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PubMed2006

[Whole sequence analysis of brain-derived neurotrophic factor gene in Asiatic Black Bear through faecal sampling].

Using our lab's improved protocol for faecal DNA extraction, the entire 753-bp DNA coding sequence of the nuclear brain-derived neurotrophic factor (BDNF) gene was cloned for the first time from Asiatic Black Bear Selenarctos thibetanus faecal samples with primers based on the reported sequence of the Malayan Bear BDNF gene. Hair was used as a positive control and the experiments were repeated several times to obtain reliable and identical results. Sequence analysis showed that the BDNF gene of Asiatic Black Bear was highly conserved compared to those of human and giant panda, with an identity of 94.5% and 98.9%, respectively. The deduced amino acid sequence of the mature protein was found to be identical to those of all the reported mammalians. According to gene sequence alignment, the giant panda appeared to be phylogenetically closer to Asiatic Black Bear than the lesser panda. This study represents the first time that a non-invasive method such as faecal sampling was used to analyze a functional nuclear BDNF gene of Asiatic Black Bear. It will not only provide important reference for the conservation and breeding of Asiatic Black Bear and open up new avenues of non-invasive sampling in the study of endangered wildlife, but also provide another molecular evidence for the study of relationship of Asiatic Black Bear and its related species.

Amino Acid Sequence↗

PubMed2005

Study of the occurrence of Vibrio vulnificus in oysters in India by polymerase chain reaction (PCR) and heterogeneity among V. vulnificus by randomly amplified polymorphic DNA PCR and gyrB sequence analysis.

The pathogenic bacterium Vibrio vulnificus is widely distributed in estuarine waters throughout the world. In this study, the presence of V. vulnificus in oysters was studied both by conventional culture and DNA-based molecular technique. Following enrichment in alkaline peptone water (APW), the bacteria were lysed and a nested polymerase chain reaction (PCR) for vvhA gene was performed. The effect of duration of enrichment on the sensitivity of detection by PCR was evaluated. The organism was isolated from 43% of samples after 18 h enrichment in APW by conventional culture method. Nested PCR amplifying a fragment of vvhA gene detected the organism in 11%, 60% and 81% of samples following 0, 6 and 18 h of enrichment. All the biochemically identified V. vulnificus strains possessed vvhA gene and belonged to biotype 1. The genetic relatedness among the strains was studied by randomly amplified polymorphic DNA (RAPD) PCR and gyrB sequence analysis. The results suggest the presence of two distinct clonal groups of V. vulnificus in oysters in India. The study demonstrates, for the first time that gyrB sequence analysis could be used to study the genetic diversity of V. vulnificus.

Amino Acid Sequence↗

PubMed2003

[Identification and molecular cloning and sequence analysis of a novel coronavirus from patients with SARS by RT-PCR].

OBJECTIVE: To investigate the etiologic agents of the SARS and develop diagnostic method for this disease. METHODS: Thirty-six nasopharyngeal aspirate specimens from 27 patients with SARS in Shenzhen were collected. The samples were aliquotted to three parts and subjected to molecular assays for human metapneumovirus, chlamydia and a novel coronavirus, which was reported recently to be the etiologic agent of SARS. Nested RT-PCR was used to amplify the RNA polymerase gene of the novel coronavirus and the PCR products were sequenced directly or after cloned to pMD18-T vector. RESULTS: Human metapneumovirus and chlamydia genes were detected in none of the specimens using the RT-PCR and nested-PCR, respectively. The novel coronavirus gene were amplified in 6 of 36 specimens, the sequence analysis indicated that this novel coronavirus is unrelated to any other coronavirus reported previously. The nucleotide and deduced amino acid alignment between this coronavirus and others was not more than 40% and 70% to 82%, respectively, while the nucleotide sequence cloned from the 6 patients were identical. CONCLUSIONS: The SARS patients in Shenzhen were infected with coronavirus and this novel coronavirus is associated with SARS. The sequence analysis indicated that the coronavirus from SARS patients in Shenzhen is the same as that identified from other areas such as Canada and Hong Kong. A specific diagnostic nested RT-PCR was developed to identify this novel coronavirus infection.

