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In the fruit fly Drosophila melanogaster , circadian rhythm was disrupted when the inner nuclear membrane protein lamin B receptor (LBR) was depleted from its clock neurons ( Proc. Natl. Acad. Sci. USA 118, e2019756118. 2021; https://doi.org/10. 1073/pnas.2019756118 and Research 6, 0139, 2023; https://doi.org/10.34133/research.0139). Ordinarily, the clock proteinPERIOD (PER) forms foci close to the inner nuclear membrane in the circadian clock's repression phase. The size, number, and location of foci near the nuclear membrane oscillate with a 24-h rhythm. When LBR was absent the foci did not form. The PER foci bring per and other clock genes close to the nuclear envelope, where their transcription is silenced. Then, in the circadian clock's activation phase, the PER protein gradually gets degraded and the foci disappear. The clock genes, including per , relocate to the nucleus interior where they resume transcription. Rhythmic re-positioning of clock genes between nucleus periphery and interior, correlates with their repression and activation in the circadian cycle. Absence of LBR disrupted this rhythm. Phosphorylation of PER promoted the formation of foci whereas dephosphorylation by protein phosphatase 2A causedthem to disappear. LBR promoted focus formation by destabilizing the catalytic subunit of protein phosphatase 2A. The lbr gene is no stranger to this journal. The first hint that vertebrate LBR is also a sterol biosynthesis enzyme, specifically, a sterol C14 reductase, was reported here ( J. Genet . 73, 33-41, 1994; https://www.ias.ac.in/article/fulltext/jgen/073/01/0033-0041). Mutations in the human Lbr gene cause a range of phenotypes--from the relatively benign Pelger-Huet anomaly to the perinatally lethal Greenberg skeletal dysplasia.Drosophila, like all insects, is a sterol auxotroph. The fly orthologue of vertebrate lbr genes encodes a protein (dLBR) that shares several properties with vertebrate LBR proteins, with one notable exception. While human LBR complemented theyeast Saccharomyces cerevisiae erg24 mutant which lacks sterol C14 reductase activity, dLBR did not ( J. Cell. Sci. 117 , 2015-28, 2004; https://doi.org/10.1242/jcs.01052). Despite not possessing sterol reductase activity, dLBR retains significant sequence homology with vertebrate LBRs which have this activity. An undergraduate summer trainee in my laboratory obtained early (unpublished) evidence that dLBR lost sterol reductase activity during evolution. She transferred adult drosophila flies to vials containing a medium made of agar, dextrose, and dried and powdered mycelium of the filamentous fungus Neurospora crassa . On medium made with wild-type mycelium, theflies mated, laid eggs, hatched larvae, and developed pupae which eclosed progeny adult flies. The life cycle was no different than on 'regular' fly food composed of agar, dextrose and yeast extract. However, on a medium made with mycelium from a sterol C14 reductase null mutant, the flies laid eggs which hatched and released larvae, but the larvae failed to pupate, and no adult progeny flies emerged. This was because the fly lacks a sterol C14 reductase. The wild-type sterol, ergosterol, is a precursor of the steroid hormone ecdysone needed for molting and metamorphosis. Can expression of vertebrate LBR in dLBR-depleted fly clock neurons restore circadian rhythm? Can expression of vertebrate LBR enable flies to complete their life cycle on mutant Neurospora medium? Does LBR regulate the vertebrate clock in a like manner? If yes, then is the sterol reductase activity dispensable in this role? These are some questions that came to my mind on a recent morning walk. The walk itself was a much-cherished outcome of my circadian clock.

