Screening a yeast promoter library leads to the isolation of the RP29/L32 and SNR17B/RPL37A divergent promoters and the discovery of a gene encoding ribosomal protein L37.

Two promoters (A7 and A23), isolated at random from the Saccharomyces cerevisiae genome by virtue of their capacity to activate transcription, are identical to known intergenic bidirectional promoters. Sequence analysis of the genomic DNA adjacent to the A7 promoter identified a split gene encoding ribosomal (r) protein L37, which is homologous to the tRNA-binding r-proteins, L35a (from human and rat) and L32 (from frogs).

Properties of promoters cloned randomly from the Saccharomyces cerevisiae genome.

Promoters were isolated at random from the genome of Saccharomyces cerevisiae by using a plasmid that contains a divergently arrayed pair of promoterless reporter genes. A comprehensive library was constructed by inserting random (DNase I-generated) fragments into the intergenic region upstream from the reporter genes. Simple in vivo assays for either reporter gene product (alcohol dehydrogenase or beta-galactosidase) allowed the rapid identification of promoters from among these random fragments. Poly(dA-dT) homopolymer tracts were present in three of five randomly cloned promoters. With two exceptions, each RNA start site detected was 40 to 100 base pairs downstream from a TATA element. All of the randomly cloned promoters were capable of activating reporter gene transcription bidirectionally. Interestingly, one of the promoter fragments originated in a region of the S. cerevisiae rDNA spacer; regulated divergent transcription (presumably by RNA polymerase II) initiated in the same region.

Isolation of the yeast gene encoding elongation factor 3 for protein synthesis.

The gene YEF-3 encoding the elongation factor 3 (EF-3) for peptide chain elongation in Saccharomyces cerevisiae has been isolated by immunoscreening of a yeast genomic library in the phage lambda gt11. The identity of the EF-3 gene was confirmed by several methods. First, a clone-encoded protein could affinity purify the antibody that specifically reacted with EF-3. Second, a recombinant fusion protein, which reacted with anti-beta-galactosidase antibody as well as with anti-EF-3 antibody, was found in the lysate of a positive clone lysogen. Third, the function of EF-3 in a yeast mutant in which the EF-3 activity is temperature-sensitive in an in vitro assay could be complemented by transformations. EF-3 protein was overproduced in a transformant which contained the EF-3 gene on a multicopy plasmid YEp-13. Southern blot analysis shows that YEF-3 is a single copy gene. The transcript unit as mapped by S1 nuclease mapping, is consistent with the size of the message determined by Northern blot analysis and shows no evidence of introns.

Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments.

We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

Identification of an altered elongation factor in temperature-sensitive mutant ts 7'-14 of Saccharomyces cerevisiae.

Postpolysomal extracts from wild-type (wt A364A) and temperature-sensitive (ts 7'-14) yeast cells were preincubated for short periods of time at the nonpermissive temperature (37-41 degrees C) prior to incubations for protein synthesis at 20 degrees C. Whereas wt A364A extracts were relatively unaffected by preincubation at the elevated temperature, mutant extracts lost their ability to translate exogenous natural mRNA and poly(U). Phe-tRNA synthetase and ribosomes from ts 7'-14 cells were not inactivated by preincubation at 37-41 degrees C, but a cytosolic component required for chain elongation, as measured by poly(U) translation, was extensively inactivated. The three elongation factors (EF-1, EF-2, and EF-3) required for chain elongation in yeast were resolved chromatographically. Only one factor, EF-3, was able to restore the poly(U)-translational activity of mutant extracts inactivated at the elevated temperature. Heat-inactivated yeast cytosols, which did not support protein synthesis with yeast ribosomes, were perfectly able to translate poly(U) with rat liver ribosomes, which require only EF-1 and EF-2. These and other experiments indicated that the genetically altered component in 7'-14 mutant cells is EF-3.

A competitive solid-phase radioimmunoassay for translational factors employing monoclonal antibodies.

Monoclonal antibodies produced by the hybridoma techniques were purified by chromatography on DEAE Affi-Gel blue, and covalently coupled to Affi-Gel 10 to purify their antigens. The purified components were used to develop a sensitive competitive radioimmune assay for the quantitative determination of translational factors, as described here with a monoclonal antibody directed against yeast elongation factor 3. Antigen was adsorbed to polyvinyl chloride plastic surfaces and a limiting concentration of monoclonal antibody necessary to bind to the adsorbed antigen was determined. Varying concentrations of purified antigen and of samples containing unknown amounts of antigen were then mixed with the limiting concentration of monoclonal antibody, prior to or at the same time as the reaction of the antibody with the surface-adsorbed antigen. The amount of monoclonal antibody that bound to the surface-adsorbed antigen was determined with a second antibody, radioactive goat anti-mouse antibody. The addition of the free antigen preparations to the monoclonal antibody served to compete for the antibody with the antigen adsorbed to the plastic surfaces. The concentration of antigen in the unknown samples was estimated from the titration curves obtained with varying concentrations of pure antigen. This technique did not require isotopic labeling, modification or derivatization of the monoclonal antibody or its antigen.

