A ras homologue of Neurospora crassa regulates morphology

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa. Received: 1 October 1996 / Accepted: 9 December 1996     

Analysis of eryBI , eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis. Received: 13 August 1997 / Accepted: 27 November 1997     

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2 , amdS , and gatA promoters

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A.␣nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells. Received: 28 June 1996 / Accepted: 7 October 1996     

Two uvs genes of Aspergillus nidulans with different functions in error-prone repair: uvsI , active in mutation-specific reversion, and uvsC , a recA homolog, required for all UV mutagenesis

Two genes of Aspergillus nidulans are known to function in UV mutagenesis, but have been assigned to different epistasis groups: uvsC, which is also required for meiosis and mitotic recombination, and uvsI, which may have no other function. To clarify their role in error-prone repair and to investigate their interaction, uvsI and uvsC single and uvsI;uvsC double mutant strains were further tested for mutagen sensitivities and characterized for effects on mutation. Spontaneous and induced frequencies were compared in forward and reverse mutation assays. All results confirmed that uvsI and uvsC are members of different epistasis groups, and demonstrated that these uvs mutants have very different defects in UV mutagenesis. The uvsI strains showed wild-type frequencies in all forward mutation tests, but greatly reduced spontaneous and UV-induced reversion of some, but not other, point mutations. In contrast, uvsC had similar effects in all assay systems: namely pronounced mutator effects and greatly reduced UV mutagenesis. Interestingly, the uvsI;uvsC double mutant strains differed from both single mutants; they clearly showed synergism for all types of reversion tested: none were ever obtained spontaneously, nor after induction by UV or EMS (ethylmethane sulfonate). Based on these results, we conclude that uvsI is active in a mutation-specific, specialized error-prone repair process in Aspergillus. In contrast, uvsC, which is now known to show sequence homology to recA, has a basic function in mutagenic UV repair in addition to recombinational repair, similar to recA of Escherichia coli. Received: 23 September 1996 / Accepted: 2 December 1996     

A meiotically reproducible chromosome length polymorphism in the ascomycete fungus Ophiostoma ulmi ( sensu lato )

We have followed the transmission of Ophiostoma ulmis.l. chromosome length polymorphisms (CLPs) into the F₂ generation to determine the reproducibility of a genome rearrangement culminating in the conversion of a 1.0 Mb chromosome into a 800 kb chromosome. The 1.0 Mb chromosome in strain CESS16K is thus far unique among O. ulmi s.l. wild-type strains, as no other wild-type strains have been observed with chromosomes smaller than 2.3 Mb. It has been previously shown that the 1.0 Mb chromosome is mitotically stable, carries at least one normally expressed gene, and is transmitted through meiosis. In this study, a series of crosses were performed to further elucidate the pattern of inheritance of the 1.0 Mb chromosome and the process of conversion of the 1.0 Mb species to 800 kb. In crosses where the 1.0 Mb chromosome was allowed to pair with itself or with the 800 kb chromosome, all progeny inherited a copy of the 1.0 Mb or 800 kb form, further demonstrating the A-type nature of these small chromosomes. When a cross was repeated between the strains CESS16K (1.0 Mb chromosome) and FG245B^r-O (no 1.0 Mb or 800 kb chromosome), the occurrence of a 800 kb chromosome was observed in 9% of the progeny. A reciprocal cross between an 800 kb strain and a strain with no 800 kb or 1.0 Mb chromosome was conducted, and a progeny strain containing a 1.0 Mb chromosome was recovered. The reproducibility and reciprocality of the 1.0 Mb to 800 kb chromosome conversion demonstrates that meiotic processes are responsible for this CLP, and that O. ulmi s.l. strains with various divergent genome architectures can remain sexually compatible. Received: 6 February 1996 / Accepted: 21 January 1997     

Tolerance of low pH in Schizosaccharomyces pombe requires a functioning pub1 ubiquitin ligase

A strain of Schizosaccharomyces pombe carrying a disrupted Na⁺/H⁺ antiporter gene (sod2::sup3-5), in addition to the common auxotrophic mutations, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media adjusted to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongated and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence of two independently segregating mutations. One, pub1 ( protein ubiquitin ligase 1), has recently been reported as an E3 protein ubiquitin ligase involved in cdc25 turnover. The second has been named elp3-1 (elongated at low pH). Genetic dissection of the original strain revealed that poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the function of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both the pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of LiCl (3 mM) in liquid culture, it was capable of partially suppressing the extreme Li⁺ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was improved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 mutation had no effect on the Li⁺ tolerance in either wild-type or sod2::ura4 backgrounds. pub1-1 cells are elongated and incapable of colony formation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 and pH␣5.5 (the normal pH of minimal medium) but can form colonies under both conditions. J227 cells are significantly longer than either single mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 background yielded a genomic clone of pub1, allowing us to define the intron-exon structure of the gene. Sequences with high homology to the predicted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd4). Based on the nature of our mutant selection, the pH-sensitive phenotype of the strains selected, and the known involvement of RSP5/NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transport processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at high and low external pH, and in the presence of toxic levels of Li⁺. Received: 12 September 1996 / Accepted: 19 December 1996     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