When a conidium (vegetative spore) or ascospore (sexually produced spore) of the filamentous fungus Neurospora crassa germinates, it produces a long narrow filamentous multinucleate cell called a hypha. Hyphae grow by elongation, they can branch, and the tips of branches can also rejoin by fusion. Growth, branching, and fusion create an interconnected web, called a mycelium, within which cytoplasmic continuity is maintained. Some researchers have focused their studies on hyphal elongation and branching, others on formation of conidia and ascospores, and still others, prominently Andre Fleißner, Nick Read, Louise Glass, and colleagues, on tip fusion. Each of these fundamental processes contributes to the development of species-characteristic mycelial morphology. Using the fluorescently tagged Neurospora proteins MAK-2 and SO, they made the startling discovery that when tips of freshly germinated and genetically identical conidia (germlings) came within 15 μm of each other, each tip took turns to send and receive a molecular signal in an oscillatory back-and-forth manner.

Dictyostelium discoideum is a species of free-living soil amoeba that feeds on bacteria that grow on decaying vegetation. Though the present account deals with D. discoideum , I use the more colloquial 'dictyostelium' in this article. In 1989, as a new PI, I began to study the response of D. discoideum amoebae to pisatin. Pisatin is the major phytoalexin of the pea plant ( Pisum sativum ). Phytoalexins are antifungal compounds made by plants in response to infection and injury. No other group has studied any dictyostelium vis-a`-vis any phytoalexin. Evidence for saying so comes from PubMed: four papers show up with the keywords 'dictyostelium', and 'phytoalexin', all from my lab. Why did we 'plough this lonely furrow' and what did we uncover?

This article describes some of the research contributions made by Prof. Ramesh Maheshwari and his colleagues at the Indian Institute of Science, Bangalore. These include (1) the understanding of the Neurospora life cycle in agricultural (sugarcane) fields, (2) identification of Neurospora mutants that trigger vegetative spore development via microcycle conidiation, and (3) isolation of wild Neurospora strains in which the essential immortality of the fungal mycelia is subverted.

Segregation distortion refers to an unusual genetic phenomenon in diploid organisms by which the two alleles at a locus in a parent are not recovered in the classical 1:1 Mendelian ratio in its offspring. The Drosophila melanogaster neogene Sd was created by a duplication breakpoint on the left arm of chromosome 2 (2L), and encodes a truncated RanGAP protein with normal GTPase activity but which mis-localizes to the nucleus and disrupts Ran gradients. Male flies carrying Sd exhibit segregation distortion for the Rsp locus on the right arm of chromosome 2 (2R). Specifically, spermatids inheriting chromosome 2 with the Sd + Rsp s(s) genotype, in heterozygous males of genotype Sd Rsp i / Sd + Rsp s(s) , fail to develop properly so that the majority of progeny (approaching 100%) carry just the Sd Rsp i chromosome. One recent paper reported novel RNAi-expressing transgenes with Sd-mimicking properties; and another reported localization of an X-linked suppressor which restores Mendelian transmission. This article highlights how Drosophila genetics resources made this possible, and the significance of these findings to nucleus-cytoplasm transactions of interest to the wider cell biology community.

The T(EB4) Nt a, T(IBj5) Nt a, and T(B362i)NtA strains were constructed by introgressing the insertional translocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma . The progeny from crosses of T(IBj5) Nt a and T(B362i) Nt A with opposite mating-type derivatives of the standard N. tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavored homokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A + mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5) Nt a and T(B362i) Nt A strains with the Eight spore (E) mutant showed an unusual ascus phenotype called 'max-4'. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 of the homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryotic progeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