Adolescent↗

PubMed1983

Molecular cloning, sequence analysis and in vitro expression of a rat tRNA gene cluster.

A rat genomic DNA fragment containing a tRNA gene cluster was isolated from a lambda phage library. Hybridization and nucleotide sequence analysis revealed the presence of a 83 bp tRNALeuCUG gene and a 72 bp tRNAAspGUG gene. Both genes possessed intact coding regions and putative transcription termination signals at their respective 3' ends. In vitro transcription analysis of the two subcloned genes in a HeLa cell S-100 system demonstrated the specific synthesis of a number of RNAs by RNA polymerase III. Studies carried out in the presence of alpha-amanitin showed that the larger RNAs are precursors for the final processed transcripts of the tRNALeu and tRNAAsp genes, respectively. Further nucleotide sequence analysis of the cluster revealed the presence of tRNAGly and a tRNAGlu pseudogenes with missing areas within their coding regions which are essential for transcription by RNA polymerase III. Within the region of DNA between the tRNALeu and tRNAAsp genes is a sequence which is 65% homologous to a region of the rat B1 element. The significance of this latter structure within the gene cluster is unknown.

Animals↗

PubMed2003

[Cloning and sequence analysis of active fragments of human thrombospondin-1].

OBJECTIVE: To lay a foundation for researches on the expression of gene engineering anti-tumor drugs. METHODS: On the basis of mRNA sequence of human thrombospondin-1(TSP-1), two pairs of primers were designed and synthesized. Two active fragments TSP-1, called TSP-1-I and TSP-1-III, were obtained by using reverse transcription-polymerase chain reaction(RT-PCR). The sizes of those two fragments were found to be 728 bp and 257 bp respectively. They were purified and then cloned into pMDT vector after transformation into Escherichia coli with recombined DNAs. RESULTS: Plasmid DNAs were isolated from transformants and digested with restriction enzyme. The results from agarose gel electrophorasis showed that these two fragments had been inserted into vector and their sizes were in accord with the expected values. Sequence analysis demonstrated that TSP-1-I is 728 bp in size and encodes a peptide containing 212 amino acid residues with relative molecular mass of 23.6 x 10(3) and the TSP-I III is 257 bp in size and encodes a peptide containing 86 amino acid residues with relative molecular mass of 9.5 x 10(3). CONCLUSION: The results from homology analysis indicate that the sequence of these two fragments is identified with that of TSP-1 of Genbank.

Adenocarcinoma↗

PubMed2006

Three type 6 hepatitis C virus subgroups among blood donors in the Yangon area of Myanmar are identified as subtypes 6m and 6n, and a novel subtype by sequence analysis of the core region.

Previously, using phylogenetic analysis of NS5b sequences, we found that three type 6 variant subgroups (M6-1, M6-2 and M6-3) exist in Myanmar. According to the new nomenclature of hepatitis C, M6-1 and M6-2 belong to subtypes 6m and 6n, respectively, but M6-3 is unassigned. In this study, we sequenced and phylogenetically analyzed the core region of these type 6 variant subgroups. Serum samples assigned as 6m or 6n by NS5b sequence were also identified as 6 m or 6n by core region analysis. The M6-3 (sample name MYAN-3E-3) remained unassigned to a subgroup based on its core region analysis. The findings of this study suggest that either the core region or the NS5b region can be analyzed for HCV subtype classification.

Base Sequence↗

PubMed1995

Sequence analysis of a 78.6 kb segment of the left end of Saccharomyces cerevisiae chromosome II.

