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Results for “plasmid evolution”

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PubMed1985

Cloning of cDNA sequences for an Artemia salina hnRNP protein: evidence for conservation through evolution.

A cDNA clone was isolated for Artemia salina protein HD40, a component of heterogenous nuclear ribonucleoproteins. Enriched Artemia 15S poly(A)+ RNA was used as a template and double-stranded cDNA sequences were inserted into the Pst I restriction endonuclease site of E. coli plasmid pBR322. Recombinant colonies were analyzed by positive hybrid selection of poly(A)+ RNA that directs the synthesis of protein HD40 in an in vitro assay. In vitro translation of the mRNA selected by recombinant clone 87HD yields a protein that is immunoprecipitated by anti-HD40 antibodies and that comigrates with authentic HD40 on gel electrophoresis. Partial proteolysis of protein HD40 and the in vitro translated product selected by clone 87HD produces the same peptide patterns. The size of the cloned insert is about 820 bp. The length of HD40 mRNA as determined by Northern blot analysis, is about 1500 nucleotides. Southern blot analysis performed with DNA of different species (plant, avian, mammal) shows cross-hybridizing bands when probed with clone 87HD DNA suggesting that the HD40 gene is evolutionarily conserved.

Animals↗

PubMed2000

Intraspecific phylogenetic congruence among multiple symbiont genomes.

Eukaryotes often form intimate endosymbioses with prokaryotic organisms. Cases in which these symbionts are transmitted cytoplasmically to host progeny create the potential for co-speciation or congruent evolution among the distinct genomes of these partners. If symbionts do not move horizontally between different eukaryotic hosts, strict phylogenetic congruence of their genomes is predicted and should extend to relationships within a single host species. Conversely, even rare 'host shifts' among closely related lineages should yield conflicting tree topologies at the intraspecific level. Here, we investigate the historical associations among four symbiotic genomes residing within an aphid host: the mitochondrial DNA of Uroleucon ambrosiae aphids, the bacterial chromosome of their Buchnera bacterial endosymbionts, and two plasmids associated with Buchnera. DNA sequence polymorphisms provided a significant phylogenetic signal and no homoplasy for each data set, yielding completely and significantly congruent phylogenies for these four genomes and no evidence of horizontal transmission. This study thus provides the first evidence for strictly vertical transmission and 'co-speciation' of symbiotic organisms at the intraspecific level, and represents the lowest phylogenetic level at which such coevolution has been demonstrated. These results may reflect the obligate nature of this intimate mutualism and indicate opportunities for adaptive coevolution among linked symbiont genomes.

Animals↗

PubMed1996

Long, polymorphic microsatellites in simple organisms.

We have examined the phylogenetic distribution of the longest, perfect microsatellites in GenBank. Despite the large contributions of model higher-eukaryotic organisms to GenBank, the selective cloning of long microsatellites from these organisms as genetic markers, and the relative lack of concentration on the microsatellites in lower eukaryotes and prokaryotes, we found that simple organisms, defined here as slime molds, fungi, protists, prokaryotes, viruses, organelles and plasmids, contributed 78 of the 375 examined sequences. These 78 simple-organism microsatellites are characterized predominantly by trinucleotide repeats, nearly half of which lie in exons, and in general show a bias towards A+T rich motifs. Simple-organism microsatellites represented more than once in GenBank displayed length polymorphisms when independent clones were compared. These facts collectively raise speculation as to the role of these 'junk' sequences in such highly economical genomes, especially when precise changes in long microsatellites are known to regulate critical virulence factors in several prokaryotes. Regardless of their biological significance, simple-organism microsatellites may provide a general source of molecular markers to track disease outbreaks and the evolution of microorganisms in unprecedented detail.

Animals↗

PubMed2003

Role of mobile DNA in the evolution of vancomycin-resistant Enterococcus faecalis.

The complete genome sequence of Enterococcus faecalis V583, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA. One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon. Three plasmids were identified, including two pheromone-sensing conjugative plasmids, one encoding a previously undescribed pheromone inhibitor. The apparent propensity for the incorporation of mobile elements probably contributed to the rapid acquisition and dissemination of drug resistance in the enterococci.

