Right Arm Of Linkage Group Research Articles

The Neurospora crassa mus-19 gene is identical to the qde-3 gene, which encodes a RecQ homologue and is involved in recombination repair and postreplication repair.

An allele called mus-19 was identified by screening temperature-sensitive and mutagen-sensitive mutants of Neurospora crassa. The mus-19 gene was genetically mapped to a region near the end of the right arm of linkage group I, where a RecQ homologue called qde-3 had been physically mapped in the Neurospora database. Complementation tests between the mus-19 mutant and the qde-3(RIP) mutant showed that mus-19 and qde-3 were the same gene. The qde-3 genes of both mutants were cloned and sequenced; and the results showed that they have mutation(s) in their qde-3 genes. The original mus-19 and qde-3(RIP) mutants are defective in quelling, as reported for other qde-3 mutants. The mutants show high sensitivity to methyl methanesulfonate, ethyl methanesulfonate, N-methyl- N'-nitro- N-nitrosoguanidine, tert-butyl hydroperoxide, 4-nitroquinoline-1-oxide, hydroxyurea and histidine. Epistasis analysis indicated that the qde-3 gene belongs both to the uvs-6 recombination repair pathway and the uvs-2 postreplication repair pathway. The qde-3 mutation has no effect on the integration of a plasmid carrying the mtr gene by homologous recombination. In homozygous crosses, the qde-3 mutant is defective in ascospore production.

Characterization and expression of a Neurospora crassa ribosomal protein gene, crp-7.

We have isolated and characterized a Neurospora crassa cytoplasmic ribosomal protein gene, named crp-7, which is found upstream of the photolyase gene. The deduced amino-acid sequence of this gene is highly homologous to the YS25 ribosomal protein of Saccharomyces cerevisiae. The crp-7 ORF consists of two exons which are separated by a short intron. The deduced polypeptide contains 87 amino acid residues and has a calculated molecular weight of 9.7 kDa. RFLP mapping showed that the crp-7 gene is located on the right arm of linkage group I. Southern blot hybridization analyses indicated that there is only one copy of the crp-7 gene in the N. crassa genome. Transcriptional elements, the Dde box, the Taq box and the CG element, that have been identified in other N. crassa ribosomal protein genes are observed in the promoter region of the crp-7 gene. The crp-7 mRNA levels were low in conidia and highest in young mycelia during vegetative growth. The mRNA levels of four r-protein genes, including the crp-7 gene, as well as the tef-1 gene encoding translational elongation factor 1 alpha, were raised following the treatment of mycelia with a low concentration of cycloheximide. This indicates that the expression of r-protein genes is under the control of so-called super-induction.

Characterization of a Neurospora crassa Photolyase-Deficient Mutant Generated by Repeat Induced Point Mutation of the phr Gene

We produced a photolyase-deficient mutant by repeat induced point mutation using the Neurospora crassa photolyase gene cloned previously. This mutation identified a new gene, phr, which was mapped on the right arm of linkage group I by both RFLP mapping and conventional mapping. To investigate the relationship between photoreactivation and dark repair processes, especially excision repair, double mutants of phr with representative repair-defective mutants of different types were constructed and tested for UV sensitivity and photoreactivation. The results show that the phr mutation has no influence on dark repair. Tests with CPD and TC(6-4) photoproduct-specific antibodies demonstrated that the phr mutant is defective in CPD photolyase and confirmed that there is no TC(6-4) photolyase activity in N. crassa. Furthermore, N. crassa photolyase is not a blue light receptor in the signal transduction that induces carotenoid biosynthesis.

Identification and linkage mapping of the phsA gene of Aspergillus nidulans, where mutation affects growth and pigmentation of colonies in a temperature- and pH-dependent way

We report the isolation and characterization of a mutant strain of the mold Aspergillus nidulans showing an altered response to environmental pH, including a reduction in its pH range for growth and the production of a melanin-like pigment at alkaline pH. We also show that the mutant strain is not detergent-sensitive and that its acid sensitivity is osmotically remediable with 0.5 M NaCl or 1.0 M sorbitol. Furthermore, the mutant phenotype is temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, with the restoration of a wild-type phenotype to the mutant strain being observed at 28°C. On the other hand, the severity of the mutant phenotype is increased at 40°C. Genetic analysis shows that this pH- and temperature-sensitive mutation, named phsA1, is located on the right arm of linkage group I of A. nidulans, between pabaA and yA, and that mutation phsA1 is recessive.

Identification and linkage mapping of the phsA gene of Aspergillus nidulans, where mutation affects growth and pigmentation of colonies in a temperature- and pH-dependent way.