We report the sequence analysis of a 78,601 bp DNA segment on the left arm of chromosome II of Saccharomyces cerevisiae. This 78.6 kb segment spans the region from the start of a subtelomeric Y' element up to the ILS1 gene. It contains 49 open reading frames (ORFs) with more than 100 amino acids length including 14 internal and five overlapping ORFs. The gene density, excluding the internal ORFs, was calculated as one ORF per 2.2 kb. Eight ORFs (PKC1, TyA, TyB, ATP1, ROX3, RPL17a, PET112 and ILS1) correspond to previously characterized genes. ORF YBL0718 was identified as CDC27; YBL0706 as TEL1. Four other ORFs show strong similarities to already known genes. The gene product of YBL0838 is 60% identical to the ribosomal protein RPL32 from rat, mouse and man. YBL0701 encodes a protein with significant similarity to the initiation factor eIF2 associated p67 glycoprotein from rat. Eight ORFs were disrupted and the resulting yeast strains analysed with respect to their phenotype.

Amino Acid Sequence↗

PubMed1987

Protein sequence analysis studies on the low molecular weight hydrophobic proteins associated with bovine pulmonary surfactant.

Lipid extracts of bovine pulmonary surfactant, which exhibit biophysical and biological activity, contain two hydrophobic proteins which have been designated surfactant protein-B (SP-B) and SP-C. Amino terminal amino acid sequence analysis of whole lipid extracts and partially purified protein fractions gave rise to three sequences, two major and one minor. The first sequence, identified as a member of the SP-B family, extended for 60 amino acids beginning with an amino terminal phe. The second polypeptide, identified as a member of the SP-C family, sequenced for 35 amino acids and had a leu amino terminus. The third minor sequence corresponded to amino acids 2-9 of SP-C (N-leu) and was designated SP-C (N-ile). Sequence analysis of cyanogen bromide peptides derived from methyl isocyanate-blocked lipid extract material produced two peptides which extended the amino acid sequence of SP-B to residue 79, which appears to be a glycine.

Amino Acid Sequence↗

PubMed1993

The effects of hypoxia and cysteamine on X-ray mutagenesis in human cells. II. hprt mRNA expression and cDNA sequence analysis of induced mutants.

Mutants at the hprt locus isolated after treatment of cells of the human lymphoblastoid cell line TK6 with X rays were examined by Northern blot and cDNA sequence analysis. In previous work, Southern blot analysis showed that approximately 25% of the mutants isolated from cultures treated with X rays displayed restriction fragment patterns indistinguishable from wild type. In addition, 38 and 48% of the mutants isolated from cultures treated with X rays under two radioprotective conditions, hypoxia or 25 mM cysteamine, respectively, had normal restriction fragment patterns. In the work presented here, Northern blot and DNA sequence analyses were used to characterize these mutants further. Mutants were classified as having normal size and amount of hprt mRNA, reduced amount or undetectable mRNA, or abnormal size message. Mutants that expressed hprt mRNA were sequenced after reverse transcription and PCR amplification. Sequence analysis is reported for 7 mutants from cultures treated in the absence of protection, 11 from cultures treated under hypoxic conditions, and 11 from cultures irradiated in cysteamine. Striking differences among the three treatment groups were not apparent. All types of mutations at both AT and GC base pairs were observed, including transitions, transversions, small deletions, and insertions. Several mutations affected RNA splicing, leading to exon skipping or inclusion of intron sequences in the final message. Approximately half (14/29) of the mutants sequenced had additions or deletions of one to several nucleotides. Also, 3/29 involved tandem DNA base changes (GG-->TT, C-->AA, AA-->CG). These observations are consistent with a mechanism involving the induction of noncoding or synthesis-blocking lesions that result in polymerase slippage or error-prone bypass synthesis. In addition, potential hotspot sites for mutation by X rays were discovered. At one site in exon 3, the same complex mutation, consisting of a G-->T transversion and a nearby six-base deletion, was detected in three independent mutants. Another mutant had a G-->C transversion at the same base, but without the deletion. At another site in exon 8, three mutations occurred at a run of three consecutive cytosines; these included a -C, a -CC, and a C-->AA. Also in exon 8, two mutations (+T, T-->C) occurred at two consecutive thymines.

