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Nucleic Acids Research, 1983, Vol. 11, No. 24 8609-8624
© 1983

MOLECULAR BIOLOGY

Molecular cloning, sequence analysis and in vitro expression of a rat tRNA gene cluster

Diane R. Makowski1, Robert A. Haas, Kevin P. Dolan2 and Dezider Grunberger3

Institute of Cancer Research, Department of Biochemistry and the Division of Environmental Sciences, College of Physicians and Surgeons, Columbia University New York, NY 10032, USA

3To whom reprint requests should be sent. This investigation was supported by Grant CA31696 and F32 CA06897 from the National Institutes of Health, DHHS, and CU 50180301, the National Foundation for Cancer Research

Received September 23, 1983. Revised November 14, 1983. Accepted November 14, 1983.

A rat genomic DNA fragment containing a tRNA gene cluster was isolated from a lambda phage library. Hybridization and nucleotide sequence analysis revealed the presence of a 83 bp tRNALeuCUG gene and a 72 bp tRNAAspGUG gene. Both genes possessed intact coding regions and putative transcription termination signals at their respective 3' ends. In vitro transcription analysis of the two subcloned genes in a HeLa cell S-100 system demonstrated the specific synthesis of a number of RNAs by RNA polymerase III. Studies carried out in the presence of {alpha}-amanitin showed that the larger RNAs are precursors for the final processed transcripts of the tRNALeu and tRNAAsp genes, respectively. Further nucleotide sequence analysis of the cluster revealed the presence of tRNAGly and a tRNAGlu pseudogenes with missing areas within their coding regions which are essential for transcription by RNA polymerase III. Within the region of DNA between the tRNALeu and tRNAAsp genes is a sequence which is 65% homologous to a region of the rat B1 element. The significance of this latter structure within the gene cluster is unknown.

1Present Address: Institute of Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.

2Present address: Dept. of Biology, New York University, New York, N.Y. 10003


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