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PubMed2002

Ordered catenation of sequence-tagged sites and multiplexed SNP genotyping by sequencing.

We describe a method for the efficient genotyping of SNPs, involving sequencing of ordered and catenated sequence-tagged sites (OCS). In OCS, short genomic segments, each containing an SNP, are amplified by PCR using primers that carry specially designed extra nucleotides at their 5'-ends. Amplification products are then combined and converted to a concatamer in a defined order by a second round of thermal cycling. The concatenation takes place because the 5'-ends of each amplicon are designed to be complementary to the ends of the presumptive neighboring amplicons. The primer sequences for OCS are chosen using newly developed dedicated software, OCS Optimizer. Using sets of SNPs, we show that at least 10 STSs can be concatenated in a predefined order and all SNPs in the STSs are accurately genotyped by one two-way sequencing reaction.

Asian People↗
PubMed1993

A sequence-specific function for the N-terminal signal-like sequence of the TonB protein.

TonB is a proline-rich protein which provides a functional link between the inner and outer membranes of Gram-negative bacteria. TonB is anchored to the inner membrane via an N-terminal signal-like sequence and spans the periplasm, interacting with transport receptors in the outer membrane. We have investigated the role of the N-terminal signal-like peptide in TonB function. Replacement of the N-terminal sequence with heterologous sequences indicates that it has at least three distinct roles in TonB function: (i) to facilitate translocation of TonB across the cytoplasmic membrane; (ii) to anchor TonB to the cytoplasmic membrane; (iii) a sequence-specific functional interaction with the ExbBD proteins.

Amino Acid Sequence↗
PubMed1992

Genomic organization and DNA sequences of human 17 beta-hydroxysteroid dehydrogenase genes and flanking regions. Localization of multiple Alu sequences and putative cis-acting elements.

Genomic 17 beta-hydroxysteroid-dehydrogenase (17-HSD) clones were isolated from a human leucocyte genomic library using cDNA encoding human placental 17-HSD as a probe. The overlapping fragments spanned more than 21 kbp containing the duplications, 6.2 kbp of each, as well as 7 kbp upstream and 1.6 kbp downstream from the duplicated sequences. 17 complete and eight partial Alu elements were clustered in this area, covering about 30% of the region, including the borders of the duplications. Each duplication contained a 17-HSD gene and a conserved region of 1.56 kbp with 98% intercopy similarity. The exon structure of the 17-HSD gene II corresponded to the known cDNA species, but both genes contained a possible promoter region with TATA, GC and inverse CAAT boxes. The 5' flanking regions contained sequences similar to the consensus sequences of cis-acting elements, defined as regulators of 17-HSD gene expression. These putative sequences included estrogen and progesterone/glucocorticoid-response elements and a cyclic-AMP regulatory element.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

tax and rex Sequences of bovine leukaemia virus from globally diverse isolates: rex amino acid sequence more variable than tax.

Bovine leukaemia virus (BLV) is an important agricultural problem with high costs to the dairy industry. Here, we examine the variation of the tax and rex genes of BLV. The tax and rex genes share 420 bases and have overlapping reading frames. The tax gene encodes a protein that functions as a transactivator of the BLV promoter, is required for viral replication, acts on cellular promoters, and is responsible for oncogenesis. The rex facilitates the export of viral mRNAs from the nucleus and regulates transcription. We have sequenced five new isolates of the tax/rex gene. We examined the five new and three previously published tax/rex DNA and predicted amino acid sequences of BLV isolates from cattle in representative regions worldwide. The highest variation among nucleic acid sequences for tax and rex was 7% and 5%, respectively; among predicted amino acid sequences for Tax and Rex, 9% and 11%, respectively. Significantly more nucleotide changes resulted in predicted amino acid changes in the rex gene than in the tax gene (P < or = 0.0006). This variability is higher than previously reported for any region of the viral genome. This research may also have implications for the development of Tax-based vaccines.

Amino Acid Sequence↗
PubMedDate not supplied

A short internal eliminated sequence with central conserved sequences interrupting the LA-MSC gene of the 81 locus in the hypotrichous ciliates Oxytricha fallax and O. trifallax.

IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytricha fallax and O. trifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT ... AATG, and thereby resembles a TBE1 transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This implies a excision mechanism for IES-LA that is distinct from those for other known Oxytricha IESs.

Animals↗
PubMed1994

Nucleotide sequence and structural organization of Yersinia pestis insertion sequence IS100.