Cloning and molecular analysis of the Arabidopsis gene Terminal Flower 1

The Arabidopsis gene Terminal Flower 1 (TFL1) controls inflorescence meristem identity. A terminal flower (tfl1) mutant, which develops a terminal flower at the apex of the inflorescence, was induced by transformation with T-DNA. Using a plant DNA fragment flanking the integrated T-DNA as a probe, a clone was selected from a wild-type genomic library. Comparative sequence analysis of this clone with an EST clone (129D7T7) suggested the existence of a gene encoding a protein similar to that encoded by the cen gene which controls inflorescence meristem identity in Antirrhinum. Nucleotide sequences of the region homologous to this putative TFL1 gene were compared between five chemically induced tfl1 mutants and their parental wild-type ecotypes. Every mutant was found to have a nucleotide substitution which could be responsible for the tfl1 phenotype. This result confirmed that the cloned gene is TFL1 itself. In our tfl1 mutant, no nucleotide substitution was found in the transcribed region of the gene, and the T-DNA-insertion site was located at 458 bp downstream of the putative polyadenylation signal, suggesting that an element important for expression of the TFL1 gene exists in this area. Received: 14 November 1996 / Accepted: 29 November 1996     

Homologous recombination involving cox2 is responsible for a mutation in the CMS-specific mitochondrial locus of Petunia

We have characterized the only mutation detected so far in S-Pcf, the mitochondrial cytoplasmic male sterility (CMS)-specific locus of petunia. This locus consists of three open reading frames (ORFs): the first contains part of atp9, an intron-less cox2 pseudogene (which does not contain the original cox2 ATG) and the unidentified reading frame urf-s; the second and third ORFs correspond to the only copies of nad3 and rps12 genes in the genome, respectively. In the cell line R13-138, which was generated from a male-sterile somatic hybrid (line SH13-138), a change in the first ORF of the S-Pcf locus has been characterized: the atp9 sequence has been lost, while exon1 of the normal copy of the cox2 gene (including the original ATG sequence) and the adjacent 5′ sequence of the petunia recombination repeat, have been introduced. The data suggest that this reorganization of mtDNA is the consequence of a homologous recombination event involving part of the cox2 coding region, and that the cox2 coding region may serve as an active site for inter- or intra-mtDNA homologous recombination. The results further suggest that in line SH13-138 (or during its maintenance in tissue culture), segregation of the S-Pcf-containing mtDNA molecules has occurred, and the mutant mtDNA is now predominant in the population. Received: 9 September 1996 / Accepted: 27 January 1997     

Characterization of a novel DNA damage-inducible gene of Saccharomyces cerevisiae , DIN7 , which is a structural homolog of the RAD2 and RAD27 DNA repair genes

A number of DNA damage-inducible genes (DIN) have been identified in Saccharomyces cerevisiae. In the present study we describe isolation of a novel gene, Din7, the expression of which is induced by exposure of cells to UV light, MMS (methyl methanesulfonate) or HU (hydoxyurea). The DNA sequence of DIN7 was determined. By comparison of the predicted Din7 amino acid sequence with those in databases we found that it belongs to a family of proteins which includes S. cerevisiae Rad2 and its Schizosaccharomyces pombe and human homologs Rad13 and XPGC; S. cerevisiae Rad27 and its S. pombe homolog Rad2, and S. pombe Exo I. All these proteins are endowed with DNA nuclease activity and are known to play an important function in DNA repair. The strongest homology to Din7 was found with the Dhs1 protein of S.␣cerevisiae, the function of which is essentially unknown. The expression of the DIN7 gene was studied in detail using a DIN7-lacZ fusion integrated into a chromosome. We show that the expression level of DIN7 rises during meiosis at a time nearly coincident with commitment to recombination. No inducibility of DIN7 was found after treatment with DNA-damaging agents of cells bearing the rad53-21 mutation. Surprisingly, a high basal level of DIN7 expression was found in strains in which the DUN1 gene was inactivated by transposon insertion. We suggest that a form of Dun1 may be a negative regulator of the DIN7 gene expression. Received: 30 May 1996 / Accepted: 26 September 1996     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis, let-395, let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch, thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position. Received: 8 May 1996 / Accepted : 27 January 1997     

PRS1 is a key member of the gene family encoding phosphoribosylpyrophosphate synthetase in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the metabolite phosphoribosyl-pyrophosphate (PRPP) is required for purine, pyrimidine, tryptophan and histidine biosynthesis. Enzymes that can synthesize PRPP can be encoded by at least four genes. We have studied 5-phospho-ribosyl-1(α)-pyrophosphate synthetases (PRS) genetically and biochemically. Each of the four genes, all of which are transcribed, has been disrupted in haploid yeast strains of each mating type and although all disruptants are able to grow on complete medium, differences in growth rate and enzyme activity suggest that disruption of PRS1 or PRS3 has a significant effect on cell metabolism, whereas disruption of PRS2 or PRS4 has little measurable effect. Using Western blot analysis with antisera raised against peptides derived from the non-homology region (NHR) and the N-terminal half of the PRS1 gene product it has been shown that the NHR is not removed by protein splicing. However, the fact that disruption of this gene causes the most dramatic decrease in cell growth rate and enzyme activity suggests that Prs1p may have a key structural or regulatory role in the production of PRPP in the cell. Received: 15 July 1996 / Accepted: 24 October 1996