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At least 721 records · Page 40Linked to original sources
PubMed2002

Change of product specificity of hexaprenyl diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations.

The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) prenyl diphosphate synthase, hexaprenyl diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes.

Alkyl and Aryl Transferases↗
PubMed2003

General method for sequence-independent site-directed chimeragenesis.

We have developed a simple and general method that allows for the facile recombination of distantly related (or unrelated) proteins at multiple discrete sites. To evaluate the sequence-independent site-directed chimeragenesis (SISDC) method, we have recombined beta-lactamases TEM-1 and PSE-4 at seven sites, examined the quality of the chimeric genes created, and screened the library of 2(8) (256) chimeras for functional enzymes. Probe hybridization and sequencing analyses revealed that SISDC generated a random library with little sequence bias and in which all targeted fragments were recombined in the desired order. Sequencing the genes from clones having functional lactamases identified 14 unique chimeras. These chimeras are characterized by a lower level of disruption, as calculated by the SCHEMA algorithm, than the library as a whole. These results illustrate the use of SISDC in creating designed chimeric protein libraries and further illustrate the ability of SCHEMA to identify chimeras whose folded structures are likely not to be disrupted by recombination.

Algorithms↗
PubMed1999

Insights into the evolutionary process of genome degradation.

Studies of noncoding and pseudogene sequence diversity, particularly in Rickettsia, have begun to reveal the basic principles of genome degradation in microorganisms. Increasingly, studies of genes and genomes suggest that there has been an extensive amount of horizontal gene transfer among microorganisms. As this inflow of genetic material does not seem generally to have resulted in genome size expansions, however, degenerative processes must be at the very least as widespread as horizontal gene transfer. The basic principles of gene degradation and elimination that are being explored in Rickettsia are likely to be of major importance for our understanding of how microbial genomes evolve.

DNA, Bacterial↗
PubMed2003

Filamentous phages linked to virulence of Vibrio cholerae.

The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXphi, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXphi relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXphi genes--including the toxin genes--and transmission of CTXphi were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXphi. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage.

Cholera↗
PubMed2001

Evolution of catabolic pathways and metabolic versatility in Pseudomonas stutzeri OX1.

Pseudomonas stutzeri OX1 is able to degrade toluene and ortho-xylene via the direct oxygenation of the aromatic ring. The genetic studies carried out suggest that the genes coding for the monooxygenase involved in the early steps of this catabolic route have been acquired by gene transfer. P stutzeri OX1 is also potentially able to utilize meta- and para-xylene as growth substrates. These two isomers are metabolized through a different pathway (TOL pathway). Both catabolic routes can be activated or inactivated by means of genomic rearrangements. The relevance of such recombination mechanisms in the evolution and the adaptability of P. stutzeri is discussed.

Biodegradation, Environmental↗
PubMed1979

Analysis of the constancy of DNA sequences during development and evolution of the nematode Caenorhabditis elegans.

In order to test for the occurrence of rearrangements in DNA during development and to assess the rate of DNA divergence during evolution, we have compared restriction fragments derived from DNA from four sources: sperm cells and somatic tissues of one strain of the nematode Caenorhabditis elegans, somatic tissues of a second strain of the same species, and whole animals of a closely related species. Restriction fragments were detected by hybridizing radioactive cloned fragments to restriction digests that had been fractionated by size on agarose gels and transferred to nitrocellulose sheets. In this way, approximately 50 BamHI restriction fragments were visualized and compared. Fragments from sperm and somatic DNAs were found to be identical; 15% differed in size between the two strains. Little cross homology was found between the two species. We conclude that, if rearrangements occur in C. elegans DNA during development, they must affect fewer than a few percent of the restriction fragments or restriction sites. The difference found between the two strains and the two species is surprisingly great.

Animals↗
PubMed1982

Differing levels of dispersed repetitive DNA among closely related species of Drosophila.

The genomic concentrations of certain middle repetitive DNA sequences vary considerably among closely related species of Drosophila. In fact, the chromosomes of D. melanogaster appear to carry approximately 3 times as much middle repetitive DNA as those of the sibling species D. simulans. Although most of the middle repetitive DNA of D. melanogaster consists of segments of "nomadic" DNA that occupy different dispersed chromosomal locations in different strains of flies, repeated DNA sequences recovered from the D. simulans genome are most often restricted to single chromosomal positions. Apparent differences in the total concentrations of middle repetitive DNA in the two species are most easily attributed to an approximately sevenfold difference in their dispersed repetitive and nomadic DNA contents. These differences may affect the relative mutation rates of these species or contribute to their reproductive isolation or both.

