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At least 541 records · Page 30Linked to original sources
PubMed1983

Cloning of a LEU gene and an ARS site of Candida maltosa.

Gene libraries of DNA from an n-alkane-assimilating yeast strain, Candida maltosa IAM12247, were constructed, using Escherichia coli plasmid vector pBR322. A LEU gene from C. maltosa was cloned, and found to complement leu- mutations in E. coli and Saccharomyces cerevisiae. In E. coli, the LEU gene in the cloned yeast DNA fragment was efficiently expressed when inserted into the vector in one orientation, while in the other orientation, it was expressed only weakly. In S. cerevisiae, the Candida LEU gene was efficiently expressed when inserted into a shuttle vector pRC3 in both orientations, suggesting that the isolated Candida DNA fragment contains a promoter sequence of Candida in front of the LEU gene, which is operative in S. cerevisiae but not in E. coli. In addition, our data suggest that the cloned LEU fragment also contains an ARS (autonomously replicating sequence) site of C. maltosa.

Candida↗
PubMed2004

The ypdI gene codes for a putative lipoprotein involved in the synthesis of colanic acid in Escherichia coli.

In Escherichia coli, the synthesis of colanic acid, an extracellular polysaccharide imminent in biofilm development, is a complicated process involving numerous genes and not yet wholly elucidated. Using a plasmid-borne E. coli K-12 gene library, we have identified a clone whose presence conferred mucoid colony phenotype onto E. coli CM2555 strain. Our results indicate that overexpression of a gene previously catalogued as ypdI, which encodes a putative lipoprotein, is responsible for this phenotype. We show that the mucoidy of ypdI -overexpressing bacteria is due to increased production of colanic acid. This phenotype depends on the function of the rcsA gene, but not on that of rcsF. These results suggest that the ypdI gene product might be an additional factor playing a role in colanic acid synthesis, indicating that this process can be even more complicated than supposed to date. However, no obvious phenotype was observed in the DeltaypdI::kan mutant cultivated under standard laboratory conditions.

Culture Media↗
PubMed1982

Isolation and analysis of genomic DNA clones encoding the third component of mouse complement.

A gene library was constructed with DNA from strain A mice by using the phage lambda vector lambda 1059. By screening with cloned cDNA for the third component of mouse complement, C3, four different C3 genomic clones were isolated from this library. Two of the recombinant phages carry insertions of 14 and 18 kilobase pairs, respectively, which together cover one complete copy of the C3 gene and several hundred nucleotides of its 5' and 3' flanking sequences. The distance from the 5' end of the gene, which includes the hexanucleotide T-A-T-A-A-A and a translation initiation codon, to its 3' end as defined by the poly(A) attachment site is 24 kilobase pairs. From the genomic DNA sequence, a signal peptide of 24 amino acid residues is predicted at the NH2 terminus of the initial translation product. The signal peptide and the next two amino acids are encoded by the first exon of this gene.

Animals↗
PubMed2001

Regulation of metalloprotease gene expression in Vibrio vulnificus by a Vibrio harveyi LuxR homologue.

Expression of the Vibrio vulnificus metalloprotease gene, vvp, was turned up rapidly when bacterial growth reached the late log phase. A similar pattern of expression has been found in the metalloprotease gene of Vibrio cholerae, and this has been shown to be regulated by a Vibrio harveyi LuxR-like transcriptional activator. To find out whether a LuxR homologue exists in V. vulnificus, a gene library of this organism was screened by colony hybridization using a probe derived from a sequence that is conserved in various luxR-like genes of vibrios. A gene containing a 618-bp open reading frame was identified and found to be identical to the smcR gene of V. vulnificus reported previously. An isogenic SmcR-deficient (RD) mutant was further constructed by an in vivo allelic exchange technique. This mutant exhibited an extremely low level of vvp transcription compared with that of the parent strain. On the other hand, the cytolysin gene, vvhA, was expressed at a higher level in the RD mutant than in the parent strain during the log phase of growth. These data suggested that SmcR might not only be a positive regulator of the protease gene but might also be involved in negative regulation of the cytolysin gene. Virulence of the RD mutant in either normal or iron-overloaded mice challenged by intraperitoneal injection was comparable to that of the parent strain, indicating that SmcR is not required for V. vulnificus virulence in mice.

Bacterial Proteins↗
PubMed1984

Amplification of a specific set of intracisternal A-particle genes in a mouse plasmacytoma.

