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Dramatic changes in the foundation of academic departments in our universities are uncommon. With the demonstration that DNA was the cellular source of genetic information, and that this information could be regulated, the field of molecular biology was born. Later, when scientists found that they could tinker with this information, the field matured. In an unusually rapid manner, molecular biology was integrated into the University of Wisconsin, Madison, in the late 1950s and early 1960s. This present article is a chronology of how it happened. What are the factors that made this transition possible in the University of Wisconsin? What lessons have we learned from this experience?

Epizootic shell disease (ESD) in American lobsters Homarus americanus is the bacterial degradation of the carapace resulting in extensive irregular, deep erosions. The disease is having a major impact on the health and mortality of some American lobster populations, and its effects are being transferred to the economics of the fishery. While the onset and progression of ESD in American lobsters is undoubtedly multifactorial, there is little understanding of the direct causality of this disease. The host susceptibility hypothesis developed here states that although numerous environmental and pathological factors may vary around a lobster, it is eventually the lobster's internal state that is permissive to or shields it from the final onset of the diseased state. To support the host susceptibility hypothesis, we conceptualized a model of shell disease onset and severity to allow further research on shell disease to progress from a structured model. The model states that shell disease onset will occur when the net cuticle degradation (bacterial degradation, decrease of host immune response to bacteria, natural wear, and resorption) is greater than the net deposition (growth, maintenance, and inflammatory response) of the shell. Furthermore, lesion severity depends on the extent to which cuticle degradation exceeds deposition. This model is consistent with natural observations of shell disease in American lobster.

A total of 70%, of the world's conventional commercial fish species are now fully exploited, overexploited, depleted or recovering from depletion. This dramatic crash in the capture world fisheries production has led to problems in foods distribution, balance of payments, employment, and ecological depletion. Public support for breeding programs with terrestrial farm animals and plants in agriculture have revolutionized this industry over the past few hundred years. However, new genetic rearing technologies to improve marine animal production through aquaculture that utilize modern biology to obtain sustainable aquaculture and preserve biodiversity provide a promise to address these problems. However aquaculture has not been subject to public discussion and approval. Public involvement, not necessarily acquiescence, provide value added in the decision making process. Public understanding and involvement involves three stages. (i) Public concern over the pool of genetic information; (ii) if aquaculture is to respond to the fisheries crises with innovation, the knowledge gap between public understanding and scientific information must be bridged; and (iii) strategies must be developed for achieving this. Release of recombinant DNA to the environment, and handling exotic species, are useful case studies. Illustrations will be given of communication bridges to the public and ways to involve the public in making policy decisions.

Harlyn O. Halvorson has played an active and defining role in the field of microbiology for more than 40 years. In this article, he reminisces on the major influence that his father, H. Orin Halvorson, had in the direction of his professional life. The two H.O. Halvorson's, often confused in the literature, became one of the only two father and son pairs to head the American Society for Microbiology in 1954 and 1977, respectively. Major scientists of the era, their research and their collaborations, are remembered in relation to their main works. Harlyn O. Halvorson's interest in bacterial and yeast spores, and especially spore germination in bacilli, is the highlight of this memoir. His scientific research thrusts, leadership at Marine Biological laboratories, Woods Hole, NACSEX (North American-Cuban Scientific Exchange) and elsewhere, his dedication to advanced students and numerous other endeavors, both scientific and political, are told here.

The identification and differentiation of bacterial strains and species are frequently carried out by the use of diagnostic biochemical profiles, serology, and the detection of restriction fragment length polymorphisms in genomic DNA. We show here that DNA restriction fragment length polymorphisms detected using a probe derived from bacteriophage M13 can discriminate between several marine Vibrio species. We have also demonstrated that individual isolates of Vibrio species can be differentiated using the M13 probe.

