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At least 253 records · Page 14Linked to original sources
PubMed2006

Fine mapping of quantitative trait nucleotides underlying thrombin-activatable fibrinolysis inhibitor antigen levels by a transethnic study.

Recent studies revisiting the association between plasma thrombin-activatable fibrinolysis inhibitor (TAFI) Ag levels and polymorphisms of the CPB2 gene (coding for TAFI) suggested that TAFI Ag levels were influenced by 2 major quantitative trait nucleotides (QTNs) in European whites. However, the strong linkage disequilibrium (LD) between CPB2 polymorphisms in European whites did not allow one to distinguish which polymorphisms could be the putative QTNs. To get a better insight into the identification of QTNs, a transethnic haplotype analysis contrasting 2 populations of African and European subjects was performed using 13 CPB2 polymorphisms. Results of the haplotype analyses suggested that 3 QTNs had independent effects and explained about 15% of the TAFI variability, consistently in the 2 populations. The lower LD observed in the African population enabled us to identify the 1583T>A SNP located in 3'UTR as one of these QTNs, whereas the -2599C>G and -2345--2344insG SNPs located in the 5' region might be the 2 other QTNs. A phylogenetic study suggested that these 3 polymorphisms occurred before the period of migration "out of Africa." Although this transethnic comparison contributed to better map the putative CPB2 QTNs, further studies are required to clarify the role of the promoter region.

3' Untranslated Regions↗
PubMed2004

Automated whole-genome multiple alignment of rat, mouse, and human.

We have built a whole-genome multiple alignment of the three currently available mammalian genomes using a fully automated pipeline that combines the local/global approach of the Berkeley Genome Pipeline and the LAGAN program. The strategy is based on progressive alignment and consists of two main steps: (1) alignment of the mouse and rat genomes, and (2) alignment of human to either the mouse-rat alignments from step 1, or the remaining unaligned mouse and rat sequences. The resulting alignments demonstrate high sensitivity, with 87% of all human gene-coding areas aligned in both mouse and rat. The specificity is also high: <7% of the rat contigs are aligned to multiple places in human, and 97% of all alignments with human sequence >100 kb agree with a three-way synteny map built independently, using predicted exons in the three genomes. At the nucleotide level <1% of the rat nucleotides are mapped to multiple places in the human sequence in the alignment, and 96.5% of human nucleotides within all alignments agree with the synteny map. The alignments are publicly available online, with visualization through the novel Multi-VISTA browser that we also present.

Animals↗
PubMed2002

Epitope mapping of mitochondrial adenine nucleotide translocase-1 in idiopathic dilated cardiomyopathy.

Mitochondrial adenine nucleotide translocase (ANT) is a specific target for the autoantibody response in idiopathic dilated cardiomyopathy (IDCM). We have undertaken an epitope analysis of ANT in IDCM by immunoblot with recombinant GST-ANT fusion proteins and with cellulose-bound decapeptides of human ANT1. Forty-five patients with IDCM, 17 patients with ischemic left ventricle dysfunction (LVD) and 20 controls were analyzed for circulating antibodies against ANT (AAb-ANT). Sixteen of the 45 (36%) IDCM patients showed AAb-ANT above controls. In immunoblots, AAb-ANT detected purified bovine heart ANT and GST-ANT1 and GST-ANT2 isoforms and, less frequently, the GST-ANT3 isoform. A construct lacking the last 146 amino acids did not react with AAb-ANT, indicating that the main epitopes are in the C-terminal 146 amino acids. Immunodetection of decapeptides covering this region shows that AAb-ANT detects at least three epitopes, demonstrating that ANT is the primary target of AAb-ANT. The most significant epitopes belong to the M2 and M3 hydrophilic loops of ANT suggesting that apart from being essential for its activity, these loops are highly immunogenic.

Animals↗
PubMed1988

Mapping of the nucleotide-binding sites in the ADP/ATP carrier of beef heart mitochondria by photolabeling with 2-azido[alpha-32P]adenosine diphosphate.

2-Azido[alpha-32P]adenosine diphosphate (2-azido[alpha-32P]ADP) has been used to photolabel the ADP/ATP carrier in beef heart mitochondria. In reversible binding assays carried out in the dark, this photoprobe was found to inhibit ADP/ATP transport in beef heart mitochondria and to bind to two types of specific sites of the ADP/ATP carrier characterized by high-affinity binding (Kd = 20 microM) and low-affinity binding (Kd = 400 microM). In contrast, it was unable to bind to specific carrier sites in inverted submitochondrial particles. Upon photoirradiation of beef heart mitochondria in the presence of 2-azido[alpha-32P]ADP, the ADP/ATP carrier was covalently labeled. After purification, the photolabeled carrier protein was cleaved chemically by acidolysis or cyanogen bromide and enzymatically with the Staphylococcus aureus V8 protease. In the ADP/ATP carrier protein, which is 297 amino acid residues in length, two discrete regions extending from Phe-153 to Met-200 and from Tyr-250 to Met-281 were labeled by 2-azido[alpha-32P]ADP. The peptide fragments corresponding to these regions were sequenced, and the labeled amino acids were identified. As 2-azido-ADP is not transported into mitochondria and competes against transport of externally added ADP, it is concluded that the two regions of the carrier which are photolabeled are facing the cytosol. Whether the two photolabeled regions are located in a single peptide chain of the carrier or in different peptide chains of an oligomeric structure is discussed.

