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At least 883 records · Page 49Linked to original sources
PubMed2004

A new cacao linkage map based on codominant markers: development and integration of 201 new microsatellite markers.

A linkage map of cacao based on codominant markers has been constructed by integrating 201 new simple sequence repeats (SSR) developed in this study with a number of isoenzymes, restriction fragment length polymorphisms (RFLP), microsatellite markers and resistance and defence gene analogs (Rgenes-RFLP) previously mapped in cacao. A genomic library enriched for (GA)(n) and (CA)(n) was constructed, and 201 new microsatellite loci were mapped on 135 individuals from the same mapping population used to establish the first reference maps. This progeny resulted from a cross between two heterozygous cacao clones: an Upper-Amazon Forastero (UPA 402) and a Trinitario (UF 676). The new map contains 465 markers (268 SSRs, 176 RFLPs, five isoenzymes and 16 Rgenes-RFLP) arranged in ten linkage groups corresponding to the haploid chromosome number of cacao. Its length is 782.8 cM, with an average interval distance between markers of 1.7 cM. The new microsatellite markers were distributed throughout all linkage groups of the map, but their distribution was not random. The length of the map established with only SSRs was 769.6 cM, representing 94.8% of the total map. The current level of genome coverage is approximately one microsatellite every 3 cM. This new reference map provides a set of useful markers that is transferable across different mapping populations and will allow the identification and comparison of the most important regions involved in the variation of the traits of interest and the development of marker-assisted selection strategies.

Cacao↗
PubMed1992

[Structure and evolution of the D17LeH80-like locus in the murine t-complex].

The t complex in the proximal part of chromosome 17 is one of the most thoroughly studied regions of the mouse genome. We determined the sequence of Tu80, a molecular clone derived from microdissected fragments of chromosome 17. The sequence data demonstrated that the total length being 324 bp, Tu80 contains an open-reading frame (ORF) of 204 bp. Two fragments were detected within the ORF, one homologous to the LINE1-element, the other to the first intron of the C epsilon gene of mouse immunoglobin. A sequence designated NOV1 was isolated from the genomic library of mouse chromosome 17. NOV1 was found to contain a B2 insert, making in structurally different from Tu80. The sequences of Tu80 and NOV1 were compared with those of LINE1 and the first intron of the C epsilon gene. The results suggested that the ancestor of the Tu80-like sequence might have arisen through illegitimate recombination between the fragments of LINE1 and the C epsilon gene. It is concluded that Tu80 and NOV1 might have resulted from duplication of the ancestral sequence and following divergence. The comparative analysis also demonstrated high degree of conservation of the LINE1 fragments in Tu80 and NOV1, as well as in the LINE1 in a number of mammalian species. Based on the structure of human, rat, rabbit and mouse LINE1 fragments, and also on that of NOV1 and Tu80, phylogenetic tree has been constructed. Its topology is consistent with the accepted phylogenetic relationships among the species studied. The data available tend to support the assumption that the ancestor for the Tu80-like sequence might have arisen not later than 27-33 million years ago.

Amino Acid Sequence↗
PubMed1991

[Molecular cloning of lipopolysaccharide genes of the Vibrio cholerae in E. coli HB101].

A genomic library of the V. cholerae 178 (Eltor biotype, Ogawa serotype) was constructed by using cosmid pHC 79 as a cloning vector. We screened the library with immune agglutination test and colonies solid phase ELISA. 13 positive recombinants which could express the O antigen of the V. cholerae lipopolysaccharide (LPS) were acquired. The LPS was then extracted from a positive recombinant PMM-VO 38 by using hot phenol-water method. It was found that purified LPS specifically reacted to antisomatic serum against the V. cholerae. The restriction endonucleases analysis showed that the molecular weight of the recombination cosmid PMM-VO 38 was about 46 kb.

Antigens, Bacterial↗
PubMed2003

High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae).

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria x ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H(e) or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12-17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.

Alleles↗
PubMed1996

Use of genomic DNA probes for the diagnosis of acute sarcocystosis in experimentally infected cattle.

