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At least 397 records · Page 22Linked to original sources
PubMed2004

Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C.

An scFv has been engineered to bind carcinoembryonic antigen (CEA) with a dissociation half-time >4 days at 37 degrees C. Two mutations responsible for this affinity increase were isolated by screening yeast surface-displayed mutant libraries by flow cytometry. Soluble expression of the mutant scFv in a yeast secretion system was increased 100-fold by screening mutant libraries for improved yeast surface display level. This scFv will be useful as a limiting case for evaluating the significance of affinity in tumor targeting to non-internalizing antigens.

Carcinoembryonic Antigen↗
PubMed1998

A chitin-binding domain in a marine bacterial chitinase and other microbial chitinases: implications for the ecology and evolution of 1,4-beta-glycanases.

To examine the ecology and evolution of microbial chitinases, especially the chitin-binding domain, one of the chitinase genes (chiA) from the marine bacterium Vibrio harveyi was analysed. The deduced amino acid sequence of ChiA is not very similar overall to other proteins, except for two regions, the putative catalytic and chitin-binding domains. Among all bacterial chitinases sequenced to date, there is no relationship between percentage similarity of catalytic domains and chitin-binding domains in pairwise comparisons, suggesting that these two domains have evolved separately. The chitin-binding domain appears to be evolutionarily conserved among many bacterial chitinases and is also somewhat similar to cellulose-binding domains found in microbial cellulases and xylanases. To investigate the role of the chitin-binding domain, clones producing versions of ChiA with or without this domain were examined. One version with the domain (ChiA1) bound to and hydrolysed chitin, whereas a truncated ChiA without the putative chitin-binding domain (ChiA2) did not bind to chitin, but it could hydrolyse chitin, although not as well. ChiA1 diffused more slowly in agarose containing colloidal chitin than ChiA2, but diffusion of the two proteins in agarose without colloidal chitin was similar. These results indicate that the chitin-binding domain helps determine the movement of chitinase along N-acetylglucosamine strands and within environments containing chitin.

Amino Acid Sequence↗
PubMed1985

[Organization of the Escherichia coli genome].

A review of literature data reveals that for the last years, the molecular biology techniques have been of an increasing use in the study of the Escherichia coli genome, having supplemented the standard genetic mapping. For the proper understanding of the Escherichia coli genome organization, recombinational events occurring in the course of evolution should be considered. The bacterial genome seems to carry traces of both "long-term" evolution, possibly responsible for appearance of the bacterial cell itself, and "current" evolution, consisting mainly of periodic genome entering by new plasmid-originated genes. It is supposed that in the process of stabilization within a genome, every new gene undergoes a stage of the "transgene", that is the gene situated in a transposon on the chromosome. In parallel with integration of new genes into the genome, some genes deleting should also take place. The formation of deletions could occur by unequal crossing over in segments of direct homologous repeats which seem to be ordinarily revealed in the experimental study of the tandem gene duplications.

Chromosome Mapping↗
PubMed2003

The gene cassette metagenome is a basic resource for bacterial genome evolution.

Lateral gene transfer has been proposed as a fundamental process underlying bacterial diversity. Transposons, plasmids and phage are widespread and have been shown to significantly contribute to lateral gene transfer. However, the processes by which disparate genes are assembled and integrated into the host regulatory network to yield new phenotypes are poorly known. Recent discoveries about the integron/gene cassette system indicate it has the potential to play a role in this process. Gene cassettes are small mobile elements typically consisting of a promoterless orf and a recombination site. Integrons are capable of acquisition and re-arrangement of gene cassettes and of the expression of their associated genes. The potential of the integron/gene cassette system is thus largely determined by the diversity contained within the cassette pool and the rate at which integrons sample this pool. We show here using a polymerase chain reaction (PCR) approach by which the environmental gene cassette (EGC) metagenome can be directly sampled that this metagenome contains both protein-coding and non-protein coding genes. Environmental gene cassette-associated recombination sites showed greater diversity than previously seen in integron arrays. Class 1 integrons were shown to be capable of accessing this gene pool through tests of recombinational activity with a representative range of EGCs. We propose that gene cassettes represent a vast, prepackaged genetic resource that could be thought of as a metagenomic template for bacterial evolution.

