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Biomedical subjects

R L Nussbaum

Publications and source records attributed to R L Nussbaum.

At least 109 records · Page 6Linked to original sources
PubMed1985

Thymidylate synthase-deficient Chinese hamster cells: a selection system for human chromosome 18 and experimental system for the study of thymidylate synthase regulation and fragile X expression.

Chinese hamster lung (CHL) V79 cells already deficient in hypoxanthine phosphoribosyltransferase were exposed to uv light and selected for mutations causing deficiency of thymidylate synthase (TS) by their resistance to aminopterin in the presence of thymidine and limiting amounts of methyl tetrahydrofolate. Three of seven colonies chosen for initial study were shown to be thymidylate synthase deficient (TS-) by enzyme assay, thymidine auxotrophy, and their inability to incorporate labeled deoxyuridine into their DNA in vivo. Complementation analysis of human X TS- hamster hybrids revealed that TS activity segregated with human chromosome 18. Southern analysis of a panel of 14 human X hamster hybrids probed with complementary DNA from mouse TS confirmed the chromosome assignment of TS to human chromosome 18; quantitative Southern blotting using unbalanced human cell lines further localized the gene to 18q21.31----qter. Another hybrid was generated that contained a human X chromosome with the Xq28 folate-dependent fragile site as its only human chromosome in a hamster TS- background. The fragile site could be easily and reproducibly expressed in this hybrid without the use of antimetabolites simply by removing exogenous thymidine from the medium. These TS-deficient cells are useful for: somatic cell genetics as a unique selectable marker for human chromosome 18, studies on regulation of the TS gene, and analysis of the fragile (X) chromosome and other folate-dependent fragile sites.

Animals↗
PubMed1984

Newborn screening for sickling hemoglobinopathies. Houston, 1976 to 1980.

Fifty of 52 infants with cord blood hemoglobin electrophoresis patterns indicative of a possible sickling hemoglobinopathy (SH) were followed up prospectively. A retrospective group of 39 children whose mothers were contacted two to four years after the birth of a child with a positive newborn screening test formed a comparison group. During an average follow-up period of 30 months, children of mothers who were informed early of a positive cord blood test were hospitalized more readily for complications of an SH. During this same period, five (6%) of 88 infants with fetal and sickle hemoglobin (FS) or fetal, sickle, and C hemoglobins (FSC) in their cord blood died, all before 6 months of age. All five deaths occurred in infants whose parents were unaware of their child's disease; four of the five deaths may have been caused by sepsis. These results alone did not prove that screening significantly reduces mortality but did show that newborn screening increases parental awareness of complications and may, therefore, improve the prognosis in sickle cell disease in infancy.

Anemia, Sickle Cell↗
PubMed1984

Organization of the HPRT gene and related sequences in the human genome.

Comparative Southern hybridization of cDNA probes to DNA from cells carrying either one or four X chromosomes has been used to distinguish sequences derived from the functional locus for hypoxanthine-guanine phosphoribosyltransferase (HPRT) on the X chromosome from four independent HPRT-like autosomal sequences in the human genome. Subfragments of cDNA were then used to orient fragments from the HPRT locus with respect to the mRNA sequence. The chromosomal origin of each of the autosomal sequences was determined by Southern analysis using DNA from a panel of human-Chinese hamster somatic cell hybrids. Two of the HPRT-like sequences were localized to chromosome 11, the third to chromosome 3, and the fourth to the region between p13 and q11 on chromosome 5. Three of these four autosomal sequences were isolated from genomic recombinant libraries and subcloned fragments from each were used as probes to study restriction fragment length polymorphisms (RFLP) at these loci. A RFLP for MspI was found at the HPRT-like locus on chromosome 5 with a 1.3-kb major allele (frequency = 0.8) and a 3.6-kb minor allele (frequency = 0.2).

Animals↗
PubMed1984

Two anonymous X-specific human sequences detecting restriction fragment length polymorphisms in region Xq26----qter.

