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Biomedical subjects

R Krishnamoorthy

Publications and source records attributed to R Krishnamoorthy.

At least 37 records · Page 2Linked to original sources
PubMed1991

A simple nonradioactive method of DNA typing for subsets of HLA-DR4: prevalence data on HLA-DR4 subsets in three diabetic population groups.

The molecular basis of eight DR4 subtypes resides in several nucleotide substitutions in the third hypervariable region of the DR beta 1 chain. The typing of DR4 subsets using the mixed lymphocyte culture (MLC) assay or allele-specific oligonucleotide hybridization is expensive, cumbersome, and requires the use of radioisotopes. We have therefore developed a rapid and safe procedure for subtyping DR4-alleles that involves selective amplification of the second exon of the DR4-DRBI gene followed by unambiguous subtype discrimination after digestion with five allele-specific endonucleases [polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)] and visualization of the polymorphic fragments with silver or ethidium bromide staining. Validity of this subtyping procedure was initially examined by the use of cell lines of known subtypes. Three groups of DR4 patients with insulin-dependent diabetes mellitus (IDDM) from Chinese, Tunisian, and Caucasian populations were subtyped and the prevalence of subtype associations with IDDM was compared.

Alleles↗
PubMed1991

Comparison of molecular epidemiological tools for Branhamella catarrhalis typing.

Twenty-one Branhamella catarrhalis strains selected for their diversity by esterase electrophoretic polymorphism, and belonging to 20 distinct zymotypes, were studied by restriction fragment length polymorphism (RFLP) of total DNA after ethidium bromide staining and of ribosomal DNA regions (ribotyping). The former analysis allowed the distinction of 20 patterns and the latter the delineation of 19 ribotypes. The three methods were correlated and showed a clonal diversity of the species. Esterase electrophoresis and Hinfl RFLP patterns after ethidium bromide staining appeared to be simple tools for use in B. catarrhalis epidemiology.

Bacterial Typing Techniques↗
PubMed1991

Origin and spread of beta-globin gene mutations in India, Africa, and Mediterranea: analysis of the 5' flanking and intragenic sequences of beta S and beta C genes.

Nucleotide polymorphisms of both the 5' flanking and intragenic regions of the human beta-globin gene were investigated by directly sequencing genomic DNA after amplification by the polymerase chain reaction in 47 subjects homozygous for the beta S or the beta C mutation. The sickle-cell mutation was found in the context of five different haplotypes defined by eight nucleotide substitutions and various structures of a region of the simple repeated sequence (AT) chi Ty. All subjects from the same geographic origin bear an identical chromosomal structure, defining the Senegal-, Bantu-, Benin-, Cameroon-, and Indian-type chromosomes. These results strengthen our previous conclusions about the multiple occurrence of the sickle-cell mutation. The Benin-type chromosome was also found among Algerian and Sicilian sickle-cell patients, whereas the Indian-type chromosome was observed in two geographically distant tribes, illustrating the spread of these sickle-cell genes. We also found that the intragenic sequence polymorphisms (frameworks) are not always in linkage disequilibrium with the BamH I polymorphism downstream from the beta-globin gene, as had been previously observed. Finally, we present a tentative phylogenetic tree of the different alleles at this locus. Some polymorphisms of this sequence might be contemporary with our last common ancestor, the great apes, that is, about 4-6 millions years old.

Africa↗
PubMed1991

HLA-DQA1 and DQB1 alleles in French and Algerian type 1 diabetic subjects.

