Cloning and functional characterization of a eucaryotic DNA photolyase gene from Neurospora crassa.

We cloned a genomic fragment of a photolyase gene from Neurospora crassa by polymerase chain reaction using synthesized oligonucleotide primers designed from the most conserved amino acid sequences among photolyases of various organisms. Using the cloned fragment as a hybridization probe we isolated a genomic fragment and cDNA clones encoding the complete photolyase gene of this organism. The amino acid sequence of the photolyase deduced from the determined nucleotide sequence indicates a protein consisting of 615 amino acid residues (Mr 69,971), which is most similar to that of Saccharomyces cerevisiae. Like yeast photolyase it contains a protruding amino terminus which is missing in photolyases of bacterial origin. Comparison of amino acids sequences among six photolyases suggests that the Neurospora crassa photolyase is more similar to photolyases of pterin type than those of deazaflavin type.     

Nucleotide sequence of the GDH gene coding for the NADP-specific glutamate dehydrogenase of Saccharomyces cerevisiae

The isolation of the Saccharomyces cerevisiae gene for NADP-dependent glutamate dehydrogenase (NADP-GDH) by cross hybridization to the Neurospora crassa am gene, known to encode for NADP-GDH is described. Two DNA fragments selected from a yeast genomic library in phage lambda gt11 were shown by restriction analysis to share 2.5 kb of common sequence. A yeast shuttle vector (CV13) carrying either to the cloned fragments complements the gdh- strain of S. cerevisiae and directs substantial overproduction of NADP-GDH. One of the cloned fragments was sequenced, and the deduced amino acid (aa) sequence of the yeast NADP-GDH is 64% homologous to N. crassa, 51% to Escherichia coli and 24% to bovine NADP-GDHs.     

Isolation and characterization of a Neurospora crassa ribosomal protein gene homologous to CYH2 of yeast.

We have isolated and characterized a Neurospora crassa gene homologous to the yeast CYH2 gene encoding L29, a cycloheximide sensitivity-conferring protein of the cytoplasmic ribosome. The cloned Neurospora gene was isolated by cross-hybridization to CYH2. It was sequenced from both cDNA and genomic clones. The coding region is interrupted by seven intervening sequences. Its deduced amino acid sequence shows 70% homology to that of yeast ribosomal protein L29 and 60% homology to that of mammalian ribosomal protein L27', suggesting that the protein has an important role in ribosomal function. The pattern of codon usage is highly biased, consistent with high translation efficiency. There is a single copy of this gene in N. crassa, and R. Metzenberg and coworkers have mapped its genetic location to the vicinity of the cyh-2 locus.     

Isolation and structural organization of the Neurospora crassa copper metallothionein gene.

The Neurospora crassa copper metallothionein gene was cloned and its complete nucleotide sequence is reported. Enriched metallothionein mRNA was used as a template for cDNA synthesis, primed by a metallothionein-specific, synthetic undecanucleotide. The sequence of the cDNA obtained allowed the synthesis of a unique 21-mer which was used to screen a genomic DNA library of N. crassa. In agreement with the published amino acid sequence, the gene codes for a polypeptide 26 amino acid residues in length. The coding region is interrupted by a small intron (94 nucleotides). The gene structure is compared with those of mammalian metallothioneins. In both cases, the coding regions are split by introns, the intron-exon boundaries, however, are in different positions. The neurospora copper metallothionein gene is, to our knowledge, the smallest gene interrupted by an intron isolated so far.     

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.     

The frost gene of Neurospora crassa is a homolog of yeast cdc1 and affects hyphal branching via manganese homeostasis

The Neurospora crassa mutant frost has a hyperbranching phenotype that can be corrected by adding Ca(2+), suggesting that characterization of this gene might clarify the mechanism of Ca(2+)-dependent tip growth. The wild-type allele was cloned by sib selection using protoplasts from arthroconidia. RFLP analysis revealed that the cloned DNA fragment mapped to the fr locus. The nucleotide sequence of genomic and cDNA was determined. The deduced amino acid sequence showed homology to the Saccharomyces cerevisiae CDC1 protein, implicated in manganese homeostasis. The fr mutant was sensitive to Mn(2+), and a revertant allele whose product differs by one amino acid was tolerant to Mn(2+). Mn(2+) depletion induced the wild-type strain to hyperbranch, resulting in a morphology similar to that of fr. The fr mutant was also sensitive to calcineurin inhibitors. These results suggest that fr is involved in Mn(2+) homeostasis and point to a role for Mn(2+) in Neurospora branching.     

