We have found that the RNA polymerase II transcription termination regions of genes from such diverse species as chickens, rabbits, and mice possess a highly conserved 20-nucleotide consensus sequence that we hypothesize is an important genetic element defining the 3' boundaries of vertebrate transcription units. To test this hypothesis we propose to determine if this sequence element is commonly observed at the 3' ends of a large number of eukaryotic structural genes. This will be done by using oligonucleotides which correspond to the consensus sequence element as hybridization probes to identify potential transcription termination sites associated with a number of other vertebrate structural gene clones. Transcription unit mapping will be used to determine if transcription terminates in restriction fragments which contain a copy of the 20-nucleotide consensus sequence element. Such a correlation would provide strong circumstantial evidence for a role of the consensus element in the process of transcription termination. In vivo functional tests of transcription termination, based on the use of recombinant adenovirus vectors or SV40-based replication vectors, will be developed to determine if the 20-nucleotide sequence element plays a functional role in the process of transcription termination. These studies will focus particularly on a functional analysis of the mouse beta-major globin and the dihydrofolate reductase terminator regions. Our analysis of the amplified dihydrofolate reductase domain has revealed evidence for the existence of three divergent transcription units located immediately in front of the dihydrofolate reductase gene: two in tandem and one embedded within another. This surprising model of transcription unit distribution and organizational arrangement will be rigorously tested for the dihydrofolate reductase locus. The generality of these features will be tested by an examination of transcription unit organization in the vicinity of the hamster CAD gene and the mouse adenosine deaminase gene. These studies should provide us with a better understanding of the distribution and organizational arrangement of transcription units in the mammalian genome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM030204-09
Application #
3277842
Study Section
Molecular Biology Study Section (MBY)
Project Start
1982-03-01
Project End
1992-02-28
Budget Start
1990-03-01
Budget End
1991-02-28
Support Year
9
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Baylor College of Medicine
Department
Type
Schools of Medicine
DUNS #
074615394
City
Houston
State
TX
Country
United States
Zip Code
77030
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Winston, J H; Hong, L; Akroyd, S et al. (1994) Diverse genetic regulatory elements are required to direct the proper tissue-specific and developmental expression of the murine adenosine deaminase gene. Adv Exp Med Biol 370:579-84
Kash, S F; Innis, J W; Jackson, A U et al. (1993) Functional analysis of a stable transcription arrest site in the first intron of the murine adenosine deaminase gene. Mol Cell Biol 13:2718-29
Mohamedali, K A; Guicherit, O M; Kellems, R E et al. (1993) The highest levels of purine catabolic enzymes in mice are present in the proximal small intestine. J Biol Chem 268:23728-33
Winston, J H; Hanten, G R; Overbeek, P A et al. (1992) 5' flanking sequences of the murine adenosine deaminase gene direct expression of a reporter gene to specific prenatal and postnatal tissues in transgenic mice. J Biol Chem 267:13472-9
Innis, J W; Moore, D J; Kash, S F et al. (1991) The murine adenosine deaminase promoter requires an atypical TATA box which binds transcription factor IID and transcriptional activity is stimulated by multiple upstream Sp1 binding sites. J Biol Chem 266:21765-72
Innis, J W; Kellems, R E (1991) A heat-labile factor promotes premature 3' end formation in exon 1 of the murine adenosine deaminase gene in a cell-free transcription system. Mol Cell Biol 11:5398-409
Guicherit, O M; Rudolph, F B; Kellems, R E et al. (1991) Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase. J Biol Chem 266:22582-7
Mifflin, R C; Kellems, R E (1991) Coupled transcription-polyadenylation in a cell-free system. J Biol Chem 266:19593-8

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