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PubMed1977

The use of nuclease P1 in sequence analysis of end group labeled RNA.

A method is described for the direct sequence analysis of 20-25 nucleotides from the termini of 5'- or 3'-end-group [32P] labeled RNA. The method involves partial endonucleolytic digestion of the labeled RNA with nuclease P1 (from Penicillium citrinum) followed by separation of the partial digestion products by two-dimensional homochromatography, the nucleotide sequence being determined by mobility shift analysis. This procedure has been applied to the sequence analysis of the terminal regions of tRNAs and of high molecular weight RNA, such as messenger RNA or viral RNA. A further application involves its use in conjunction with snake venom phosphodiesterase to determine the sequence of 5'-end group labeled oligonucleotides, containing modified bases, derived from T1 or pancreatic RNase digestion of tRNA.

Base Sequence↗

PubMed1993

An OspA serotyping system for Borrelia burgdorferi based on reactivity with monoclonal antibodies and OspA sequence analysis.

A total of 136 Borrelia burgdorferi sensu latu strains from various biological sources (ticks, human skin, and cerebrospinal fluid) and geographical sources (Europe and North America) were investigated by Western blot (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). On the basis of the differential reactivities of these monoclonal antibodies, seven OspA serotypes were defined. As determined by 16S rRNA sequence analysis, these serotypes correlated well with recently delineated genospecies: serotype 1 corresponds to B. burgdorferi sensu strictu, serotype 2 corresponds to group VS461, and serotypes 3 to 7 correspond to Borrelia garinii sp. nov. (G. Baranton, D. Postic, I. Saint Girons, P. Boerlin, J.-C. Piffaretti, M. Assous, and P. A. D. Grimont, Int. J. Syst. Bacteriol. 42:378-383, 1992). Antigenic differences were confirmed by partial sequence analysis of OspA of representatives of each serotype. Comparative sequence analysis suggested that serotype 5 OspA resulted from genetic recombination of serotype 4 and 6 ospA genes. Serotype 2 (group VS461) was most prevalent among European skin isolates (49 of 62 isolates). Among all B. garinii strains included in this study, serotype 6 was most frequently found in ticks and only rarely in human skin and cerebrospinal fluid, whereas serotypes 4 and 5 were isolated from patients but never from ticks. Our data suggest different pathogenic potentials and organotropisms of distinct OspA serotypes and raise the question of true antigenic variation among B. garinii strains.

Amino Acid Sequence↗

PubMed1989

RNA sequence analysis of a perinatal lethal osteogenesis imperfecta mutation.

The perinatal lethal form of osteogenesis imperfecta often results from mutations which disrupt stable assembly, delay secretion, and cause excessive posttranslational modification of type I procollagen molecules. One such mutation was efficiently characterized by an indirect method of RNA sequence analysis. The mutation initially was localized in procollagen by mapping the distribution of abnormal posttranslational modification within the triple helical domain of mutant molecules. Total RNA was isolated from osteogenesis imperfecta cells in culture, cDNA was synthesized using alpha 1(I) and alpha 2(I) specific primers, and fragments of cDNA suspected to harbor the mutation were amplified by the polymerase chain reaction technique and then cloned in M13 vectors. Sequence analysis of the amplified cDNA revealed a new, heterozygous Gly----Val substitution at residue 256 of the triple helical domain of alpha 1(I) chains produced by the perinatal lethal osteogenesis imperfecta cells. The nature and location of the mutation were confirmed by sequence analysis of amplified genomic DNA. A Gly----Val substitution has not previously been associated with the lethal form of osteogenesis imperfecta, and this mutation has the most amino-terminal location within the alpha 1(I) chain triple helical domain reported to date.

Alleles↗

PubMed1994

Characterization and sequence analysis of a Streptomyces rochei A2 endoglucanase-encoding gene.

A 7-kb fragment of Streptomyces rochei A2 chromosomal DNA was cloned into pAT153 and shown to confer endoglucanase (EglS) activity on Escherichia coli cells. In E. coli clones, the EglS was secreted into the periplasm. Deletion analysis revealed that an 827-bp fragment was enough for the enzymatic activity. Sequence analysis showed that the 827-bp fragment codes for the catalytic domain of the enzyme. The complete sequence of the gene (eglS) is 1149-bp long. A signal peptide, a catalytic domain and a cellulose-binding domain were identified from the nucleotide sequence, and the EglS found to belong to the family H of cellulase catalytic domains. These conclusions were substantiated by determination of the N-terminal sequence of the purified protein and zymogram analysis, which revealed protein species with a molecular mass equal to that deduced from the nt sequence analysis.

Amino Acid Sequence↗

PubMed2005

Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr diversification by sequence analysis.

