Identification and functional analysis of a chondrocyte-specific internal promoter of the type III collagen gene / Yejia Zhang.

Format:

Book

Manuscript

Thesis/Dissertation

Author/Creator:

Zhang, Charley Yejia.

Contributor:

Adams, Sherrill L., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Cell and Molecular Biology.

Academic Dissertations as Topic.

Medical Subjects:

Cell and Molecular Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Cell and molecular biology.

Cell and molecular biology--Penn dissertations.

Physical Description:

ix, 73 pages : illustrations ; 29 cm

Production:

1998.

Summary:

Type III collagen is not found in normal growth cartilage. However, we demonstrated previously that embryonic chick chondrocytes contain an unusual transcript derived from the type III collagen gene that initiate at an internal site within intron 23. Use of this internal start site results in replacement of exons 1-23 with a chondrocyte-specific exon (exon 23A) and a change in the translational reading frame; thus the alternative transcript does not encode type III collagen. We have isolated a genomic DNA fragment containing exon 23A by screening a chicken genomic DNA library. The sequences preceding exon 23A did not contain any recognizable promoter elements, in that there was no TATA box or CCAAT box. Thus we initially performed RNase protection and primer extension assays, to confirm that no additional sequences were present at the 5$\sp\prime$ end of the alternative transcript. We subsequently demonstrated that a DNA fragment consisting of 518 bp of intron 23 and 41 bp of exon 23A is highly active in chondrocytes, but appears to be repressed in fibroblasts. Thus we have isolated an internal promoter of the type III collagen gene that appears to be responsible for production of the 4 kb alternative transcript.

Analysis of 5$\sp\prime$ end deletion mutants of the internal promoter suggested that the sequences between nucleotides $-$145 and $-$64 are responsible for determining the chondrocyte-preferential activity of this promoter. We subsequently used linker substitution mutagenesis and electrophoretic mobility shift assays to demonstrate that the transcription factor AP1 binds to a site at $-$122 to $-$116 that is responsible in part for the high level of activity of this promoter in chondrocytes, and also contributes to the low activity of the promoter in skin fibroblasts. Furthermore, we have identified an element between $-$73 and $-$56 that is responsible for repression of transcriptional activity in fibroblasts. One transcription factor that binds this site is similar or identical to the previously identified factor LBP-1.

Notes:

Supervisor: Sherrill L. Adams.

Thesis (Ph.D. in Cell and Molecular Biology) -- University of Pennsylvania, 1998.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 98-30024.

OCLC:

187471094