Source: UNIV OF CALIFORNIA (VET-MED) submitted to
UNIQUE PROMOTER REGULATING TROPHOBLASTIC DIFFERENTIATION
 
PROJECT DIRECTOR: Conley, A. J.
 
PERFORMING ORGANIZATION
POPULATION HEALTH AND REPRODUCTION
UNIV OF CALIFORNIA (VET-MED)
DAVIS,CA 95616
 
NON TECHNICAL SUMMARY:
 
OBJECTIVES: We have identified a unique palindromic promoter that dries expression of CYP17 and possibly other genes involve din the differentiation of the trophectoderm of, and the establishment of pregnancy by, pre-attachment porcine blastocysts. This project will: 1. Define the core sequence required for promoter activitya nd binding of nuclear transacting proteins that activate expression of porcine CYP17 and, 2. Clone and characterize the 66 kD transcription factor that activates the core promoter of CYP17 expressed in the trophectoderm of pre-attachment porcine blastocysts.
 
APPROACH: The basal promoter recently identified in the upstream region of the porcine CYP17 gene will be completely characterized by mutagenesis, and its activity detrmined by reporter assays. Binding by proteins in nuclear extracts will be examined by gel shift and Southwestern analyses, and the precision of transcription initiation will be verified by nuclease protection assays. The 66 kD protein that recognizes this element will be cloned either directly from an expresison library, by using a yeast one hybrid approach or by protein purificaiton and micro-sequencing techniques. Its expressionwill be further characterized in porcine trophoblast by gel shift, Southwestern blotting and in siut hybridization. These data will provide basic information on molecular mechanisms controlling trophoblastic differentiation that is essential for the successful establishment of pregnancy in domestic livestock.
 
CRIS NUMBER: 0180820 SUBFILE: CRIS
PROJECT NUMBER: CA-V*-PHR-6562-H SPONSOR AGENCY: CSREES
PROJECT TYPE: HATCH PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Jan 1, 1999 TERMINATION DATE: Dec 31, 2003

GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
311351010804.225%
311351010904.225%
311351011024.225%
311351010204.225%

CLASSIFICATION HEADINGS
KA311 - Animal Diseases
S3510 - Swine, live animal
F1020 - Physiology
F1080 - Genetics
F1102 - Mycology
F1090 - Immunology
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks


RESEARCH EFFORT CATEGORIES
BASIC (N/A)%
APPLIED 100%
DEVELOPMENTAL (N/A)%

KEYWORDS: differentiation; promoters (genetics); genetic regulation; animal genetics; in situ hybridization; swine; pregnancy; transcriptional regulation; transcription; gene expression; binding proteins; blastocysts; dna sequences; reporter genes; protein binding; protein purification; protein characterization; mutagenesis; gene analysis

PROGRESS: Jan 1, 1999 TO Dec 31, 2003
Trophoblastic differentiation is essential for the establishment and successful completion of pregnancy. This process proceeds in porcine pre-attachment blastocysts with the expression of the CYP17 gene that encodes for the enzyme 17 -hydroxylase/17,20-lyase cytochrome P450 (P450c17). The expression of this enzyme, exclusively in the trophectoderm layer, is associated with trophoblastic differentiation and is essential for conceptus survival in utero. We have cloned the entire CYP17 gene in the domestic pig, and have begun defining sequence elements in the regulatory region that are unique in driving trophoblastic expression. The specific aims of the project are: 1) To define the core sequence of the palindromic promoter driving CYP17 expression in porcine trophoblast, 2) To clone transcription activating protein(s) associated with the core palindromic promoter. Results We have defined a novel, non-TATA promoter element directing the assembly of a transcriptional complex at a position on the gene that is uniquely used for the expression of CYP17 in the porcine trophectoderm. The essential core element exists within a 34 base pair fragment of the porcine CYP17 gene, 142 bp 5' of the transcription start site utilized in gonadal and adrenal tissues. It has extensive homology to previously described 'initiator elements', but with a very different core structure. Site-directed mutagenesis experiments, together with gel shift and southwestern blotting experiments have defined the essential nucleotide bases associated with promoter activity. These do not correspond to those identified as core for initiator elements. Finally, we have only just begun screening of expression libraries, with a plan to use our defined promoter element, and inactive point mutants, as probes to clone the protein(s) that bind and thereby modulate the activity of his unique trophoblastic porcine CYP17 promoter. These experiments will completed, and the final goal of the project pursued further, even though the current project will be officially terminated with this report.

IMPACT: 1999-01-01 TO 2003-12-31 The placenta is the organ that determines the growth rate and well-being of the fetus, and diseases or conditions such as the large calf syndrome associated with cloned animals are believed to arise from derangements of placental development. The research described adds to our understanding of the transcriptional and other regulatory mechanisms that are unique to the placenta and therefore are likely to provide insight into diseases afflicting offspring before and after birth.

PUBLICATION INFORMATION: 1999-01-01 TO 2003-12-31
No publications reported this period

PROJECT CONTACT INFORMATION
NAME: (N/A)
PHONE: (N/A)
FAX: (N/A)