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PubMed1998

cDNA sequence analysis, chromosomal assignment and expression pattern of the gene coding for integral membrane protein 2B.

The complete cDNA of the mouse integral membrane protein 2B gene (Itm2b) was determined by sequence analysis of expressed sequence tag (EST) clone L26775 and a clone isolated from a cDNA library of the osteogenic stromal cell line MN7 (Mathieu et al., 1992. Calcif. Tissue Int. 50, 362-371) and by 5' rapid amplification of cDNA ends (RACE). Alignment of different mouse ESTs confirmed the entire sequence. Northern blot analysis of different neonatal and adult mouse tissues showed that Itm2b is ubiquitously expressed. There are three mRNAs with different lengths in neonatal as well as in adult tissues, originating from alternative polyadenylation by usage of one consensus and two additional variant polyadenylation signals. The cDNA sequence of the human Itm2b homolog (ITM2B) was assembled using data from available human ESTs. Both the mouse and the human gene code for a protein of 266 amino acids (aa) that is homologous to a previously described integral membrane protein, Itm2A, of which the expression is restricted to osteo- and chondrogenic tissues. Itm2A and Itm2B belong to a family of type II integral membrane proteins, which contains a third member, Itm2C (Deleersnijder et al., 1996. J. Biol. Chem. 271, 19475-19482). The human ITM2B and mouse Itm2b genes were previously mapped as unknown ESTs to conserved syntenic regions Homo sapiens 13q12-13 and Mus musculus 14.

Adaptor Proteins, Signal Transducing↗

PubMed1994

Nucleotide sequence analysis of two 5-nitroimidazole resistance determinants from Bacteroides strains and of a new insertion sequence upstream of the two genes.

DNA sequence analysis of regions from plasmid pIP417 and chromosome BF8 which encode 5-nitroimidazole resistance in Bacteroides strains allowed the identification of two open reading frames corresponding to new genes, nimA (528 bp) and nimB (492 bp). Either gene may confer 5-nitroimidazole resistance to susceptible strains of Bacteroides. The encoded polypeptides have deduced molecular masses of 20.1 and 18.6 kDa, respectively, and share about 73% identity and 85% similarity. A new insertion sequence (IS) element named IS1168 lies 14 bases upstream of the nimA gene. The complete sequence of IS1168 was determined. A similar IS exists 12 bp upstream of the nimB gene. About 60% of the BF8 IS element was also sequenced and shown to be almost identical to IS1168.

Bacteroides↗

PubMed1994

Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin variants, including two PIQ0 alleles and one deficient PIM allele.

We have investigated the molecular basis of 15 new alpha 1-antitrypsin (alpha 1AT) variants. Phenotyping by isoelectric focusing (IEF) was used as a screening method to detect alpha 1AT variants at the protein level. Genotyping was then performed by sequence analysis of all coding exons, exon-intron junctions, and the hepatocyte-specific promoter region including exon Ic. Three of these rare variants are alleles of clinical relevance, associated with undetectable or very low serum levels of alpha 1AT:the PI*Q0saarbruecken allele generated by a 1-bp C-nucleotide insertion within a stretch of seven cytosines spanning residues 360-362, resulting in a 3' frameshift and the acquisition of a stop codon at residue 376; a point mutation in the PI*Q0lisbon allele, resulting in a single amino acid substitution Thr68(ACC)-->Ile(ATC); and an in-frame trinucleotide deletion delta Phe51 (TTC) in the highly deficient PI*Mpalermo allele. The remaining 12 alleles are associated with normal alpha 1AT serum levels and are characterized by point mutations causing single amino acid substitutions in all but one case. This exception is a silent mutation, which does not affect the amino acid sequence. The limitation of IEF compared with DNA sequence analysis, for identification of new variants, their generation by mutagenesis, and the clinical relevance of the three deficiency alleles are discussed.

Alleles↗

PubMed1986

Development of a superior strategy for computer-assisted nucleotide sequence analysis.

A new strategy for high-resolution nucleotide sequence analysis has been developed. The strategy involves an exhaustive tree-searching algorithm which examines all possible combinations of short regions of sequence alignments, followed by culling of unsuitable sequence relationships. The new algorithm can detect sequence homologies invisible to existing algorithms, and is capable of detecting all possible sequence relationships.

