PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “plasmid evolution”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,243 records · Page 69Linked to original sources
PubMed1987

Fusion of trpB and trpA of Escherichia coli yields a partially active tryptophan synthetase polypeptide.

The separate alpha and beta polypeptides of the tryptophan synthetase of bacteria are represented in fungi by a fusion polypeptide in which the first third is homologous to bacterial alpha chains and the remainder is homologous to bacterial beta chains. In the yeast polypeptide, a short nonhomologous "connector" joins the two homologous segments. The chromosomal order of all bacterial genes that specify tryptophan synthetase beta and alpha chains, respectively, is trpB-trpA. Fusion of these genes in their present arrangement would result in the synthesis of a polypeptide with a segmental order, N-beta-alpha-C, opposite that observed in fungi. To investigate possible explanations for the apparent transposition that occurred in the evolution of the fungal gene we have made two fusions of trpB and trpA of Escherichia coli in their natural orientation. We find that the fusion proteins are synthesized but both are less active catalytically than the wild type bacterial protein. In addition, the fusion proteins associate abnormally, they are activated only slightly by wild type alpha or beta 2, and they are less sensitive than the wild type protein to inhibition by antibodies to alpha or beta 2. The fusion proteins have normal substrate affinities. Our findings suggest that the altered structures of the fusion proteins affect catalytic ability and the locations of the alpha and/or beta chain combining sites. This structural distortion may have prevented the natural selection of direct gene fusions during the course of the fungal gene's evolution.

Base Sequence↗
PubMed1987

Crucial role of the connecting region joining the two functional domains of yeast tryptophan synthetase.

We constructed a hybrid plasmid expressing yeast tryptophan synthetase in Escherichia coli. Several deletion variants lacking the A or B domains of this polypeptide (recognized by their homology to the alpha and beta subunits of prokaryotic tryptophan synthetase) showed no enzymatic activity and failed to substitute for the corresponding E. coli subunits. To examine the role of a presumed interdomain connecting region in the yeast enzyme, we constructed a variant lacking 18 amino acids in that region. The variant polypeptide was completely inactive. Replacing 14 of the 18 missing amino acids with a segment having a different sequence partially restored activity. A spontaneous revertant was characterized and shown to have a duplication of 16 amino acid residues in this region; the activity of the duplication polypeptide was better than that of the 14-residue replacement. If confirmed by additional studies, our finding that the length of the connecting region is more critical than its sequence has implications for understanding the origin of gene fusions during evolution as well as for designing artificial fusions.

Amino Acid Sequence↗
PubMed1980

On procaryotic gene expression in eucaryotic systems.

Numerous types of interaction between pro- and eucaryotes exist in nature, from the endosymbiosis of some bacteria with unicellular organisms and insects to the complex systems of bacterial flora associated with the skin and intestines of animals and man, and nitrogen-fixation and crown-gall tumor induction in plants. Until recently, such interactions were not thought to include genetic transfer, but an increasing body of evidence points to the probability of similar naturally-occurring exchanges with wide-ranging implications for evolution and genetic manipulation. Experiments to elucidate the possible effects of procaryotic genes in eucaryotic systems have included in vitro and in vivo studies with both plant and animal systems, for instance the translation of bacterial messenger RNAs in the wheat germ and rabbit reticulocyte systems and the introduction of bacterial genes into plant protoplasts, animal cells and whole organisms. In the present paper we have tried to summarize the results of experiments involving the uptake, replication, transcription, translation and integration of procaryotic genes in various eucaryotic systems and to discuss the implications of such findings for basic research as well as for possible biomedical applications. Awareness of the possibility of procaryotic-eucaryotic genetic interactions may help to elucidate unresolved questions in pathology, such as possible involvement of the intestinal flora in carcinogenesis, as well as to provide valuable probes of eucaryotic control mechanisms and new approaches in agricultural genetic engineering.

Animals↗
PubMed1993

Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications.

Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor. These 12 conserved Lys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis. These Lys-->Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysK and cysM. One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E. coli NK3 transformed with the Lys-49 mutant. These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase. This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the beta-carbon of amino acids.

Amino Acid Sequence↗
PubMed1984

Polar encapsidation of adenovirus DNA: evolutionary variants reveal dispensable sequences near the left ends of Ad3 genomes.

