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[Use of gene fusion in the study of the interaction of ribosomal protein L45 with the Saccharomyces cerevisiae ribosome].

Cytoplasmic ribosomes from all cells have a set of highly acidic proteins (proteins A; pI less than 4.0). Proteins A are indispensable for ribosomal function since they mediate the interaction between GTP dependent translational factors and the ribosome. From gene cloning and DNA sequence analysis, during the past few years the primary structure of several prokaryotic and eukaryotic proteins A has been deduced. When their primary structure is compared, it is evident that these proteins have three well defined domains: firstly the aminus terminal domain, comprising the first 40 to 50 amino acids; secondly, the central domain, rich in non-polar amino acids and a high percent of alpha helix; and thirdly, the alpha carboxyl terminal domain, relatively acidic and highly conserved throughout evolution. In Saccharomyces cerevisiae the four genes coding for proteins A L45, L44, L44' and A1 have been cloned and sequenced. This enabled us to study the physiological role of each of the three domains in proteins A. Plasmid YEp45-1 encompassing a 596 base pairs stretch of the L45 genomic DNA fused to the LacZ gene was constructed and used to transform yeast cells. The chimeric gene expressed an hybrid protein in which the covalently linked beta-galactosidase enzyme follows after the first 75 amino acids from the alpha amino terminal domain of protein A L45. After cell fractionation of yeast transformants, beta-galactosidase activity was found specifically bound to the 80S ribosome and polysome particles. From these results we conclude that the amino terminal domain of protein L45 interacts to the ribosome. We postulate, by inference, that the carboxyl terminal domain of proteins A interacts with the GTP dependent cytoplasmic translational factors.

Amino Acid Sequence↗
PubMed1998

The epidemiology of Salmonella typhimurium in cattle: plasmid profile analysis of definitive phage type (DT) 204c.

During the period 1979-1991, Salmonella Typhimurium DT 204c was the cause of a major epidemic of salmonellosis in calves in the UK. Plasmid profile analysis of DT 204c isolates from England and Wales commenced in 1986 and isolates from all subsequent incidents were examined by this technique. Forty-three different plasmid profile types (PPTs) were detected, of which the commonest, designated type E, constituted 44.6-80.2% of the annual incidents during the study period. Some PPTs, e.g., F and P, were detected throughout most years of the study, whereas PPTs O and 6 persisted for short periods. Until 1984, most isolates were resistant to neomycin, but the subsequent predominant PP type E was sensitive to this antibacterial agent. It was concluded that during the epidemic there was an evolution of new genotypes, of which only some persisted; again, antibacterial resistance genes may be acquired or lost. The study demonstrated the value of PP typing for epidemiological studies.

Animals↗
PubMed1994

Extrachromosomal resistance in gram-negative organisms: the evolution of beta-lactamase.

beta-Lactamases are the major defense used by bacteria to overcome the effects of penicillins, cephalosporins and related beta-lactam antibiotics. In the antibiotic era, the enzymes have evolved to become more prevalent, to appear in new hosts, to be expressed at higher levels, to be acquired by plasmids and to change catalytic properties to increase affinity for what were meant to be nonhydrolysable substrates or to reduce affinity for beta-lactamase inhibitors.

Genes, Bacterial↗
PubMed1992

Cloning and sequencing of a plasmid-mediated erythromycin resistance determinant from Staphylococcus xylosus.

A 2.3-kb DNA fragment cloned from plasmid pCH200, the largest (52 kb) of four plasmids detected in Staphylococcus xylosus, was found to confer resistance to 14-membered ring macrolides in Bacillus subtilis and Staphylococcus aureus. DNA-sequence analysis of the fragment revealed the presence of an open-reading frame, the deduced product of which was identical to one of the two ATP-binding domains encoded by the macrolide/streptogramin-B-resistance gene msrA of Staphylococcus epidermidis. The observation that a polypeptide homologous to the C-terminus of MsrA is capable of mediating erythromycin resistance in the absence of the N-terminal region is of significance both to the evolution and functional activity of members of the ATP-binding transport super-gene family.

Amino Acid Sequence↗
PubMed1995

Differential DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters.