Base Sequence↗
PubMed1998

The path from the RNA world.

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA --> RNP --> protein, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the "introns-first" theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a "eukaryotic-like" genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome --> eukaryotic-like DNA genome --> prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome --> eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA --> RNP --> protein. A likely molecular mechanism, "plasmid transfer," is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history.

Archaea↗
PubMed2000

Parallel evolution of virulence in pathogenic Escherichia coli.

The mechanisms underlying the evolution and emergence of new bacterial pathogens are not well understood. To elucidate the evolution of pathogenic Escherichia coli strains, here we sequenced seven housekeeping genes to build a phylogenetic tree and trace the history of the acquisition of virulence genes. Compatibility analysis indicates that more than 70% of the informative sites agree with a single phylogeny, suggesting that recombination has not completely obscured the remnants of ancestral chromosomes. On the basis of the rate of synonymous substitution for E. coli and Salmonella enterica (4.7 x 10(-9) per site per year), the radiation of clones began about 9 million years ago and the highly virulent pathogen responsible for epidemics of food poisoning, E. coli O157:H7, separated from a common ancestor of E. coli K-12 as long as 4.5 million years ago. Phylogenetic analysis reveals that old lineages of E. coli have acquired the same virulence factors in parallel, including a pathogenicity island involved in intestinal adhesion, a plasmid-borne haemolysin, and phage-encoded Shiga toxins. Such parallel evolution indicates that natural selection has favoured an ordered acquisition of genes and the progressive build-up of molecular mechanisms that increase virulence.

DNA, Bacterial↗
PubMed2006

Static recipient cells as reservoirs of antibiotic resistance during antibiotic therapy.

How does taking the full course of antibiotics prevent antibiotic resistant bacteria establishing in patients? We address this question by testing the possibility that horizontal/lateral gene transfer (HGT) is critical for the accumulation of the antibiotic-resistance phenotype while bacteria are under antibiotic stress. Most antibiotics prevent bacterial reproduction, some by preventing de novo gene expression. Nevertheless, in some cases and at some concentrations, the effects of most antibiotics on gene expression may not be irreversible. If the stress is removed before the bacteria are cleared from the patients by normal turnover, gene expression restarts, converting the residual population to phenotypic resistance. Using mathematical models we investigate how static recipients of resistance genes carried by plasmids accumulate resistance genes, and how specifically an environment cycling between presence and absence of the antibiotic uniquely favors the evolution of horizontally mobile resistance genes. We found that the presence of static recipients can substantially increase the persistence of the plasmid and that this effect is most pronounced when the cost of carriage of the plasmid decreases the cell's growth rate by as much as a half or more. In addition, plasmid persistence can be enhanced even when conjugation rates are as low as half the rate required for the plasmid to persist as a parasite on its own.

Anti-Bacterial Agents↗
PubMed2005

Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1.

We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host's immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group.

Amino Acid Sequence↗
PubMed2002

Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii.

To determine whether the widespread clinical use of beta-lactams has been selective for Citrobacter freundii-derived alleles of plasmid ampC genes, we generated a Bayesian consensus phylogeny of the published ampC sequences and compared the MICs of 16 beta-lactam antibiotics for Escherichia coli strains containing cloned copies of the C. freundii ampC alleles. We found that for the majority of compounds investigated, there has been essentially no increase in beta-lactam resistance conferred by those alleles. We also found that ampC alleles from the chromosomes of two beta-lactam-sensitive C. freundii strains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing beta-lactam resistance in E. coli as were the plasmid-borne alleles from beta-lactam-resistant clinical isolates. These results suggest that selection for increased resistance to beta-lactam antibiotics has not been a significant force directing the evolution of the C. freundii ampC alleles found in beta-lactam-resistant clinical isolates.

