Riyo Kunisawa

Isolation of the putative structural gene for the lysine-arginine-cleaving endopeptidase required for processing of yeast prepro-α-factor

S. cerevisiae kex2 mutants are defective for the production of two biologically active secreted peptides: killer toxin and the mating pheromone, alpha-factor. Both molecules are excised from larger precursor polypeptides. In normal cells, the alpha-factor precursor is core-glycosylated and proteolytically processed intracellularly. In kex2 mutants, however, prepro-alpha-factor is not proteolytically cleaved and is secreted in a highly glycosylated form. All kex2 mutants examined (three independent alleles) lack a Zn++-sensitive membrane-associated endopeptidase with specificity for cleaving on the carboxyl side of a pair of basic residues. Absence of this activity cosegregates with the other phenotypes of a kex2 lesion in genetic crosses. The normal KEX2 gene was isolated by complementation of three of the phenotypes conferred by the kex2-1 mutation. The cloned DNA, either on a multicopy plasmid or integrated into the genome, restores both enzymatic activity in vitro and the normal pattern of proteolytic processing and glycosylation of prepro-alpha-factor in vivo. Gene dosage effects suggest that KEX2 is the structural gene for the endopeptidase.

A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle.

Probes derived from cDNAs encoding isozymes of rat protein kinase C (PKC) were used to screen the genome of the budding yeast S. cerevisiae. A single gene (PKC1) was isolated that encodes a putative protein kinase closely related to the alpha, beta, and gamma subspecies of mammalian PKC. Deletion of PKC1 resulted in recessive lethality. Cells depleted of the PKC1 gene product displayed a uniform phenotype, a characteristic of cell division cycle (cdc) mutants, and arrested cell division at a point subsequent to DNA replication, but prior to mitosis. Unlike most cdc mutants, which continue to grow in the absence of cell division, PKC1-depleted cells arrested growth with small buds. PKC1 may regulate a previously unrecognized checkpoint in the cell cycle.

A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae

MATa cells carrying an sst2 mutation are unable to recover from the G1-specific cell cycle arrest induced by the mating pheromone alpha factor. The KSS1 gene, when overexpressed, suppresses this adaptation defect. KSS1 overexpression also suppresses the recovery defect manifested by cells expressing an alpha factor receptor lacking its 136 amino acid cytoplasmic tail. Because SST2 product and the receptor tail contribute independently to events that allow recovery from pheromone-induced growth arrest, KSS1 function defines a third independent process that promotes desensitization. The KSS1 gene encodes an apparent protein kinase homologous to the CDC28 (S. cerevisiae) and cdc2+ (S. pombe) gene products. The recovery-promoting activity of the KSS1 gene requires a functional WHI1 gene, which encodes a yeast homolog to animal cyclins, suggesting that the KSS1 and WHI1 proteins act in the same growth control pathway.

A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae.

Exposure of a haploid yeast cell to mating pheromone induces transcription of a set of genes. Induction is mediated through a cis-acting DNA sequence found upstream of all pheromone-responsive genes. Although the STE12 gene product binds specifically to this sequence element and is required for maximum levels of both basal and induced transcription, not all pheromone-responsive genes are regulated in an identical manner. To investigate whether additional factors may play a role in transcription of these genes, a genetic screen was used to identify mutants able to express pheromone-responsive genes constitutively in the absence of Ste12. In this way, we identified a recessive, single gene mutation (mot1, for modifier of transcription) which increases the basal level of expression of several, but not all, pheromone-responsive genes. The mot1-1 allele also relaxes the requirement for at least one other class of upstream activating sequence and enhances the expression of another gene not previously thought to be involved in the mating pathway. Cells carrying mot1-1 grow slowly at 30 degrees C and are inviable at 38 degrees C. The MOT1 gene was cloned by complementation of this temperature-sensitive lethality. Construction of a null allele confirmed that MOT1 is an essential gene. MOT1 residues on chromosome XVI and encodes a large protein of 1,867 amino acids which contains all seven of the conserved domains found in known and putative helicases. The product of MOT1 is strikingly homologous to the Saccharomyces cerevisiae SNF2/SW12 and RAD54 gene products over the entire helicase region.

Multiple Ca2+/calmodulin-dependent protein kinase genes in a unicellular eukaryote.

We purified a Ca2+/calmodulin (CaM)‐dependent protein kinase (CaM kinase) from the yeast Saccharomyces cerevisiae with properties similar to mammalian type II CaM kinases. Degenerate oligonucleotides designed on the basis of the amino acid sequence of tryptic peptides from the 55 kd subunit of the yeast CaM kinase were used to isolate its gene from a set of lambda gt11‐yeast genomic DNA phage clones initially selected by the ability to bind 125I‐labelled yeast CaM. The cloned gene (CMK1) encodes an open reading frame that is homologous to the sequences of vertebrate type II CaM kinases. Several criteria demonstrated that the CMK1 gene product is the 55 kd polypeptide. Neither over‐production (11‐fold) nor complete elimination of the CMK1 gene product had any detectably deleterious effect on yeast cell growth. Extracts from cmk1 delta cells, which lacked detectable p55 using an antiserum raised against a Staphylococcus aureus protein A‐CMK1 fusion protein, possessed significant residual Ca2+/CAM‐dependent protein kinase activity. Using the CMK1 gene as a probe at low stringency, a second gene (CMK2) encoding another CaM‐dependent protein kinase with striking sequence similarity to CMK1 was cloned. Deletion of CMK2, or both CMK1 and CMK2, was not lethal, although loss of CMK2 caused a slow rate of spore germination.