Base Sequence↗

PubMed1994

Molecular cloning and sequence analysis of the cDNA encoding porcine acrosin inhibitor.

A full-length cDNA encoding the porcine acrosin inhibitor has been isolated from a boar seminal vesicle cDNA library. Nucleotide sequence analysis of the 667-bp cDNA predicts a precursor protein of 97 amino acid residues, which includes a 26-residue signal peptide and a 71-residue secreted protein. The predicted amino acid sequence of the mature protein agrees completely with that of the sperm-associated acrosin inhibitor determined by conventional amino acid sequence analysis. However, the asparagine/aspartic acid and glutamine/glutamic acid substitutions, as reported in the seminal plasma counterpart, have not been observed. Southern blot analysis shows only a single hybridizing band with three different restriction endonucleases, suggesting the presence of a single copy of the acrosin inhibitor gene in the porcine genome.

Acrosin↗

PubMed1997

Sequence analysis of the nucleocapsid gene of measles virus isolates from South Africa identifies a new genotype.

Sequence analysis was performed on 20 measles virus (MV) isolates from South Africa, five of which were obtained between 1986 and 1989 and 15 isolates collected during the 1994/95 measles season. A 590 bp fragment of the carboxyl terminus of the nucleocapsid (N) was amplified by PCR and subjected to sequence and phylogenetic analysis. Comparison of the South African MV strains with those previously described revealed that at least two distinct groups of wild-type (wt) MV exist, one of which has been circulating since 1986. The major genotype (I) was represented by the more recent isolates which showed three characteristic amino acid substitutions. Furthermore, three vaccine-like viruses with sequences very similar to the Edmonston wt strain were identified. Phylogenetic analysis of 100 MV strains allowed the assignment of new definitions for MV genotypes and subgroups. Employing these definitions, the majority of South African isolates analysed here formed a new genotype.

Amino Acid Sequence↗

PubMed2001

Relationships within the Proteobacteria of plant pathogenic Acidovorax species and subspecies, Burkholderia species, and Herbaspirillum rubrisubalbicans by sequence analysis of 16S rDNA, numerical analysis and determinative tests.

Sequence data for 16S rDNA of the type strains of Acidovorax avenae subsp. avenae, A. avenae subsp. cattleyae, A. avenae subsp. citrulli, A. konjaci and Herbaspirillum rubrisubalbicans were compared with GenBank library accessions of Burkholderia spp., Comamonas sp., Ralstonia solanacearum and Variovorax sp. Maximum Parsimony analysis produced two clusters: 1. Acidovorax spp., Comamonas sp., and Variovorax sp. (all in the Comamonadaceae), and 2. Burkholderia spp., Ralstonia solanacearum, and Herbaspirillum rubrisubalbicans. Maximum Likelihood analysis produced only one cluster (of the Comamonadaceae). Using nutritional and laboratory tests, all Acidovorax spp., Burkholderia spp., and Herbaspirillum rubrisubalbicans were discriminated in distinct clusters at the species level, and could be identified by selected determinative tests. There were no phenotypic tests constituted as a circumscription of the genera and which permitted the allocation of strains to genera. Strain identification as species allowed allocation to genera only by inference. The nomenclatural implications of these data are discussed.

Base Sequence↗

PubMed2001

Genomic sequence analysis tools: a user's guide.

The wealth of information from various genome sequencing projects provides the biologist with a new perspective from which to analyze, and design experiments with, mammalian systems. The complexity of the information, however, requires new software tools, and numerous such tools are now available. Which type and which specific system is most effective depends, in part, upon how much sequence is to be analyzed and with what level of experimental support. Here we survey a number of mammalian genomic sequence analysis systems with respect to the data they provide and the ease of their use. The hope is to aid the experimental biologist in choosing the most appropriate tool for their analyses.

Internet↗

PubMed1995

Characterization of relevant membrane antigens by two-dimensional immunoblot and n-terminal sequence analysis in patients with myocarditis.