Animals↗
PubMed2001

Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51.

Bloom's syndrome (BS) is an autosomal recessive disorder that predisposes individuals to a wide range of cancers. The gene mutated in BS, BLM, encodes a member of the RecQ family of DNA helicases. The precise role played by these enzymes in the cell remains to be determined. However, genome-wide hyper-recombination is a feature of many RecQ helicase-deficient cells. In eukaryotes, a central step in homologous recombination is catalyzed by the RAD51 protein. In response to agents that induce DNA double-strand breaks, RAD51 accumulates in nuclear foci that are thought to correspond to sites of recombinational repair. Here, we report that purified BLM and human RAD51 interact in vitro and in vivo, and that residues in the N- and C-terminal domains of BLM can independently mediate this interaction. Consistent with these observations, BLM localizes to a subset of RAD51 nuclear foci in normal human cells. Moreover, the number of BLM foci and the extent to which BLM and RAD51 foci co-localize increase in response to ionizing radiation. Nevertheless, the formation of RAD51 foci does not require functional BLM. Indeed, in untreated BS cells, an abnormally high proportion of the cells contain RAD51 nuclear foci. Exogenous expression of BLM markedly reduces the fraction of cells containing RAD51 foci. The interaction between BLM and RAD51 appears to have been evolutionarily conserved since the C-terminal domain of Sgs1, the Saccharomyces cerevisiae homologue of BLM, interacts with yeast Rad51. Furthermore, genetic analysis reveals that the SGS1 and RAD51 genes are epistatic indicating that they operate in a common pathway. Potential roles for BLM in the RAD51 recombinational repair pathway are discussed.

Adenosine Triphosphatases↗
PubMed2003

Determination of transmembrane topology of the Escherichia coli natural resistance-associated macrophage protein (Nramp) ortholog.

The natural resistance-associated macrophage protein (Nramp) defines a conserved family of secondary metal transporters. Molecular evolutionary analysis of the Nramp family revealed the early duplication of an ancestral eukaryotic Nramp gene, which was likely derived from a bacterial ortholog and characterized as a proton-dependent manganese transporter MntH (Makui, H., Roig, E., Cole, S. T., Helmann, J. D., Gros, P., and Cellier, M. F. (2000) Mol. Microbiol. 35, 1065-1078). Escherichia coli MntH represents a model of choice to study structure function relationship in the Nramp protein family. Here, we report E. coli MntH transmembrane topology using a combination of in silico predictions, genetic fusion with cytoplasmic and periplasmic reporters, and MntH functional assays. Constructs of the secreted form of beta-lactamase (Blam) revealed extra loops between transmembrane domains 1/2, 5/6, 7/8, and 9/10, and placed the C terminus periplasmically; chloramphenicol acetyltransferase constructs indicated cytoplasmic loops 2/3, 6/7, 8/9, and 10/11. Two intra loops for which no data were produced (N terminus, intra loop 4/5) both display composition bias supporting their deduced localization. The extra loops 5/6 and 6/7 and periplasmic exposure of the C terminus were confirmed by targeted reporter insertion. Three of them preserved MntH function as measured by a disk assay of divalent metal uptake and a fluorescence assay of divalent metal-dependent proton transport, whereas a truncated form lacking transmembrane domain 11 was inactive. These results demonstrate that EcoliA is a type III integral membrane protein with 11 transmembrane domains transporting both divalent metal ions and protons.

Amino Acid Sequence↗
PubMed2001

Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions.

The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.

Biological Assay↗
PubMed2004

Retroelement dynamics and a novel type of chordate retrovirus-like element in the miniature genome of the tunicate Oikopleura dioica.

Retrotransposable elements have played an important role in shaping eukaryotic DNA, and their activity and turnover rate directly influence the size of genomes. With approximately 15,000 genes within 65-75 megabases, the marine tunicate Oikopleura dioica, a nonvertebrate chordate, has the smallest and most compact genome ever found in animals. Consistent with a massive elimination of retroelements, only one apparently novel clade of non-long terminal repeat (non-LTR) retrotransposons was detected within 41 megabases of nonredundant genomic sequences. In contrast, at least six clades of non-LTR elements were identified in the less compact genome of the tunicate Ciona intestinalis. Unexpectedly, Ty3/gypsy-related Tor LTR retrotransposons presented an astonishing level of diversity in O. dioica. They were generally poorly or apparently not corrupted, indicating recent activity. Both Tor3 and Tor4b families bore an envelope-like open reading frame, suggesting possible horizontal acquisition through infection. The Tor4b envelope-like gene might have been obtained from a paramyxovirus (RNA virus). Tor3 and Tor4b are phylogenetically clearly distinct from vertebrate retroviruses (Retroviridae) and are more reminiscent of certain insect and plant sequences. Tor elements potentially represent a so far unknown, ancient type of infectious retroelement in chordates. Their distribution and transmission dynamics in tunicates and other chordates deserve further study.