Two clones of 1.4 and 4.33 kilobase pairs (kbp) DNA inserts, were selected from a Sarcocystis cruzi sporozoite genomic library constructed in bacteriophage lambda gt10. These clones strongly hybridized with sporozoite and merozoite DNA and were evaluated as probes for detection of merozoite DNA in clinical samples. Of five calves in the experiment, four were each orally dosed with approximately 200,000 S. cruzi sporocysts; one calf served as non-infected control. Subsequently, blood was collected from the calves twice weekly for 3.5 months and fractionated into buffy coats, polymorphonuclear cells, and plasma. Total cellular DNA extracted from these fractions was dot blotted on nylon membranes and hybridized with the probes radiolabeled with [alpha-32P]dATP. The probes detected merozoites on Day 22 post infection in the buffy coats and intermittently from Day 25-39 in the granulocyte fraction. Parasitemia (i.e. merozoites in blood) was also detected by indirect fluorescent antibody technique (IFAT) and direct microscopy, Diagnosis of sarcocystosis in cattle using genomic DNA probes by dot blot hybridization provides an alternative method of detecting parasitemia that is more rigorous than the other two tests (IFAT, direct microscopy) which rely on morphology of the merozoite and visualization by the examiner. As probes detected merozoite DNA in the granulocyte fraction, polymorphonuclear cells may be involved in the pathogenesis of S.cruzi; however this hypothesis requires further study.

Animals↗
PubMed1995

Genomic organization of a mouse type I activin receptor.

We have characterized the genomic organization of a mouse type I activin receptor. Using the mouse tsk7L cDNA, 4 overlapping lambda clones containing the activin receptor IA (ActRIA) gene were isolated from a mouse 129 Sv genomic library. The mouse ActRIA gene is encoded by 10 exons and spans approximately 40 kb. The size of the introns was determined and the intron/exon boundaries were sequenced. Primer extension analysis of the 5' non-translated region using RNA from different organs or tissues revealed a strong transcription start site 68 nucleotides upstream of the ATG. Knowledge of the structure of the ActRIA gene is essential for the production of ActRIA deficient mice by homologous recombination.

Activin Receptors↗
PubMed2002

Isolation, characterization, and localization of beta-tubulin genomic clones of three Drosophila montium subgroup species.

Genomic libraries were constructed from three Drosophila species, namely Drosophila auraria, Drosophila serrata, and Drosophila kikkawai, belonging to the Drosophila montium subgroup of the Drosophila melanogaster species group. Clones containing beta-tubulin specific sequences were isolated, characterized by restriction endonuclease digestions and Southern hybridizations, and mapped by in situ hybridization on the polytene chromosomes of the species studied. The distribution of the beta-tubulin loci was found to be similar in D. montium species and D. melanogaster.

Animals↗
PubMed1994

Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis.

A gene whose overexpression can endow Saccharomyces cerevisiae cells with resistance to HM-1 killer toxin was cloned from an S. cerevisiae genomic library. This gene, designated HKR1 (Hansenula mrakii killer toxin-resistant gene 1), contains a 5.4-kb open reading frame. The predicted amino acid sequence of the protein specified by HKR1 indicates that the protein consists of 1,802 amino acids and is very rich in serine and threonine, which could serve as O-glycosylation sites. The protein also contains two hydrophobic domains at the N-terminal end and in the C-terminal half, which could function as a signal peptide and transmembrane domain, respectively. Hkr1p is found to contain an EF hand motif of the calcium-binding consensus sequence in the C-terminal cytoplasmic domain. Thus, Hkr1p is expected to be a calcium-binding, glycosylated type I membrane protein. Southern and Northern (RNA) analyses demonstrated that there is a single copy of the HKR1 gene in the S. cerevisiae genome, and the transcriptional level of HKR1 is extremely low. Gene disruption followed by tetrad analysis showed that HKR1 is an essential gene. Overexpression of the truncated HKR1 encoding the C-terminal half of Hkr1p made the cells more resistant to HM-1 killer toxin than the full-length HKR1 did, demonstrating that the C-terminal half of Hkr1p is essential for overcoming the effect of HM-1 killer toxin. Furthermore, overexpression of HKR1 increased the beta-glucan content in the cell wall without affecting in vitro beta-glucan synthase activity, suggesting that HKR1 regulates beta-glucan synthesis in vivo.

Amino Acid Sequence↗
PubMed2001

Nitrogen fixation genes in an endosymbiotic Burkholderia strain.