In myocarditis an antigen-specific immune response to cardiac epitopes has been demonstrated by several investigators. In 25 patients with histologically proven myocarditis, autoantibodies to cardiac tissue were observed in 65% by immunofluorescence assay. By immunoblot analysis 46% of sera reacted with cardiac tissue. These antibodies were preferentially directed against proteins with molecular weights of 43 and 67 kDa. Many of these antibodies have been demonstrated to be cytolytic to isolated cardiocytes in vitro. However, their corresponding antigens on heart muscle cells still remains unclear. To characterize the epitopes recognized by the autoantibodies, isoelectric focusing followed by SDS-PAGE was used to separate the complex mixture of proteins from human heart. Immunoblot analysis of antigens revealed proteins in the range of 30-67 kDa at isoelectric points of 6.5-8.5 to be of particular interest. Five of these proteins were identified by immunoblot analysis and two were identified by n-terminal sequence analysis (Edman Degradation) as creatine kinase and a sarcomeric isoenzyme. As creatine kinase is an intracellularly located enzyme, the pathogenetic role of the autoantibodies directed against it remains to be elucidated further.

Adult↗

PubMed1998

The V(D)J recombination activating protein RAG2 consists of a six-bladed propeller and a PHD fingerlike domain, as revealed by sequence analysis.

The RAG1 and RAG2 proteins play a crucial role in V(D)J recombination by cooperating to make specific double-stranded DNA breaks at a pair of recombination signal sequences (RSSs). However, the exact function they perform has heretofore remained elusive. Using a combination of sensitive methods of sequence analysis, we show here that the active core region of the RAG2 protein, confined to the first three quarters of its sequence, is in fact composed of a six-fold repeat of a 50-residue motif which is related to the kelch/mipp motif. This motif, which forms a four-stranded twisted antiparallel beta sheet, is arranged in a circular formation like blades of a propeller or turbine. Given the known properties of the beta-propeller fold in mediating protein-protein interactions, it is proposed that this six-laded propeller structure of the RAG2 active core would play a crucial role in the tight complex formed by the RAG1 and RAG2 proteins and RSSs. Moreover, the presence of a plant homeodomain finger-like motif in the last quarter of the RAG2 sequence suggests a potential interaction of this domain with chromatin components.

Amino Acid Sequence↗

PubMed2000

Sequence analysis of measles virus hemagglutinin isolated in Argentina during the 1997-1998 outbreak.

Sequence analysis was performed on 50 measles viruses (MV) isolated in Argentina. Forty-six were obtained during the current outbreak (1997-1998), three from the previous outbreak (1991) and one sporadic case (1994). A 377-bp fragment of the hemagglutinin (H) gene was directly amplified by RT-PCR from nasopharyngeal secretions. Nucleotides 8152 to 8417 were sequenced and subjected to phylogenetic analysis. Multiple silent changes and point mutations were found in all MVs. In 1991, substitutions affected the third base in codons resulting in silent changes. In 1994 an A-->C substitution at position 8321 changed amino acids 351 (Leu-->Ile). In 1997-1998, an A-->G substitution at position 8339 changed amino acids 357 (Val-->Ile). In 3/46 viruses, guanine deletion at position 8205 changed the reading frame and insertion of an extra cytosine at nucleotide 8235 shifted it back to the original frame. Phylogenetic analysis revealed that viruses leading to the last two major outbreaks are clustered into two separate branches. MVs that prevailed until 1994 were related to genotype C1 and MVs of the current outbreak to D6. Random drift mutations rendered a 0.5 ratio of nonsilent over silent mutations in most of the MVs analyzed. However, in those showing a reading frame shift, the ratio was greater than 1, suggesting that it was driven by immune selection.

Amino Acid Sequence↗

PubMed1999

Comparison of line probe assay (LIPA) and sequence analysis for detection of HIV-1 drug resistance.

