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Biomedical subjects

D Patterson

Publications and source records attributed to D Patterson.

At least 19 recordsLinked to original sources
PubMed1992

Human chromosomal localization of genes encoding the gamma 1 and gamma 2 subunits of the gamma-aminobutyric acid receptor indicates that members of this gene family are often clustered in the genome.

The gamma-aminobutyric acid (GABA) receptors are the major inhibitory neurotransmitter receptors in the brain and the site of action of a number of important pharmacological agents including barbiturates, benzodiazepines, and ethanol. The gamma 1 and gamma 2 subunits have been shown to be important in mediating responses to benzodiazepines, and a splicing variant of the gamma 2 subunit, gamma 2L, has been shown to be necessary for ethanol actions on the receptor, raising the possibility that the gamma 2 gene may be involved in human genetic predisposition to the development of alcoholism. We have assigned the human genes encoding the gamma 1 and gamma 2 subunits of the GABAA receptor to chromosomes 4 and 5, respectively, by PCR amplification of human-specific products from human-hamster somatic cell hybrid DNAs. Using panels of chromosome-specific natural deletion hybrids, we have further localized the gamma 1 gene (GABRG1) to 4p14-q21.1 and the gamma 2 gene (GABRG2) to 5q31.1-q33.2. These data indicate that the gamma 1 gene may be clustered together with the previously mapped alpha 2 and beta 1 genes on chromosome 4 and that the gamma 2 gene may be close to the previously localized alpha 1 gene on chromosome 5. To further examine the latter possibility the alpha 1 gene was mapped using the chromosome 5 deletion hybrids and shown to be within the same region as the gamma 2 gene, 5q31.1-q33.2. A PCR-based screening strategy was used to isolate a 450-kilobase human genomic yeast artificial chromosome clone containing both the alpha 1 and gamma 2 genes. Pulsed-field gel restriction mapping of the yeast artificial chromosome indicates that the two genes are within 200 kilobases of each other. The data presented here provide further evidence for the nonrandom organization of the human genome by demonstrating that members of the GABAA receptor gene family often occur in small gene clusters widely distributed in the genome.

Base Sequence↗
PubMed1992

Effect of simvastatin on high density lipoprotein subfractions and apolipoproteins in type IIa hypercholesterolemia.

Changes in plasma concentrations of high density lipoproteins (HDL) and triglycerides may partly explain the ability of cholesterol-lowering drugs to decrease the incidence of coronary heart disease. We measured the response of fasting plasma lipids, lipoproteins, and apolipoproteins in 46 subjects with Type IIa hypercholesterolemia treated with simvastatin for 3 months. The initial dose of simvastatin (10 mg/day) was subsequently increased up to 40 mg/day if the plasma cholesterol concentration had not fallen below 5.2 mmol/l. Plasma concentrations of HDL cholesterol and of the apolipoproteins AI and AII were increased by simvastatin. The increase in HDL cholesterol (9%) was due to increases in both subfractions (HDL2 17%; HDL3 7%), changes that would be consistent with a beneficial effect on cardiovascular risk. Simvastatin decreased plasma triglyceride concentrations by 25%. Plasma total cholesterol concentrations fell by 35% after 3 months of treatment; this fall was proportional to the initial concentration and was due almost entirely to a 45% fall in low density lipoprotein cholesterol. In contrast, plasma concentrations of lipoprotein Lp(a) were not affected by simvastatin.

Anticholesteremic Agents↗
PubMed1992

A single base change at a splice acceptor site leads to a truncated CAD protein in Urd-A mutant Chinese hamster ovary cells.

