Harlyn O. Halvorson

A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides.

Presecretory signal peptides of 39 proteins from diverse prokaryotic and eukaryotic sources have been compared. Although varying in length and amino acid composition, the labile peptides share a hydrophobic core of approximately 12 amino acids. A positively charged residue (Lys or Arg) usually precedes the hydrophobic core. Core termination is defined by the occurrence of a charged residue, a sequence of residues which may induce a beta-turn in a polypeptide, or an interruption in potential alpha-helix or beta-extended strand structure. The hydrophobic cores contain, by weight average, 37% Leu: 15% Ala: 10% Val: 10% Phe: 7% Ile plus 21% other hydrophobic amino acids arranged in a non-random sequence. Following the hydrophobic cores (aligned by their last residue) a highly non-random and localized distribution of Ala is apparent within the initial eight positions following the core: (formula; see text) Coincident with this observation, Ala-X-Ala is the most frequent sequence preceding signal peptidase cleavage. We propose the existence of a signal peptidase recognition sequence A-X-B with the preferred cleavage site located after the sixth amino acid following the core sequence. Twenty-two of the above 27 underlined Ala residues would participate as A or B in peptidase cleavage. Position A includes the larger aliphatic amino acids, Leu, Val and Ile, as well as the residues already found at B (principally Ala, Gly and Ser). Since a preferred cleavage site can be discerned from carboxyl and not amino terminal alignment of the hydrophobic cores it is proposed that the carboxyl ends are oriented inward toward the lumen of the endoplasmic reticulum where cleavage is thought to occur. This orientation coupled with the predicted beta-turn typically found between the core and the cleavage site implies reverse hairpin insertion of the signal sequence. The structural features which we describe should help identify signal peptides and cleavage sites in presumptive amino acid sequences derived from DNA sequences.

Distinct repressible mRNAs for cytoplasmic and secreted yeast invertase are encoded by a single gene

We have studied regulation of invertase putative structural genes (SUC) in S. cerevisiae and the synthetic relationship between secreted, glycosylated invertase (E.C.3.2.1.26) and the cytoplasmic, nonglycosylated form of the enzyme. Using immunoprecipitation and gel electrophoresis, we have analyzed invertase polypeptides and glycopeptides synthesized in vitro and in vivo. Analysis of size-fractionated mRNA from a SUC2 strain has shown that three mature, catabolite-repressible mRNA species direct the in vitro synthesis of three invertase polypeptides that have differing molecular weights. Two of these polypeptides, P63 and P62 (63 and 62 kd), are larger than the polypeptides of the secreted enzyme and are cotranslationally processed by microsomal membranes in vitro to yield secreted invertase glycopeptides (GP90 and GP87). The smallest polypeptide, P60 (60 kd), which comigrates electrophoretically with cytoplasmic invertase, is not processed. Posttranslationally, a microsomal-membrane detergent extract removes approximately 20 aminoacids from P62 but not from P60. In vitro translations of mRNAs from a genetically confirmed suc3 mutant strain, from the parental SUC3 strain and from derivative meiotic segregants have shown that the three polypeptides (and therefore three mRNA species) are encoded by one gene. Analysis of in vivo radiolabeled invertase from the same SUC3 and suc3 strains has verified that the SUC3 locus contains the structural gene for secreted and cytoplasmic invertase. Through the derepressed synthesis of multiple primary or processed transcripts, the SUC2 and SUC3 genes are regulated to produce multiple invertase polypeptides. The larger two polypeptides appear to be processed and secreted to yield glycosylated invertase, while the smallest remains in the cytoplasm.

