Altered RNA turnover in carcinogenesis. The diagnostic potential of modified base excretion.

Excretion of urinary modified nucleosides is frequently elevated in patients with oncogenic disease. Increases of urinary pseudouridine excretion are now demonstrated in patients with a variety of brain tumors. The potential use of urinary modified base excretion as a cancer marker is discussed and possible sources of the elevated nucleosides are detailed. The specific steps in RNA metabolism that result in increased levels of RNA nucleoside excretion are poorly understood. This knowledge will be necessary to understand the molecular mechanism and the clinical significance of urinary nucleoside excretion in treatment and diagnosis of oncogenic disease.

Purification of plasmid-expressed proteins which lack functional assay systems.

A general method for the purification of proteins whose genes are cloned into plasmid vectors, but whose biochemical and functional characteristics are unknown, is described. A cell-free transcription-translation system from Escherichia coli K-12 is used to synthesize in vitro radiolabeled protein expressed from recombinant plasmid vectors. The radiolabeled proteins are then fractionated and used as markers for the purification of nonradiolabeled proteins without recourse to functional assays. Biochemical analysis of the purified proteins can reveal information about their cellular localization, binding parameters, and physical, enzymatic, or regulatory properties. This information complements in vivo genetic analysis with the goal of identifying the gene and the function of its protein product. An example using this technique in which the product of the usg gene in the hisT operon of E. coli has been purified and biochemically characterized is described.

Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I.

The RNA modification enzyme, tRNA pseudouridine synthase I has been isolated in 95% purity from an Escherichia coli strain harboring a multicopy plasmid with a 2.3-kilobase pair insert from the hisT operon. Its molecular size, amino acid composition, and amino-terminal sequence correspond to those predicted by the structure and expression of the hisT gene. Enzyme activity, as measured by a 3H release assay, is unaffected by pretreatment of tRNA pseudouridine synthase I with micrococcal nuclease and is optimized by the addition of a monovalent cation and thiol reductant. The activity is inhibited by all tRNA species tested, including substrates, modified tRNAs, nonsubstrates, or tRNAs containing 5-fluorouridine. Binding of tRNA pseudouridine synthase I occurs with both substrate and nonsubstrate tRNAs and does not require a monovalent cation. Our findings are consistent with a multistep mechanism whereby tRNA pseudouridine synthase I first binds nonspecifically and then forms transient covalent adducts with tRNA substrates. In the absence of other proteins, purified tRNA pseudouridine synthase I forms psi at all three modification sites known to be affected in hisT mutants. The 36.4-kDa polypeptide product of the gene adjacent to hisT, whose translation is linked to that of tRNA pseudouridine synthase I, is not a functional subunit for tRNA pseudouridine synthase I activity, nor is it a separate synthase acting at one of the three loci.

The hisT-purF region of the Escherichia coli K-12 chromosome. Identification of additional genes of the hisT and purF operons.

A 9.7-kilobase pair segment of the Escherichia coli chromosome spanning the hisT and purF loci has been characterized. Six genes were identified in this region by complete DNA sequence analysis, in vivo expression in maxicells, and RNA transcript analysis. S1 nuclease analysis has demonstrated that some of these genes are part of the hisT or purF operons. Two of the newly identified genes, dedA and dedB, were localized immediately downstream of hisT in the hisT operon. Two other genes, denoted dedC and dedD, have been localized between the hisT and purF operons. The other two genes, dedE and dedF flank the purF gene. dedE has been previously described as the first gene in the purF operon (Makaroff, C.A., and Zalkin, H. (1985) J. Biol. Chem. 260, 10378-10387). dedF was localized downstream from purF and is part of the purF operon. In addition, dedF is homologous to the ubiX gene of Salmonella typhimurium. Adjacent to dedF is the E. coli homologue of the S. typhimurium argT locus encoding the lysine/arginine/ornithine-binding protein. All of the genes in this region of the chromosome were found to be transcribed in a counter-clockwise direction on the E. coli map which revises the direction of purF transcription.

Effect of retinoic acid and phorbol-12-myristate-13-acetate on glycosyltransferase activities in normal and transformed cells.

