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At least 217 records · Page 12Linked to original sources
PubMed2004

Isolation of mutations with dumpy-like phenotypes and of collagen genes in the nematode Pristionchus pacificus.

The nematode Pristionchus pacificus was developed as a satellite system in evolutionary developmental biology and forward and reverse genetic approaches allow a detailed comparison of various developmental processes between P. pacificus and Caenorhabditis elegans. To facilitate map-based cloning in P. pacificus, a genome map was generated including a genetic linkage map of approximately 300 molecular markers and a physical map of 10,000 BAC clones. Here, we describe the isolation and characterization of more than 40 morphological mutations that can be used as genetic markers. These mutations fall into 12 Dumpy genes and one Roller gene that represent morphological markers for all six P. pacificus chromosomes. Using an in silico approach, we identified approximately 150 hits of P. pacificus collagen genes in the available EST, BAC-end, and fosmid-end sequences. However, 1:1 orthologs could only be identified for fewer than 20 collagen genes.

Animals↗
PubMed2003

On selecting markers for association studies: patterns of linkage disequilibrium between two and three diallelic loci.

Association studies depend on linkage disequilibrium (LD) between a causative mutation and linked marker loci. Selecting markers that give the best chance of showing useful levels of LD with the causative mutation will increase the chances of successfully detecting an association. This report examines the variation in the extent of LD between a disease locus and one or two diallelic marker loci (termed single nucleotide polymorphisms or SNPs). We use a simulation method based on the neutral coalescent in a population of variable size to find the distribution of LD as a function of allele frequencies, the recombination rate, and the population history. Given that LD exists, the allele frequencies determine if a site will be useful for detecting an association with the disease mutation. We show that there is extensive variation in LD even for closely linked loci, implying that several markers may be needed to detect a disease locus. The distribution of LD between common variants is strongly influenced by ancestral population size. We show that in general, best results will be obtained if the frequencies of marker alleles are at least as large as the frequency of the causative mutation. Haplotypes of two or more SNPs generally have a higher probability than individual SNPs of showing useful LD with a disease mutation, although exceptions are described.

Alleles↗
PubMed1990

Transactivation of anthocyanin biosynthetic genes following transfer of B regulatory genes into maize tissues.

The C1, B and R genes regulating the maize anthocyanin biosynthetic pathway encode tissue-specific regulatory proteins with similarities to transcriptional activators. The C1 and R regulatory genes are usually responsible for pigmentation of seed tissues, and the B-Peru allele of B, but not the B-I allele, can substitute for R function in the seed. In this study, members of the B family of regulatory genes were delivered to intact maize tissues by high velocity microprojectiles. In colorless r aleurones or embryos, the introduction of the B-Peru genomic clone or the expressed cDNAs of B-Peru or B-I resulted in anthocyanin-producing cells. Luciferase produced from the Bronze1 anthocyanin structural gene promoter was induced 100-fold when co-introduced with the expressed B-Peru or B-I cDNAs. This quantitative transactivation assay demonstrates that the proteins encoded by these two B alleles are equally able to transactivate the Bronze1 promoter. Analogous results were obtained using embryogenic callus cells. These observations suggest that one major contribution towards tissue-specific anthocyanin synthesis controlled by the various alleles of the B and R genes is the differential expression of functionally similar proteins.

Alleles↗
PubMed1991

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Animals↗
PubMed1994

Human immunodeficiency viruses regulated by alternative trans-activators: genetic evidence for a novel non-transcriptional function of Tat in virion infectivity.

Thirteen genetically altered HIV-1 proviruses were created. These various genomes can be segregated into three groups: (i) a set of tat(-) viruses that have a functional HTLV-I Tax inserted in nef; (ii) a set of tat(-) viruses with Gal4 binding sites inserted in U3 and a Gal4-VP16 cDNA inserted in nef; and (iii) a set of tat(+) HIV genomes that are 5' and 3' TAR(-) and are Gal4-binding-site(+) in U3 and Gal4-VP16(+) in nef. We found that viruses in groups (i) and (ii), although tat(-), were fully complemented for viral gene expression based on quantitative measurements of viral protein synthesis and on the visualization by electron microscopy of the proper assembly of morphologically correct virions. Interestingly, group (i) and (ii) virions were defective in a spreading cytopathic infection when assayed in T-lymphocytes. Group (iii) viruses, although capable of producing intact Tat protein, also could not use Tat for transcription/gene expression because of the TAR(-) genotype. However, this class of viral genomes produced viruses that were highly infectious and cytopathic in primary and in continuously propagated T-lymphocytes. These three groups of viruses are all transcriptionally Tat-TAR independent. Their distinct differences in infectivity/cytopathicity provide genetic evidence that Tat provides a transcriptionally independent function in determining infectivity and cytopathicity in the setting of a spreading viral infection. Given that all HIV virions normally contain four intact copies of TAR RNA, our findings suggest a re-examination of whether Tat could be a virion-TAR-associated protein and the possible implications of this for virus infectivity/cytopathicity.