Resolution of α, β and γ DNA of Saccharomyces cerevisiae with the antitumor drug cis-Pt(NH3)2Cl2. Evidence for preferential drug binding by GpG sequences of DNA☆

Abstract This paper reports further studies on the separation of DNAs with the antitumor drug cis-Pt(NH3)2Cl2. cis-Pt(NH3)2Cl2 permits resolution of the three DNA components from whole Saccharomyces cerevisiae in CsCl gradients, avoids pelleting of mitochondrial (β) DNA and does not require a critical molar ratio of platinum drug to DNA-P. However, the difficulty in removing all of the DNA-bound platinum may limit its preparative use. The linear relationship between the increase in buoyant density of platinized double-stranded DNA and its G + C content is employed to calculate a G + C content of 41.2% and 45.8% for α and γ DNA, respectively, using a value of 20% G + C for β DNA. In parallel experiments, we find that poly(dG)·poly(dC), which contains sequential guanine bases, exhibits an unexpectedly large buoyant density increase with cis-Pt(NH3)2 Cl2, while the buoyant density increase of poly[d(G-C)]is markedly retarded, indicating an effect of nucleotide base sequence on DNA separation. The trans platinum compound, which has no antitumor properties, separates DNAs on the basis of G + C content in a similar fashion, but does not preferentially increase the buoyant density of poly(dG)·poly(dC).

Location and arrangement of genes coding for ribosomal RNA in Saccharomyces cerevisiae

Abstract The 140 genes for ribosomal RNA (rDNA) in Saccharomyces cerevisiae occur in clusters of from 10 to 30 cistrons. Approximately 84 rDNA genes have been mapped to chromosome I by comparing the amount of rDNA in a strain monosomic (2 n − 1) for chromosome I with a related diploid (2 n ) strain. The monosomic strain showed an approximate 30% decrease from the diploid in both the amount of heavy satellite DNA and the amount of hybridizable rRNA. It is proposed that three to ten clusters of rDNA are located throughout chromosome I.

Biological Implications of the Viking Mission to Mars

A central purpose of Viking was to search for evidence that life exists on Mars or may have existed in the past. The missions carried three biology experiments the prime purpose of which was to seek for existing microbial life. In addition the results of a number of the other experiments have biological implications: (1) The elemental analyses of the atmosphere and the regolith showed or implied that the elements generally considered essential to terrestrial biology are present. (2) But unexpectedly, no organic compounds were detected in Martian samples by an instrument that easily detected organic materials in the most barren of terrestrial soils. (3) Liquid water is believed to be an absolute requisite for life. Viking obtained direct evidence for the presence of water vapor and water ice, and it obtained strong inferential evidence for the existence of large amounts of subsurface permafrost now and in the Martain past. However it obtained no evidence for the current existence of liquid water possessing the high chemical potential required for at least terrestrial life, a result that is consistent with the known pressure-temperature relations on the planets surface. On the other hand, the mission did obtain strong indications from both atmospheric analyses and orbital photographs that large quantities of liquid water flowed episodically on the Martian surface 0.5 to 2.5 G years ago.The three biology experiments produced clear evidence of chemical reactivity in soil samples, but it is becoming increasingly clear that the chemical reactions were nonbiological in origin. The unexpected release of oxygen by soil moistened with water vapor in the Gas Exchange experiment together with the negative findings of the organic analysis experiment lead to the conclusion that the surface contains powerful oxidants. This conclusion is consistent with models of the atmosphere. The oxidants appear also to have been responsible for the decarboxylation of the organic nutrients that were introduced in the Label Release experiment. The major results of the GEX and LR experiments have been simulated at least qualitatively on Earth. The third, Pyrolytic Release, experiment obtained evidence for organic synthesis by soil samples. Although the mechanism of the synthesis is obscure, the thermal stability of the reaction makes a biological explanation most unlikely. Furthermore, the response of soil samples in all three experiments to the addition of water is not consistent with a biological interpretation.The conditions now known to exist at and below the Martian surface are such that no known terrestrial organism could grow and function. Although the evidence does not absolutely rule out the existence of favourable oases, it renders their existence extremely unlikely. The limiting conditions for the functioning of terrestrial organisms are not the limits for conceivable life elsewhere, and accordingly one cannot exclude the possibility that indigenous life forms may currently exist somewhere on Mars or may have existed sometime in the past. Nevertheless, the available information about the present Martian environment puts severe constraints and presents formidable challenges to any putative Martian organisms. The Martian environment in the past, on the other hand, appears to have been considerably less hostile biologically, and it might possibly have permitted the origin and transient establishment of a biota.

