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PubMed1989

Mapping of the gene encoding the multifunctional protein carrying out the first three steps of pyrimidine biosynthesis to human chromosome 2.

The CAD gene encodes a trifunctional protein that carries the activities of the first three enzymes (carbamyl phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase) of de novo pyrimidine biosynthesis. Genomic fragments of the human CAD gene have been obtained by screening a human genomic library in bacteriophage lambda using a Syrian hamster cDNA clone as a probe. These human genomic clones have been used to assign the CAD gene to human chromosome 2 using in situ hybridization to human metaphase chromosomes and Southern blot hybridization analysis of DNA isolated from a panel of Chinese hamster/human hybrid cells. In situ hybridization analysis has allowed further localization of this gene to the chromosomal region 2p21-p22.

Amidohydrolases↗
PubMed1989

Differential expression within a family of novel wound-induced genes in potato.

Wounding in higher plants leads to an increased synthesis of specific messenger RNAs. A cDNA clone complementary to a wound-induced message from potato tubers was used to isolate a lambda clone from a genomic library of Solanum tuberosum var. Maris Piper. DNA sequence analysis has shown that this single genomic clone contains two novel wound-induced genes, called win1 and win2, organised in close tandem array. The coding sequences of these two genes are highly homologous and are interrupted by a single intron. However, the sequences of the introns and flanking regions have diverged widely. Win1 and win2 encode cysteine-rich proteins of 200 and 211 amino-acids, respectively, which show striking homologies to several chitin-binding proteins. Southern analysis of genomic DNA has shown that win1 and win2 are members of a small multi-gene family which is estimated to have a minimum of five members per haploid genome of Maris Piper and appears to be conserved within the Solanaceae. We have shown by Northern analysis and S1 mapping that the two genes exhibit differential organ-specific expression after the wounding of a potato plant.

Amino Acid Sequence↗
PubMed2002

Microsatellite markers isolated in olive ( Olea europaea L.) are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars.

We have isolated and sequenced 52 microsatellites or simple sequence repeats (SSRs) from nearly 60 positive clones obtained from two 'Frantoio' olive genomic libraries enriched in (AC/GT) and (AG/CT) repeats, respectively. The repeat-containing fragments obtained from genomic DNA restricted with Tsp509I were separated using a biotinylated probe bound to streptavidin-coated paramagnetic beads. Fragments were then cloned into lambda ZAPII vector and sequenced. Thirty of the 36 primer pairs which gave correct re-amplification in the source genome were used to assay the polymorphism of 12 olive cultivars, namely four well-known cultivars ('Coratina', 'Frantoio', 'Leccino', 'Pendolino') and eight ancient cultivars grown locally near Lake Garda ('Casaliva', 'Favarol', 'Fort', 'Grignan', 'Less', 'Raza', 'Rossanel', 'Trep'). The local cultivars were each re- presented by two to four long-lived individuals. The analysis was carried out using (33)P-labelled primers and 6% polyacrylamide sequencing gels. All except two microsatellites showed polymorphism, the number of alleles varying from 1 to 5. The average genetic diversity ( H) was 0.55. The power of discrimination ( PD) was 0.60. All cultivars, including the local ones, were easily separated from each other. Variations in the SSR pattern were observed among individual plants of the same cultivar in four out of the eight local cultivars analysed. Several primer pairs (17%) amplified more than one locus.

Journal Article↗
PubMed1994

Expression of two variants of the human mu opioid receptor mRNA in SK-N-SH cells and human brain.

A partial mu opioid receptor gene was isolated from a human genomic library using a mouse delta opioid receptor cDNA as a probe. Using information from this genomic clone and the published human mu receptor, MOR1, a cDNA was isolated from SK-N-SH mRNA that codes for a variant of the MOR1 mRNA, MOR1A. The presence of MOR1A is also shown in human brain using RT-PCR. MOR1A differs from MOR1 in that the 3' terminal intron has not been removed. An in-frame termination codon is found four amino acids after the 5' consensus splice site, making MOR1A eight amino acids shorter than MOR1. Both receptors show similar ligand binding and coupling to cAMP in CHO-K1 cells. The C-terminal differences between MOR1 and MOR1A could have effects on receptor coupling or receptor transport and localization.

