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Biomedical subjects

T B Shows

Publications and source records attributed to T B Shows.

At least 307 records · Page 17Linked to original sources

The mucolipidoses: identification by abnormal electrophoretic patterns of lysosomal hydrolases.

The human mucolipidoses (ML) are characterized by abnormal activities and abnormal electrophoretic patterns of fibroblast lysosomal hydrolases. These altered mobility patterns can be used to confirm the clinical diagnosis of the four mucolipidoses. The mobility patterns of one nonlysosomal and seven lysosomal enzymes were tested in fibroblasts from two ML I (sialidosis type 2, infantile), fifteen ML II (I-cell disease), eight ML III (pseudohurler polydystrophy), and one ML IV patients. A single sialidosis type 2, juvenile, line was also examined. Characteristic mobility patterns were found which identify each of the four mucolipidoses. Both the ML I and sialidosis type 2 juvenile lines displayed anodal mobility patterns, but distinct differences between the two disorders were observed. Lysosomal hydrolases from ML II lines demonstrated reduced activities or had altered mobilities. Differing electrophoretic patterns demonstrated the presence of at least two groups within the clinical phenotype diagnosed as ML II, indicating heterogeneity. The ML III lines showed normal electrophoretic patterns for most lysosomal hydrolases. The ML IV line expressed normal mobilities for every enzyme studied, with a single exception. The electrophoretic patterns of only beta-hexosaminidase, acid phosphatase-2, alpha-galactosidase, and esterase A4 were sufficient to identify and distinguish the different mucolipidosis types. Electrophoretic variation was also seen in liver but not kidney extracts from three ML II patients. beta-Hexosaminidase and alpha-mannosidase B secreted into the medium by ML II and ML III fibroblasts had mobility patterns different from normal and from their intracellular patterns. These data suggest that the mucolipidoses are genetically distinct with heterogeneity within them.

Diagnosis, Differential↗

Assignment of the glyoxalase II gene (HAGH) to human chromosome 16.

The human and rodent forms of glyoxalase II (Hydroxyacylglutathione hydrolase, HAGH) can readily be separated by starch gel electrophoretic procedures. Fifty-one human-rodent somatic cell hybrid clones were examined for their human HAGH and for human enzyme markers whose genes are encoded on each autosome and the X chromosome. Sixteen clones were also examined for their human karyotypes. Human glyoxalase II segregated only with chromosome 16, demonstrating that the gene is located on this chromosome.

Animals↗

Assignment of LIPA, associated with human acid lipase deficiency, to human chromosome 10 and comparative assignment to mouse chromosome 19.

The genetics of lysosomal acid lipase (LIP) has been investigated in human-Chinese hamster and mouse-Chinese hamster somatic cell hybrids. Cellulose acetate electrophoresis of human fibroblast extracts demonstrated that LIP activity consists of three isozymes. A deficiency of LIP activity has been observed in Wolman's disease (WD), cholesterol ester storage disease (CESD), and I-cell disease (ICD); this deficiency was associated with only one LIP isozyme, LIPA. We have demonstrated concordant segregation between human LIPA and human chromosome 10 and its enzyme marker glutamate oxaloacetate transaminase-1 (GOT1) in cell hybrid clones. Previous evidence suggested the different mutations associated with WD and CESD to be in the structural gene which we assign to human chromosome 10, while a different gene, involved in the processing of LIPA, is altered in ICD. These results indicate that several types of gene products are involved in the final expression of LIPA. In mouse-Chinese hamster hybrid clones, mouse Lip-1 (homologous to human LIPA) was assigned to chromosome 19. Previously, mouse Got-1 has been assigned to chromosome 19. Thus, the LIPA-GOT1 linkage groups has remained intact during the 80 X 10(6) years of evolution that separates humans and mice.

Animals↗

The proopiocortin (adrenocorticotropin/beta-lipoprotein) gene is located on chromosome 2 in humans.

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related genes sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDHI), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin and ACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.

Animals↗

Leukocyte and fibroblast interferon genes are located on human chromosome 9.

At least eight leukocyte interferon genes (IFL) and the single fibroblast interferon gene (IFF) have been located on chromosome 9 in humans. In somatic cell hybrids of human and mouse cells containing a normal complement of mouse parental cell chromosomes but reduced numbers of human chromosomes, the human leukocyte and fibroblast interferon DNA sequences were present only when human chromosome 9 was also present.