Insertion sequence IS100 was localized on a 9.5-kb plasmid of Yersinia pestis and was shown to be specific for Y. pestis and serotype I strains of Y. pseudotuberculosis. The nucleotide sequence of IS100 isolated from this plasmid was determined. The element, which was flanked by 5-bp direct repeats, contained 1953 bp including imperfect inverted terminal repeats of 52 and 61 bp long (43 bp were identical). Two open reading frames encoding potential polypeptides of 340 and 252 amino acids were identified on one DNA strand. Nucleotide sequence as well as deduced polypeptide sequences of IS100 were homologous to those of IS21, IS232 and IS640.

Base Sequence↗
PubMed1991

The ompH gene of Yersinia enterocolitica: cloning, sequencing, expression, and comparison with known enterobacterial ompH sequences.

We have recently described a previously uncharacterized outer membrane protein of Salmonella typhimurium and Escherichia coli and cloned and sequenced the corresponding gene, the ompH gene, of S. typhimurium (P. Koski, M. Rhen, J. Kantele, and M. Vaara, J. Biol. Chem. 264:18973-18980, 1989). We report here the cloning, sequencing, and expression of the corresponding gene of Yersinia enterocolitica. It is significantly homologous to the ompH genes of E. coli and S. typhimurium (homology percentages, 65 and 64%, respectively), has a promoter region strongly homologous to the E. coli 17-bp class consensus promoter, and encodes a protein consisting of 165 amino acids (22 of which form the signal sequence). The plasmid-borne Y. enterocolitica ompH was found to be expressed both in the E. coli host and in minicells. The isolated outer membrane of Y. enterocolitica was shown to contain OmpH. The homology of the Y. enterocolitica OmpH protein is 66% with E. coli OmpH and 64% with S. typhimurium OmpH. All OmpH proteins have almost identical hydrophobic profiles, charge distributions, and predicted secondary structures. Because yersiniae are considered rather distant relatives of E. coli and S. typhimurium in the Enterobacteriaceae family, these results might indicate that most or all strains of the family Enterobacteriaceae have OmpH proteins remarkably homologous to those now sequenced.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence of rice dwarf phytoreovirus genome segment 2: completion of sequence analyses of rice dwarf virus.

The complete nucleotide sequence of rice dwarf phytoreovirus genome segment 2 (S2) was determined to be 3,512 nucleotides long with one open reading frame initiating at nucleotide 15 and terminating at nucleotide 3363. The encoded polypeptide was predicted to have 1,116 residues with a relative molecular weight of 123 kD. Comparison of S2 of two isolates showed they had identical lengths and 97 and 98.3% nucleotide and amino acid sequence identities, respectively. A search of the Swiss-Prot data base (R 22.0) failed to find any proteins with significant homology to the S2-encoded protein. Determination of the nucleotide sequence of the S2 has completed the sequence determination of the genome of rice dwarf virus. Homology searches made for proteins encoded by each of the genomic segments showed that the polypeptide encoded by S11 has similarity to histone H1 protein and VP6 of blue tongue virus, indicating it might possess nucleic acid binding properties.

Amino Acid Sequence↗
PubMed1993

PRAD1/cyclin D1 proto-oncogene: genomic organization, 5' DNA sequence, and sequence of a tumor-specific rearrangement breakpoint.

PRAD1 (previously D11S287) is a putative proto-oncogene at 11q13, activated by overexpression through gene rearrangement or gene amplification in several types of human tumors including parathyroid adenomas, centrocytic lymphomas and other B-cell tumors with t(11;14), and breast cancers. PRAD1 (also CCND1) encodes cyclin D1, which may regulate the G1-S phase transition in the cell cycle. Here, we report the cloning and characterization of the chromosomal PRAD1/cyclin D1 gene and the sequence of its promoter region. The gene spans about 15 kb and has 5 exons; its promoter region has Sp1 binding sites and no obvious TATA box, characteristics of housekeeping genes and growth-regulating genes. Furthermore, an E2F binding motif present close to the major transcription start site may be involved in cell cycle-dependent expression of this gene. We also report the sequence of DNAs spanning joining regions of a reciprocal parathyroid hormone/PRAD1 gene rearrangement in a parathyroid adenoma. Comparison with normal sequences suggests that the rearrangement was not a simple break-and-ligate event, but rather involved multiple steps, including two microdeletions and a microinversion. Very short sequences conserved near the breakpoints and symmetrical elements in the eventually inverted DNA segment might have played a role in this illegitimate complex recombination, which may have similarities with a constitutional translocation in Duchenne muscular dystrophy.

Adenoma↗
PubMed1995

Cloning of salicylate hydroxylase gene and catechol 2,3-dioxygenase gene and sequencing of an intergenic sequence between the two genes of Pseudomonas putida KF715.

