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At least 1,585 records · Page 88Linked to original sources
PubMed1997

A deletion mutation at the ep locus causes low seed coat peroxidase activity in soybean.

The Ep locus severely affects the amount of peroxidase enzyme in soybean seed coats. Plants containing the dominant Ep allele accumulate large amounts of peroxidase in the hourglass cells of the sub-epidermis. Homozygous recessive epep genotypes do not accumulate peroxidase in the hourglass cells and are much reduced in total seed coat peroxidase activity. To isolate the gene encoding the seed coat peroxidase and to determine whether it corresponds to the Ep locus, a cDNA library was constructed from developing seed coats and an abundant 1.3 kb peroxidase transcript was cloned. The corresponding structural gene was also isolated from a genomic library. Sequence analysis shows that the seed coat peroxidase is translated as a 352 amino acid precursor protein of 38 kDa. Processing of a putative 26 amino acid signal sequence results in a mature protein of 326 residues with a calculated mass of 35 kDa and a pl of 4.4. Using probes derived from the cDNA, genomic DNA blot hybridization and polymerase chain reaction analysis detected polymorphisms that distinguished EpEp and epep genotypes. Co-segregation of the polymorphisms in an F2 population from a cross of EpEp and epep plants shows that the Ep locus encodes the seed coat peroxidase protein. Comparison of Ep and ep alleles indicates that the recessive gene lacks 87 bp of sequence encompassing the translation start codon. Analysis by RNA blot hybridization shows that epep plants have drastically reduced amounts of peroxidase transcript compared with EpEp plants. The peroxidase mRNA is abundant in seed coat tissues of EpEp plants during the late stages of seed maturation, and could also be detected in root tissues, but not in the flower, embryo, pod or leaf. The results indicate that the lack of peroxidase accumulation in seed coats of homozygous recessive epep plants is due to a mutation of the structural gene that reduces transcript abundance.

Amino Acid Sequence↗
PubMed1996

Human gene encoding prostacyclin synthase (PTGIS): genomic organization, chromosomal localization, and promoter activity.

The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13. 11-q13.13. The 1.5-kb sequence of the 5'-upstream of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-gamma response element, GATA, NF-kappaB, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5'-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3'-untranslated region as probe indicated that the sizes of the 3'-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs.

Animals↗
PubMed1989

Major sperm protein genes from Onchocerca volvulus.

Nematode spermatozoa, unlike their mammalian counterparts, are nonflagellated crawling cells. The pseudopod of these cells contains the major sperm protein (MSP) which comprises more than 15% of the protein in the sperm. MSP is presumed to function as a cytoskeletal element involved in motility. An Ascaris MSP cDNA sequence was used as a probe to identify and isolate Onchocerca volvulus MSP clones from a lambda gt11 genomic library. Two clones, OVGS-1 (765 bp) and OVGS-2 (1765 bp), were characterized by restriction endonuclease mapping and sequence analysis. Both genomic clones contain MSP protein coding regions of 99 and 282 bp separated by an intervening sequence of 153 bp. The genes OVGS-1 and OVGS-2 are 95% similar in nucleotide sequence in the protein coding regions, but only 79% similar in their intron sequences. A number of potential regulatory sequences in the flanking regions and at the exon/intron junctions of the O. volvulus MSP genes are in good agreement with consensus sequences in other eukaryotic cells. The nucleotide sequence of the O. volvulus MSP genes were over 80% similar to the Ascaris MSP cDNA sequence and 79% similar to the Caenorhabditis MSP-3 cDNA. The predicted amino acid sequence of the O. volvulus MSPs were 96% similar to each other, 90-91% similar to Ascaris MSP and 81-82% similar to Caenorhabditis MSP-3. These results offer evidence that the MSP sequences have been highly conserved throughout nematode evolution but are variable in their genomic organization and the presence of introns.

Amino Acid Sequence↗
PubMed1988

Genomic structure of the human mitochondrial aldehyde dehydrogenase gene.

We have isolated and characterized four overlapping clones from two cosmid human genomic libraries, which span about 90 kilobase pairs (kbp) and contain the entire human mitochondrial aldehyde dehydrogenase (ALDH2) gene. Restriction maps of the genomic clones were elucidated utilizing cDNA probes and specific oligonucleotide probes. The organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The ALDH2 gene is about 44 kbp in length and contains at least 13 exons which encode 517 amino acid residues. Except for the signal NH2-terminal peptide, which is absent in the mature enzyme, the amino acid sequence deduced from the exons coincided with the reported primary structure of human liver ALDH2 (J. Hempel, R. Kaiser, and H. Jörnvall, 1985, Eur. J. Biochem. 153: 13-28). Several introns contain Alu repetitive sequences. A TATA-like sequence (TTATAAAA) and a CAAT-like sequence (GTCATCAT) are located 473 and 515 bp, respectively, upstream from the translation initiation codon. Primer extension and S1 nuclease mapping were performed to characterize the 5'-region of the gene.