Alternative Splicing↗
PubMed1991

Mouse ferritin H multigene family is polymorphic and contains a single multiallelic functional gene located on chromosome 19.

Multiple ferritin H subunit sequences are present in the genome of higher vertebrates, but it is not yet known with certainty if more than one is expressed. In this paper, we provide evidence that there is only one functional ferritin H gene in the mouse. We screened a mouse genomic library using a mouse ferritin H cDNA as a probe and characterized five clones. These genomic clones proved to contain three pseudogenes and two allelic forms of a unique functional gene. These two alleles differed by only two point mutations in the promoter and three in the first intron and by a 31-bp insertion in the first intron. They were equally expressed when transiently transfected in HeLa cells. These five genomic clones account for all the bands observed on a Southern blot of mouse genomic DNA hybridized with a ferritin H cDNA, and these bands present a restriction fragment length polymorphism between various representatives of the genus Mus. Using a DNA panel prepared from the backcross progeny (C57BL/6 X Mus spretus)F1 X C57BL/6, we localized the functional ferritin H gene (Fth) in region B of mouse chromosome 19 and established cen-Ly-1-Fth-Pax-2 as the most likely gene order, thus defining a conserved syntenic fragment with human chromosome 11q.

Alleles↗
PubMed2000

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene.

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.

Amino Acid Sequence↗
PubMed1982

Structure of two human alpha-tubulin genes.

The ability of a chicken alpha-tubulin cDNA probe to cross-hybridize with human DNA under stringent conditions has been exploited to screen two independently constructed human genomic libraries. Nine clones were isolated, accounting for 60% of the bands observed in a whole genomic Southern blot of human DNA. Two clones were selected for further analysis by restriction mapping, orientation experiments using 3'- or 5'-specific probes, and electron microscopy of heteroduplexes. One clone, 2 alpha, contains an alpha-tubulin-specific region of 5.0 kilobases that includes three intervening sequences. The second clone, 19 alpha, contains an alpha-tubulin-specific region of 5.4 kilobases and has somewhat diverged 5' and 3' ends. Clone 19 alpha has only two intervening sequences that correspond to the first two in clone 2 alpha. However, these intervening sequences differ in size between clones 2 alpha and 19 alpha and show no detectable sequence homology. The sum of the lengths of sequences in either clone that hybridize to the cDNA probe accounts for essentially the entire length of the cDNA molecule.

Cloning, Molecular↗
PubMed1992

Genomic structure and chromosomal mapping of the mouse N-cadherin gene.

N-cadherin is a member of the cadherin cell-cell adhesion receptor family that includes P-, E-, and R-cadherin and liver cell adhesion molecule (L-CAM). In this study, we determined the structure of the mouse N-cadherin gene by analyzing overlapping genomic clones obtained from a mouse genomic library. This gene consists of 16 exons that disperse over greater than 200 kilobases of genomic DNA. This large size of the N-cadherin gene, compared with its cDNA (4.3 kilobases), is ascribed to the fact that the first and second introns are 34.2 kilobases and greater than 100 kilobases long, respectively. When the N-cadherin gene was compared with that of L-CAM and P-cadherin, the exon-intron boundaries were found to be fully conserved between them, except that the P-cadherin first exon includes the first and second exons of the other two genes. Also, the second intron, which is equivalent to the first intron in P-cadherin, is exceptionally large and this structural feature is conserved in all of these genes. An interesting feature of the N-cadherin gene is that this gene has an extra 16th exon that is almost identical to the other exon, 100% in the coding region and 99% in the 3' untranslated region in the nucleotide level. We also determined the chromosomal localization of the N-cadherin gene by interspecific backcross analysis and found that this gene is localized in the proximal region of mouse chromosome 18. The E- and P-cadherin genes are tightly linked and located on chromosome 8 in this species. Thus, N-cadherin is unlinked to these other cadherin loci.