Intracisternal A-particles (IAPs) derive from endogenous retrovirus-like genes that are present in ca. 1,000 copies per haploid mouse genome. A particular IAP sequence variant that represents only 2% of the endogenous genes was found to be the predominant transcript in five out of seven plasmacytoma lines examined, and these genes have been amplified two- to fourfold in MOPC 315. Restriction enzyme digests of normal and tumor DNAs revealed a set of conserved sites within the population of amplified genes which could be used to generate a consensus restriction enzyme map of the amplified IAP genome. Molecular clones of IAP genes related to those amplified in MOPC 315 were isolated from gene libraries of MOPC 315 and MOPC 104E DNAs. Each clone was characterized for the presence of the restriction enzyme sites conserved in the amplified genes. Of 26 clones isolated from MOPC 315 DNA, 14 contained all of the conserved sites, whereas none of the clones isolated from MOPC 104E DNA contained all of the sites. These results suggested that approximately one-half of the genes isolated from MOPC 315 DNA were members of the amplified IAP gene population, representing newly acquired proviruses in the tumor DNA. Unique cellular flanking sequences were isolated from four of the MOPC 315 clones and four of the MOPC 104E clones. Probes from three of the MOPC 315 clones detected restriction fragments that were unique to MOPC 315 DNA and equivalent in size to the cloned gene, demonstrating that these three IAP genes had integrated into new sites in the plasmacytoma DNA. The other five clones were found to be endogenous to the BALB/c genome. Thus, IAPs represent a significant source of insertional mutations for any cell expressing the retrovirus-like particles. Furthermore, these results suggest that only one or a small number of endogenous IAP sequences are activated to give the homogeneous population of amplified genes.

Animals↗
PubMed1989

Expression and organization of BP74, a cyclic AMP-regulated gene expressed during Dictyostelium discoideum development.

We have characterized a cDNA and the corresponding gene for a cyclic AMP-inducible gene expressed during Dictyostelium development. This gene, BP74, was found to be first expressed about the time of aggregate formation, approximately 6 h after starvation. Accumulation of BP74 mRNA did not occur in Dictyostelium cells that had been starved in fast-shaken suspension cultures but was induced in similar cultures to which cyclic AMP pulses had been added. The BP74 cDNA and gene were characterized by DNA sequence analysis and transcriptional mapping. When the BP74 promoter region was fused with a chloramphenicol acetyltransferase reporter gene and reintroduced into Dictyostelium cells, the transfected chloramphenicol acetyltransferase gene displayed the same developmentally regulated pattern of expression as did the endogenous BP74 gene, suggesting that all of the cis-acting elements required for regulated expression were carried by a 2-kilobase cloned genomic fragment. On the basis of sequence analysis, the gene appeared to encode a protein containing a 20-residue hydrophobic sequence at the amino-terminal end and 26 copies of a 20-amino-acid repeat.

Amino Acid Sequence↗
PubMed2001

Single-chain antibody against the common epitope of mutant p53: isolation and intracytosolic expression in mammalian cells.

The peptide epitope FRHSVV is cryptic in wild-type p53 and is exposed in many types of mutant p53 molecules isolated from various tumors. Mutant p53 marked by this epitope abrogates a tumor-suppressor function of wild-type p53 and possibly contributes to the transforming potential of other oncogenic processes. We report here the construction of a single-chain scFv antibody gene library derived from the mRNA of a mouse immunized with the epitope peptide FRHSVV which mimics the common epitope in p53 mutant protein molecules. The scFv was presented by phage display. The selected antibody gene, named ME1, was found to bind to the mutant p53 protein but not to the wild-type p53 protein. Preliminary studies show that the ME1 gene is expressed in the cytosol of mammalian cells. These findings suggest that the ME1 single-chain antibody may be useful as a tool for clarifying the role of mutant p53 in tumor transformation, especially in cells heterozygous in p53, and possibly for gene therapy of tumors.

Amino Acid Sequence↗
PubMed2000

Gene sam1 encoding adenosylmethionine synthetase: effects of its expression in the fission yeast Schizosaccharomyces pombe.

By screening gene libraries of Schizosaccharomyces pombe with a DNA fragment encoding part of the Saccharomyces cerevisiae S-adenosylmethionine synthetase (SAMS), we isolated the fission yeast sam1 gene. Its sequence exhibits good homology to SAMSs of other organisms and reveals the motifs characteristic for SAMSs. SAMS activity and sam1 mRNA levels decrease when cells enter stationary phase. In haploid strains, gene sam1 is essential for growth; if weakly expressed, cells mate and sporulate at a reduced rate. Strains overexpressing sam1 exhibit methionine-sensitive growth. This methionine-induced growth inhibition is partially relieved by adenine. We assume that methionine reduces the level of one or several adenine nucleotides by a SAMS-mediated mechanism. Intracellular SAM levels increase drastically by exogenously added methionine. This increase predicts that mutants exhibiting methionine revertible phenotypes can be indicative for mutations in proteins exhibiting SAM-dependent functions. In agreement with this prediction, we show that mutant pmt2-5 has this phenotype and that gene pmt2 encodes a potential SAM-dependent enzyme.