Germination of spores of Bacillus cereus T and Bacillus subtilis 168 was inhibited by the trypsin inhibitors leupeptin and tosyllysine chloromethyl ketone (TLCK) and by the substrates tosylarginine methyl ester (TAME), benzoyl-L-arginine-p-nitroanilide (L-BAPNA) and D-BAPNA. Potencies of these inhibitory compounds were estimated by finding the concentration which inhibited 50% germination (ID50), as measured by events occurring early (loss of heat resistance), at an intermediate stage [dipicolinic acid (DPA) release], and late in germination (decrease in optical density). In B. cereus T, all the compounds inhibited early and late events with the same ID50. In B. subtilis, TAME inhibited early and late events at the same ID50, but all other inhibitors had a lower ID50 for late events than for early events. This suggests that a trypsin-like enzyme activity is involved at two sequential stages in the germination of B. subtilis spores, one occurring at or before the loss of heat resistance and one at or before the decrease in optical density. Different trypsin-like activities were detected in broken dormant spores and germinated spores of B. cereus T and in germinated spores of B. subtilis by means of three chromogenic substrates: benzoyl-L-phenylalanyl-L-valyl-L-arginine-p-nitroanilide (L-PheVA), L-BAPNA and D-BAPNA. Separation of extracts of germinated spores on non-denaturing polyacrylamide gels showed that in both species the substrates were hydrolysed by three distinct enzymes with different electrophoretic mobilities. The three enzymes had different Ki values for the above inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)

Spores of a Bacillus subtilis mutant temperature sensitive in deoxyribonucleic acid (DNA) replication proceeded through outgrowth at the nonpermissive temperature to the same extent as the wild-type parent spores. In contrast, the DNA synthesis inhibitor novobiocin completely prevented spore outgrowth while displaying a marginal effect on logarithmic growth during one generation time. Inhibition of outgrowth by novobiocin occurred in the absence of DNA replication, as demonstrated in an experiment with spores of the temperature-sensitive DNA synthesis mutant at the restrictive temperature. Novobiocin inhibited the initial rate of ribonucleic acid synthesis to the same extent in germinated spores and in exponentially growing cells. A novobiocin-resistant mutant underwent normal outgrowth in the presence of novobiocin. Therefore, novobiocin inhibition was independent of its effect on chromosome replication per se.

We describe the cloning of a 6.0-kb PstI fragment of the Bacillus subtilis genome which contains much of the ilvBC-leu gene cluster. This plasmid clone and two others that had been previously isolated were characterized physically and genetically to permit the construction of a physical map of this region that is correlated to the genetic map.

Sporulation in Saccharomyces cerevisiae occurs in the absence of a exogenous nitrogen source. Thus, the internal amino acid pool and the supply of nitrogen compounds from protein and nucleic acid turnover must be sufficient for new protein synthesis. Since sporulation involves an increased rate of protein turnover, an investigation was conducted of the changes in the specific activity of various proteinases. A minimum of 30% of the vegetative proteins was turned over during the course of sporulation. There was a 10- to 25-fold increase in specific activity of various proteinases, with a maximum activity around 20 h after transfer into the sporulation medium. The increase in activities was due to de novo synthesis since inhibition of protein synthesis by cycloheximide blocks both an increase in proteinase activities and sporulation. There was no increase observed in proteinase activities of nonsporogenic cultures (a and alpha/alpha strains) inoculated into the sporulation medium, suggesting that the increase in proteinase activities is "sporulation specific" and not a consequence of step-down conditions. The elution patterns through diethylaminoethyl-Sephadex chromatography of various proteinases extracted from T0 and T18 cells were similar, and no new species was observed.

To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.

The germination of Bacillus cereus T spore suspensions is partially prevented by several inhibitors of trypsin-like enzymes. Leupeptin, antipain, and tosyl-lysine-chloromethyl ketone are effective inhibitors, whereas chymostatin, elastatinal, and pepstatin are inactive. A synthetic substrate of trypsin, tosyl-arginine-methyl ester, also inhibits germination. Its inhibitory effect decreases as a function of incubation time in the presence of spores and is abolished by previous hydrolysis with trypsin. Germinating, but not dormant, spore suspensions hydrolyze tosyl-arginine-methyl ester; its hydrolysis is insensitive to chloramphenicol, sulfhydryl reagents, and EDTA. A crude extract of germinated B. cereus spores contains a trypsin-like enzyme whose activity, as measured by hydrolysis of benzoyl-arginine p-nitroanilide, is sensitive to germination-inhibitory compounds such as leupeptin, tosyl-arginine-methyl ester, and tosyl-lysine-chloromethyl ketone. Spore suspensions exposed to the above inhibitors under germination conditions lose only part of their heat resistance and some 10 to 30% of their dipicolinic acid content. Part of the germinating spore population becomes "phase grey" under phase optics. Based on a study of the inhibition of germination by protease inhibitors and the activity of a protease in germination spores and spore extracts, it is suggested that the activity of a trypsin-like enzyme may be involved in the mechanism of the breaking of dormancy in spores of B. cereus T.

