We describe the isolation and the sequence of the gene for the bifunctional enzyme dihydrofolate reductasethymidylate synthase (DHFR-TS; EC 1.5

We determined the nucleotide sequences of three mouse tRNA His genes and a tRNAGIl gene present in two different A clones. One A clone contained two tRNA His genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHIs and a tRNAGIy gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHiS if posttranscriptional modifications are not considered. Notably, the three tRNAHiS genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.Mammalian tRNA genes are reiterated and dispersed in the genome. Individual members of some mammalian tRNA gene families vary in respect to coding-or flanking-region sequences or both (26,37,41,45). Similar types of variation are also seen in tRNA genes isolated from amphibians (6), insects (e.g., see references 1, 10, 20), and yeasts (reviewed in reference 16). To obtain a detailed understanding of the mechanisms by which tRNA gene expression is regulated, it is necessary to determine the effects of intrafamilial sequence variation on gene activity.The efficiency of transcription, as measured by in vitro assays, is one level at which sequence variation can modulate tRNA gene activity. Eucaryotic tRNA genes contain highly conserved sequences within the coding regions that are absolutely required for transcription (4,14,19,42). These "internal" control regions appear to be sites of interaction of specific transcription factors with the gene (12,13,24,43,47 MATERIALS AND METHODSIsolation of X clones containing tRNAHiS genes. A recombinant DNA library containing DBA-2 mouse genomic DNA cloned in phage A Charon 4A (17) was plated, and the plaques were transferred to cellulose nitrate filters (BA85; Schleicher & Schuell, Inc., Keene, N.H.) by the method of Benton and Davis (2). Filters were hybridized with 5 x 106 cpm of 32P-labeled tRNAHi_specific probe ...

We determined the nucleotide sequences of three mouse tRNAHis genes and a tRNAGly gene present in two different lambda clones. One lambda clone contained two tRNAHis genes 600 base pairs (bp) apart in opposite orientations. The other clone contained a tRNAHis and a tRNAGly gene 569 bp apart in the same orientation. The coding regions of the three tRNAHis genes were identical to sequenced mammalian tRNAHis if posttranscriptional modifications are not considered. Notably, the three tRNAHis genes and a fourth gene previously sequenced by us contained within the flanking regions, various amounts of short, conserved 5' leader sequences and 3' trailer sequences directly abutting the coding regions. Otherwise the flanking regions were not homologous. Deletion mutants of one of the tRNAHis genes were constructed which contained 228, 99, 9, and 3 bp of the wild-type 5'-flanking region, respectively. Deletion of 5'-flanking sequences from positions -9 to -4 reduced transcriptional activity substantially (ca. fivefold) in a HeLa cell S-100 lysate. This effect was independent of the vector sequences in the deletion clone, implying that the region from -4 to -9 of the intact gene contains a positive modulatory element for transcription in vitro. The deletion mutant containing 3 bp of wild-type 5'-flanking sequence also had a greatly reduced ability to inhibit the transcription of a second tRNA gene in a competition assay. Thus, the normal 5'-flanking region influences the ability of the gene to form stable complexes with transcription factors. These data further indicate that a mammalian transcription extract is sensitive to 5'-flanking-region effects if a suitable tRNA gene is assayed.