Three genes specifically expressed during phosphate deficiency in<Emphasis Type="Italic"> Pholiota nameko</Emphasis> strain N2 encode hydrophobins

We previously isolated 31 cDNAs corresponding to pdi [inorganic phosphate (Pi) deficiency-inducible] genes through the differential screening of a cDNA library constructed from the mycelium of Pholiota nameko strain N2 cultured in Pi-depleted medium. Among the cDNAs, pdi251, pdi263 and pdi315 were analyzed here. The deduced amino acid sequences of pdi251, pdi263 and pdi315 showed high similarity to fungal hydrophobins and genes corresponding to these cDNAs encoding P. nameko hydrophobins were designated pnh1, pnh2 and pnh3, respectively. PNH1, PNH2 and PNH3 had a conserved spacing of eight cysteine residues in hydrophobins and a hydropathy pattern characteristic of class I hydrophobins. Phylogenetic analysis showed that PNH1, PNH2 and PNH3 were phylogenetically similar and significantly related to the hydrophobin POH1 specifically expressed in fruiting bodies of Pleurotus ostreatus. Northern blot analysis indicated that, under conditions of Pi deficiency, pnh2 and pnh3 were also induced in strains N4 and N301.Communicated by U. Kück     

Structure and expression of a phosphate deficiency-inducible ribonuclease gene in<Emphasis Type="Italic"> Pholiota nameko</Emphasis>

Thirty-one cDNAs corresponding to pdi genes [inorganic phosphate (Pi) deficiency-inducible genes] were previously isolated through the differential screening of a cDNA library constructed from the mycelium of Pholiota nameko. Among the cDNAs, pdi370 was analyzed here. The deduced amino acid sequence showed high similarity to fungal ribonucleases (RNases) and contained two signature sequences conserved in T2 family RNases: CAS1 and CAS2. Genomic DNA harboring the pdi370 gene was isolated from a genomic library of P. nameko. Sequence analysis showed that the pdi370 gene is interrupted by 16 introns and that the promoter region contains two cis-acting sequences found in Pi deficiency-induced genes from Saccharomyces cerevisiae, together with several known functional elements, such as a TATA box. RNase activity in the mycelium and culture filtrate increased 5.6-fold and 5.2-fold, respectively, under Pi-deficient conditions. Staining for RNase activity showed that at least four RNases are induced and secreted under the conditions. The N-terminal sequence of one of them agreed with that of the pdi370 gene product.Communicated by J. Heitman     

Regulation of synthesis and secretion of acid and alkaline phosphatases in Neurospora crassa

Summary We show that N. crassa represses the production of acid phosphatase at pH higher than 8.0, irrespective of the carbon source used, whereas production was stimulated by sucrose at slightly acidic pH. The same profile of acid phosphatase production was observed in the pho-2A, pho-3A, nuc-1A, nuc-2A and preg ^c mutant strains. We also show that acid phosphatase synthesized by the preg ^c mutant strain grown on high phosphate medium has pronounced differences when compared to the enzyme synthesized by the wild-type strain grown on low phosphate medium in terms of heat stability, steady-state kinetic properties and DEAE-cellulose chromatography. In addition, the synthesis and/or secretion of only phosphate-repressible alkaline phosphatase is affected by mutations in acu-1, and acu-5 and acu-7 genes. These results, which indicate distinct pathways for the synthesis and secretion of acid and alkaline phosphatases in N. crassa, contradict the dosage titration model proposed by Metzenberg et al. (1974) whereby the synthesis of these enzymes should occur through a single hierarchical regulatory circuit as a response to phosphate starvation.     

Identification and linkage mapping of the genes for the putative homeodomain protein (hox1) and the putative pheromone receptor protein homologue (rcb1) in a bipolar basidiomycete, Pholiota nameko

In the current studies, we sequenced and characterized the gene for the homeodomain protein (hox1) in a bipolar mushroom, Pholiota nameko, which is a putative homologue of A mating type genes in the tetrapolar basidiomycete, Coprinopsis cinerea. We also sequenced and characterized the gene for the pheromone receptor (rcb1) in P. nameko, which is a putative homologue of the B mating type genes in C. cinerea. Restriction fragment length polymorphism (RFLP) and linkage analyses indicated that the both genes are present as a single locus on the different chromosome. Moreover, in P. nameko, the hox1 gene was mapped to the A mating type locus in linkage group I. However, rcb1 was not linked to the A mating type locus and was mapped to the other linkage group. These results strongly suggest that hox1 regulates with incompatibility in the bipolar mushroom, and that rcb1 may not affect the mating function in P. nameko. This is the first report regarding the structure of the mating type genes in bipolar mushrooms.     