Retinoic acid was found to increase the activity of cytidine monophosphosialic acid:lactosylceramide sialyltransferase activity in a nontransformed clonal hamster cell line, NIL 8, and a virally transformed clone, NIL 8-HSV. The potent tumor promoter phorbol-12-myristate-13-acetate (PMA) had no significant effect on sialyltransferase activity in NIL 8 cells but stimulated this activity almost 6-fold when added to NIL 8-HSV cells. There was a synergistically additive effect on sialyltransferase activity when PMA was added to NIL 8 cells in concert with retinoic acid. On the other hand neither PMA nor retinoic acid had an appreciable effect on two other glycosyltransferases measured, uridine diphospho-N-acetylgalactosamine:globotriaosylceramide N-acetylgalactosaminyl-transferase and uridine diphosphogalactose:asialoagalactofetuin galactosyltransferase. Examination of sialyltransferase activity in a human epidermoid carcinoma cell line showed a large increase in enzyme activity in response to retinoic acid administration. Two nontransformed hamster cell lines had less basal sialyltransferase activity but also showed marked elevations after retinoic acid treatment. It is proposed that one of the molecular mechanisms underlying the biological effects of retinoic acid and PMA may be an increase in sialyltransferase activity. Possible regulatory mechanisms are discussed.

A program for the identification of tRNA-like structures in DNA sequence data.

A computer algorithm has been developed which identifies tRNA genes and tRNA-like structures in DNA sequences. The program searches the sequence string for specific base positions that correspond to the invariant and semi-invariant bases found in tRNAs. The tRNA nature of the sequence is confirmed by the presence of complementary base pairing at the tRNA's calculated 5' and 3' ends (which in situ constitutes the amino-acyl stem region). The program achieves greater than 96% accuracy when run against known tRNA sequences in the Genbank database. The program is modular and is readily modified to allow searching either a file or database. The program is written in "C" and operates on a D.E.C. Vax 750. The utility of the algorithm is demonstrated by the identification of a distinctive tRNA structure in an intron of a published bovine hemoglobin gene.

Structural features of the hisT operon of Escherichia coli K-12.

The DNA sequence of a 2,3-kilobase segment of the E. coli hisT operon was determined. Analysis of the sequence indicated that the upstream gene in the operon encodes a 36,364-dalton polypeptide, which runs aberrantly on SDS-polyacrylamide gels. The distal hisT gene encodes the tRNA modification enzyme, pseudouridine synthase I, which was shown to have a polypeptide molecular mass of 30,399 daltons. The DNA sequence was consistent with the phenotypes and hisT expression of mutant operons. Analysis of the sequence and genetic complementation experiments demonstrated that the upstream and hisT genes are evolutionarily, structurally, and functionally unrelated; however, translation signals for the two genes overlap, which is consistent with genetic evidence suggesting translational coupling. Codon usage in the upstream gene is radically different from the hisT gene and may underlie the differential expression observed from the operon. Gene-inactivation experiments and S1-mapping of in vivo transcripts indicated that the operon contains an additional upstream gene. S1-mapping experiments also confirmed the presence of an internal promoter, which might be stringently controlled. Taken together, these results show that the structure of the hisT operon is complex and suggest that the operon might be regulated at several levels.

hisT is part of a multigene operon in Escherichia coli K-12.

The Escherichia coli K-12 hisT gene has been cloned, and its organization and expression have been analyzed on multicopy plasmids. The hisT gene, which encodes tRNA pseudouridine synthase I (PSUI), was isolated on a Clarke-Carbon plasmid known to contain the purF gene. The presence of the hisT gene on this plasmid was suggested by its ability to restore both production of PSUI enzymatic activity and suppression of amber mutations in a hisT mutant strain. A 2.3-kilobase HindIII-ClaI restriction fragment containing the hisT gene was subcloned into plasmid pBR322, and the resulting plasmid (designated psi 300) was mapped with restriction enzymes. Complementation analysis with different kinds of hisT mutations and tRNA structural analysis confirmed that plasmid psi 300 contained the hisT structural gene. Enzyme assays showed that plasmid psi 300 overproduced PSUI activity by ca. 20-fold compared with the wild-type level. Subclones containing restriction fragments from plasmid psi 300 inserted downstream from the lac promoter established that the hisT gene is oriented from the HindIII site toward the ClaI site. Other subclones and derivatives of plasmid psi 300 containing insertion or deletion mutations were constructed and assayed for production of PSUI activity and production of proteins in minicells. These experiments showed that: (i) the proximal 1.3-kilobase HindIII-BssHII restriction fragment contains a promoter for the hisT gene and encodes a 45,000-dalton polypeptide that is not PSUI; (ii) the distal 1.0-kilobase BssHII-ClaI restriction fragment encodes the 31,000-dalton PSUI polypeptide; (iii) the 45,000-dalton polypeptide is synthesized in an approximately eightfold excess compared with PSUI; and (iv) synthesis of the two polypeptides is coupled, suggesting that the two genes are part of an operon. Insertion of mini-Mu d1 (lac Km) phage into plasmid psi 300 confirmed that the hisT gene is the downstream gene in the operon.