Repeated passage of adenovirus type 3 in HeLa cells has led to a novel stock of variant genomes, characterized by deletions and substitutions of DNA sequences within the left-end 750 base pairs. This heterogeneous stock retains few if any parental genomes--the majority of variants appear viable. Analysis of viable variants with deleted sequences reveals the 182 nucleotides proximal to the left-end inverted terminal repeat (136-318 bp) are not required for Ad3 infectivity in cultured human cell lines nor for maintenance of viral DNA encapsidation polarity.

Adenoviruses, Human↗
PubMed1988

Transcriptional activation by the SV40 AP-1 recognition element in yeast is mediated by a factor similar to AP-1 that is distinct from GCN4.

The consensus recognition element for the mammalian transcription factor AP-1 is very similar to that of the transcriptional activator GCN4. Here, we show that the AP-1 recognition element (ARE) found in the SV40 enhancer can activate transcription from a heterologous promoter in S. cerevisiae. This activation, however, is not dependent on the presence of GCN4 as evidenced by ARE-dependent transcription in a gcn4 yeast strain. A previously unknown yeast transcription factor that is probably responsible for this activation was identified and highly purified. The yeast factor, designated yAP-1, shares remarkably similar biochemical and DNA-binding characteristics with mammalian AP-1. These data suggest that the yeast and mammalian AP-1 are evolutionarily conserved and perhaps functionally related. Also note-worthy is that GCN4 can bind to a GCN4 recognition element (GCRE) and to the ARE with approximately equal affinities; yAP-1, however, has a much lower affinity for the GCRE than the ARE, suggesting that yAP-1 can discriminate between these elements in vivo.

Antibodies, Monoclonal↗
PubMed1993

Cloning and characterization of a novel zinc-finger protein-encoding cDNA from the mouse eye lens.

Zinc fingers (Zf) are a common structural motif found in many nucleic acid-binding proteins. In an effort to identify potential transcription factors in the mouse eye lens, we have isolated a Zf-containing clone from a newborn mouse lens cDNA library. The clone, named pMLZ-4, is 4.5 kb in length and contains an open reading frame of 1073 bp. The putative pMLZ-4 protein consists of a short, N-terminal acidic domain followed by twelve tandemly arrayed Zf of the C2H2 variety. The remaining 3.2 kb of the cDNA comprises the 3'-untranslated region. PCR analysis detected the presence of pMLZ-4 RNA in liver, heart, kidney, spleen and brain of newborn mice. Hybridization of pMLZ-4 to genomic DNA from a number of species of vertebrates revealed the presence of homologous sequences only in mouse and rat. Unexpectedly, the probe also hybridized to a single band in yeast DNA digested with EcoRI. NIH3T3 cells were stably transformed with a construct that over-expresses the pMLZ-4 mRNA. The stably transformed cells did not differ in appearance from untransformed cells, and an analysis of proteins from transformed and untransformed cells failed to detect any differences resulting from over-expression of the pMLZ-4 mRNA.

3T3 Cells↗
PubMed2000

Sugar uptake and utilisation in Streptomyces coelicolor: a PTS view to the genome.

Our research group is studying the phosphotransferase system (PTS) of Streptomyces coelicolor, which, in other bacteria, is centrally involved in carbon source uptake and regulation. We have surveyed the public available S. coelicolor genome sequence produced by the ongoing genome sequencing project for pts gene homologues (http://www.sanger.ac.uk/Projects/S_coelicolor/). Three genes encoding homologues of the general PTS components enzyme I (ptsI), HPr (ptsH), and enzyme IIA(Crr) (crr; IIA(Glc)-homologue) and six genes encoding homologues of sugar-specific PTS components were identified. The deduced primary sequences of the sugar-specific components shared significant similarities to PTS permeases of the mannitol/fructose family and of the glucose/sucrose family. A model is presented, in which possible functions of the novel described PTS homologues are discussed.

Amino Acid Sequence↗
PubMed1997

Dynamic bacterial genome organization.

Recently completed projects of sequencing chromosomal fragments and entire chromosomes, as well as physical mapping of genomes, have opened novel inroads to the understanding of the biology of bacterial genomes. From these studies one may draw some conclusions. (i) The organization of orthologous genes on the bacterial chromosome is not conserved during evolution. (ii) The bacterial genome is more complex and also more flexible than hitherto thought. Genetic elements are sometimes part of the chromosome, while at other times they are independent elements or parts of alternative replicons (e.g. large plasmids). Such replicons, carrying essential genes, now seem to deserve the designation 'secondary chromosomes'. A study of the regulation of replication and segregation of these essential genetic elements will be of great interest.