Meiotic silencing by unpaired DNA (MSUD), an RNAi-mediated gene silencing process, is efficient in crosses made in the Neurospora crassa standard Oak Ridge (OR) genetic background. However, MSUD was decidedly less efficient when the OR-derived MSUD testers were crossed with many wild-isolated strains (W), suggesting that either sequence heterozygosity in tester x W crosses suppresses MSUD, or that OR represents the MSUD-conducive extreme in the range of genetic variation in MSUD efficiency. Our results support the latter model. MSUD was less efficient in near-isogenic crosses made in the novel N. crassa B/S1 genetic background, and in N. tetrasperma strain 85. Possibly, in B/S1 and 85, additional regulatory cues, absent from OR, calibrate the MSUD response. A locus in distal chromosome 1R appears to underlie the OR vs. B/S1 difference. Repeat-induced point mutation (RIP) destroys duplicated genes by G:C to A:T mutation of duplicated DNA sequences. Chromosome segment duplications ( Dp s) dominantly suppress RIP, possibly by titrating out the RIP machinery. In Dp x N crosses, the Dp -borne genes cannot pair properly, hence efficient MSUD, as in OR, silences them and renders the crosses barren. We speculate that the increased productivity engendered by inefficient MSUD enables small duplications to escape RIP., (Copyright © 2019 Giri et al.)

The vertebrate inner nuclear membrane protein, lamin B receptor, has an N-terminal ~200 residue nucleoplasmic domain (NTD), and a ~420 residue C-terminal domain (CTD) that anchors the NTD to the INM. Chen et al (2016) showed the NTD interacts with Xist long noncoding RNA to effect X chromosome inactivation in female mammals. Tsai et al (2016) showed the CTD has sterol reductase activity that is essential for viability. And Nikolakaki et al (2017) proposed a model to interconnect these disparate functions of this chimeric protein. It amuses me now to think back to 24 years ago, when I was concerned that these domains might have come together in a cloning artifact.

When two lineages derived from a common ancestor become reproductively isolated (e.g. Neurospora crassa and N. tetrasperma), genes that have undergone mutation and adaptive evolution in one lineage can potentially become dysfunctional when transferred into the other, since other genes have undergone mutation and evolution in the second lineage, and the derived alleles were never 'tested' together before hybrid formation. Bateson (1909), Dobzhansky (1936), and Muller (1942) recognized that incompatibility between the derived alleles could potentially make the hybrid lethal, sterile, or display some other detriment. Alternatively, the detrimental effects seen in crosses with the hybrids may result from the silencing of ascus-development genes by meiotic silencing by unpaired DNA (MSUD). Aberrant transcripts from genes improperly paired in meiosis are processed into single-stranded MSUD-associated small interfering RNA (masiRNA), which is used to degrade complementary mRNA. Recently, backcrosses of N. crassa / N. tetrasperma hybrid translocation strains with wild-type N. tetrasperma were found to elicit novel ascus dysgenesis phenotypes. One was a transmission ratio distortion that apparently disfavoured the homokaryotic ascospores formed following alternate segregation. Another was the production of heterokaryotic ascospores in eight-spored asci. Lewis (1969) also had reported sighting rare eight-spored asci with heterokaryotic ascospores in interspecific crosses in Sordaria, a related genus. Ordinarily, in both Neurospora and Sordaria, the ascospores are partitioned at the eight-nucleus stage, and ascospores in eight-spored asci are initially uninucleate. Evidently, in hybrid crosses of the family Sordariaceae, ascospore partitioning can be delayed until after one or more mitoses following the postmeiotic mitosis.

During ascogenesis in Neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. We had previously created novel TNt strains by introgressing four Neurospora crassa insertional translocations (EB4, IBj5, UK14-1, and B362i) into N. tetrasperma. We now show that crosses of all the TNt strains with single-mating-type derivatives of the standard N. tetrasperma pseudohomothallic strain 85 (viz. T Nt a x 85A or T Nt A x 85a) can produce rare eight-spored asci that contain heterokaryotic ascospores, or ascospores with other unexpected genotypes. Our results suggest that these rare asci result from the interposition of additional mitoses between the post-meiotic mitosis and the partitioning of nuclei into ascospores, leading to the formation of supernumerary nuclei that then generate the heterokaryotic ascospores. The rare asci probably represent a background level of ascus dysgenesis wherein the partitioning of ascospores becomes uncoupled from the post-meiotic mitosis. Ordinarily, the severest effect of such dysgenesis, the production of mating-type heterokaryons, would be suppressed by the N. crassa tol (tolerant) gene, thus explaining why such dysgenesis remained undetected thus far.