The allelic variations of the regulatory operon agr (groups I-IV) and the cap polymorphism (capsular types 5 and 8) were used as a typing scheme for rapid strain designation in Staphylococcus aureus. In combining 10 agr subgroups resolved by restriction fragment length polymorphism (RFLP) analysis with the two cap polymorphisms 12 types could be defined. To assess whether this type designation is informative for the population structure of the species S. aureus, agr and cap types were determined in clonal lineages defined by pulsed-field gel electrophoresis (PFGE) of a collection of 219 isolates. agr groups and cap types were both linked to certain clone complexes. However, little correlation was found between the two polymorphic loci. By PFGE cluster analysis 11 prevalent and 52 sporadic clones were defined. Most of the prevalent clones (9/11) could be discriminated by agr/cap typing. Thus, this technique allows a first subdivision of isolates and an inter-center comparable designation of S. aureus clones preceding a more detailed clonal analysis by PFGE or multi-locus sequence typing (MLST). To get insight into agr diversification, sequence analysis of the variable and conserved part of agr from selected S. aureus clones was performed. Strains of agr-I displayed the highest sequence divergence on the nucleotide and amino acid level, suggesting an early diversification of this group. When analyzing the relationship between the four agr interference groups we could show: (i) one intermediate between agr-I and agr-IV alleles; (ii) agr-IV sequences seem to bridge the agr-I and -III groups and (iii) two cases of horizontal transfer of the variable gene cassette from an agr-I strain to an agr-II strain. Thus, stepwise evolutionary progression and rare events of recombination were evident in the diversification of the agr locus.

Alleles↗

PubMed1998

Sequence-based diagnosis of tyrosinase-related oculocutaneous albinism: successful sequence analysis of the tyrosinase gene from blood spots dried on filter paper.

BACKGROUND: A large number of mutations of the tyrosinase gene result in oculocutaneous albinism (OCA). Therefore, at present, sequence analysis of the tyrosinase gene has become necessary to diagnose OCA patients and their relatives. OBJECTIVE: The aim of this study was to facilitate the sequence-based diagnosis of tyrosinase-related OCA by using small amounts of the patient's blood. METHODS: Blood spots dried on filter papers were used as sources of genomic DNA. The exons and flanking regions of the tyrosinase gene were amplified by polymerase chain reaction (PCR) and were directly sequenced in both directions. RESULTS: We successfully amplified all exons of the tyrosinase gene by PCR and were able to characterize compound heterozygous mutations of R278X and + delta C310 in the patient's gene. CONCLUSION: Recent advances of PCR-related technology allowed us to use fairly limited samples of blood for sequence analysis of the tyrosinase gene.

Albinism, Oculocutaneous↗

PubMed1999

Identification and grouping of Dichelobacter nodosus, using PCR and sequence analysis.

Dichelobacter nodosus is the causative agent of ovine foot rot, a disease that is a constant economic burden for many Western sheep ranches. Vaccination is one method of treating foot rot. A higher and more specific immune response is observed when monovalent vaccines are used to treat foot rot, as compared to multivalent vaccines, which incorporate all 10 major New Zealand D. nodosus serogroups. There is no single assay for specifically identifying and grouping D. nodosus for the purpose of incorporating only the desired serogroup(s) in a vaccine. A polymerase chain reaction (PCR)-based assay was used to specifically identify and group D. nodosus from a foot rot lesion. Identification and grouping was determined by predicted fragment size analysis and nucleotide sequence information. The PCR approach vastly improves the accuracy in identifying and grouping D. nodosus from a foot rot lesion.

Animals↗

PubMed1997

Sequence analysis of HIV-1 group O from Norwegian patients infected in the 1960s.

Three Norwegians, a couple and their daughter, died from AIDS in 1976 after up to 10 years of clinical manifestations of HIV infection (Lindboe et al., 1986, Acta Pathol. Microbiol, Immunol. Scand. 94, 117-123; Frøland et al., 1988, Lancet i, 1344-1345). We here demonstrate the presence of HIV DNA in autopsy materials from the father and the daughter. In phylogenetic analysis, the obtained sequences of the HIV pol and vif genes clustered with the HIV-1 group O clade. The genotyping was confirmed by detection of antibodies against HIV-1 group O in blood samples from the father and the mother. That these and other early isolates of HIV-1 are very similar to the presently circulating viruses and not intermediates between the present subtypes, verifies that the latest common ancestor of HIV-1 existed long before the emergence of the present epidemic. The presence of HIV-1 group O 30 years ago suggests that the limited spread of these viruses, compared to HIV-1 group M viruses, is not due to a later emergence of the group O viruses.

