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PubMed1996

Generation of expressed sequence tags and sequence-tagged sites as physical landmarks in the mosquito, Aedes aegypti, genome.

Twenty-three clones were developed as expressed sequence tags (ESTs) and sequence-tagged sites (STSs) that provide broad coverage of the mosquito, Aedes aegypti, genome at an average spacing of about 7.2 cM. Each of these clones had been mapped previously as a restriction fragment length polymorphism (RFLP) marker. Nineteen of these clones represent anonymous cDNAs and 4 clones represent known genes. Forty-seven percent of the anonymous cDNAs showed significant deduced amino acid sequence similarity to previously described sequences from a wide variety of organisms. STSs developed from RFLPs will provide effective tools for rapid screening of YAC and cosmid libraries, and will therefore, provide anchor points for contig construction for any region in the A. aegypti genome. Also, they facilitate the simultaneous integration of physical and genetic mapping data for A. aegypti.

Aedes↗
PubMed2004

The phylogeny of the social wasp subfamily Polistinae: evidence from microsatellite flanking sequences, mitochondrial COI sequence, and morphological characters.

BACKGROUND: Social wasps in the subfamily Polistinae (Hymenoptera: Vespidae) have been important in studies of the evolution of sociality, kin selection, and within colony conflicts of interest. These studies have generally been conducted within species, because a resolved phylogeny among species is lacking. We used nuclear DNA microsatellite flanking sequences, mitochondrial COI sequence, and morphological characters to generate a phylogeny for the Polistinae (Hymenoptera) using 69 species. RESULTS: Our phylogeny is largely concordant with previous phylogenies at higher levels, and is more resolved at the species level. Our results support the monophyly of the New World subgenera of Polistini, while the Old World subgenera are a paraphyletic group. All genera for which we had more than one exemplar were supported as monophyletic except Polybia which is not resolved, and may be paraphyletic. CONCLUSION: The combination of DNA sequences from flanks of microsatellite repeats with mtCOI sequences and morphological characters proved to be useful characters establishing relationships among the different subgenera and species of the Polistini. This is the first detailed hypothesis for the species of this important group.

Animals↗
PubMed2005

Pigs in sequence space: a 0.66X coverage pig genome survey based on shotgun sequencing.

BACKGROUND: Comparative whole genome analysis of Mammalia can benefit from the addition of more species. The pig is an obvious choice due to its economic and medical importance as well as its evolutionary position in the artiodactyls. RESULTS: We have generated approximately 3.84 million shotgun sequences (0.66X coverage) from the pig genome. The data are hereby released (NCBI Trace repository with center name "SDJVP", and project name "Sino-Danish Pig Genome Project") together with an initial evolutionary analysis. The non-repetitive fraction of the sequences was aligned to the UCSC human-mouse alignment and the resulting three-species alignments were annotated using the human genome annotation. Ultra-conserved elements and miRNAs were identified. The results show that for each of these types of orthologous data, pig is much closer to human than mouse is. Purifying selection has been more efficient in pig compared to human, but not as efficient as in mouse, and pig seems to have an isochore structure most similar to the structure in human. CONCLUSION: The addition of the pig to the set of species sequenced at low coverage adds to the understanding of selective pressures that have acted on the human genome by bisecting the evolutionary branch between human and mouse with the mouse branch being approximately 3 times as long as the human branch. Additionally, the joint alignment of the shot-gun sequences to the human-mouse alignment offers the investigator a rapid way to defining specific regions for analysis and resequencing.

Animals↗
PubMed1993

Exclusion of mutations in the gene for type III collagen (COL3A1) as a common cause of intracranial aneurysms or cervical artery dissections: results from sequence analysis of the coding sequences of type III collagen from 55 unrelated patients.

