Xinnan Niu

Publication Date

12-1999

Advisor(s) - Committee Chair

Claire Rinehart, Sigrid Jacobshagen, Cheryl Davis

Comments

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Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

Inhibition of genetic information flow can be accomplished by using antisense and truncated gene strategies. Generally speaking, the antisense and truncated gene sequences should correspond to a partial sequence of a target gene. By inhibiting the expression of a particular gene by antisense RNAs or disrupting the functional protein complex by addition of a truncated gene product, one can gain an understanding of the gene’s function. In this study, the glnA gene, encoding glutamine synthetase, was chosen as a positive control to study the function of the cysZ gene, which has a putative sulfate transport function. Three different sizes of glnA genes (glnA1139, glnA452, and glnA182) and one size of cysZ gene (cysZ726) were cloned and subcloned in the antisense or truncated gene orientations into the expression vector pAV. Escherichia. coli strain JM109 DE-3 cells then were transformed with the pAV vector carrying either the antisense or truncated glnA and cysZ genes. The results of this study show that both of the glnA and cysZ genes of E. coli JM109 DE-3 cells were inhibited after induction and expression of antisense or truncated glnA and cysZ products. Hence, the results of this study support the use of antisense or truncated gene technology as a valuable research tool in the analysis of target gene function and structure. Since it has been proposed that an ani-mRNA, which is complementary to the 5’ end of the mRNA, can inhibit translation maximally, all of the antisense glnA and cysZ genes used in this study contain a sequence complementary to the Shine-Dalgarno sequence (ribosome-binding region). It has been proposed that the formation of a duplex of RNAs at this position or location would efficiently inhibit the binding of ribosomes to the SD sequence. The results of the present study are consistent with this hypothesis, in that the target genes were inhibited by corresponding antisense RNAs. However, the most efficient inhibition of target genes by antisense RNAs were those constructs that were of a length short enough to prevent the formation of secondary structures that would decrease the formation of duplex RNAs. The most efficient disruption of target protein function by a truncated gene was obtained by those constructs that were truncated at the functional domain while keeping the interaction domain intact. During this study, it also was observed that the induced E. coli strain JM109 DE-3 cells could grow quickly after a long lag phase. This result indicates that there may be a second gene involved in glutamine synthetase activity. In the E. coli database, there is a gene (b1297) identified as a putative glutamine synthetase. It is possible that the expression of this gene rescues the transformed E. coli strain JM109 DE-3 cells after inactivation of the major GS gene by the antisense or truncated glnA gene.

The results of this research also strongly suggest that the cysZ gene (putative sulfate transport) plays a key role in E. coli growth and that the cysZ gene may be involved in the metabolism or transport of sulfate. The question of whether or not the cysZ gene is indeed involved in actual sulfate transport will require further investigation.

Disciplines

Biology | Life Sciences

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