Biomedical subjects

A Hajra

Publications and source records attributed to A Hajra.

25 records · Page 2Linked to original sources

PubMed1993

Identification of yeast artificial chromosomes containing the inversion 16 p-arm breakpoint associated with acute myelomonocytic leukemia.

We report the cloning of the chromosome 16 p-arm breakpoint involved in inversion 16(p13;q22) associated with subtype of acute myelomonocytic leukemia (AMML) M4Eo. Inter-Alu polymerase chain reaction (PCR) products from a series of interspecific somatic cell hybrids that contain only small portions of the human chromosome 16 p-arm were generated for use as fluorescent in-situ hybridization (FISH) probes. When applied to patient cells, rapid and unambiguous identification of the inversion resulted. Using FISH analysis, cosmid clones associated with the hybrids were identified that bracketed the p-arm breakpoint. A repeat-free fragment of one of these cosmids (35B11) when used as probe on Southern blots from pulsed-field gels identified rearranged macrorestriction fragments in patient DNA. Yeast artificial chromosomes (YACs) were isolated using sequences derived from cosmids flanking 35B11 in a cosmid contig. Of 4 YACs so identified, 3 were shown by FISH to cross the inversion-16 p-arm breakpoint. Therefore, the breakpoint has been molecularly cloned, and identified as being within these 3 YACs. These clones will facilitate the unraveling of the genetic events associated with inversion-16 and are available tools with immediate clinical application.

Base Sequence↗

PubMed1992

Detection of human DNA mutations with nonradioactive, allele-specific oligonucleotide probes.

We describe a method of detecting human DNA mutations with nonradioactive, biotinylated allele-specific oligonucleotide probes. This method can detect seven different mutations in the butyrylcholinesterase, cystic fibrosis, and N-acetyltransferase genes under identical assay conditions. This indicates that it may be used to detect mutations responsible for a wide variety of genetic diseases and pharmacogenetic conditions. The method involves first amplifying selected DNA fragments by the polymerase chain reaction and dot blotting the amplified DNA in duplicate onto small nitrocellulose squares. Each dot blot is then hybridized in individual wells containing a tetramethylammonium chloride solution with short biotinylated probes specific for either the normal or mutant allele. Successfully hybridized probes are detected by a simple colorimetric reaction using an avidin-alkaline phosphatase conjugate, which yields a very strong, clear signal. DNA from homozygous normal or mutant individuals hybridizes only to the normal- or mutant-specific probes respectively, while DNA from heterozygous individuals hybridizes equally well with both probes. These results can be easily interpreted to assign a genotype to the sample DNA. This method is amenable to automation, and may be useful in clinical laboratories for diagnosis of a wide variety of DNA mutations responsible for unusual reactions to drugs and environmental chemicals.

Alleles↗

PubMed1990

Phenotypic and molecular biological analysis of human butyrylcholinesterase variants.

Our laboratory has recently shown that several variant forms of human butyrylcholinesterase, associated with unusual sensitivity to succinylcholine, are caused by specific mutations within the structural DNA coding for this enzyme. Atypical (dibucaine-resistant) butyrylcholinesterase is caused by a point mutation at nucleotide position 209(GAT-- greater than GGT), which changes aspartate 70 to glycine. One fluoride-resistant variant family has a point mutation at nucleotide 728(ACG-- greater than ATG), which changes threonine 243 to methionine. Another type of fluoride-resistant variant has a point mutation at nucleotide 1169(GGT-- greater than GTT), which changes glycine 390 to valine. One type of silent phenotype is due to a frame-shift mutation at nucleotide position 351(GGT-- greater than GGAG). A polymorphic site at nucleotide position 1615 (GCA/ACA), coding for Ala/Thr, accounts for the quantitative K-variant, which causes an approximate one-third reduction of activity, if Thr occupies that position at codon 539. Examples are given to illustrate the advantages of using a combination of the new DNA analytical techniques, including: the use of allele-specific probes, with the standard serum cholinesterase phenotyping methods. More accurate typing of patients with certain variants is now possible; pedigree analysis will be aided by the improved methodology.

Alleles↗

PubMed1990

Transgene-induced mutation of the murine steel locus.