Some alleles of the HLA-DQB1 and DQA1 loci are preferentially associated with susceptibility to type 1 (insulin-dependent) diabetes mellitus (IDDM). Analysis of the HLA-DQ genetic profile may therefore become important for the screening of subjects at risk of IDDM. However ethnic variations in the genetic profile can occur and require background knowledge of the HLA-DQ allelic distribution before screening campaigns. In the present work, HLA-DQA1 and DQB1 genes have been analyzed, after PCR amplification of the genomic DNA, in French and Algerian control subjects (a total of 148) and diabetic patients (a total of 107). Allelic distributions have been investigated in view of a) possible inter-ethnic differences; b) identification of risk and protective alleles and c) the prevalence of DQB1 aspartate 57 negative and DQA1 arginine 52 positive alleles in control and diabetic groups. The DQB1 allelic distribution was similar in both control groups; alleles negative for aspartate at position 57 were 48% in French and 50% in Algerian. In both diabetic groups, the prevalence of alleles negative for aspartate at position 57 was significantly higher: 91% (French) and 81% (Algerian) (p less than 0.001). A majority of patients were homozygote for DQB1 Asp 57 negativity: 83% (French) and 63% (Algerian). The highest relative risk was associated with HLA-DQB1 0201/0302 heterozygosity. The HLA-DQA1 allelic distribution was also similar in French and Algerian controls. Alleles positive for arginine (ARG+) at position 52 were 50% (French) and 57% (Algerian) of controls. In both diabetic groups the prevalence of alleles positive for arginine at position 52 was significantly higher: 78% (French) and 84% (Algerian).(ABSTRACT TRUNCATED AT 250 WORDS)

Algeria↗
PubMed1991

Asthma and HLA system.

Study of atopic asthma provides an excellent model for our understanding of the complex molecular genetics of human immune responsiveness. Family and population studies have suggested that multiple genetic factors influence global and specific immune responsiveness to allergens. Global response of IgE production appears to be controlled by a dominant "atopy gene" unlinked to HLA class II associated Ir genes, whereas the specific immune response in terms of IgE and IgG antibodies against precise allergens is unequivocally linked to the HLA class II molecules. Other genetic factors/polygenic component may influence the bronchial hyperreactivity observed among asthmatics. Not the least, the genetics of the "releasability" of mediators of inflammation from basophils should also be considered in the genetic studies of asthma. Given this overall complexity of genetics of asthma, we primarily focused our attention to the genetics of a defined subgroup of asthmatic patients. We have chosen to study three well-defined clinical subsets (atopic asthma with and without aspirin sensitization, and nonatopic asthma with aspirin sensitization) in comparison to age and sex matched control groups for their HLA class II polymorphism at the DNA level. Use of in vitro DNA amplification and allele specific restriction enzymes allowed us to define that a particular combination of a specific DQ and DPB alleles is highly prevalent among asthmatic patients with aspirin sensitization and this is exclusively in an atopic context (R.R. = 54). Intriguingly, an identical combination of HLA alleles are similarly enriched among patients with coeliac disease. Further studies are required to understand such an intriguing similarities of molecular markers between these apparently two unrelated disorders.

Alleles↗
PubMed1990

HLA-DQB 1 codon 57 and genetic susceptibility to type 1 (insulin-dependent) diabetes mellitus in French children.

Variations in the incidence rate of Type 1 (insulin-dependent) diabetes mellitus might relate to ethnic-specific genetic backgrounds. HLA-DQB 1 alleles were typed in 75 French diabetic children and 85 matched control children. Enzymatically-amplified DQB 1 exon 2 was hybridized with oligoprobes specific for the six most common alleles. Alleles coding for an Asp residue at position 57 in the DQ beta chain are strongly negatively associated with Type 1 diabetes in the French population. Nevertheless, one of the diabetic children was an Asp 57 homozygote (DQB 1.2/3.1). Among alleles coding for a residue other than Asp at position 57, alleles 3.2 and 2 (Ala 57) are positively associated with diabetes but not allele 1.1 (Val 57) which is less frequent in diabetic children than in control children. Heterozygosity for 2/3.2 alleles is the genotype most strongly associated with diabetes (Odds ratio = 52.9). Large comparative population studies will be necessary to determine whether the frequency of DQB 1 alleles positively associated with Type 1 diabetes (2 and 3.2) in a given ethnicity is related to its incidence rate in the same population.

Alanine↗
PubMed1990

Correlation between ribosomal DNA polymorphism and electrophoretic enzyme polymorphism in Yersinia.