The STA2 and MEL1 genes of Saccharomyces cerevisiae are idiomorphic

Summary The STA2 (glucoamylase) gene of Saccharomyces cerevisiae has been mapped close to the end of the left arm of chromosome II. Meiotic analysis of a cross between a haploid strain containing STA2, and another strain carrying the melibiase gene MEL1 (which is known to be at the end of the left arm of chromosome II) produced parental ditype tetrads only. Since there is no significant DNA sequence similarity between the STA2 and MEL1 genes, or their respective flanking regions, we conclude that these two genes are carried by separate non-hybridizing sequences of chromosomal DNA, either of which can reside at the end of the left arm of chromosome II. By analogy with the mating-type locus of Neurospora crassa, we suggest that the STA2 and MEL1 genes are idiomorphs with respect to one another.     

Cloning and expression on a multicopy vector of five invertase genes of Saccharomyces cerevisiae

Summary Six unlinked loci for invertase structural genes are known in the yeast Saccharomyces cerevisiae: SUC1-SUC5 and SUC7. These genes are similar in structure and expression but not identical. Different yeast strains possess none, one or several of these genes.We have isolated the genes SUC1-SUC5, subcloned them into the multicopy vector YEp24 and compared the expression of the five SUC genes in one recipient strain. SUC2 was isolated by transformation of a suc0 strain with a gene pool and complementation to sucrose fermentation. SUC4 was cloned from a minipool of chromosomal fragments which were shown to contain SUC4 by Southern hybridization. SUC1, SUC3 and SUC5 were isolated using the method of plasmid eviction. A plasmid containing regions flanking SUC4 was integrated next to these SUC genes. The plasmid together with the SUC genes were then cut out of the chromosome using an appropriate restriction endonuclease.The length of chromosomal DNA fragments containing the different SUC genes were 4.8 kb for SUC1, 5.2 kb for SUC2, 4.8 kb for SUC3, 12.8 kb for SUC4 and 17.2 kb for SUC5.Fragments containing the complete SUC genes and the sequences controlling their expression were subcloned into YEp24 and transformed into a strain without any active invertase gene. Invertase activity of transformants was measured after growth repressing (8% glucose) and derepressing (2% raffinose) conditions. As expected from results with strains carrying the individual SUC genes in a chromosomal location, the SUC genes were expressed to a different extent.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthdayThis work was supported by Deutsche Forschungsgemeinschaft     

Cloning and functional characterization of a fatty acid synthase component FAS2 gene from Saccharomyces kluyveri

A gene coding the α subunit of fatty acid synthase (FAS2) was isolated from the budding yeast Saccharomyces kluyveri. Nucleotide sequence analysis indicated that this gene, termed Sk-FAS2, coded a protein having an amino acid sequence 83% identical to the FAS2 protein of S. cerevisiae (Sc-FAS2). The Sk-FAS2 gene was able to functionally complement an S. cerevisiae fas2 disruptant. This Sk-FAS2-expressing strain was found to produce larger amounts of C18 than C16, in contrast to the Sc-FAS2-expressing fas2 strain. In addition, fusion genes of Sk-FAS2 and Sc-FAS2 were transformed into a fas2-disrupted strain of S. cerevisiae, and fatty acid analysis of these transformants suggested that the region containing the acyl carrier protein and β-ketoacyl reductase domains of yeast FAS2 protein play an important role in determining carbon chain length of fatty acids.GenBank Submission: The nucleotide sequences reported in this paper have been submitted to GenBank Nucleotide Sequence Database under the accession number AB115969.     

Either one of the two yeast EF-1α genes is required for cell viability

Summary Two genes,TEF1 andTEF2, encode the protein elongation factor EF-1 in the yeastSaccharomyces cerevisiae. We have generated yeast haploid strains containing eitherTEF1 orTEF2 interrupted by insertion of a large piece of foreign DNA. Cells which contain either one functional copy of the EF-1 genes are viable. In contrast, attempts to isolate a yeast haploid strain with bothTEF1 andTEF2 inactivated have failed suggesting that the double gene disruption is a lethal event.     

Physical mapping of the Schizosaccharomyces pombe histone genes

The histone-encoding genes in Schizosaccharomyces pombe were physically mapped by hybridisation to filters containing cosmid and P1 genomic libraries. The H2A.2 gene and the H2A.1-H2B.1 gene pair mapped between the ade6 and rikl genes on chromosome III. The three H4–H3 gene pairs were mapped to three different regions by a H4.1 probe. Southern analysis of clones from each region revealed the positions of the three H4–H3 gene paris. H4.1–H3.1 was localised to chromosome I between the mei2 and rad1 genes; H4.2–H3.2 mapped between rad3 and cdc2 on chromosome II; H4.3–H3.3 was localised to a region between the nuc1 and puc1 genes on chromosome II.     