The plasmid-borne pheBA operon of Pseudomonas putida strain PaW85 allows growth of the host cells on phenol. The promoter of this operon is activated by the chromosomally encoded LysR-type regulator CatR, in the presence of the inducer cis,cis-muconate. cis,cis-muconate is an intermediate of catechol degradation by the chromosomally encoded ortho or beta-ketoadipate pathway. The catBC operon encodes two enzymes of the beta-ketoadipate pathway and also requires CatR and cis,cis-muconate for its expression. The promoters of the pheBA and catBC operons are highly homologous, and since both respond to CatR, it is likely that the pheBA promoter was recruited from the ancestral catBC promoter. Gel shift assays and DNase I footprinting have shown that the pheBA promoter has a higher binding affinity for CatR than the catBC promoter. Like the catBC promoter, the pheBA promoter forms two complexes (C1 and C2) with CatR in the absence of cis,cis-muconate, but only forms a single complex (C2) in the presence of cis,cis-muconate. Like the catBC promoter CatR repression binding site (RBS) and activation binding site (ABS) arrangement, the pheBA promoter demonstrates the presence of a 26 bp segment highly homologous to the RBS that is protected by CatR from DNase I digestion in the absence of the inducer. An additional 16 bp sequence, similar to the catBC promoter ABS, is protected only when the inducer cis-cis-muconate is present. The binding of CatR in absence of cis,cis-muconate bends the catBC and pheBA promoter regions to significantly different degrees, but CatR binding in the presence of cis,cis-muconate results in a similar degree of DNA bending. The evolutionary implications of the interactions of CatR with these two promoters are discussed.

Adipates↗
PubMed2002

[In vitro molecular evolution of humanized mouse ScFv specific against human cross-linked fibrin].

In early work at our laboratory, a mouse ScFv All specific against human cross-linked fibrin was picked out by using phage displaying technique, and it was useful in prepareing targeting thrombolytic agent. To reduce its immunogenicity, All was humanized according to the surface reshaping approach. In order to increase the affinity and specifity of humanized ScFv, the Mixed HCDR3 and LCDR3 library was constructed and selected, and five different humanized ScFv with better affinity or specifity than humanized ScFv were identified, but the affinity of these five humanized scFvs was still lower than parent mouse scFv All. In this study, in order to further increase the affinity of humanized scFv, the identified five different CDR3 mutant of humanized ScFvs were mutated and reassembled with the methods of error-prone PCR and DNA shuffling, then the shuffled ScFvs were cloned into plasmid pHB-1 HSCFV to construct ScFv library of 10(5). The ScFv library was displayed on the surface of phage and panned with antigen DD, then five ScFvs with better affinity or specifity were identified by using ScFv-alkaline phosphatase detection system which was established in our laboratory. Based on identified five better ScFv, the second round error-prone PCR and DNA shuffling program was taken out, a ScFv library of 10(6) was constructed and selected as before. And four humanized ScFv that had evidently better affinity than parent mouse ScFv was identified. This work laid a foundation for further research of targeting thrombolitic agent with low immunogenicity.

Animals↗
PubMed1982

Analysis of chromosomal homologies between two species of the subgenus Sophophora: D. miranda and D. melanogaster using cloned DNA segments.

Chromosomal homology between two species of the subgenus Sophophora, D. miranda and D. melanogaster, belonging to the obscura and the melanogaster group respectively, was probed by DNA in situ cross hybridizations. A set of recombinant plasmids with inserts derived from the D. melanogaster genome were cross hybridized to the D. miranda karyotype. Vice versa, recombinant Lambda phages isolated from a genomic D. miranda library were localized in D. miranda and probed for localization in D. melanogaster. In the main, the results support the homology relations proposed on the basis of cytogenetic data. However, the location of both tandemly repetitive genes tested, 5S RNA genes and the histone genes, is not in accordance with expectation. The 5S RNA genes, when probed with the D. melanogaster plasmid 12D8 (Artavanis-Tsakonas et al., 1977), were found to occur at two sites in both, D. miranda and D. pseudoobscura.

