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PubMed1998

Sequence analysis of the Rhizobium etli ribose kinase gene rbsK and its phylogenetic position.

DNA sequence analysis of a 1878-bp DNA fragment located downstream from the Rhizobium etli ptsN gene revealed the presence of an open reading frame coding for a protein of 300 amino acids. This protein is homologous to members of the PfkB subfamily of carbohydrate and carbohydrate phosphate kinases. Since the highest homology is observed with the ribokinases of Escherichia coli, Haemophilus influenzae and Bacillus subtilis, the isolated gene was named the R. etli rbsK gene. The eubacterial ribokinases form a cluster distinct from the cluster of ribokinase proteins of the archaebacteria Methanobacterium thermoautotrophicum, Methanococcus jannaschii and Sulfolobus solfoataricus, which form a more divergent group of proteins. R. etli RbsK has a molecular mass of 30.6 kDa and a calculated isoelectric point of 4.5. No homologues of Escherichia coli ORF284 and ORF90 were found downstream from R. etli ptsN.

Amino Acid Sequence↗

PubMed2000

Identification and sequence analysis of the core protein genes of bovine adenovirus 2.

The DNA sequence of the genome of bovine adenovirus type 2 (BAdV-2) was determined between map units 42.5 and 50. By sequence analysis and homology search, the genes of five structural proteins were identified within this region: the penton base protein (III; partial sequence), the major core protein precursor (pVII), the minor core protein (V), the mu core protein precursor (pX) and the hexon associated protein precursor (pVI; partial sequence). The putative polypeptides were compared to their known counterparts from other adenoviruses. The existence of protein V and the presence and structure of certain protease cleavage recognition sites confirmed BAdV-2 as a member of the genus Mastadenovirus.

Amino Acid Sequence↗

PubMed1992

Nucleotide sequence analysis of human hypoxanthine phosphoribosyltransferase (HPRT) gene deletions.

We have determined the nucleotide sequences of 10 intragenic human HPRT gene deletion junctions isolated from thioguanine-resistant PSV811 Werner syndrome fibroblasts or from HL60 myeloid leukemia cells. Deletion junctions were located by fine structure blot hybridization mapping and then amplified with flanking oligonucleotide primer pairs for DNA sequence analysis. The junction region sequences from these 10 HPRT mutants contained 13 deletions ranging in size from 57 bp to 19.3 kb. Three DNA inversions of 711, 368, and 20 bp were associated with tandem deletions in two mutants. Each mutant contained the deletion of one or more HPRT exon, thus explaining the thioguanine-resistant cellular phenotype. Deletion junction and donor nucleotide sequence alignments suggest that all of these HPRT gene rearrangements were generated by the nonhomologous recombination of donor DNA duplexes that share little nucleotide sequence identity. This result is surprising, given the potential for homologous recombination between copies of repeated DNA sequences that constitute approximately a third of the human HPRT locus. No difference in deletion structure or complexity was observed between deletions isolated from Werner syndrome or from HL60 mutants. This suggests that the Werner syndrome deletion mutator uses deletion mutagenesis pathway(s) that are similar or identical to those used in other human somatic cells.

Base Sequence↗

PubMed1992

Comparative sequence analysis of the catB gene from Clostridium butyricum.

Sequence analysis of the Clostridium butyricum chloramphenicol acetyltransferase (CAT) gene, catB, showed that it encoded a CAT monomer of 219 amino acids with a molecular weight of 26,114. Comparison of the deduced amino acid sequence of the CATB monomer to those of sixteen other CATs showed that it was most closely related to the CATQ monomer from Clostridium perfringens.

Amino Acid Sequence↗

PubMed2003

Sequence analysis reveals new membrane anchor of reaction centre-bound cytochromes possibly related to PufX.

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Amino Acid Sequence↗

PubMed1974

Deoxynucleotide substitution: a new technique for sequence analysis of RNA.