The mitochondrial genome of the basidiomycete Agrocybe aegerita: molecular cloning,physical mapping and gene location

Summary The mtDNA of a wild-type strain of Agrocybe aegerita was purified from mitochondria isolated by cellular fractionation. A representative library was constructed in E. coli by molecular cloning at the HindIII restriction site of pBR322. Southern hybridizations between total DNA of the fungal strain and cloned mitochondrial insert probes were used to establish the restriction map of the mtDNA molecule. Its size was assessed at about 80 500 bp. Four structural genes (for Cox 1, Cox 2, Atp 6, and Atp 8) were located on the map by heterologous hybridizations with oligonucleote probes specific for yeast mitochondrial genes. The location of the genes coding for the large and the small RNAs of the mitochondrial ribosome was determined by hybridization with the E. coli rrnB operon. A comparison of A. aegerita mtDNA organization with that of both phylogenetically close and distant fungi is discussed.     

Alteration of the cytochrome c oxidase subunit 2 gene in the [exn-5] mutant of Neurospora crassa

Summary The maternally inherited [exn-5] mutant of Neurospora crassa is characterized by its slow-growth rate and deficiency of cytochrome aa ₃ relative to wildtype strains. We have determined the DNA sequence of the COXI and COXII genes of the mutant, which encode subunits 1 and 2 of cytochrome c oxidase, respectively. No changes in the DNA sequence of the COXI gene relative to the corresponding wild-type gene were found. In the region of the COXII gene we found two alterations, one a C to T transition eight base pairs upstream of the coding sequence and the second within the coding sequence for subunit 2 affecting amino acid 27 of the precursor polypeptide (amino acid 15 of the mature polypeptide). The altered codon in [exn-5] specifies an isoleucine residue rather than the wild-type threonine residue. The corresponding position in subunit 2 sequences of all other organisms examined is conserved either as a threonine or a serine residue. Thus, we consider it likely that the mutation directly affecting the coding sequence of the polypeptide is responsible for the [exn-5] phenotype. Analysis of serially passaged heterokaryons constructed between wild-type and [exn-5] shows that both mutations segregate with the [exn-5] phenotype. Examination of mitochondrial translation products in [exn-5] revealed a deficiency of subunit 2, as well as the presence of a polypeptide that corresponds to a previously described precursor of subunit 1 that accumulates in a COXI mutant of N. crassa, [mi-3]. We propose possible relationships between [exn-5], [mi-3], and the nuclear su-1 ^[mi-3] allele, which suppresses both mutations.     

Analysis of Opi1p repressor mutants

Opi1p is the only known repressor protein specific to the phospholipid biosynthetic pathway. Opi1p is required for repression in response to inositol and choline supplementation. However, the mechanism of Opi1p repression is not completely understood. In part, this is because previously identified opi1 mutants contained nonsense mutations and thus provided little insight into the mechanism of Opi1p function. We have recently reported isolating novel opi1 mutants (rum and dim mutants) that contain missense mutations. Here, we show that these opi1 mutants produce Opi1p product at levels comparable to a wild-type strain. However, these mutants mis-regulate expression of two target genes, INO2-HIS3 and INO1-lacZ, and are also defective in autoregulation. An opi1-S339F mutant is particularly interesting because it completely eliminated autoregulation, but only abated regulation of an INO1-lacZ reporter. Two of the mutations in OPI1 (V343Q and S339F) provide genetic evidence for an interaction between Opi1p and the Ino2p activator since they reside in a region of Opi1p recently shown to interact with Ino2p in vitro. A third mutation (L252F) resides in a region of Opi1p with no known function.     

Isolation and characterization of Pioneer1, a novel Chlamydomonas transposable element

During the course of this study a novel family of Chlamydomonas mobile elements has been identified in natural isolate strain 224. The first member of this class to be characterized, a 2.8-kb element named Pioneer1, was trapped in an intron of the nitrate reductase structural gene, NIT1. This element has been cloned and completely sequenced and found to be unusual in structure. Pioneer elements are present in a very low-copy number of three per genome in strain 224. The copy number increased by one upon transposition of Pioneer1. Hybridization of Pioneer1 to a variety of Chlamydomonas strains confirmed that this element differed from previously described Chlamydomonas transposons. It also indicated that related elements are present in low-copy number in natural isolate strains 356 and S1D2, but not in the most commonly used laboratory strains 137c and 21 gr. For these reasons, members of the Pioneer family might prove useful as insertional mutagens.     