Cytoplasmic Leucyl-Trna Synthetase of Neurospora Crassa Is Not Specified by the Leu-5 Locus

We generated a lambda gt11 Neurospora crassa cDNA library and screened the library for the cytoplasmic leucyl-tRNA synthetase (cyto LeuRS) clones using cyto LeuRS specific antibody. Two clones, lambda NCLRSC1 and lambda NCLRSC2, were obtained which have inserts of approximately 2 kbp and approximately 1.3 kbp, and which overlap by about 0.6 kbp. The following lines of evidence indicate that lambda NCLRSC1 and lambda NCLRSC2 encode parts of cyto LeuRS. (1) Antibodies affinity purified using either of the fusion proteins encoded by lambda NCLRSC1 or lambda NCLRSC2 inhibit cyto LeuRS activity. Thus, the fusion protein and cyto LeuRS share immunological determinants. (2) The same antibodies also react with an approximately 115-kDa protein, which comigrates with purified cyto LeuRS, in immunoblots of total N. crassa proteins. We used the cDNA clones to probe a N. crassa genomic DNA library and isolated two genomic DNA clones. Partial sequence analysis of cDNA and genomic DNA clones shows a methionine initiated open reading frame, which includes a stretch of amino acid residues that are highly conserved and that are at the ATP binding site in aminoacyl-tRNA synthetases. Using the cloned DNA as probe, we show that the cyto LeuRS mRNA is approximately 3900 nucleotides long. Finally, we have used restriction fragment length polymorphism mapping to show that the cyto LeuRS gene resides on the far right of linkage group II and not on linkage group V where the leu-5 mutation, which was previously reported to specify cyto LeuRS, is located.     

Isolation and characterization of the adenylate cyclase structural gene of Neurospora crassa.

A single gene (nac) encoding an adenylate cyclase was cloned from the genomic DNA library of Neurospora crassa, using the DNA fragment encoding the catalytic domain of adenylate cyclase of Saccharomyces cerevisiae as a probe. The open reading frame of this gene (6900 base pairs) was interrupted three time by introns. The protein encoded consists of 2300 amino acids and has adenylate cyclase activity. N. crassa adenylate cyclase has a high degree of homology with the catalytic domains of yeast and bovine brain adenylate cyclases.     

Duplication-induced mutation of a new Neurospora gene required for acetate utilization: properties of the mutant and predicted amino acid sequence of the protein product.

A cloned Neurospora crassa genomic sequence, selected as preferentially transcribed when acetate was the sole carbon source, was introduced in extra copies at ectopic loci by transformation. Sexual crossing of transformants yielded acetate nonutilizing mutants with methylation and restriction site changes within both the ectopic DNA and the normally located gene. Such changes are typical of the duplication-induced premeiotic disruption (the RIP effect) first described by Selker et al. (E. U. Selker, E. B. Cambareri, B. C. Jensen, and K. R. Haack, Cell 51:741-752, 1987). The mutants had the unusual phenotype of growth on ethanol but not on acetate as the carbon source. In a cross to the wild type of a mutant strain in which the original ectopic gene sequence had been removed by segregation, the acetate nonutilizing phenotype invariably segregated together with a RIP-induced EcoRI site at the normal locus. This mutant was transformed to the ability to use acetate by the cloned sequence. The locus of the mutation, designated acu-8, was mapped between trp-3 and un-15 on linkage group 2. The transcribed portion of the clone, identified by probing with cDNA, was sequenced, and a putative 525-codon open reading frame with two introns was identified. The codon usage was found to be strongly biased in a way typical of most Neurospora genes sequenced so far. The predicted amino acid sequence shows no significant resemblance to anything previously recorded. These results provide a first example of the use of the RIP effect to obtain a mutant phenotype for a gene previously known only as a transcribed wild-type DNA sequence.     