Animals↗
PubMed2005

Expression and purification of histidine-tagged bacteriophage T7 DNA polymerase.

The formation of inclusion bodies is a frequent consequence of high-level production of foreign protein in the cytoplasm of Escherichia coli. This phenomenon is also observed with bacteriophage T7 gene 5 protein, the phage-encoded subunit of T7 DNA polymerase, if expression is based on the T5 promoter/lac operator transcription-translation system present in a vector with ColE1 origin of replication. To avoid tedious procedures for recovering protein from insoluble aggregates, we studied the expression of T7 gene 5 protein using a series of E. coli strains, and optimized the yield of soluble, histidine-tagged (His-tagged) protein by varying the respective growth conditions (temperature, amount of inducer isopropyl-beta-d-thiogalactopyranoside, and presence of organic osmolytes). Although the expression levels in three different strains (BL21, SG13009, and XL1-Blue) were almost comparable with a given set of growth conditions, the yields of soluble protein differed markedly. The largest quantities of soluble, His-tagged T7 gene 5 protein were achieved using "cloning strain" XL1-Blue which benefitted significantly from the presence of sorbitol and glycine betaine-in contrast to the expression strains BL21 and SG13009. Purification of His-tagged T7 gene 5 protein was achieved using single-step metal-affinity chromatography that yielded large amounts of highly active polymerase (97% homogeneity). The application of this expression/purification approach represents not only a useful method to purify large quantities of T7 DNA polymerase for structural investigations but also, provides a fast and efficient protocol for the parallel purification of T7 DNA polymerase variants, e.g., for automated screenings or directed evolution experiments.

Bacteriophage T7↗
PubMed2005

Group-beneficial traits, frequency-dependent selection and genotypic diversity: an antibiotic resistance paradigm.

The evolution of group-beneficial traits potentially allows the survival of 'cheaters' that would otherwise be unfit. Here we describe experimental work on group-beneficial traits and the consequences of frequency-dependent selection in the context of bacterial antibiotic resistance. We constructed a 'self-limited antibiotic resistant' (SLAR) strain of Escherichia coli in which a TEM-1 beta-lactamase was anchored to the inner membrane. In pairwise competition experiments between the SLAR strain and ampicillin-sensitive strains, only the SLAR strain survived in the presence of ampicillin. We also constructed a 'shared antibiotic resistant' (SAR) strain in which TEM-1 beta-lactamase protected both the SAR strain and nearby sensitive cells, thus acting as a model for a genetically defined group-beneficial trait. In pairwise competition experiments of the SAR strain against two different sensitive strains of E. coli, we found that the sensitive strains maintained themselves at frequencies of 5-12% in the presence of ampicillin. When the relative cost of the SAR strain was lowered, its equilibrial frequency rose. Sensitive strains also arose from pure cultures of the SAR strain. In these cases, too, the sensitive 'cheaters' were maintained in ampicillin at frequencies comparable to those observed in the previous competitions. These results suggest that traits which benefit other group members can permit survival of genotypes that otherwise would be eliminated by natural selection, and allow the maintenance of greater genetic variation upon which evolution can operate.

Ampicillin↗
PubMed1992

Multiple repetitive elements and organization of the lux operons of luminescent terrestrial bacteria.

The complete nucleotide sequences of the luxA to luxE genes, as well as the flanking regions, were determined for the lux operons of two Xenorhabdus luminescens strains isolated from insects and humans. The nucleotide sequences of the corresponding lux genes (luxCDABE) were 85 to 90% identical but completely diverged 350 bp upstream of the first lux gene (luxC) and immediately downstream of the last lux gene (luxE). These results show that the luxG gene found immediately downstream of luxE in luminescent marine bacteria is missing at this location in terrestrial bacteria and raise the possibility that the lux operons are at different positions in the genomes of the X. luminescens strains. Four enteric repetitive intergenic consensus (ERIC) or intergenic repetitive unit (IRU) sequences of 126 bp were identified in the 7.7-kbp DNA fragment from the X.luminescens strain isolated from humans, providing the first example of multiple ERIC structures in the same operon including two ERIC structures at the same site. Only a single ERIC structure between luxB and luxE is present in the 7-kbp lux DNA from insects. Analysis of the genomic DNAs from five X. luminescens strains or isolates by polymerase chain reaction has demonstrated that an ERIC structure is between luxB and luxE in all of the strains, whereas only the strains isolated from humans had an ERIC structure between luxD and luxA. The results indicate that there has been insertion and/or deletion of multiple 126-bp repetitive elements in the lux operons of X.luminescens during evolution.