Low frequency of the CYP21A2 deletion in ethnic Chinese (Taiwanese) patients with 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder which causes more than 90% of CAH cases due to defects in the steroid 21-hydroxylase gene (CYP21A2). The frequency of large mutations was determined in 200 ethnic Chinese (i.e., Taiwanese) CAH patients belonging to 200 families with different clinical forms of CYP21A2 deficiency over 10 years of molecular diagnoses. For a large-gene deletion (or conversion) and the CYP21A2 deletion identification, a PCR product covering the TNXB gene and the 5′-end of the CYP21A2 gene with TaqI endonuclease digestion was analyzed by electrophoresis on agarose gels. For CYP21A2 mutational analysis, secondary PCR amplification of the amplification-created restriction site method was applied. From the results of the analysis, we found that large-gene deletions (or conversions) occurred in 7.5% of the alleles including three different types of the chimeric CYP21A1P/CYP21A2 genes and the haplotype of IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation). The CYP21A2 deletion occurred in 2.0% of the alleles which contained three types of the chimeric TNXA/TNXB genes with two novel ones. We concluded that the CYP21A2 deletion in the ethnic Chinese (Taiwanese) patients exhibits a low occurrence, with the haplotype of the IVS2-12A/C>G in combination with the 707-714del mutation (without the P30L mutation) being prevalent among large gene deletions or conversions.     

Analyzing time-dependent microarray data using independent component analysis derived expression modes from human macrophages infected with F. tularensis holartica

The analysis of large-scale gene expression profiles is still a demanding and extensive task. Modern machine learning and data mining techniques developed in linear algebra, like Independent Component Analysis (ICA), become increasingly popular as appropriate tools for analyzing microarray data. We applied ICA to analyze kinetic gene expression profiles of human monocyte derived macrophages (MDM) from three different donors infected with Francisella tularensis holartica and compared them to more classical methods like hierarchical clustering. Results were compared using a pathway analysis tool, based on the Gene Ontology and the MeSH database. We could show that both methods lead to time-dependent gene regulatory patterns which fit well to known TNFα induced immune responses. In comparison, the nonexclusive attribute of ICA results in a more detailed view and a higher resolution in time dependent behavior of the immune response genes. Additionally, we identified NFκB as one of the main regulatory genes during response to F. tularensis infection.     

Okadaic Acid,a Phosphatase Inhibitor,Enhances the Phorbol Ester-Induced Interleukin-1B Expression via an AP-1-Mediated Mechanism

Protein kinase C (PKC)-activating phorbol esters are known to induce the expression of several genes in monocytic cells. As the effect of serine-threonine kinases, such as PKC, is often counteracted by specific protein phosphatases, we have now examined the role of phosphatases in the regulation of the phorbol ester (PM A)-induced interleukin-B(IL-1 B) gene expression in theTHP-1 monocytic leukaemia cell line. Okadaic acid (OA) is a potent tumour promoter, the function of which is based on its activity to inhibit the serine/threonine specific phosphatases 1 and 2A (PPI and PP2A, respectively). Thus, it mimicks or potentiates the action of PKC activators in several cell types. Our data demonstrate that alone OA induced a very weak expression of IL-B mRNA, but it strongly enhanced the PMA-induced IL-1BS expression. To analyse the site of action of OA, the cells were transiently transfected with a chloramphenicol acetyl transferase (CAT) –reporter plasmid containing the AP-1 binding site as the enhancer. Alone, OA was a weak inducer of CAT–activity in these cells, but again it strongly enhanced the PMA-induced response. Similar data were obtained with cells transfected with a reporter plasmid containing the PMA-responsive element (containing a putative AP-1 binding site) of the IL-B gene. Thus, these data indicate that the PMA-induced AP-1 enhancer activity, which is required for the expression of the IL–lB gene, is controlled in these cells by PPI and/or PP2A. As OA did not synergize with PMA in the induction of expression of genes encoding the AP-1 proteins (c–fos, c–jun, junB), it is likely that OA potentiates the AP-1 enhancer activity by its effect on protein phosphorylation.     

The putative LEF-1 proteins from two distinct Choristoneura fumiferana multiple nucleopolyhedroviruses share domain homology to eukaryotic primases

We have identified the lef-1 genes from two multiple nucleopolyhedroviruses that infect natural populations of Choristoneura fumiferana. The lef-1 genes in both viruses are directly upstream and in the opposite orientation of their respective ecdysteroid UDP-glucosyltransferase (egt) genes. This gene organization pattern is similar to that found in the genomes of AcMNPV and of OpMNPV. As well, the coding regions and putative protein sequences share a high degree of similarity. Alignment of the predicted amino acid sequences of all known baculovirus lef-1 genes suggests that the LEF-1 proteins have a relatively high degree of conservation, particularly at four identified and distinct domains. Moreover, LEF-1 proteins bear clear similarity to some eukaryotic primases, predominately at three of the four domains where certain amino acids are absolutely conserved.