We have previously reported the isolation and characterization of mutant Chinese hamster ovary (CHO-K1) cells of the Urd-A complementation group, which require uridine for growth, are deficient in the activities of the first three enzymes of de novo UMP biosynthesis, and produce markedly reduced amounts of a truncated form of the multifunctional protein CAD, which contains these three enzyme activities. We report here that a single base change of G to A at a highly conserved RNA splice acceptor site is responsible for the phenotype of this mutant. In addition to a small amount of apparently normal CAD mRNA, this mutation causes production of two alternative forms of CAD mRNA in the mutant, one that includes the intron just prior to the mutation and one that excludes the exon just after the mutation. The affected splice site is located at the intron-exon boundary just preceding the exon that encodes the beginning of the aspartate transcarbamylase (ATCase) domain of the CAD protein. Both intron inclusion and exon exclusion during RNA processing introduce a translation stop codon upstream of the region encoding this domain, resulting in the production of the truncated CAD protein seen in the Urd-A mutant. This mutation also results in markedly decreased levels of CAD mRNA and protein in the mutant.

Amino Acid Sequence↗
PubMed1992

Gene-dosage mapping of 30 DNA markers on chromosome 21.

Using a slot-blot method for the dosage of single-copy sequences, the copy numbers of 30 chromosome 21 markers were assessed in the blood DNA of 11 patients with partial trisomy or monosomy 21 and in the DNA of a patient-derived human-hamster hybrid cell line carrying a microduplication of chromosome 21. The physical order of these markers on chromosome 21 was thereby determined.

Aneuploidy↗
PubMed1992

Sequence-tagged sites (STSs) for a set of mapped markers on chromosome 21.

Sequence tagged sites (STSs) have been proposed as a "common language" for comparing physical and genetic maps of the human genome produced by a variety of techniques. We have produced 44 STSs from 38 mapped loci on human chromosome 21. The STSs represent most of the loci designated as genetic reference or ordered physical framework markers, along with a number of others chosen to span all regions of 21q. Of the STSs, 12 are from gene segments, including 4 from exons of the APP gene encoding the amyloid beta protein precursor, and 32 mark anonymous DNA loci. These STSs make each of the corresponding loci readily accessible to the research community without the need for exchange of clones. These sites also represent multiple start points for the isolation of YAC clones that should permit overlapping the entire chromosome 21 long arm as cloned DNA.

Animals↗
PubMed1992

Integrating maps of chromosome 21.

The past year has seen major progress in the construction of various types of maps of human chromosome 21. Perhaps more significantly, the chromosome 21 research community is making very significant progress on integration of these maps through the use of common resources and increased collaboration and communication.

Chromosome Mapping↗
PubMed1992

Isolation, characterization, and regional mapping of microclones from a human chromosome 21 microdissection library.

Thirty-four unique-sequence microclones were isolated from a previously described microdissection library of human chromosome 21 and were regionally mapped using a cell hybrid mapping panel which consists of six cell hybrids and divides chromosome 21 into eight regions. The mapping results showed that the microclones were unevenly distributed along chromosome 21, with the majority of microclones located in the distal half portion of the long arm, between 21q21.3 and 21qter. The number of unique-sequence clones began to decrease significantly from 21q21.2 to centromere and extending to the short arm. This finding is consistent with those reported in other chromosome 21 libraries. Thus, it may be inferred that the proximal portion of the long arm of chromosome 21 contains higher proportions of repetitive sequences, rather than unique sequences or genes. The microclones were also characterized for insert size and were used to identify the corresponding genomic fragments generated by HindIII. In addition, we demonstrated that the microclones with short inserts can be efficiently used to identify YAC (yeast artificial chromosome) clones with large inserts, for increased genomic coverage for high-resolution physical mapping. We also used 200 unique-sequence microclones to screen a human liver cDNA library and identified two cDNA clones which were regionally assigned to the 21q21.3-q22.1 region. Thus, generation of unique-sequence microclones from chromosome 21 appears to be useful to isolate and regionally map many cDNA clones, among which will be candidate genes for important diseases on chromosome 21, including Down syndrome, Alzheimer disease, amyotrophic lateral sclerosis, and one form of epilepsy.

Base Sequence↗
PubMed1991

Chromosome locations of the MYB related genes, AMYB and BMYB.