Base Sequence↗

PubMed1995

Macintosh sequence analysis software. DNAStar's LaserGene.

The analysis of information in nucleotide and amino acid sequence data from an investigator's own laboratory, or from the ever-growing worldwide databases, is critically dependent on well planned and written software. Although the most powerful packages previously have been confined to workstations, there has been a dramatic increase over the last few years in the sophistication of the programs available for personal computers, as the speed and power of these have increased. A wide choice of software is available for the Macintosh, including the LaserGene suite of programs from DNAStar. This review assesses the strengths and weaknesses of LaserGene and concludes that it provides a useful and comprehensive range of sequence analysis tools.

Minicomputers↗

PubMed1996

Sequence analysis of VP7 gene of two Nigerian rotavirus strains.

The complete nucleotide sequences of gene 9 (VP7) of rotavirus strains MGH66 and RHIB55 isolated in northern and southern Nigeria, respectively, were determined. The sequence of either strain was 1062 nucleotides along with two potential glycosylation sites and two in-phase initiation codons encoding a protein of 326 amino acids provided the first ATG codon was utilised. Comparison of the deduced amino acid sequences of VP7 of the strains with that of published sequences of serotype G1 strains and a representative strain of each of serotypes 2-6 and 8-14 revealed > or = 91.41% and > or = 81.60% homology, respectively. The stool sample obtained from a diarrhoeic child in Maiduguri containing strain MGH66 was classified by polymerase chain reaction (PCR) technique as possessing a dual infection specificity of VP7 serotypes G1 and G3. The nucleotide sequencing, however, revealed the dual infection specificity of VP7 serotypes G1 and G8. The implications of nucleotide sequence analysis for serotyping of rotavirus strains originating from different geographical regions and for vaccine development are discussed.

Amino Acid Sequence↗

PubMed2003

Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes.

Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and b-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40-100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 b-strands. We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens. The M. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M. leprae genome compared to the M. tuberculosis genome.

Amino Acid Sequence↗

PubMed1991

Identification of the porcine paramyxovirus LPMV matrix protein gene: comparative sequence analysis with other paramyxoviruses.

The complete nucleotide sequence of the gene encoding the matrix protein (M) of the porcine paramyxovirus LPMV has been determined. The gene is 1376 nucleotides long including 5' and 3' non-coding sequences with a protein-coding sequence of 1107 nucleotides. The deduced protein, containing 369 amino acids with a calculated Mr of 41,657, is hydrophobic overall with a net positive charge of +17.5. Comparative sequence analysis revealed high amino acid homology to other paramyxovirus M proteins, with the highest degree of identity (46%) with the human mumps virus. This is strong evidence that the porcine paramyxovirus LPMV is a genuine member of the paramyxovirus genus.

Amino Acid Sequence↗

PubMed1996

Cloning and sequence analysis of the Rhizobium etli ccmA and ccmB genes involved in c-type cytochrome biogenesis.

In this paper we describe the sequence analysis of two Rhizobium etli genes (ccmA and ccmB) which are believed to participate in the transport of the haem moiety to the periplasm in other bacterial species. The characterized DNA region was isolated by complementation of a R. etli Tn5mob induced mutant (CFN4201) which was affected in the production of c-type cytochromes. Sequence analysis of this region identified three open reading frames, two were identified as the ccmA and ccmB genes. The predicted protein sequence of ccmA showed significant homology with ATP binding proteins of the ABC-type transporter systems, while ccmB encodes for a hydrophobic protein probably associated with the ccmA gene product. The Tn5mob insertion in CFN4201 strain was located in the carboxy terminus of CcmB. Restriction mapping of the EcoRI fragment containing the Tn5mob insertion showed that it involved a deletion of approximately 1.5 kb. Mutagenesis of the wild-type region with a miniMu transposon and complementation analysis showed that the mutation in ccmB, and not the deletion, was responsible for the phenotype of CFN4201 strain and that ccmA and ccmB are independent transcription units. We found that a region located downstream of ccmB is reiterated twice, one near the chromosomal ccmA-ccmB locus while the second in plasmid e. Finally, CFN4201 membranes had detectable levels of C1 apoprotein which did not contain bound haem. This data could suggest that haem binding to the apoprotein occurs after the translocation of the apo form of c1 to the membrane.

Amino Acid Sequence↗

PubMed1987

Molecular cloning and nucleotide sequence analysis of several naturally occurring HPV-5 deletion mutant genomes.