Adenosine Diphosphate↗
PubMed1992

Intracellular amplification and expression of a synthetic analog of rotavirus genomic RNA bearing a foreign marker gene: mapping cis-acting nucleotides in the 3'-noncoding region.

cDNAs were constructed to encode plus- or minus-sense analogs of gene 9 RNA of porcine rotavirus strain OSU in which the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was flanked by the 5'-terminal 44 nucleotides (nt) and 3'-terminal 35 nt of the authentic rotavirus gene. Transfection of plus-sense gene-9-CAT RNA into rotavirus-infected cells resulted in its amplification and in the efficient expression of CAT; this was greatly enhanced by the presence of a 5' cap structure. Amplification was ablated by omitting the rotavirus superinfection or by removing the 3'-terminal 35-nt rotavirus sequence from the RNA. This result indicated that amplification depended both on rotavirus proteins supplied in trans and on cis-acting rotavirus sequences. Minus-sense or double-stranded gene-9-CAT RNA was essentially inactive, indicating that synthetic RNAs can be introduced into the rotavirus replicative cycle in vivo only when provided in the plus sense. However, incorporation of the CAT-bearing RNA into infectious rotavirus was not detected. Two heterologous rotaviruses, the simian RRV and chicken Ch2 strains, efficiently complemented the OSU-based gene-9-CAT RNA, even though the Ch2 strain was only 50%-66% related in the noncoding regions. Mutational analysis of the 35-nt 3'-noncoding region showed that the 3'-terminal 12 or 17 nt were sufficient for reduced (12% or 23%, respectively) levels of amplification, whereas inclusion of the 3'-terminal 19 nt fully restored amplification. Thus, the 3'-terminal cis-acting signals required for amplification include the 7-nt-terminal consensus sequence together with 12 nt of adjoining, less-well-conserved sequence.

Animals↗
PubMed2001

Binding sites for Rev and ASF/SF2 map to a 55-nucleotide purine-rich exonic element in equine infectious anemia virus RNA.

The equine infectious anemia virus (EIAV) Rev protein (ERev) negatively regulates its own synthesis by inducing alternative splicing of its mRNA. This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. When Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. The interaction of ERev with its cis-acting RNA response element, the RRE, is also essential for nuclear export of intron-containing viral mRNAs that encode structural and enzymatic gene products. The primary ERev binding site and the manner in which ERev interacts with RNA or cellular proteins to exert its regulatory function have not been defined. We have performed in vitro RNA binding experiments to show that recombinant ERev binds to a 55-nucleotide, purine-rich tract proximal to the 5' splice site of exon 3. Because of its proximity to the 5' splice site and since it contains elements related to consensus exonic splicing enhancer sequences, we asked whether cellular proteins recognize the EIAV RRE. The cellular protein, ASF/SF2, a member of the serine- and arginine-rich family of splicing factors (SR proteins) bound to repeated sequences within the 55-nucleotide RRE region. Electrophoretic mobility shift and UV cross-linking experiments indicated that ERev and SR proteins bind simultaneously to the RRE. Furthermore, in vitro protein-protein interaction studies revealed an association between ERev and SR proteins. These data suggest that EIAV Rev-induced exon skipping observed in vivo may be initiated by simultaneous binding of Rev and SR proteins to the RRE that alter the subsequent assembly or catalytic activity of the spliceosomal complex.

Alternative Splicing↗
PubMed1985

Nucleotide sequence and transcriptional mapping of the yeast pet56-his3-ded1 gene region.

Genes of the baker's yeast Saccharomyces cerevisiae are densely clustered on 16 linear chromosomes. Here, I characterize a 1.8 kb region of chromosome XV containing the entire structural gene for the histidine biosynthetic enzyme imidazoleglycerolphosphate (IGP) dehydratase (his3) as well as the promoter sequences and 5'-proximal mRNA coding regions for the adjacent genes. The his3 gene encodes several mRNA species averaging 820 bases in length, all of which contain an open reading frame of 219 codons. The location of this open reading frame coincides with the his3 gene as defined by functional criteria, suggesting that the primary translation product of yeast IGP dehydratase has a molecular weight of 23,850. Phenotypic analysis of mutations constructed in vitro indicate that one of the adjacent genes (pet56) is required for mitochondrial function, whereas the other gene (ded1) is essential for cell viability. The pet56 and his3 genes are transcribed divergently from initiation sites that are separated by only 192 bp. Transcription of the ded1 gene is initiated only 130 bp beyond the 3'-end of the his3 mRNA coding region. These results suggest that these unrelated genes are located extremely close together and that the spacer regions between them consist largely of promoter and terminator sequences.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400.