Conformational alterations in the proximal portion of the yeast invertase signal peptide do not block secretion

SummaryVarious amino acid insertions have been introduced into the proximal portion of the signal sequence of secreted yeast invertase. The altered invertase genes have been reintroduced into yeast and monitored for their ability to direct synthesis of secreted invertase in vivo. The insertions should alter the signal polypeptide local secondary structure as predicted by the Chou and Fasman rules (1978). Secretion of these altered invertase polypeptides is not blocked by the amino acid insertions.

Regulation of galactokinase (GAL1) enzyme accumulation in Saccharomyces cerevisiae.

SummaryThe regulation of GAL1 RNA and enzyme synthesis has been investigated in Saccharomyces cerevisiae. We have shown that the induction of GAL10 and GAL1 RNAs is coordinate. GAL1 RNA transcripts appear within 4.5 to 6 min and galactokinase synthesis within 6 to 9 min. Steady-state RNA levels were reached within 50 min and the steady-state rate of galactokinase enzyme synthesis within 40–50 min. From these kinetic studies, the initial induction of GAL1 enzyme activity is apparently under transcriptional control. In addition, during early induction, two galactokinase enzyme activities were detected; a major stable form and a minor unstable form.

Deletions within E. coli plasmids carrying yeast rDNA

Deletions occur in recombinant DNA plasmids that contain yeast ribosomal DNA (rDNA) inserted into the E. coli plasmids pSC101 and pMB9. Deletions within a pMB9 plasmid containing an insert longer than one tandem rDNA repeat apparently are due to homologous recombination because (1) all of the independently derived deletion products of this plasmid lost one complete rDNA repeat (8.6 kb) and retained only a single copy of the segment repeated at the ends of the original insert and (2) deletions were detected only when the insert had terminal redundancy. Deletions also occur within a pSC101 plasmid containing a tandem duplication of a segment (4.7 kb) including both pSC101 DNA and rDNA. Once again these deletions appear to be due to the presence of a duplicated region because all deletion products have lost one complete repeat. Deletions within both of these plasmids took place in both rec+ and recA- host cells, but occurred more frequently in rec+ cells. Oligomerization of the deletion products also occurred in both hosts and was more frequent in rec+ cells.

Use of disomic strains to study the arrangement of ribosomal cistrons in saccharomyces of ribosomal cistrons inSaccharomyces cerevisiae

SummaryDisomic strains ofSaccharomyces cerevisiae were studied by DNA-rRNA hybridization to examine the arrangement of rRNA cistrons on yeast chromosomes as well as to identify a disomic strain which was enriched for rRNA cistrons. Four of the five disomic strains tested showed a per cent hybridization lower than wild type. Two of these strains were found to be disomic for more than one chromosome. A slight increase in the per cent hybridization was observed with DNA isolated from one disomic strain. It was concluded that some chromosomes inSaccharomyces cerevisiae had few if any rRNA cistrons suggesting that the rRNA cistrons are non randomly distributed over the genome. From DNA-tRNA hybridization experiments, evidence for the presence of tyrosine tRNA genes on chromosomes VI was obtained.

Cloning of randomly sheared DNA fragments from a φ105 lysogen of Bacillus subtilis identification of prophage-containing clones

A gene bank for Bacillus subtilis has been developed by cloning randomly sheared DNA fragments of a B. subtilis (phi 105) lysogen DNA in Escherichia coli employing the pMB9 plasmid vector. The DNA was inserted by the oligo(dA)-oligo(dT) method, and the average insert size of the cloned DNA was 7 kilobase pairs (kb). Three clones have been identified which carry DNA from the phi 105 prophage. None of these clones contain the phage-chromosome junction.