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Results for “alignment chaining method”

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PubMed2000

Amplification of IgG VH and VL (Fab) from single human plasma cells and B cells.

Amplification of human immunoglobulin has many potential applications such as analysis of clonality, isolation of immunogenic antigens and antigen-specific immunotherapy. Here we describe a method for amplification of human immunoglobulin heavy and light chains from single B lymphocytes or plasma cells. Cells are isolated by FACS, and Ig is amplified by semi-nested RT-PCR. The method is versatile, sensitive and reliable: it provides appropriately paired heavy and light chains, requiring as little as 2 days to produce amplified Fab DNA from human tissues.

B-Lymphocytes↗

PubMedDate not supplied

Ribosomal RNA sequences of Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis: phylogenetic construction and structural correspondence.

The microsporidian species Enterocytozoon bieneusi, Septata intestinalis and Ameson michaelis were compared by using sequence data of their rRNA gene segments, which were amplified by polymerized chain reaction and directly sequenced. The forward primer 530f (5'-GTGCCATCCAGCCGCGG-3') was in the small subunit rRNA (SSU-rRNA) and the reverse primer 580r (5'-GGTCCGTGTTTCAAGACGG-3') was in the large subunit rRNA (LSU-rRNA). We have utilized these sequence data, the published data on Encephalitozoon cuniculi and Encephalitozoon hellem and our cloned SSU-rRNA genes from E. bieneusi and S. intestinalis to develop a phylogenetic tree for the microsporidia involved in human infection. The higher sequence similarities demonstrated between S. intestinalis and E. cuniculi support the placement of S. intestinalis in the family Encephalitozoonidae. This method of polymerized chain reaction rRNA phylogeny allows the establishment of phylogenetic relationships on limiting material where culture and electron microscopy are difficult or impossible and can be applied to archival material to expand the molecular phylogenetic analysis of the phylum Microspora. In addition, the highly variable region (E. coli numbering 590-650) and intergenic spacer regions in the microsporidia were noted to have structural correspondence, suggesting the possibility that they are coevolving.

AIDS-Related Opportunistic Infections↗

PubMed1994

Bacillus DNA in fossil bees: an ancient symbiosis?

We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences. These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp. Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B. firmus, B. subtilis, and B. circulans. These four extant Bacillus spp. are commonly isolated from abdominal tissue of stingless bees. The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P. dominicana.

Animals↗

PubMed2003

Non-radiolabelled PCR consensus primers and automatic sequencing enable rapid identification of tumor-specific V(H) CDR3 in aggressive B-cell malignancies.

Even though the results of current therapy are improved for B-cell acute lymphoblastic leukemia (B-ALL) and Burkitt's lymphoma (BL), prognosis of relapsed mature B-ALL and BL still remain extremely poor. In this study, we investigated the possibility of applying the use of non-radiolabelled PCR consensus primers and automatic sequencing for the rapid identification of the tumor-specific V(H) CDR3 nucleotide sequence, in mature B-ALL and BL. RNA was extracted from four consecutive, unselected samples from BL cases and three consecutive, unselected samples from mature B-ALL cases. The feasibility of the identification of the tumor-specific V(H) CDR3 nucleotide sequence was then assessed by using non-radiolabelled PCR consensus primers with automatic sequencing. The tumor-specific V(H) CDR3 nucleotide sequence was successfully identified for all seven patients (3 mature B-ALL and BL). The time required was substantially lower than that of the other methods previously published, despite the poor quality of some of the samples. The procedure showed rapidity, reliability and reproducibility. The characteristics of the methodology applied widen the possibility of developing anti-idiotypic therapeutic strategies, even in these B-cell malignancies.

Automation↗

PubMed1996

Identification and characterization of up-regulated genes during chondrocyte hypertrophy.

Chondrocyte hypertrophy involves de novo acquisition and/or increased expression of certain gene products including, among others, type X collagen, alkaline phosphatase, and matrix metalloproteinases. To analyze further the genetic program associated with chondrocyte hypertrophy, we have employed a modification of the polymerase chain reaction-mediated subtractive hybridization method of Wang and Brown (Wang and Brown [1991] Proc. Natl. Acad. Sci 88:11505). Cultures of hypertrophic tibial chondrocytes and nonhypertrophic sternal cells were used for poly A+ RNA isolation. Among 50 individual cDNA fragments isolated for up-regulated hypertrophic genes, 18 were tentatively identified by their similarities to entries in the GenBank database, whereas the other 32 showed no significant similarity. The identified genes included translational and transcriptional regulatory factors, ribosomal proteins, the enzymes transglutaminase and glycogen phosphorylase, type X collagen (highly specific for hypertrophic cartilage matrix), gelsolin, and the carbohydrate-binding protein galectin. Two of these, transglutaminase and galectin, were cloned and were further characterized. The chondrocyte transglutaminase revealed previously in hypertrophic cartilage by immunochemical methods appears to be the chicken equivalent of mammalian factor XIIIa (showing 75% overall protein similarity). The chicken chondrocyte galectin is a variant of mammalian galectin-3. Galectins are known to bind to components found in hypertrophic cartilage, and factor XIIIa is known to crosslink some of the same components, possibly modifying them for calcification and/or removal.

