completely sequenced prokaryotic genomes . Inferences on the biological roles of these G-proteins have been obtained by their classification into known functional subfamilies . We have identified 497 G-proteins in 42 genomes . Seven small GTP-binding protein homologues have been identified in prokaryotes with at least two of the diagnostic sequence motifs of G-proteins conserved . The translation factors have the largest representation (234 sequences) and are found to be ubiquitous, which is consistent with their critical role in protein synthesis . The GTP_OBG subfamily comprises of 79 sequences in our dataset . A total of 177 sequences belong to the subfamily of GTPase of unknown function and 154 of these could be associated with domains of known functions such as cell cycle regulation and t-RNA modification . The large GTP-binding proteins and the alpha-subunit of heterotrimeric G-proteins are not detected in the genomes of the prokaryotes surveyed . Science, 2003 Aug 8, 301(5634), 790 - 3 Prokaryotic chromosomes and disease; Hacker J et al.; Recent insights into bacterial genome organization and function have improved our understanding of the nature of pathogenic bacteria and their ability to cause disease . It is becoming increasingly clear that the bacterial chromosome constantly undergoes structural changes due to gene acquisition and loss, recombination, and mutational events that have an impact on the pathogenic potential of the bacterium . Even though the bacterial genome includes additional genetic elements, the chromosome represents the most important entity in this context . Here, we will show that various processes of genomic instability have an influence on the many manifestations of infectious disease. Biochim Biophys Acta, 2003 Aug 18, 1605(1-3), 67 - 82 A novel membrane-bound respiratory complex from Desulfovibrio desulfuricans ATCC 27774; Pires RH et al.; In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm . The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized . In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774 . This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers . The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6 . Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain . EPR studies of the as-isolated and dithionite-reduced complex support the presence of a {3Fe-4S}(1+/0) center and at least four {4Fe-4S}(2+/1+) centers . Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr) . The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC . Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS . The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes. Structure (Camb), 2003 Aug, 11(8), 961 - 72 The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy; Ireton GC et al.; Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway . The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors . The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively . The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase . The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates. Chin Med Sci J, 2002 Jun, 17(2), 68 - 72 The coexpression of the preS1 (1-42) and the core (1-144) antigen of HBV in E . coli; Zhao Y et al.; OBJECTIVE: To study the therapeutic T cell vaccine for the treatment of chronic hepatitis B by improving the cellular immunization of HBsAg vaccine with the coexpression of the preS1 (1-42) and the Core (1-144) antigen of HBV in E . coli . METHODS: The genes of HBcAg (1-144) and preS1 (1-42) were amplified and fused by PCR . This fused gene was inserted in the prokaryotic expression vector pET-11d and expressed in E . coli . RESULTS: It was showed by SDS-PAGE that the protein molecular weight of the coexpression product was about 20 kD, 20% of all bacteria protein . The monoclonal antibodies against core and preS1 antibody could react with this fused protein by Western-blot technique respectively . The fused gene was verified by sequencing . Under the immune electron microscopy, this fused protein is typical particle of HBcAg but in an aggregated form . CONCLUSION: The results might aid for studying T cell immunotherapeutic vaccine for chronic hepatitis B. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2003 Apr, 25(2), 181 - 4 {Gene clone and its characteristics on band 7-like protein in Plasmodium falciparum FCC1/HN}; Zhang L et al.; OBJECTIVE: To identify and clone the gene named pfstom gene which encoding the protein belonging to band 7 family and to do primary research on its function . METHODS: Based on the finished data in international public malaria database, coding sequence of pfstom cDNA was obtained by RT-PCR from FCC1/HN . Its phylogenetic profiles and the homogeny were analyzed by some softwares . After Prokaryotic expression, C terminal of Pfstom protein was expressed by Pet30a system . Recombinant Pfstom protein was used to immol/Lunize rabbit and then serum was harvested and the IgG was purified for Western blot . RESULTS: The coding sequence of pfstom is 1,125 bp which encoding 374 amino acids with C-terminal fragment being homogenous to stomatin-like protein which belongs to band 7 family . Phylogenetic profiles analysis revealed its homogeny to stomatin . Western blot showed its stage-specific expression in trophozoite . CONCLUSION: Pfstom belongs to band-7 family . It was expressed specifically in trophozoite in erythrocyte stage of plasmodium falciparum . It was not expressed in ring stage . And it is membrane-related protein . All these results provided the foundation for further research on pfstom. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Jun, 24(3), 250 - 3 {Cloning, expression and purification of neural specific HuD cDNA}; Chen JH et al.; OBJECTIVE: To prokaryoticly express and purify HuD protein and its RNA recognition motifs . METHODS: HuD protein was prokaryoticly expressed and purified by molecular cloning technology . Its biologic activity was testified by Western Blot . RESULTS: Purified HuD protein and its RNA recognized motifs were observed . CONCLUSIONS: The result might aid for basic research and clinical application. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2002 Jun, 24(3), 246 - 9 {The cloning and prokaryotic expression of human tumor necrosis factor like protein}; Hu XY et al.; OBJECTIVE: To identify and clone the gene encoding human TNFLP (tumor necrosis factor like protein) for some functional study on TNFLP . METHODS: The full-length cDNA of TNFLP was isolated from fetal brain cDNA library . Several kinds of software were used to analyze nucleotide sequence and amino acid sequence of TNFLP . TNFLP mRNA distribution was identified by Northern blot . TNFLP-C and TNFLP-N were expressed in E . coli with GST expression system . RESULTS: The cDNA of human TNFLP was 2,112 bp, which encoded protein of 208 amino acid . Hydrophobility analysis found there were two hydrophobility regions of human TNFLP . TNFLP-C (112-207 amino acid) and mouse TNF-alpha were homologous . The identity of their amino acid sequence was 42% . Moreover, both of them had a motif-TYKRL . TNFLP was located in chromosome 16 . Human TNFLP was widely expressed in various human tissues . Northern blot showed TNFLP was highly expressed in heart, brain and spleen, only one transcript can be seen . GST-TNFLP-C and GST-TNFLP-N fusion proteins were obtained . CONCLUSIONS: Tissue expression spectrum of TNFLP and prokaryotic expression of TNFLP have been done, which establish the base for the functional analysis of TNFLP. Microbiology, 2003 Aug, 149(Pt 8), 2243 - 50 Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome; Miyata S et al.; Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes . Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects . Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene . Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations . The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity . Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity . This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome. J Biol Chem, 2003 Oct 17, 278(42), 40771 - 7 Epub 2003 Aug 06. In vivo assembly of phage phi 29 replication protein p1 into membrane-associated multimeric structures; Serrano-Heras G et al.; The mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown . In the case of the Bacillus subtilis phage 29, the viral protein p1 enhances the rate of in vivo viral DNA replication . Previous work showed that p1 generates highly ordered structures in vitro . We now show that protein p1, like integral membrane proteins, has an amphiphilic nature . Furthermore, immunoelectron microscopy studies reveal that p1 has a peripheral subcellular location . By combining in vivo chemical cross-linking and cell fractionation techniques, we also demonstrate that p1 assembles in infected cells into multimeric structures that are associated with the bacterial membrane . These structures exist both during viral DNA replication and when 29 DNA synthesis is blocked due to the lack of viral replisome components . In addition, protein p1 encoded by plasmid generates membrane-associated multimers and supports DNA replication of a p1-lacking mutant phage, suggesting that the pre-assembled structures are functional . We propose that a phage structure assembled on the cell membrane provides a specific site for 29 DNA replication. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2000 Jun, 22(3), 227 - 31 {Coexpression of the preS1(1-42) and the Core(1-144) antigen of HBV in E . coli}; Zhao Y et al.; OBJECTIVE: To study the therapeutic T cell vaccine for the treatment of chronic hepatitis B . METHODS: The genes of HBcAg (1-144) and preS1 Ag (1-42) were amplified and fused by PCR . This fusion gene was inserted in the prokaryotic expression vector pET-11d and expressed in E . coli . RESULTS: It was showed by SDS-PAGE that the protein molecular weight of the coexpression product was about 20,000, twenty percent of all bacteria protein . The monoclone antibody against Core and preS1 antigen could react with this fused protein by Western-blot technique respectively . The fused gene was verified by sequencing . Under the immune electron microscopy, this fused protein is a typical particle of HBcAg but in an aggregated form . CONCLUSIONS: The results may aid for studying T cell immunotherapeutic vaccine to chronic hepatitis B. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 2000 Dec, 22(6), 521 - 4 {Identification