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At least 1,639 records · Page 91Linked to original sources
PubMed2000

Investigation of Schizosaccharomyces pombe as a cloning host for human telomere and alphoid DNA.

The fission yeast Schizosaccharomyces pombe (Sch. pombe) has been proposed as a possible cloning host for both mammalian artificial chromosomes (MACs) and mammalian genomic libraries, due to the large size of its chromosomes and its similarity to higher eukaryotic cells. Here, it was investigated for its ability to form telomeres from human telomere sequence and to stably maintain long stretches of alphoid DNA. Using linear constructs terminating in the telomere repeat, T2AG3, human telomere DNA was shown to efficiently seed telomere formation in Sch. pombe. Much of the human telomeric sequence was removed on addition of Sch. pombe telomeric sequence, a process similar to that described in S. cerevisiae. To investigate the stability of alphoid DNA in fission yeast, bacterial artificial chromosomes (BACs) containing 130 and 173 kb of alphoid DNA were retrofitted with the Sch. pombe ars1 element and ura4+ marker using Cre-lox recombination. These alphoid BACs were found to be highly unstable in Sch. pombe deleting down to less than 40 kb, whilst control BACs of 96 and 202 kb, containing non-repetitive DNA, were unrearranged. Alphoid DNA has been shown to be sufficient for human centromere function, and this marked instability excludes Sch. pombe as a useful cloning host for mammalian artificial chromosomes. In addition, regions containing repetitive DNA from mammalian genomes may not be truly represented in libraries constructed in Sch. pombe.

Base Sequence↗
PubMed1987

Molecular cloning of a gene that is necessary for G1 progression in mammalian cells.

We have cloned a human cDNA that complements the mutation of ts11, a temperature-sensitive (ts) mutant of the BHK hamster cell line that at the nonpermissive temperature is blocked in progression through the G1 phase of the cell growth cycle. After transfecting human chromosomal DNA into ts11 cells and selecting for cells that had acquired a non-ts phenotype, we screened a genomic library constructed in the EMBL3 lambda vector from a secondary non-ts transformant and isolated a recombinant phage containing human DNA sequences that were uniformly present in primary and secondary non-ts transformants. Genomic probes that recognized an mRNA of about 2 kilobases in human cells were used to isolate from a cDNA expression library two cDNA plasmids that could efficiently transform ts11 cells to a non-ts phenotype. Sequencing of one of these cDNAs revealed a single open reading frame, which could encode a 540 amino acid protein. The ts11 gene has at least two other homologs in human DNA and thus it appears to be part of a small gene/pseudogene family. Experiments with serum-synchronized cells indicate that the expression of the ts11 gene, which is necessary for G1 progression, is itself cell-cycle regulated, being induced in approximately mid-G1.

Amino Acid Sequence↗
PubMed1990

Murine ferritin heavy chain: isolation and characterization of a functional gene.

A mouse liver genomic library screened with a full-length cDNA encoding murine ferritin heavy chain (mFHC) [Torti et al., J. Biol. Chem. 263 (1988) 12638-12644] yielded a functional genomic clone mFHC. The genomic clone isolated included a region of approximately 3 kb containing four exons and three introns. Sequence comparisons of the mouse genomic clone with other genomic clones from rat, human and chicken showed a high degree of similarity among species in the coding regions. Introns and flanking sequences were less conserved. However, comparison of mFHC promoter elements with FHC genes from other species revealed common elements. Analysis of the genomic structure of FHC suggested the presence of pseudogenes. S1 nuclease analysis, however, confirmed that this mouse clone, when transfected into human MRC-5 fibroblasts, was transcribed, indicating that this clone contains an FHC functional gene.

Animals↗
PubMed1992

The CCS1 gene from Saccharomyces cerevisiae which is involved in mitochondrial functions is identified as IRA2 an attenuator of RAS1 and RAS2 gene products.

The ccs1-1 mutation of Saccharomyces cerevisiae, which has been previously described, is associated with an increase in cytochrome content, in respiration, and in ATP synthesis. In addition, this mutation leads to the same phenotype as cells de-regulated in the cAMP pathway. From a yeast genomic library, we have isolated a DNA fragment in a recombinant plasmid pCD1 which complements the ccs1-1 mutation. Homologous integration of this DNA in the genome occurs at the CCS1 locus. An 11 kb of the DNA insert is necessary for complementation. Sequencing part of the fragment identifies CCS1 as the IRA2 gene. The IRA2 gene is known to encode an attenuator of RAS gene product activity which stimulates the GTPase activity of the RAS proteins. This result underlines the involvement of cAMP-dependent phosphorylation in mitochondrial function. We present the sequence of 1 kb DNA upstream of the putative ATG of the IRA2/CCS1 gene product which is devoid of an ORF and could contain several regulatory sites.