The salicylate hydroxylase can convert the salicylate to catechol, and catechol 2,3-dioxygenase catalizes the conversion of catechol to 2-hydroxymuconic semialdehyde. A salicylate hydroxylase gene and a catechol 2,3-dioxygenase have been cloned from chromosomal DNA of P. putida KF715. The two genes have different promoters. An open reading frame with 339 nucleotides preceded by a putative ribosome-binding sequence (GGAGG) was identified in the intergenic sequence between salicylate hydroxylase gene and catechol 2,3-dioxygenase gene of P. putida KF715 and its sequence analyzed. This open reading frame can encode a polypeptide of molecular weight 13 kDa containing 112 amino acids, whose sequence exhibited 87% homology with that of ferredoxin encoded in NAH7 of P. putida PpG7 and significant homology with those of redox components in phenol hydroxylase, benzoate 1,2-dioxygenase, toluate 1,2-dioxygenase, xylene monooxygenase, and toluene 4-monooxygenase.

Amino Acid Sequence↗
PubMed1993

Sequence-tagged NotI sites of human chromosome 21: sequence analysis and mapping.

We isolated and analyzed 19 NotI site-containing clones specific for human chromosome 21. The overall process consisted of selective isolation of NotI site-containing clones from flow-sorted chromosome 21 libraries, selection of independent clones by their restriction patterns and nucleotide sequences, and assignment of the clones to chromosome 21. Sequence analysis showed that the regions around the NotI sites had features typical of CpG islands, such as extremely high GC content, high-frequency appearance of CpG dinucleotide sequences, and lack of methylation of these CpGs. PCR conditions for these extremely G + C-rich templates were optimized to establish these NotI sites as sequence-tagged sites.

Base Sequence↗
PubMed1993

Nucleotide sequence, expression, and chromosomal mapping of Mrp and mapping of five related sequences.

We isolated and characterized a genomic clone for the mouse MARCKS-related protein, or MRP, also known as F52 and MacMARCKS. A 3699-bp plasmid contained 407 bp of 5'-flanking region, 186 bp of 5'-untranslated region, 600 bp of protein coding region, 784 bp of a single intron, 746 bp of 3'-untranslated region, and 976 bp of 3'-flanking region. The position of the single intron was identical to the intron position in all known MARCKS mRNAs. When the plasmid containing the genomic sequences was transfected into fibroblasts lacking endogenous Mrp expression, the 407 bp of promoter conferred high-level expression of the full-length, spliced mRNA. The putative promoter was therefore functional; however, despite tissue-specific regulation and transcriptional induction in some cells in a manner similar to that seen with MARCKS expression, the promoters were highly dissimilar at the level of primary sequence (37% identity over 407 bp). Mrp mapped to a position on mouse chromosome 4 that was closely linked to the Lck locus. Numerous additional species that hybridized to the MRP cDNA were noted on Southern blotting of mouse genomic DNA. Five related loci were labeled Mrp-rs1 through Mrp-rs5 (for Mrp-related sequences) and were mapped to mouse chromosomes 10, 17, 15, 13, and X, respectively. Three of these related sequences have been cloned, and all appear to represent pseudogenes.

3T3 Cells↗
PubMed1997

The complete mitochondrial DNA sequence of the white rhinoceros, Ceratotherium simum, and comparison with the mtDNA sequence of the Indian rhinoceros, Rhinoceros unicornis.

The complete nucleotide sequence of the mitochondrial genome of the white rhinoceros, Ceratotherium simum, was determined. The length of the reported sequence is 16,832 nucleotides. This length can vary, however, due to pronounced heteroplasmy caused by differing numbers of a repetitive motif (5'-CG-CATATACA-3') in the control region. The 16,832 nucleotide sequence presented here is the longest version of the molecule and contains 35 copies of this motif. Comparison between the complete mitochondrial sequences of the white and the Indian (Rhinoceros unicornis) rhinoceroses allowed an estimate of the date of the basal evolutionary divergence among extant rhinoceroses. The calculation suggested that this divergence took place approximately 27 million years before present.

Animals↗
PubMed1996

Flanking sequences of an Alu source stimulate transcription in vitro by interacting with sequence-specific transcription factors.

An Alu source gene, called the EPL Alu, was previously isolated by a phylogenetic strategy. Sequences flanking the EPL Alu family member stimulate its RNA polymerase III (Pol III) template activity in vitro. One cis-acting element maps within a 40-nucleotide region immediately upstream to the EPL Alu. This same region contains an Ap1 site which, when mutated, abolishes the transcriptional stimulation provided by this region. The flanking sequence, as assayed by gel mobility shift, forms sequence-specific complexes with several nuclear factors including Ap1. These results demonstrate that an an ancestral Alu source sequence fortuitously acquired positive transcriptional control elements by insertion into the EPL locus, thereby providing biochemical evidence for a model which explains the selective amplification of Alu subfamilies.