Bacteria↗
PubMed2001

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.

Animals↗
PubMed2001

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

Acetyltransferases↗
PubMed2002

The Clostridium ramosum IgA proteinase represents a novel type of metalloendopeptidase.

Clostridium ramosum is part of the normal flora in the human intestine. Some strains produce an IgA proteinase that specifically cleaves human IgA1 and the IgA2m(1) allotype. This prolylendopeptidase was purified from a broth culture supernatant, and N-terminal sequences of the native protein and tryptic fragments thereof were determined. A fragment of the iga gene encoding the IgA proteinase was isolated using degenerate primers in PCR, and the complete gene was obtained by inverse PCR. The identity of the iga gene was confirmed by heterologous expression in Escherichia coli. The deduced amino acid sequence indicated a signal peptide of 30 residues and a secreted proteinase of 133,828 Da. A typical Gram-positive cell wall anchor motif was identified in the C terminus. The presence of a putative zinc-binding motif His-Glu-Phe-Gly-His together with inhibition studies indicate that the proteinase belongs to the zinc-dependent metalloproteinases. However, the sequence of the C. ramosum IgA proteinase shows no overall similarity to other proteins except for significant identity around the zinc-binding motif with family M6 of metalloendopeptidases, and the unique sequence of the IgA proteinase in this area presumably establishes a new subfamily. The GC percentage of the iga gene is significantly higher than that for the entire genome of C. ramosum, suggesting that the gene was acquired recently in evolution.

Alanine↗
PubMed2005

Hoxd13 and Hoxa13 directly control the expression of the EphA7 Ephrin tyrosine kinase receptor in developing limbs.

Hoxa and Hoxd genes, related to the Drosophila Abd-B gene, display regionally restricted expression patterns and are necessary for the formation of the limb skeletal elements. Hox genes encode transcription factors, which are supposed to control the expression of a series of downstream target genes, whose nature has remained largely elusive. Several genes were identified that are differentially expressed in relation to Hox gene activity; few studies, however, explored their direct regulation by Hox proteins. Ephrin tyrosine kinase receptors and ephrins have been proposed as Hox targets, and recently, evidence was gained for their role in limb development. The expression of the EphA7 gene in developing limbs was shown to correlate with the expression of Hoxa13 and Hoxd13; however, its direct regulation by these genes has never been assessed. We have characterized the EphA7 promoter region and show that it contains multiple binding sites for paralog group 13 Hox proteins. We found that one of these sites is bound in vivo by HOXA13 and HOXD13 and by endogenous Hoxd13 in developing mouse limbs. Moreover, we show that HOXD13 and HOXA13 activate transcription from the EphA7 promoter and that a mutation of the HOXA13/HOXD13 binding site was sufficient to abolish activation. Conversely, the HOXD13(147L) mutation, identified in patients displaying a novel brachydactyly-polydactyly syndrome, does not bind to in vivo, and fails to transactivate the EphA7 promoter. These results establish that EphA7 is a direct downstream target of Hoxd13 and Hoxa13 during limb development, thus providing further insight into the regulatory networks that control limb patterning.

Amino Acid Sequence↗
PubMed2001

Translational misreading: a tRNA modification counteracts a +2 ribosomal frameshift.

Errors during gene expression from DNA to proteins via transcription and translation may be deleterious for the functional maintenance of cells. In this paper, extensive genetic studies of the misreading of a GA repeat introduced into the lacZ gene of Escherichia coli indicate that in this bacteria, errors occur predominantly by a +2 translational frameshift, which is controlled by a tRNA modification involving the MnmE and GidA proteins. This ribosomal frameshift results from the coincidence of three events: (1) decreased codon-anticodon affinity at the P-site, which is caused by tRNA hypomodification in mnmE(-) and gidA(-) strains; (2) a repetitive mRNA sequence predisposing to slippage; and (3) increased translational pausing attributable to the presence of a rare codon at the A-site. Based on genetic analysis, we propose that GidA and MnmE act in the same pathway of tRNA modification, the absence of which is responsible for the +2 translational frameshift. The difference in the impact of the mutant gene on cell growth, however, indicates that GidA has at least one other function.