Amino Acid Sequence↗
PubMed1999

Inhibition of bacterial cell wall-induced leukocyte recruitment and hepatic granuloma formation by TGF-beta gene transfer.

Intraperitoneal injection of streptococcal cell walls (SCW) into Lewis rats results in dissemination of SCW to the liver, spleen, bone marrow, and peripheral joints. The uptake of SCW by Kupffer cells in the liver initiates a chain of events largely mediated by T lymphocytes and macrophages. Local synthesis and secretion of cytokines and growth factors in response to the persistent SCW lead to the evolution and maintenance of a chronic T cell-dependent granulomatous response and result in granuloma formation and irreversible hepatic fibrosis. In an attempt to impede the development of the chronic granulomatous lesions in the liver, we injected a plasmid DNA encoding TGF-beta 1 i.m. to the SCW animals to determine the effect of TGF-beta 1 gene transfer on the course of liver inflammation and fibrosis. A single injection of plasmid DNA encoding TGF-beta 1 resulted in virtual abolition of the development of the SCW-induced hepatic granuloma formation and matrix expansion. TGF-beta 1 DNA not only reduced key proinflammatory cytokines including TNF-alpha, IL-1 beta, IFN-gamma, and IL-18, but also inhibited both CXC and CC chemokine production, thereby blocking inflammatory cell recruitment and accumulation in the liver. Moreover, TGF-beta 1 gene delivery inhibited its own expression in the liver tissue, which is otherwise up-regulated in SCW-injected animals. Our study suggests that TGF-beta 1 gene transfer suppresses hepatic granuloma formation by blocking the recruitment of inflammatory cells to the liver, and thus may provide a new approach to the control of hepatic granulomatous and fibrotic diseases.

Animals↗
PubMed2002

Mobility of a restriction-modification system revealed by its genetic contexts in three hosts.

The flow of genes among prokaryotes plays a fundamental role in shaping bacterial evolution, and restriction-modification systems can modulate this flow. However, relatively little is known about the distribution and movement of restriction-modification systems themselves. We have isolated and characterized the genes for restriction-modification systems from two species of Salmonella, S. enterica serovar Paratyphi A and S. enterica serovar Bareilly. Both systems are closely related to the PvuII restriction-modification system and share its target specificity. In the case of S. enterica serovar Paratyphi A, the restriction endonuclease is inactive, apparently due to a mutation in the subunit interface region. Unlike the chromosomally located Salmonella systems, the PvuII system is plasmid borne. We have completed the sequence characterization of the PvuII plasmid pPvu1, originally from Proteus vulgaris, making this the first completely sequenced plasmid from the genus Proteus. Despite the pronounced similarity of the three restriction-modification systems, the flanking sequences in Proteus and Salmonella are completely different. The SptAI and SbaI genes lie between an equivalent pair of bacteriophage P4-related open reading frames, one of which is a putative integrase gene, while the PvuII genes are adjacent to a mob operon and a XerCD recombination (cer) site.

Amino Acid Sequence↗
PubMed2005

Improved solubility of TEV protease by directed evolution.

The efficiency and high specificity of tobacco etch virus (TEV) protease has made it widely used for cleavage of recombinant fusion proteins. However, the production of TEV protease in E. coli is hampered by low solubility. We have subjected the gene encoding TEV protease to directed evolution to improve the yield of soluble protein. Libraries of mutated genes obtained by error-prone PCR and gene shuffling were introduced into the Gateway cloning system for facilitated transfer between vectors for screening, purification, or other applications. Fluorescence based in vivo solubility screening was carried out by cloning the libraries into a plasmid encoding a C-terminal GFP fusion. Mutant genes giving rise to high GFP fluorescence intensity indicating high levels of soluble TEV-GFP were subsequently transferred to a vector providing a C-terminal histidine tag for expression, purification, and activity tests of mutated TEV. We identified a mutant, TEV(SH), in which three amino acid substitutions result in a five-fold increase in the yield of purified protease with retained activity.