Adhesins, Bacterial↗

PubMed1981

Physical and functional mapping of Tn2603, a transposon encoding ampicillin, streptomycin, sulfonamide, and mercury resistance.

A map of cleavage sites for restriction endonuclease EcoRI, BamHI, HindIII, and SalI on Tn2603, a transposon encoding resistance to ampicillin, streptomycin, sulfonamide, and mercury, was constructed by an analysis of restriction cleavage patterns of plasmid pMK1.::Tn2603 and its deletion derivative. By cloning the fragments generated from pMK1.::Tn2603 with these restriction endonucleases to a pACYC184 plasmid vehicle, the regions necessary for expression of resistance were located on the restriction cleavage map of Tn2603. Ampicillin, streptomycin, and sulfonamide-resistance genes were mapped in a cluster on the region between the center and the right and the mercury-resistance gene was located to the left of the map. The final functional map of Tn2603 was compared with those of Tn4 and Tn21 and the evolutional relationships between them were discussed.

Chromosome Mapping↗

PubMed1996

RP4::Mu3A-mediated in vivo cloning and transfer of a chlorobiphenyl catabolic pathway.

Chromosomal DNA fragments encoding the ability to utilize biphenyl as sole carbon source (Bph+) were mobilized by means of plasmid RP4::Mu3A from strain JB1 (tentatively identified as Burkholderia sp.) to Alcaligenes eutrophus CH34 at a frequency of 10(-3) per transferred plasmid. The mobilized DNA integrated into the recipient chromosome or was recovered as catabolic prime plasmids. Three Bph+ prime plasmids were transferred from A. eutrophus to Escherichia coli and back to A. eutrophus without modification of the phenotype. The transferred Bph+ DNA segments allowed metabolism of biphenyl, 2-, 3- and 4-chlorobiphenyl, and diphenylmethane. Genes involved in biphenyl degradation were identified on the prime plasmids by DNA-DNA hybridization and by gene cloning. Bph+ prime plasmids were transferred to Burkholderia cepacia, Pseudomonas aeruginosa, Comamonas testosteroni and A. eutrophus and the catabolic genes were expressed in those hosts. Transfer of the plasmid to the 3-chlorobenzoate-degrading bacterium Pseudomonas sp. B13 allowed the recipient to mineralize 3-chlorobiphenyl. Other catabolic prime plasmids were obtained from JB1 by selection on m-hydroxybenzoate and tyrosine as carbon sources. 16S rRNA sequence data demonstrated that the in vivo transfer of bph was achieved between bacteria belonging to two different branches of the beta-Proteobacteria.

Alcaligenes↗

PubMed1993

Characterization of a cta/CDE operon-like genomic region encoding subunits I-III of the cytochrome c oxidase of the cyanobacterium Synechocystis PCC 6803.

Strong heterologous hybridization of a synthetic oligonucleotide probe of 17 bp originally used to clone subunit I of the Paracoccus denitrificans cytochrome c oxidase (M. Raitio, T. Jalli and M. Saraste (1987) EMBO J. 6, 2825-2833) to a single band was observed on Southern blots of Anacystis nidulans R2 (Synechococcus PCC 7942), Synechocystis PCC 6803, and Nostoc Mac PCC 8002 chromosomal DNA digests. Six pooled gene banks prepared from Synechocystis PCC 6803 contained regions that hybridized to the oligonucleotide (probe C) which is specifically directed toward the putative Cu-binding site VWAHHMY of subunit I. Two of these gene banks were transformed into Escherichia coli and screened for colonies hybridizing to probe C. Several clones were recovered, and one type of plasmid was identified from each gene bank. The two (overlapping) plasmids were called pDAUV1 and pDAUV2. A restriction map of the plasmids showed that the overlapping region contained an 80 bp PvuI-KpnI fragment binding to probe C. The two clones together permitted sequencing of the entire gene for cytochrome c oxidase subunit I from Synechocystis PCC 6803. Further systematic sequencing of approximately 1000 bp upstream and downstream each of the ctaD (subunit I) gene revealed the presence of two genes encoding subunits II (ctaC gene) and III (ctaE gene) due to conspicuous similarities to homologous genes from other cytochrome c oxidase-containing organisms. Yet, no indications of genes encoding additional subunits of the oxidase were found within the region sequenced.