By introgressing Neurospora crassa translocations into N. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes (i.e., [T + N]) or a duplication and its complementary deficiency (i.e., [Dp + Df]). The [T + N] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [Dp + Df]. Self-cross of either heterokaryon produces [T + N] and [Dp + Df] progeny. Occasionally during N. tetrasperma ascus development, a pair of smaller homokaryotic ascospores replaces a heterokaryotic ascospore. Crosses with the Eight-spore mutant increase such replacement, and can generate asci with eight homokaryotic ascospores, either 4T + 4N from alternate segregation, or 4Dp + 4Df from adjacent-1 segregation. Crosses of some of the introgressed translocation strains with normal sequence N. tetrasperma produced more Dp than T or N homokaryotic progeny. We suggest this is due to an insufficiency for a presumptive ascospore maturation factor, which increases the chance that, in asci with > 4 viable ascospores, none properly mature. Since only four viable ascospores (Dp or [Dp + Df]) share the limiting factor following adjacent-1 segregation, whereas four to eight ascospores compete for it following alternate segregation, this would explain why Dp homokaryons outnumber T and N types, whereas the heterokaryons are not as affected. We believe that this novel form of transmission ratio distortion is caused by a Bateson-Dobzhansky-Muller Incompatibility (BDMI) triggered by an N. crassa gene in the N. tetrasperma background. Heterokaryons tend not to out-cross, and crosses of Dp strains are barren, thus the BDMI impedes interspecies gene flow., (Copyright © 2016 Giri et al.)

Introgression is the transfer of genes or genomic regions from one species into another via hybridization and back-crosses. We have introgressed four translocations (EB4, IBj5, UK14-1, and B362i) from Neurospora crassa into N. tetrasperma. This enabled us to construct two general types of heterokaryons with mat-A and mat-a nuclei of different genotypes: one type is [T + N] (with one translocation nucleus and one normal sequence nucleus), and the other is [Dp + Df] (with one nucleus carrying a duplication of the translocation region and the other being deleted for the translocation region). Self-crossing these heterokaryons again produced [T + N] and [Dp + Df] progeny. From conidia (vegetative spores) produced by the heterokaryotic mycelia, we obtained self-fertile (heterokaryotic) and self-sterile (homokaryotic) derivative strains. [T + N] heterokaryons produced homokaryotic conidial derivatives of both mating types, but [Dp + Df] heterokaryons produced viable conidial homokaryons of only the mating type of the Dp nucleus. All four [T + N] heterokaryons and three [Dp + Df] heterokaryons produced both self-sterile and self-fertile conidial derivatives, but the [Dp(B362i) + Df(B362i)] heterokaryons produced only self-sterile ones. Conceivably, the Df(B362i) nuclei may be deleted for a nucleus-limited gene required for efficient mitosis or nuclear division, and whose deficit is not complemented by the neighboring Dp(B362i) nuclei. A cross involving Dp(EB4) showed repeat-induced point mutation (RIP). Because RIP can occur in self-crosses of [Dp + Df] but not [T + N] heterokaryons, RIP alteration of a translocated segment would depend on the relative numbers of [Dp + Df] vs. [T + N] ancestors., (Copyright © 2015 Giri et al.)