Amino Acid Sequence↗

PubMed2000

A molecular tool for species identification and benzimidazole resistance diagnosis in larval communities of small ruminant parasites.

This report describes a molecular method for determining in a first step the generic composition of a nematode community and in a second step, the resistance of each species to benzimidazole (BZ). We first established a polymerase chain reaction (PCR) linked to a restriction fragment length polymorphism strategy using the isotype 1 beta-tubulin gene. This method overcame the limitations of morphological identification of larval stages of trichostrongylid nematode species. Geographically distant isolates from the three main gastrointestinal species in temperate zones, Teladorsagia circumcincta, Haemonchus contortus, and Trichostrongylus colubriformis, were distinguished using this method. We then used an allele-specific PCR (AS-PCR) to detect mutations of residue 200 of the beta-tubulin, which is implicated in BZ resistance. The sequences of several samples confirmed the BZ-resistance genotype determined by AS-PCR. The ability to process large numbers of samples simultaneously makes this PCR-based strategy particularly suitable for epidemiological studies. It may also be useful for monitoring the emergence of resistant alleles in nematode communities.

Animals↗

PubMed2005

Immunoglobulin mutational status detected through single-round amplification of partial V(H) region represents a good prognostic marker for clinical outcome in chronic lymphocytic leukemia.

The immunoglobulin (Ig) mutational status in B-cell chronic lymphocytic leukemia (CLL) distinguishes two subsets of patients with different prognosis. Ig status detection is commonly performed with a panel of V(H) family-specific primers. Although this method detects clonal VDJ rearrangement in virtually all cases, it is technically cumbersome and therefore not widely used clinically. Here, we describe a simple and rapid method to establish the mutational status of IgV(H) in CLL. The method is based on a consensus V(H) FR2 primer, used in both polymerase chain reaction (PCR) and sequencing reactions. Overall, monoclonal B-cell populations were detected in 163 of 189 CLL patients (86%). The prognostic value of IgV(H) mutational status was then evaluated by analyzing survival in 146 CLL cases using different V(H) homology cutoffs. CLL prognostic groups were best separated by the classical 98% cutoff: median survival was 127 and 206 months in unmutated and mutated CLL cases, respectively (P = 0.0023). V(H) FR2 consensus and V(H) family PCR were compared in 41 cases, correctly assigning all cases by both methods. Therefore, we suggest a sequential strategy to detect immunoglobulin mutational status in CLL patients by first using the approach described in this study followed by alternative V(H) family-specific PCRs for negative cases.

Adult↗

PubMed1999

Identification of a 3 kb Alu-mediated BRCA1 gene rearrangement in two breast/ovarian cancer families.

Most of the hereditary breast cancers are attributed to constitutive alterations of either BRCA1 or BRCA2 genes; nonetheless, germline mutations of these genes in 'high risk' families are found less frequently than expected from linkage data. Recent findings suggest that major genomic rearrangements of the BRCA1 gene might account for at least some of the apparently mutation negative cases. We studied 60 affected probands belonging to families with a strong history of breast and/or ovarian cancer who scored negative for BRCA1 gene mutations by PTT and SSCP analysis. DNA was analysed by the Southern blotting procedure using three different restriction enzymes, and two probes obtained by RT-PCR of the 5' and 3' BRCA1 coding sequence. A 3 kb deletion encompassing exon 17 and causing a frameshift mutation was identified in two independently ascertained families. RT-PCR and long-range DNA PCR were employed to characterize the rearrangement that was finally shown to be the result of a recombination between two very similar Alu repeats. This type of mutation is not identified by the conventional methods of mutation detection which are based on PCR amplification of single exons. Therefore, further search for gene rearrangements is needed to better define the proportion of 'high risk' families that might be explained by gross genomic alterations of the BRCA1 gene.

Alu Elements↗

PubMed1994

Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

Animals↗

PubMed2003

Human cytomegalovirus glycoprotein N (gpUL73-gN) genomic variants: identification of a novel subgroup, geographical distribution and evidence of positive selective pressure.

