Horizons in biochemistry and biophysics最新文献

Metallothioneins are a family of ubiquitous, cysteine rich proteins, whose amino acidic and genomic sequences have been highly conserved during evolution. MT synthesis is induced by heavy metals, glucocorticoids and a bacterial lipopolysaccharide in vivo and in vitro. MT forms stable complexes with heavy metals. One MTIIA gene, four MTI class genes and five pseudogenes have been isolated in humans. The cluster of MT genes is located on chromosome 16. The cloned, transfected genes retain metal inducibility. The first 150 bp of the 5' flanking region of mouse and human MT genes are essential for transcription and metal regulation. Two control regions have been identified. The distal region, between -151 and -78 is essential for efficient transcription and binding of cellular factor(s) which regulates MT gene expression. In Menkes' disease, a lethal X-linked recessive disorder, copper accumulates intracellularly bound to MT. Low doses of copper induce MT synthesis in Menkes' fibroblasts, but not in normal controls. Transfection experiments using the mouse MTI promoter fused to CAT show that the effect of copper in MT transcription is in trans. Menkes' cells are more sensitive to copper than normal controls and respond to copper poisoning by synthesizing two heat-shock like proteins. A mutation affecting copper transport or metabolism is discussed.

The X-linked human Pgk gene has been cloned and partially characterized, and some preliminary results have been obtained regarding active vs. inactive gene comparisons of chromatin structure and methylation patterns. As yet we can say nothing definitive about what role, if any, these differences may play in X inactivation. The studies showing that DNA from the inactive X chromosome in mature somatic cells does not function in transformation of the Hprt gene strongly imply modification of the inactive X chromosome at the DNA level. However, methylation studies with the Hprt, Gd Pgk genes have revealed a complexity of methylation patterns including hypermethylation of parts of the active X gene. Resolution of just what difference is critical in expression, differentiating between cause and effect, and extrapolating to the spreading and initiation aspects of X inactivation are still, unfortunately, long-range goals. The Pgk system may be of special value in unraveling some of these difficult questions. A unique autosomal Pgk locus exists and should allow an informative comparison between an X-linked housekeeping gene and an autosomal, tissue-specific gene encoding proteins of identical enzymatic function. The proximity of Pgk to the X-inactivation control center may be useful in identifying the starting point of this very important event in early mammalian development.

Human HPRT deficiency leads to two major forms of human disease. Partial enzyme deficiency results in gouty arthritis, while an almost complete deficiency leads to the Lesch-Nyhan disease. The latter is characterized by severe neurological dysfunction in addition to gouty arthritis, including retardation, choreoathetosis and aggressive and compulsive self-mutilation. The biochemical basis for the neurological symptoms is not understood. The human and mouse cDNA (RNA copy) genes have been isolated and sequenced. In addition, the amino acid sequence of the human protein has been directly determined. The human and mouse proteins differ at 7 amino acids out of the total, (including the N terminal methionine, which is processed off during maturation) of 218. There are 42 out of 654 nucleotide differences between the human and mouse genes in the amino acid coding region. The mouse genomic structure has been determined. It has 9 exons and 8 introns with a total size of approximately 36 kb. The human gene is very similar with identical intron-exon junction points and approximately the same total gene size. Both mouse and human presumed promotor region at the 5' end, lack a recognizable promotor in the form of a "TATAA" box and are very G-C rich, though not the same. This may be a feature of most "housekeeping" genes. HPRT gene point mutations in three gouty arthritis and one Lesch-Nyhan patient have been identified by peptide sequencing. Six gross gene rearrangements have been identified in Lesch-Nyhan HPRT genes. However it is likely that most mutations are point mutations or small deletions. So far all gene mutations identified are different from all others. The gene has been engineered into retrovirus vehicles which allows its efficient introduction into a wide variety of cells, including mouse marrow stem cells. This may allow treatment of Lesch-Nyhan patients as a model of gene therapy.