Cloning and expression of a beta tubulin gene of Physarum polycephalum

A beta tubulin gene of Physarum polycephalum has been isolated from a genomic library in the phage EMBL4. Southern-blot hybridization to genomic DNA indicates that the cloned DNA is derived from the betB1 locus of the beta tubulin gene family. A tubulin-specific subfragment of the phage DNA was used as a hybridization probe to construct a restriction map of the betB1 locus. The probe consisted of the almost complete coding region of the 5' half of the tubulin gene, interrupted by one intron. The derived amino acid sequence of this subclone deviates from the protein sequence for Physarum amoebal beta tubulin (amino acids 4-207) in two of 207 amino acids. We used both recA and recBC sbcB bacterial host strains, which have been recommended for cloning of instability-conferring sequences of the Physarum genome, but were unable to subclone the 3' part of the gene from the phage DNA. Primer-extension analysis indicates that the betB gene is expressed in the vegetatively proliferating amoebal and plasmodial stages of the life cycle as well as in differentiating (sporulating) plasmodia.     

Molecular Cloning of a Gene (Cfp) Encoding the Cytoplasmic Filament Protein P59nc and Its Genetic Relationship to the Snowflake Locus of Neurospora Crassa

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurospora crassa ribosomal DNA: sequence of internal transcribed spacer and comparison with N. intermedia and N. sitophila

Using [32P]DNA probes from a clone containing 17S, 5.8S and 26S rRNA of Neurospora crassa, the remainder of the repeat unit (RU) for ribosomal DNA (rDNA) has been cloned. Combining restriction analysis of the cloned DNA and restriction digests of genomic DNA, the RU was found to be 8.7 kb. The nucleotide sequence was determined for the internal transcribed spacer (ITS) regions one and two, for 5.8S rRNA and for portions of 17S and 26S rRNAs immediately flanking the ITS regions, and compared to the corresponding region of Saccharomyces carlsbergensis. In addition, a comparative restriction analysis of two other Neurospora species was performed using twelve restriction endonucleases. Genomic DNA blots of rDNA from N. intermedia and N. sitophila revealed rDNA RUs of 8.4 kb. The majority of differences in restriction patterns were confined to sequences outside the mature rRNA regions. However, one SmaI recognition site was found in 26S rRNA of N. crassa and N. sitophila but not in N. intermedia.     

Vegetative incompatibility in the het-6 region of Neurospora crassa is mediated by two linked genes

Non-self-recognition during asexual growth of Neurospora crassa involves restriction of heterokaryon formation via genetic differences at 11 het loci, including mating type. The het-6 locus maps to a 250-kbp region of LGIIL. We used restriction fragment length polymorphisms in progeny with crossovers in the het-6 region and a DNA transformation assay to identify two genes in a 25-kbp region that have vegetative incompatibility activity. The predicted product of one of these genes, which we designate het-6(OR), has three regions of amino acid sequence similarity to the predicted product of the het-e vegetative incompatibility gene in Podospora anserina and to the predicted product of tol, which mediates mating-type vegetative incompatibility in N. crassa. The predicted product of the alternative het-6 allele, HET-6(PA), shares only 68% amino acid identity with HET-6(OR). The second incompatibility gene, un-24(OR), encodes the large subunit of ribonucleotide reductase, which is essential for de novo synthesis of DNA. A region in the carboxyl-terminal portion of UN-24 is associated with incompatibility and is variable between un-24(OR) and the alternative allele un-24(PA). Linkage analysis indicates that the 25-kbp un-24-het-6 region is inherited as a block, suggesting that a nonallelic interaction may occur between un-24 and het-6 and possibly other loci within this region to mediate vegetative incompatibility in the het-6 region of N. crassa.     