Aldehyde Dehydrogenase↗
PubMed1994

Isolation and characterization of hypervariable sequences from tsetse fly genome.

A Glossina brevipalpis Newstead genomic library, constructed using a Charomid 9-36 vector, was used to isolate putative clones that hybridize to polymorphic regions of the tsetse genome. Five types of probes, that reveal individual DNA polymorphic in humans and higher animal species, were used to screen 300 tsetse Charomid clones; 15% of the clones hybridized with at least one probe. Twenty four recombinants were further characterized by Southern blotting hybridization using DNA isolated from individual tsetse fly from Glossina morsitans centralis Machado. Two classes of DNA profiles were obtained upon hybridization with the recombinant inserts. The first, termed the "multilocus profile" displayed a complex DNA pattern of multiple components whilst the second, referred to as the "single locus profile" revealed one or two bands in each individual. Of the 24 recombinant inserts tested, 13 were multilocus probes and the remainder were single locus probes, each of which hybridized to a single location when G. m. centralis DNA had been cleaved with EcoRI. These single locus probes revealed a low level of genetic variability among individual flies from an inbred colony. The hybridization profiles using multilocus and single locus probes were also obtained on DNA from individual Glossina palpalis palpalis Robineau-Desvoidy and Glossina palpalis gambienis Vanderplank and some of the G. brevipalpis recombinant clones also detected multilocus profiles in honey bees and man.

Animals↗
PubMed2003

The human mitochondrial translation initiation factor 2 gene (MTIF2): transcriptional analysis and identification of a pseudogene.

Mitochondrial translation initiation factor 2 (MTIF2) is nuclear-encoded and functions in mitochondria to initiate the translation of proteins encoded by the mitochondrial genome. To gain insight into mechanisms that regulate MTIF2 gene expression, the genomic copy and the 5' and 3' flanking regions of MTIF2 were isolated using a combination of genomic library screening and polymerase chain reaction (PCR). MTIF2 is approximately 33.5-kb long and contains 16 exons, confirming data from the Human Genome Project. With RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE), we mapped the transcription start point in human heart tissue to a cytosine residue 296 bp upstream from the translation initiation site. The region surrounding the transcription start point contains consensus binding sites for transcription factors Sp1, nuclear respiratory factor 2 (NRF-2) and estrogen receptor, while enhancer binding sites were identified upstream. Promoter constructs were prepared in a luciferase reporter vector and transiently transfected into 293T cells. The minimal promoter gave an expression level 3.5x higher than the SV40 control (P=0.001), while the construct containing the minimal promoter plus the enhancer region gave a 3.8x higher level of expression compared to the control (P<0.001). We also discovered a pseudogene of MTIF2 and mapped it to chromosome 1p13-12.

Base Sequence↗
PubMed1996

Structure and expression of the mouse oxytocin receptor gene.

To determine the structure of the mouse oxytocin receptor (OTR) gene, we have screened a mouse genomic library and confirmed cDNA sequence with rapid amplification of cDNA ends. Southern blot using human OTR cDNA probes indicated that the mouse genome has a single copy of the gene. The predicted amino acid sequence is 91% identical to human OTR. The gene contains 4 exons and 3 introns. Exons 1 and 2 contain the 5' untranslated region, with exons 3 and 4 encoding the amino acids of the receptor. Intron 3 interrupts the coding at the same location, after transmembrane domain 6, as in OTR genes of other species. The promoter region lacks an apparent TATA box but contains multiple putative interleukin-response elements and estrogen responsive elements. Expression of OTR gene in the uterus during pregnancy reached maximum at day 20 gestation. The information obtained from the mouse OTR gene facilitates further comparative and biochemical analysis for protein structure-function relationships and gene regulatory mechanisms.

Amino Acid Sequence↗
PubMed1997

Structure and chromosomal mapping of the mouse P2X3 gene.