Base Sequence↗
PubMed1986

Highly adherent mutants of SV40-transformed mouse fibroblasts: genetic and biochemical characterization.

Mutants of SV40-transformed mouse fibroblasts have been isolated that have greatly increased cell-substratum adherence. The adherent phenotype (COL-) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild-type and mutant cells were found with respect to the protein content of the substrate-attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+-chelating agent. However, the mutants yielded 2.5-10-fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6-thioguanine, which suggests X-linkage.

Animals↗
PubMed1995

Suppression of tumorigenicity, but not invasion, in glioblastoma/HeLa cell hybrids.

Somatic cell hybrids between SNB-19 human glioblastoma cells and human D98OR HeLa cells were produced and analyzed for their ability to form tumors in nude mice and to invade reconstituted extracellular matrix (Matrigel). Whereas both the SNB-19 and D98OR HeLa parental cells form tumors, four of six hybrid lines did not form tumors, even after periods up to six months, suggesting that each cell type can complement the tumorigenicity of the other. SNB-19 cells showed high rates of Matrigel invasion at all cell densities examined, whereas D98OR HeLa cells showed lower rates of invasion that were further reduced at high cell density. All six hybrid cell lines displayed a combination of these properties: at low cell density, the hybrids showed high rates of invasion, similar to the SNB-19 cells, but the invasion rate diminished at higher cell densities, similar to the D98OR HeLa cells. Taken together, these results provide new experimental evidence that several distinct genetic changes are involved in generating the tumorigenic and invasive phenotype of glioblastoma cells.

Animals↗
PubMed1975

Assembly of the tail of bacteriophage T4.

The protein products of at least 21 phage genes are needed for the formation of the tail of bacteriophage T4. Cells infected with amber mutants defective in these genes are blocked in the assembly process. By characterizing the intermediate structures and unassembled proteins accumulating in mutant-infected cells, we have been able to delineate most of the gene-controlled steps in tail assembly. Both the organized structures and unassembled proteins serve as precursors for in vitro tail assembly. We review here studies on the initiation, polymerization, and termination of the tail tube and contractile sheath and the genetic control of these processes. These studies make clear the importance of the baseplate; if baseplate formation is blocked (by mutation) the tube and sheath subunits remain essentially unaggregated, in the form of soluble subunits. Seventeen of the 21 tail genes specify proteins involved in baseplate assembly. The genes map contiguously in two separate clusters, one of nine genes and the other of eight genes. Recent studies show that the hexagonal baseplate is the end-product of two independent subassembly pathways. The proteins of the first gene cluster interact to form a structure which probably represents one-sixth of the outer radius. The products of the other gene cluster interact to form the central part of the baseplate. Most of the phage tail precursor proteins appear to be synthesized in a nonaggregating form; they are converted to a reactive form upon incorporation into preformed substrate complexes, without proteolytic cleavage. Thus reactive sited are limited to growing structures.

Centrifugation, Density Gradient↗
PubMed2004

Cloning and functional characterization of the SUR2/SYR2 gene encoding sphinganine hydroxylase in Pichia ciferrii.

Saccharomyces cerevisiae sphinganine C4-hydroxylase encoded by the SUR2 gene catalyses the conversion of sphinganine to phytosphingosine. We isolated the SUR2 gene from Pichia ciferrii using nucleotide sequence homology to S. cerevisiae SUR2 to study hydroxylation of sphinganine in the sphingoid base overproducing yeast P. ciferrii. A positive clone was confirmed by nucleotide sequencing. A syringomycin-E resistance phenotype of a S. cerevisiae sur2-null mutant was complemented by expression of the cloned P. ciferrii SUR2 gene. Restoration of phytosphingosine production in the complemented strain was also confirmed, indicating that the cloned gene is a functional homologue of S. cerevisiae SUR2. .

Amino Acid Sequence↗
PubMed2005

A novel yeast genomic DNA library on a geneticin-resistance vector.

We have constructed a Saccharomyces cerevisiae genomic library utilizing a novel centromere-containing vector bearing the TRP1 and KANMX4 G418-resistance selectable markers. Random Sau3AI genomic DNA fragments with an average size of 6.4 kb were introduced into a unique BamHI site in the vector's polylinker. Six independent library pools were generated and the characteristics of these pools were determined. This library can be used to introduce low-copy genomic DNA fragments into any strain that is not G418-resistant, making it useful for low-copy overexpression analysis or complementation of mutant phenotypes in strains that lack a suitable auxotrophy marker or only exhibit phenotypes on rich, undefined, growth media.

Aldose-Ketose Isomerases↗
PubMed1988

A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis.

