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At least 613 records · Page 34Linked to original sources
PubMed1980

Mutations preventing transpositions of yeast mating type alleles.

Homothallic strains of Saccharomyces cerevisiae can switch from one mating type to the other as often as every cell division. The conversion of mating type alleles (from MATa to MATalpha or vice versa) depends on other, unexpressed copies of a or alpha information that can be transposed to MAT. Previously, "inconvertible" mutations within MATalpha and MATa have been described that block the excision of the MAT allele. In this paper we describe two cis-acting mutations that also impair mating type switching and lie very near, but outside, the MAT locus. Both "stuck" mutations, stk1 and stk2, diminish the efficiency of converting MATa to MATalpha to less than 10% of normal. The stk1 mutation also slightly reduces conversion of MATalpha to MATa, whereas stk2 has no discernible effect. Unlike the inconvertible MATalpha-inc and MATa-inc mutations within MAT, the stk mutations are not replaced by wild-type sequences after the "stuck" cells occasionally switch to the opposite mating type. Because these mutations are not "healed" by mating type conversions, they must lie in sequences outside of the transposable mating type information. These results indicate that the efficient replacement of MAT alleles depends on sequences both within and adjacent to the MAT locus. Among subclones of homothallic stk MATa strains, approximately 2% show "illegal" transpositions of mating type genes. In these colonies the silent copy of alpha information at the HMLalpha locus has been converted to a, without any change of MATa or the silent a copy at HMRa. Such conversions of the unexpressed library genes are not found in wild-type homothallic strains that can switch mating type efficiently, but they are found in MATa-inc and MATalpha-inc strains. It appears that all of the cis-acting mutations within or adjacent to mating type result in these unusual switches of mating type information at HML and HMR.

Alleles↗
PubMed1985

Molecular structures of mitochondrial-DNA-like sequences in human nuclear DNA.

Two lambda phage clones carrying mitochondrial-DNA-like (mtDNA-like) sequences isolated from a human gene library were named Lm E-1 and Lm C-2, and their DNA structures were characterized. Lm E-1 contains about 0.4 kb DNA homologous to the 5' portion of the mitochondrial 16S ribosomal RNA (rRNA) gene and Lm C-2, a 1.6 kb DNA homologous to the 3' portion of the 12S rRNA gene and to almost all of the 16S rRNA gene. Comparisons of their nucleotide sequences with those of the corresponding regions of the human mtDNA revealed no detectable DNA rearrangement and their homologies to the human mtDNA are 84% and 80%, respectively. There are neither terminal repeats in the nuclear mtDNA-like sequences nor duplications of the nuclear DNAs flanking the mtDNA-like sequences. Evolutionary relationship between these two human nuclear mtDNA-like sequences and the human and bovine mtDNAs is discussed.

Animals↗
PubMed1997

cis-trans isomerization of unsaturated fatty acids: cloning and sequencing of the cti gene from Pseudomonas putida P8.

Transposon mutants of Pseudomonas putida P8 were generated by applying a mini-Tn5 mutagenesis system. The mutants obtained were checked for their ability to tolerate increased temperatures and elevated phenol concentrations. Approximately 5,800 transposon mutants were used to generate a pool of 600 temperature-sensitive strains; one of these strains was identified as being damaged in its ability to perform cis-trans isomerization of fatty acids. A gene library of P. putida P8 was constructed and screened by using as a probe sequences immediately adjacent to the mini-Tn5 insertion. A DNA fragment that complemented the mutation was isolated and cloned. The corresponding gene, termed cti, is located close to the methionine synthase locus (metH) in P. putida P8. A cti-carrying fragment integrated into a plasmid also conferred the ability for cis-trans isomerization to Escherichia coli; the cti gene was completely sequenced, and the amino acid sequence was deduced.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2.