Meiotic silencing by unpaired DNA is a presumed RNAi-mediated elimination of the transcripts of any gene that is not properly paired with a homolog in meiosis. Wild-isolated strains of Neurospora crassa can be classified into three types based on the apparent strength of meiotic silencing of the bml (β-tubulin) and mei-3 genes in crosses of the wild strains with the tester strains ::Bml(r) and ::mei-3. Crosses with "OR" and "Sad" type wild-isolates, respectively, did or did not silence both the genes, whereas crosses with the "Esm" type silenced bml, but not mei-3(+). Presumably, bml is more sensitive to silencing than mei-3, and silencing is strongest in crosses with the OR type, weakest (or non-evident) in crosses with the Sad type, and of intermediate strength in crosses with Esm type. Now, while constructing recombinant inbred lines from the Sad type wild strains Bichpuri-1 a and Spurger-3 A, we found that although crosses of Bichpuri-1 a and Spurger-3 A, and of most of their f1 progeny, with the ::Bml(r) and ::mei-3 testers were consistently and reproducibly Sad type, those of later generation strains of each line could show Sad, Esm, or a variably expressive Sad/Esm type. Since any genotype found in a later generation is, in principle, obtainable in the f1, the observed transition from an apparently stable Sad phenotype to an apparently unstable Sad/Esm phenotype appears not to be due to genotype differences between the generations. Therefore, the Sad versus Esm difference may have a genotype-independent basis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Meiotic silencing by unpaired DNA is a presumed RNAi-mediated elimination of the transcripts of any gene that is not properly paired with a homolog in meiosis. Eighty wild-isolated strains of Neurospora crassa were classified into three types based on the apparent strength of meiotic silencing of the bml (β-tubulin) and mei-3 genes in crosses with the ::Bml(r) and ::mei-3 tester strains. "OR" and "Sad" type wild-isolates, respectively, did or did not silence both the genes, whereas the "Esm" type (68 strains) silenced bml but not mei-3(+), suggesting an intermediate strength of silencing. Many wild strains, especially of the Esm type, gave fertile crosses with strains bearing the 148 kbp chromosome segment duplication Dp(EB4) but their crosses with the 405 kbp Dp(IBj5) were barren. Larger Dps might include more genes whose sensitivity to silencing is comparable with that of bml, consequently their crosses with the OR and Esm type strains would be about equally barren, whereas small Dps, with presumably few or no bml-like genes suffer little gene silencing in crosses with Esm and Sad strains and thus remain fertile. Using lollipop-shaped asci as indicators of silencing in crosses of the ::act tester with Esm type strains, and white-spored asci in Neurospora tetrasperma self-crosses heterozygous for an ectopic insertion of the asm-1 gene, we found that meiotic silencing was expressed more strongly in the early but not in the late perithecia of these crosses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Repeat-induced point mutation (RIP) is a sexual stage-specific mutational process of Neurospora crassa and other fungi that alters duplicated DNA sequences. Previous studies from our laboratory showed that chromosome segment duplications (Dps) longer than (approx.) 300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps less than 200 kbp did not individually suppress RIP, they could do so in homozygous and multiply heterozygous crosses, provided the sum of the duplicated DNA exceeds (approx.) 300 kbp. Here we demonstrate suppression of RIP in a subset of progeny carrying the normally sub-threshold 154 kbp Dp(R2394) from a cross of T(R2394) to the wild isolated Carrefour Mme. Gras strain (CMG). Thus, the CMG strain contains a factor that together with Dp(R2394) produces a synthetic RIP suppressor phenotype. It is possible that the factor is a cryptic Dp that together with Dp(R2394) can exceed the size threshold for titration of the RIP machinery and thereby causes RIP suppression.