P2X3 is one of seven cloned ATP-gated non-selective cation channels. We have isolated a full-length mouse P2X3 gene from a phage lambda-129/Sv genomic library. The gene consists of 12 exons spanning a locus of approximately 40 kb. No significant similarities have been found between the genomic organisation of the mouse P2X3 gene and genes encoding other ion channels. The encoded mouse P2X3 protein consists of 397 amino acids and shows 99% identity with rat P2X3. Using RNase protection and primer extension assays, multiple transcription initiation sites have been mapped in the mouse P2X3 promoter to a region 162-168 bp upstream of the translation initiation codon. The P2X3 gene has been mapped to mouse chromosome 2p by fluorescence in situ hybridisation. The RAG locus-associated gene T160 is located 1.8 kb upstream of the transcription start site of mouse P2X3 gene. The promoter region of the mouse P2X3 gene lacks a conventional TATA and CCAAT consensus sites, and initiator elements. P2X3 is the first member of the P2X gene family to be completely characterised.

Amino Acid Sequence↗
PubMed2001

From Arabidopsis to rice genomics: a survey of French programmes.

During the last ten years, Arabidopsis thaliana has become the most favoured plant system for the study of many aspects of development and adaptation to adverse conditions and diseases. The sequencing of the Arabidopsis thaliana genome is nearly completed with more than 90% of the sequence being released in public databases. This is the first plant genome to be analysed and it has revealed a tremendous amount of information about the nature of the genes it contains and its largely duplicated organisation. French groups have been involved in Arabidopsis genomics at several steps: EST (expressed sequence tags) sequencing, construction and ordering (physical mapping of chromosomes) of a YAC (yeast artificial chromosomes) library, genomic sequencing. In parallel an extensive programme of functional genomics is being undertaken through the systematic analysis of insertional mutants. This information provides a support for analysing other more economically important plant genomes such as the rice genome and constitutes the beginning of a systematic investigation on plant gene functions and will promote new strategies for plant improvement.

Arabidopsis↗
PubMed1997

Analysis of the mosquito lysosomal aspartic protease gene: an insect housekeeping gene with fat body-enhanced expression.

In the fat body of insects with cyclic egg maturation, lysosomes play a critical role in the termination of vitellogenesis by selectively degrading the secretory machinery involved in the massive production of yolk protein precursors. To investigate this fat body-specific lysosomal activity in the mosquito, a cathepsin D-like aspartic protease (LAP) was previously purified and its cDNA cloned. Here we report the isolation of the AaLAP gene from an Aedes aegypti genomic library. The transcribed region of the gene is comprised of five exons, spanning 1904 base pairs. Restriction fragment length polymorphism (RFLP) and genomic clone analyses show this gene to be single copy and polymorphic. Primer extension analysis revealed two putative transcription start sites (TSS). The extension products corresponding to the distal and proximal TSSs were present in both pre- and vitellogenic fat bodies, suggesting that both TSSs are involved in housekeeping as well as tissue-specific expression of this gene. TATA box-like and arthropod initiator sequences, hallmarks of regulated genes, were present near each putative TSS. Several sequences resembling binding sites for liver- and fat body-specific transcription factors were identified within 1 kb upstream and downstream of the gene. Significantly, direct binding for the C/EBP and GATA families of transcription factors was demonstrated in vitro by electrophoretic mobility shift assays (EMSA). Three sequences located upstream of AaLAP resembled the Drosophila melanogaster yolk protein fat body enhancer (Dm Yp FBE). Potential hormone-response elements were also recognized in the gene; however, they did not bind the mosquito ecdysteroid receptor/Ultraspiracle heterodimer in EMSA experiments, indicating that these sequences may interact with different nuclear receptors.

Aedes↗
PubMed1999

Microsatellite loci in eelgrass Zostera marina reveal marked polymorphism within and among populations.

Using an enriched genomic library, we developed seven (CT)n/(GA)n microsatellite loci for eelgrass Zostera marina L. Enrichment is described and highly recommended for genomes in which microsatellites are rare, such as in many plants. A test for polymorphism was performed on individuals from three geographically separated populations (N = 15/population) and revealed considerable genetic variation. The number of alleles per locus varied between five and 11 and the observed heterozygosities for single loci ranged from 0.16 to 0.81 within populations. Mean allele lengths were markedly different among populations, indicating that the identified loci will be useful in studying population structure in Z. marina. As the frequency of the most abundant multilocus genotype within populations was always < 1%, these loci have sufficient resolving power to address clone size in predominantly vegetatively reproducing populations.

Genetic Variation↗
PubMed1998

Cloning, expression, purification, and characterization of the acid alpha-mannosidase from Trypanosoma cruzi.