Monoclonal antibody specific for yeast elongation factor 3.

Hybridomas have been prepared by fusing mouse myeloma (P3 X 63 Ag8) cells with spleen cells of mice immunized with a yeast fraction enriched with respect to non-ribosomal translational components. Cloned hybridoma lines were grown in the form of ascites tumors, and the monoclonal antibodies produced were purified from the ascites fluid by chromatography on DEAE-Affi-Gel Blue. One of the antibodies, from a hybridoma cell line designated as PSH-1, inhibited the translation of natural mRNA and poly(U) and polysomal chain elongation in a cell-free protein-synthesizing system from yeast. Resolution and partial purification of the elongation factors indicated that the monoclonal antibody from PSH-1 did not interact with EF-1 or EF-2 but reacted with and inactivated EF-3, the 125 000 molecular weight additional elongation factor specifically required with yeast ribosomes. The EF-3 purified from the cytosol by immunoaffinity chromatography was comparable to that prepared by ion-exchange chromatography. Evidence was obtained which indicated that EF-3 was essential for the translation of natural mRNA as well as poly(U), was associated with polysomes but not ribosomal subunits, and was required for every cycle in the elongation phase of protein synthesis.

The effect of non-permissive temperature on met-tRNA synthetase in Saccharomyces cerevisiae temperature-sensitive mutant ts 7-45.

The effects of incubation of yeast spheroplasts at elevated temperature (40 degrees C) on a number of activities involved in protein biosynthesis have been examined in preparations obtained from wild-type cells (wt A364A ) and a temperature-sensitive mutant (ts 7-45) derived from it. With wild-type cells, preincubation of spheroplasts at the elevated temperature had little or no effect on the following: the ribosomal subunit-polysome pattern; the translation of exogenous natural mRNA in postpolysomal extracts devoid of endogenous mRNA; the translation of poly(U) in postpolysomal extracts; the incorporation of methionine into 40 S preinitiation and 80 S initiation complexes; the synthesis of Met-tRNA in postribosomal (cytosol) extracts; and the formation of eIF-2 X GTP X Met-tRNAf ternary complex in the cytosol. With temperature-sensitive spheroplasts that had not been preincubated at the elevated temperature, the concentration of free, native 40 S subunits appeared to be lower and that of 60 S subunits higher than in wild-type cells; translation of exogenous natural mRNA in postpolysomal extracts was somewhat lower than in wild-type preparations, but all of the other reactions and components measured were comparable to those in wild-type preparations. Preincubation of temperature-sensitive spheroplasts at 40 degrees C resulted in: a further decrease in the level of 40 S subunits; disaggregation of polysomes; loss of ability to translate natural mRNA but not poly(U); decreased ability to form 40 S preinitiation intermediates; and production of an activity, found in the cytosol, that inhibited Met-tRNA synthetase reversibly. The inhibitor had the characteristics of a protein and did not appear to be a proteinase, nuclease, or nucleotidase.

Studies on the effect of ethanol on eukaryotic protein synthesis in vitro.

The effects of varying concentrations of ethanol on reactions involved in protein biosynthesis have been examined using a cell-free system from Chinese hamster ovary cells that actively translates natural mRNAs in order to detect those components most sensitive to alcohol. Ethanol, at relatively low concentrations (0.2 M or lower) inhibited the translation of endogenous polysomal mRNAs and, in mRNA-depleted extracts, of exogenous natural mRNA. Ethanol markedly inhibited leucyl-tRNA synthetase, and it inhibited Phe- and Glu-tRNA synthetases to some extent, but had only a small effect on several other aminoacyl-tRNA synthetases, elongation factors 1 and 2, ribosomes, or the formation of eukaryotic initiation factor 2 . GTP . Met-tRNAr ternary complex. Methanol inhibited slightly the translation of mRNA and Leu-tRNA synthetase, but isobutyl alcohol and isopropyl alcohol strongly depressed these activities. Ethanol inhibited the interaction of leucine with Leu-tRNA synthetase competitively, whereas isobutyl alcohol and acetaldehyde inhibited the leucine interaction in a noncompetitive manner. Leu-tRNA synthetase from Chinese hamster ovary cells was more sensitive to ethanol than that from yeast.