We performed detailed DNA sequencing analysis on type III collagen cDNA from 58 patients with either intracranial artery aneurysms or cervical artery dissections. The 58 patients were of seven different nationalities; among the patients were three pairs of relatives, so that 55 were unrelated, and of these, 29 had at least one blood relative with either an intracranial artery aneurysm or a cervical artery dissection. The age of the patients at the time of diagnosis ranged from 15 to 68 years (mean +/- SD = 40.3 +/- 11.0). The study group consisted of 25 males and 33 females. The analysis covered 3,232 nucleotides of significant (nonredundant) sequences per allele; therefore, we analyzed as many as 355,520 nucleotides. Mutations in the coding sequences for the triple-helical domain of type III collagen were excluded in 40 individuals with intracranial aneurysms and 18 individuals with cervical artery dissections. Direct sequencing of polymerase chain reaction products allowed mutations to be excluded with a high degree of confidence. Mutations that markedly decreased expression from one allele were also excluded in 42 of the 58 individuals, since the presence of both bases at one or more polymorphic sites in the 42 patients showed that two alleles were transcribed. The results indicated that mutations in the gene for type III procollagen (COL3A1) are not a common cause of either intracranial artery aneurysms or cervical artery dissections.

Adolescent↗
PubMed2004

Construction and evaluation of cDNA libraries for large-scale expressed sequence tag sequencing in wheat (Triticum aestivum L.).

A total of 37 original cDNA libraries and 9 derivative libraries enriched for rare sequences were produced from Chinese Spring wheat (Triticum aestivum L.), five other hexaploid wheat genotypes (Cheyenne, Brevor, TAM W101, BH1146, Butte 86), tetraploid durum wheat (T. turgidum L.), diploid wheat (T. monococcum L.), and two other diploid members of the grass tribe Triticeae (Aegilops speltoides Tausch and Secale cereale L.). The emphasis in the choice of plant materials for library construction was reproductive development subjected to environmental factors that ultimately affect grain quality and yield, but roots and other tissues were also included. Partial cDNA expressed sequence tags (ESTs) were examined by various measures to assess the quality of these libraries. All ESTs were processed to remove cloning system sequences and contaminants and then assembled using CAP3. Following these processing steps, this assembly yielded 101,107 sequences derived from 89,043 clones, which defined 16,740 contigs and 33,213 singletons, a total of 49,953 "unigenes." Analysis of the distribution of these unigenes among the libraries led to the conclusion that the enrichment methods were effective in reducing the most abundant unigenes and to the observation that the most diverse libraries were from tissues exposed to environmental stresses including heat, drought, salinity, or low temperature.

Expressed Sequence Tags↗
PubMed1996

DNA sequencing of the MHC class II region and the chromosome 6 sequencing effort at the Sanger Centre.

The human Major Histocompatibility Complex (MHC) is located on the short arm of chromosome 6 (6p21.3) and spans about 4 Mb. According to different gene families the MHC is subdivided into a class I, class II and class III region and many of its gene products are associated with the immune system and the susceptibility to various diseases. To date, we have sequenced about 40% (400 kb) of the class II region between HLA-DP and HLA-DQ and a coordinated effort to sequence the entire MHC is well underway. Analysis of the sequence revealed several novel genes and provides new insights into the molecular organisation and evolution of the MHC. All our data are publicly available via the MHC database (MHCDB) which allows rapid access, retrieval and display in the context of other MHC associated data. MHCDB is online available at (http:(/)/www.hgmp.mrc.ac.uk/) and, together with all our sequences also via anonymous ftp (ftp.icnet.uk/icrf-public).

Animals↗
PubMedDate not supplied

Identification of human cytomegalovirus target sequences in the human immunodeficiency virus long terminal repeat. Potential role of IE2-86 binding to sequences between -120 and -20 in promoter transactivation.