Algal Proteins↗
PubMed2004

Properties of unpaired DNA required for efficient silencing in Neurospora crassa.

The presence of unpaired copies of a gene during meiosis triggers silencing of all copies of the gene in the diploid ascus cell of Neurospora. This phenomenon is called meiotic silencing and on the basis of genetic studies appears to be a post-transcriptional gene silencing (PTGS) mechanism. Previously, meiotic silencing was defined to be induced by the presence of a DNA region lacking an identical segment in the homologous chromosome. However, the determinants of unpaired DNA remained a mystery. Using the Ascospore maturation-1 (Asm-1) gene, we defined what needs to be "unpaired" to silence a gene. For efficient silencing, an unpaired region of DNA needs to be of a sufficient size and contain homology to the reporter transcript. The greater the size of the loop and the larger the homology to the reporter transcript, the better the resulting meiotic silencing is. Conversely, regions not containing homology to the transcript, e.g., intergenic regions, did not silence the reporter. Surprisingly, unpaired fragments lacking a canonical promoter silenced the reporter. Additionally, we detected the unpairing-dependent loss of a transcript during meiotic silencing. Our observations further support a PTGS mechanism for meiotic silencing and offer insight into the evolutionary consequences resulting from this novel meiotic checkpoint.

Alleles↗
PubMed1992

Comparison of the genomic organizations of the rat grp78 and hsc73 gene and their evolutionary implications.

GRP78, a 78-kDa protein localized in the endoplasmic reticulum, is a member of the HSP70 protein family. However, unlike hsp70 which is intronless and belongs to a multigene family, grp78 is a single copy gene and contains intervening sequences. In this aspect, the grp78 gene resembles more closely that of the constitutively expressed heat shock cognate gene, hsc73. In this report, we compare the grp78 and hsc73 genes and show that: (1) the intron/exon junctions of the two genes are not conserved, but occur at similar positions; (2) while the protein sequences are highly conserved, being 62% identical, the conservation is concentrated at discrete regions over the N-terminal three-fourths of the protein; (3) this conservation represents the ATP binding and structural domains shared among the members of the HSP70 family; (4) the C-terminus is highly divergent and likely represents domains specific for the individual family members; and (5) sequences partially homologous to the grp78 5' UTR and signal sequence, which targets GRP78 into the ER, are found within an additional first intron of hsc73. The evolutionary implications of these two genes are discussed.

Amino Acid Sequence↗
PubMed1995

Identification of a novel class of mammalian Fc gamma receptor.

A cDNA encoding an Ig receptor that conferred the ability to bind erythrocytes sensitized with IgG2, but not IgG1, was cloned by screening a cattle alveolar macrophage library, made in the vector pCDM8, expressed in COS-7 cells. A search of the PIR database indicated a greater level of similarity with human Fc alpha R than with any other FcR. The percentage of identical amino acids was 41% and nucleotides 56%. This high similarity is between the extracellular and transmembrane domains; the cytoplasmic tails are unrelated. Similarities with human Fc gamma RI, Fc gamma RII, Fc gamma RIII, Fc epsilon RI, or bovine Fc gamma RII were less than 28%. COS-7 cells transfected with the cloned plasmid bound erythrocytes specifically sensitized with IgG2 but not with IgG1. In tests with heat-aggregated bovine Igs, IgG2 purified from serum bound to transfected COS-7 cells but IgG1 from serum and IgA from tracheobronchial secretions did not. Human serum IgA also failed to bind to transfected COS-7 cells although it did bind to bovine neutrophils and monocytes. Only aggregated bovine IgG2 inhibited the binding of IgG2-sensitized erythrocytes to COS-7 cells transfected with the plasmid. These observations indicate that the FcR bound IgG2 specifically and was not a receptor for IgA. Thus, this receptor, which we have named bovine Fc gamma 2R, represents a novel class of mammalian Fc gamma R, the evolution of which is likely to have been influenced by the truncated hinge of the bovine IgG2 molecule. Analysis of genetic divergence of the genes encoding bovine and human Fc gamma R and Fc alpha R indicated that the novel bovine gene and the human Fc alpha R gene probably evolved from a common ancestor, which is not shared by other Fc gamma R.

Amino Acid Sequence↗