We report the isolation and characterization of a mutant strain of the mold Aspergillus nidulans showing an altered response to environmental pH, including a reduction in its pH range for growth and the production of a melanin-like pigment at alkaline pH. We also show that the mutant strain is not detergent-sensitive and that its acid sensitivity is osmotically remediable with 0.5 M NaCl or 1.0 M sorbitol. Furthermore, the mutant phenotype is temperature-remediable with respect to pigmentation, extent of conidiation and growth diameter, with the restoration of a wild-type phenotype to the mutant strain being observed at 28 degrees C. On the other hand, the severity of the mutant phenotype is increased at 40 degrees C. Genetic analysis shows that this pH- and temperature-sensitive mutation, named phsA1, is located on the right arm of linkage group I of A. nidulans, between pabaA and yA, and that mutation phsA1 is recessive.

Characterization of Neurospora crassa albino mutants that were previously unassigned to locus

Neurospora crassa mutant hunts by various groups have identified albino (al) mutants that map to the al-1 - al-2 region on the right arm of linkage group I. The cloning of the al-1 and al-2 genes (Schmidhauser et al. 1990 Mol. Cell. Biol. 10:5064-5070, Schmidhauser et al. 1994 J. Biol. Chem. 269:12060-12066) allows assignment of locus to the above mutants by DNA mediated transformation. Analysis of phytoene desaturases from different organisms indicates at least three types of enzymes as defined by the number of desaturation steps carried out (Sandmann 1994 J. Plant Physiol. 143:444-447). The N. crassa phytoene desaturase, the al-1 gene product, introduces four double bonds converting phytoene to lycopene. Of the three intermediates in this reaction sequence two are colored. The occurrence of visibly distinguishable albino alleles in N. crassa has been noted (Perkins 1989 Fungal Genetics Newsl. 36:63). Assignment of locus to N. crassaalbino alleles represents a first step in the functional characterization of N. crassa carotenogenic loci.

Correlation of the physical and genetic maps of the centromeric region of the right arm of linkage group III of Neurospora crassa.

We have cloned three linked genes serine-1 (ser-1), proline-1 (pro-1) and acetate-2 (ace-2) that lie near the centromere on the right arm of linkage group III (LGIIIR) of Neurospora crassa. The ser-1 gene was cloned by sib selection. A chromosomal walk that spans 205 kilobases (kb) was initiated from ser-1. Complementation analysis with clones isolated during the walk allowed identification of the pro-1 and ace-2 genes. Restriction fragment length polymorphism analysis has confirmed the localization of ser-1, pro-1 and ace-2 to the centromeric region of LGIIIR. Genetically, we measured 1% recombination between ser-1 and pro-1 and 2% recombination between pro-1 and ace-2. Physical distances for these intervals were 114 kb from ser-1 to pro-1 and 36 kb from pro-1 to ace-2. Thus, for the pro-1 to ace-2 interval we calculate a physical/genetic correlation of 18 kb/map unit (mu) whereas, in the immediately adjacent, centromere-proximal interval from ser-1 to pro-1, we calculate 114 kb/mu. This provides evidence for a centromere effect, a decrease in recombination frequency as one approaches the centromere.

FK506-binding protein of Neurospora crassa (NcFKBP) mediates sensitivity to the immunosuppressant FK506; resistant mutants identify two loci

Growth of Neurospora crassa wild-type is inhibited by micromolar concentrations of the immunosuppressive macrolide FK506. Spontaneous and induced mutations that confer resistance to FK506 identified two loci, fkr-1 and fkr-2. They map on the right arm of linkage group V on either side of inl with fkr-1 being centromere proximal. Allele fb (fkr-2) lacks immunodetectable N. crassa FK506-binding protein (NcFKBP). This demonstrates that the sensitivity of N. crassa towards FK506 is mediated by NcFKBP. FK506-binding proteins have been shown to be highly conserved, i.e., found in all eukaryotic cells tested, and to exhibit peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro. Possible functions for the loci are discussed. Apart from the resistance to FK506 no other mutant phenotype was detected not even in double mutants that lacked NcFKBP as well as cyclophilin. Cyclophilin mediates the cytotoxic effect of the immunosuppressive drug Cyclosporin A and is also characterized by PPIase activity in vitro. Both FK506-resistant alleles studied exhibit incomplete dominance in forced heterokaryons. A mechanism is proposed to explain this dominance especially in view of the NcFKBP-deficient allele, fb.

Ribosomal DNA is a site of chromosome breakage in aneuploid strains of Neurospora.

