Activation of 2-aminofluorene by cultured plant cells.

Cultured tobacco plant cells activated 2-aminofluorene to an agent mutagenic to Salmonella typhimurium strain TA98. The plant activation of 2-aminofluorene is heat-inactivated and may not involve solely cytochrome P-450. The kinetics of activation demonstrated both time- and concentration-dependent responses.

Cell interactions and regulation of cell type in the yeast Saccharomyces cerevisiae.

Examination of the control of cell type in yeast at the molecular level and understanding of the biochemical basis of the cell-cell interactions involved in the mating process are clearly entering an extremely productive and exciting period. The tools and opportunities are now available to answer fundamental questions with regard to the mechanism of differential gene expression in eukaryotic cells by using cloned a-specific, alpha-specific, and haploid-specific genes as the probes. Basic questions concerning eukaryotic chromosome structure and organization can be addressed by elucidating the properties of the SIR/MAR regulators and their mode of action. Furthermore, the availability both of cloned MAT, HML, and HMR regions and of the HO gene will provide the material for unravelling the enzymology of the DNA transposition that occurs during mating type interconversion. The isolation of the structural genes for the pheromones and mutations that block pheromone production will provide useful information on how extracellular hormones are synthesized, processed, and secreted by eukaryotic cells. Moreover, the apparent mode of action of the phermonones through cyclic AMP as an intracellular "second messenger," and the genetic and biochemical tractability of yeast cells, may allow tracing of the entire pathway of hormonal regulation of a eukaryotic cell division cycle. These and other studies of the developmental biology of yeast cells will provide more important insights into fundamental aspects of the genetic control of developmental processes in eukaryotic cells.

Saccharomyces cerevisiae STE14 gene is required for COOH-terminal methylation of a-factor mating pheromone.

Saccharomyces cerevisiae a-factor is a dodecapeptide pheromone in which the carboxyl group of the COOH-terminal cysteine residue is methyl-esterified and the sulfhydryl side chain is conjugated in thioether linkage to a farnesyl moiety. We found that MAT a ste14 mutant cells secreted a biologically inactive form of a-factor which had more hydrophilic character than the wild-type pheromone. The authentic pheromone could be metabolically labeled with [methyl-3H]methionine, and the resulting COOH-terminal methyl ester could be removed by mild alkaline hydrolysis. In contrast, a-factor secreted by ste14 mutants did not incorporate a base-labile 3H-methyl moiety. Base treatment converted the normal pheromone into a form which was biologically inactive and which comigrated with the ste14 form of the peptide upon thin-layer chromatography. These results indicate that STE14 gene function is required for COOH-terminal methylation of a-factor.

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture.

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO2 (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 µg per g fresh weight.Elevated CO2 (1%-5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO2 1(-1)) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1(-1) CO2, 2% O2, and dark respiration ranged from 29 to 44 µmol CO2 mg(-1) Chl h(-1). Photosynthesis was inhibited by O2. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C3 plants) was due to low RuBPcase activity relative to that of C3 leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO2.

Control of yeast cell types by mobile genes: a test.

The yeast Saccharomyces cerevisiae changes cell types by switching the alleles of the mating type locus (MAT) from a to alpha and vice versa. In the cassette model, these switches, --e.g, from a to alpha--occur when a replica of silent alpha information (an alpha "cassette") replaces the resident a cassette at the mating type locus and is thereby expressed. We have identified a mutation in the locus postulated to be the silent alpha information (HMLalpha) and find that a mutation is introduced into the mating type locus as a result of interconversion: HMLalpha(-) MATalpha cells switch to MATa and then to MATalpha(-). The MATalpha(-) mutation leads to defective mating and behaves like some previously identified MATalpha(-) mutations. These observations satisfy the prediction of the cassette and controlling element models that genetic information is transmitted from HMLalpha to the mating type locus.

Healing of mat mutations and control of mating type interconversion by the mating type locus in Saccharomyces cerevisiae.

Homothallic yeasts switch cell types (mating types a and alpha) at high frequency by changing the alleles of the mating type locus, MATa and MATalpha. We have proposed in the cassette model that yeast cells contain silent MATa and MATalpha blocs ("cassettes"), copies of which can be substituted at the mating type locus for the resident information. The existence of silent cassettes was originally proposed to explain efficient switching of a defective MATalpha locus (matalpha) to a functional MATalpha locus. We report here that this "healing" of mat mutations is a general property of the mating type interconversion system and is not specific to the class of matalpha mutations studied earlier: a defective MATa (mata1) switches readily to MATa and various matalpha loci switch readily to MATalpha. These observations satisfy the prediction of the cassette model that all mutations within MATa and MATalpha be healed. These studies also identify MAT functions that control the switching process: the same functions known to promote sporulation and prevent mating in a/alpha cells also inhibit the switching system in a/alpha cells. Finally, we present additional characterization of a natural variant of MATalpha, MATalpha-inc [Takano, I., Kusumi, T. & Oshima, Y. (1973) Mol. Gen. Genet. 126, 19-28] that is insensitive to switching. Our observation that MATalpha-inc acts in cis suggests that it may be altered in a site concerned with excision of MATalpha-inc or its replacement by another cassette.

Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae.

MAT alpha haploids with mutations in the STE13 or KEX2 gene, and MATa haploids with mutations in the STE6 or STE14 gene, do not mate with wild-type cells of the opposite mating type. We found that such mutants were able to mate with partners that carry mutations (sst1 and sst2) that cause cells to be supersensitive to yeast mating pheromone action. Mating ability of MAT alpha ste13 and MAT alpha kex2 mutants could also be restored by adding normal MAT alpha cells to mating mixtures or by adding just the appropriate purified pheromone (alpha-factor). Therefore, the mating deficiencies caused by the ste13 and kex2 lesions, and by inference, the ste6 and ste14 mutations, appear to result only from secretion of an insufficient amount of pheromone or a nonfunctional pheromone.

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels.

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO(2) (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO(2) levels (5%). Photoautotrophy was indicated by the requirement for CO(2) and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C(4)Amaranthus species. The cells showed high dark respiration rates and had lower net CO(2) fixation under high O(2) conditions. Dark CO(2) fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO(2) conditions showed the unique properties of a high RuBPcase activation level in ambient CO(2) and a stable ability to show net CO(2) fixation in 21% O(2) conditions.

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Specificity of cotranslational amino-terminal processing of proteins in yeast.

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.