Two anonymous X-specific sequences isolated from a genomic library of flowsorted X chromosomal DNA were selected for study because they revealed restriction fragment length polymorphisms in the region Xq26----qter. One sequence, DXS10, detected a two-allele TaqI polymorphic system with allele frequencies of 0.33 and 0.67. The other, 4D-8, defined an MspI polymorphism with allele frequencies of 0.18 and 0.82. DXS10 is tightly linked to the hypoxanthine phosphoribosyltransferase (HPRT) locus with recombination distance theta = O cM at LOD = 5.55 (95% probability limit theta less than 15 cM). DXS10 maps to Xq26 but is not contained within the HPRT locus itself. 4D-8 shows no detectable linkage to the HPRT locus, with maximum likelihood estimate for theta = 50 cM and a LOD score of -2.61 at theta = 5 cM. These two polymorphisms provide additional chromosomal loci for gene mapping by linkage at the distal end of the long arm of the human X chromosome.

Base Sequence↗
PubMed1984

The Gardner syndrome. Significance of ocular features.

Gardner syndrome is a dominantly inherited familial cancer syndrome characterized by intestinal polyposis, bony hamartomata, and various soft tissue tumors. The risk of malignancy during adult life is essentially 100%, but as yet no phenotypic marker nor biochemical or serological linkage have been useful to identify the presence of the gene in early life. We studied three families in which multiple and bilateral patches of congenital hypertrophy of the retinal pigment epithelium are related uniquely to other phenotypic features of the Gardner gene. This readily identifiable characteristic may be useful to identify early in life individuals at risk for malignancy. We also suggest that the Gardner syndrome may be genetically heterogeneous.

Adult↗
PubMed1984

Morbidity of sickle cell trait at high altitude.

A Caucasian with sickle cell trait (54% A, 43% S hemoglobin) had symptoms and signs of chronic hemolytic anemia as well as splenic infarction while living at very high altitude (in excess of 2,800 m). Trips from sea level back to high altitude repeatedly precipitated pulmonary infarctions. All symptoms and evidence of hemolysis resolved at low altitude.

Adult↗
PubMed1984

A case of myocardial infarction in a patient with Werner's syndrome.

We report on a case of premature atherosclerosis in a patient with short stature, dysmorphic features, mild diabetes mellitus, hypertriglyceridemia, Mönckeberg's sclerosis, and the general appearance of premature aging, compatible with the diagnosis of Werner's Syndrome. The patient was referred to us after suffering a myocardial infarction at the age of 26.

Journal Article↗
PubMed1984

Human chromosomal assignments for 14 argininosuccinate synthetase pseudogenes: cloned DNAs as reagents for cytogenetic analysis.

There are multiple, processed, dispersed pseudogenes for human argininosuccinate synthetase. Chinese hamster X human somatic cell hybrids were used to map DNA fragment groups corresponding to the single expressed gene and 14 pseudogene loci. Each chromosomal assignment was confirmed using hybrids containing very few human chromosomes and/or by demonstrating monosomic or trisomic dosage in human cell lines with chromosomal abnormalities. Pseudogenes were mapped to chromosomes 2cen-p25, 3q12-qter, 4q21-qter, 5 (two loci), 6, 7, 9p13-q11, 9q11-q22, 11q, 12, Xp22-pter, Xq22-q26, and Ycen-q11. DNA fragments from the expressed gene were mapped to 9q34-qter in agreement with the previous assignment for enzyme activity. A high-frequency restriction fragment length polymorphism mapped to 9q11-q22. The analyses emphasized the feasibility of using chromosomally abnormal human cell lines for confirmation and regionalization of gene-mapping assignments made using somatic-cell hybrids. Conversely, cloned DNA probes, once mapped and characterized, can be very valuable for determining the chromosomal composition of interspecies hybrids and the dosage of loci in human cells. The argininosuccinate synthetase cDNA is a convenient reagent for dosage analysis of 15 human loci on 11 different chromosomes. Improved reagents could be designed that would simplify Southern blot patterns by eliminating overlapping DNA fragments and providing a single DNA fragment for each locus.