Ordinarily, RIP-induced erg-3 mutant Neurospora crassa ascospores and their erg(+) siblings do not differ in stability during long-term storage. Consequently, the frequency of RIP-induced erg-3 mutants remains about constant regardless of the time that has elapsed between ascospore harvest and germination. We found, however, that RIP-induced erg-3 mutants were apparently selectively lost with time from among the ascospores stored from a cross with the wild-isolated Carrefour Mme. Gras strain from Haiti. The Haitian strain was also found to exert a dominant suppression of meiotic silencing by unpaired DNA. Similar loss of RIP-induced erg-3 mutant ascospores was seen among the stored ascospores from a subset of crosses heterozygous for the semi-dominant Sad-1 or Sad-2 suppressors of meiotic silencing. Our results suggest that crosses suppressed in meiotic silencing can compromise the stability during storage of ascospores that inherit RIP-induced mutations., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In Neurospora crassa, crosses between normal sequence strains and strains bearing some translocations can yield progeny bearing a duplication (Dp) of the translocated chromosome segment. Here, 30 breakpoint junction sequences of 12 Dp-generating translocations were determined. The breakpoints disrupted 13 genes (including predicted genes), and created 10 novel open reading frames. Insertion of sequences from LG III into LG I as translocation T(UK8-18) disrupts the eat-3 gene, which is the ortholog of the Podospora anserine gene ami1. Since ami1-homozygous Podospora crosses were reported to increase the frequency of repeat-induced point mutation (RIP), we performed crosses homozygous for a deficiency in eat-3 to test for a corresponding increase in RIP frequency. However, our results suggested that, unlike in Podospora, the eat-3 gene might be essential for ascus development in Neurospora. Duplication-heterozygous crosses are generally barren in Neurospora; however, by using molecular probes developed in this study, we could identify Dp segregants from two different translocation-heterozygous crosses, and using these we found that the barren phenotype of at least some duplication-heterozygous crosses was incompletely penetrant.

The Neurospora crassa fmf-1 mutation exerts an unusual 'perithecium-dominant' developmental arrest; fmf-1 x fmf-1+ cross becomes arrested in perithecial development regardless of whether the mutant participates in the cross as the male or female parent. We localized fmf-1 to the LG IL genome segment between the centromere-proximal breakpoint of the chromosome segment duplication Dp(IL)39311 and the centromere. By mapping crossovers with respect to RFLP markers in this region we further localized fmf-1 to an approximately 34-kb-genome segment. Partial sequencing of this segment revealed a point mutation in the gene NCU 09387.1, a homologue of the Schizosaccharomyces pombe ste11+ regulator of sexual development. The fmf-1 mutation did not complement a NCU 09387.1 deletion mutation, and transformation with wild-type NCU 09387.1 complemented fmf-1. S. pombe Ste11 protein (Ste11p) is a transcription factor required for sexual differentiation and for the expression of genes required for mating pheromone signalling in matP and matM cells. If FMF-1 also plays a corresponding role in mating pheromone signalling in Neurospora, then protoperithecia in an fmf-1 x fmf-1+ cross would be unable to either send or receive sexual differentiation signals and thus become arrested in development.

Studies on Neurospora chromosome segment duplications (Dps) performed since the publication of Perkins's comprehensive review in 1997 form the focus of this article. We present a brief summary of Perkins's seminal work on chromosome rearrangements, specifically, the identification of insertional and quasiterminal translocations that can segregate Dp progeny when crossed with normal sequence strains (i.e., T x N). We describe the genome defense process called meiotic silencing by unpaired DNA that renders Dp-heterozygous crosses (i.e., Dp x N) barren, which provides a basis for identifying Dps, and discuss whether other processes also might contribute to the barren phenotype of Dp x N and Dp x Dp crosses. We then turn to studies suggesting that large Dps (i.e., >300 kbp) can allow smaller gene-sized duplications to escape another genome defense process called repeat-induced point mutation (RIP), possibly by titration of the RIP machinery. Finally, we assess whether in natural populations dominant RIP suppressor Dps provide an "RIP-free" niche for evolution of new genes following the duplication of existing genes.

Repeat-induced point mutation (RIP) is a hypermutational process that alters duplicated DNA sequences in Neurospora crassa. In previous studies, five of six large ( > 100 kb) chromosome segment duplications (Dp's) examined were shown to dominantly suppress RIP in smaller (< 5 kb) duplications. The suppressor duplications were > 270 kb, whereas the lone non-suppressor duplication was approximately 117 kb. We have now screened another 33 duplications and found 29 more suppressors and four more non-suppressors. All 22 suppressor duplications whose size could be estimated were > 270 kb, whereas two newly identified non-suppressor duplications examined were 140-154 kb. RIP was suppressed in a subset of crosses heterozygous for more than one ordinarily non-suppressor duplication. These results strengthen the hypothesis that large duplications titrate out the RIP machinery and suggest the "equivalence point" for the titration is close to 300 kb.