Amino Acid Sequence↗
PubMed1994

Replacement of the Saccharomyces cerevisiae RPR1 gene with heterologous RNase P RNA genes.

Phylogenetic studies of yeast nuclear RNase P RNA genes have shown a striking conservation of secondary structure for the Saccharomyces and Schizosaccharomyces RNase P RNAs, yet much of the primary sequence and many substructures vary among the RNAs examined. To investigate which sequences and structural features can be varied and still allow function in a heterologous organism, RNase P genes from several yeast species were tested for the ability to substitute for the Saccharomyces cerevisiae RNA. The RNase P genes from Saccharomyces carlsbergensis and Saccharomyces kluyveri could act as the sole source of RNase P RNA within S. cerevisiae cells, whereas the genes from Saccharomyces globosus and Schizosaccharomyces pombe could not. Although heterologous RNase P RNAs were synthesized by the cells in all cases, the RNAs that complemented tended to be processed from longer precursor transcripts into mature-sized RNase P RNA, while the RNAs that did not complement tended to accumulate as the longer precursor form. The results identified sequences and structures in the RNA that are not essential for interaction with species-specific proteins, processing or localization, and suggested other positions that may be candidates for such processes.

Base Sequence↗
PubMed2001

Genomic approaches to typing, taxonomy and evolution of bacterial isolates.

The current literature on bacterial taxonomy, typing and evolution will be critically examined from the perspective of whole-genome structure, function and organization. The following three categories of DNA band pattern studies will be reviewed: (i) random whole-genome analysis; (ii) specific gene variation and (iii) mobile genetic elements. (i) The use of RAPD, PFGE and AFLP to analyse the whole genome will provide a skeleton of polymorphic sites with exact genomic positions as whole-genome sequence data become available. (ii) Different genes provide different levels of evolutionary information for determining isolate relatedness depending on whether they are highly variable (prone to recombination events and horizontal transfer), housekeeping genes with only a small number of single nucleotide differences between isolates or part of the rrn multigene family that is prone to intragenomic recombination and concerted evolution. Comparative analyses of these different gene classes can provide enhanced information about isolate relatedness. (iii) Mobile genetic elements such as insertion sequences, transposons, plasmids and bacteriophages integrate into the bacterial genome at specific (e.g. tRNA genes) or non-specific sites to alter band patterns produced by PFGE, RAPD or AFLP. From the literature it is not clear what level of genetic element duplication constitutes non-relatedness of isolates. A model is presented that incorporates all of the above genomic characteristics for the determination of isolate relatedness in taxonomic, typing and evolutionary studies.

Bacteria↗
PubMed1992

A tyrosyl-tRNA synthetase binds specifically to the group I intron catalytic core.

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns in mitochondria. Here, we show that CYT-18 binds strongly to diverse group I introns that have minimal sequence homology and recognizes highly conserved structural features of the catalytic core of these introns. Inhibition experiments indicate that the intron RNA and tRNA(Tyr) compete for the same or overlapping binding sites in the CYT-18 protein. Considered together with functional analysis, our results indicate that the CYT-18 protein promotes splicing by binding to the intron core and stabilizing it in a conformation required for catalytic activity. Furthermore, the specific binding of the synthetase suggests that the group I intron catalytic core has structural similarities to tRNAs, which could reflect either convergent evolution or an evolutionary relationship between group I introns and tRNAs.

Base Sequence↗
PubMed2003

Bacterial sex: playing voyeurs 50 years later.

The concept of chromosomes with a ring structure was born during the early studies of bacterial sexuality, and the discovery of fertility factors- episomes or plasmids-provided much later the key tools for gene cloning and biotechnology. But the plasmid-mediated transfer of antibiotic and other resistances, as well as pathogenicity, has served bacteria well in their own adaptive evolution.

Bacteriology↗
PubMed2000

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants.

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.

Amino Acid Substitution↗