OBJECTIVE: Because of the important medical consequences of human cytomegalovirus (HCMV) infection in human immunodeficiency virus (HIV)-infected individuals, we wanted to understand the molecular interactions that occur during co-infection. Specifically, in this study, we wanted to identify the transactivating target sequences on the HIV long terminal repeat (LTR) that responded to HCMV infection. STUDY DESIGN/METHODS: In this study, we transfected the HIV-LTR into human fibroblasts and then mapped the regulation of this promoter following HCMV infection and co-transfection with the HCMV immediate-early (IE) gene product IE2-86. In addition, we examined IE2-86 binding to specific sequences in the HIV-LTR by electrophoretic mobility shift assay. RESULTS: Our results documented that HCMV and IE2-86 could transactivate the HIV-LTR. In mapping the regions of the HIV-LTR that IE2-86 transactivates, we identified discrete target sequences between -120 and -20 that are the major transactivating regions for the IE2-86-mediated effects and determined that IE2-86 could specifically bind to several discrete sequences within this region of the HIV-LTR. CONCLUSIONS: Our discovery of the binding of IE2-86 to the HIV-LTR, coupled with its ability to transactivate the HIV-LTR and induce cellular transcription factors, points to potential molecular mechanisms used by HCMV to upregulate the HIV life cycle and, consequently, exacerbate the conditions observed in individuals co-infected with HCMV and HIV.

Base Sequence↗
PubMed2004

[Sequencing and analyzing of expressed sequence tags (ESTs) of pig fat tissue].

In this paper,the ESTs of pig fat tissue were sequenced and analyzed using the large-scale DNA sequencing method, 7790 high quality ESTs were gained,from which 4354 genes were obtained using cluster analysis with the STACK-PACK software, including 3609 singlet genes and 745 multicopy genes. The 4354 candidate genes were compared with BLASTN to the nr library (e =1e-10), of which 2712 were known genes, containing 1 987 singlet genes and 725 multicopy genes,there are 2 109 unknown genes and new ESTs. Based on the results of BlastN and the index of GenBank Accenssion No., the known gene expression profile of pig fat were constructed. It showed that the genes participating in metabolism held the highest proportion in the 7 sorts, and in some aspects showed the hearty metabolism activity of fat tissue. 257 total reads of the Major Histocompatibility Complex (MHC) genes in the pig fat tissue were found,including 181 singlet genes and 44 multicopy genes. They account for 44.9% of the cell and organism defense, and 5.4% of all known genes,from this we can see that the MHC has a fairly high expression degree. Comparing all ESTs related with MHC (257reads) with known pig BAC we found the partial sequences (approximately 200 bp) of the ESTs distributing to every exon of. We can predict that the ESTs in MHC contain approximately 200 bp highly consistent regions and every exon possesses one of these regions. Although these MHC sequences in different BAC have different protein domains,they are highly consistent and related with immune functions. When the MHC genes transfer to the next generation,they can duplicate repeatly and inherit stably.

Adipose Tissue↗
PubMed2005

Simple modifications of the standard DNA sequencing protocol allow for sequencing through siRNA hairpins and other repeats.

RNAi is a relatively new but powerful technology used for monitoring gene silencing in many species. The RNA corresponding to the gene of interest can be delivered into cells using various protocols, but the approach where both sense and antisense strands are located in the same transcript seems to be the most promising. However, the DNA sequencing step is a challenge due to the formation of a strong hairpin structure. In this work we present a very simple modification to the standard DNA sequencing protocol that allows sequencing through such difficult regions. The modification does not require any additional enzymatic or chemical manipulations. It simply adds a 5-min heat-denaturation step at 98 degrees C, prior to addition of a Taq mix. The inclusion of this step is very effective in sequencing through many different categories of difficult templates. Other examples of the implementation of this step have been described previously and more detailed information on heat-denaturation of plasmid DNAs has been presented.

RNA, Small Interfering↗
PubMed1991

Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites.