It is possible to replace in a normal transcription reaction catalyzed by E.coli RNA polymerase one of the four precursors by the corresponding deoxynucleoside triphosphate. These deoxynucleotide-substituted RNA's offer interesting prospects for nucleotide sequence analysis. Indeed by the use of U(2)-RNase with dG-RNA, or pancreatic RNase with dC-RNA or dU-RNA, base specific cleavage can be obtained at any of the four residues. In this way overlap of at least six residues in length can be obtained for any site in the RNA. The technique offers also great benefit for solving the sequence of the more difficult T(1)-oligonucleotides. Some examples in the sequence analysis of SV40 DNA-Hind fragments are reported.

Animals↗

PubMed2003

Sequence analysis of a functional Drosophila centromere.

Centromeres are the site for kinetochore formation and spindle attachment and are embedded in heterochromatin in most eukaryotes. The repeat-rich nature of heterochromatin has hindered obtaining a detailed understanding of the composition and organization of heterochromatic and centromeric DNA sequences. Here, we report the results of extensive sequence analysis of a fully functional centromere present in the Drosophila Dp1187 minichromosome. Approximately 8.4% (31 kb) of the highly repeated satellite DNA (AATAT and TTCTC) was sequenced, representing the largest data set of Drosophila satellite DNA sequence to date. Sequence analysis revealed that the orientation of the arrays is uniform and that individual repeats within the arrays mostly differ by rare, single-base polymorphisms. The entire complex DNA component of this centromere (69.7 kb) was sequenced and assembled. The 39-kb "complex island" Maupiti contains long stretches of a complex A+T rich repeat interspersed with transposon fragments, and most of these elements are organized as direct repeats. Surprisingly, five single, intact transposons are directly inserted at different locations in the AATAT satellite arrays. We find no evidence for centromere-specific sequences within this centromere, providing further evidence for sequence-independent, epigenetic determination of centromere identity and function in higher eukaryotes. Our results also demonstrate that the sequence composition and organization of large regions of centric heterochromatin can be determined, despite the presence of repeated DNA.

Animals↗

PubMed1994

Comparison of the rotavirus nonstructural protein NSP1 (NS53) from different species by sequence analysis and northern blot hybridization.

The nucleotide sequence of gene 5 encoding the rotavirus nonstructural protein NSP1 (NS53) of 6 strains (EW, EHP, RRV, I321, OSU, and Gottfried) was determined and compared to 6 previously reported strains (SA11, UK, RF, Hu803, DS-1, and Wa). The 12 rotavirus strains were derived from a total of five separate species (murine, bovine, simian, porcine, and human). Gene sizes ranged from 1564 to 1611 nucleotides in length and the deduced protein sequences were found to be 486 to 495 amino acids in length. Comparisons of NSP1 amino acid sequences showed identities ranging from 36 to 92%. This diversity was most evident between strains from different species. Phylogenetic analysis revealed a clustering of NSP1 sequences according to species origin with the exception that the human and porcine strains were included in a single grouping. Northern blot hybridizations using additional rotavirus strains from the five species confirmed the grouping found by sequence analysis. The species specificity of NSP1 is consistent with the hypothesis that NSP1 plays a role in host range restriction.

Amino Acid Sequence↗

PubMed1983

An efficient method for the sequence analysis of oligodeoxyribonucleotides.

Modifications of the chemical method of DNA sequence analysis that permit rapid and reliable sequence determination of single-stranded oligodeoxyribonucleotides as short as 4 nucleotides in length are reported. The principal changes made were increasing the level of chemical modification and optimizing the conditions for recovery of the chemically modified oligodeoxyribonucleotides. This method includes two approaches to the removal of [gamma-32P]ATP from 32P-labeled oligodeoxyribonucleotides and is especially useful in the determination of the sequence of chemically synthesized oligodeoxyribonucleotides, which are generally between 4 and 20 nucleotides in length.

Animals↗

PubMed2004

Rapid genome evolution revealed by comparative sequence analysis of orthologous regions from four triticeae genomes.