The acid alpha-mannosidase of Trypanosoma cruzi is a broad-specificity hydrolase involved in the catabolism of glycoconjugates, presumably in the digestive vacuole. We have cloned the alpha-mannosidase gene from a T.cruzi epimastigote genomic library. The alpha-mannosidase gene was determined to be single copy by Southern analysis, and similar sequences were not detected in genomic digests of either Trypanosoma brucei or Leishmania donovani. The coding region was subcloned into the Pichia pastoris expression vector pPICZ, and alpha-mannosidase activity was detected in the medium of induced cultures. The recombinant alpha-mannosidase demonstrated a pH optimum, inhibition by swainsonine, Km, and substrate specificity consistent with the characteristics of the alpha-mannosidase previously purified from T.cruzi epimastigotes. The recombinant enzyme was purified 103-fold from the culture medium of Pichia pastoris and had a native molecular mass of 359 kDa by gel filtration. A combination of SDS-PAGE, deglycosylation with endo H, and NH2-terminal sequencing indicates that the enzyme is originally synthesized as a homodimeric polypeptide that is subsequently cleaved to form a heterotetramer composed of 57 and 46 kDa subunits. A polyclonal antibody raised to the recombinant enzyme was shown to immunoprecipitate the alpha-mannosidase from T.cruzi cell extracts and will be used in future immunolocalization studies.

Amino Acid Sequence↗
PubMed2003

Cloning and expression of Bartonella henselae sucB gene encoding an immunogenic dihydrolipoamide succinyltransferase homologous protein.

Immunoscreening of a ZAP genomic library of Bartonella henselae strain Houston-1 expressed in Escherichia coli resulted in the isolation of a clone containing 3.5 kb BamHI genomic DNA fragment. This 3.5 kb DNA fragment was found to contain a sequence of a gene encoding a protein with significant homology to the dihydrolipoamide succinyltransferase of Brucella melitensis (sucB). Subsequent cloning and DNA sequence analysis revealed that the deduced amino acid sequence from the cloned gene showed 66.5% identity to SucB protein of B. melitensis, and 43.4 and 47.2% identities to those of Coxiella burnetii and E. coli, respectively. The gene was expressed as a His-Nus A-tagged fusion protein. The recombinant SucB protein (rSucB) was shown to be an immunoreactive protein of about 115 kDa by Western blot analysis with sera from B. henselae-immunized mice. Therefore the rSucB may be a candidate antigen for a specific serological diagnosis of B. henselae infection.

Acyltransferases↗
PubMed1993

Identification and mapping of the gene encoding the glycoprotein complex gp82-gp105 of human herpesvirus 6 and mapping of the neutralizing epitope recognized by monoclonal antibodies.

Monoclonal antibodies (MAbs) 2D4, 2D6, and 13D6 against human herpesvirus 6 (HHV-6) variant A strain GS recognized virion envelope glycoprotein complex gp82-gp105 and neutralized the infectivity of HHV-6 variant A group isolates. A 624-bp genomic fragment (82G) was identified from an HHV-6 strain GS genomic library constructed in the lambda gt11 expression system by immunoscreening with MAb 2D6. Rabbit antibodies against the fusion protein expressed from the genomic insert recognized glycoprotein complex gp82-gp105 from HHV-6-infected cells, thus confirming that the genomic fragment is a portion of the gene(s) that encodes gp82-gp105. This genomic insert hybridized specifically with viral DNAs from HHV-6 variant A strains GS and U1101 under high-stringency conditions but hybridized with HHV-6 variant B strain Z-29 DNA only under low-stringency conditions. DNA sequence analysis of the insert revealed a 167-amino-acid single open reading frame with an open 5' end and a stop codon at the 3' end. Hybridization studies with HHV-6A strain U1102 DNA localized the gp82-gp105-encoding gene to the unique long region near the direct repeat at the right end of the genome. To locate the neutralizing epitope(s) recognized by the MAbs, a series of deletions from the 3' end of the gene were constructed with exonuclease III, and fusion proteins from deletion constructs were tested for reactivity with MAbs in a Western immunoblot assay. Sequencing of deletion constructs at the reactive-nonreactive transition point localized the epitope recognized by the three neutralizing MAbs within or near a repeat amino acid sequence (NIYFNIY) of the putative protein. This repeat sequence region is surrounded on either side by two potential N-glycosylation sites and three cysteine residues.

Amino Acid Sequence↗
PubMedDate not supplied

[Cloning and structural analysis of genes coding for tumor necrosis factor and lymphotoxin in rabbits].