The killer system of Kluyveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which block the expression of the killer character have been isolated. These mutations reside in a single chromosomal gene which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a disrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutation of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutation of S. cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.

Cloning, Molecular↗
PubMed1991

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes.

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.

Arginine↗
PubMed1992

DNA transformations of Candida tropicalis with replicating and integrative vectors.

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.

Alcohol Dehydrogenase↗
PubMed1993

A series of yeast/Escherichia coli lambda expression vectors designed for directional cloning of cDNAs and cre/lox-mediated plasmid excision.

A series of Saccharomyces cerevisiae/Escherichia coli lambda/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from lambda. Features of six are described, and two designated lambda PG15 and lambda AD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters, ADH2, PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator. Unique EcoRI and XhoI restriction sites in the intervening polylinker make these lambda vectors compatible for directional cloning of 'ZAP'-synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in both S. cerevisiae and E. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the lambda vector is mediated by the activity of the cre-encoded enzyme upon lox sequences flanking the plasmid and adjoining the lambda arms. The plasmids contain the yeast 2 microns origin and E. coli pBR322 origin, the URA3 or TRP1 yeast selectable markers, and ampicillin-resistance marker in E. coli. The usefulness of the lambda PG15 and the lambda AD5 cloning vectors was demonstrated by constructing large Neurospora crassa cDNA libraries. The lambda PG15-N. crassa library was used to infect purE, purC and trpC mutants of E. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning of cDNAs is discussed.

Bacteriophage lambda↗
PubMed1995

The red ade mutants of Kluyveromyces lactis and their classification by complementation with cloned ADE1 or ADE2 genes from Saccharomyces cerevisiae.

Seventy-six red adenine mutants of Kluyveromyces lactis were isolated. By complementation they could be assigned to two groups with 31 and 45 mutants. Transformation of several strains from each group with plasmids containing the Saccharomyces cerevisiae ADE1 or ADE2 gene showed that the largest group was ade2 and the other group was ade1. Several previously isolated 'ade1' mutants were classified to either group and given new gene and allele numbers. ADE1 was localized at chromosome III, closely linked to the mating type gene, making it a convenient marker for mating type. ADE2 was localized at chromosome V.

Adenine↗
PubMed2003

Isolation and characterization of the TRP1 gene from the yeast Yarrowia lipolytica and multiple gene disruption using a TRP blaster.

The TRP1 gene encoding N-(5'-phosphoribosyl)-anthranilate isomerase was isolated from the yeast Yarrowia lipolytica, in which only a few genetic marker genes are available. The Y. lipolytica TRP1 gene (YlTRP1) cloned by complementation of Y. lipolytica trp1 mutation was found to be a functional homologue of Saccharomyces cerevisiae TRP1. Since YlTRP1 could be used for counterselection in medium containing 5-fluoroanthranilic acid (5-FAA), we constructed TRP blasters that contained YlTRP1 flanked by a direct repeat of a sequence and allowed the recycling of the YlTRP1 marker. Using the TRP blasters the sequential disruption of target genes could be carried out within the same strain of Y. lipolytica. The nucleotide sequence of the YlTRP1 gene has been deposited at GenBank under Accession No. AF420590.

Aldose-Ketose Isomerases↗
PubMed1995

Expression, purification, and characterization of the human squalene synthase: use of yeast and baculoviral systems.

We have cloned and utilized a cDNA corresponding to the human squalene synthase gene to generate active enzyme from yeast and baculoviral expression systems. Expression of human squalene synthase in yeast resulted in production of active enzyme in cellular lysates. The presence of the active human enzyme, however, was insufficient to rescue growth of spores defective in yeast squalene synthase function, suggesting that structural differences in the yeast and human enzymes may affect localization or folding of the protein. Expression of the human enzyme in Sf-9 insect cells after infection with recombinant baculovirus encoding the human squalene synthase gene resulted in detection of substantial enzymatic activity in cell lysate preparations. Following extraction from the Sf-9 cells, the human enzyme was purified to near homogeneity utilizing a series of ion-exchange chromatography steps with an overall yield of purified protein of approximately 5 mg per liter of Sf-9 cell culture. The purified enzyme was characterized through steady-state kinetic and physical measurements and the kinetic constants are consistent with values observed for other squalene synthases. Zaragozic acid C was found to be a competitive inhibitor with respect to farnesyl pyrophosphate and has a Kis value of 250 pM (@ [NADPH] = 5 mM). Inhibition experiments with zaragozic acid C at low (approximately 0.5 x Km) and high (approximately 10 x Km) concentrations of NADPH indicated that the inhibitor does not bind in the enzyme's NADPH binding domain. These studies demonstrate that the human enzyme can be prepared from baculovirus-infected Sf-9 cells in a catalytically active configuration and in sufficient quantities to allow for further biochemical, kinetic, and structural characterization.

Amino Acid Sequence↗