Animals↗

The insulin gene is located on the short arm of chromosome 11 in humans.

The human insulin gene has been previously localized to chromosome 11. We have analyzed the human DNA sequences present in a human-mouse somatic cell hybrid line possessing a translocation involving human chromosomes 11 and X. These data indicate that the human insulin gene is located on the short arm of chromosome 11 in the region p13 leads to pter.

Animals↗

Genes for growth hormone, chorionic somatommammotropin, and growth hormones-like gene on chromosome 17 in humans.

The human genes for growth hormone (GH), chorionic somatomammotropin (CSH), and a third growth hormone-like gene (GHL) have been located on chromosome 17 in humans. DNA fragments of 2.6, 2.8, and 9.5 kilobase pairs containing GH, CSH, and GHL, respectively, were identified in human genomic DNA, and a 7.5-kilobase DNA fragment related to growth hormone DNA sequences was found in mouse cells. In somatic hybrids of human and mouse cells containing reduced numbers of human chromosomes, but a normal complement of mouse chromosomes, the mouse, 7.5-kolobase DNA fragment was always present, whereas the 2.6-, 2.8-, and 9.5-kilobase human fragments were present only when human chromosome 17 was also present.

Animals↗

Branched-chain aminotransferase deficiency in Chinese hamster cells complemented by two independent genes on human chromosomes 12 and 19.

Branched-chain aminotransferase (BCT) catalyzes the reversible transamination of the branched-chain alpha-keto acids to the branched-chain L-amino acids. Since branched-chain L-amino acids (L-isoleucine, L-leucine, and L-valine) are essential for cell growth, cells which lack BCT were unable to proliferate in media containing alpha-keto acids in place of the corresponding L-amino acids. CHW-1102, a Chinese hamster cell line, lacks BCT and does not grow in alpha-keto acid media. Somatic cell hybrids were made by the fusion of CHW-1102 (HPRT-) with several human cell lines and isolated on HAT medium. Growth assays of hybrid clones on alpha-keto acid selection media independent of the HAT selection medium indicated two cell hybrid phenotypes: either (1) the hybrid clone, like the parental CHW-1102, could not utilize alpha-keto acid media, or (2) the hybrid could proliferate on all three alpha-keto acid media. The ability of hybrid cells to proliferate on alpha-keto acid media correlated with the presence of either of two human genes which independently complemented the Chinese hamster deficiency. Two human genes. BCT1 assigned to chromosome 12 and BCT2 assigned to chromosome 19, were demonstrated to code for the expression of two molecular forms of BCT.

Amino Acids, Branched-Chain↗

Expression of H1 histone genes in mouse-human somatic cell hybrids.

The synthesis of H1 histones was studied in nine mouse-human somatic cell hybrid clones containing reduced numbers of human chromosomes. The entire human genome could be accounted for karyologically and by the use of functional assays for specific enzyme markers encoded by human chromosomes. Chromatographic resolution and peptide mapping of species-specific H1 histones failed to reveal human H1 histones to a level of about 1% of total in the nine clones. In addition to the species-specific extinction of human H1 histones, effects were seen on the quantity of mouse H1 histone subtypes produced in four of the nine clones. The remaining five clones produced H1 histones qualitatively and quantitatively identical with those of the mouse parent, which was common to all nine clones. The results suggest at least two levels of control for H1 histone gene expression.

Animals↗

Gene transfer and gene mapping in mammalian cells in culture.

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.

Animals↗

Somatic cell genetics of adenosine deaminase expression and severe combined immunodeficiency disease in humans.

