High level expression in Saccharomyces cerevisiae of an artificial gene encoding a repeated tripeptide aspartyl-phenylyalanyl-lysine.

A chemically synthesized gene, which encodes a 64 or 128 times-repeated tripeptide, aspartyl-phenylalanyl-lysine, has been cloned onto the yeast expression vector pAM82 containing the PHO5 promoter. The artificial gene (LAP gene) contains the untranslated leader sequence of the E. coli lipoprotein gene (lpp) with its transcription terminator sequence. When yeast AH22 cells transformed by recombinant plasmid containing repeated tripeptide gene were derepressed in low phosphate medium, the artificial polypeptides were synthesized to the amounts of about 30% of the total cell protein. SDS-polyacrylamide gel electrophoresis and immunoblot analysis indicated that the artificial polypeptides synthesized in yeast have molecular weights ranging from about 30,000 and 60,000 and have immunoreactivity with the artificial polypeptides expressed in E. coli. The artificial popypeptides in whole cell extract were insoluble and seem to be synthesized as insoluble aggregates. Electron microscopy showed the presence of inclusion bodies in the cell. These polypeptides can be hydrolyzed to tripeptides with trypsin or chymotrypsin. These properties along with the high expression and easy separation may make the artificial polypeptides a potential raw material for the production of an artificial sweetener, Aspartame.

Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine.

Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL. These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E. coli chromosome. Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes. The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations. The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALys, was impaired in asuE mutant strains, presumably at the 2-thiolation step.

Isolation and characterization of antisuppressor mutations in Escherichia coli.

Nonsense mutations in lacI have been shown to be useful as indicators of the efficiency of nonsense suppression. From strains containing supE and a lacI nonsense mutation, selection for LacI- mutants has resulted in the isolation of four antisuppressor mutations. Tn10 insertions linked to these mutations were isolated and used to group the four mutations into three loci. The asuA1 and asuA2 mutations are linked to trp, reduce suppression by supE approximately twofold, and affect a variety of suppressors. The asuB3 mutation was mapped by P1 cotransduction to rpsL but does not confer resistance to streptomycin. The asuC4 mutation reduced suppression by supE by 95% and was shown biochemically to result in the loss of two pseudouridine modifications from the 3' side of the anticodon stem and loop of tRNA2Gln. This mutation is linked to purF, suggesting that it is a new allele of hisT.

Changing postdoctoral career patterns for biomedical scientists.

Between 1973 and 1977 the total number of Ph.D.'s holding postdoctoral appointments in the biomedical sciences increased at a rate of more than 550 individuals (12.5 percent) per year. During this same period the total number of doctorates awarded each year in these disciplines showed very little change. The postdoctoral growth can be attributed to substantial increases in both the numbers of recent graduates taking postdoctorals and the length of stay on these appointments. The lack of alternative employment opportunities has contributed heavily to the postdoctoral buildup. Continued growth is likely to have important consequences for biomedical research and research training.

Isopentenyladenosine deficient tRNA from an antisuppressor mutant of Saccharomyces cerevisiae.

We have isolated a mutant of Saccharomyces cerevisiae that contains 1.5% of the normal tRNA complement of isopentenyladenosine (i6A). The mutant was characterized by the reduction in efficiency of a tyrosine inserting UAA nonsense suppressor. The chromatographic profiles of tRNATyr and tRNASer on benzoylated DEAE-cellulose are consistent with the loss of i6A by these species. Transfer RNA from the mutant exhibits 6.5% of the cytokinin biological activity expected for yeast tRNA. Transfer RNAs from the mutant that normally contain i6A accept the same levels of amino acids in vitro as the fully modified species. With the exception of i6A, the level of modified bases in unfractionated tRNA from the mutant appears to be normal. The loss of i6A apparently affects tRNA's role in protein synthesis at a step subsequent to aminoacylation.

Isolation and identification of cytokinins from Euglena gracilis transfer ribonucleic acid.

Three ribonucleosides responsible for cytokinin activity in Euglena gracilis var Bacillaris tRNA have been isolated and identified as 6-(3-methyl-2-butenylamino)-9-beta-D-ribofuranosylpurine, 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-D-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine. The structures of these compounds were assigned on the basis of their chromatographic properties and ultraviolet and mass spectra which were identical with those of the corresponding synthetic compounds. The elution profiles of cytokinin bioassay activity and of 35S radioactivity suggest the presence of a trace amount of 6-(3-methyl-2-butenylamino)-2-methylthio-9-beta-D-ribofuranosylpurine.

Subcellular localization of cytokinins in transfer ribonucleic Acid.

Evidence on the localization of cytokinins in chloroplast tRNA was obtained by comparison of Euglena gracilis var. bacillaris light-grown and dark-grown wild type cultures and chloroplast-bleached mutant strains. The several cytokinins characteristic of tRNA were separated by Sephadex LH-20 column chromatography of the hydrolysates and were quantitatively determined by tobacco bioassays of the eluates. The results indicate that 6-(3-methyl-2-butenylamino)-9-beta-d-ribofuranosylpurine (i(6) A) is formed in both the cytoplasmic and chloroplast tRNA, whereas 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9-beta-d- ribofurano-sylpurine (c-io(6)A) is produced mainly in the cytoplasmic tRNA and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-beta-d- ribofurano-sylpurine (ms(2)io(6)A) is localized exclusively in chloroplast tRNA. The restriction of the methiolation reaction to the chloroplast is supported by results of radioisotope experiments showing that (35)S-labeled MgSO(4) is incorporated into ms(2)io(6)A in the wild type cultures, but not in the chloroplast-bleached mutant strains.