Amino Acid Sequence↗
PubMed1982

KpnI families of long, interspersed repetitive DNAs in human and other primate genomes.

KpnI restriction of DNAs from all anthropoid primates studied releases a conspicuous series of segments representing families of long, interspersed repetitive DNAs termed here the KpnI 1.2, 1.5, 1.8 and 1.9 kb families. Human KpnI 1.2 to 1.9 kb segments representative of these families were isolated and separately cloned in the KpnI site of a plasmid pBK5, specially constructed for this purpose. The KpnI clones did not cross-hybridize with cloned, primate alphoid sequences, suggesting that the KpnI families represent sequences separate and distinct from the alphoid DNAs. Secondary restriction analyses of cloned KpnI segments demonstrated microheterogeneity among individual members within the same KpnI family. Autoradiograms of capuchin monkey, AGM and human DNA cleaved with HaeIII, AluI or RsaI and hybridized to various cloned human KpnI sequences demonstrated a remarkable conservatism and relative simplicity in the organization of the KpnI families in the genomes of these widely divergent primates. The KpnI 1.2 kb and 1.5 kb families occur in high frequency (15%) among all plaques in two recombinant human genome libraries. Evidence is presented suggesting that the bulk of the KpnI families occur in the genome as clusters or congeries of higher molecular weight segments (greater than 2 kb) containing sequences homologous to the low molecular weight segments (1.2 to 1.9 kb).

Animals↗
PubMed1985

Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.

Full length cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from rat and man have been isolated and sequenced. Many GAPDH gene-related sequences have been found in both genomes based on genomic blot hybridization analysis. Only one functional gene product is known. Results from genomic library screenings suggest that there are 300-400 copies of these sequences in the rat genome and approximately 100 in the human genome. Some of these related sequences have been shown to be processed pseudogenes. We have isolated several rat cDNA clones corresponding to these pseudogenes indicating that some pseudogenes are transcribed. Rat and human cDNAs are 89% homologous in the coding region, and 76% homologous in the first 100 base pairs of the 3'-noncoding region. Comparison of these two cDNA sequences with those of the chicken, Drosophila and yeast genes allows the analysis of the evolution of the GAPDH genes in detail.

Amino Acid Sequence↗
PubMed2005

Insights into the genomes of archaea mediating the anaerobic oxidation of methane.

The anaerobic oxidation of methane is a globally significant process which is mediated by consortia of yet uncultivated methanotrophic archaea (ANME) and sulfate-reducing bacteria. In order to gain deeper insights into genome characteristics of the different ANME groups, large-insert genomic libraries were constructed using DNA extracted from a methanotrophic microbial mat growing in the anoxic part of the Black Sea, and from sediments above gas hydrates at the Hydrate Ridge off the coast of Oregon. Analysis of these fosmid libraries with respect to archaeal 16S rRNA gene diversity revealed a single ANME-1b ribotype for the Black Sea libraries, whereas the sequences derived from the Hydrate Ridge library phylogenetically affiliated with the ANME-2a, ANME-2c and ANME-3 group. Genome walking for ANME-1b resulted in a contiguous 155 kb composite genome fragment. The comparison of a set of four genomic fragments belonging to the different ANME groups revealed differences in the rRNA operon structure and the average G+C content, with the ANME-2c contig showing the highest divergence within the set. A detailed analysis of the ANME contigs with respect to genes putatively involved in the anaerobic oxidation of methane led to the identification of: (i) a putative N5,N10-methenyltetrahydromethanopterin cyclohydrolase gene, (ii) a gene cluster supposedly encoding a novel type of heterodisulfide reductase/dehydrogenase complex and (iii) a gene cluster putatively encoding a new type of CO dehydrogenase/acetyl-CoA synthase enzyme complex.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a fish alpha 2-adrenoceptor.

