An Integrated Sequence-Structure Database incorporating matching mRNA sequence, amino acid sequence and protein three-dimensional structure data.

We have constructed a non-homologous database, termed the Integrated Sequence-Structure Database (ISSD) which comprises the coding sequences of genes, amino acid sequences of the corresponding proteins, their secondary structure and straight phi,psi angles assignments, and polypeptide backbone coordinates. Each protein entry in the database holds the alignment of nucleotide sequence, amino acid sequence and the PDB three-dimensional structure data. The nucleotide and amino acid sequences for each entry are selected on the basis of exact matches of the source organism and cell environment. The current version 1.0 of ISSD is available on the WWW at http://www.protein.bio.msu.su/issd/ and includes 107 non-homologous mammalian proteins, of which 80 are human proteins. The database has been used by us for the analysis of synonymous codon usage patterns in mRNA sequences showing their correlation with the three-dimensional structure features in the encoded proteins. Possible ISSD applications include optimisation of protein expression, improvement of the protein structure prediction accuracy, and analysis of evolutionary aspects of the nucleotide sequence-protein structure relationship.     

Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism

Hybrid molecules containing DNA sequences complementary to bovine pituitary mRNA were constructed in the Pst I site of pBR322 by the dC . dG tailing technique. Recombinant plasmids containing bovine prolactin (bPRL) sequences were amplified in bacteria and identified by hybridization to purified [32P]bPRL cDNA sequences. Nucleotide sequence analysis was performed on the inserts from two of the positive clones. One clone, pBPRL72, contained a 982-base pair insert that included 67 nucleotides of the 5'-untranslated region, the complete coding region of the preprolactin protein (690 nucleotides), and the entire 3'-untranslated region (150 nucleotides) of bPRL mRNA. The nucleotide sequence analysis of clone pBPRL72 predicted the sequence of a 30-amino acid signal peptide and confirmed the published amino acid sequence of the protein with one exception. A comparison of the pBPRL72 cDNA sequence with a second bPRL clone, pBPRL4, revealed four silent nucleotide differences. Three of the base changes occurred in the third position of amino acid codons, and one occurred in the 3'-noncoding region. The sequence polymorphism suggests the existence of alleles or multiple loci for bPRL that do not alter the protein structure.     

Complete nucleotide sequence of a porcine HSP70 gene

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M69100.     

The nucleotide sequence of the rodC operon of Bacillus subtilis

The rodC1 mutation of Bacillus subtilis is a temperature-sensitive marker which affects the levels of teichoic acid synthesis and the cellular morphology. We have determined the nucleotide sequence of the bicistronic operon which contains the rodC gene and the nucleotide sequence of the rodC1 mutant allele. The temperature-sensitive phenotype of the rodC mutant is the result of a single base-pair change. A cytosine to thymine transition in the non-coding strand results in the replacement of a serine residue in the wild-type protein with a phenylalanine residue in the mutant protein. The other gene in the operon, the rodD gene, appears to be equivalent to the gtaA gene which encodes uridine diphosphate-glucose poly-(glycerol phosphate) alpha-glucosyl transferase, an enzyme involved in techoic acid synthesis. This is the first nucleotide sequence analysis of both the wild-type and mutant alleles of a morphogene in B. subtilis.     

Allele-specific polymerase chain reaction: a method for amplification and sequence determination of a single component among a mixture of sequence variants

A new technique is described for amplifying individual alleles in a mixture of two or more alleles by the polymerase chain reaction (PCR) to determine their nucleotide sequence. This technique involves amplifying and separating target sequences by the PCR-mediated single-strand conformation polymorphism (PCR-SSCP) method, isolating each polymorphic DNA strand, and amplifying it by a second-stage PCR for its sequence determination. By this technique, the sequence of a minor constituent (approximately 3%) can be determined accurately.     