Amino Acid Sequence↗

PubMed2006

SCALEs: multiscale analysis of library enrichment.

We report a genome-wide, multiscale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves (i) growth selections on a mixture of several different plasmid-based genomic libraries of defined insert sizes or SCALEs, (ii) microarray studies of enriched plasmid DNA, and a (iii) mathematical multiscale analysis that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multigene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in Escherichia coli by monitoring, in parallel, a population of >10(6) genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

DNA Fragmentation↗

PubMed1987

Molecular characterization of the resolvase gene, res, carried by a multicopy plasmid from Clostridium perfringens: common evolutionary origin for prokaryotic site-specific recombinases.

Clostridium perfringens strain CPN50 harbours a 10.2 kb plasmid known as pIP404 which, in addition to a set of UV-inducible genes involved in bacteriocin production, carries res, a gene probably encoding a site-specific recombinase. The RES protein is highly homologous to the resolvases of transposons from both Gram-negative and Gram-positive bacteria as well as enzymes involved in site-specific DNA inversion. A likely role for the RES protein would be to stabilize pIP404 by reducing the number of plasmid multimers resulting from homologous recombination. A putative resolution site for RES action was found overlapping the res promoter. Phylogenetic analysis of the primary structures of ten site-specific recombinases suggested a common descent and showed the RES protein to be closest to the resolvase encoded by Tn917 from Streptococcus faecalis.

Amino Acid Sequence↗

PubMed2002

[Construction and homologous detection of a DNA plasmid library specific for Z chromosome of quail].

A simple method was used to adapt a standard light microscope for the collection of quail Z chromosomes from mitotic-metaphase spreads. The microisolated chromosomes were subjected to proteinase K treatment in a collection drop to release DNA, which was then amplified using a degenerate oligonucleotide-primed PCR (DOP-PCR) strategy. Size distributions of the PCR products were analyzed by agarose gel electrophoresis, and smears of DNA revealed that ranged in size from 200-1 400 bp, without any evidence of preferential amplification. The second-round PCR products were cloned into pBluescript plasmids to construct a Z chromosome-specific DNA library. The size range of the cloned inserts was 200-1 400 bp. Using inserted fragments from the library as probes, chromosome painting was performed on quail chromosomes. The results showed that Z chromosomes of quail were completely covered by strong signals and there were little signals on other chromosomes. It was indicated that inserted DNA of the library was specific to the Z chromosome of quail. The library can be used as chromosome painting probe to detect conserved syntenic groups on the chromosomes of other related species and study mechanisms of sex-chromosomes evolution in birds.

Animals↗

PubMed1986

Broad geographical distribution of homologous erythromycin, kanamycin, and streptomycin resistance determinants among group D streptococci of human and animal origin.

The Emr, Kmr, and Smr determinants of the Streptococcus faecalis R plasmid pJH1 were cloned in Streptococcus sanguis with a streptococcal plasmid vector, pVA380-1. Each cloned determinant was used as a probe in hybridization reactions with dot blots containing plasmid-enriched DNA from 91 group D streptococcal isolates resistant to erythromycin, kanamycin, and streptomycin; the isolates were obtained from animal and human sources in a variety of geographical locations. Nearly 70% of the strains contained DNA that hybridized to each of the three resistance determinants from pJH1. Five plasmids mediating resistance to erythromycin, kanamycin, and streptomycin were examined in more detail. These plasmids varied in size between 26 and 105 kilobase pairs (kbp) and exhibited very different EcoRI restriction patterns. However, each plasmid contained the resistance determinants on a single 13- to 20-kbp EcoRI fragment. Southern blot hybridizations and additional restriction endonuclease digests revealed extensive DNA sequence homology and virtually indistinguishable restriction endonuclease maps within a 9- to 11-kbp region of each plasmid which included the resistance determinants.

