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M Opper

Publications and source records attributed to M Opper.

At least 19 recordsLinked to original sources
PubMed1994

Biochemical properties of recombinant human beta-glucuronidase synthesized in baby hamster kidney cells.

The cDNA sequence encoding human beta-glucuronidase [Oshima, Kyle, Miller, Hoffmann, Powell, Grubb, Sly, Troplak, Guise and Gravel (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 685-689] was expressed in baby hamster kidney (BHK) cells. After purification from the culture supernatant in one step by use of immunoaffinity chromatography, the biochemical properties of the enzyme were examined. With a pH optimum of 4.0, a Km of 1.3 mM and thermal stability up to 68 degrees C, this protein has characteristics very similar to those described for beta-glucuronidase from human placenta [Brot, Bell and Sly (1978) Biochemistry 17, 385-391. However, the recombinant product has several structural properties not previously reported for beta-glucuronidase isolated from natural sources. First, recombinant beta-glucuronidase is synthesized as a tetramer consisting of two disulphide-linked dimers. As can be inferred from the cDNA sequence, the enzyme possesses five cysteine residues after cleavage of the signal peptide. By introducing a C-terminal truncation, we eliminated the last cysteine at position 644. In the mutant, covalent linkage between two monomers is no longer observed, indicating that Cys-644 is involved in intermolecular disulphide-bond formation. The functional role of the disulphide bond remains elusive, as it was shown that (i) intracellular transport of the mutant is not impaired and (ii) it is still able to form an enzymically active tetramer. A second feature that has not previously been observed for beta-glucuronidase from any origin is the existence of two enzymically active species for recombinant beta-glucuronidase, when examined by gel filtration on a TSK 3000 column. With apparent molecular masses of 380 kDa and 190 kDa we propose that they represent tetramers and dimers respectively. Partial N-terminal sequencing and electrophoresis under denaturing conditions revealed that the dimers consist of subunits that have been proteolytically processed at their C-terminus losing 3-4 kDa in peptide mass. Controlled proteolysis demonstrates that the enzyme's overall protein backbone as well as its activity are resistant to a number of proteases. Only the C-terminal portion is susceptible to protease action, and the disulphide-linked form is readily converted into non-disulphide-bonded subunits. Pulse-chase analysis shows that human beta-glucuronidase remaining intracellular in BHK cells after synthesis undergoes a similar proteolytic processing event, i.e. a reduction in mass of 3-4 kDa.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Molecular basis for the regulation of cell fate by the lethal (2) giant larvae tumour suppressor gene of Drosophila melanogaster.

Tumour suppressor genes act as recessive determinants of cancer. Their function is required for normal cell growth and differentiation during development. When both alleles of these developmental genes are inactivated, cell growth becomes unrestricted. In Drosophila, a series of genes have been identified which when mutated produce tissue-specific tumours. Of these the lethal(2)giant larvae (l(2)gl) gene is the best studied. Homozygous l(2)gl mutations cause the development of malignant tumours in the brain and the imaginal discs. Genomic DNA from the l(2)gl locus has been cloned, introduced back into l(2)gl mutant animals by P-element-mediated transformation and shown to restore normal development. The nucleotide sequence of the l(2)gl gene (13.1 kb) has been determined, as well as the sequences of the two classes of transcripts. These transcripts encode two polypeptides of 127 kDa and 78 kDa, respectively. Both proteins have been immunologically identified. Analyses of the spatial distribution of both l(2)gl transcripts and proteins revealed that during early embryogenesis the l(2)gl gene is uniformly expressed in all cells and tissues. In late embryos, the l(2)gl expression becomes gradually restricted to tissues presenting no morphological or neoplastic alteration in the mutant animals. Further mosaic experiments pointed out that the critical period for the establishment of tumorigenesis is limited to early embryogenesis at a time when the l(2)gl expression is most intense in all cells.

Animals↗
PubMed1987

Structure of the l(2)gl gene of Drosophila and delimitation of its tumor suppressor domain.

We have previously cloned lethal(2)giant larvae, a tumor-suppressor gene of Drosophila that normally controls cell proliferation and/or differentiation in the optic centers of the brain and the imaginal discs. Here we describe the structure of the l(2)gl genes as determined by sequencing genomic and cDNA clones. The structure of the cDNAs indicates the use of alternative splicing, either in the 5' untranslated exons or in the 3' coding exons. Thus the gene encodes two putative proteins of 1161 and 708 amino acids, p127 and p78, respectively, differing at their C termini. A 3'-truncated l(2)gl transposon that leaves the coding sequence of p78 intact but deletes 141 residues of p127 was capable of suppressing tumor formation in l(2)gl-deficient animals. These results suggest that the putative p78 protein is effective in controlling cell proliferation and/or differentiation.

Amino Acid Sequence↗