The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida F1 and have been named bphA, bphE, bphF, and bphG. From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase. Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a [2Fe-2S] cluster. Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding. Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified. Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.

Amino Acid Sequence↗
PubMed1998

Nucleotide sequence and chromosomal mapping of the 5S rDNA repeat of the crustacean Proasellus coxalis.

In this investigation we analysed the 5S rRNA genes of the isopod crustacean Proasellus coxalis, 5S rDNA hybridization of digested genomic DNA and amplification by PCR demonstrate that these genes are organized in tandem repeats of 589 bp, 120 of which represent the coding sequence and 469 the spacer sequence. Proasellus coxalis is the first crustacean species in which 5S rRNA genes have been found tandemly arranged without being linked to other repeated genes. The PCR product has been used as a probe in FISH to locate the 5S rRNA genes on two chromosome pairs of the P. coxalis karyotype. Comparison of the 5S rRNA sequence of this species with previously published sequences of six other crustacean species shows the existence of a good correlation between phylogenetic relationships and sequence identity.

Animals↗
PubMed2000

Organization, nucleotide sequence, and chromosomal mapping of a tandemly repeated unit containing the four core histone genes and a 5S rRNA gene in an isopod crustacean species.

A tandemly repeated unit of 6553 bp containing a copy of the four core histone genes H2B, H2A, H3, and H4, and also a 5S rRNA gene, was amplified by PCR from genomic DNA of the isopod crustacean Asellus aquaticus. The linkage between 5S rRNA genes and histone genes has been so far observed in only one other organism, the anostrac crustacean Artemia salina. The gene cluster was cloned and sequenced. The histone genes, in their 3' flanking region, have the interesting feature of possessing two different mRNA termination signals, the stem-loop structure and the AATAAA sequence. A part of the PCR product was used as a probe in FISH experiments to locate the gene cluster on an inter-individually variable number of chromosomes from 6 to 12 per diploid cell, always in a terminal position and never associated with the heterochromatic areas. Fluorescence in situ hybridization (FISH) was also performed on preparations of released chromatin and the reiteration level of the gene cluster was determined as approximately 200-300 copies per haploid genome.

Animals↗
PubMed1999

Mapping HLA for single nucleotide polymorphisms.

Knowledge of DNA sequence variation may help us understand how genetic variability gives rise to functional variability and, in so doing, revolutionize the development of strategies to combat and prevent disease. Single nucleotide polymorphisms (SNPs) are stable, inherited, biallelic, single base pair differences which are present in the human genome at a density of 1 to 10 per 1,000 nucleotides. It is anticipated that SNPs will account for much of the functional heterogeneity in gene expression and protein activity exhibited in the human population. Susceptibility to or protection from a number of diseases, particularly those of autoimmune etiology, has been associated with specific alleles of the human leukocyte antigen (HLA) complex. Interestingly, the precise molecular defects in the HLA genes are unknown and the notion that non-HLA genes, within the same chromosomal region, are involved remains a formal possibility. We have determined the nucleotide sequence of a contiguous 2.2 Mbp segment of chromosome six that includes all of the HLA class I region, and have identified over 10,000 SNPs therein. Because of the derivative knowledge of gene and SNP content and position, the scientific community is now uniquely poised to identify disease-contributory SNPs that lie within the MHC.

HLA Antigens↗
PubMed1989

The molecular cloning of the gene encoding the Escherichia coli 75-kDa helicase and the determination of its nucleotide sequence and gentic map position.

A previously unreported DNA unwinding enzyme, referred to as the 75-kDa helicase, was recently purified from Escherichia coli cell extracts and biochemically characterized (Wood, E. R., and Matson, S. W. (1987) J. Biol. Chem. 262, 15269-15276). In order to initiate the genetic analysis of the 75-kDa helicase, the gene encoding this enzyme was cloned. DNA sequencing confirmed the identity of the gene since the predicted amino acid sequence of the encoded polypeptide precisely matched the sequence of the first 27 NH2-terminal amino acid residues of the 75-kDa helicase as determined by peptide sequencing. The predicted amino acid sequence of the 75-kDa helicase is similar in several regions to the amino acid sequences of two other E. coli helicases, Rep protein and helicase II. The gene encoding the 75-kDa helicase was mapped to 22 min on the E. coli chromosome. We propose that this newly defined locus be referred to as helD, and, to avoid confusion with other E. coli helicases with a similar molecular size, we propose that the 75-kDa helicase be referred to as helicase IV.

Adenosine Triphosphatases↗
PubMedDate not supplied

[DNA fingerprinting: hypervariable loci and genetic marking].

The paper contains a short review of the data on some possibilities of the DNA fingerprinting technology in genetic analysis. The main principles and the story of this new method are described in brief. The peculiar structure of the hypervariable genetic loci and some features of the multiallelic markers based on highly polymorphic mini-satellites are discussed.

Animals↗