Amino Acid Sequence↗
PubMed1983

Isolation and characterization of a human interleukin 2 gene.

An interleukin 2 (IL-2) gene was isolated from a Charon 4A human gene library. Electron microscopic examination of 15 heteroduplexes formed between the genomic DNAs and the IL-2 cDNAs demonstrated that the size of the IL-2 gene is about 5.1 +/- 0.5 kb and that there are at least two introns in this gene. Nucleotide sequence of the 5' flanking region of the IL-2 gene showed a homology with that of the corresponding region of the human immune interferon gene.

Base Sequence↗
PubMed1991

Isolation of ribosomal RNA operons of Streptomyces lividans and sequence analysis of a 5S-rRNA gene.

Three clones containing rRNA genes have been isolated from a gene library of Streptomyces lividans. Two clones carried entire but different rRNA-operons. The third clone comprised the 3'-end of a 23S-rRNA gene, the entire 5S-rRNA gene and the spacer region between them. The nucleotide sequence starting within the 23S-rRNA gene beyond the putative transcription terminator downstream the 5S-rRNA gene has been determined (EMBL acc. no X58874).

Base Sequence↗
PubMed1992

Characterization of the gene encoding human pituitary-specific transcription factor, Pit-1.

Pit-1 is a pituitary-specific transcription factor that binds to and transactivates promoters of growth hormone- and prolactin-encoding genes. A chromosomal gene related to human Pit-1 isolated from human gene libraries was over 14 kb long and split into six exons. All of the splice donor and acceptor sites conformed to the GT/AG rule. The gene was mapped to human chromosome region 3p11.

Base Sequence↗
PubMedDate not supplied

Mitochondrial DNA sequences in the nuclear genome of a locust.

The endosymbiotic theory of the origin of mitochondria is widely accepted, and implies that loss of genes from the mitochondria to the nucleus of eukaryotic cells has occurred over evolutionary time. However, evidence at the DNA sequence level for gene transfer between these organelles has so far been limited to a single example, the demonstration that a mitochondrial ATPase subunit gene of Neurospora crassa has an homologous partner in the nuclear genome. From a gene library of the insect, Locusta migratoria, we have now isolated two clones, representing separate fragments of nuclear DNA, which contain sequences homologous to the mitochondrial genes for ribosomal RNA, as well as regions of homology with highly repeated nuclear sequences. The results suggest the transfer of sequences between mitochondrial and nuclear genomes, followed by evolutionary divergence.

Animals↗
PubMed1995

Mutant farnesyltransferase beta subunit of Saccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit.

The protein farnesyltransferase (PFT) beta-subunit gene of Saccharomyces cerevisiae, DPR1, was randomly mutagenized by PCR to construct a mutant DPR1 gene library on a high-copy plasmid. The library was screened for suppression of the temperature sensitivity conferred by a mutation in the protein geranylgeranyltransferase type I (PGGT-I) beta-subunit gene, CAL1. A mutant DPR1 gene was identified whose product contained a single amino acid change of Ser-159 to Asn. This mutant gene also suppressed a cal1 disruption even on a low-copy plasmid, suggesting that the product (designated S159N) can substitute for PGGT-I beta subunit in vivo. Its ability to act as a PFT is not drastically reduced, since the mutant gene still complemented a dpr1 disruption. Results of in vitro assays demonstrate that the mutant enzyme has increased activity to farnesylate, a substrate for PGGT-I. On the other hand, the ability to farnesylate its own substrate is reduced. The increased ability to utilize the PGGT-I substrate is due to its increased affinity for the protein substrate. In addition, the mutant enzyme shows a severalfold increase in the sensitivity to a peptidomimetic inhibitor that acts as a competitor of the protein substrate. These results point to the importance of the beta subunit of PFT for the binding of a protein substrate and demonstrate that Ser-159 of DPR1 product is critical for its substrate specificity.

Alkyl and Aryl Transferases↗
PubMed1994

Molecular cloning and sequence analysis of the proBA operon from an extremely thermophilic eubacterium Thermus thermophilus.