In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

Amino Acid Sequence↗
PubMed2005

Multicopy suppression screen in the msb3 msb4 Saccharomyces cerevisiae double mutant, affected in Ypt/RabGAP activity.

The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase-activating protein (GAP) family. They are essential to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into four groups: vesicular transport proteins (Sec7p, Vps35p, and Uso1p), a protein involved in cell division (Sap155p), a molecular chaperon (Ssz1p), and a protein associated with the 25S proteasome (Cic1p).

Actins↗
PubMed1991

Structure of rabbit butyrylcholinesterase gene deduced from genomic clones and from cDNA with introns.

1. Three clones were isolated from a rabbit genomic library. They covered the entire coding sequence of the rabbit BChE gene. The positions of splice sites between exons 2, 3, and 4 are identical to those found in the human gene (Arpagaus et al., 1990). Exon 2 covers 83% of the coding sequence. This contrasts with the small size of exon 3 (167 bp) and large size of introns 2 and 3 (greater than 20 kb each). The active-site serine at position 198 is found in a highly conserved region. Aspartic acids in positions 91 and 170 are conserved in human and rabbit, and one of them could be involved in the calytic triad. Aspartic acid 70, present in the anionic site of human BChE, is also conserved in rabbit BChE. The coding sequences of human and rabbit BChE are 89% identical over 744 bp around the active-site serine. 2. In addition to the genomic clones, one cDNA clone (BNY1) was isolated. This cDNA was unusual in that it contained intronic sequences. The insert of 1 kb contained 167 coding bases homologous to the nucleotide sequence 1434 to 1600 of human cDNA and corresponded to exon 3 of the BChE gene. On each side of this coding region, consensus sequences of intron-exon boundaries were found. 3. The presence of large-size transcripts in Northern blots and the existence of a cDNA copy of unprocessed mRNA found in the BNY1 clone suggest a slow processing of transcripts. A genomic sequence unspliced in a cDNA of Torpedo AChE could give a transmembrane domain (Sikorav et al., 1988); the corresponding sequence in rabbit BChE gene, also found in a cDNA, had no homology with Torpedo AChE but could be translated in a hydrophobic C-terminal domain if maintained in mature mRNA.

Amino Acid Sequence↗
PubMed1992

Rabbit lung surfactant protein A gene: identification of a lung-specific DNase I hypersensitive site.

Expression of the gene encoding pulmonary surfactant protein A, SP-A, is lung specific and developmentally and hormonally regulated. Previously, we observed that SP-A gene transcription is initiated in fetal rabbit lung after day 21 of gestation and reaches maximal levels by day 28. In the present study, a cDNA specific for rabbit SP-A was used to isolate the SP-A gene from a rabbit genomic library. A 7.6-kb fragment containing the entire structural gene and approximately 380 bp of 5'-flanking DNA was isolated and characterized. The transcription initiation site, mapped by primer extension analysis, was localized 23 bp downstream of a putative TATA element. The structural gene is composed of five exons and four introns. The first exon encodes the 5'-untranslated region of the mRNA; the translation initiation site is in exon II, and exon V contains the two polyadenylation sites that give rise to the 2.0- and 3.0-kb species of SP-A mRNA. A potential adenosine 3',5'-cyclic monophosphate (cAMP)-regulatory element (CRE) was identified at -261 bp, and sequences with homology to glucocorticoid-regulatory element (GRE) half-sites were found at -150 and -190 bp upstream of the transcription initiation site and within the first intron. A DNase I hypersensitive site was identified in genomic DNA isolated from 21- and 28-day fetal and adult rabbit lung tissues. This site was mapped within the 5'-flanking region of the SP-A gene, at approximately -80 to -180 bp upstream of the transcription initiation site. The absence of this hypersensitive site in genomic DNA of liver, kidney, and heart tissues suggests that altered chromatin structure may serve a role in lung-specific SP-A gene expression. The presence of this tissue-specific DNase I hypersensitive site in lung nuclei from 21-day gestational age fetal rabbits suggests that the SP-A gene may exist in an accessible conformation prior to the time of transcription initiation.

Amino Acid Sequence↗
PubMed1993

Cloning and structural analysis of genomic DNA for human renal dipeptidase.

