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" SEQUENCING, CLONING, IN VITRO AND IN VIVO EXPRESSION OF MAMMALIAN TRANSFER-RNA GENES "
M. A. Ahmed
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Latin Dissertation
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| Language of Document
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English
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| Record Number
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1112423
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| Doc. No
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TLpq303481717
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| Main Entry
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M. A. Ahmed
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| Title & Author
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SEQUENCING, CLONING, IN VITRO AND IN VIVO EXPRESSION OF MAMMALIAN TRANSFER-RNA GENES\ M. A. Ahmed
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| College
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The University of Oklahoma
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| Date
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1987
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| student score
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1987
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| Degree
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Ph.D.
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| Page No
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235
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| Abstract
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The complete nucleotide sequence of a 2.8 kb mouse genome DNA fragment containing both a tRNAlle and tRNASer gene has been determined. The tRNAlle gene coding sequence is identical to the sequence reported for the mouse tRNAlle, and the tRNASer gene coding sequence is identical to the sequence of rat liver tRNASer. Both genes are transcribed independently under in vitro conditions, and termination of transcription occurs within a cluster of 7T residues located 11 nucleotides downstream of the 3 terminus. By deleting a large portion of the 5 flanking region of both genes, the importance of the 5 flanking region for transcription of both tRNA genes was demonstrated. The transcription and processing data for both tRNA genes is consistent with an ordered tRNA precursor processing scheme in which the removal of the 5 extension is followed by the subsequent removal of the 3 trailer sequence. In an earlier work, it had been reported that the nucleotide sequence of a human tRNAAsn gene differed from the major mammalian tRNAAsn, since this gene contained an A rather than a G at position 47. Using the in vitro mutagenesis technique, the A{47} was converted to G{47} so that it corresponded to the major tRNAAsn gene. The results of in vitro and in vivo transcription experiments with both tRNAAsn genes indicate that both genes are transcribed and processed at the same rate and that the 5 terminus is processed before the formation of the mature 3 terminus. In vivo expression studies in the Xenopus oocyte system demonstrated no discernible difference in the rate of transport of the mature transcripts of the two tRNAAsn genes from the nucleus, and were consistent with the hypothesis that the change at position 47 is not involved either in the transcription, processing, or transport of transcripts. In a final series of experiments an attempt was made to express the human tRNAAsn gene in an in vivo yeast system. Although these yeast cells over-expressed a yeast tRNA gene which was also encoded on the yeast-E. coli shuttle vector, they failed to express the human tRNAAsn gene. These results suggest, first, that the internal control regions of tRNAAsn gene are not sufficient to initiate in vivo transcription and, second, that specific, unknown extragenic regions are required to initiate tRNA gene transcription in yeast.
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| Subject
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Biochemistry
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Pure sciences
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