In wild-type strains of Neurospora crassa, the rDNA is located at a single site in the genome called the nucleolus organizer region (NOR), which forms a terminal segment on linkage group (LG) V. In the quasiterminal translocation strain T(I;V)AR190, most of the right arm of LG I moved to the distal tip of the NOR, and one or a few rDNA repeat units are moved to the truncated right arm of LG I. I report here that, in partial diploid strains derived from T(I;V)AR190, large terminal deletions result from chromosome breakage in the NOR. In most of these partial diploids, chromosome breakage is apparently frequent and the breakpoints occur in many parts of the NOR. The rDNA ends resulting from chromosome breakage are "healed" by the addition of new telomeres. Significantly, the presence of ectopic rDNA creates a new site of chromosome breakage in the genome of partial diploids. These results raise the possibility that, under certain conditions, rDNA is a region of fragility in eukaryotic chromosomes.

Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa.

We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

Coordiante expression of ribosomal protein genes inNeurospora Crassaand identification of conserved upstream sequences

The relative levels of rRNAs and ribosomal proteins are coordinately regulated by growth rate and carbon nutrition in Neurospora crassa. However, little is known about the mechanisms involved. To investigate the transcriptional regulation of ribosomal protein genes in N. crassa, we cloned and sequenced a ribosomal protein gene (crp-3). The inferred crp-3 protein sequence shares 89% and 83% homology at its N-terminus with the yeast rp51 and the human S17 ribosomal proteins respectively. The crp-3 gene contains two introns, neither of which are conserved in position with the RP51 or the S17 genes. The crp-3 gene is present in a single copy and was mapped by RFLP analysis to the right arm of linkage group IV, near the cot-1 locus. Sequence comparisons of the upstream regions of the three sequenced crp genes revealed several common features. These include a 'Taq box' (consensus: ARTTYGACTT) at -39, a CG repeat (consensus: CCCRCCRRR) at -65, and a major transcription initiation site embedded in a purine rich region flanked by an upstream pyrimidine rich sequence. Using four N.crassa ribosomal protein genes as probes, we demonstrated that the levels of the four ribosomal protein mRNAs were closely coordinated during a nutritional downshift from sucrose to quinic acid.

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosylmethionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere- spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.

Determination of the inactivating alterations in two mutant alleles of the Neurospora crassa cross-pathway control gene cpc-1.

cpc-1 is the locus specifying what is believed to be the major trans-activating transcription factor that regulates expression of amino acid biosynthetic genes subject to cross-pathway control in Neurospora crassa. Mutants altered at this locus are incapable of the global increase in gene expression normally seen in response to amino acid starvation. Using polymerase chain reaction methodology we have cloned and sequenced the inactive mutant allele, cpc-1 (CD15). The cpc-1 (CD15) mutation was found to be a single base pair deletion in codon 93 of the cpc-1 structural gene. A second, presumed lethal, allele, cpc-1 (j-5), also was investigated. Northern analyses with strains carrying the cpc-1 (j-5) allele revealed that no cpc-1 mRNA is produced. Southern and genetic analyses established that the cpc-1 (j-5) mutation involved a chromosomal rearrangement in which a break occurred within the cpc-1 locus, normally resident on linkage group VI; a small fragment from the left arm of linkage group VI, containing the cpc-1 promoter region and ylo-1, was translocated to the right arm of linkage group I. Other studies indicate that the cpc-1 locus itself is not essential for viability. Lethality previously attributed to the cpc-1 (j-5) mutation is due instead to the production of progeny that are deficient for essential genes in an adjoining segment of linkage group VI. Molecular characterization of cpc-1 (j-5) x ylo-1 pan-2 duplication progeny indicated that cpc-1 is normally transcribed towards the linkage group VI centromere.

ANeurosporacrassa ribosomal protein gene, homologous to yeastCRY1, contains sequences potentially coordinating its transcription with rRNA genes

We have isolated and sequenced a Neurospora crassa ribosomal protein gene (designated crp-2) strongly homologous to the rp59 gene (CRY1) of yeast and the S14 ribosomal protein gene of mammals. The inferred sequence of the crp-2 protein is more homologous (83%) to the mammalian S14 sequence than to the yeast rp59 sequence (69%). The gene has three intervening sequences (IVSs) two of which are offset 7 bp from the position of IVSs in the mammalian genes. None correspond to the position of the IVS in the yeast gene. Crp-2 was mapped by RFLP analysis to the right arm of linkage group III. The 5' region of the gene contains three copies of a sequence, the Ribo box, previously shown to be required for transcription of both 5S and 40S rRNA genes. We speculate that the Ribo box may coordinate ribosomal protein and rRNA gene transcription.

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-2 encoding the 57-kDa polypeptide and comparison to vma-1.