Genes, coding for tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta), have been cloned from the rabbit genomic library. The two genes are tandemly arranged and separated only by 1 kb of DNA as previously observed in human and mouse genomes. We have sequenced the entire rabbit lymphotoxin gene (LT) and calculated the amino acid sequence of the rabbit LT whose cDNA is not yet cloned. We also analyzed the upstream sequences of this gene and revealed a number of recognition sites for the known transcriptional factors. The rabbit TNF gene comprised in the cloned genomic region has been sequenced earlier.

Amino Acid Sequence↗
PubMed1994

Structure of the L-histidine decarboxylase gene.

Two species of L-histidine decarboxylase (HDC) mRNA were found in the KU-812-F basophilic cell line, but only the 2.4-kilobase (kb) one encodes the functional HDC (Mamune-Sato, R., Yamauchi, K., Tanno, Y., Ohkawara, Y., Ohtsu, H., Katayose, D., Maeyama, K., Watanabe, T., Shibahara, S., and Takishima, T. (1992) Eur. J. Biochem. 209, 533-539). The 3.4-kb one encodes a truncated HDC protein and is also found in human leukemia-derived cell lines HEL and KCL-22. To clarify the mechanisms that regulate transcription of the HDC gene and generate the two species of mRNA, we have isolated genomic DNA clones coding for the HDC from human genomic libraries. Structural analysis of the isolated clones revealed that the human HDC gene is composed of 12 exons spanning approximately 24 kb. Genomic DNA blot analysis suggested that HDC is encoded by a single copy gene. The structural analysis also demonstrated that the heterogeneity of the HDC mRNA is caused by an insertion of the seventh intron sequence and alternative use of the splicing acceptor site at the 12th exon. The transcription start site of the HDC gene and the nucleotide sequences of the promoter and first exon regions were determined. We found a TATA-like sequence, a GC box, four CACC boxes, four GATA consensus sequences, and six leader-binding protein-1 binding motifs in the promoter region of the HDC gene.

Base Sequence↗
PubMed1993

Subunit VIa of yeast cytochrome c oxidase is not necessary for assembly of the enzyme complex but modulates the enzyme activity. Isolation and characterization of the nuclear-coded gene.

COX13, the nuclear gene for cytochrome c oxidase subunit VIa of Saccharomyces cerevisiae, has been isolated in two steps. First, the partial amino acid sequence information of the subunit was used to design two degenerate oligodeoxynucleotide primers to amplify part of the gene in a polymerase chain reaction. Next, the amplified product was used to screen a yeast genomic library in order to obtain the entire gene and its flanking sequences. COX13 is present as a single copy gene per haploid genome. Alignment of the N-terminal sequence of mature, subunit VIa with the amino acid sequence deduced from the DNA sequence indicates that subunit VIa is synthesized as a precursor comprised of a leader sequence of 9 amino acid residues and a mature polypeptide of 120 amino acid residues. The mature polypeptide shares 34% identical amino acid residues with the human subunit isoform VIa-L. Sequence analysis of the 3'-flanking region of COX13 revealed that the gene is located 599 base pairs downstream of CDC55, a gene which has been mapped to the left arm of chromosome VII. Null mutants of COX13, generated by gene replacement, showed a slightly reduced growth rate on nonfermentable carbon sources. Heme spectra and analysis of immunopurified cytochrome c oxidase from a null strain demonstrated that the enzyme is fully assembled without subunit VIa. At low ionic strength, cytochrome c oxidase missing subunit VIa was more active, whereas at high ionic strength, it was less active than the enzyme complex in which subunit VIa was present. In addition, distinct effects of ATP on the activity of the null and wild type enzyme were found. The results suggest that ATP interacts specifically with subunit VIa and thereby modulates the cytochrome c oxidase activity.

Adenosine Triphosphate↗
PubMed1990

Molecular analysis of the human MHC class I region using yeast artificial chromosome clones.

The cloning of large genomic fragments corresponding to the major histocompatibility complex (MHC) class I region provides the necessary framework for a better understanding of its organization and for the localization of new genes involved in MHC-associated disease. Two human genomic libraries constructed in yeast artificial chromosomes (YACs) have been prepared using complete Not I or Mlu I digestion of source DNA. From these libraries three YAC clones with inserts belonging to the MHC class I region have been isolated. They correspond to exact copies of three genomic fragments of 210, 145, and 50 kilobases (kb), respectively and have been precisely located in the restriction map of the region. Detailed rare-cutter restriction maps of the inserts have been generated. Within these clones we have demonstrated the presence of two class I genes, one of which is HLA-E, and of at least three Hpa II tiny fragment (HTF) islands, corresponding to three putative new transcribed sequences. End clones, which are of particular interest in the extension and refinement of the regional map, have been rescued by systematic subcloning of purified YACs.

Base Sequence↗