Animals↗
PubMed1983

Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid.

Interspecific somatic cell hybrids were constructed between a Chinese hamster lung cell line deficient in hypoxanthine phosphoribosyltransferase and two lymphoblastoid cultures (GM 4025 and GM 3200) from unrelated males affected with the fragile (X) syndrome. Thirteen independent colonies survived selection in hypoxanthine-azaserine, while only one colony survived selection in hypoxanthine-aminopterin-thymidine. One hybrid formed from GM 4025 was found to contain a human X chromosome as the only detectable human chromosome in the majority of cells analyzed. Induction of fragile (X) expression in this hybrid at frequencies up to 20% was achieved by treatments with 5-fluoro-2'-deoxyuridine (5 X 10(-8) M or 1 X 10(-7) M) or methotrexate (5 X 10(-6) or 1 X 10(-5) for 12 h. Use of the somatic cell hybrid system may allow study of the fragile (X) from different patients on a homogeneous xenogeneic background and may provide a better system for characterization of the fragile (X) at the biochemical and molecular level.

Animals↗
PubMed1983

Miller-Dieker syndrome: lissencephaly and monosomy 17p.

Miller-Dieker syndrome, which includes lissencephaly and a characteristic phenotypic appearance, has been reported to have an autosomal recessive pattern of inheritance. However, we have found abnormalities of chromosome 17 in two of three unrelated patients with this syndrome, one with a ring chromosome 17 and the other with an unbalanced translocation resulting in partial monosomy of 17p13. A review of the literature revealed five additional patients in three families, who had Miller-Dieker syndrome and an abnormality of 17p. Thus, we propose that monosomy of distal 17p may be the cause of Miller-Dieker syndrome in some patients.

Abnormalities, Multiple↗
PubMed1983

A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man.

Using cloned cDNA sequences of murine and human hypoxanthine phosphoribosyltransferase (HPRT: IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8), we have identified and characterized a three-allele restriction-fragment-length polymorphism for the restriction endonuclease BamHI at the human HPRT locus. The alleles are expressed phenotypically on Southern blots as three distinct pairs of fragments that hybridize to HPRT cDNA: (i) a 22-kilobase (kb)/25-kb pair, (ii) a 12-kb/25-kb pair, and (iii) a 22-kb/18-kb pair. In addition to fragments from the HPRT locus, sequences recognized by both HPRT cDNA probes are also present on at least two autosomes in the human genome. Allele frequencies in an unselected Caucasian population are 0.77 for the 22-kb/25-kb allele. 0.16 for the 12-kb/25-kb allele, and 0.07 for the 22-kb/18-kb allele, resulting in an average heterozygosity of 38% in females in this population. This polymorphism should facilitate gene mapping by linkage in this region of the human X chromosome.

Alleles↗
PubMed1983

Human hypoxanthine-guanine phosphoribosyltransferase. Detection of a mutant allele by restriction endonuclease analysis.

We have developed a method for the direct analysis of a hypoxanthine-guanine phosphoribosyltransferase (HPRT) allele associated with a deficiency of enzyme activity and an early onset of gout. The functionally abnormal enzyme coded for by this mutant allele (HPRTToronto) differs from the normal enzyme by an arginine-to-glycine substitution at position 50. A single base change in the codon for arginine 50 can explain this substitution. Direct analysis of this point mutation is based on the observation that it abolishes a Taq I recognition site in HPRT DNA. As predicted, DNA from individuals with the HPRTToronto allele exhibited an abnormal restriction pattern when digested with Taq I and probed with HPRT complimentary DNA: a normal 2.0-kb fragment is replaced by a 4.0-kb fragment. The 4.0/2.0-kb restriction fragment variation was used to detect the HPRTToronto allele in a heterozygote that was otherwise normal with respect to the classical techniques used to diagnose heterozygosity in HPRT deficiency.