1. Pigment granule aggregation in specialized cells (melanophores) from the skin of teleost fishes has been shown to be mediated by receptors with an alpha 2-adrenoceptor pharmacology. We now report the cloning of the alpha 2-F, a fish skin alpha 2-receptor from the cuckoo wrasse (Labrus ossifagus). 2. Degenerate oligonucleotides corresponding to conserved regions of the human alpha 2-adrenoceptor subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated subtypes were used in a polymerase chain reaction (PCR) with cDNA prepared from mRNA isolated from the skin of the cuckoo wrasse. An 876 base pair (bp) product was obtained that was homologous with that of the human alpha 2-adrenoceptor and was used to screen a genomic library from the cuckoo wrasse. 3. A clone (pTB17BS) consisting of approximately 5 kb of genomic DNA was obtained which contained the nucleotide sequence of the initial PCR product. In addition, it contained an open reading frame that encoded a protein of 432 amino acids and approximately 2 kb of 5'untranslated sequence. The deduced amino acid sequence of this protein showed 47-57% identity with the human alpha 2-adrenoceptors and thus appeared to encode a fish alpha 2-adrenoceptor. 4. In the 5'-untranslated region of the gene, nucleotide sequences were present suggesting that transcription of the alpha 2-F might be regulated by cyclic AMP, calcium and/or steroids. 5. The alpha 2-F was expressed in COS-7 cells and radioligand binding studies were performed with [3H]-rauwolscine. The binding was of high affinity and it was saturable with a KD of 0.8 +/- 0.1 nM and a Bmax of 5.7 +/- 1.0 pmol mg-1 of protein.6. Competition curves for the displacement of specific [3H]-rauwolscine binding showed the following order of potency: for agonists, medetomidine > clonidine >p-aminoclonidine> B-HT 920> (- )-noradrenaline;for antagonists, rauwolscine > atipamezole > yohimbine > phentolamine > prazosin.7. These results show that alpha2-F has characteristics of both the human alpha2-CIO and alpha2-C4 and that it might represent an ancestral alpha2-adrenoceptor subtype.

Amino Acid Sequence↗
PubMed2006

An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae.

The polyketide toxin cercosporin plays a key role in pathogenesis by fungal species of the genus Cercospora. The bacterium Xanthomonas campestris pv. zinniae is able to rapidly degrade this toxin. Growth of X. campestris pv. zinniae strains in cercosporin-containing medium leads to the breakdown of cercosporin and to the formation of xanosporic acid, a nontoxic breakdown product. Five non-cercosporin-degrading mutants of a strain that rapidly degrades cercosporin (XCZ-3) were generated by ethyl methanesulfonate mutagenesis and were then transformed with a genomic library from the wild-type strain. All five mutants were complemented with the same genomic clone, which encoded a putative transcriptional regulator and an oxidoreductase. Simultaneous expression of these two genes was necessary to complement the mutant phenotype. Sequence analysis of the mutants showed that all five mutants had point mutations in the oxidoreductase gene and no mutations in the regulator. Quantitative reverse transcription-PCR (RT-PCR) showed that the expression of both of these genes in the wild-type strain is upregulated after exposure to cercosporin. Both the oxidoreductase and transcriptional regulator genes were transformed into three non-cercosporin-degrading bacteria to determine if they are sufficient for cercosporin degradation. Quantitative RT-PCR analysis confirmed that the oxidoreductase was expressed in all transconjugants. However, none of the transconjugants were able to degrade cercosporin, suggesting that additional factors are required for cercosporin degradation. Further study of cercosporin degradation in X. campestris pv. zinniae may allow for the engineering of Cercospora-resistant plants by using a suite of genes.

Ascomycota↗
PubMed1996

Cloning and structural analysis of the melanin biosynthesis gene SCD1 encoding scytalone dehydratase in Colletotrichum lagenarium.