Given the lack of effective vaccines to control Streptococcus suis infection and the lack of a rapid and reliable molecular diagnostic assay to detect its infection, a polyclonal antibody was raised against the whole-cell protein of S. suis type 2 and used to screen an S. suis gene library in an effort to identify protective antigen(s) and antigens of diagnostic importance. A clone that produced a 45-kDa S. suis-specific protein was identified by Western blotting. Restriction analysis showed that the gene encoding the 45-kDa protein was present on a 1.6-kb pair DraI region on the cloned chromosomal fragment. The nucleotide sequence contained an open reading frame that encoded a polypeptide of 448 amino acid residues with a calculated molecular mass of 48.8 kDa, in close agreement with the size observed on Western blots. A GenBank database search revealed that the derived amino acid sequence is homologous to the sequence of glutamate dehydrogenase (GDH) protein isolated from various sources, including conserved motifs and functional domains typical of the family 1-type hexameric GDH proteins, thus placing it in that family. Because of these similarities, the protein was designated the GDH of S. suis. Hybridization studies showed that the gene is conserved among the S. suis type 2 strains tested. Antiserum raised against the purified recombinant protein was reactive with a protein of the same molecular size as the recombinant protein in S. suis strains, suggesting expression of the gene in all of the isolates and antigenic conservation of the protein. The recombinant protein was reactive with serum from pigs experimentally infected with a virulent strain of S. suis type 2, suggesting that the protein might serve as an antigen of diagnostic importance to detect S. suis infection. Activity staining showed that the S. suis GDH activity is NAD(P)H dependent but, unlike the NAD(P)H-dependent GDH from various other sources, that of S. suis utilizes L-glutamate rather than alpha-ketoglutarate as the substrate. Highly virulent strains of S. suis type 2 could be distinguished from moderately virulent and avirulent strains on the basis of their GDH protein profile following activity staining on a nondenaturing gel. We examined the cellular location of the protein using a whole-cell enzyme-linked immunosorbent assay and an immunogold-labeling technique. Results showed that the S. suis GDH protein is exposed at the surface of intact cells.

Animals↗
PubMed1999

Antibody production without animals.

Research always depended on animals or directly on human beings for the generation of antibodies with new specificities. With the opportunity to clone the human antibody repertoire, it is now also feasible to tap the human immune system and to search for new antibody specificities in antibody gene libraries [1, 2]. Phage display technology has fostered these approaches as it allows even rare specificities to be identified in libraries of a complexity of 10(8) to 10(10) [3, 4]. While most of the work in this field is still based on immune libraries, that is, B-cells derived from immunised animals or human individuals, more recent approaches allow even the necessity for immunisation to be by-passed. Synthetic antibody libraries exist of a complexity comparable to the natural immune system of mammals, so that theoretically every imaginable antibody may be detected and then used either for diagnostic or therapeutic purposes [5-7]. Thus, these new gene technology approaches make it possible to generate antibodies completely independently of animals. It may be that animals will only be used in the future to study the immune system of the animal.

Animal Welfare↗
PubMed1990

Molecular biology of type A endogenous retrovirus.