Human cytomegalvirus (HCMV) ORF UL73 is a polymorphic locus, encoding the viral glycoprotein gpUL73-gN, a component of the gC-II envelope complex. The previously identified gN genomic variants, denoted gN-1, gN-2, gN-3 and gN-4, were further investigated in this work by analysing a large panel of HCMV clinical isolates collected from all over the world (223 samples). Sequencing and phylogenetic analysis confirmed the existence of the four gN genotypes, but also allowed the identification of a novel subgroup belonging to the gN-3 genotype, which was designated gN-3b. The number of non-synonymous (d(N)) and synonymous (d(S)) nucleotide substitutions and their ratio (d(N)/d(S)) were estimated among the gN genotypes to evaluate the possibility of positive selection. Results showed that the four variants evolved by neutral (random) selection, but that the gN-3 and gN-4 genotypes are maintained by positive selective pressure. The 223 HCMV clinical isolates were subdivided according to their geographical origin, and four main regions of gN prevalence were identified: Europe, China, Australia and Northern America. The gN variants were found to be widespread and represented within the regions analysed without any significant difference, and no new genotype was detected. Finally, for clinical and epidemiological purposes, a rapid and low-cost method for genetic grouping of the HCMV clinical isolates was developed based on the RFLP revealed by SacI, ScaI and SalI digestion of the PCR-amplified UL73 sequence. This technique enabled us to distinguish all four gN genomic variants and also their subtypes.

Amino Acid Sequence↗

PubMed1998

The C4b-binding protein-protein S interaction is hydrophobic in nature.

C4b-binding protein (C4BP) is a major regulatory molecule of the complement system. By forming a non covalent complex with the anticoagulant cofactor protein S (PS), it also plays an important role in blood coagulation. C4BP is composed of one beta-chain and seven alpha-chains that are essentially built from complement control protein (CCP)-modules. Our group has previously reported that the first (N-terminal) CCP module of the beta-chain (betaCCP1) contains the entire binding site for protein S. We now investigate further the binding of protein S to C4BP and show that the complex formation is essentially dependent on hydrophobic forces with minor contribution from electrostatic interactions. This result is in agreement with homology modeling experiments carried out in conjunction with inter-species sequence comparison and theoretical enumeration of potential binding sites. These methods pinpoint a solvent exposed hydrophobic cluster at the surface of the betaCCP1 module that is of crucial importance for the binding process.

Amino Acid Sequence↗

PubMed2001

Lactic acid bacterial diversity in the traditional mexican fermented dough pozol as determined by 16S rDNA sequence analysis.

The lactic acid bacteria diversity of pozol, a Mexican fermented maize dough, was studied using a total DNA extraction and purification procedure and PCR amplification of 16S rDNA for gram-positive and related bacterial groups. Thirty-six clones were obtained and sequenced to 650 nucleotides. These partial sequences were identified by submission to the non-redundant nucleotide database of NCBI. The identified sequences were aligned with reference sequences of the closest related organisms. This analysis indicated that only 14 sequences were unique clones and these were identified as Lactococcus lactis, Streptococcus suis, Lactobacillus plantarum, Lact. casei, Lact. alimentarium, and Lact. delbruekii and Clostridium sp. Two non-ribosomal sequences were also detected. Unlike other environments analyzed with this molecular approach where many unidentified microorganisms are found, the identity of most sequences could be established as lactic acid bacteria, indicating that this is the main group among the gram-positive bacteria in pozol. Use of this molecular method permitted detection of lactic acid bacteria different from those previously isolated and identified by culture techniques

Clostridium↗

PubMed2005

Correlation of a resistance-associated Rdl mutation in the German cockroach, Blattella germanica (L), with persistent dieldrin resistance in two Danish field populations.

Danish Blattella germanica (L) populations carry the resistance-associated mutation A302S within the Resistance to dieldrin (Rdl) gene. The mutation has remained in field populations long after the discontinuation of dieldrin for cockroach control. The mutation has also persisted in our laboratory strains with high and intermediate frequencies for more than 8 years without selection. The toxicity of dieldrin was tested by topical application to male cockroaches in the susceptible strain DPIL-SUS and two field strains, Zo960302 and Su960304, which were 1270- and 15-fold resistant to dieldrin at LD50. We report the sequencing of exon 7 of the B. germanica Rdl gene and the finding of the putative resistance-associated A302S mutation. We have developed and implemented a PCR-based diagnostic method with the detection of a restriction endonuclease polymorphism, which allows for easy discrimination of susceptible, resistant and heterozygote genotypes. The frequency of the resistance-associate allele A302S was 0.97 and 0.38 in the Zo960302 and Su960304 populations, respectively. The cockroach Rdl A302S allele confers high dieldrin resistance in homozygotes and intermediate resistance in heterozygotes, and its presence is responsible for the persisting dieldrin resistance in Danish populations of B. germanica.

Amino Acid Sequence↗

PubMed1995

Heterogeneity among 16S-23S rRNA intergenic spacers of species within the 'Streptococcus milleri group'.

