The dynamics of mitochondrial plasmids in a Hawaiian population of Neurospora intermedia

We analysed the distribution of mitochondrial plasmids among 82 Neurospora intermedia isolates from Hawaii; 74% of the isolates carried the neutral circular plasmid Han-2, whereas 38% contained the linear senescence-causing plasmid kalDNA. The distributions of the two plasmids are independent. There is no significant difference between the Kauaian population of 1972 and that of 1976. To further examine the reasons for this frequency distribution we studied the transmission of both Hawaiian plasmids through the maternal parent in a large series of crosses using non-Kalilo isolates as conidial parents. Plasmids can be lost during the sexual cycle. The Han-2 plasmid is transmitted more efficiently than kalDNA. No clear cases of autonomous or non-autonomous plasmid suppression were observed, so loss can be considered accidental. One Kalilo strain proved to be ineffectual as a maternal parent, and this reduced its ability to transmit kalDNA to the next generation. The dynamic balance of plasmids in natural populations over time is probably a result of the interplay of many forces, including those described in this work and those from several other studies on Neurospora plasmids.     

Relationship of histidine sensitivity to DNA damage and stress induced responses in mutagen sensitive mutants of Neurospora crassa

Summary Previous work in other laboratories has shown that several mutagen sensitive mutants of Neurospora crassa are extremely sensitive to low levels of histidine in the culture medium. We have shown that wild type Neurospora accumulates nicks or breaks in the DNA in the presence of histidine. The number of nicks accumulating in histidine sensitive mutants is found to increase in relation to their sensitivity to histidine. Although these nicks can be repaired by both wild type and histidine sensitive mutants when histidine is removed from the medium, a steady state number of nicks exists as long as histidine is present. We suggest that the presence of these nicks or breaks induces an increase in recombination in these possibly recombination defective mutants and that this is the source of the high level of histidine sensitivity. We speculate on the mechanisms by which histidine induces this DNA damage. This report also shows that several polypeptides are induced by the wild type organism in the presence of histidine. Some of these polypeptides are also induced during other stress situations, such as heat shock and DNA damage due to ultraviolet irradiation. Two of the histidine induced proteins cannot be induced by any of the histidine sensitive mutants.     

Preparation of a cell-free translation system from a wild-type strain of Neurospora crassa

Summary We describe the preparation of an in vitro translation system from a wild-type strain of Neurospora crassa. The system is capable of supporting efficient and faithful translation of native and in vitro transcribed eukaryotic messages. The translation products have minimal background and can be clearly analyzed by SDS-polyacrylamide gel electrophoresis. The method of preparation of the lysate is simple, fast and reproducible. The procedure should be readily applicable to other filamentous fungi.     

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.     

Cloning and characterization of the yeast RAD1 homolog gene (mus-38) from Neurospora crassa: evidence for involvement in nucleotide excision repair

A Neurospora crassa gene encoding a product with homology to the Saccharomyces cerevisiae Rad1 nucleotide excision repair (NER) protein was isolated by degenerate PCR. The predicted protein consists of 892 amino acids with a molecular weight of 100.4 kDa, and 32–37% identity to the XPF/ERCC4 protein family. The homolog was mapped to the left arm of linkage group I, the location of the mus-38 gene. Subsequently, gene inactivation and complementation studies identified the RAD1 homolog as mus-38. Immunological assays showed that the mus-18 (UV-specific endonuclease) and mus-38 strains have partial and normal UV-damage excision activities, respectively, but removal of thymine dimers and TC (6-4) photoproducts is abolished in the mus-18 mus-38 double mutant. The double mutant also was synergistically more sensitive to UV than either single mutant. The data suggest that mus-38 may participate in a different NER pathway from that involving the mus-18 gene. Received: 27 November 1997 / 28 January 1998     

Cold-sensitive mutation in Neurospora crassa affecting the production of 17S ribosomal RNA from ribosomal precursor RNA

Summary The cold-sensitive mutant strain of Neurospora crassa, crib-1 (PJ30201) has a conditional defect in ribosome biosynthesis. At 10 °C this mutant underproduces the small (37S) ribosomal subunit. Experiments were done to study rRNA synthesis in crib-1 after a shift from the permissive (25 °C) to the nonpermissive temperature and vice versa. The results showed that the primary cold-sensitive defect in crib-1 is in the function, rather than the synthesis, of a molecule needed for the production of the 17S rRNA component of the 37S subunit. Pulse-labeling experiments showed that at 10 °C crib-1 synthesizes the 2.4-Mdal pre-rRNA molecule which contains the sequences for the 17S, 5.8S and 25S rRNA species, but that processing of this molecule is defective such that relatively few stable 17S rRNA molecules accumulate.     

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