Intracisternal A particles (IAPs) are retrovirus-like structures consistently observable in a variety of mouse tumor cells such as myeloma and hybridoma and in early embryonic cells derived from rodents but nothing is known of their infectivity. Mouse IAPs contain a gag-like protein, a reverse transcriptase and a polyadenylated RNA molecule (IAP RNA). DNA sequences complementary to IAP RNA (IAP genome) are interspersedly present in rodent such as mice, rats, Chinese hamsters and Syrian hamsters at several hundred to a thousand copies per haploid genome. Molecularly cloned IAP genomes from two species Mus and Syrian hamster were 6 to 8 kb in length with LTRs of about 0.4 kb long. The nucleotide sequence of the Syrian hamster IAP genome, H18, predicted a typical LTR-gag-prt-pol-env-LTR structure, although many stop codons were present in the region corresponding to env. The comparison of the deduced amino acid sequences of the pol region showed IAP (type A), mouse mammary tumor virus (MMTV) (type B), and squirrel monkey retrovirus (SMRV) (type D) genomes to be closely related. By using a DNA fragment encoding the pol region of the Syrian hamster IAP genome, human endogenous retroviruses termed HERV-K, were cloned from a fetal human liver gene library. Typical HERV-K genome was 9.5 kb in length having LTRs of about 1.0 kb. The HERV-K provirus could encode gag (666 codons), prt (334 codons), pol (937 codons), and env (618 codons) genes. HERV-K was shown to be closely related to types A, B and D retroviruses. The HERV-K genomes are present at about 50 copies per haploid human genome. In several human tumor cell lines, the HERV-K genome was expressed as 8.8 kb poly(A)+ RNA which appeared to be a full-size transcript of this genome. In the human breast cancer cell line T47D, stimulation of HERV-K genome expression was observed following female steroids treatment. In a detailed investigation on the organization of HERV-K proviruses in human genome, we found repetitive sequences homologous to the LTR region of the HERV-K genome. They were about 630 bp in length with an A rich tail at 3' end and found to be a SINE type nonviral retroposon. These elements were present at 4,000 to 5,000 copies per haploid human genome.

Animals↗
PubMed2004

Single domain antibodies derived from dromedary lymph node and peripheral blood lymphocytes sensing conformational variants of prostate-specific antigen.

The importance of the lymphocyte source to generate hybridomas or to construct antibody gene libraries from which to identify potent monoclonal antibodies is understudied. However, the few comparative studies that exist seem to favor the lymph node tissue as a B-cell source. Here the peripheral blood and lymph node lymphocytes of a dromedary immunized with prostate-specific antigen (PSA) have been employed to clone two independent gene banks of the variable domains of heavy-chain antibodies (i.e. the VHHs). Several PSA-specific VHHs were retrieved after panning of these phage-displayed VHH libraries. Some of them were derived from the same B-cell lineage, possibly reflecting the restricted primary repertoire of heavy-chain antibodies. Other binders originated from different B-cell lineages and apparently converged toward a striking homologous amino acid sequence motif in their CDR3. This illustrates the strong somatic hypermutation and stringent antigen-driven selection ongoing in these animals. Although the various antigen binders exhibit a broad range of kinetic rate constants for their interaction with the PSA, leading to equilibrium constants from 70 pM to 100 nM, no significant difference existed between the binders from the two B-cell sources. The VHHs of both libraries were categorized in three groups based on nonoverlapping epitopes. Some of these VHHs could inhibit and others could enhance the proteolytic activity of the antigen. Remarkably, VHHs seem to sense or induce conformational changes on different PSA isoforms, a feature that might be exploited to study the PSA conformational flexibility and to discriminate the stages of prostate cancer.

Amino Acid Sequence↗
PubMed2003

Molecular characterisation of a male-specific serine/threonine phosphatase from Oesophagostomum dentatum (Nematoda: Strongylida), and functional analysis of homologues in Caenorhabditis elegans.

A male-specifically expressed sequence tag was used as a probe to screen adult male Oesophagostomum dentatum (Nematoda; Strongylida) gene libraries. The cDNA clones isolated coded for a serine/threonine protein phosphatase with approximately 85% identity to two Caenorhabditis elegans proteins implicated in reproduction. However, the genomic structures for the two species were distinct, in that the O. dentatum gene contained seven introns, whereas the C. elegans homologues contained three (two of which were conserved between the two nematodes). The promoters of all three nematode genes contained two putative GATA motifs separated by six to seven nucleotides and located within 100 nucleotides of the predicted transcriptional start site. RNA interference (RNAi) experiments in C. elegans, targetting the two homologues, revealed a consistent reduction in the number of progeny produced by treated worms, indicating a functional role in reproduction. Expression of green fluorescent protein, directed by the putative promoters for the C. elegans phosphatase genes, was analysed in transgenic C. elegans. The present results suggest that there is a significant degree of conservation between O. dentatum and C. elegans in the features and function of the serine/threonine protein phosphatase characterised, which should have implications for detailed investigations into molecular reproductive processes of some parasitic nematodes.

