Kalilo plasmids are a family of four distinct members with individual global distributions across species

Kalilo is a linear 9-kb plasmid, isolated originally from Hawaiian strains of the heterothallic fungus Neurospora intermedia. Its properties include terminal inverted repeats, two ORFs coding for a presumptive DNA and an RNA polymerase, and the ability to cause senescence in its original host and in the closely related species Neurospora crassa. We have examined natural isolates alleged to contain plasmids homologous to kalilo. Most of these isolates do in fact contain plasmids with so close an identity to kalilo as to be certain relatives. We found a new case of kalilo in Neurospora tetrasperma from Moorea-Tahiti, and a new case of LA-kalilo (previously found only in N. tetrasperma) in N. crassa from Haiti. A previously unreported, substantially shorter, kalilo variant has been found in three geographically separate isolates of the heterothallic species Neurospora discreta. Therefore, if the previously reported kalilo variant from the genus Gelasinospora is included, in all there are four members of the kalilo plasmid family. The main differences between these plasmids are in the terminal inverted repeats (TIRs). The phylogeny of the TIR sequences is largely congruent with that of nuclear DNA in the species in which they are found, suggesting that the plasmids are related by vertical descent throughout the evolution of these species. However, there are two cases of a plasmid found in a heterothallic and a pseudohomothallic species in the same global area; these cases might have arisen from more recent horizontal transmission or introgression. Received: 14 July / 17 September 1999     

Analysis of conversion tracts associated with recombination events at the am locus of Neurospora crassa

In cross B163, heteroallelic am ¹ am ⁶ and heterozygous for both conventional genetic flanking markers and closer molecular markers, we previously found that the majority on flanker exchanges were remote from events that generated prototrophic recombinants. We report here that natural polymorphisms distinguishing the parents of cross B163 also include sequences within and closely flanking am. Segregation of these markers in B163 prototrophs confirms that the majority of meiotic recombination events at am resulted from gene conversion. Conversion of am ⁶, the distal allele, is more frequent than conversion of am ¹. Twelve percent of tracts in am ⁶ convertants were discontinuous while 30% of continuous tracts converting am ⁶ extend less than 741 bp. Received: 8 September 1997 / 15 April 1998     

Chromosome pairing and meiotic recombination in Neurospora crassa spo11 mutants

Some organisms, such as mammals, green plants and fungi, require double-strand breaks in DNA (DSBs) for synapsis of homologous chromosomes at pachynema. Drosophila melanogaster and Caenorhabditis elegans are exceptions, achieving synapsis independently of DSB. SPO11 is responsible for generating DSBs and perhaps for the initiation of recombination in all organisms. Although it was previously suggested that Neurospora may not require DSBs for synapsis, we report here that mutation of Neurospora spo11 disrupts meiosis, abolishing synapsis of homologous chromosomes during pachynema and resulting in ascospores that are frequently aneuploid and rarely viable. Alignment of homologues is partially restored after exposure of spo11 perithecia to ionising radiation. Crossing over in a spo11 mutant is reduced in two regions of the Neurospora genome as expected, but is unaffected in a third.     

Characterization of hch, the Podospora anserina homolog of the het-c heterokaryon incompatibility gene of Neurospora crassa

The het-c locus controls heterokaryon formation in Neurospora crassa. It is subject to balancing selection operating to maintain polymorphism at that locus in natural populations. We have isolated hch, the het-c homolog from the related species Podospora anserina (hch for het-c homolog), in order to determine if this gene also functions as a het gene in that species. The het-c and hch sequences are highly similar but differ in the region defining allele specificity in N. crassahet-c. Analysis of hch variability in 11 natural P. anserina isolates with different het genotypes revealed no polymorphism. This suggested that hch does not function as a het gene. However, heterologous expression of the N. crassa het-c ^PA allele in P. anserina triggers a growth defect reminiscent of the het-c incompatibility reaction. Received: 23 November 1999 / 7 March 2000     

Neurosprora crassa RAD5 homologue, mus-41, inactivation results in higher sensitivity to mutagens but has little effect on PCNA-ubiquitylation in response to UV-irradiation

The DNA replication machinery stalls at damaged sites on DNA. Postreplicaton repair (PRR) is a system to avoid cell death in such circumstances of deadlock. In Saccharomyces cerevisiae, the Rad6/Rad18 heterodimer plays pivotal roles in PRR. It promotes translesion synthesis via the monoubiquitylation of the DNA sliding clamp, PCNA. Ubc13/Mms2/Rad5 can extend the ubiquitin chain from this monoubiquitylated PCNA with a non-canonical lysine 63-linked ubiquitin-chain, resulting in an error-free mode of bypass. In this study, we identified and characterized the RAD5 homolog in Neurospora crassa, which we named mus-41. A mus-41 mutant was sensitive to several DNA-damaging agents including UV and MMS. Genetic analyses indicated that uvs-2 (RAD18 homolog) was epistatic to mus-41, suggesting a role for mus-41 in postreplication repair. Additionally, it was shown that mus-41 has a role independent from TLS gene upr-1 (REV3 homolog) and works in the error-free pathway, indicating that the function of mus-41 as a RAD5 homolog is also conserved in N. crassa. However, mus-41 is not essential for the ubiquitylation of PCNA that is detected in the wild-type background, suggesting that there is another ubiquitin ligase catalyzing ubiquitylation of PCNA in response to UV in N. crassa.     

Generation of new mutants of nmr,the negative-acting nitrogen regulatory gene of Neurospora crassa,by repeat induced mutation

Summary The repeat induced point mutation (RIP) phenomenon has been used to generate new mutants of nmr, the negative nitrogen regulatory gene in Neurospora crassa. The wild-type nmr gene was cotransformed along with the hygromycin B resistance gene into wild-type cells by selecting for hygromycin B resistance. Following purification of primary transformants using microconidia, crosses to wild-type. Detailed analyses of some of the progeny revealed that we had generated authentic nmr mutants at high frequency. The polymerase chain reaction was used to amplify and clone a fragment of a mutagenized nmr copy from one of the mutants. The nucleotide sequence analysis showed that 14% of the guanine residues have been converted into adenines, resulting in numerous missense and nonsense mutations. The newly created nmr mutants were found suitable for use as host strains in transformation experiments.     

Bialaphos resistance as a dominant selectable marker in Neurospora crassa

Summary Conidia of Neurospora crassa are sensitive to the herbicide bialaphos at concentrations of 160 mg/1 in Westergaard's or Fries' minimal media. Plasmid pJA4 was constructed by inserting a truncated bar gene from Streptomyces hygroscopicus fused to the his-3 promoter from N. crassa into pUC19. The bar gene in plasmid pJA4 confers resistance to bialaphos when transformants are selected on a medium containing bialaphos. The bar gene can be used as an additional dominant selectable marker for transformation of fungi.