Amino Acid Substitution↗
PubMed1997

Molecular evolution of multiply-antibiotic-resistant staphylococci.

Methicillin-resistant Staphylococcus aureus (MRSA) is an intractable nosocomial pathogen. The chemotherapeutic intransigence of this organism stems from its predilection to antimicrobial resistance as a consequential response to selective pressures prevailing in the clinical environment. MRSA isolates are frequently resistant to all practicable antimicrobials except the glycopeptide, vancomycin. Although antimicrobial resistance sometimes arises via chromosomal mutation, the emergence of multiply-antibiotic-resistant staphylococci is primarily due to the acquisition of pre-existent resistance genes; such determinants can be encoded chromosomally or by plasmids and are often associated with transposons or insertion sequences. Clinical staphylococci commonly carry one or more plasmids, ranging from small replicons that are phenotypically cryptic or contain only a single resistance gene, to larger episomes that possess several such determinants and sometimes additionally encode systems that mediate their own conjugative transmission and the mobilization of other plasmids. The detection of closely related plasmids, elements and/or genes in other hosts, including coagulase-negative staphylococci and enterococci, attests to interspecific and intergeneric genetic exchange facilitated by mobile genetic elements and DNA transfer mechanisms. The extended genetic reservoir accessible to staphylococci afforded by such horizontal gene flux is fundamental to the acquisition, maintenance and dissemination of staphylococcal antimicrobial resistance in general, and multiresistance in particular.

Drug Resistance, Microbial↗
PubMed2003

Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria.

Gram-positive spore-forming entomopathogenic bacteria can utilize a large variety of protein toxins to help them invade, infect, and finally kill their hosts, through their action on the insect midgut. These toxins belong to a number of homology groups containing a diversity of protein structures and modes of action. In many cases, the toxins consist of unique folds or novel combinations of domains having known protein folds. Some of the toxins display a similar structure and mode of action to certain toxins of mammalian pathogens, suggesting a common evolutionary origin. Most of these toxins are produced in large amounts during sporulation and have the remarkable feature that they are localized in parasporal crystals. Localization of multiple toxin-encoding genes on plasmids together with mobilizable elements enables bacteria to shuffle their armory of toxins. Recombination between toxin genes and sequence divergence has resulted in a wide range of host specificities.

Bacillus↗
PubMed2000

Heat shock protein 60 sequence comparisons: duplications, lateral transfer, and mitochondrial evolution.

Heat shock proteins 60 (GroEL) are highly expressed essential proteins in eubacterial genomes and in eukaryotic organelles. These chaperone proteins have been advanced as propitious marker sequences for tracing the evolution of mitochondrial (Mt) genomes. Similarities among HSP60 sequences based on significant segment pair alignment calculations are used to deduce associations of sequences taking into account GroEL functional/structural domain differences and to relate HSP60 duplications pervasive in alpha-proteobacterial lineages to the dynamics of lateral transfer and plasmid integration. Multiple alignments with consensuses are determined for 10 natural groups. The group consensuses sharpen the similarity contrasts among individual sequences. In particular, the Mt group matches best with the classical alpha-proteobacteria and closely with Rickettsia but significantly worse with the rickettsial groups Ehrlichia and Orientia. However, across broad protein sequence comparisons, there appears to be no consistent prokaryote whose protein sequences align best with animal Mt genomes. There are plausible scenarios indicating that the nuclear-encoded HSP60 (and HSP70) sequences functioning in Mt are results of lateral transfer and are probably derived from an alpha-proteobacterium. This hypothesis relates to the plethora of duplicated HSP60 sequences among the classical alpha-proteobacteria contrasted with no duplications of HSP60 among other clades of proteobacterial genomes. Evolutionary relations are confounded by differential selection pressures, convergence, variable mutational rates, site variability, and lateral gene transfer.

Bacteria↗