Naturally occurring suppressor mutants derived from tRNATrp genes have never been identified in S. cerevisiae. Oligonucleotide-directed mutagenesis was used to generate potential ochre and opal suppressors from a cloned tRNATrp gene. In vitro transcription analyses show the ochre suppressor form of the gene, TRPO, accumulates precursors and tRNA in amounts comparable to the parent. The opal suppressor, TRPOP, accumulates 4-5 fold less tRNA. Both forms of the gene are processed and spliced in vitro to produce tRNAs with the expected base sequences. The altered genes were subcloned into yeast vectors and introduced into yeast strains carrying a variety of amber, ochre, and opal mutations. When introduced on a CEN vector, neither ochre nor opal suppressor forms show suppressor activity. Deletion of the CEN region from the clones increases the copy number to 10-20/cell. The opal suppressor form shows moderate suppressor activity when the gene is introduced on this vector, however, the ochre suppressor form exhibits no detectable biological activity regardless of gene copy number. Northern blot analyses of the steady state levels of tRNATrp in cells containing the high copy-number clones reveal 20-100% increases in the abundance of tRNATrp.

To monitor in vivo transcription and chromatin structure of yeast tRNA genes, we constructed a synthetic tRNA gene that can be used as a reporter. Constructs in which this synthetic tRNA gene is combined with different flanking regions can be integrated into the genome as single copies. The artificial tRNA gene is tagged by the insertion of an intron-like sequence that cannot be spliced out from the precursor and transcripts can thus be identified and quantitated. By several criteria, the artificial tRNA gene behaves like a resident tRNA gene. By measuring the accessibility towards DNaseI in chromatin, we found that the artificial tRNA gene exhibits the same characteristic pattern as resident tRNA genes. Three DNaseI-sensitive sites across the transcribed part of the gene and the immediate flanking regions reflect the formation of the stable transcription complex; positioned nucleosomes are observed in the upstream flanking region. We are confident that the system we have established will prove useful for studying regulatory aspects of tRNA gene expression as well as aspects of pre-tRNA processing and splicing.

We have investigated the transcriptional regulation of the human embryonic zeta-globin gene promoter. First, we examined the effect that deletion of sequences 5' to zeta-globin's CCAAT box have on zeta-promoter activity in erythroid cell lines. Deletions of sequences between -116 and -556 (cap = 0) had little effect while further deletion to -84 reduced zeta-promoter activity by only 2-3-fold in both transiently and stably transfected erythroid cells. Constructs containing 67, 84 and 556 bp of zeta-globin 5' flanking region linked to a beta-galactosidase reporter gene (lacZ) and hypersensitive site -40 (HS-40) of the human alpha-globin gene cluster were then employed for the generation of transgenic mice. LacZ expression from all constructs, including a 67 bp zeta-globin promoter, was erythroid-specific and most active between 8.5 and 10.5 days post-fertilisation. By 16.5 days gestation, lacZ expression dropped 40-100-fold. These results suggest that embryonic-specific activation of the human zeta-globin promoter is conferred by a 67 bp zeta-promoter fragment containing only a CCAAT and TATA box.

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