Base Sequence↗
PubMed1988

Isolation, characterization and sequencing of a novel repetitive DNA from the mung bean Vigna radiata.

A family of highly reiterated, small (approx. 300 bp) sequences has been identified in DNA of the mung bean Vigna radiata. The members are extensively interspersed throughout the chromosomes with some clustering. They also occur extrachromosomally. There is no tissue-specificity to the repeat family but it is highly species-specific. The repetitive DNA hybridizes to total RNA as well as to polyadenylated RNA isolated from germinated mung beans. It has analogy with the human AluI family in the mode of isolation, size, genomic distribution, copy number and transcribability though they do not share any sequence homology. A repetitive DNA clone was selected from a shotgun genomic library of mung bean DNA in pBR322. The average copy number of the cloned repeat is estimated to be 8 x 10(4) per haploid genome, and thus constitutes approx. 5% of the total mung bean genome. The genomic organization and transcription of the cloned repeat is reported. Sequencing of the cloned repetitive DNA reveals the presence of the number of direct and inverted repeats and some short palindromic sequences.

Base Sequence↗
PubMed1999

Wide distribution of short interspersed elements among eukaryotic genomes.

Most short interspersed elements (SINEs) in eukaryotic genomes originate from tRNA and have internal promoters for RNA polymerase III. The promoter contains two boxes (A and B) spaced by approximately 33 bp. We used oligonucleotide primers specific to these boxes to detect SINEs in the genomic DNA by polymerase chain reaction (PCR). Appropriate DNA fragments were revealed by PCR in 30 out of 35 eukaryotic species suggesting the wide distribution of SINEs. The PCR products were used for hybridization screening of genomic libraries which resulted in identification of four novel SINE families. The application of this approach is illustrated by discovery of a SINE family in the genome of the bat Myotis daubentoni. Members of this SINE family termed VES have an additional B-like box, a putative polyadenylation signal and RNA polymerase III terminator.

Animals↗
PubMed1994

Human thrombopoietin: gene structure, cDNA sequence, expression, and chromosomal localization.

Thrombopoietin (TPO), a lineage-specific cytokine affecting the proliferation and maturation of megakaryocytes from committed progenitor cells, is believed to be the major physiological regulator of circulating platelet levels. Recently we have isolated a cDNA encoding a ligand for the murine c-mpl protooncogene and shown it to be TPO. By employing a murine cDNA probe, we have isolated a gene encoding human TPO from a human genomic library. The TPO locus spans over 6 kb and has a structure similar to that of the erythropoietin gene (EPO). Southern blot analysis of human genomic DNA reveals a hybridization pattern consistent with a single gene locus. The locus was mapped by in situ hybridization of metaphase chromosome preparations to chromosome 3q26-27, a site where a number of chromosomal abnormalities associated with thrombocythemia in cases of acute myeloid leukemia have been mapped. A human TPO cDNA was isolated by PCR from kidney mRNA. The cDNA encodes a protein with 80% identity to previously described murine TPO and is capable of initiating a proliferative signal to murine interleukin 3-dependent BaF3 cells expressing the murine or human TPO receptor.

Amino Acid Sequence↗
PubMed1998

Structure and expression of the orphan nuclear receptor SHP gene.

To determine the organization of the orphan nuclear receptor SHP gene (Seol, W., Choi, H.-S., and Moore, D.D. (1996) Science 272, 1336-1339), genomic clones were isolated from human and mouse genomic libraries. The SHP gene was composed of two exons interrupted by a single intron spanning approximately 1.8 kilobases in human and 1.2 kilobases in mouse. Genomic Southern blot analysis and fluorescence in situ hybridization of human metaphase chromosomes indicated that the SHP gene is located at the human chromosome 1p36.1 subband. The 5'-flanking regions of human and mouse SHP genes were highly conserved, showing 77% homology in the region of approximately 600 nucleotides upstream from the transcription start site. Primer extension analysis was carried out to determine the transcription start site of human SHP to 32 nucleotides downstream of a potential TATA box. The human SHP gene was specifically expressed in fetal liver, fetal adrenal gland, adult spleen, and adult small intestine. As expected from this expression pattern, the activity of the mouse SHP promoter measured by transient transfection was significantly higher in the adrenal-derived Y1 cells than HeLa cells.