A 3.6 kb DNA fragment carrying the Thermus thermophilus proBA region, which encodes the first two steps in the proline biosynthetic pathway, was cloned from the Thermus thermophilus gene library, and its complete nucleotide sequence was determined. The deduced amino acid sequence of gamma-glutamyl kinase (40,657 Da), the product of proB gene, and gamma-glutamyl phosphate reductase (48,747 Da), the product of proA gene, showed 44.1% and 44.4% identity to those of Escherichia coli, respectively. The termination codon of the proB gene and the initiation codon of the proA gene overlapped by 2 bp. A possible transcriptional termination structure was found downstream of the proA gene but not downstream of the proB gene. These results indicate that the proBA genes of T. thermophilus form a single operon as in E. coli.

Aldehyde Oxidoreductases↗
PubMed1989

In vivo cloning of Pseudomonas aeruginosa genes with mini-D3112 transposable bacteriophage.

The transposition properties of the Pseudomonas aeruginosa mutator bacteriophage D3112 were exploited to develop an in vivo cloning system. Mini-D replicon derivatives of D3112 were constructed by incorporating broad host range plasmid replicons between short terminal D3112 sequences. These elements were made with small replication regions from the RK2, Sa, and pVS1 plasmids and selectable genes for tetracycline, carbenicillin, kanamycin, and gentamicin resistance. Some of the mini-D replicons also contain the RK2 oriT origin-of-transfer sequence, which allows them to be mobilized by conjugation to many different species of gram-negative bacteria. These elements were used to clone DNA by preparing lysates from P. aeruginosa cells harboring an inducible D3112 cts prophage and a mini-D replicon plasmid. These lysates were used to infect sensitive P. aeruginosa recipients and select recombinant plasmids as drug-resistant transductant colonies. These transductants form a gene library from which particular clones can be selected, such as by their ability to complement specific mutations. This system was used to clone nine different genes from the PAO chromosome. The ability of this system to precisely identify a gene was demonstrated by isolating clones of the argF+ and cys-59+ genes. Restriction maps of clones of these genes, which have different amounts of flanking DNA, located the positions of these genes. The sizes of the chromosomal DNA segments from 10 individual clones examined ranged from 6 to 21 kilobases (kb), with an average of about 10 kb. This is consistent with the approximately 40-kb DNA-packaging size of the D3112 phage.

Bacteriophages↗
PubMed1996

Molecular cloning of a novel gene, dtsR, which rescues the detergent sensitivity of a mutant derived from Brevibacterium lactofermentum.

Several strains of Corynebacterium and Brevibacterium are known for their ability to secrete large amounts of amino acids, especially L-glutamate. We focused on the mechanism of L-glutamate secretion triggered by a detergent, namely polyoxyethylenesorbitan monopalmitate (PESP). A mutant strain, AJ11060, derived from Brevibacterium lactofermentum ATCC 13869 indicates the sensitivity to PESP. A multicopy suppresser gene that compliments the sensitivity of AJ11060 to the detergent was derived from a gene library of B. lactofermentum AJ12036. A 2855-bp DNA fragment was cloned and sequenced. An open reading frame was found that coded for the rescuer gene of the sensitivity to PESP of AJ11060 and was designated dtsR. The expression of the dtsR gene in B. lactofermentum was confirmed by using anti-DtsR antibody. The deduced DtsR protein indicated significant homology with some biotin enzymes such as the beta chain of propionyl-CoA carboxylase from rat (48.3%) and human (48.7%), or a 12S chain of methylmalonyl-CoA carboxyltransferase from Propionibacterium freudenreichii (43.1%).

Amino Acid Sequence↗
PubMed2001

Screening from a subtracted embryonic chick hindbrain cDNA library: identification of genes expressed during hindbrain, midbrain and cranial neural crest development.

The vertebrate hindbrain is segmented into a series of transient structures called rhombomeres. Despite knowing several factors that are responsible for the segmentation and maintenance of the rhombomeres, there are still large gaps in understanding the genetic pathways that govern their development. To find previously unknown genes that are expressed within the embryonic hindbrain, a subtracted chick hindbrain cDNA library has been made and 445 randomly picked clones from this library have been analysed using whole mount in situ hybridisation. Thirty-six of these clones (8%) display restricted expression patterns within the hindbrain, midbrain or cranial neural crest and of these, twenty-two are novel and eleven encode peptides that correspond to or are highly related to proteins with previously uncharacterised roles during early neural development. The large proportion of genes with restricted expression patterns and previously unknown functions in the embryonic brain identified during this screen provides insights into the different types of molecules that have spatially regulated expression patterns in cranial neural tissue.

Amino Acid Sequence↗