Amino Acid Sequence↗
PubMed1988

Cloning and expression in Escherichia coli of the perfringolysin O (theta-toxin) gene from Clostridium perfringens and characterization of the gene product.

The gene encoding perfringolysin O, the thiol-activated hemolysin from Clostridium perfringens (ATCC 13124), was cloned and expressed in Escherichia coli. A gene library of C. perfringens chromosomal DNA was constructed in bacteriophage lambda EMBL3. A recombinant was identified that produced a hemolysin that was inhibited by cholesterol and was tentatively identified as perfringolysin O. Subcloning experiments localized the perfringolysin O gene (pfo) to a 1.8-kilobase region on the cloned chromosomal fragment. E. coli which carried a plasmid subclone of pfo (pRT1B) expressed perfringolysin O and secreted it into the periplasm. The amino-terminal sequence of the pfo gene product was identical with that determined for perfringolysin O purified from C. perfringens, indicating that E. coli correctly removed the signal peptide during secretion. Purification of the pfo product was accomplished by high-resolution gel filtration and anion-exchange chromatography. Analysis of the pfo product by sodium dodecyl sulfate gel electrophoresis showed that it comigrated with authentic perfringolysin O; both had an estimated molecular weight of 54,000. Two-dimensional tryptic peptide maps of the pfo product and of authentic perfringolysin O purified from C. perfringens were identical. The hemolytic activity of the pfo product was similar to that of authentic perfringolysin O; one hemolytic unit (HU) of the cloned gene product or authentic perfringolysin O corresponded to approximately 1 ng or a hemolytic activity of 10(6) HU per mg.

Amino Acid Sequence↗
PubMed1988

Localization of the kdsA gene with the aid of the physical map of the Escherichia coli chromosome.

The isolation and analysis of two recombinant plasmids containing the kdsA gene from Escherichia coli chromosomal gene libraries is reported. The subfragments obtained from the inserts correspond to the fragment pattern around coordinate 1,282 kilobases of the physical map of the E. coli chromosome (Kohara et al. Cell 50:495-508, 1987). The kdsA gene has been located at coordinates 1,282 through 1,283 kilobases, corresponding to min 26.7 in the classical map coordinates. The kdsA gene is transcribed from this position toward the nearby nar gene.

Aldehyde-Lyases↗
PubMed2000

Growth and migration markers of rat C6 glioma cells identified by serial analysis of gene expression.

Tumors derived from rat C6 cell implants into rat brain exhibit similar morphological characteristics and degree of vascularization to human glioblastomas. To establish a molecular basis for C6 gliosarcoma malignancy, we have constructed a molecular profile of the most abundantly expressed genes, using serial analysis of gene expression (SAGE). Sequence tags (1168) representing 738 individual transcripts were collected and tag-to-gene mapping was carried out using the UniGene data set for rat. Differentially expressed C6 transcripts were identified by comparison of tags collected for C6 cells with a similar number (1002) of tags from a rat primary astrocyte library. Genes found to be expressed at increased levels in C6 cells are associated with cell surface interactions, migration, or metastasis formation and proliferation. These include the receptor for hyaluronan-mediated motility (RHAMM), S-100 related protein 42A, galectin I, preproenkephalin, osteopontin, autocrine motility factor, alpha-tubulin, ad1 antigen, and cofilin. In addition, a tag with no database match probably representing a previously uncharacterized transcript was differentially expressed in C6 cells. Transcripts showing reduced expression in C6 cells relative to astrocytes included the extracellular matrix glycoprotein osteonectin/SPARC (secreted protein, acidic, rich in cysteine), actin-binding proteins thymosins beta-4 and beta-10, the cysteine protease inhibitor cystatin C, the actin-gelling protein SM22/transgelin, and ferritin-H. SAGE results were confirmed by Northern blot for all transcripts tested, reaffirming the value of the SAGE technique for expression profiling in cancer biology.

