Hans Küntzel

Phospholipase C binds to the receptor-like GPR1 protein and controls pseudohyphal differentiation in Saccharomyces cerevisiae.

The hormone receptor-like protein Gpr1p physically interacts with phosphatidylinositol-specific phospholipase C (Plc1p) and with the Gα protein Gpa2p, as shown by two-hybrid assays and co-immune precipitation of epitope-tagged proteins. Plc1p binds to Gpr1p in either the presence or absence of Gpa2, whereas the Gpr1p/Gpa2p association depends on the presence of Plc1p. Genetic interactions between the null mutations plc1Δ,gpr1Δ, gpa2Δ, and ras2Δsuggest that Plc1p acts together with Gpr1p and Gpa2p in a growth control pathway operating in parallel to the Ras2p function. Diploid cells lacking Gpr1p, Plc1p, or Gpa2p fail to form pseudohyphae upon nitrogen depletion, and the filamentation defect of gpr1Δ and plc1Δ strains is rescued by activating a mitogen-activated protein kinase pathway via STE11-4 or by activating a cAMP pathwayvia overexpressed Tpk2p. Plc1p is also required for efficient expression of theFG(TyA)::lacZ reporter gene under nitrogen depletion. In conclusion, we have identified two physically interacting proteins, Gpr1p and Plc1p, as novel components of a nitrogen signaling pathway controlling the developmental switch from yeast-like to pseudohyphal growth. Our data suggest that phospholipase C modulates the interaction of the putative nutrient sensor Gpr1p with the Gα protein Gpa2p as a downstream effector of filamentation control.

Glucose-induced cAMP signaling in Saccharomyces cerevisiae is mediated by the CDC25 protein

Functional mapping of the cell cycle START gene CDC25 has revealed two domains which are dispensable for viability (germination and growth in glucose media), but are essential for sporulation and differentially involved in glucose‐induced cAMP signaling. The transient rise of cAMP is completely prevented by various deletions within the amino‐terminal half (α domain) of the CDC25 gene product. In contrast, the deletion of the carboxy‐terminal 38 residues (β2 domain) results in a rapid, but persisting, rise of cAMP. Our data suggest that the α domain of the CDC25 protein is involved in glucose signal transduction, whereas the β2 domain is required for downregulating the cAMP control chain.

Mitochondrial tRNA gene clusters in Aspergillus nidulans: Organization and nucleotide sequence

Abstract The organization of tRNA genes on the circular 32 kb mitochondrial genome of the ascomycete Aspergillus nidulans has been studied by gel transfer hybridization and by DNA sequencing. Most of the tRNA genes are tightly clustered within two regions (1 kb each) flanking the split gene for the large ribosomal subunit RNA. The upstream cluster contains nine genes, the downstream cluster eleven genes. The twenty tRNA genes are on the same strand as the two rRNA genes and are separated from each other by AT-rich spacer sequences, usually consisting of only a few nucleotides. Two tRNA genes ( leul and ala ) are joined end to end. The occurrence of two tRNA Gty genes is the first exception to the observation that in mitochondria all four-codon families are read by a single tRNA. Both genes are adjacent and show extensive sequence homology, suggesting relatively recent origin by gene duplication. The product of glyl has a U in the wobble position as do all other tRNA gene products specific for four-codon families, whereas the gly2 product, which has a rare A in the same position, should read only the codon GGU. The products of metl and thr have an A and G in positions 18 and 55, respectively, like the mitochondrial tRNA fMet and tRNA Thr of Neurospora crassa. Other unusual features are the replacement of the invariant G-C pair at positions 53 and 61 by A-T in met2, glyl and gly2 , the replacement of the invariant T at position 8 by A in phe and G in pro and the deletion of a nucleotide at position 9 in ser2 .

Nuclear migration in Saccbaromyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein

SummaryWe have isolated a novel gene (NUM1) with unusual internal periodicity. The NUM1 gene encodes a 313 kDa protein with a potential Ca2+ binding site and a central domain containing 12 almost identical tandem repeats of a 64 amino acid polypeptide. num1-disrupted strains grow normally, but contain many budded cells with two nuclei in the mother cell instead of a single nucleus at the bud neck, while all unbudded cells are uninucleate: This indicates that most G2 nuclei divide in the mother before migrating to the neck, followed by the migration of one of the two daughter nuclei into the bud. Furthermore, haploid num1 strains tend to diploidize during mitosis, and homozygous num1 diploid or tetraploid cells sporulate to form many budded asci with up to eight haploid or diploid spores, respectively, indicating that meiosis starts before nuclear redistribution and cytokinesis. Our data suggest that the NUM1 protein is involved in the interaction of the G2 nucleus with the bud neck.

Anatomy of Amplified Mitochondrial DNA in "Ragged" Mutants of Aspergillus amstelodami: Excision Points within Protein Genes and a Common 215 bp Segment Containing a Possible Origin of Replication

SummaryThe extranuclearly-inherited ragged growth phenotype (Rgd) of Aspergillus amstelodami is always accompanied by excision and head-to-tail amplification of mtDNA sequences. In one mutant strain (Rgd1) the amplified mtDNA segment (rgd1 DNA, monomeric length 0.9 kb) maps downstream of the large subunit ribosomal RNA gene (Region 1), whereas in all other strains analyzed the amplified sequences (rdg3-7DNA) are located in Region 2 between genes coding for cytochrome b and ATPase subunit 6. The various region 2 sequences differ in lengths (1.5 to 2.7 kb) but have in common a 215 bp sequence mapping between an. unidentified protein gene (corresponding to URF4 of human mtDNA) and an arginine tRNA gene. This common sequence may contain an origin of replication, because a looped-out hairpin structure similar to that of yeast and human mitochondrial origin sequences can be formed. Furthermore, Region 2 DNA suppresses replication of Region 1 DNA, indicating that the former group of molecules contains the more efficient origin. The nucleotide sequence of the rgd6 repeat unit starts and ends within protein genes of mtDNA, and no homologies were found between heads and tails or their flanking sequences.