The 16S-23S rRNA intergenic spacer has been suggested as a suitable region of the bacterial genome from which to derive useful taxonomic information, particularly with regard to identification at the species level. To investigate this approach as an aid to the identification of the three species comprising the 'Streptococcus milleri group' (SMG), the spacers of isolates of Streptococcus intermedius, Streptococcus anginosus and Streptococcus constellatus were amplified by PCR and length polymorphisms determined by agarose gel electrophoresis. Phenotypically atypical isolates which had been identified presumptively as belonging to these three species were also included. Spacers from two representatives of each spacer length found within the three SMG species were sequenced. 16S-23S rRNA intergenic spacer length polymorphisms allowed discrimination between S. anginosus (350 bp or 450 bp amplification product) and S. constellatus (380 bp amplification product), species that are difficult to differentiate phenotypically. S. intermedius (330 bp or 450 bp amplification product) and S. anginosus (350 bp or 450 bp amplification product) were not reliably distinguished by this method but are phenotypically distinct. Sequencing data demonstrated that the spacers had a central region of highly variable length flanked by conserved regions which included a single tRNA(Ala) gene. Polymorphism in the length of the 16S-23S spacer determined by PCR provides a rapid and useful adjunct to strain identification for S. anginosus and S. constellatus, which are not readily differentiated phenotypically.

Base Sequence↗

PubMed1999

Analysis of porcine cytomegalovirus DNA polymerase by consensus primer PCR.

We used a consensus primer PCR method to amplify a region of herpesviral DNA-directed DNA polymerase gene using degenerate primers for initial characterization of the porcine cytomegalovirus (PCMV) genome. The sequence of the PCR product from PCMV DNA template and its alignment with other herpesvirus DNA polymerase counterparts showed that both conserved amino acid residues and conservative amino acid substitutions are in parallel. Phylogenetic analysis revealed that PCMV should be included in the clade comprising human herpesvirus 6 and 7, rather than human and mouse cytomegaloviruses, in Betaherpesvirus subfamily.

Amino Acid Sequence↗

PubMed1994

Nucleotide sequence of the core region of hepatitis C virus in Pakistan and Bangladesh and the geographic characterisation of hepatitis C virus in south Asia.

A large number of complete and partial hepatitis C virus (HCV) sequences have been reported and classified into several genotypes, although none have been reported from South Asia. We have determined and evaluated partial sequences in the core region of HCV obtained from patients with chronic hepatitis in Pakistan and Bangladesh. Nucleotide sequences from these viruses show significant homology with the Japanese HCV-TR isolate (91.7%-97.9%) and low homology with other Japanese, American, and UK isolates including HCV-1, HC-J4, HC-J6, HC-J8, and E-b1 (79.3%-86.2%). The homologies of their deduced amino acids sequence with HCV-1, HC-J4, HC-J6, HC-J8, E-b1, and HCV-TR were 84.3%-89.8%, 85.0-87.9%, 84.1%-86.9%, 84.3%-87.0%, 90.2%-93.1%, and 89.8%-93.5%, respectively. These results suggest that our clones might be classified into the same genotype as HCV-TR. Further analysis using molecular evolutionary methods strongly supported the classification of these sequences with the HCV-TR genotype. Moreover, we could not detect any isolates which were closely related to our clones or HCV-TR in countries outside the South Asian area. These data further support the association of HCV genotypes with distinct geographic regions.

Amino Acid Sequence↗

PubMed1996

Strongylus asini (Nematoda, Strongyloidea): genetic relationships with other Strongylus species determined by ribosomal DNA.

Genomic DNA was isolated from adult Strongylus asini collected from zebra. The second ribosomal transcribed spacer (ITS-2) was amplified and sequenced using polymerase chain reaction (PCR) based techniques. The DNA sequence was compared with previously published data for 3 related Strongylus species. A PCR-linked restriction fragment length polymorphism method allowed the 4 species to be differentiated unequivocally. The ITS-2 sequence of S. asini was found to be more similar to those of S. edentatus (87.1%) and S. equinus (95.3%) than to that of S vulgaris (73.9%). This result confirms that S. Asini and S vulgaris represent separate species and supports the retention of the 4 species within 1 genus.

Animals↗

PubMed1999

Towards rice genome scanning by map-based AFLP fingerprinting.

Map-based DNA fingerprinting with AFLP markers provides a fast method for scanning the rice genome. Three hundred AFLP markers identified with ten primer combinations were mapped in two rice populations. The genetic maps were aligned and almost full coverage of the rice genome was obtained. The transferability of AFLP markers between indica x japonica and indica x indica crosses was tested. The chromosomes were divided into DNA Fingerprint Linkage Blocks (DFLBs) defined by specific AFLP markers. Using these blocks, the degree of similarity or divergence within specific chromosome regions was calculated for nine varieties. Applications of map-based fingerprinting for biodiversity studies and maker-assisted selection are discussed.

Chromosome Mapping↗