Nitrogen fixation by unicellular blue-green algae

SummaryThe ability of some unicellular blue-green algae to grow at the expense of N2 under aerobic conditions has been confirmed and the distribution of this property in the Chroococcaceae has been investigated. It appears to be confined to strains with spherical cells enclosed by the multilaminate sheaths characteristic of the genus Gloeocapsa. Only two unicellular blue-green algae of this type are now available in pure culture; and their properties are so similar that they may well be independent isolates of the same species.No differences in structure between cells grown with nitrate and N2 could be detected, either by light or by electron microscopy; under both conditions of growth, the population consists entirely of vegetative cells. These two Gloeocapsa strains can therefore maintain a functional nitrogenase system in vegetative cells that are freely exposed to air.

Heterocyst formation and nitrogenase synthesis in Anabaena sp.

SummaryWhen filaments from a culture of Anabaena sp. growing photoautotrophically with nitrate as a nitrogen source are placed in a nitrate-free mineral medium and incubated anaerobically in the light, the formation of heterocysts and the synthesis of nitrogenase both begin after a lag of about 24 hours. During the lag period, about 70% of the phycocyanin is destroyed. Under an atmosphere of N2-CO2, the nitrogenase activity rises to a peak value, and then falls markedly as growth at the expense of N2 begins. Phycocyanin synthesis resumes concomitantly with growth. Under an atmosphere of Ar-CO2, the formation of heterocysts and the synthesis of nitrogenase proceed to higher levels than those observed under N2-CO2, and the nitrogenase level is thereafter maintained. Under these conditions, neither growth nor resynthesis of phycocyanin occurs, and phycocyanin eventually falls to about 10% of its initial level in the filaments; however, growth can be promptly initiated if N2 is admitted to the system. The implications of these findings are discussed.

Studies on the role of carotenoid pigments in a chemoheterotrophic bacterium, corynebacterium poinsettiae

SummaryThe hypothesis that colored carotenoids can protect chemoheterotrophic microorganisms from damage by visible light has been investigated.nCorynebacterium poinsettiae, a bacterium that forms the three carotenoid pigments lycoxanthin, cryptoxanthin and spirilloxanthin, was used as test organism. Non-pigmented cells, in which the normal carotenoids were largely replaced by the colorless C40 polyene, phytoene, were obtained by two methods: isolation of a mutant with a block in carotenoid synthesis; and cultivation of the parent strain in the presence of diphenylamine, a specific chemical inhibitor of carotenoid synthesis.Comparative studies of the effects of visible light on dye-sensitized pigmented and non-pigmented cells showed that non-pigmented cells can be rapidly killed by exposures which are without effect on pigmented cells. Both physiological and genetic suppression of pigment synthesis produce photosensitivity. The non-pigmented mutant is killed by ultraviolet light at the same rate as the pigmented parent strain, indicating that the acquired photosensitivity of the former is specific for visible light.

Complete nucleotide sequence of the gene encoding the regulatory subunit of 3',5'-cyclic AMP-dependent protein kinase from the yeast Saccharomyces cerevisiae.

The amino acid sequence of twenty residues from the N-termlnus of the regulatory subunit of cAMP-dependent protein kinase purified from the yeast Saccharomyces cerevisiae was reported several years ago (1). We prepared three corresponding ollgonucleotldes: one 23-base mixed sequence (128-fold degenerate) probe and two unique (45and 60-base) probes that accommodate the apparent codon usage bias of £. cerevisiae (2,3). The three synthetic probes were used to screen a yeast genomlc DNA library In the vector YCp50 (kindly provided by M. Rose). Two plasmlds were obtained that, by restriction endonuclease cleavage site mapping, contained a common segment of genomic DNA. Digests of this common region (Bglll, EcoRV, Ms£l, m i . Sau3A, ̂ p_hl, Xbal) were sub-cloned into M13mpl8 and M13mpl9 (4) and were sequenced on both strands by standard dideoxy chain termination methods (5). The open reading frame present encodes a 416-residue polypeptide that, aside from the Initiator methionlne, commences with a perfect match (underlined) to the N-termlnal sequence of the purified protein. Hybridization of the cloned gene to Southern blots of genomic DNA digests (6), to whole yeast chromosomes separated by orthogonal fieldalternation gel electrophoresls (7), and to polyA RNA species fractionated by by gel electrophoresis (8), demonstrated that the gene is a unique single-copy locus, resides on chromosome IX, and encodes a transcript of 1595 bases. Two mutations (bcyl and sral) known to affect the level of regulatory subunit (9,10) also map on chromosome IX and probably represent lesions In REG1. Computer comparison (ALIGN program of Intelligenetlcs, Inc.) indicates that the C-termlnal two-thirds of the yeast REG1 protein shares 40% Identity with both mammalian type I and type II regulatory subunlts (11,12). Homology is especially conserved around the residues known to be involved in cAMP binding (asterisks). Like the type II subunit, the predicted yeast protein has a potential phosphorylatlon site (P) in the domain corresponding to the hinge region thought to be the major site of Interaction with the catalytic subunit (13). The yeast REG1 protein can be phosphorylated In vitro by both the yeast (14) and bovine (1) catalytic subunit and appears to be phosphorylated In vivo (15).

Mutations of Anacystis nidulans that affect cell division

SummaryFollowing nitrosoguanidine treatment of the blue-green alga Anacystis nidulans strain 6311, mutants which grow predominantly as short or long filaments are commonly produced. Cytological study shows that the filaments are multinucleate, coenocytic structures. Such mutations are therefore best interpreted as ones that impair cell division in a normally unicellular organism.