The identification of HIV strains that are resistant to antiretroviral drugs, which emerge during a patient's therapy or are already present in infected individuals prior to treatment, is of increasing importance for the clinical management of HIV-infected persons. Two different methods were compared for the genotypic detection of resistance development in the HIV-1 reverse transcriptase (RT) gene, the commonly used sequence analysis, and the commercially available RT-line immunoprobe assay (LIPA), which can detect mutations at six separate codons of the RT gene, which are known to confer resistance to certain nucleoside inhibitors. Eighty serum samples from HIV-1-infected persons, some of whom were receiving antiretroviral therapy, were investigated in parallel by sequencing as well as by LIPA. LIPA results agreed with sequence data in the vast majority of the cases. However, in 40% of the samples, LIPA failed to yield evaluable results for one or more of the codon positions. In particular, LIPA detection rate was low at codon 41 (75%), whereas at codons 69/70, 74, 184, and 215 results were obtained from 90%-95% of the samples. A number of mutations in the vicinity of the respective codons were detected by sequencing, and these may have been responsible for the LIPA hybridization failure. There remained a number of samples, however, where no explanation for the lack of hybridization could be derived from sequence data. Our results indicate that the use of the LIPA does not eliminate the need for sequence analysis for detection of drug-resistant HIV strains.

Adult↗

PubMed1991

Sequence analysis of a new hepatitis A virus naturally infecting cynomolgus macaques (Macaca fascicularis).

A new isolate of hepatitis A virus (HAV), CY-145, was isolated from stool specimens obtained from cynomolgus macaques naturally infected with this agent. Sequence analysis of the capsid region of the genome indicated that this virus differed from other sequenced HAV strains by about 20% at the nucleotide level and 7% at the amino acid level. Two amino acid residues (residues 70 of VP3 and 102 of VP1), previously identified as constituting an immunodominant site and conserved in all sequenced HAVs, were changed in the CY-145 virus. Sequence analysis of a second cynomolgus HAV isolate (CY-55), which came from a different geographical location, showed the same amino acid replacement at these two sites. In addition both isolates had an amino acid substitution at the VP3-VP1 cleavage site. These data suggest that the cynomolgus HAV differs genetically and antigenically from all other sequenced HAVs.

Amino Acid Sequence↗

PubMed2002

Characterization of transthyretin variants in familial transthyretin amyloidosis by mass spectrometric peptide mapping and DNA sequence analysis.

Transthyretin (TTR) is a 127-amino acid residue transport protein. In plasma, TTR exists as a tetramer and binds the hormone thyroxine and the retinol-binding protein-vitamin A complex. Amino acid substitutions in TTR are hypothesized to destabilize the tetramer and cause the protein to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with extracellular deposition of wild-type TTR, its variants or fragments as amyloid fibrils in various tissues and organs. A definitive diagnosis of ATTR depends on the detection and identification of TTR variants. Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS), in combination with trypsin digestion, have been shown to be powerful tools in characterizing TTR variants. Typically, TTR or its tryptic digest is analyzed by MALDI-TOF MS, liquid chromatography ESI MS, or both. Analysis of tryptic digests by MALDI-TOF MS does not provide enough sequence coverage in TTR to identify all possible modifications. To improve sequence coverage, aliquots of immunoprecipitated TTR samples were digested with trypsin, lysyl endopeptidase Lys-C, or endoproteinase Asp-N. Identification of the peptides from each digest by MALDI-TOF MS provided preliminary information about the sites and mass shifts due to amino acid substitutions from genetic mutations and to posttranslational modifications. The location and identity of the modifications in the variant proteins were then confirmed by tandem mass spectrometry, accurate mass measurements, and direct DNA sequence analysis. Using these methodologies, we achieved 100% sequence coverage. The detection of two nonpathologic variants (Thr119Met and Gly6Ser) and four pathologic variants (Phe64Leu, Asp38Ala, Phe44Ser, and previously unreported Trp41Leu) are described as illustrations of this approach.

Adult↗

PubMed2002

[Cloning and sequence analysis of a novel TT virus varian].