Adenine Phosphoribosyltransferase↗
PubMed1981

Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Saccharomyces cerevisiae.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was purified 12 000-fold to homogeneity from yeast by a three-step procedure including acid precipitation, anion-exchange chromatography, and guanosine 5' -monophosphate affinity chromatography. The enzyme is a dimer consisting of two, probably identical, subunits of Mr 29 500. The enzyme recognized hypoxanthine and guanine, but not adenine or xanthine, as substrates. An antiserum against both native and denatured enzyme has been raised and shown to be specific for the enzyme. The antiserum has no affinity for Chinese hamster or human HPRT but does recognize subunits of yeast HPRT as well as some cyanogen bromide fragments of the enzyme.

Epitopes↗
PubMed1980

Sporadic occurrence of Duchenne muscular dystrophy: evidence for new mutation.

The mechanism of inheritance of sporadically occurring Duchenne muscular dystrophy has been investigated in 42 families, using carrier detection methods and genetic evaluation. Our studied find that serum CPK detects 72% of female carriers of DMD. Quantitative LDH and/or its isozymes were not found to be a useful means of carrier detection, as reported by Roses et al. (1977). Employing family pedigree data and CPK carrier testing, we determined by Bayesian methods the probability that the mother and maternal grandmother in these families were DMD carriers. These studies revealed 23 families (Category I) with no evidence for DMD carriers, 11 families (Category II) in which the mother was found to be a carrier, and 3 families (Category III) in which both mother and maternal grandmother were found to be carriers. Nineteen of the 42 families have a greater than 83% probability that the sporadic DMD case arose by mutation in a maternal gamete. This finding is in good agreement with the theoretically expected 1/3 of DMD cases arising by new mutation.

Chromosomes↗
PubMedDate not supplied

Somatic cell hybrid studies of fragile (X) expression in a carrier female and transmitting male.

We have extended our previous studies of fra(X) expression in somatic cell hybrids to the analysis of a carrier female with a low level of expression and to an unaffected, transmitting male who shows no expression in lymphocytes or lymphoblasts. Optimum conditions for fra(X) expression was treatment with 10(-8) M FUdR for 16 hours. In recent experiments, addition of 2.2 mM caffeine 2 hours before harvest was found to increase expression consistently. Two clones from the carrier female containing the fra(X) chromosome but not the normal X showed expression of 2-4%, indicating that expression in heterozygous females is not influenced by the presence or absence of the normal X. Expression rate was increased to 20% by exposure to FUdR plus caffeine. Analysis of hybrids containing only the fra(X) in an inactive state, and after reactivation by 5-azacytidine, showed no change in the frequency of expression. A hybrid clone from the nonexpressing, transmitting male containing only the X and chromosome 13, showed expression ranging from 2% without caffeine to 12% with caffeine in three different experiments. The ability to induce fra(X) expression in hybrid from this nonexpressing male may be explained in one of several ways: 1) a second mutation has occurred, 2) an autosomal suppressor locus was lost, or 3) the hamster genome or unusually short cell cycle lowers the threshold for expression, particularly in the presence of caffeine.

Animals↗
PubMedDate not supplied

Caffeine enhances fragile (X) expression in somatic cell hybrids.

Yunis and Soreng recently demonstrated enhanced expression of common fragile sites as and of fra(X) when 2.2 mM caffeine is added to FUdR treated lymphocyte cultures 6 hours before harvest. We failed to replicate this finding for fra(X) expression in lymphocytes. However, we do find a consistent increase in expression levels in somatic cell hybrids between a Chinese hamster cell line and 3 unrelated individuals with the fra(X) mutation when caffeine is present for the last 2 hours before harvest. This was particularly true for "low-expressing" cell lines, in which a 4-6 fold increase was observed. Using a thymidylate synthase deficient hybrid which could be blocked in the S phase by thymidine deprivation, we found that caffeine significantly reduced the recovery time from thymidine release to mitosis. This produced the highest level of fra(X) expression (48%) only one hour after release from thymidine deprivation. These results show that caffeine does enhance fra(X) expression in at least some cell types. The effect is probably indirect, inhibiting the mitotic delay usually associated with DNA damage.

Animals↗