Animals↗

PubMed1978

Genetic mapping of tyramine oxidase and arylsulfatase genes and their regulation in intergeneric hybrids of enteric bacteria.

The genes for arylsulfatase (atsA) and tyramine oxidase (tynA) have been mapped in Klebsiella aerogenes by P1 transduction. They are linked to gdhD and trp in the order atsA-tynA-gdhD-trp-pyrF. Complementation analysis using F' episomes from Escherichia coli suggested an analogous location of these genes in E. coli, although arylsulfatase activity was not detected in E. coli. P1 phage and F' episomes were used to create intergeneric hybrid strains of enteric bacteria by transfer of the ats and tyn genes between K. aerogenes, E. coli, and Salmonella typhimurium. Intergeneric transduction of the tynK gene from K. aerogenes to an E. coli restrictionless strain was one to two orders less frequent than that of the leuK gene. The tyramine oxidase of E. coli and S. typhimurium in regulatory activity resemble very closely the enzyme of K. aerogenes. The atsE gene from E. coli was expressed, and latent arylsulfatase protein was formed in K. aerogenes and S typhimurium. The results of tyramine oxidase and arylsulfatase synthesis in intergeneric hybrids of enteric bacteria suggest that the system for regulation of enzyme synthesis is conserved more than the structure or function of enzyme protein during evolution.

Arylsulfatases↗

PubMed1989

[Organization of Bsp-repeats in the fox genome].

Bsp repeats comprise approximately 1% of silver for total DNA and are preferentially localized in centromeric regions. Two of Bsp fragments cloned earlier, such as non-homologous rsV1 and rsV3, make up together a Bsp unit (680 bp) and possess a set of sites known to have regulatory functions in eucaryotic genomes. In this work, tandem organization of Bsp repeats is demonstrated. A stretched Bsp sequence (approximately 1460 bp, dimeric form) flanked by BamHI sites was cloned and its restriction map was plotted. With use of rsV1 and rsV3 probes the new sequence exhibited linked structure: rsV1-rsV3-rsV1-rsV3-rsV1. Blot-hybridization with the restriction fragments of fox total DNA suggested hierarchical order of Bsp clustes in the genome. It is supposed that the processes of selective amplification of individual fragments had been of real importance during evolution of Bsp repeats.

Animals↗

PubMed1994

Homology between Borrelia burgdorferi OspC and members of the family of Borrelia hermsii variable major proteins.

Synthesis of the Borrelia burgdorferi outer surface protein C (OspC) is quite variable. We have cloned and sequenced the ospC gene from B. burgdorferi isolate CA-11.2A, a clone in which ospC expression varies. The 5' flanking region of the gene contains at least two consensus promoter regions, as well as two large overlapping inverted repeats. Sequence comparison to other OspC proteins indicated that the CA-11.2A OspC is as closely related to OspC from two different genospecies of Lyme disease spirochetes as it is to OspC from the prototype B. burgdorferi strain, B31. Comparisons of the OspC amino acid (aa) sequence with those in aa sequence databases revealed partial identity with the variable major proteins Vmp3 and Vmp24 of B. hermsii, a causative agent of tick-borne relapsing fever. An ospC probe hybridized to B. hermsii restriction fragments and linear plasmids that also were recognized by the vmp3 and vmp24 probes. OspC and these Vmp appear to be related, but their synthesis is regulated differently in the two species of spirochetes. This represents a fascinating example of the evolution of the number, position, regulation and perhaps function of homologous genes in two related pathogens. These parameters may relate to characteristic properties of the pathogens and their separate tick vectors.

Amino Acid Sequence↗

PubMed1990

Evolution of a telomere associated satellite DNA sequence in the genome of Drosophila tristis and related species.