A Bacillus subtilis plasmid capable of producing phenotypic erythromycin resistance was compared with an Eryr staphylococcal plasmid. The two plasmids did not interfere with the sporulation process in B. subtilis, in contrast to chromosomal erythromycin mutations.

A single structural gene, SUC2, encodes both secreted and cytoplasmic invertase in Saccharomyces cerevisiae. It is known that the unprocessed polypeptides which differ by a secretion signal sequence are encoded by separate mRNAs. This unusual transcriptional organization raises the question as to the degree to which the transcripts can be independently regulated. To define a system for studying this problem, we examined invertase transcription after various physiological perturbations of cells: rapid catabolite derepression, heat shock, and cell cycle arrest. With each treatment, fluctuations in mRNA levels for both cytoplasmic and secreted invertase were observed. We concluded that (i) catabolite-derepressed synthesis of the mRNAs occurs rapidly after a drop in glucose, is a sustained response, and does not require de novo protein synthesis; (ii) heat shock transcription of both invertase mRNAs is, in contrast, a brief and transient response requiring de novo protein synthesis; and (iii) alpha-mating hormone treatment (G1 phase arrest and release) results in regular and coordinated synthesis of both mRNAs midway between rounds of histone mRNA synthesis. We propose that invertase mRNA regulation involves constitutively synthesized transcriptional factors (observed during catabolite derepression) and transient factors (observed during heat shock and possibly during synchronous growth). Moreover, the mRNA levels for secreted and cytoplasmic invertase can be independently regulated.

Trypsin-like enzymes were studied in dormant, activated, and germinated spores of Bacillus cereus T. Dormant spores contained two heat-labile enzyme activities. One was extractable with 2 M KCl and hydrolyzed azo-albumin. The second, a trypsinlike activity, was not extractable with 2 M KCl and hydrolyzed benzoyl-L-arginine-p-nitroanilide. Because of their heat instability, these two enzyme activities are probably not involved in the germination of heat-activated spores. Upon germination of heat-treated spores, a trypsinlike protease which was not detected in intact dormant spores was activated or exposed. This enzyme, when measured in intact germinated spores, hydrolyzed benzoyl-DL-arginine-p-nitroanilide but not azo-albumin and was inhibited in situ by sulfhydryl-blocking reagents such as p-chloromercuribenzoic acid and Hg2+. There was a correlation between the inhibition of germination and enzymatic activity by sulfhydryl-blocking reagents. The enzyme was also inhibited by leupeptin, tosyl-L-lysine chromoethyl ketone, and tosyl-L-arginine methyl ester. Good correlation existed between the inhibition of germination and enzymatic activity by these agents. Electron micrographs showed that in the presence of trypsin inhibitors, the spores did not lose their cortex. The protein extracts of the inhibited spores formed a somewhat different electrophoretic pattern in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the protein extracts of dormant or germinated spores.

Phosphate uptake in sporulating cultures of Saccharomyces cerevisiae has been found to occur approximately 2 h after the transfer to sporulation medium. Early ribonucleic acid synthesis begins at approximately 4 h and continues to 8 h. Incorporation of phosphate into acid-extractable precursor pools parallels phosphate uptake. In triple-labeling experiments it was observed that the breakdown of vegetatively synthesized ribonucleic acid is not a significant source of precursors for ribonucleic acid synthesis during sporulation. The majority of the ribonucleic acid made in a 10-min period during sporulation does not migrate on gels with precursor or mature ribosomal ribonucleic acid.