Bread wheat (Triticum aestivum) is an allohexaploid species, consisting of three subgenomes (A, B, and D). To study the molecular evolution of these closely related genomes, we compared the sequence of a 307-kb physical contig covering the high molecular weight (HMW)-glutenin locus from the A genome of durum wheat (Triticum turgidum, AABB) with the orthologous regions from the B genome of the same wheat and the D genome of the diploid wheat Aegilops tauschii (Anderson et al., 2003; Kong et al., 2004). Although gene colinearity appears to be retained, four out of six genes including the two paralogous HMW-glutenin genes are disrupted in the orthologous region of the A genome. Mechanisms involved in gene disruption in the A genome include retroelement insertions, sequence deletions, and mutations causing in-frame stop codons in the coding sequences. Comparative sequence analysis also revealed that sequences in the colinear intergenic regions of these different genomes were generally not conserved. The rapid genome evolution in these regions is attributable mainly to the large number of retrotransposon insertions that occurred after the divergence of the three wheat genomes. Our comparative studies indicate that the B genome diverged prior to the separation of the A and D genomes. Furthermore, sequence comparison of two distinct types of allelic variations at the HMW-glutenin loci in the A genomes of different hexaploid wheat cultivars with the A genome locus of durum wheat indicates that hexaploid wheat may have more than one tetraploid ancestor.

Amino Acid Sequence↗

PubMed1996

Sequence analysis and characterization of stutter products at the tetranucleotide repeat locus vWA.

The PCR amplification of tetranucleotide short tandem repeat (STR) loci typically produces a minor product band 4 bp shorter than the corresponding main allele band; this is referred to as the stutter band. Sequence analysis of the main and stutter bands for two sample alleles of the STR locus vWA reveals that the stutter band lacks one repeat unit relative to the main allele. Sequencing results also indicate that the number and location of the different 4 bp repeat units vary between samples containing a typical verses low proportion of stutter product. The results also suggest that the proportion of stutter product relative to the main allele increases as the number of uninterrupted core repeat units increases. The sequence analysis and results obtained using various DNA polymerases appear to support the slipped strand displacement model as a potential explanation for how these stutter products are generated.

Alleles↗

PubMed2002

Rapid assignment of nucleotide sequence data to allele types for multi-locus sequence analysis (MLSA) of bacteria using an adapted database and modified alignment program.

A novel database and modified alignment program is described which provides a fast and accurate procedure for assigning nucleotide sequences to allele types for multi-locus sequence analysis (MLSA). The database has between 40 and 160 alleles per organism including Neisseria meningitidis, Streptococcus pneumoniae, Staphylococcus aureus and Haemophilus influenzae. The database directly compares the query nucleotide sequence against all alleles within the database and this system reduces the time taken for the analysis of nucleotide sequence data and assignment of alleles for subsequent sequence analysis.

Alleles↗

PubMed1985

A putative Ca2+-binding protein: structure of the light subunit of porcine calpain elucidated by molecular cloning and protein sequence analysis.

cDNA clones specific for the light subunit of porcine calpain I have been isolated from a porcine kidney cDNA library. The complete primary structure of the light subunit has been revealed by nucleotide sequence analysis of the cDNA clones isolated and amino acid sequence analysis of peptides isolated from the purified mature protein. We found that the light subunit contains two distinct domains. Domain I, the amino-terminal half, has two unusually long, paired polyglycyl sequences and may serve as a binding site to the heavy subunit. Domain II, the carboxyl-terminal half, is a region highly homologous to the putative Ca2+-binding domain of the heavy subunit of chicken calpain elucidated recently. This region has four potential Ca2+-binding sites, each having the "E-F hand" structure. Our results suggest that the Ca2+-mediated proteolytic activity of calpain is controlled through the cooperative and/or sequential actions of multiple Ca2+-binding sites present in both two-subunit molecules, heavy and light subunits of calpain.

Amino Acid Sequence↗

PubMed1996

Biological testing versus sequence analysis for determination of drug resistance of human immunodeficiency virus.

Resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) is associated with one or more of five mutations in the reverse transcriptase (RT) segment of the polymerase gene ("AZT-specific mutations"). Therefore, sequence analysis of the proviral DNA, derived directly from the blood, is considered to replace the biological test. Additional arguments are non-cultivatable viral strains, the universality of the sequence analysis in combination therapy, and the suspicion that the cultivated virus does not represent the predominant viral variant in the blood. In this investigation, 21 strains of human immunodeficiency virus type 1 (HIV-1) were comparatively analysed by molecular and biological testing. For 12 strains, the homology of the RT gene segment between the predominant provirus in the blood and the cultivated virus was ascertained (99.72% homology). 11 strains from untreated patients or patients treated no longer than 5 months were free from AZT-specific mutations and proved to be sensitive. 10 strains from patients treated for 17 to 57 months displayed 2-4 AZT-specific mutations. However, it was not possible to correlate the degree of sensitivity to the number or the pattern of the mutations. Suppression of AZT resistance by strain-specific sequences in other parts of the gene are discussed as the reason for that discrepancy. Remarkably, the productivity of resistant virus strains could be drastically enhanced by non-inhibiting concentrations of the drug.

DNA, Viral↗

PubMed2004

Description of 'Candidatus Helicobacter heilmannii' based on DNA sequence analysis of 16S rRNA and urease genes.

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as 'Helicobacter heilmannii' or 'Gastrospirillum hominis'. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with 'Candidatus Helicobacter suis', thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name 'Candidatus Helicobacter heilmannii' is proposed.

Adult↗

PubMed1991

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster.

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. Only one of the X-ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X-ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and "point mutations." Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the break-point with one of the repeats deleted.

Alcohol Dehydrogenase↗

PubMed2004

Specific detection and typing of Mycoplasma synoviae strains in poultry with PCR and DNA sequence analysis targeting the hemagglutinin encoding gene vlhA.

Mycoplasma synoviae is a major pathogen of chickens and turkeys, causing economic losses to the poultry industry worldwide. In this study, we validated and applied polymerase chain reaction (PCR) and DNA sequence analysis on the N-terminal end of the hemagglutinin encoding gene vlhA as an alternative for the detection and initial typing of field strains of M. synoviae in commercial poultry. PCR primers were tested against isolates of M. synoviae from various sources along with other avian mycoplasma and other bacterial species. The vlhA gene-targeted PCR assay was highly specific in the identification of M. synoviae, with a detection limit of 4.7 x 10(2) color changing units/ml. DNA sequence analysis of amplified products was also conducted to validate the potential for typing M. synoviae strains using the N-terminal region of the vlhA gene. To evaluate the test, we applied the PCR assay to tracheal swabs collected from chickens challenged with M. synoviae strain K1968 and compared the results to the serologic detection. The PCR assay was also evaluated directly on tracheal samples collected from commercial layers. Overall, this vlhA gene-targeted PCR is a useful tool for detection and initial typing of M. synoviae and can be applied in the preliminary identification of M. synoviae isolates directly from clinical samples.

Agglutination Tests↗

PubMed1986

Covalently linked sequencing primer linkers (splinkers) for sequence analysis of restriction fragments.

A new method for direct sequence analysis of DNA restriction fragments uses synthetic covalently linked complementary oligodeoxynucleotides, as universal sequencing primer linkers (splinkers). These splinkers were designed to contain an inverted repeat sequence which forms a double-stranded hairpin structure with a known restriction site. The splinkers were characterized by their ability to be self-ligating (dimerized) and by their restriction digest product analysis of both the monomer and dimer. They can also be ligated to dephosphorylated DNA restriction fragments which contain the appropriate end. This was evidenced by mobility shifts of the splinker-ligated restriction fragments. The splinker-ligated restriction fragments, after denaturation, form a single-stranded DNA template containing an inverted repeat sequence (from splinker) at one terminus. The splinker is thus suitably oriented and serves as a primer for dideoxy nucleotide (nt) sequencing catalyzed by either Klenow fragment of Escherichia coli DNA polymerase I or avian myeloblastosis virus reverse transcriptase. As demonstrated for both pBR322 and phi X174, release of the primer extension strand by digestion at the splinker restriction site results in a ladder of labelled fragments which corresponds to a unique nt sequence.

Base Composition↗