We have isolated and characterized a gene,fdh, from Neurospora crassa which is developmentally regulated and which produces formate dehydrogenase activity when expressed in Escherichia coli. The gene is closely linked (less than 0.6 kb apart) to the ku-S gene encoding mitochondrial leucyl-tRNA synthetase; the two genes are transcribed convergently from opposite strands. The expression patterns of these genes differ:fdh mRNA is found only during conidiation and early germination and is not detectable during mycelial growth, while ku-5 mRNA appears during germination and mycelial growth. The structure of thefdh gene was determined from the sequence of cDNA and genomic DNA clones and from mRNA mapping studies. The gene encodes a 375-amino-acid-long protein with sequence similarity to NAD-dependent dehydrogenases of the E. coli 3-phosphoglycerate dehydrogenase (serA gene product) subfamily. In particular, there is striking sequence similarity (52% identity) to formate dehydrogenase from Pseudomonas sp. strain 101. All of the residues thought to interact with NAD in the crystal structure of the Pseudomonas enzyme are conserved in the N. crassa enzyme. We have further shown that expression of the N. crassa gene in E. coli leads to the production of formate dehydrogenase activity, indicating that the N. crassa gene specifies a functional polypeptide.

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EMBL accession no. X54752 We are developing vectors based on the halobacterial plasmid pGRB1 (1) for use in H. halobium. To identify a region that could serve as a site for cloning without affecting plasmid maintenance, 212 pGRB1 transformants of H. halobium RI were screened for spontaneous insertion events in the plasmid. One derivative, pMPK29, was obtained, and the nucleotide sequence of the insert and target site was determined by dideoxy sequencing of both strands (Fig. 1). The 1068 bp insert has features typical of insertion elements, including a large open reading frame (334 aa) and a 15 bp inverted repeat at the ends of the element. A direct repeat of 7 bp flanks the element at the insertion site. No significant sequence homology was found between this and other halobacterial insertion elements, including ISHI (2), ISHJ. 8 (3), ISH2 (4), ISH26 (5), ISH50 (6), ISH5J (7), and ISH SI (8). Furthermore, no correspondence was observed with the restriction enzyme maps of ISH24 (9), ISH27, and ISH28 (10) or with the limited terminal sequence available for ISH24 (9) and ISH27 (11). The insertion in pMPK29 therefore represents a new H. halobium insertion sequence, which we designate ISHII. pGRB1 and related 1.8 kbp plasmids (12-15) contain an 1 kbp open reading frame and a region of four conserved hexanucleotide repeats 500-550 bp upstream. The insertion of ISHII in pMPK29 (position 189 of pGRBI, numbered as in (12)) does not interrupt the open reading frame or alter the spacing among the hexanucleotide repeats, consistent with a role of these regions in plasmid maintenance.

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNALU gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNAI"U gene. Extracts from HeLa or CV1 cells transcribed both tRNALeU genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNALu.Surprisingly, when the same tRNAI"U genes were introduced into CV1 cells, only pre-tRNAsLu were produced. The pre-tRNAsI"u made in vivo were of the same size and contained the same 5'-leader and 3'-trailer sequences as did pre-tRNAsLu made in vitro. Furthermore, the pre-tRNAsI"u made in vivo were processed to mature tRNAIeU when incubated with HeLa cell extracts. A tRNALeU gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNALu in CV1 cells is blocked at the level of 5'-and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAslu synthesized in CV1 cells are not transported to the appropriate site.