A recombinant cosmid clone was isolated from a library created from cosmid pQB79-1 and Bacillus subtilis DNA, and a 15 kb BamHI fragment derived from the cloned insert was transferred to the vector pHV33. The recombinant clone, pRC12, was capable of complementing eight auxotrophic markers in the spoIIA-tyrA region of the B. subtilis chromosome (map positions 205-210). It also complemented eight of nine markers in the spoIIA locus. The exception, spoIIA176, is the most distal marker from lysine. Although pRC12 failed to complement sporulation defects in spoVA or spoIVA (spoIIA+) strains, subclones of pRC12, lacking a functional spoIIA gene, did complement these mutations. pRC12 inhibited sporulation in a spo+ recE strain, possibly due to the presence of multiple functional spoIIA genes. Both the original cosmid and pRC12 were unstable in Escherichia coli and B. subtilis. Antibiotic selection of the vector resulted in extensive deletion of the insert, while selection for insert function in B. subtilis invariably led to loss of the chloramphenicol resistance vector function.

When a strain of Saccharomyces cerevisiae monosomic for chromosome I and initially deficient for 25% of the genes coding for ribosomal RNA is repeatedly subcultured, the number of these genes increases to and remains stable at the number in the wild type. This strain shows 2:2; viable: inviable first division segregation and hemizygosity for the ade1 gene (a chromosome I marker), evidence that the strain is still monosomic for chromosome I. The increase in the number of genes coding for ribosomal RNA in yeast may be analogous to the magnification of the ribosomal RNA genes in Drosophila melanogaster bobbed mutants.

We examined the genetic system responsible for transcriptional regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum, EC 3.1.3.2]) in Saccharomyces cerevisiae at the molecular level by analysis of previously isolated and genetically well-defined regulatory gene mutants known to affect APase expression. These mutants identify numerous positive- (PHO4, PHO2, PHO81) and negative-acting (PHO80, PHO85) regulatory loci dispersed throughout the yeast genome. We showed that the interplay of these positive and negative regulatory genes occurs before or during APase gene transcription and that their functions are all indispensible for normal regulation of mRNA synthesis. Biochemical evidence suggests that the regulatory gene products they encode are expressed constitutively. More detailed investigation of APase synthesis is a conditional PHO80(Ts) mutant indicated that neither PHO4 nor any other protein factor necessary for APase mRNA synthesis is transcriptionally regulated by PHO80. Moreover, in the absence of PHO80, the corepressor, presumed to be a metabolite of Pi, did not inhibit their function in the transcriptional activation of APase.

Blunt-end palindromic DNA linkers with a central restriction site have been designed for the multiple reading frame insertion (abbreviated MURFI) of a sense or nonsense codon into DNA. We have utilized an amber MURFI linker, 5'CTAG TCTAGA CTAG3' to disrupt the lacZ gene, yielding truncated beta-galactosidase proteins. Conditional disruption of the tetr gene in E. coli has also been demonstrated. Nonsense codon MURFI linkers permit conditional fusion of multiple gene products while sense codon linkers can add structural elements (e.g. beta-turn, cationic segment, hydrophobic segment) or a desired amino acid to a protein (e.g. methionine, cysteine). Shotgun or alternatively site-directed insertion of the symmetric linkers is possible. The over-all length of the linker may be adjusted to retain the original reading frame, matching nucleotide additions or subtractions at recipient DNA sites. If a linker restriction site occurs elsewhere in the target DNA, single linker copies may still be inserted using non-phosphorylated linkers.

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.

A hybrid molecule constructed from Escherichia coli plasmid pMB9 and a fragment of Bacillus subtilis 168 deoxyribonucleic acid functions in cells of leu-E. coli, converting them to leucine prototrophy, but fails to survive in strains of B. subtilis 168.