Preparation of Escherichia coli tRNAs terminating of modified nucleosides by the use of CTP(ATP):tRNA nucleotidyltransferase and polynucleotide phosphorylase.

Two procedures were investigated for the modification of tRNAs at the 3'-terminal nucleoside. The first involved the incubation of an enzymatically abreviated tRNA (tRNA-C-COH) with appropriate nucleoside triphosphates in the presence of CTP(ATP):tRNA nucleotidyltransferase from Escherichia coli and yeast. The E. coli enzyme did not utilize 2'- or 3'-deoxyadenosine 5'-triphosphate as substrates, but affected incorporation of the 2'- and 3'-O-methyladenosine triphosphates onto tRNA-C-Cou to the extent of 30 and 37%, respectively. Although incorporation of the deoxynucleotides could not be effected using the E. coli enzyme, yeast CTP(ATP:tRNA nucleotidyltransferase produced the desired tRNAs in yields of 45-65%. The second modification procedure involved incubation of tRNA-C-COH with (appropriately blocked) nucleoside diphosphates in the presence of polynucleotide phosphorylase. This procedure afforded the tRNAs terminating in 2'- and 3'-deoxyadenosine in yields of 4% (and the yield of the former was increased to 36% when the incubation was carried out in the presence of 20% methanol). The yields of tRNAs terminating in 2'- and 3'-O-methyladenosing produced by this procedure were 55 and 17%, respectively. Because only single isomers of most of the tRNAs terminating in 2'- and 3'-deoxy- and O-methyladenosine are aminoacylated, attempts were made to obtain the other isomericaminoacyl-tRNA by enzymatic introduction of chemically preaminoacylated nucleotides onto tRNA-C-COH. Although incubation of tRNA-C-COH with three aminoacylated nucleoside 5'-triphosphates and E. coli CTP(ATP):tRNA nucleotidyltransferase did not result in production of the desired tRNAs to a detectable extent, incubation with 2'-deoxy-3'-O-L-phenylalanyladenosine 5'-diphosphate and polynucleotide phosphorylase afforded E. coli tRNA terminating with the corresponding aminoacylated deoxynucleoside.

Genetic analysis of a transposable suppressor gene in Saccharomyces cerevisiae.

We have demonstrated in Saccharomyces cerevisiae the transposition of a gene coding for an efficient ochre (UAA) suppressor from a centromere-linked site on chromosome III to two new sites in the yeast genome. One site is on chromosome VI, very close to, if not allelic with, SUP11, one of eight genes coding for a tyrosine-inserting suppressor. The second site is on chromosome III, unlinked to the centromere and distal to the mating type locus. This site is very close to those mapped for the recessive lethal amber suppressors, SUP-RL1 and SUP61.

Indirect selection for auxotrophic mutants of Saccharomyces cerevisiae using the antibiotic netropsin.

The small basic oligopeptide antibiotic, netropsin, can be successfully employed as an effective counterselecting agent in Saccharomyces cerevisiae. The use of the drug results in approximately a 35-fold enrichment of auxotrophic mutants in a mutagenized culture of yeast. The experimental procedure is quite simple and less time consuming than other presently used methods for indirect mutant selection in yeast.

Transfer ribonucleic acid biosynthesis. Substrate specificity of ribonuclease P.

Bacteriophage T4 synthesizes proline and serine tRNA species which are derived from a common precursor RNA. The processing of this precursor RNA involves the replacement of a U-A-A terminus in serine tRNA by C-C-A prior to precursor cleavage. In the present work we have examined in detail the cleavage of T4 proline-serine precursor RNA by the previously identified ribonuclease P. Ribonuclease P accurately cleaves precursor RNA terminating in either C-C-A or U-A-A to generate the 5' termini characteristic of both mature tRNA species. These cleavages do not depend solely on the nucleotide sequence of the precursor RNA since isolated oligonucleotides spanning the cleavage sites are not substrates for the enzyme. Two types of experiments show that RNase P kinetically favors precursor RNA ending C-C-A over that ending U-A-A. Isolated preparations of precursor RNA containing the C-C-A sequence were cleaved more rapidly by RNase P than precursor RNA ending U-A-A. In addition, the serine tRNA generated by limited cleavage of a mixed population of precursor RNA ending C-C-A or U-A-A was enriched 3-fold in the C-A-A sequence relative to the starting material. Bacteriophage T4 proline-serine precursor RNA, in contrast to other tRNA precursors, accumulates in measurable amounts in wild type cells. This accumulation would appear to be a consequence of the requirement for the generation of the C-C-A sequence prior to RNase P cleavage. The enzymic specificity of RNase P in vitro therefore reflects the in vivo pathway for serine tRNA biosynthesis, where the C-C-A sequence is synthesized while the serine tRNA sequence is still a part of the large precursor RNA.