Three deletion mutants of naturally occurring human papillomavirus type 5 (HPV-5) were molecularly cloned into phage vectors. The nature of these deletions was characterized initially by restriction endonuclease mapping and electron microscopic heteroduplex analysis and ultimately by nucleotide sequence analysis. The sizes of the deletions are 353, 1329, 1571, and 2267 bp and map to the late gene region of the HPV-5 genome. The 80 nucleotides immediately adjacent to the deletions exhibit no significant detectable sequence homologies or symmetries and therefore were probably not formed by the sequence-dependent events of homologous recombination or site-specific recombination.

Base Sequence↗

PubMed1988

Isolation and sequence analysis of amyloid protein AA from a patient with cystic fibrosis.

This study represents the first sequence analysis of an amyloid fibril protein from a patient with cystic fibrosis. Although chronic infections are a hallmark of cystic fibrosis, secondary amyloidosis is a rare complication, and during the past 20 years, only 16 cases of amyloidosis in patients with cystic fibrosis have been reported. We examined amyloid fibrils isolated from the spleen of a 25-year-old man who had a history of a chronic cough since infancy and a diagnosis of cystic fibrosis at age 6 years. After solubilization in 6 mol/L guanidine and purification by gel filtration, the major component of the amyloid fibrils was a homogeneous 8000 dalton protein that reacted positively with antiserum to human amyloid A (AA) protein. Complete protein sequence analysis was carried out by using the whole protein and fragments obtained by treatment of the protein with cyanogen bromide, lysyl endopeptidase, and carboxypeptidase. The protein contained 76 residues and showed minor heterogeneity when compared with other AA protein sequences. The cystic fibrosis AA protein represents a product of the SAA-specific cDNA clone now known to be the alpha-allelic form of SAA1 in which valine is present at position 52 and alanine is at position 57.

Adult↗

PubMed1995

Sequence analysis of the rat jun-D gene.

A rat jun-D genomic clone was isolated from a rat liver lambda EMBL3 library. Sequence analysis revealed a high sequence similarity with mouse jun-D, and relatively low similarity with human and chicken jun-D. In both the trans-activation and DNA-binding domains, however, the amino-acid sequences were well conserved among rat, mouse, human and chicken.

Amino Acid Sequence↗

PubMed2001

Direct genotyping and nucleotide sequence analysis of VS1 and VS2 of the Omp1 gene of Chlamydia trachomatis from Moroccan trachomatous specimens.

To determine the range of ocular strains of Chlamydia trachomatis circulating in southern Morocco, where trachoma is endemic, and to compare the value of the molecular methods for genotyping C. trachomatis, ocular specimens were subjected to a direct Omp1 PCR-restriction fragment length polymorphism (RFLP)-based analysis and direct sequencing. PCR-RFLP analysis shows that the Ba genotype represents the most frequent one (63%), followed by genotype A (45%), whereas no B or C genotypes were identified among the 53 out of 108 specimens that were strongly positive in the Omp1 CT1-CT5 PCR. Our results further show that the notion of interfamily and intrafamily transmission is very likely. To confirm the genotype identity of C. trachomatis as determined by PCR-RFLP, 16 selected specimens were sequenced across variable sequence 1 (VS1) and 2 (VS2). No discrepancies were found between PCR-RFLP typing and the genotype identity confirmed by nucleotide sequencing of the PCR product. Our results clearly indicate that both molecular methods of typing chlamydiae (i.e., PCR-RFLP and sequencing) are important and have specific applications for clinical epidemiological purposes. This is the case for individuals infected with more than one clonal population of C. trachomatis. The unambiguous nucleotide sequencing therefore defines an important epidemiologic descriptor for the infected patient whether the source is from a clonal population of organisms or whether it represents a more dynamic process of strain dominance or genetic change. Furthermore, Omp1 genotyping affords the necessary approach to epidemiologic investigations in areas of the world endemic for trachoma, where only one or two serovars are known to predominate.

Bacterial Outer Membrane Proteins↗

PubMed1997

Molecular characterization of avian reoviruses using nested PCR and nucleotide sequence analysis.

A nested polymerase chain reaction (PCR) with subsequent nucleotide sequence analysis identified and differentiated avian reoviruses (ARVs). PCR products amplified from the S1 gene segment of ARV of USA isolates were 738 and 342 bp, respectively. PCR products were conformed by Southern and dot blot hybridizations. The amplified cDNA fragments were cloned into the pUC18 vector and subjected to DNA sequencing. The nucleotide and deduced amino acid sequences of four USA (S1133, 1733, 2408, and CO8) and two Australian isolates (RAM-1 and SOM-4) were compared. Results of paired difference analysis and a predicted dendrogram revealed that USA isolates were closely related, but different from, Australian isolates. The deduced amino acid sequences of the N-terminal region of ARV sigma C showed a heptapeptide repeat of hydrophobic residues in all ARV isolates.

Amino Acid Sequence↗