We have examined the regulation of repressible acid phosphatase (APase; orthophosphoric-monoester phosphohydrolase [acid optimum], EC 3.1.3.2) in Saccharomyces cerevisiae at the physiological and molecular levels, through a series of repression and derepression experiments. We demonstrated that APase synthesis is tightly regulated throughout the growth phase and is influenced by exogenous and endogenous Pi pools. During growth in a nonlimiting Pi medium, APase is repressed. When external Pi becomes limiting, there is a biphasic appearance of APase mRNA and enzyme. Our data on APase mRNA half-lives and on the flux of intracellular Pi and polyphosphate during derepression are consistent with a mechanism of transcriptional autoregulation for the biphasic appearance of APase mRNA. Accordingly, preculture concentrations of Pi control the level of corepressor generated from intracellular polyphosphate degradation. When cells are fully derepressed, APase mRNA levels are constant, and the maximal linear accumulation rate of APase is observed. A scheme to integrate phosphorus metabolism and phosphatase regulation in S. cerevisiae is proposed.

In vivo, ribosomal RNA of Saccharomyces cerevisiae is transcribed from the light strand of gamma DNA. In vitro, gamma DNA is transcribed with equal efficiency by both of the yeast nuclear RNA polymerases, polymerase I and polymerase II; however the RNA products synthesized by the two enzymes differ from each other both in size and in the relative composition of guanine and cytosine. RNA synthesized by polymerase I hybridizes preferentially to the light strand, while that synthesized by polymerase II hybridizes equally well to either strand. Selective transcription of the light strand of the ribosomal DNA also occurs when high-molecular-weight total nuclear DNA is used as template.

An approx. 14-kb Sau3A fragment of Bacillus subtilis DNA containing the aroC and ser-22 genes has been isolated. Gene aroC is expressed in both B. subtilis and Escherichia coli and appears to contain its own promoter, allowing complementation in B. subtilis. However, expression in E. coli is dependent on insert orientation, so the direction of transcription can be deduced. The level of dehydroquinase-specific activity, encoded by the cloned aroC gene, is raised 30- to 40-fold in both E. coli and B. subtilis. The clones are stable in both E. coli and B. subtilis but appear to have undergone several large deletions during their construction.

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.

A 7.9-kilobase (kb) chromosomal fragment was cloned from a mercury-resistant Bacillus sp. In Escherichia coli, in the presence of a second plasmid carrying functional transport genes, resistance to HgCl2 and to phenylmercury acetate (PMA) was expressed. Shortening the cloned fragment to 3.8 kb abolished resistance to PMA but not to HgCl2. In Bacillus subtilis, the 3.8-kb fragment produced mercuric reductase constitutively but did not produce resistance to HgCl2 or to PMA.

Studies were undertaken to understand the control of synthesis, stability and modification of UDP galactose epimerase and DNA-dependent RNA polymerase during sporulation of Saccharomyces cerevisiae. When a pre-induced culture of an inducible strain (wild type) is transferred to sporulation medium, the epimerase is inactivated to an undetectable level within 16 hours. Surprisingly, the addition of cycloheximide, a protein synthesis inhibitor, during sporulation stabilizes the epimerase activity. However, in a constitutive strain, the epimerase continues to be synthesized de novo during sporulation. Since the enzyme is synthesized during both vegatative growth and sporulation constitutively, the controls for synthesis of epimerase must be similar under these physiologically different conditions. After chromatography on DEAE Sephadex, there is no change observed in the elution patterns of RNA polymerase forms extracted from acetate growth vegetative cells, sporulating cells or from mature asci ; in all cases RNA polymerase consists of three forms, Ib, II and III. However, single spore suspension obtained from asci by treatment with zymolase contains a new form with chromatographic properties similar to those of form Ia. Our data suggests that form Ia may be a modification product of from Ib.

The synthesis of repressible acid phosphatase in Saccharomyces cerevisiae was examined under conditions of blocked derepression as described by Toh-e et al. (Mol. Gen. Genet. 162:139-149, 1978). Based on a genetic and biochemical analysis of the phenomenon these authors proposed a new regulatory model for acid phosphatase expression involving a simultaneous interaction of regulatory factors in the control of structural gene transcription. We demonstrate here that under growth conditions that fail to produce acid phosphatase the enzyme is readily inactivated. Furthermore, we demonstrate under these conditions the production of acid phosphatase mRNA which is active both in vitro and in vivo in the synthesis of enzyme. This eliminates any step prior to translation of acid phosphatase polypeptide as an explanation for the phenomenon. We interpret our results for the block in appearance of acid phosphatase as a result of both deaccelerated growth and cellular biosynthesis during derepression, accompanied by an enhanced instability of the enzyme.