The MYB related loci, AMYB and BMYB, were localized to specific human chromosome regions by Southern blot analysis of their segregation patterns in a panel of rodent-human hybrid DNAs using radiolabeled AMYB and BMYB probes. The AMYB locus was present in hybrids retaining the chromosome region 8cen----8q22 and was absent in hybrids which had lost this chromosome region. The presence of the BMYB locus in rodent-human hybrids correlated with, and only with, chromosome region Xq13. Chromosomal in situ hybridization refined the localization of AMYB to region 8q22-23 and confirmed the localization of BMYB to region Xq13. Chromosome region 8q22 is involved in recurrent translocations in malignant lymphoma and in acute myeloid leukemia (AML-M2); therefore AMYB is a candidate for involvement in such translocations. A region on Xq13 is also involved in chromosomal abnormalities in acute myeloid leukemia and myelodysplasias.

Chromosome Aberrations↗
PubMed1991

Isolation of a yeast artificial chromosome spanning the 8;21 translocation breakpoint t(8;21)(q22;q22.3) in acute myelogenous leukemia.

The 8;21 translocation is one of the most common specific rearrangements in acute myelogenous leukemia. We have identified markers (D21S65 and a Not I boundary clone, Not-42, referred to as probe B) flanking the chromosome 21 translocation breakpoint (21q22.3) that demonstrate physical linkage in normal genomic DNA, by using at least three restriction endonucleases (Not I, Sac II, and BssHII), and that are located not more than 250-280 kilobases apart. Pulsed-field gel analysis of DNA from somatic cell hybrids containing the 8;21 translocation chromosomes demonstrates rearrangement of these markers. A 470-kilobase yeast artificial chromosome, YAC-Not-42, has been isolated that contains both probes. Mapping of lambda subclones constructed from YAC-Not-42 suggests that greater than 95% (25/26 probes tested) of the yeast artificial chromosome DNA is located on the proximal (D21S65) side of the breakpoint. In situ hybridization studies using metaphase chromosomes from five acute myelogenous leukemia patients with the 8;21 translocation confirmed these results and demonstrated the translocation of probe B to the derivative chromosome 8. A chromosome walk of approximately 39 kilobases from probe B has allowed identification of the breakpoint in DNA from a somatic cell hybrid containing the derivative chromosome 8. Since probe B contains conserved DNA sequences and is in close proximity to the translocation breakpoint, it may represent a portion of the involved gene on chromosome 21.

Blotting, Southern↗
PubMed1991

Cloning and in vivo expression of the human GART gene using yeast artificial chromosomes.

Two Yeast Artificial Chromosomes (YACs) were isolated each with a full-length copy of the human gene that encodes the trifunctional protein containing phosphoribosylglycinamide synthetase (GARS), phosphoribosylglycinamide formyltransferase (GART) and phosphoribosylaminoimidazole synthetase (AIRS). The YACs were characterized by restriction mapping and by in situ hybridization of cosmid subclones containing the YAC ends to human metaphase chromosomes. One of the YACs contains co-cloned non-contiguous DNA whereas the other appears to have a single 600 kbp insert from 21q22.1, the location of the GART gene. A restriction map of the gene was obtained from two cosmid subclones which together span the 40 kb gene. The gene is functional when YAC DNA is transferred into GARS- or GARS-and-AIRS-deficient Chinese Hamster Ovary cells. The gene transfer was carried out both by lipofection using purified yeast DNA and by fusion between yeast spheroplasts and the hamster cells. Restriction analysis of DNA from cell lines whose purine auxotrophy was complemented by the YAC showed that with either method a complete and unrearranged copy of the gene can be transferred. The majority of the fusion cell lines appear to contain at least 80% of the YAC.

Acyltransferases↗
PubMed1991

Molecular characterization of a patient with del(1)(q23-q25).

We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23-1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23-q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000-16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21-1q25.

Abnormalities, Multiple↗
PubMed1991

Isolation of a human cDNA encoding amidophosphoribosyltransferase and functional complementation of a CHO Ade-A mutant deficient in this activity.