The somatic cell hybrid method has been used to study the number and different types of human genes involved in the expression of adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) in normal cells and cells from a patient with ADA-deficient severe combined immunodeficiency disease (SCID). Genetic and biochemical characterization of ADA in SCID and the ADA tissue-specific isozymes in normal human cells indicates that additional genes, besides the ADA structural gene on chromosome 20, are involved in ADA expression. Human chromosome 6 encodes a gene, ADCP-1, whose presence is necessary for the expression of an ADA-complexing protein in human-mouse somatic cell hybrids [Koch, G. & Shows, T. B. (1978) Proc. Natl. Acad. Sci. USA 75, 3876-3880]. We report the identification of a second gene, ADCP-2, on human chromosome 2, that is also involved in the expression of the ADA-complexing protein. The data indicate that these two ADCP genes must be present in the same cell for that cell to express the complexing protein. Human-mouse somatic cell hybrids, in which the human parental cells were fibroblastss from an individual with ADA-deficient SCID, also required human chromosomes 2 and 6 to express the ADA-complexing protein, indicating that neither ADCP-1 nor ADCP-2 is involved in the ADA deficiency in SCID. The SCID-mouse hybrid cells expressed no human ADA even when human chromosome 20 had been retained. The deficiency of human ADA in these hybrids maps to human chromosome 20, and therefore is not due to the repression or inhibiton of ADA or its product by unlinked genes or gene products. We propose that the expression of the polymeric ADA tissue isozymes in human cells requires at least three genes: ADA on chromosome 20, ADCP-1 on chromosome 6, and ADCP-2 on chromosome 2. A genetic scheme is presented and the different genes involved in ADA expression and their possible functions are discussed.

Adenosine Deaminase↗

DNA-mediated cotransfer of unlinked mammalian cell markers into mouse L cells.

Purified DNA from three different types of mammalian cells was precipitated with calcium phosphate and added to mouse L cells deficient in thymidine kinase (TK). Donor DNA was prepared from three cell lines: (a) mouse cells transfected with UV-inactivated herpes simplex virus (HSV) type 1, or a purified fragment of HSV carrying the TK gene (b) human HeLa cells, and (c( CHO, a cell line derived from Chinese hamster ovaries. Several hypoxanthine-aminopterin-thymidine resistant colonies were isolated from each experiment. The origin of the TK that is expressed in these cells was studied by polyacrylamide gel electrohporesis, isoelectric focusing, or heat stability. The TK in all instances was of the donor origin. To determine the extent of gene transfer we have assayed the CHO and HeLa DNA transfectants for galactokinase (GALK), a marker closely linked to TK, and 25 other isozymes representing a large number of different chromosomes. No cotransfer of GALK was observed, indicating that the size of the transferred DNA segment is limited. We observed that, in one instance, esterase-D, an unlinked marker of Chinese hamster origin, was transferred along with TK. These experiments indicate that nonselected markers can be transferred by this method, although at a low efficiency.

Animals↗

Bioautographic visualization of aminoacylase-1: assignment of the structural gene ACY-1 to chromosome 3 in man.

A bioautographic assay was developed for the visualization of aminoacylase-1 (N-acylamino acid aminohydrolase, ACY-1; EC 3.5.1.14) after zone electrophoresis. Bioautography and species differences in electrophoretic mobility of ACY-1 made it possible to investigate the chromosome assignment of the gene for human ACY-1 using human--mouse somatic cell hybrids. Human ACY-1 segregated concordantly with beta-galactosidase-A (beta GALA; EC 3.2.1.23) but showed discordant segregation with 32 other markers representing 23 linkage groups. The beta GALA gene has been previously assigned to chromosome 3. From this evidence and confirming chromosome analyses, ACY-1 has been assigned to chromosome 3. A genetic polymorphism in the electrophoretic mobility of ACY was observed in mouse strains, demonstrating that this enzyme can be mapped in genetic crosses of Mus musculus.

Amidohydrolases↗

Genetics of cell fusion: human chromosome 10 assignment of a gene (FUSE) that promotes polykaryocyte formation.

FUSE, a human gene which promotes polykaryocyte formation, has been identified and examined in cocultivation assays between rat XC cells and human-mouse hybrids retaining different combinations of human chromosomes. Polykaryocyte formation was never detected when parental cells of hybrids were cocultivated with XC cells. Somatic genetic synteny analysis employing different hybrid sets demonstrated that FUSE was coexpressed with the chromosome 10 markers glutamate oxaloacetate transaminase (GOTs) and an external membrane protein (EMP-130). Cytogenetic analysis confirmed this assignment to human chromosome 10. FUSE was expressed by hybrids made with both human leukocytes and fibroblasts from several individuals, indicating the gene is found in different tissues and may be ubiquitous. Only XC cells were involved in polykaryocyte formation as demonstrated by 33258 Hoechst staining and the absence of heteropolymers between rat and cell hybrid multimeric enzymes. Evidence suggests that the gene FUSE produces a nondiffusible and noninfectious product that is associated with the human-mouse hybrid surface.

Animals↗