Appressoria of the phytopathogenic fungus Colletotrichum lagenarium contain melanin, which has been implicated as an important factor in the penetration of host plants. A cDNA clone containing the melanin biosynthetic gene encoding scytalone dehydratase (SCD1) from C. lagenarium was identified by hybridization with a heterologous cDNA probe from Magnaporthe grisea. The cDNA clone was used to identify a cosmid containing SCD1 in a genomic library of C. lagenarium, and the nucleotide sequence was determined for both the cDNA and genomic clones. The SCD1 gene contained one open reading frame composed of 188 codons and two deduced introns of 57 and 67 nucleotides. The deduced amino acid sequence of the N-terminal region of SCD1 showed high similarity to the amino acid sequence of scytalone dehydratase from Cochliobolus miyabeanus. A plasmid containing the SCD1 gene transformed the melanin-deficient mutant 9201Y (Scd-) to the wild phenotype but did not complement the conditional scytalone dehydratase-deficient mutant C. lagenarium 8015. Genomic DNA analysis indicated that the SCD1 gene is a single locus in C. lagenarium. Transcripts of the SCD1 gene were detected 2 h after the start of conidial germination.

Amino Acid Sequence↗
PubMed1997

A single 13-kilobase divergent locus in the Kaposi sarcoma-associated herpesvirus (human herpesvirus 8) genome contains nine open reading frames that are homologous to or related to cellular proteins.

Two small fragments of a novel human gammaherpesvirus genome known as Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) have been shown to be present in virtually all AIDS and non-AIDS KS lesions, as well as in body cavity-based lymphomas (BCBL) and in multicentric Castleman's disease. We have extended those studies by identifying and sequencing a third fragment of HHV-8 DNA encoding a viral thymidylate synthetase (TS) gene. Use of this viral TS fragment as a probe led to the identification and mapping of a cluster of overlapping phage lambda clones from a BCBL tumor DNA genomic library that spanned 48 kb on the left-hand side of the HHV-8 genome between the equivalents of open reading frame 6 (ORF6) and ORF31 of herpesvirus saimiri (HVS). DNA sequencing of a 17-kb segment encompassing a gammaherpesvirus divergent locus (DL-B) between ORF11 and ORF17 revealed the presence of nine viral ORFs with predicted gene products related to cellular proteins. These include the complete TS gene and a dihydrofolate reductase (DHFR) gene, four novel cytokine genes (encoding viral interleukin-6, viral MIP-1A, viral MIP-1B, and BCK) that have not previously been found to be encoded by a virus, and a bcl-2 homolog. This region in HHV-8 also contains the T1.1 abundant lytic cycle nuclear RNA gene and encompasses two genes (or exons) encoding proteins with C4HC3 zinc finger domains of the PHD/leukemia-associated protein subtype. The latter are related to the spliced immediate-early IE1 protein of the gamma-2 class herpesvirus bovine herpesvirus type 4 and a similar motif found in HVS ORF12. Although genes for TS and DHFR enzymes are also encoded by HVS (ORF70 and ORF2), both occur at different genomic loci than in HHV-8, and the HHV-8 DHFR protein is much farther diverged from human DHFR than is the HVS version, implying that they were probably acquired as host cell cDNAs by independent evolutionary events. Transcripts from the IE1-A, IE1-B, DHFR, and MIP-1B genes were all detected by Northern blot hybridization analysis in a BCBL cell line at 12 h after induction with butyrate but were not present before induction, indicating that these are all primarily lytic cycle genes. We conclude that the DL-B locus of gammaherpesviruses displays considerably more variability that previously appreciated and that expression of many of these genes is likely to have important implications for HHV-8 biology and therapy.

AIDS-Related Opportunistic Infections↗
PubMed1989

Molecular cloning and expression of a fifth muscarinic acetylcholine receptor.