ANCAC: amino acid, nucleotide, and codon analysis of COGs -- a tool for sequence bias analysis in microbial orthologs

ABSTRACT: BACKGROUND: The COG database is the most popular collection of orthologous proteins from many different completely sequenced microbial genomes. Per definition, a cluster of orthologous groups (COG) within this database exclusively contains proteins that most likely achieve the same cellular function. Recently, the COG database was extended by assigning to every protein both the corresponding amino acid and its encoding nucleotide sequence resulting in the NUCOCOG database. This extended version of the COG database is a valuable resource connecting sequence features with the functionality of the respective proteins. RESULTS: Here we present ANCAC, a web tool and MySQL database for the analysis of amino acid, nucleotide, and codon frequencies in COGs on the basis of freely definable phylogenetic patterns. We demonstrate the usefulness of ANCAC by analyzing amino acid frequencies, codon usage, and GC-content in a species- or function-specific context. With respect to amino acids we, at least in part, confirm the cognate bias hypothesis by using ANCAC's NUCOCOG dataset as the largest one available for that purpose thus far. CONCLUSIONS: Using the NUCOCOG datasets, ANCAC connects taxonomic, amino acid, and nucleotide sequence information with the functional classification via COGs and provides a GUI for flexible mining for sequence-bias. Thereby, to our knowledge, it is the only tool for the analysis of sequence composition in the light of physiological roles and phylogenetic context without requirement of substantial programming-skills.     

The nucleotide sequence of bovine MHC class IIDQB andDRB genes

The nucleotide sequences of most of the exons and parts of the introns of twoBoLA-DQB genes and twoBoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The twoDQB genes probably represent true alleles. Based on the exons sequenced, bothDQB genes and one of theDRB genes seem to be functional. The otherDRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occured in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease association in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30002–M30014. Address correspondence and offprint requests to: M. A. M. Groenen.     

The genome sequence DataBase

The Genome Sequence DataBase (GSDB) is a database of publicly available nucleotide sequences and their associated biological and bibliographic information. Several notable changes have occurred in the past year: GSDB stopped accepting data submissions from researchers; ownership of data submitted to GSDB was transferred to GenBank; sequence analysis capabilities were expanded to include Smith-Waterman and Frame Search; and Sequence Viewer became available to Mac users. The content of GSDB remains up-to-date because publicly available data is acquired from the International Nucleotide Sequence Database Collaboration databases (IC) on a nightly basis. This allows GSDB to continue providing researchers with the ability to analyze, query and retrieve nucleotide sequences in the database. GSDB and its related tools are freely accessible from the URL: http://www.ncgr.org     

Cloning and complete sequence of an HLA-A2 gene: analysis of two HLA-A alleles at the nucleotide level

The gene for the HLA-A2 antigen has been cloned from the human lymphoblastoid cell line 721. Comparison of this sequence with the published sequence for HLA-A3 permits the examination of two alleles at the extremely polymorphic HLA-A locus. A high degree of sequence conservation was seen in both coding and noncoding DNA, 97.2% and 94.5%, respectively. Interestingly, the 3' untranslated region was the most conserved, with 99.5% homology. The polymorphism of the HLA-A antigens results from a high proportion of amino acid substitutions relative to the total nucleotide changes in exons 2 and 3. Unlike the clustered differences seen in this region on comparison of two H-2K alleles of mouse, nucleotide substitutions between the HLA-A2 and A3 alleles are evenly distributed. Substitutions at silent sites and within introns were used to calculate an intra-allelic divergence time of at least 10 to 15 million years for these two HLA-A alleles.     

Structure, sequence, and polymorphism of the Lyt-2 T cell differentiation antigen gene

We have determined the nucleotide sequence and genomic organization of the mouse Lyt-2 T lymphocyte differentiation antigen gene. This gene consists of five exons and four introns, and the organization roughly parallels the protein domains. Alternative splicing to include or exclude exon IV (encoding part of the cytoplasmic tail) results in two forms of mRNA and accounts for the difference in size between the alpha- and alpha'-chains of Lyt-2. The gene structure provides further evidence for the evolutionary relationship between Lyt-2 and immunoglobulin genes. Comparison of the nucleotide sequence of the Lyt-2.1 and Lyt-2.2 alleles shows a high degree of conservation, but indicates that a single nucleotide change and consequent amino acid substitution in the variable region-like domain accounts for the serologic difference between these two alleles.     

Nucleotide sequence of the Escherichia coli mutH gene.