Split gene for mitochondrial 24S ribosomal RNA of neurospora crassa

The 60 kb circular mitochondrial genome of N. crassa has previously been shown to contain a single transcription unit for 17S and 24S rRNA mapping within the largest Eco RI fragment E1 (19.6 kb). This fragment was isolated from uncloned mitochondrial DNA and further analyzed by cleavage with restriction endonucleases Hind II, Hind III, Bam HI, Pvu II and BgI I, and by electron microscopy of rRNA/DNA hybrids. The resulting map shows a 2.3 kb intervening sequence interrupting the gene for 24S rRNA. The main part (2.7 kb) of this gene is separated from the 17S rRNA gene by a 5 kb segment which contains several transfer RNA genes. This segment is much longer than the putative 1 kb spacer sequence within the 32S precursor molecule for both rRNAs, suggesting a second splicing event in that region.

High frequency transfer of species specific mitochondrial DNA sequences between members of the aspergillaceae.

SummaryThe mitochondrial genome of Aspergillus nidulans var. echinulatus is approximately 20% larger than that of the closely related species Aspergillus nidulans (Eidam) Winter. Restriction enzyme mapping and electron microscopy has revealed that the size difference is due to the presence of six inserted sequences in the former. With the exception of a small number of species specific restriction sites and the six insertions/deletions, the two mitochondrial genomes appear identical. Protoplast fusion between the two species followed by selection of extranuclear drug resistance markers resulted in the isolation of recombinant mitochondrial genomes in an A. nid. var. echinulatus background. Restriction maps of the hybrid genomes indicated that three of the additional sequences found in A. nid. var. echinulatus could be transferred to the A. nidulans nuclear background without loss or detectable alteration. The nature of the additional mitochondrial DNA and high frequency transfer of certain species specific sequences is discussed with reference to studies in yeast and Neurospora crassa.

Specificity of mitochondrial and cytoplasmic ribosomes from Neurospora crassa in poly‐U dependent cell free systems

Two different types of ribosomes have been isolated from Neurospora crassa: cytoplasmic 77 s and mitochondrial 73 s ribosomes [l] . The cytoplasmic ribosomes are structurally related to 80 s ribosomes from eucaryotic organisms whereas mitochondrial ribosomes resemble procaryotic 70 s particles [2-41 . A functional relationship between mitochondrial and bacterial ribosomes is suggested by the observation that both ribosomes are sensitive to chloramphenicol and resistent to cycloheximide, in contrast to the cytoplasmic ribosomes from Neurospora [5,6] . Another functional difference between ribosomes from procaryotic and eucaryotic organisms is their specific interaction with peptide chain elongation factors. It has been demonstrated that heterologous Poly-U dependent systems containing ribosomes and supernatant factors from different organisms are active only when systems within the procaryotic class or within the eucaryotic class are interchanged; systems combining ribosomes from bacteria with supernatant enzymes from higher cells, and vice versa, are inactive [7] . To find out whether mitochondrial ribosomes belong to the procaryotic class when tested for their specificity in chain elongation, poly-U dependent cell-free systems from Neurospora mitochondria and cytoplasm were combined with each other and with systems from L?. coli and rat liver. Part of these results has been presented at the 6th FEBS meeting in Madrid (1969) [8] . 2. Methods

CDC25‐dependent induction of inositol 1,4,5‐trisphosphate and diacylglycerol in Saccharomyces cerevisiae by nitrogen

The addition of ammonium sulfate to starved yeast cells leads to a 3‐ to 4‐fold rapid increase of the second messengers inositol 1,4,5‐trisphosphate (IP3) and diacylglycerol (DAG), the products of phosphoinositide‐specific phospholipase C (PI‐PLC). This response is reduced by dissecting the RAS‐activating Cdc25 protein, and is completely abolished by the cdc25‐1 mutation even at permissive temperature. Starved cdc25‐1 mutant cells have a strongly reduced IP3 content, but an at least 10‐fold increased DAG level compared to the isogenic wild‐type strain. NH4 does not stimulate cAMP synthesis, and glucose does not induce IP3 and DAG. Our data suggest that the Cdc25 protein controls a nitrogen‐specific signalling pathway involving the effector PI‐PLC, in addition to the glucose‐induced activation of adenylyl cyclase (AC).

The Genetic Apparatus of Mitochondria from Neurospora and Yeast

The function, biogenesis and genetic autonomy of mitochondria has remained a central topic for biologists and biochemists since the early speculations at the end of the 19th century (Altmann, 1890). The discovery of mitochondrial DNA in the late 1950 (Chevremont et al., 1959; Nass and Nass, 1962) initiated a new phase of research which resulted in a logarithmic growth of literature on mitochondrial biogenesis and in a parallel production of review articles (Gibor and Granick, 1964; Wilkie, 1964; Roodyn and Wilkie, 1968; Wagner, 1969; Nass, 1969a; Schatz, 1970).