In partially purified preparations of the vacuolar ATPase from Neurospora crassa, the two most prominent components are polypeptides of Mr = 70,000 and 60,000. We previously reported the isolation of the gene vma-1, which encodes the Mr = 70,000 polypeptide, and presented evidence that the polypeptide contains the site of ATP hydrolysis (Bowman, E. J., Tenney, K., and Bowman, B. J. (1988) J. Biol. Chem. 263, 13994-14001). We now report the isolation of a gene (designated vma-2), that encodes the Mr = 60,000 polypeptide. Analysis of the DNA sequence shows that the polypeptide has 513 amino acids and a molecular mass of 56,808 daltons (and will thus be referred to as the 57-kDa polypeptide). It is fairly rich in polar amino acids and has no apparent membrane-spanning domains. The vma-2 gene contains five short introns (55-71 bases), all clustered in the 5' end of the coding region. The gene maps to the right arm of linkage group II, near 5 S RNA gene 3. Thus, it is unlinked to vma-1 and to other known ATPase genes in N. crassa. The 57-kDa polypeptide shows 25% amino acid sequence identity with the vma-1 gene product. It shows essentially the same degree of similarity (25-28%) to both the alpha and beta subunits of F0F1 ATPases. Analysis of specific regions of the 57-kDa polypeptide, however, suggests it may have a function like that of the alpha subunit in F0F1 ATPases. The data indicate that all four types of ATPase polypeptides have evolved from a common ancestor and that the vacuolar-type ATPases have a structure surprisingly similar to that of the F0F1 ATPases.

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans.

We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.

Genetic mapping of the 5S rRNA gene cluster of the nematode Caenorhabditis elegans.

We have identified a restriction fragment length difference (RFLD) affecting the genomic sequences immediately flanking the 5S rRNA gene cluster in the Bristol and Bergerac strains of the nematode Caenorhabditis elegans. We have used this RFLD as a molecular marker to follow the segregation of the 5S rRNA gene cluster through a series of two- and three-factor interstrain crosses. Our results show that the 5S rRNA gene cluster maps between unc-76 and dpy-21 on the right arm of linkage group V. This genetic localization provides a linkage group V "landmark" with which to localize other cloned sequences by in situ hybridization.

Beta-glucuronidase mutants of the nematode Caenorhabditis elegans.

Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta-glucuronidase.

Growth and macromolecular synthesis phenotypes of a heat-sensitive mutant strain, rip-1, of Neurospora crassa

A heat-sensitive mutant of Neurospora crassa, strain 4M(t), was isolated using ultraviolet-light mutagenesis followed by the inositol-less death enrichment technique. The heat-sensitivity is the result of a single gene mutation which maps to the distal end of the right arm of linkage group II. The mutation defines the rip-1 gene locus. Both conidial germination and mycelial extension are inhibited in the mutant at 35 degrees C and above (the nonpermissive temperature) but prolonged incubation at that temperature is not lethal to either cell type. Analysis of the lateral mycelial growth rates of wild type and of the rip-1 mutant at a variety of temperatures between 10 and 40 degrees C indicated that the maximal growth rate occurs at 35 degrees C in the wild type, and at 25 degrees C in the rip-1 strain. The rip-1 mutant grows 239-times slower at 35 degrees C than at 25 degrees C, whereas the wild type grows 1.4-times faster. Temperature shift-up experiments showed that even 3 h at 20 degrees C is not sufficient to allow germination at 37 degrees C, thereby showing that the mutant cannot accumulate enough heat-sensitive product at the permissive temperature to contribute to germination at 37 degrees C. The reciprocal temperature shift-down experiments showed that the molecular events at 37 degrees C may be qualitatively useful for germination after shifting to 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Temperature-sensitive Mutant of Neurospora crassa that Affects Mycelial Growth and Morphology

A temperature-sensitive mutant was isolated that conditionally affected the vegetative growth phase of the asexual developmental cycle of Neurospora crassa. The formation of aerial hyphae, conidiation and the initial steps of conidial germination were not temperature-sensitive in this strain. However, germ tube elongation was blocked at the restrictive temperature. The growth rate of the vegetative hyphae was greatly reduced and their morphology was altered when the hyphae grew on the surface of agar medium at 34 °C. These properties were not temperature-sensitive when the hyphae grew within the agar medium. In liquid medium, vegetative growth was temperature-sensitive when low concentrations of conidia (less than 104 ml−1) were used to inoculate the cultures. After a prolonged incubation at 34 °C, the cells died. The requirement for high cell densities for survival and growth in liquid medium at 34 °C could be overcome by adding histidine to the medium, but on agar medium containing histidine, the mutant strain still grew colonially at 34 °C. In addition to these temperature-sensitive properties, the mutant strain expressed a very strong circadian conidiation rhythm when grown on agar medium at 22 °C. The temperature-sensitive growth properties of the mutant strain and its ability to conidiate rhythmically reverted simultaneously to the wild-type phenotype. In forced heterokaryons, the mutant allele was recessive to the wild-type allele. The mutant allele was mapped on the right arm of linkage group IV.