The product of the steel locus is essential for normal development of three distinct populations of stem cells--the neural crest-derived melanoblasts, germ cells, and blood cell precursors. Many mutant alleles at steel are lethal in homozygotes and produce coat color dilution in heterozygotes. We have identified a transgenic mouse with diluted pigmentation that closely resembles that of steel heterozygotes. We have demonstrated that the site of transgene insertion is genetically linked to the phenylalanine hydroxylase locus on mouse chromosome 10. In addition, the chromosome carrying the transgene fails to complement the recessive lethality of the Sl allele of steel and the pigmentation defect of the Slpan allele. The data indicate that the inserted transgene has disrupted the steel locus. The resulting allele, designated Sltg, provides a molecular tag for isolation of the steel gene, as well as a new allele for characterization of this developmentally important locus.

Animals↗

PubMed1989

Identification of the structural mutation responsible for the dibucaine-resistant (atypical) variant form of human serum cholinesterase.

A point mutation in the gene for human serum cholinesterase was identified that changes Asp-70 to Gly in the atypical form of serum cholinesterase. The mutation in nucleotide 209, which changes codon 70 from GAT to GGT, was found by sequencing a genomic clone and sequencing selected regions of DNA amplified by the polymerase chain reaction. The entire coding sequences for usual and atypical cholinesterases were compared, and no other consistent base differences were found. A polymorphic site near the C terminus of the coded region was detected, but neither allele at this locus segregated consistently with the atypical trait. The nucleotide-209 mutation was detected in all five atypical cholinesterase families examined. There was complete concordance between this mutation and serum cholinesterase phenotypes for all 14 heterozygous and 6 homozygous atypical subjects tested. The mutation causes the loss of a Sau3A1 restriction site; the resulting DNA fragment length polymorphism was verified by electrophoresis of 32P-labeled DNA restriction fragments from usual and atypical subjects. Dot-blot hybridization analysis with a 19-mer allele-specific probe to the DNA amplified by the polymerase chain reaction distinguished between the usual and atypical genotypes. We conclude that the Asp-70----Gly mutation (acidic to neutral amino acid substitution) accounts for reduced affinity of atypical cholinesterase for choline esters and that Asp-70 must be an important component of the anionic site. Heterogeneity in atypical alleles may exist, but the Asp-70 point mutation may represent an appreciable portion of the atypical gene pool.

Base Sequence↗

PubMed1986

Zellweger syndrome: diagnostic assays, syndrome delineation, and potential therapy.

Patients with the cerebrohepatorenal syndrome of Zellweger lack peroxisomes and certain peroxisomal enzymes such as dihydroxyacetone phosphate acyltransferase in their tissues. Deficiency of this enzyme, which is necessary for glycerol ether lipid synthesis, provides a biochemical method for recognizing patients with subtle manifestations of Zellweger syndrome and suggests the utility of exogenous ether lipid precursors as a therapeutic strategy for these children. We describe the results of glycerol ether lipid supplementation to two children, one with classic Zellweger syndrome and 9% of control fibroblast dihydroxyacetone phosphate acyltransferase activity, and one with mild facial manifestations, wide sutures, hypotonia, developmental delay, hepatomegaly, peripheral retinal pigmentation, and 50% of control fibroblast dihydroxyacetone phosphate acyltransferase activity. An increase in erythrocyte plasmalogen levels following therapy was clearly demonstrated in the milder patient, and neither patient showed evidence of toxicity. Evaluation of therapy by comparison to the usual clinical course of Zellweger syndrome was not helpful because of the variability and incomplete documentation of 90 previously reported cases. The literature survey did provide criteria for classic Zellweger syndrome, which include hypotonia with or without deformation of limbs, large fontanels and split sutures, prominent forehead, flattened facial profile with hypoplastic supraorbital ridges, anteverted nares, highly arched palate, cryptorchidism or labial hypoplasia, hepatomegaly or elevated liver enzymes, peripheral pigmentation of the retina, renal cortical cysts, and characteristic neuropathology involving decreased myelinization, abnormal neuronal migration, and sudanophilic macrophages. Less severe patients, as exemplified by our case 2 and others from the literature, will not have all the classic features and can be recognized only by a growing panel of biochemical indicators. Our patient studies illustrate the complexity of designing comprehensive therapy for Zellweger-like conditions, suggest other diseases that may involve peroxisomal alterations, and emphasize the need for multicenter, collaborative studies to evaluate biochemical heterogeneity and therapy of peroxisomal disorders.

Adrenoleukodystrophy↗

PubMed1975

Kala-azar.

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Adolescent↗