A cDNA of 2149 base pairs with an incomplete open reading frame (ORF) encoding amino acids 1-516 of a 531-amino acid protein highly homologous to muscarinic receptors was cloned from a rat brain cDNA library. The complete ORF was then deduced from a DNA fragment cloned from a rat genomic library. This ORF was subcloned into the eukaryotic expression vector p91023(B) under control of the adenovirus major late promoter and co-transfected with the thymidine kinase selection marker into muscarinic receptor-negative, thymidine kinase-negative murine L cells. Stable transformants were selected and tested for acquisition of muscarinic receptors by following appearance of specific binding sites for the muscarinic ligand [3H] N-methylscopolamine. Two cell lines, LM5.36 and LM5.40, were cloned and shown to express typical muscarinic receptor sites, thus confirming that the newly cloned ORF encodes a muscarinic receptor, the rat M5 muscarinic acetylcholine receptor. Tests for activities showed it to stimulate phosphoinositide hydrolysis in intact cells, without affecting positively or negatively adenylyl cyclase activity. The M5 receptor contains two putative glycosylation sites at its amino terminus and, based on hydropathicity analysis, is predicted to span the plasma membrane seven times. Like 17 other receptors of this class, the M5 receptor has 19 conserved amino acids, among which are 4 prolines located in the 4th, 5th, 6th, and 7th predicted transmembrane regions, conferring possible bends to these helices, and 2 cysteines, one in the 1st and the other in the 2nd extracellular loop, possibly providing for a disulfide bond. Similarity in amino acid composition and in patterns of antagonist binding and biologic effects suggest the M5 receptor to be M1-like.

Adenylyl Cyclases↗
PubMed1984

[Cloning of human genes].

The fears concerning potential hazards of man made recombinant DNA have been dismissed and the burden of compelling unnecessary safety regulations has been cleared away. The deciphering of the entire human genome at the nucleotide level is no longer an impossible task. It is based upon the utilization of cloned fragments of DNA used as specific probes. The present catalogue of cloned human genes is still small, but it will expand rapidly in the near future. There are two categories of probes: those corresponding to know genes, and those consisting of random "anonymous" DNA sequences present in genomic libraries. The increasing number of cloned probes will allow us to saturate progressively the entire genome. Medicine will benefit from this progress, first of all with regard to pre-natal and post-natal diagnosis of some genetic diseases and in the field of cancer research.

Burkitt Lymphoma↗
PubMed1994

Cloning and analysis of a constitutive heat shock (cognate) protein 70 gene inducible by L-glutamine.

An intronless gene encoding a protein of 652 amino acid residues with an M(r) of 71,266, showing between 79% and 59% identity in nucleotide sequence with heat shock protein 70 (HSP 70) genes of Bremia lactucae (a parasitic Oomycete of lettuce) and a wide range of organisms that include humans, was isolated from the nonparasitic Oomycete Achlya klebsiana. While the gene appears to be constitutively expressed, L-glutamine augmented its expression particularly under conditions of nutritional stress. L-Glutamine enhanced the transcription of a 2.4-kilobase poly(A)+ RNA simultaneously in the same way as it elevated the cellular level of the HSP 70-like protein. A polyclonal antibody (affinity-purified) raised in rabbit against the purified monomeric (M(r) 120,000) form of an NAD-specific glutamate dehydrogenase (Yang, B., and LéJohn, H.B. (1994) J. Biol. Chem. 269, 4506-4512) immunoprecipitated the HSP 70-like protein, and it was used to study the kinetics of induction of this stress-related protein and the effect of proteinase inhibitors on its metabolism. By using as probes four partial length cDNA clones, nine overlapping DNA fragments of the organism's genome carrying the HSP 70-like protein gene were isolated from a genomic library. The nucleotide sequence of the gene, including its boundaries, was determined by using these genomic clones. The 5'-untranslated boundary of the gene displayed the classical nucleotide arrangement of heat shock elements as well as CCAAT and TATA box motifs. Within the coding region are the typical conserved amino acid heat shock protein signatures 1 and 2 at the predicted locations. By primer extension and S1 nuclease protection mapping system, we estimated that the gene is probably transcribed into a message of 2.2 kilobases.

Amino Acid Sequence↗