Microbial type III polyketide synthases (PKSs) have revealed remarkable mechanistic as well as functional versatility. Recently, a type III PKS homolog from Azotobacter has been implicated in the biosynthesis of resorcinolic lipids, thus adding a new functional significance to this class of proteins. Here, we report the structural and mutational investigations of a novel type III PKS protein from Neurospora crassa involved in the biosynthesis of resorcinolic metabolites by utilizing long chain fatty acyl-CoAs. The structure revealed a long hydrophobic tunnel responsible for its fatty acyl chain length specificity resembling that of PKS18, a mycobacterial type III PKS. Structure-based mutational studies to block the tunnel not only altered the fatty acyl chain specificity but also resulted in change of cyclization pattern affecting the product profile. This first structural characterization of a resorcinolic lipid synthase provides insights into the coordinated functioning of cyclization and a substrate-binding pocket, which shows mechanistic intricacy underlying type III PKS catalysis.

Crosses involving the Adiopodoumé strain of Neurospora crassa are defective for repeat-induced point mutation (RIP), a genome defense mechanism of fungi. We show here that the Adiopodoumé strain possesses an incompletely penetrant and variably expressive dominant suppressor of RIP (Srp) that maps to an approximately 34-kbp genome segment that is approximately 26 kbp proximal to mat on linkage group IL. Gene disruption experiments revealed that Srp is the upr-1 allele of Adiopodoumé (upr-1(Ad)) that is contained within this segment. The upr-1 gene codes for the catalytic subunit of the translesion DNA polymerase-zeta (Pol-zeta) and it is unusually polymorphic in Neurospora. That the upr-1 gene contains upstream ORFs that overlap with the main ORF is potentially relevant to the incomplete penetrance and variable expressivity of the suppressor. Crosses between heterokaryons that contain upr-1(Ad) and strains that prevent mating events involving nuclei that contain upr-1(Ad) yielded no progeny in which RIP had occurred, consistent with the idea that the suppressor encoded by upr-1(Ad) is diffusible. The potential involvement of the Pol-zeta subunit in two functions, translesion DNA synthesis and RIP regulation, might account for the rapid evolution of its gene in Neurospora.

Pol zeta, Pol eta, Pol iota, Pol kappa and Rev1 are specialized DNA polymerases that are able to synthesize DNA across a damaged template. DNA synthesis by such translesion polymerases can be mutagenic due to the miscoding nature of most damaged nucleotides. In fact, many mutational and hypermutational processes in systems ranging from yeast to mammals have been traced to the activity of such polymerases. We show however, that the translesion polymerases are dispensable for repeat-induced point mutation (RIP) in Neurospora crassa. Additionally, we demonstrate that the upr-1 gene, which encodes the catalytic subunit of Pol zeta, is a highly polymorphic locus in Neurospora.

Fungi, animals, and single-celled organisms belonging to the choanozoans together constitute the supergroup Opisthokonta. The latter are considered crucial in understanding the evolutionary origin of animals and fungi. The choanozoan Corallochytrium limacisporum is an enigmatic marine protist of considerable interest in opisthokontan evolution. Several isolates of the organism were obtained from a coral reef lagoon in the Lakshadweep group of islands of the Arabian Sea. The capability of these cultures to grow on media containing inorganic nitrogen sources prompted us to examine the possible presence of fungal signatures, namely the enzyme alpha-aminoadipate reductase (alpha-AAR) involved in the alpha-aminoadipate (AAA) pathway for synthesizing lysine and ergosterol, in one of the isolates. These features, as well as the sterol C-14 reductase gene involved in the sterol pathway of animals and fungi, were detected in the organism. Phylogenetic trees based on the alpha-AAR gene suggested that Corallochytrium limacisporum is a sister clade to fungi, while those based on the C-14 reductase gene did not adequately resolve whether the organism was more closely related to fungi or animals. While many studies indicate that Corallochytrium is a sister clade to animals, we suggest that further studies are required to examine whether this protist is in fact more closely related to fungi rather than to animals.

