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Degree

Doctor of Philosophy, Case Western Reserve University, Biology, 1990.

Abstract

A malic enzyme (L-malate, NADP⁺-oxidoreductase (decarboxylating), EC 1.1.1.40) cDNA clone derived from the goose uropygial gland was used to isolate homologous cDNA clones from duck liver and chicken liver cDNA libraries. These clones, in turn, were used to isolate and partially characterize additional cDNA clones derived from embryonic chick hepatocytes cultured in the presence of both insulin and thyroid hormone. One of these clones, pCME5, contains an insert of ∼2000 bp and represents >95 % of chicken malic enzyme mRNA. The availability of malic enzyme cDNA clones facilitated the isolation and partial characterization of a series of genomic clones constituting part of the malic enzyme locus in the chicken. These clones span ∼100 kb of nonoverlapping genomic DNA. Gaps exist between some clones, hence the exact length of the transcription unit is unknown. The 3′-most genomic clone, λCME1, contains a consensus sequence for polyadenylation, thus likely encodes the 3′ end of the mRNA. The 5′-most 254 nt of the cDNA are encoded by two exons that are separated by an intron of at least 39 kb. Hybridization and sequencing analyses of the genomic clones suggest the presence of at least 11 exons, thus at least 10 introns, within the malic enzyme gene The 5′-most genomic clone, λCME245, was shown by primer-extension and S1 nuclease analyses to encode the 5′end of the mRNA. Transcription initiates at multiple sites in liver and hepatocytes, but primarily at a site corresponding to the 5′ end of the cDNA in pCME5. Sequence analysis of the putative promoter region revealed the absence of "CCAAT box" and "TATA box" homologies, consistent with multiple sites of transcription initiation. The 5′ flanking region also lacks putative binding sites for the transcription factor Sp1. The 5′ flanking region contains distinct sequences that exhibit (a) similarity to T₃-response elements found in other T₃-responsive genes, (b) similarity to the AP-2 sites within the genes encoding rat prolactin and tyrosine aminotransferase, and (c) identity to the AP-2 site of human proenkephalin. Also identified were a putative binding site for the transcription factor AP-1 and a poly(pyrcdotpur) sequence which may be involved in the regulation of transcription.

Subject Headings

Biology, Molecular

Keywords

gene encoding malic enzyme chicken

Advisor

Alan G Goodridge

Pages

127p.

Document number: case1054664842
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=case1054664842

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