OBJECTIVE: To clone the DNA of a TT virus (TTV) variant isolated from a patient with elevated alanine transaminase (ALT) of unknown etiology, and conduct sequence analysis. METHODS: The long fragment of TTV DNA was amplified by nested PCR and then cloned into pGEM-T vector. A clone named 56-B containing 3.2 kb TTV DNA was selected for sequence analysis besides homology analysis with other 5 TTV variants retrieved from GenBank, and phylogenetic analysis was carried out by maximum likelihood method. RESULTS: The nucleotide identities of 56-B with the other 5 TTV strains TA278, JA10, US35, SANBAN and TUS01 were 42.4%, 48.2%, 47.9%, 49.8 % and 61.7 % respectively, and the corresponding amino acid identities were even lower. Phylogenetic analysis showed that 56-B was far from other TTV strains in genetic distance that ranged from 0.344 to 0.458. However, the sequences in the 5'- and 3'-end were still much conservative. CONCLUSION: The isolated 56-B showed high heterogeneity in genetic background and was therefore quite distinct from other TTV strains as a novel TTV variant that represents a new TTV genotype.

Cloning, Molecular↗

PubMed2001

Nucleotide sequence analysis of pRS2 and pRS3, two small cryptic plasmids from Oenococcus oeni.

Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2.

Base Sequence↗

PubMed1987

Alterations in the phenotype of plant cells studied by NH(2)-terminal amino acid-sequence analysis of proteins electroblotted from two-dimensional gel-separated total extracts.

Phenotypic alterations induced by the cytokinin 6-benzylaminopurine in cell suspensions of Nicotiana plumbaginifolia were studied at the level of the NH(2)-terminal sequence of the constituent proteins. Total protein extracts were separated by classical two-dimensional PAGE, and the proteins were recovered by electroblotting onto support materials allowing direct gas-phase sequence analysis of the immobilized proteins. The systems used consist of an efficient electrotransfer buffer (50 mM Tris borate, pH 8.3) in combination with either glass-fiber sheets to which poly(4-vinyl-N-methylpyridinium iodide) is adsorbed or with membranes of polyvinylidene difluoride. The former is an improved version of our previously reported Polybrene-coated glass-fiber sheets and was found to be at least twice as efficient as the polyvinylidene difluoride blots. Thirteen proteins were selected for analysis. They were either induced, repressed, or independent of cytokinin. Ten proteins yielded a sequence, ranging from 10 to 38 residues. Three of the studied Nicotiana proteins show a degree of homology higher than 85% with the amino acid sequences of other eukaryotic proteins-triose-phosphate isomerase, Mn superoxide dismutase, and (1,3)-beta-glucanase. The latter enzyme was repressed by the plant hormone. This study demonstrates that proteins associated with phenotypic variations in cells can now be sequenced by a straightforward procedure involving two-dimensional gel separation of total cellular proteins, recovery by electroblotting, and gas-phase sequence analysis of the immobilized proteins.

Journal Article↗

PubMed2004

Comparative sequence analysis of a gene-dense region among closely related species of Drosophila melanogaster.

Comparative sequence analysis among closely related species is essential for investigating the evolution of non-coding sequences, which evolve more rapidly than protein-coding sequences. We sequenced the cytogenetic map 56F10-16, a gene-dense region of D. simulans and D. sechellia, closely related species to D. melanogaster. About 57 kb of the genomic sequences containing 19 genes were annotated from each species according to the corresponding region of the D. melanogaster genome. The order and orientation of genes were perfectly conserved among the three species, and no transposable elements were found. The rate of nucleotide substitutions in the non-coding sequences was lower than that at the fourfold-degenerate sites, implying functional constraints in the non-coding regions. The sequence information from three closely related species, allowed us to estimate the insertions and the deletions that may have occurred in the lineages of D. simulans and D. sechellia using the D. melanogaster sequence as an outgroup. The number of deletions was twice that of insertions for the introns of D. simulans. More remarkably, the deletion outnumbered insertions by 7.5 times for the intergenic sequences of D. sechellia. These results suggest that the non-coding sequences have been shortened by deletion biases. However, the deletion bias was lower than that previously estimated for pseudogenes, suggesting that the non-coding sequences are already rich in functional elements, possibly involved in the regulation of gene expression including transcription and pre-mRNA processing. These features of non-coding sequences may be common to other gene-dense regions contributing to the compactness of the Drosophila genome.

Animals↗