Effects of retinoic acid on protein synthesis in cultured melanoma cells.

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G1 phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis analysis of L-[35S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.

Analysis of temperature-sensitive mutant ts 187 of Saccharomyces cerevisiae altered in a component required for the initiation of protein synthesis.

Postpolysomal extracts have been prepared from wild type haploid Saccharomyces cerevisiae cells (wt A364A) and from a temperature-sensitive mutant strain (ts 187, gene prt 1). The extracts, prepared via spheroplasts and depleted of endogenous mRNA with nuclease, translate exogenous natural mRNA and polyuridylic acid. The activity of wt A364A with respect to translation of yeast mRNA, poly(U)-dependent synthesis of polyphenylalanine which measures elongation components, reactions involved in the initiation of protein synthesis, and termination and release of polypeptides, is not significantly affected when spheroplasts are incubated at 39 degrees C for relatively short periods of time, prior to the preparation of the cell-free system. With extracts obtained from ts 187 cells, preincubation of spheroplasts at 39 degrees C prior to the preparations of the cell-free system markedly decreases the ability to translate natural mRNA but not poly(U). Compared to extracts from unheated spheroplasts, the following activities in ts 187 extracts from spheroplasts preincubated at 39 degrees C are not significantly affected: activation of methionine and methionylation of tRNAMet; formation of (eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex; binding of mRNA to 40S preinitiation intermediate containing Met-tRNAf, and joining of 60S subunits to form the 80S initiation complex; elongation factor 1- and elongation factor 2-dependent elongations reactions; and termination and release of completed polypeptide chains. However, the interaction between the [eukaryotic initiation factor 2.Met-tRNAf.GTP] ternary complex and 40 S subunits, to form the 40 S preinitiation complex, is drastically inhibited by treatment of the spheroplasts at 39 degrees C.

Studies on the mechanism of action of a eukaryotic codon-dependent factor specific for initiator Met-tRNAf and ribosomal 40 S subunits.

A putative eukaryotic initiation factor purified from rat liver cytosol promotes the ApUpGp- and Mg2+-dependent, GTP-independent binding of initiator Met-tRNAf to ribosomal 40 S subunits. The isotopically-labeled factor binds specifically to 40 S subunits to form a binary complex which can then bind Met-tRNAf in the presence of ApUpGp. When 60 S subunits are added, an 80 S complex is formed which contains Met-tRNAf but not the factor. The Met-tRNAf bound to 80 S ribosomes reacts quantitatively with puromycin. Purified 40 S subunits exist in the form of monomers and dimers. The factor converts monomers, which appear to contain one molecule of tRNA, to a form that does not contain tRNA and dimerizes readily. Deacylated tRNA inhibits dimerization and, in the presence of the factor, converts dimers to the monomeric form; thus, the factor appears to act in a reversible manner. This protein factor may play a role in the removal of tRNA from 40 S subunits, which could be generated as a consequence of chain termination, a prerequisite to the bindig of Met-tRNAf.

The effect of cessation of growth on protein synthesis in a mutant of Chinese hamster ovary cells with a temperature-sensitive leucyl-tRNA synthetase.

A temperature-sensitive mutant of Chinese hamster ovary cells with an altered leucyl-tRNA synthetase fails to grow and to incorporate amino acids into protein properly at or near the non-permissive temperature. This mutant was used to determine whether cessation of growth at the elevated temperature affected elongation factor EF-1, since the activity of EF-1 is markedly lower in non-growing cells in stationary phase than in rapidly-growing cells in exponential phase. Cell-free extracts prepared from cells maintained at 39 degrees C for 24 h showed a marked decrease in the ability to translate natural mRNAs, compared to cells incubated at 34 degrees C. However, the ability to translate poly(U), which requires elongation factor EF-1 (and EF-2), was not affected. Analyses of activities involved in the initiation of protein synthesis and in the activation of amino acids revealed that, with the exception of leucyl-tRNA synthetase, the rest of the components required for translation also appeared to be relatively stable even after 24 h at the elevated temperature. The effects of elevated temperature on cell-free extracts were also investigated. The results were similar to those obtained with intact cells; that is, except for leucyl-tRNA synthetase which was rapidly inactivated in vitro at 39 degrees C, other aminoacyl-tRNA synthetases and translational components involved in chain initiation and elongation were relatively stable. Thus, no change in EF-1 activity was detected as a result of arrested cell growth, an inherent lability of the elongation factor, or metabolic degradation as a consequence of a rapid turnover rate in the absence of protein synthesis.