Animals↗
PubMed1983

Mammalian keratin gene families: organisation of genes coding for the B2 high-sulphur proteins of sheep wool.

We have isolated two genomic clones containing three B2 high-sulphur keratin genes from a sheep genomic library constructed in Charon 4A. These genes do not contain intervening sequences. Two genes, encoding the B2A and B2D proteins are closely linked in the genome, being separated by 1.9 kb, and are transcribed in the same direction. Although there is extensive sequence conservation in the 5' non-coding and coding regions, the 3' non-coding regions diverge both in length and sequence. Within the 5' non-coding region adjacent to the initiating AUG there is a highly conserved 18 bp sequence which is also present in another gene coding for a member of a different, unrelated high-sulphur keratin family. In the B2A-B2D intergene region, tightly linked to the B2D gene, there is a putative, divergently transcribed gene.

Amino Acid Sequence↗
PubMed1986

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants.

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.

Cloning, Molecular↗
PubMed2003

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida.

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1983

Genomic organization, DNA sequence, and expression of chicken embryonic histone genes.

We have isolated and characterized in detail 15 lambda Charon 4A recombinant bacteriophage containing histone genes from a chicken genomic library. Restriction enzyme-mapping analysis and Southern hybridization to sequenced, homologous histone probes indicate that these genes are not tandemly reiterated within the chicken genome; they usually reside in clusters even though there is no unique array of genes that appears to constitute a typical cluster. Chicken H4 and H1 genes were identified within the genomic recombinants and subsequently sequenced. Extensive regions of homology exist in the 5'- and 3'-flanking regions of the chicken H4 gene when compared to H4 genes from other organisms. In addition to the well documented histone-specific domains, two previously unreported regions of homology lie 5' to this gene: an octanucleotide and a pentanucleotide sequence lying 59 and 116 nucleotides upstream from the H4 gene CAP site, respectively. The H1 gene sequence predicts that the H1 polypeptide is 217 amino acids in length. The 5'-flanking domain of this gene contains, in addition to the transcriptional initiation site and the ATA box, two unusual sequences: one is a nonamer which resides 29 nucleotides upstream from the "ATA" box and is conserved in both the chicken and sea urchin H1 genes, while the other is a GC-rich repetitive sequence element. The majority of the chicken histone genes among the 15 unique lambda recombinant clones are expressed almost exclusively during in ovo development (i.e. from at least 4 days postfertilization up to hatching, about 20-21 days postfertilization) and appear not to be associated with any particular tissue type.

Amino Acid Sequence↗
PubMed1990

Cloning of a species-specific DNA probe from Onchocerca gibsoni.

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.

Animals↗
PubMed1985

Cloning of Physarum actin sequences in an exonuclease-deficient bacterial host.

A genomic library of Physarum was constructed in the replacement vector EMBL3. Efficient propagation of the recombinant phages occurred only on the recBC-sbcB- host Escherichia coli CES200, which is deficient in the exonucleases I and V. Thirteen different recombinants with actin-related sequences were detected and 10 were purified from 90,000 plaques (the equivalent of 6 Physarum genomes) on strain CES200. Comparison of the plating efficiencies of the library and the actin-related isolates suggests that palindromic DNA sequences are responsible for the instability of Physarum DNA in E. coli. In one of these isolates, lambda PpA10, and in a 2.81-kilobase subclone of that isolate in plasmid pBR322, a deletion of 360 base pairs was detected that led to stable propagation of the recombinant DNA molecules in Rec+ E. coli. Electron microscopic analysis of the 2.81-kilobase fragment, after denaturation and self-hybridization, revealed secondary structures consistent with "foldback" structures. Restriction and DNA blot analysis of lambda PpA10 suggest that the unstable DNA segment is in close proximity to, if not part of, the previously defined actin-gene locus ardA.

Actins↗
PubMed1990

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene.

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of a new member of the melanocyte-stimulating hormone receptor family.