Base Sequence↗
PubMed1993

Nucleotide sequence of the coat protein gene of pelargonium leaf curl virus and comparison of the deduced coat protein amino acid sequence with those of other tombusviruses.

The sequence of 1,787 nucleotides (nts) in the genomic RNA of pelargonium leaf curl virus (PLCV) was determined. It included the entire coat protein (cp) gene (nts 585 to 1,754), 558 nts of the 3' end of the putative RNA polymerase gene, 26 nts of an intercistronic region between the two genes and 33 nts downstream of the stop codon of the cp gene. The cp gene was cloned into the expression vector pET8c and expressed in E. coli. The deduced cp amino acid sequence of PLCV was compared with those of five other tombusviruses. The closer the degree of serological relatedness between two viruses, the more similarity was found in their cp amino acid sequences not only in the protruding domains, but also in their random and shell domains and in the arm regions. Nucleic acid hybridization tests, cp amino acid comparisons and serological tests all suggest the same order of sequence for the relationships in the tombusvirus group.

Amino Acid Sequence↗
PubMed1993

Partial sequence of the gene for a serine protease from a halophilic archaeum Haloferax mediterranei R4, and nucleotide sequences of 16S rRNA encoding genes from several halophilic archaea.

A part of the gene coding for a halophilic serine protease from a halophilic archaeum Haloferax mediterranei R4 was amplified by PCR and its 672 nucleotide sequence was determined. Tentative translation to the amino acid sequence suggested that the enzyme was quite similar to halolysin produced by another halophilic archaeum strain 172P1. Nucleotide sequences of 16S rRNA encoding genes from 9 halophilic archaea were determined. Alignment of 19 sequences known so far showed that there are more than 20 positions carrying bases or deletions specific for each halobacterial genus: Halobacterium, Haloarcula, Haloferax, and Halococcus.

Amino Acid Sequence↗
PubMed1999

Partial sequence of a sponge mitochondrial genome reveals sequence similarity to Cnidaria in cytochrome oxidase subunit II and the large ribosomal RNA subunit.

A 2550-bp portion of the mitochondrial genome of a Demosponge, genus Tetilla, was amplified from whole genomic DNA extract and sequenced. The sequence was found to code for the 3' end of the 16S rRNA gene, cytochrome c oxidase subunit II, a lysine tRNA, ATPase subunit 8, and a 5' portion of ATPase subunit 6. The Porifera cluster distinctly within the eumetazoan radiation, as a sister group to the Cnidaria. Also, the mitochondrial genetic code of this sponge is likely identical to that found in the Cnidaria. Both the full COII DNA and protein sequences and a portion of the 16S rRNA gene were found to possess a striking similarity to published Cnidarian mtDNA sequences, allying the Porifera more closely to the Cnidaria than to any other metazoan phylum. The gene arrangement, COII-tRNALys-ATP8-ATP6, is observed in many Eumetazoan phyla and is apparently ancestral in the metazoa.

Amino Acid Sequence↗
PubMed2003

Sequence variations of simple sequence repeats on chromosome-4 in two subspecies of the Asian cultivated rice.

Computational screening of the chromosome-4 sequence of the rice cultivar Nipponbare ( Oryza sativa L. japonica) revealed 1,844 tandem simple sequence repeats (SSRs) or microsatellites with SSR motifs >/=20 bp and repeated unit length of 1-6 base pairs. Thus SSRs occur once in every 18.8 kb, on the average, on the chromosome with one SSR per 23.8 kb and 16 kb on the short and long arms, respectively. No SSR was detected in the core region of the centromere. Poly(AT)(n) repeats represented the most abundant and length polymorphic class of SSRs on the chromosome, but it did not occur in the exons. GC-rich trinucleotide repeats were most abundant in the coding regions, representing 71.69% of the SSRs identified in the exons. Two hundred and twenty four SSRs were associated with the repetitive DNA sequences, most of them were poly(AT)(n) tracts. Sequence variations of SSRs between two cultivars, representing the two subspecies of the Asian cultivated rice indica and japonica, were identified, revealing that divergence and convergence of the two subspecies could be traced by the analysis of SSRs. These results provide a great opportunity for SSR-based marker development and comparative genome analysis of the two subspecies of the Asian cultivated rice.

Base Composition↗