A genomic DNA for human renal dipeptidase was isolated from a human genomic library using probes for human renal dipeptidase cDNA. The human renal dipeptidase gene, containing ten exons and nine introns, had a total length of approx. 6 kbp. The DNA sequence of these exons was slightly different from that of the human renal dipeptidase cDNA reported by Adachi et al. [1]. From the results of a comparison of the deduced amino acid sequence of each exon with various mammalian renal dipeptidases, the fourth exon was found to be highly conserved (90%).

Amino Acid Sequence↗
PubMed1995

Cloning of ascidian homeobox genes provides evidence for a primordial chordate cluster.

In order to isolate genes important in controlling embryonic development in Tunicates, a genomic library from the ascidian Ciona intestinalis was screened with a degenerate oligodeoxyribonucleotide encoding the third helix of Antennapedia-type homeoboxes. Fourteen C. intestinalis homeobox genes, corresponding to several classes of homeodomains, have been identified. Five of the isolated homeoboxes show their highest homology to members of the Vertebrate HOX clusters. mRNAs for two of the isolated homeoboxes are present in unfertilized C. intestinalis eggs.

Amino Acid Sequence↗
PubMed2005

A genome-wide overexpression screen in yeast for small-molecule target identification.

We describe a multicopy gene suppression screen of drug sensitivity in Saccharomyces cerevisiae that facilitates the identification of cellular targets of small molecules. An array of yeast transformants harboring a multicopy yeast genomic library was screened for resistance to growth inhibitors. Comparison of array growth patterns for several such inhibitors allowed the differentiation of general and molecule-specific genetic suppressors. Specific resistance to phenylaminopyrimidine (1), an inhibitor identified from a kinase-directed library, was associated with the overexpression of Pkc1 and a subset of downstream kinases. Components of two other pathways (pheromone response/filamentous growth and Pho85 kinase) that genetically interact with the PKC1 MAPK signaling cascade were also identified. Consistent with the suppression screen, inhibitor 1 bound to Pkc1 in yeast cell lysate and inhibited its activity in vitro. These results demonstrate the utility of this approach for the rapid deconvolution of small-molecule targets.

Drug Evaluation, Preclinical↗
PubMed2004

Isolation and characterization of an aspartic proteinase gene from cowpea (Vigna unguiculata L. Walp.) .

A cowpea (Vigna unguiculata cv. EPACE-1) aspartic proteinase (AP) gene was isolated by genomic Library screening. Sequence analysis shows that this AP gene follows the same pattern of intron/exon number and organization as the other isolated plant AP genes, which are distinct from other solved AP genes. Northern blot analysis revealed that cowpea AP accumulates in leaves and stems but not in roots, indicating tissue-specific expression. An increased accumulation of transcripts during senescence suggests enzyme involvement in this process.

Amino Acid Sequence↗
PubMed1992

Genomic organization of the human retinoic acid receptor beta 2.

Recently three isoforms of the mouse retinoic acid receptor (mRAR beta 1, mRAR beta 2, mRAR beta 3) have been described, generated from the same gene (Zelent et al., 1991). The isoforms differ in their 5'-untranslated (5'-UTR) and A region, but have identical B to F regions. The N-terminal variability of mRAR beta 1/beta 3 is encoded in the first two exons (E1 and E2), while exon E3 includes N-terminal sequences of the mRAR beta 2 isoform. We have determined the structure of the human RAR beta 2 gene, using a genomic library from K562 cells. The open reading frame is split into eight exons: E3 contains sequences for the N-terminal A region and E4 to E10 encode the common part of the receptor, including the DNA-binding domain and ligand-binding domain. Corresponding to other nuclear receptors, both 'zinc-fingers' of the DNA-binding domain are encoded separately in two exons and the ligand-binding domain is assembled from five exons.

Amino Acid Sequence↗
PubMed2002

Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni.

A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii. To understand the source of thymidine synthesis in Hbt. salinarum, a genomic library of Hbt. salinarum was constructed and used to complement a Hfx. volcanii thyA deletion mutation. The Hbt. salinarum ORF that complemented the thyA mutation shares sequence homology with ORFs found in numerous microorganisms that lack a thyA gene, including the recently discovered thyX of Helicobacter pylori. We also show that a homolog of the Hbt. salinarum ORF is present in Campylobacter jejuni and is able to complement an Escherichia coli thyA mutant under oxygen-limiting conditions.

Campylobacter jejuni↗