We report here the isolation of a human cDNA encoding the first step in de novo purine biosynthesis, amidophosphoribosyltransferase (PRAT). The human PRAT cDNA was isolated by complementation of a Saccharomyces cerevisiae ade4 mutant deficient in PRAT enzymatic activity. The identity of the isolated cDNA, designated pAdeA-3, was confirmed by several independent methods. Genomic DNA sequences homologous to pAdeA-3 show coordinate segregation with the hypoxanthine nutritional requirement in Chinese hamster ovary (CHO) cell Ade-A-human hybrids, segregants of these hybrids, and irradiation reduction hybrids. The PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT cDNA after insertion into a mammalian expression vector was capable of correcting the PRAT enzyme deficiency in CHO Ade-A mutants. This correction was monitored by both cell-free PRAT assays and in vivo phosphoribosylformylglycinamide (FGAR) accumulation studies. FGAR accumulation is a classic method for assessment of the early steps of purine nucleotide biosynthesis. Two of the isolated transformants, designated PRAT-1 and PRAT-2, exhibited 22% and 53%, respectively, of wild-type CHO K1 PRAT enzymatic activity using a cell-free enzyme assay. These same two transformants plus an additional transformant, designated PRAT-13, showed FGAR accumulations of 150%, 260%, and 140%, respectively, compared to the levels of accumulation seen in CHO K1. Transformants PRAT-1 and PRAT-2 both contained a mRNA species recognized by the PRAT cDNA of identical size to a mRNA species in human fibroblasts homologous to the PRAT cDNA. This observation, along with the functionality of the cDNA in both yeast and CHO cells deficient in PRAT activity, suggests the isolated cDNA is full length.

Amidophosphoribosyltransferase↗
PubMed1991

Expression of a human cDNA encoding a protein containing GAR synthetase, AIR synthetase, and GAR transformylase corrects the defects in mutant Chinese hamster ovary cells lacking these activities.

The isolation of a human cDNA encoding the multifunctional protein containing GAR synthetase, AIR synthetase, and GAR transformylase by functional complementation of purine auxotrophy in yeast has been reported. Chinese hamster ovary (CHO) cell mutant purine auxotrophs deficient in GAR synthetase (Ade-C) or AIR synthetase plus GAR transformylase (Ade-G) activities were transfected with this human GART cDNA subcloned into a mammalian expression vector. This restored 49-140% of the activities of GAR synthetase, AIR synthetase, and GAR transformylase in transfected cells when compared to wild-type CHO K1 parental cells. Study of one stably expressing transfectant, AdeC2, revealed that the human GART cDNA was incorporated into the CHO genome. The enzyme activities appear to be associated with an expressed protein of 110 kDa, very similar to that of purified human GART trifunctional enzyme. The Ade-C mutant shows reduced amounts of GART mRNA compared to CHO K1 and a protein of apparently reduced size, results consistent with the purine requirement and enzyme deficiency observed in the mutant. These experiments provide definitive evidence that the human GART cDNA encodes and can direct the production of active human GART trifunctional protein in mammalian cells. They also provide important evidence that the Ade-C and Ade-G mutants of CHO cells are defective in this gene.

Acyltransferases↗
PubMed1991

Mapping of a locus correcting lack of phosphoribosylaminoimidazole carboxylase activity in Chinese hamster ovary cell Ade-D mutants to human chromosome 4.

The human phosphoribosylaminoimidazole (AIR) carboxylase locus has been until this report one of the genes encoding purine biosynthetic enzymes that had not been assigned to an individual human chromosome. Characterization of Chinese hamster ovary (CHO) cell mutant Ade-D showed that the cell line was unable to produce IMP and accumulated AIR. CHO Ade-D cells were fused with normal human lymphocytes utilizing inactivated Sendai virus and the resulting hybrid cell lines were selected for purine prototrophy. Cytogenetic analysis showed a 100% concordance value for chromosome 4. Two of the isolated subclones contained only the long arm of chromosome 4 translocated onto a CHO chromosome, providing evidence for a regional assignment of the Ade-D gene to the long arm of chromosome 4. Two of the subclones containing chromosome 4 were subjected to the BrdU visible light segregation. All of the isolated purine auxotrophic cell lines showed a loss of the q arm of chromosome 4. The localization of the Ade-D locus to the long arm of chromosome 4 may reveal further clustering of the mammalian purine genes since the Ade-A locus has previously been regionally assigned to 4pter-q21.

Animals↗