A total of nine gerJ mutants have now been isolated in Bacillus subtilis. All are defective in their spore germination properties, being blocked at an intermediate (phase grey) stage. The dormant spores are sensitive to heating at 90 degrees C and two of the mutants (generated by transposon insertion) produce spores sensitive at 80 degrees C. The spores of these two more extreme mutants had a visibly defective cortex when studied by electron microscopy, as did some of the other mutants. During sporulation, the acquisition of spore resistance properties and the appearance of the sporulation-specific penicillin-binding protein PBP5* were delayed. A strain probably carrying a lacZ fusion to the gerJ promoter demonstrated increased expression between t2 and t4. We propose that the gerJ locus is involved in the control of one or more sporulation-specific genes.

Bacteria resistant to cadmium or mercury or both were isolated from the Great Sippewissett Marsh (Cape Cod, Mass.) and from Boston Harbor. Many of these metal-resistant isolates were gram-positive aerobic sporeformers, although not necessarily isolated as spores. Although several of the isolated strains bore plasmids, cadmium and mercury resistances appeared to be, for the most part, chromosomally encoded. DNA sequence homology of the gram-positive cadmium- and mercury-resistant isolates was not demonstrable with metal resistance genes from plasmids of either gram-positive (pI258) or gram-negative (pDB7) origin. Cadmium resistance of all the marsh isolates tested resulted from reduced Cd2+ transport. On the other hand, three cadmium-resistant harbor isolates displayed considerable influx but no efflux of Cd2+. Hg-resistant strains detoxified mercury by transforming Hg2+ to volatile Hg0 via mercuric reductase.

Saccharomyces cerevisiae strain FH4C carries a single invertase structural gene, SUC2, but produces distinct invertase mRNAs and polypeptides for the secreted and cytoplasmic forms of the enzyme. The two major invertase cell-free translation products are polypeptides of 60,000 daltons (p60) and 62,000 daltons (p62) and correspond to the nonglycosylated cytoplasmic form of invertase and the precursor of glycosylated secreted invertase, respectively. This paper describes amino acid sequence and peptide map analyses of invertase polypeptides. The peptide maps demonstrate that p62, p60, and the in vivo secreted polypeptide have significant structural homology. Sequence analysis, however, revealed differences between p62 and p60 at their amino termini. p62 contains an amino-terminal signal sequence of 19 amino acid residues that is specifically cleaved during secretion in a cell-free system to generate the secreted 87,000-dalton invertase glycopeptide gp87. This signal sequence is not present in p60. p60 synthesis begins with a methionine which can be aligned with a methionine at residue 21 in p 62. During translation, the p60 initiator methionine is removed and the newly generated amino terminus is acetylated. Based on peptide map similarities, partial amino-terminal sequence data, and common genetic origin, it is suggested that p60 and p62 have identical amino acid sequences carboxy-terminal to the p60 initiator methionine (residue 21 of p62). The reciprocal correlations of signal sequence with secretion and absence of signal sequence with cytoplasmic localization provide proof of the signal hypothesis for secreted proteins. Two mechanisms are proposed for the derivation of p60 and p62 from a single structural gene: alternative promoter sites, and differential processing of a single primary transcript.

The activities of the nuclear RNA polymerases I and II have been investigated in cells of Sacharomyces cerevisiae at G(1), S, early G(2), and late G(2) stages of the cell cycle. The results show that the ratio of the activities of the two enzymes is different at these four stages, indicating that the two RNA polymerases are regulated independently during the cell cycle. The specific activity of the RNA polymerase I remains constant but that of RNA polymerase II increases sharply in cells at the beginning of the G(2) phase. These results are compatible with a pattern of continuous synthesis of the RNA polymerase I and step-wise synthesis of the RNA polymerase II during the yeast cell cycle.

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.

The 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.