A highly repetitive satellite DNA sequence from the genome of Drosophila tristis with a length of 181 bp has been cloned in the pUC plasmid. The sequence hybridizes to the telomeres of all chromosomes but the Y of D. tristis and produces a ladderlike hybridization pattern with filterbound genomic DNA of D. tristis digested with Eco RI or Pst I with the hybridization bands at fragment lengths in multiples of 181 bp. A similar pattern is found when the genomic DNA comes from D. ambigua or, though less clear, from D. microlabis. Additional bands appear in the zones of high fragment lengths, too. In D. obscura and D. kitumensis, however, the 181 bp sequence is found in fragments with a length of a few kb only. The 181 bp sequence is tandemly arranged in the genome of D. tristis and has a copy number of about 82,000 per haploid genome (i.e. 10 per cent of the total DNA). A sequence comparison among four independently cloned copies of the family from D. tristis and another homologous sequence from D. obscura, found by chance, shows a one to six per cent variation in basepair composition. However, low divergence (only one per cent) between two copies of D. tristis and between the one of D. obscura and one of D. tristis was observed, and high divergence (six per cent) between these two pairs. This is discussed and explained as the evolutionary consequence of an existing homogenization process by unequal crossing over.

Animals↗

PubMed1998

Evidence for growth of strains of the plant epiphytic bacterium Erwinia herbicola and transconjugation among the bacterial strains in guts of the silkworm Bombyx mori.

Growth of plant epiphytic bacteria Erwinia herbicola and Pseudomonas syringae in guts of the silkworm, Bombyx mori, was studied. Fifth instar silkworm larvae were fed artificial diets supplemented with these bacteria for 6 to 12 h followed by uncontaminated diets. At 1, 3, and 6 days after feeding, bacteria were isolated from insect guts and feces. A much larger population of E. herbicola was detected in the samples collected 3 and 6 days after the inoculation than in samples collected after 1 day, indicating that these bacteria grew in the insect gut, while P. syringae was unable to survive. Transconjugation between E. herbicola strains in the insect gut was also examined. First, either a donor or a recipient strain was fed to the insects in artificial diets containing the bacteria during 12 h, and then pairing strains were fed during 12 h after starvation for 12 h. The conjugative plasmid pBPW1::Tn7 was transferred into recipient cells at very high frequencies (10(-1)/recipient after 3 days and 10(-3) after 6 days) in insect guts. Indigenous plasmids of E. herbicola mobilized RSF1010 plasmid into recipient cells at frequencies of 10(-4) in insect guts. These transconjugants were detected in the feces of the insects. Thus, plasmid-mediated gene transfer among the epiphytic bacteria in insect guts was demonstrated. The results obtained suggest that in insecta gene transfer may play an important role in the evolution of plant epiphytic bacteria.

Animals↗

PubMed2000

p53 intronic point mutation, aberrant splicing and telomeric associations in a case of B-chronic lymphocytic leukaemia.

We report a case of chronic lymphocytic leukaemia (CLL) with telomeric associations and a p53 intronic point mutation. Karyotypic analysis revealed clonal and non-clonal telomeric associations, accompanied by clonal cytogenetic abnormalities and also in isolation. The p53 mutation, which occurred at the invariant base pair -2 of the splice acceptor site in intron 7 resulted in the abolition of correct splicing of exon 7 to exon 8. Multiple aberrant splice products were characterized, all of which differed from wildtype in the DNA binding domain. Fluorescence in situ hybridization demonstrated that the clone retained two copies of the p53 gene and wild-type p53 transcript was detected on cloning of reverse transcriptase polymerase chain reaction (RT-PCR) product, indicating that one wild-type allele remained. However, a plasmid clone with correct splicing at the exon 7/8 boundary, but with a 21 bp deletion in exon 8, was also found at low frequency. This finding indicates clonal evolution, resulting in complete loss of wild-type p53. The intronic point mutation was not present in DNA extracted from cervical tissue indicating that it was a leukaemic phenomenon. This is the first case of an intronic point mutation to be reported in CLL. This mutation led to chaotic p53 expression and, interestingly, occurred in a case showing telomeric associations, a rare phenomenon in B-CLL.

Amino Acid Sequence↗

PubMed2004

Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae.

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.

Adaptation, Physiological↗