Base Sequence↗

PubMed1989

Molecular cloning, sequence analysis, and functional expression of a novel growth regulator, oncostatin M.

Oncostatin M is a polypeptide of Mr approximately 28,000 that acts as a growth regulator for many cultured mammalian cells. We report the cDNA and genomic cloning, sequence analysis, and functional expression in heterologous cells of oncostatin M. cDNA clones were isolated from mRNA of U937 cells that had been induced to differentiate into macrophagelike cells by treatment with phorbol 12-myristate 13-acetate, and a genomic clone was also isolated from human brain DNA. Sequence analysis of these clones established the 1,814-base-pair cDNA sequence as well as exon boundaries. This sequence predicted that oncostatin M is synthesized as a 252-amino-acid polypeptide, with a 25-residue hydrophobic sequence resembling a signal peptide at the N terminus. The predicted oncostatin M amino acid sequence shared no homology with other known proteins, but the sequence of the 3' noncoding region of the cDNA contained an A + T-rich stretch with sequence motifs found in the 3' untranslated regions of many cytokine and lymphokine cDNAs. Oncostatin M mRNA of approximately 2 kilobase pairs was detected in phorbol 12-myristate 13-acetate-treated U937 cells and in activated human T cells. Transfection of cDNA encoding the oncostatin M precursor into COS cells resulted in the secretion of proteins with the structural and functional properties of oncostatin M. The unique amino acid sequence, expression by lymphoid cells, and growth-regulatory activities of oncostatin M suggest that it is a novel cytokine.

Amino Acid Sequence↗

PubMed2000

Nucleotide sequence analysis of the entire coding regions of virulent and avirulent strains of viral haemorrhagic septicaemia virus.

The complete nucleotide sequences of the six open reading frames (N, P, M, G, NV and L) and the intergenic regions were determined for two virulent freshwater strains and two avirulent marine strains of viral haemorrhagic septicaemia virus (VHSV). Sequence analysis of 10,845 nucleotides revealed > 97.2% nucleotide sequence similarity and > 98.6% amino acid similarity, confirming the close genetic relationship between marine and freshwater strains of VHSV. Moreover, as few as 10 amino acid substitutions were identically substituted between the marine and freshwater strains investigated. The results of this study suggest that only a limited number of amino acid residues may be involved in the determination of VHSV virulence for salmonids and further highlights the potential risk that marine strains may pose to freshwater aquaculture.

Animals↗

PubMed2006

Comparative sequence analysis of major envelope protein gene (B2L) of Indian orf viruses isolated from sheep and goats.

Orf virus (ORFV), the type species of Parapoxvirus, is responsible for contagious ecthyma in sheep and goats. In the present report, sequence analysis of major envelope gene (B2L) of four Indian orf virus isolates originating two each from sheep and goats was carried out. These recent isolates belonged to different outbreaks that occurred in Kumaon hills and adjoining plains during 2004-2005. Preliminary screening of the scab samples was carried out by diagnostic PCR. Full-length B2L gene encoding for immunogenic major envelope protein from all the four ORFV isolates was amplified by PCR and the amplicons (1206 bp) were cloned and sequenced. Comparative sequence analysis revealed an open reading frame of 1137 nucleotides (nt) encoding a polypeptide of 378 amino acids (aa). Indian isolates were highly related amongst themselves with sequence identity of over 97% at the nt and aa level. Further, they showed 97-98% sequence identity with sequences of other ORFV isolates from around the world; while 94-95 and 82.7-83.8% sequence identity was observed, respectively, with pseudocowpox and bovine papular stomatitis viruses--the other members of the genus. Phylogenetic analysis also showed that these Parapoxviruses from sheep and goats are closely related to other orf viruses reported worldwide.

