Date of Award

1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Simon H. Chang

Abstract

Phosphofructokinase (ATP:Fructose-6-phosphate 1-phospho-transferase, EC 2.7.1.11, PFK) catalyzes the phosphorylation of fructose 6-phosphate to generate fructose 1,6-bisphosphate, the rate-limiting step of glycolytic pathway. Li et al. (1990) have identified two cDNAs for the rabbit muscle PFK with an identical coding regions but different 5$\sp\prime$ untranslated regions (UTRs). In this dissertation research, six cDNAs for the rabbit muscle PFK mRNAs were cloned. These cDNAs are different in their 5$\sp\prime$ UTRs but have an identical open reading frame. The transcription initiation sites of two downstream promoters of this gene have been determined. A proximal promoter (Pa) is TATA-less and 86 bp upstream of the ATG start codon. The mRNA expressed from this promoter has been detected in skeletal muscle and heart tissues. The second promoter (Pb) is skeletal muscle specific and produces two mRNAs (mRNA-b1 and mRNA-b2) by alternative splicing. Part of the 5$\sp\prime$ UTR in mRNA-b1 is spliced out to yield mRNA-b2. A fourth mRNA (mRNA-c) is also skeletal muscle specific. An additional two mRNAs (mRNA-d1 and -d2) are produced by alternative splicing. An exon of 106 bp was retained in mRNA-d1, but not in mRNA-d2. Interestingly, mRNA-d1 was detected in skeletal muscle, heart, and brain, but not in liver and kidney tissues. Its splicing variant, mRNA-d2 is detected in all five tissues examined. Therefore, the 106 bp exon is retained in a tissue-specific manner. Since the 5$\sp\prime$ ends of mRNA-c, -d1, and -d2 are upstream of promoters a and b, these mRNAs can not be produced by these promoters. Therefore, rmPFK expresses its six mRNAs by at least three promoters and alternative splicing. During the course of this research, I have developed a protocol for primer extension at higher temperature. In primer extension using M-MLV reverse transcriptase, a secondary structure of the RNA template was found to be problematic. I therefore optimized conditions for primer extension using thermostable DNA polymerase from Thermus thermophilus at high temperature. The primer extension at high temperature (65$\sp\circ$C) using this enzyme reduced the artifact caused by the secondary structure in the RNA template.

Pages

137

Share

COinS