A new member of the G protein-coupled receptor superfamily has been isolated from an ovine genomic library with a probe generated by the application of the PCR technique, using cDNA synthesized on a mRNA template isolated from the ovine pars tuberalis. This genomic clone encodes a novel receptor of 325 amino acids with seven transmembrane domains. These domains share homology with other members of this family, but the best homology is with the recently cloned human MC-1 (50% in the transmembrane domains) and MC-3 (69% in the transmembrane domains) MSH receptors and the human ACTH (42% in the transmembrane domains) receptor. When this receptor was expressed in Cos7 cells, it was able to bind a potent analogue of alpha-MSH, [Nle4,D-Phe7]-alpha-MSH (NDP-MSH), with high affinity. This binding could be displaced by pro-opiomelanocortin-derived and related peptides, with the order of potency NDP-MSH > alpha-MSH = ACTH > beta-MSH and with no effect of gamma-MSH, delta-MSH or beta-endorphin. The expressed receptor was demonstrated to be functionally coupled to the adenylate cyclase second messenger pathway, with alpha-MSH, beta-MSH and ACTH stimulating cyclic AMP production. The amount of the mRNA for this receptor was found to be very low. The tissue distribution of this receptor could only be observed using the reverse transcription-PCR technique and the receptor was found to be present in a number of somatic tissues. These data indicate that this is a new and distinct member of the melanocortin receptor family.

Amino Acid Sequence↗
PubMed2006

Characterization and functional analysis of a pollen-specific gene st901 in Solanum tuberosum.

A pollen-specific gene, sb401, which was isolated from a cDNA library of in vitro geminated pollen of the diploid potato species Solanum berthaultii, belongs to the class of genes expressed late during pollen development. Using sb401 as a probe, a pollen-specific gene st901 was isolated from the genomic library of a potato species Solanum tuberosum cv. Desiree. Sequencing and RT-PCR analysis showed that the st901 genomic gene is 2,889 bp long, contains three exons and two introns, and encodes a putative polypeptide of 217 residues. The predicted protein sequence contains four imperfect repeated motifs of V-V-E-K-K-N/E-E; the core sequence of the repeats (K-K-N/E-E) resembles a microtubule-binding domain of the microtubule-associated protein MAP1B from mouse. The examination of a promoter-reporter construct in transgenic potato plants revealed that the st901 is expressed exclusively in mature pollen grains, which is consistent with the results of Northern blot and RT-PCR. For analysis of the function of st901, transgenic plants harboring antisense copies of st901 cDNA driven by a native st901 promoter were generated. Suppression of st901 gene in potato resulted in aberrant pollen at maturation and pollen viability of transgenic plants ranged from 4.4 to 14.8%, while that of control plants were more than 90%. These results strongly suggest that st901 has an essential role in pollen development.

Amino Acid Sequence↗
PubMed1986

Human cardiac myosin heavy chain genes. Isolation of a genomic DNA clone and its characterization and of a second unique clone also present in the human genome.

A gene sequence coding for myosin heavy chain (MHC) of human cardiac muscle was isolated by screening a human genomic library with a 32P-labelled 1.1kb SacI restriction fragment from a previously characterized cDNA clone specifying the light meromyosin and 3' untranslated region of mRNA encoding rabbit cardiac alpha-MHC. The DNA of this human genomic clone (lambda HCMHC8) hybridized much more strongly than did other clones isolated under similar, low stringency conditions both to the rabbit cDNA probe and to mRNA isolated from rat cardiac, but not skeletal, muscle tissue. Probe made from a DNA restriction fragment of lambda HCMHC8 hybridized a single 31S band of human ventricular mRNA. This size is identical to that of cardiac MHC mRNA of other species. Heteroduplex analysis showed hybridization of lambda HCMHC8 with exon segments in a rabbit cardiac MHC genomic clone (lambda MHC alpha 12/1). It also showed that lambda HCMHC8 spanned 14 kb of DNA and contained exon segments estimated to code for two-thirds of a MHC including the carboxylic acid terminus. By rescreening the library under more stringent conditions, where only DNA sequences having strong homology to cardiac MHC genes would be expected to hybridize, clones having restriction maps overlapping lambda HCMHC8 were isolated together with a unique clone (lambda HCMHC9). DNA gel blot hybridization of human genomic DNA with lambda HCMHC8 probe at medium stringency gave a pattern of restriction fragments similar to the restriction map of lambda HCMHC8. A weaker set of bands also appeared which corresponded in pattern to the map of lambda HCMHC9.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