Amino Acid Sequence↗

PubMed1994

Sequence analysis of the 190-kDa antigen-encoding gene of Rickettsia conorii (Malish 7 strain).

The complete nucleotide (nt) sequence of the gene (rompA) encoding the 190-kDa immunodominant surface antigen (rOmpA) of Rickettsia conorii (Malish 7 strain) was determined. Sequence analysis revealed an ORF of 6063 nt encoding a deduced protein of 203,247 Da. Ten consecutive highly conserved repeat units, located at the 5' end of rompA, spanned 2.2 kb. Two types of repeats could be identified: type I of 225 bp, and type II of 216 bp. The order and number of repeats differed from those reported for R. conorii (Kenya tick typhus strain), R. akari (Kaplan strain) and R. rickettsii (R strain). Alignment of the R. conorii (Malish 7 strain) rompA gene with its R. rickettsii homolog revealed 95% nt sequence similarity. The conservation of rompA across several pathogenic spotted fever group (SFG) rickettsial species suggests that it may be a potential candidate for use as a subunit vaccine for SFG rickettsial diseases.

Amino Acid Sequence↗

PubMed1996

Hepatitis B nucleotide sequence analysis: linking an outbreak of acute hepatitis B to contamination of a cryopreservation tank.

An epidemiological investigation indicated that six patients treated in a haematology unit who developed acute hepatitis B may have been infected as a result of contamination of a liquid nitrogen bone marrow storage tank. The clinical details are described elsewhere (Tedder et al., 1995); we describe the virological methods used to support the findings. HBV DNA was amplified from sera using a nested PCR with primers for the surface gene, and a region encompassing precore, the 3' end of X, and the 5' end of core. HBV DNA was also extracted from the frozen detritus in the liquid nitrogen storage tank. After equilibration, the aqueous material was filtered, co-precipitated with albumin and polyethylene glycol and the HBV DNA extracted by phenol-chloroform and ethanol precipitation. Direct nucleotide sequence analysis indicated that four patients were infected with HBsAg subtype adw viruses which carried novel amino acid substitutions at codons 145 and 146 of the X gene. HBV DNA extracted from the storage tank detritus contained identical sequences. The samples from two other patients, subtype ayw, did not contain the novel sequence changes in X and had other sequence differences. These findings linked conclusively the four patients as a cluster and the rescue of HBV-DNA sequences from the contaminated storage tank by the method described confirmed this as the common source of infection. Two other HBsAg-positive patients were excluded from the cluster by sequence analysis. Demonstration of infection by this route has implications for the safe storage of bone marrow and other related biological materials.

Acute Disease↗

PubMed1998

Discovery of the tail tube gene of bacteriophage Mu and sequence analysis of the sheath and tube genes.

The nucleotide sequence was determined for 2.75 kbp of phage Mu DNA encoding the contractile tail sheath protein L. N-terminal sequence analysis of Mu tail tube and sheath proteins identified the open reading frame just downstream of gene L as the tube gene. This clustering and order of the sheath and tube genes appear to be common among the myoviridae. Database homology searches revealed high similarity between the Mu sheath and tube proteins and two proteins in a Haemophilus influenzae Mu-like prophage, suggesting that they are the sheath and tube proteins of that prophage.

Amino Acid Sequence↗

PubMed2004

Cloning and sequence analysis of gene oipA encoding an outer membrane protein of human Helicobacter pylori.

AIM: To construct a recombinant E. coli strain that would highly express the proinflammatory outer membrane protein of human Helicobacter pylori (H pylori). METHODS: The oipA DNA was amplified by PCR, inserted into pET-32a, and transformed into Top10 E. coli strain. This recombinant plasmid of Top10 was sent out for nucleotide sequence analysis. Finally this sequence AF479754 was compared with HP0638 and JHP0581. RESULTS: The sequence of the aim gene was obtained. It had 924 base pairs. The identity was 95.32% against HP0638, 95.02% against JHP0581, which was higher than the identity between HP0638 and JHP0581. CONCLUSION: Although the aim gene was obtained, but it was different from the published sequence of GenBank. It is not clear what makes this difference. Maybe it is because different strain was used or because there were some variations. So more researches are required to prove it.