Ribosomal DNA (rDNA) polymorphism was compared with electrophoretic enzyme polymorphism for the intra- and interspecies differentiation of Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. aldovae, Y. frederiksenii and Y. kristensenii. DNA from 90 strains previously classified into six zymotypes (Y. enterocolitica and Y. frederiksenii) and into distinct enzyme electrophoretic patterns (the four other species) was digested with EcoRI or HindIII and analysed by Southern blotting. The six species were clearly differentiated from each other. In Y. enterocolitica, the subclassification of biotype 1 into zymotypes 1A and 1B was also reflected in the rDNA and the four other bio-zymotypes gave four different classes of restriction pattern. In Y. frederiksenii, both EcoRI and HindIII gave five distinct riboclasses which correlated with the zymotypes. In the four other species, the phenotype polymorphism appeared to be better correlated with the restriction fragment length polymorphism data in some enzymes than others. The data demonstrate that the inter- and intraspecies classification by rDNA polymorphism using two restriction enzymes is similar to that based on electrophoretic enzyme polymorphism. The analysis could be refined for taxonomic and epidemiological purposes by using other restriction enzymes.

DNA, Bacterial↗
PubMed1990

Locus assignment of human alpha globin mutations by selective amplification and direct sequencing.

We describe a simple approach for molecular characterization and locus assignment of structural mutants by direct sequencing of enzymatically amplified DNA selective to alpha 1 and alpha 2 globin gene regions. Nucleotide substitution of two structural variants (Stanleyville II alpha 2(78Lys) and J Mexico alpha 2(54Glu) were determined and their encoding loci were specified. The amplified segment encompasses sequences upstream of the CAAT box to downstream of the Poly(A) addition signal. Hence all of the alpha globin structural variants and most of the nondeletion alpha thalassaemic mutants should be characterizable by this approach.

Africa↗
PubMed1989

A simple, sensitive method of analyzing bacterial ribosomal DNA polymorphism.

A rapid DNA extraction procedure and random primer labelling of Escherichia coli ribosomal RNA with cloned reverse transcriptase have been used to establish a simple and highly sensitive method for studying ribosomal DNA polymorphism in bacteria. Examples of inter- and intraspecies differentiation of bacteria are given and potential applications in bacterial epidemiology and taxonomy are discussed.

Acinetobacter↗
PubMed1989

Effect of ligand-affinity differences of human hemoglobin variants on electrophoretic behavior and their isolation and functional characterization.

A natural sulfated polysaccharide (agaropectin), contained in crude agar, can be used as a medium for electrophoretic separation of hemoglobin mutants, constituting a particular class of protein-ligand interactions. Mutations which either modify the electrostatic charge at the surface of the hemoglobin molecule or not, have been studied according to their putative interaction with the medium. Using conformational specificities of the hemoglobin molecule, we have also demonstrated that isoelectric focusing on a polyacrylamide gel in the absence of heme ligands represents a useful, convenient and rapid procedure for isolating silent Hb variants in their native form, provided that they exhibit an abnormal Bohr effect.

Calcium↗
PubMed1989

The analysis of five carbenicillin-hydrolysing enzymes by electrophoretic methods.

Five carbenicillin-hydrolysing enzymes (carbenicillinases, or CARB), PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3, CARB-4 and CARB-5, and the beta-lactamase PSE-2 were compared by analysing their isoelectric points (pI), electrophoretic mobilities (mR) and titration curves (pH gradient electrophoresis). The pI determined by isoelectric focusing were 4.3 (CARB-4), 5.3 (PSE-4/CARB-1), 5.7 (PSE-1/CARB-2), 5.75 (CARB-3), 6.1 (PSE-2) and 6.35 (CARB-5). Their mR were estimated by zone electrophoresis as congruent to 26 for PSE-1 (CARB-2), CARB-3 and CARB-5, congruent to 30 for PSE-2, congruent to 33 for PSE-4 (CARB-1) and congruent to 61 for CARB-4. Titration curve analyses indicated that (1) PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5 are closely related variants differing by a few amino acid substitutions; (2) the qualitative titration curve of CARB-4 is different from those of PSE-4 (CARB-1), PSE-1 (CARB-2), CARB-3 and CARB-5, although their patterns are somewhat similar; and (3) PSE-2 has no structural relationship with any of the other carbenicillin-hydrolysing enzymes or carbenicillinases (CARB) studied. Electrophoretic methods, and in particular titration curve determination combined with other physicochemical and enzymatic data, allowed a rapid comparison of the molecular structures of the beta-lactamases, and hence their classification.

Acinetobacter↗
PubMed1989

Analysis of the molecular relatedness of four extended spectrum beta-lactamases (SHV-2, SHV-3, SHV-4 and SHV-5) by comparative protein titration curves.