Animals↗
PubMed1983

Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria.

A cosmid cloning system has been developed which is useful for the construction of genomic libraries and the introduction of clones into a broad range of bacterial species. The cosmids pMMB33 and pMMB34 allow selective cloning into their unique BamHI site of 36-kb DNA fragments generated by BamHI, Sau3A and MboI partial digestion. This selective cloning is achieved by a strategy that avoids formation of polycosmids without a dephosphorylation step. It uses two unique recognition sites within the vectors for endoncleases that generate blunt-ended DNA fragments for the preparation of left and right cosmid "arms". An alternative method that uses the unique EcoRI and SstI sites and dephosphorylation of the cosmid arms prior to BamHI digestion is also outlined and discussed. The DNA is first cloned with either vector into a rec- E. coli strain, where clones can be maintained stably, and can then be introduced by mobilization into a wide range of Gram-negative species to permit the study of gene expression and complementation. Because mobilization is much more efficient than transformation, the vector has the advantage that it can be transferred between bacterial species that specify different restriction systems, where transformation appears to be inefficient. The vectors have been used to generate gene libraries from the chromosomal DNA of several Pseudomonas and a Thiobacillus species. The genes specifying myo-inositol transport from Pseudomonas strain JD34 have been cloned with this system.

Cloning, Molecular↗
PubMed1998

Trans activation of the Escherichia coli ato structural genes by a regulatory protein from Bacillus megaterium: potential use in polyhydroxyalkanoate production.

A Bacillus megaterium genomic fragment, which encoded an activator homologous to sigma 54 regulators and which was capable of activating Escherichia coli ato genes in trans, was detected in a gene library of B. megaterium screened for beta-ketothiolase activity. The fragment presented only one complete open reading frame (ORF1), which encoded a protein of 398 amino acids. The recombinant plasmid complemented mutations in the Escherichia coli atoC regulatory gene. The constitutive expression of the E. coli ato operon mediated by ORF1 could be useful for the synthesis of polyhydroxyalkanoates with different flexibility properties by recombinant E. coli strains.

Amino Acid Sequence↗
PubMed1996

Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries.

Ideal gene therapy vectors would be delivered intravenously to transfect only specific cells. Existing vectors only transfect cells in vivo in a manner determined by blood flow and the site of introduction. As a general and systematic approach for generating cell-targeting ligands for gene therapy vectors, we have used peptide-presenting phage libraries to select peptides that bind and enter several different cell types. Because of their small size, cell-binding peptides such as these could be incorporated into biological or physical gene therapy vectors. In addition, peptide-presenting phage themselves may also be candidates for gene therapy vectors.

Amino Acid Sequence↗
PubMed2002

A flavoprotein encoded in Selenomonas ruminantium is characterized after expression in Escherichia coli.

Selenomonas ruminantium is an obligate anaerobe that is very important for the provision of vitamin B12 to ruminants, which are particularly dependent upon this cofactor. One important use for vitamin B12 in anaerobic bacteria is for the utilization of glycerol as carbon source. A new flavoprotein has been found expressed by Escherichia coli from a plasmid created as part of a gene library of S. ruminantium. The 2.5-kb fragment of chromosomal DNA responsible for protein expression contains parts of two operons. Only one polypeptide (the flavoprotein) encoded by the S. ruminantium DNA is produced in E. coli in large amounts. The gene for the flavoprotein has been identified and is probably transcribed as part of an operon involved in glycerol metabolism in S. ruminantium. The flavoprotein has been purified and its molecular properties have been examined. Sequence analysis showed that this protein is a divergent member of the family of nitroreductases. Pure protein is a homodimer with a molecular weight of 44,500, containing one molecule of FMN per dimer. Like other nitroreductases, this protein forms a complex with pyridine nucleotide (NADPH), but unlike other nitroreductases, it fails to be reduced in this complex at a biologically significant rate. It has none of the common catalytic properties of other members of the nitroreductase family.