Bacterial Proteins↗
PubMed1994

Organization and evolution of naphthalene catabolic pathways: sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NAH7 plasmid.

The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmid NAH7 was determined. This segment contains three large open reading frames designated nahQ', nahE, and nahD. The first of these is the 3' end of an open reading frame that has no known function, the second (993 bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), and the third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031). This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segment from the dox (dibenzothiophene oxidation) operon of Pseudomonas sp. strain C18, which encodes a pathway analogous to that encoded by NAH7. However, 84 bp downstream from nahD, the last gene in the nah operon, this homology ends. This 84-bp sequence at the downstream end of nah and dox homology has 76% homology to a sequence that occurs just upstream of the nah promoter in NAH7. These directly repeated 84-bp sequences thus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.

Amino Acid Sequence↗
PubMed2002

Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells.

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.

Biological Evolution↗
PubMed2000

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.

Bacterial Toxins↗
PubMedDate not supplied

[Cloning of the DNA fragment of a transgenic mouse containing an integrated recombinant plasmid].

The "plasmid rescue" method has been used to isolate the recombinant plasmid pMA3 fragment and flanking sequences from the transgenic mouse genome containing the fragment in integrated form. The "rescued" plasmid, pMAR1, lacks all virus sequences and retains only those regions of pBR322 that are responsible for the plasmid replication and Escherichia coli ampicillin resistance. The plasmid pMA3 deletion has occurred at its integration into the mouse genome after microinjection into the zygote. The integrated fragment of the plasmid is adjacent to the genome repeated sequence that is highly conservative in evolution.

Animals↗
PubMed1997

On the evolution of the single-subunit RNA polymerases.

Many eukaryotic nuclear genomes as well as mitochondrial plasmids contain genes displaying evident sequence similarity to those encoding the single-subunit RNA polymerase (ssRNAP) of bacteriophage T7 and its relatives. We have collected and aligned these ssRNAP sequences and have constructed unrooted phylogenetic trees that demonstrate the separation of ssRNAPs into three well-defined and nonoverlapping clusters (phage-encoded, nucleus-encoded, and plasmid-encoded). Our analyses indicate that these three subfamiles of T7-like RNAPs shared a common ancestor; however, the order in which the groups diverged cannot be inferred from available data. On the basis of structural similarities and mutational data, we suggest that the ancestral ssRNAP gene may have arisen via duplication and divergence of a DNA polymerase or reverse transcriptase gene. Considering the current phylogenetic distribution of ssRNAP sequences, we further suggest that the origin of the ancestral ssRNAP gene closely paralleled in time the introduction of mitochondria into eukaryotic cells through a eubacterial endosymbiosis.

Amino Acid Sequence↗
PubMed1994

A kalilo-like linear plasmid in Louisiana field isolates of the pseudohomothallic fungus Neurospora tetrasperma.

Two Louisiana strains of Neurospora tetrasperma contain a linear plasmid (LA-kalDNA) with a restriction map identical to the Hawaiian Neurospora intermedia senescence plasmid, kalDNA, but with termini 100 nucleotide pairs shorter. One of these strains also bore a circular plasmid similar to the Hawaiian circular plasmid Hanalei-2. One species probably acquired both plasmids from the other by horizontal transfer, at a time sufficiently distant for sequence divergence to take place. Many LA-kalDNA-bearing derivative strains senesced, but this plasmid does not guarantee senescence. Furthermore, LA-kalDNA does not insert into mtDNA. One senescent strain showed no LA-kalDNA. The plasmids are effectively transmitted via the pseudohomothallic sexual cycle. Single mating-type derivatives transmit plasmids maternally.

Biological Evolution↗