Six beta-lactamases from Klebsiella pneumoniae, five of which (SHV-1, SHV-2, SHV-3, SHV-4 and SHV-5) were plasmid-encoded and one which (beta 1a GN 11-03) was chromosomally-encoded, were compared by analysis of their isoelectric points (pI), electrophoresis mobilities (MF) and titration curves or pH gradient electrophoresis. Four groups were defined by their pI and MF, namely SHV-1 and SHV-2 (pI = 7.6, MF approximately 14), SHV-3 and beta 1a GN 11-03 (pI = 7.0, MF approximately 20), SHV-4 (pI = 7.8 MF approximately 12) and SHV-5 (pI = 8.2, MF approximately 5). The titration curves of SHV-1 and SHV-2 enzymes on the one hand, and SHV-3 and beta 1a GN 11-03 on the other were completely superimposable for the whole of the pH gradient (3.5-10), indicating strongly similarity. Conservative amino-acid substitutions could account for the differences in the substrate spectra of the purified enzymes. The differences observed between the titration curves of the enzymes SHV-1/SHV-3, SHV-1/beta 1a GN 11-03, SHV-2/SHV-3 and SHV-5/SHV-4 pairs were consistent with the replacement of a basic amino-acid residue in the former enzyme of each pair by an acidic residue in the latter. Similarly, the titration curves of SHV-1/SHV-4 and of SHV-2/SHV-4 pairs may suggest the replacement of an acidic amino-acid in the former beta-lactamases by a neutral amino-acid in the latter of each pair. However, the presence of several self-cancelling or neutral substitutions is also possible. In contrast, when SHV-1 and TEM-1 (pI = 5.4 MF approximately 45) were titrated together, no structural relationship could be inferred.

Chromosomes, Bacterial↗
PubMed1989

A gene conversion located 5' to the A gamma gene in linkage disequilibrium with the Bantu haplotype in sickle cell anemia.

Cloning and sequencing of the gamma-globin gene of a sickle cell anemia patient homozygous for the Bantu haplotype has revealed a gene conversion that involves the replacement of an A gamma sequence by a G gamma sequence in the promoter area of the A gamma gene. This event is similar to another gene conversion believed to be responsible for the very high homology between gamma-globin genes, suggesting that the promoter area of these genes is prone to this type of genetic rearrangement. Further analysis demonstrated that the chromosome bearing this gene conversion has a very high frequency among Bantu chromosomes and a very low or nil frequency in other haplotypes linked to the beta s gene. No correlation was found between the G gamma/A gamma ratio and the presence of the gene conversion among Bantu haplotype patients, thus excluding a portion of the gamma gene sequence in the determination of this ratio.

Anemia, Sickle Cell↗
PubMed1989

Nucleotide variations in the 3' A gamma enhancer region are linked to beta-gene cluster haplotypes and are unrelated to fetal hemoglobin expression.

Molecular cloning and sequence analysis of a nondeletion form of Sicilian beta o hereditary persistence of fetal hemoglobinemia (HPFH) (mutation in IVS2 nt1 position) homozygous for haplotype III revealed the presence of four sequence variations: C----T at -158 5' to G gamma, T----C at +2285, C----A at +2476, and A----G at +2676, all 3' to A gamma. The latter three variations in the putative A gamma enhancer are identical to those observed in the case of Seattle HPFH. However, a severe beta o-thalassemia case from Algeria (mutation in IVS1 nt1 position), also homozygous for haplotype III, revealed the same nucleotide variation, albeit an inefficient HbF production. We conclude that the variations in the A gamma enhancer element do not play a role in the regulation of HbF production. To assess both the linkage of these sites with the beta-cluster haplotype and the extent of the polymorphism, we examined several black and Mediterranean chromosomes, by PCR amplification followed by both EspI digestion and oligonucleotide hybridization. Our data indicate that these sequence variations in the enhancer element are absent in Mediterranean haplotypes I, V, and VII but are consistently associated with Mediterranean haplotypes II, III, and IX, as well as with the black beta c-associated haplotype. The common feature of all the latter haplotypes is the presence of a polymorphic PvuII site between A gamma and psi beta, which is thus in linkage disequilibrium with the variations in the A gamma enhancer.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