Amino Acid Sequence↗
PubMed1993

Zebrafish Mhc class II alpha chain-encoding genes: polymorphism, expression, and function.

Its small size and short generation time renders the zebrafish (Brachydanio rerio) an ideal vertebrate for immunological research involving large populations. A prerequisite for this is the identification of the molecules critical for an immune response in this species. In earlier studies, we cloned the zebrafish genes coding for the beta chains of the class I and class II major histocompatibility complex (Mhc) molecules. Here, we describe the cloning of the zebrafish alpha chain-encoding class II gene, which represents the first identification of a class II A gene in teleost fishes. The gene, which is less than 3 kilobases (kb) distant from one of the beta chain-encoding genes, is approximately 1.2 kb long and consists of four exons interrupted by very short (< 200 base pairs) introns. Its organization is similar to that of the mammalian class II A genes, but its sequence differs greatly from the sequence of the latter (36% sequence similarity). Among the most conserved parts is the promoter region, which contains X, Y, and TATA boxes with high sequence similarity to the corresponding mammalian boxes. The observed striking conservation of the promoter region suggests that the regulatory system of the class II genes was established more than 400 million years ago and has, principally, remained the same ever since. Like the DMA, but unlike all other mammalian class II A genes, the zebrafish gene codes for two cysteine residues which might potentially be involved in the formation of a disulfide bond in the alpha 1 domain. The primary transcript of the gene is 1196 nucleotides long and contains 708 nucleotides of coding sequence. The gene is expressed in tissues with a high content of lymphoid/myeloid cells (spleen, pronephros, hepatopancreas, and intestine). The analyzed genomic and cDNA sequences are probably derived from different loci (their overall sequence similarity in the coding region is 73% and their 3' untranslated regions are highly divergent from each other). The genes are apparently functional. Comparison of genes from different zebrafish populations reveals high exon 2 variability concentrated in positions coding for the putative peptide-binding region. Phylogenetic analysis suggests that the zebrafish class II A genes stem from a different ancestor than the mammalian class II A genes and the recently cloned shark class II A gene.

Amino Acid Sequence↗
PubMed2000

Chemical genetics: ligand-based discovery of gene function.

Chemical genetics is the study of gene-product function in a cellular or organismal context using exogenous ligands. In this approach, small molecules that bind directly to proteins are used to alter protein function, enabling a kinetic analysis of the in vivo consequences of these changes. Recent advances have strongly enhanced the power of exogenous ligands such that they can resemble genetic mutations in terms of their general applicability and target specificity. The growing sophistication of this approach raises the possibility of its application to any biological process.

Animals↗
PubMed2001

Molecular characteristics of non-small cell lung cancer.

We used hierarchical clustering to examine gene expression profiles generated by serial analysis of gene expression (SAGE) in a total of nine normal lung epithelial cells and non-small cell lung cancers. Separation of normal and tumor, as well as histopathological subtypes, was evident by using the 3,921 most abundant transcript tags. This distinction remained when only 115 highly differentially expressed tags were used. Furthermore, these 115 transcript tags clustered into groups suggestive of the unique biological and pathological features of the different tissues examined. Adenocarcinomas were characterized by high-level expression of small airway-associated or immunologically related proteins, whereas squamous cell carcinomas overexpressed genes involved in cellular detoxification or antioxidation. The messages of two p53-regulated genes, p21(WAF1/CIP1) and 14-3-3final sigma, were consistently underexpressed in the adenocarcinomas, suggesting that the p53 pathway itself might be compromised in this cancer type. Gene expression patterns observed by SAGE were consistent with results obtained by quantitative real-time PCR or cDNA array analyses by using a total of 43 lung tumor and normal samples. Thus, although derived from only a few tissue libraries, gene expression profiles obtained by using SAGE most likely represent an unbiased yet distinctive molecular signature for the most common forms of human lung cancer.

Base Sequence↗