By examining a large database of amino acid sequences of antibodies of various specificities, we have found that many antibodies of distinctly different specificities assemble identical VL domains with different VH domains. In contrast, rarely is the same VH domain found in sets of antibodies of different specificities. We identified additional sets of antibodies of different specificities and identical sequences covering amino acid residues VH 1 to 94 and VL 1 to 95. In addition, there were segments of additional antibodies for which complete sequences were not available, but identities were seen in VL CDR1, VL CDR2, and VL CDR3 up to the VL-JL junction. The finding that there are many identical VL 1 to 95 segments with different VH 1 to 94 sequences, and vice versa, raises important questions as to the role of VH in influencing the conformation of VL and, conversely, the role of VL in influencing the conformation of VH. Evidence is also cited indicating that a single amino acid change may seriously disrupt site structure and in some instances abolish binding. Our findings suggest that it will be important in the future to investigate further conformational effects on antibody structure by using X-ray crystallography, nuclear magnetic resonance spectroscopy, or other methods, to obtain a better understanding of the functions and topography of antibody-combining sites.

Amino Acid Sequence↗
PubMed1991

The N-terminal amino acid sequence of phenol hydroxylase contains a dinucleotide-binding sequence motif.

The N-terminal sequence of phenol hydroxylase from Trichosporon cutaneum was determined by Edman degradation of the integral protein and of fragments obtained by hydroxylamine cleavage and by digestion with Staphylococcus V8 protease. A continuous sequence of 80 residues from the N terminus was determined: TKYSESYCDV10, LIVGAGPAGL20 MAARVLSEYV30 RQKPDLKVRI40 IDKRSTKVYN50 GQADGLQCRT60 LESLKNLRLA70 DKIXSEXNDM80. A single N-terminal sequence was detected, suggesting two identical subunits in the dimeric enzyme. We suggest the occurrence of an FAD-binding site near the N terminus. The C-terminal sequence is -LSTA, as determined by carboxypeptidase digestion.

Amino Acid Sequence↗
PubMed1990

DNA sequencing separations in capillary gels on a modified commercial DNA sequencing instrument.

DNA sequencing separations of standard DNA fragments of known sequence have been achieved in small diameter capillary gels electrophoresed and analyzed in parallel in a modified commercial DNA sequencer instrument. DNA sequencing in terms of base-calling accuracy is comparable to conventional slab gels; however, the separations in the capillary were performed somewhat faster and required less sample than those in the slab gel. Advantages of this approach vs. separations on conventional slab gels are discussed.

Base Sequence↗
PubMed1987

Multiple species of bovine adrenodoxin mRNA. Occurrence of two different mitochondrial precursor sequences associated with the same mature sequence.

Bovine adrenodoxin mRNA is found to consist of several distinct mRNA species which can be divided into two sets. Each set utilizes at least three of four separate poly(A)+ addition sites providing an explanation of the three sizes of adrenodoxin mRNA (1.75, 1.4, and 0.95 kilobases) previously observed in bovine adrenocortical RNA by this laboratory (Okamura, T., John, M.E., Zuber, M.X., Simpson, E.R., and Waterman, M.R. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5705-5709). The two sets are distinguished from one another by a unique 5' sequence leading to two different amino acid sequences (approximately 10% homology) for the precursor portion of this nuclear encoded, mitochondrial protein. A common mature adrenodoxin sequence is encoded by both sets of mRNA. One set of RNAs is 10-fold more abundant than the other, but the levels of both sets can be induced by treatment of primary bovine adrenocortical cell cultures with adrenocorticotropin. The biological significance of these two types of adrenodoxin precursor sequences remains obscure.

Adrenal Cortex↗
PubMed1988

The sequence specificity of bleomycin damage in three cloned DNA sequences that differ by a small number of base substitutions.

The DNA sequence specificity of the cancer chemotherapeutic agent, bleomycin, has been investigated in three clones of human alpha RI-DNA. The three 340-base pair alpha RI-DNA sequences were almost identical in their nucleotide sequence enabling the study of subtle effects of base substitutions on bleomycin cleavage. By utilizing densitometer scanning and statistical analysis of the degree of bleomycin DNA cleavage, we found 17 significant differences between the three DNA sequences. Eleven of these differences could be attributed to base substitutions close to the dinucleotide cleavage site. However, six of the differences were at positions two or more base pairs from the base substitution sites. The significant differences were up to 12 base pairs from base substitutions. It is proposed that these long range effects are due to base substitutions causing microvariation in the DNA structure to which bleomycin cleavage is sensitive.