The complete nucleotide sequence of mutH gene from E. coli has been determined. Based on the deduced amino acid sequence, the MutH protein has a molecular weight of 25.4 kdaltons in agreement with the previous estimates based on SDS-polyacrylamide gel electrophoresis of the purified protein. Deletion analysis of the DNA sequences upstream of mutH has identified the promoter region for this gene. Two independently isolated temperature sensitive alleles of the mutH gene have also been sequenced. One mutation results in an amino acid change at position 27 (thr to leu) while the other occurs at position 156 (asp to asn).     

Variation in the sequence and modification state of the human insulin gene flanking regions.

The nucleotide sequence of a highly repetitive sequence region upstream from the human insulin gene is reported. The length of this region varies between alleles in the population, and appears to be stably transmitted to the next generation in a Mendelian fashion. There is no significant correlation between the length of this sequence and two types of diabetes mellitus. We observe variation in the cleavability of a BglI recognition site downstream from the human insulin gene, which is probably due to variable nucleotide modification. This presumed modification state appears not to be inherited, and varies between tissues within an individual and between individuals for a given tissue. Both alleles in a given tissue DNA sample are modified to the same extent.     

Comparative analysis of sequence variability in the upstream regulatory region of the HLA-DQB1 gene

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X74230-X74239.     

Nucleotide sequence of the BALB/c H-2T region gene,T3 d

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M75875.     

The isolation and sequence of the chromosomal gene and regulatory regions of Ly-6A.2

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M73552.     

Nucleotide sequence of a cDNA clone of the horse (Equus caballus) DRA gene

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60100. Clone pL33EA is available upon request.     

Nucleotide sequence of the yeast regulatory gene GAL80

The GAL80 gene in Saccharomyces cerevisiae encodes a negative regulatory protein for the set of inducible genes involving metabolism of galactose and melibiose. We have determined the nucleotide sequence of GAL80 and its flanking regions and assigned the 5' end of its mRNA to the sequence. The deduced coding sequence for GAL80 protein contains 1305 nucleotides and the calculated molecular weight of the peptide chain is 48309. The 5' end of the GAL80 mRNA maps about 67 nucleotides upstream from the translation initiating ATG. We have also determined the nucleotide sequence of uninducible alleles GAL80S-0, GAL80S-1 and GAL80S-2, and found single base substitution in each of these mutant genes which would lead to alteration of amino acid in GAL80 protein.     

Nucleotide sequence of the tumor necrosis factor: a gene in seven different inbred mouse strains

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers L22359-L22365.     

Genotyping of Streptococcus thermophilus strains isolated from traditional Egyptian dairy products by sequence analysis of the phosphoserine phosphatase (serB) gene with phenotypic characterizations of the strains

Aims: To develop a new, simplified genotyping method for examining the genetic diversity of Streptococcus thermophilus strains isolated from traditional Egyptian fermented dairy products and to characterize phenotypic traits of those strains related to their potential use in bioprocessing applications. Methods and Results: A novel, simplified approach was developed for genotyping Strep. thermophilus involving the analysis of nucleotide sequence variations within a housekeeping gene encoding the phosphoserine phosphatase (SerB). Using this method, it was possible to identify ten genotypes involving diverse serB alleles among 54 Strep. thermophilus isolates cultured from Egyptian dairy products. These isolates harboured five de novo serB alleles that have not been detected in other Strep. thermophilus strains, deposited in a multilocus sequence typing (MLST) database. To assess distinct genotypes of the organism with phenotypic traits relevant to their potential use in industry, Strep. thermophilus strains were all subjected to a series of phenotypic characterizations. The strains were found to exhibit phenotypic diversity in terms of their ability to ferment lactose and galactose, express urease activity, produce exopolysaccharides and develop acidity. Conclusions: The analysis of nucleotide sequence variations within the serB gene could serve as a suitable tool for probing diverse genotypes of Strep. thermophilus. Streptococcus thermophilus isolates associated with traditional Egyptian dairy products show high degree of genetic and phenotypic diversity. Significance and Impact of the Study: This study presents a novel, simplified procedure based on serB nucleotide sequencing for genotyping Strep. thermophilus. It also provides a pool of phenotypically diverse Strep. thermophilus cultures, from which certain strains could be selected for use in bioprocessing applications including the preparation of fermented dairy products.