R D Miller

University of Missouri - St. Louis, Saint Louis, Michigan, United States

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Publications (4)20.48 Total impact

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    ABSTRACT: We have sequenced 4.2 kb of the 5' flanking region of the human beta-glucuronidase gene, compared this sequence to the 5' upstream sequence reported for the murine gene, determined the transcription start sites of the human gene, and studied expression of human minigene deletion constructs in COS cells. The 200 bp immediately 5' to the translation initiation codon have a high G + C content (72%) and contain no TATA box, two properties commonly associated with "housekeeping genes." The sequence 5' to -200 bp contains seven Alu repetitive elements which account for more than 50% of this flanking sequence. From deletion analysis of minigene constructs, 200 bp of 5' sequence appeared sufficient for maximal expression in transfected COS cells. S1 nuclease protection analysis showed that transcription initiates from a cluster of sites around -30 bp in all tissues examined. In some cases, a low but detectable level of transcription also initiates 126 bp upstream of the ATG. Inspection of the sequence surrounding both start sites revealed some similarity to the recently described "initiator" transcriptional control element (S.T. Smale and D. Baltimore (1989), Cell 57: 103-113). Comparison of the 5'flanking sequence with that available from the murine beta-glucuronidase gene reveals only one 28-bp highly conserved region, which surrounds the -126 start site.
    Genomics 09/1991; 10(4):1009-18. · 3.01 Impact Factor
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    ABSTRACT: We have isolated a cosmid clone that contains GUSB, the human gene encoding beta-glucuronidase. The 21-kb gene contains 12 exons ranging from 85 to 376 bp in length. Exon 6 corresponds to the 153-bp deletion in the shorter of two types of cDNAs reported earlier, supporting the hypothesis that this cDNA arose by alternate splicing leading to exon skipping. The insert contains 4.2 kb of sequence upstream from the first exon and 6 kb 3' of the last exon. The clone expresses human beta-glucuronidase in stably transformed rat XCtk- cells. Comparison of the human gene organization with that recently reported for the murine beta-glucuronidase gene revealed that the intron/exon boundaries are identical. In the splice junctions, the most highly conserved regions are those identified as consensus sequences, and these are at least as highly conserved as bases encoding the translated portion of the gene.
    Genomics 07/1990; 7(2):280-3. · 3.01 Impact Factor
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    ABSTRACT: A cDNA for rat liver beta-glucuronidase was isolated, its sequence determined and its expression after transfection into COS cells studied. The deduced amino acid sequence of the rat liver clone showed 77% homology with that from the cDNA for human placental beta-glucuronidase and 47% homology with that deduced from the cDNA for Escherichia coli beta-glucuronidase. Several differences were found between the cDNA from rat liver and that previously reported from rat preputial gland. Only one change leads to an amino acid difference in the mature enzyme. A chimeric clone was constructed by using a fragment encoding the first 18 amino acid residues of the signal sequence from the human placental cDNA clone and a fragment from the rat clone encoding four amino acid residues of the signal sequence, all 626 amino acid residues of the mature rat enzyme, and all of the 3' untranslated region. After transfection into COS cells the chimeric clone expressed beta-glucuronidase activity that was specifically immunoprecipitated by antibody to rat beta-glucuronidase. The Mr value of 76,000 of the expressed gene product was characteristic of the glycosylated rat enzyme. It was proteolytically processed in COS cells to Mr 75,000 6 h after metabolic labelling. At least 50% of the expressed enzyme was secreted at 60 h post-transfection, but the secreted enzyme did not undergo proteolytic processing. These results provide evidence that the partial cDNA isolated from a rat liver library contains the complete coding sequence for the mature rat liver enzyme and that the chimeric signal sequence allows normal biosynthesis and processing of the transfected rat liver enzyme in COS cells.
    Biochemical Journal 04/1988; 250(2):547-55. · 4.65 Impact Factor
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    ABSTRACT: We report here the cDNA sequence for human placental beta-glucuronidase (beta-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. We also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH2-terminal amino acid sequence determined for human spleen beta-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human beta-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human beta-glucuronidase, demonstrate the existence of two populations of mRNA for beta-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.
    Proceedings of the National Academy of Sciences 03/1987; 84(3):685-9. · 9.81 Impact Factor

Publication Stats

128 Citations
20.48 Total Impact Points

Institutions

  • 1991
    • University of Missouri - St. Louis
      Saint Louis, Michigan, United States
  • 1990
    • Washington University in St. Louis
      San Luis, Missouri, United States
  • 1988
    • St Louis University Hospital
      San Luis, Missouri, United States