The size and extent of four Neurospora crassa duplications, Dp(AR17), Dp(IBj5), Dp(OY329), and Dp(B362i), was determined by testing the coverage of RFLP markers. The first three duplications were all > approximately 350 kb and have been shown in earlier studies to act as dominant suppressors of repeat-induced point mutation (RIP) in gene-sized duplications, possibly via titration of the RIP machinery. Dp(B362i), which is only approximately 117 kb long, failed to suppress RIP. RIP suppression in gene-sized duplications by large duplications was demonstrated using another test gene, dow, and supposedly applies generally. Crosses homozygous for Dp(AR17) or Dp(IBj5) were as barren as heterozygous crosses. Barrenness of the heterozygous but not the homozygous crosses was suppressible by Sad-1, a semidominant suppressor of RNAi-dependent meiotic silencing by unpaired DNA. A model is proposed in which large duplications recessively suppress semidominant Sad-1 mutations. The wild-isolated Sugartown strain is hypothesized to contain a duplication that confers not only dominant suppression of RIP but also a barren phenotype, which is linked (9%) to supercontig 7.118 in LG VII.

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1-0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

The pseudohomothallic fungus Neurospora tetrasperma is naturally resistant to the antibiotic hygromycin. We discovered that mutation of its erg-3 (sterol C-14 reductase) gene confers a hygromycin-sensitive phenotype that can be used to select transformants on hygromycin medium by complementation with the N. crassa erg-3+ and bacterial hph genes. Cotransformation of hph with PCR-amplified DNA of other genes enabled us to construct strains duplicated for the amplified DNA. Using transformation we constructed self-fertile strains that were homoallelic for an ectopic erg-3+ transgene and a mutant erg-3 allele at the endogenous locus. Self-crosses of these strains yielded erg-3 mutant ascospores that produced colonies with the characteristic morphology on Vogel's sorbose agar described previously for erg-3 mutants of N. crassa. The mutants were generated by repeat-induced point mutation (RIP), a genome defense process that causes numerous G:C to A:T mutations in duplicated DNA sequences. Homozygosity for novel recessive RIP-deficient mutations was signaled by self-crosses of erg-3-duplication strains that fail to produce erg-3 mutant progeny. Using this assay we isolated a UV-induced mutant with a putative partial RIP defect. RIP-induced mutants were isolated in rid-1 and sad-1, which are essential genes, respectively, for RIP and another genome defense mechanism called meiotic silencing by unpaired DNA.

Repeat-induced point mutation (RIP) in Neurospora results in inactivation of duplicated DNA sequences. RIP is thought to provide protection against foreign elements such as retrotransposons, only one of which has been found in N. crassa. To examine the role of RIP in nature, we have examined seven N. crassa strains, identified among 446 wild isolates scored for dominant suppression of RIP. The test system involved a small duplication that targets RIP to the easily scorable gene erg-3. We previously showed that RIP in a small duplication is suppressed if another, larger duplication is present in the cross, as expected if the large duplication competes for the RIP machinery. In two of the strains, RIP suppression was associated with a barren phenotype--a characteristic of Neurospora duplications that is thought to result in part from a gene-silencing process called meiotic silencing by unpaired DNA (MSUD). A suppressor of MSUD (Sad-1) was shown not to prevent known large duplications from impairing RIP. Single-gene duplications also can be barren but are too short to suppress RIP. RIP suppression in strains that were not barren showed inheritance that was either simple Mendelian or complex. Adding copies of the LINE-like retrotransposon Tad did not affect RIP efficiency.