Base Sequence↗
PubMed1987

Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase.

cDNA clones for rat acyl-CoA oxidase were isolated. The 3.8-kilobase mRNA sequence of the enzyme was completely covered by two overlapping clones. The composite cDNA sequence consisted of 3741 bases and contained a 1983-base open reading frame which encodes a polypeptide of 661 amino acid residues. Two species of acyl-CoA oxidase cDNA were identified. They differed in their coding nucleotide sequences, only within a small region. They contained the same number of nucleotides and can be translated in a common reading frame. They are 55% and 50% homologous in the above region at the nucleotide and the amino acid levels, respectively. Both types of cDNA were isolated from a library constructed from mRNA of a single rat, thereby suggesting the occurrence of two species of acyl-CoA oxidase in each rat. The amino terminus of the enzyme was determined to be N-acetylmethionine, which corresponds to the initiator methionine, thus confirming the absence of a terminal presequence. We reported previously that a purified preparation of the enzyme contained three polypeptide components, A, B, and C, and suggested that components B and C are produced by a proteolytic cleavage of component A (Osumi, T., Hashimoto, T., and Ui, N. (1980) J. Biochem. (Tokyo) 87, 1735-1746). We located components B and C on the amino- and the carboxyl-terminal sides of component A. Possible functional significances of several stretches of amino acids of the enzyme are discussed, based on the sequence comparison data between rat and yeast acyl-CoA oxidases.

Acyl-CoA Oxidase↗
PubMed1987

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level.

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.

Amino Acid Sequence↗
PubMed1984

The nucleotide sequence of the aroF gene of Escherichia coli and the amino acid sequence of the encoded protein, the tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase.

The translated sequence of aroF, the first structural gene of the tyrosine operon of Escherichia coli, has been determined. The 1068 nucleotides encode the 356 amino acids that form the subunit of the dimeric tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. The primary structure of this enzyme has been confirmed by automated Edman degradation of peptide fragments produced by cleavage with cyanogen bromide, limited trypsin digestion, Staphylococcus aureus strain V8 protease, or mild acid hydrolysis. The amino acid sequence of this enzyme is compared with the sequence of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, deduced from the aroG DNA sequence (Davies, W. D., and Davidson, B. E. (1982) Nucleic Acids Res. 10, 4045-4058).

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1982

Nucleotide sequence of Escherichia coli purF and deduced amino acid sequence of glutamine phosphoribosylpyrophosphate amidotransferase.

The Escherichia coli gene purF, coding for 5-phosphoribosylamine:glutamine pyrophosphate phosphoribosyltransferase (amidophosphoribosyltransferase) was subcloned from a ColE1-purF plasmid into pBR322. Amidophosphoribosyltransferase levels were elevated more than 5-fold in the ColE1-purF plasmid-bearing strain compared to the wild type control, and a further 10- to 13-fold elevation was observed in several pBR322 derivatives. The nucleotide sequence of a 2478-base pair PvuI-HinfI fragment encoding purF was determined. The purF45 structural gene codes for a 56,395 Mr protein chain having 504 amino acid residues. Methionine-1 is removed by processing in vivo leaving cysteine as the NH2-terminal residue. The deduced amino acid sequence was confirmed by comparisons with the NH2-terminal amino acid sequence determined by automated Edman degradation (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536) and amino acid analyses of CNBr peptides including a 4-residue peptide from the CO2H terminus of the enzyme. Nucleotide sequences characteristic of bacterial promoter-operator regions were identified in the 5' flanking region. The coding region appears to be preceded by a 277-297 nucleotide mRNA leader. A deletion removing the putative promoter-operator region results in defective purF expression.

Amidophosphoribosyltransferase↗