Bacterial Outer Membrane Proteins↗

PubMed2005

[Molecular detection and sequence analysis of hepatitis A virus (HAV) in two outbreaks in 2004 in North East Hungary].

INTRODUCTION: Hepatitis A virus (HAV) is the most important cause of acute infectious hepatitis worldwide. In Hungary, the reported number of HAV infections decreasing in the last decades, however, in every year approximately 500-800 new cases occur. In Hungary, particularly in North East region not only sporadic cases but also outbreaks of HAV are happen from time to time. Serology is routinely used laboratory method for diagnosis of HAV infections, although, there was no direct molecular detection and sequence analysis for the circulating HAV strains in Hungary. AIMS: Author's aims were to detection and genetic characterization of hepatitis A virus in outbreaks of hepatitis by molecular methods for reason of molecular epidemiology in Hungary. MATERIALS AND METHODS: Sera samples from symptomatic patients were tested from two acute hepatitis outbreaks in two settlements (Hajdúböszörmény and Kázsmárk) in North East Hungary in 2004 by enzim-immunoassay (EIA) and reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Sera in 58 (100%) and 4 (28.6%) symptomatic patients were positive in outbreaks of Hajdúböszörmény and Kázsmárk by HAV IgM EIA, however, 4 (57.1%) and 2 (66.6%) HAV IgM positive samples were positive by RT-PCR. By sequence analysis, outbreaks caused by the same hepatitis A virus which belongs to genotype I, subtype IA. These viruses had 98.4% nucleotide identity to IT-SCH-00 virus detected in year 2000 in Italy the closest match in GenBank. CONCLUSIONS: Methods of molecular biology give new opportunity for surveillance of infectious diseases in public health. Firstly characterized hepatitis A viruses in Hungary show that the subtype IA have an important epidemiological role in outbreaks. It is also suggested that genotype IA HAV play a part in sporadic HAV cases in endemic region in Hungary, too.

Adolescent↗

PubMed2004

[Analysis and evaluation of phage amplified biologically assay, DNA sequencing analysis and single-strand conformational polymorphism for the drug susceptibility testing of Mycobacterium tuberculosis].

OBJECTIVE: To rapidly identify drug-resistant Mycobacterium tuberculosis using phenotypic and genotypic methods and to evaluate the clinical significance of rapid phenotypic susceptibility test by phage amplified biologically assay (PhaB). METHODS: PhaB, DNA sequencing and polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) were performed on the 91 rifampicin (RFP)-resistant strains (including 82 multi-drug resistant Mycobacterium tuberculosis strains), 42 RFP-susceptible strains, 75 ofloxacin (OFLX)-resistant strains and 40 OFLX-susceptible strains at the same time. RESULTS: The results obtained using PhaB assay, DNA sequencing and PCR-SSCP were compared with the absolute concentration method. For RFP susceptibility, the accordance, sensitivity and specificity of PhaB were 93%, 92% and 95% respectively; the accordance, sensitivity and specificity of DNA sequencing were 93%, 90% and 100% respectively; those of PCR-SSCP were 90%, 86% and 100% respectively. For OFLX susceptibility, the accordance, sensitivity and specificity of PhaB assay were 95%, 95% and 95%; those of DNA sequencing were 80%, 71% and 98% respectively; those of PCR-SSCP were 75%, 63% and 98% respectively. CONCLUSIONS: PhaB assay is a low-cost, rapid, and sensitive method and shows high accordance with absolute concentration technology. It can give drug susceptibility test results within 48 - 96 h, and is a promising technology in clinical laboratory.

Antibiotics, Antitubercular↗