Spontaneous Mutation at the Mtr Locus in Neurospora: The Molecular Spectrum in Wild-Type and a Mutator Strain

Sequence analysis of 34 mtr mutations has yielded the first molecular spectrum of spontaneous mutants in Neurospora crassa. The great majority of the mutations are base substitutions (48%) or deletions (35%). In addition, sequence analysis of the entire mtr region, including the 1472-base pair open reading frame and 1205 base pairs of flanking DNA, was performed in both the Oak Ridge and Mauriceville strains of Neurospora, which are known to be divergent at the DNA level. Sixteen sequence differences between these two strains have been found in the mtr region, with 13 of these in DNA flanking the open reading frame. The differences consisted of base substitutions and small frameshifts at monotonic runs. This set of sequence differences has allowed a comparison of mutations in unselected DNA to those mutations that produce a phenotypic signal. We have isolated a mutator strain (mut-1) of Neurospora in which the spontaneous mutation rate at various loci is as much as 80-fold higher than in the non-mutator (wild type). Twenty-one mtr mutations in the mutator background have been sequenced and compared to the non-mutator spectrum, revealing a striking increase in -1 frameshift mutations. These frameshifts occur exclusively within or adjacent to monotonic runs and can be explained by small slippage events during DNA replication. This argues for a role of the mut-1 gene in this process.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suco and N. crassa inv strains transformed with pNC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suco (pNC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa, although S. cerevisiae suc+ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI-restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv- to Inv+ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

Suppression of the cr-1 mutation in Neurospora crassa.

We have cloned a DNA fragment, which hybridized with the adenylate cyclase gene (CYR1) of Saccharomyces cerevisiae, from genomic DNA libraries of Neurospora crassa. The cr-1 mutation was able to be suppressed by introducing this DNA fragment on a cosmid vector, judging from recovery of the adenylate cyclase activity and the abnormal morphology.     

Transformation by integration in Podospora anserina. III. Replacement of a chromosome segment by a two-step process

Summary We have developed in Podospora anserina a two-step procedure for DNA sequence replacement through transformation which might be applicable to other filamentous fungi. Targeting of transforming DNAs to their homologous locus is achieved provided a cosmid vector is used. Southern blot analysis of genomic DNAs from a set of transformants is presented. The data confirm that cosmids integrate into the chromosome through mostly homologous recombination which leads to a duplicated sequence separated by the vector. This event was found to be unstable in crosses. We show that this instability is due to the frequent excision of the vector together with the selective marker and one copy of the duplication, either the resident or foreign sequence. The two sequences can be distinguished because they exhibit restriction fragment length polymorphism. Therefore, Podospora anserina treats duplications occurring through transformation in a way differing from that exhibited by Neurospora crassa and Ascobolus immersus.     

球毛壳菌（Chaetomium globosum）过氧化物膜蛋白过敏原基因克隆、序列分析及原核表达

以球毛壳菌cDNA文库中获得过氧化物膜蛋白（pero）基因片段（GenBank Accn:BP099709）为基础,用RACE 技术获得该基因的全长cDNA序列。序列长747bp,由412bp的3′RACE产物和508bp的5′RACE产物拼接而成。开放阅读框501bp,编码166个氨基酸,蛋白分子量为17.5kD,理论等电点为5.75。利用cDNA两侧非编码区序列作引物克隆出该基因的DNA序列,序列分析表明该基因由2个内含子和3个外显子组成。ClustalX多序列比对表明：该基因与粗糙脉孢菌（Neurospora crassa）的过氧化物膜蛋白过敏原同源性最高（83%）。将pero基因编码区克隆到原核表达载体pET28a中,构建成表达质粒pET28a-pero并转化大肠杆菌BL21,IPTG诱导后SDS-PAGE检测表达情况,结果发现在21kD处有一特异性融合蛋白带,大小与预期相符,说明该基因已经在大肠杆菌中表达。克隆的cDNA序列、DNA序列及推测的氨基酸序列在GenBank登录(登录号分别为AY555771,AY584753,AAS66898)。