Structure and regulation of the gene encoding the alpha-subunit of GTP-binding regulatory protein Gz.

Format:

Book

Thesis/Dissertation

Author/Creator:

Zhang, Boyuan.

Contributor:

University of Pennsylvania.

Language:

English

Subjects (All):

Pathology.

Molecular biology.

0307.

0571.

Local Subjects:

0307.

0571.

Physical Description:

138 pages

Contained In:

Dissertation Abstracts International 57-11B.

System Details:

Mode of access: World Wide Web.

text file

Summary:

The alpha-subunit of the GTP-binding regulatory protein (G protein) Gz, alpha-z, exhibits a pattern of expression restricted primarily to cells of neuronal and hematopoietic origin. However, the molecular mechanisms governing the transcription of alpha-z gene have not been previously elucidated. In this thesis, the rat alpha-z gene structure was characterized and the regulatory elements directing gene expression were defined by reporter gene assays. The rat alpha-z gene comprises three exons and two large introns, and the 5$\sp{\prime}$-flanking sequence conforms to a CpG island. S1 nuclease protection and primer extension assays showed that transcription of the gene is initiated principally at two closely spaced sites, $-676$ and $-673,$ relative to the translation initiation codon.

To define elements of gene structure controlling expression of alpha-z, rat basophilic leukemia-2H3 (RBL-2H3) cells were transiently transfected by electroporation with up to 1.5 kilobase pairs of the 5$\sp{\prime}$-flanking region inserted into a vector encoding the human growth hormone. Measurements of growth hormone secreted into the medium demonstrated that the 1.5-kilobase-pair 5$\sp{\prime}$-flanking sequence contains a significant level of promoter activity. Progressive truncations of the 5$\sp{\prime}$-flanking sequence led to a doubling of reporter gene activity, indicating removal of an inhibitory sequence between $-1493$ and $-1058,$ followed by an abrupt decline in activity when a region between $-769$ and $-710$ (35-93 base pairs from the transcription start site(s)), deemed the basal promoter, was removed. This 59-base-pair basal promoter is highly (G+C)-rich and lacks an apparent TATA box or initiator (Inr) element homologies but does contain two conserved elements for transcription factor Sp1. GATA binding sequences, although found in the 5$\sp{\prime}$-flanking region, were irrelevant to expression, in spite of the fact that these regulatory elements are important in the regulated expression of other mast cell- and megakaryocyte-specific genes.

In contrast to the strict cell type-specificity of alpha-z mRNA expression, the alpha-z promoter showed activity in Rat-1a fibroblasts which do not express the endogenous alpha-z gene. Furthermore, no element in the 5$\sp{\prime}$-flanking region contributed to expression of the gene in a cell-specific context. These data demonstrate that the 5$\sp{\prime}$-flanking region of alpha-z gene contains a promoter that maintains the basal transcription but does not function in a cell-specific manner. These data also suggest that a CpG island gene such as that encoding alpha-z, whose expression is relatively specific, may retain features of a housekeeping (or constitutive) gene but develop novel mechanism(s) to regulate tissue specificity.

Notes:

Source: Dissertation Abstracts International, Volume: 57-11, Section: B, page: 6862.

Thesis (Ph.D.)--University of Pennsylvania, 1996.

Local Notes:

School code: 0175.

ISBN:

9780591205275

Access Restriction:

Restricted for use by site license.