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J K Winkler

Publications and source records attributed to J K Winkler.

8 recordsLinked to original sources
PubMed1990

Altered regulation of protein disulfide isomerase in cells resistant to the growth-inhibitory effects of transforming growth factor beta 1.

A murine keratinocyte cell line that is resistant to the growth-inhibitory effects of transforming growth factor beta 1 (TGF beta 1) was examined for differential gene expression patterns that may be related to the mechanism of the loss of TGF beta 1 responsiveness. Cells that were resistant to the growth-inhibitory effects of TGF beta 1 (KCR cells) were derived from K-ras-transformed BALB/MK keratinocytes (KC cells). Using a subtractive hybridization procedure with KC and KCR mRNAs, we isolated a complementary DNA clone for murine protein disulfide isomerase (PDI). The mRNA for PDI is inhibited by TGF beta 1 treatment in the parental KC cells, but not in the TGF beta 1-resistant KCR cells. Similar PDI down-regulation also occurs in other TGF beta-sensitive cells, but not in a human pancreatic carcinoma cell line which is insensitive to the growth-inhibitory effects of TGF beta 1. The results suggest that misregulation of PDI, an important component of co- and posttranslational modification systems, may be involved in the mechanism by which some cells escape from the growth-inhibitory effects of TGF beta 1.

Animals↗
PubMed1988

Molecular cloning and nucleotide sequence for the complete coding region of human UMP synthase.

The last two steps in the de novo biosynthesis of UMP are catalyzed by orotate phosphoribosyltransferase (OPRT; orotidine-5'-phosphate:pyrophosphate phosphoribosyltransferase; EC 2.4.2.10) and orotidine-5'-monophosphate decarboxylase (orotidine-5'-phosphate carboxy-lyase; EC 4.1.1.23). In mammals these two activities are found in a single, bifunctional protein called UMP synthase. A human T-lymphoblastic cell cDNA library constructed in lambda gt10 was screened with a UMP synthase-specific rat cDNA probe. Human UMP synthase cDNAs were isolated and then used to select UMP synthase gene fragments. The complete coding sequence of the mRNA for UMP synthase was determined by analysis of overlapping cDNA and genomic fragments. One of the cDNAs appears to have been synthesized from an incompletely or alternatively processed form of the UMP synthase mRNA. This cDNA lacks a poly(A) tail and has an extended 3'-nontranslated region that hybridizes with larger forms of the UMP synthase mRNA. The UMP synthase protein is composed of 480 amino acids with a molecular weight of 52,199. The two activities of UMP synthase reside in distinct domains encoded by the 3' and 5' halves of the mRNA. The COOH-terminal 258 amino acids of the human UMP synthase protein contain the orotidine-5'-monophosphate decarboxylase catalytic domain. This region is highly homologous to the mouse orotidine-5'-monophosphate decarboxylase sequence. The NH2-terminal 214 amino acids contain the OPRT domain. There is amino acid homology between this protein domain and specific regions of the Escherichia coli OPRT. The human OPRT domain also contains the putative catalytic site common to other human phosphoribosyltransferases.

Amino Acid Sequence↗
PubMed1988

Analysis of UMP synthase gene and mRNA structure in hereditary orotic aciduria fibroblasts.

Hereditary orotic aciduria is an autosomal recessive disease in which there is a severe deficiency in the activity of the de novo pyrimidine pathway enzyme uridine 5'-monophosphate (UMP) synthase. UMP synthase is a bifunctional enzyme containing the two activities orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase, which catalyze the two-step conversion of orotic acid to UMP. Cell lines from three orotic aciduria patients have been characterized for UMP synthase gene and mRNA content. Restriction-enzyme analysis of DNA from the deficient cells revealed no changes in the gene structure compared with normal cell DNA structure. The amount of UMP synthase mRNA was not decreased, nor was there a detectable difference in the size of the UMP synthase mRNA in the deficient cells. Analysis of the mRNA by hybridization with a nearly full-length UMP synthase cDNA followed by S1 nuclease digestion showed no alteration in the mRNA structure. The UMP synthase activity of the deficient cells ranges from 2% to 7% of the normal cell level. The activity can be significantly increased by growing the deficient cells in barbituric acid. Our data indicate that UMP synthase gene transcription in the orotic aciduria cells produces the expected amount of a stable, correctly processed mRNA. The mRNA appears to code for a mutant enzyme that has reduced stability or altered kinetic properties.

Carboxy-Lyases↗