Chromosome segment duplications in Neurospora crassa: barren crosses beget fertile science

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 × N). We describe the genome defense process called meiotic silencing by unpaired DNA that renders Dp‐heterozygous crosses (i.e., Dp × N) barren, which provides a basis for identifying Dps, and discuss whether other processes also might contribute to the barren phenotype of Dp × N and Dp × 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.     

A factor in a wild isolated <Emphasis Type="BoldItalic">Neurospora crassa</Emphasis> strain enables a chromosome segment duplication to suppress repeat-induced point mutation

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 ~300 kbp can dominantly suppress RIP, presumably by titration of the RIP machinery, and that although Dps <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 ~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.     

Evidence for dominant suppression of repeat-induced point mutation (RIP) in crosses with the wild-isolatedNeurospora crassa strains Sugartown and Adiopodoume-7

A convenient assay to score repeat-induced point mutation (RIP) inNeurospora employs theerg-3 locus as a mutagenesis target. Using this assay we screened 132 wild-isolatedNeurospora crassa strains for ability to dominantly suppress RIP. RIP was exceptionally inefficient in crosses with the wild isolates Sugartown (P0854) and Adiopodoume-7 (P4305), thereby suggesting the presence of dominant RIP suppressors in these strains. In other experiments, we found no evidence for dominant RIP suppression by theSpore killer haplotypesSk-2 andSk-3.     

Repeat-induced point mutation (RIP) in crosses with wild-isolated strains of Neurospora crassa: evidence for dominant reduction of RIP

Seventy-one wild-isolated strains of Neurospora crassa were examined for their ability to support repeat-induced point mutation (RIP) in the erg-3 locus. RIP was exceptionally inefficient but detectable in crosses with the strain FGSC 430 from Adiopodoume, Ivory Coast. We could find no consistent differences in ascospore yields when wild isolates identified as "low-RIP" or "high-RIP" strains were crossed with strains bearing the segmental duplication Dp(IIIR > [I; II])AR17. This suggested that RIP may not be responsible for the barren phenotype of crosses involving segmental duplication strains.     

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.     

Titration of repeat-induced point mutation (RIP) by chromosome segment duplications in <Emphasis Type="Italic">Neurospora crassa</Emphasis>

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 ∼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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This article is dedicated to the memory of Robert L. Metzenberg.     

Isolation and characterization of a new gene, sre , which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ⁻ strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ⁻ mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells. Received: 20 January 1998 / Accepted: 23 April 1998     

Genes encoding chimeras of<Emphasis Type="Italic">Neurospora crassa erg-3</Emphasis> and human TM7SF2 proteins fail to complement Neurospora and yeast sterol C-14 reductase mutants

The human gene TM7SF2 encodes a polypeptide (SR-1) with high sequence similarity to sterol C-14 reductase, a key sterol biosynthetic enzyme in fungi, plants and mammals. In Neurospora and yeast this enzyme is encoded by theerg-3 anderg24 genes respectively. In an effort to demonstrate sterol C-14 reductase activity for SR-1 we constructed six recombinant genes coding for chimeras of the Neurosporaerg-3 and SR-1 protein sequences and tested them for complementation of the Neurosporaerg-3 mutant. To our surprise, all the chimeras failed to complementerg-3. A few of the chimeric proteins were also tested against the yeasterg24 mutant, but again there was no complementation. We discuss some reasons that might account for these unexpected findings     

Premeiotic disruption of the Neurospora crassa malate synthase gene by native and divergent DNAs

Summary Repeat-induced point mutation (RIP) has been used to generate new mutations in the previously uncharacterised gene for malate synthase in Neurospora crassa. Molecular clones carrying the am (NADP-glutamate dehydrogenase) gene and the malate synthase gene from either N. crassa or Aspergillus nidulans have been introduced into Neurospora as ectopic duplicate copies by transformation, selecting for the am function in a deletion host. A number of meiotic progeny derived from these transformants were unable to use acetate as sole carbon source, yielded no detectable malate synthase activity and demonstrated extensive cytosine methylation of their duplicated sequences. The new locus has been designated acu-9 and has been assigned to linkage group VII.     

Evidence for non-random spatial distribution of meiotic exchanges in Podospora anserina: comparison between linkage groups 1 and 6.

Summary In Podospora anserina, positive and very efficient chiasma interference is observed. However, its modalities are different for the two linkage groups 1 (LG1) and 6 (LG6) studied here.In the right arm of LG1, two zones exist in which always occurs only one crossing-over. They are formed independently each other. Moreover, the genetic map consists of clusters of genes located near the centromere and at the limit between the two interference zones. It is postulated that this structure of the map results from the localization of crossing-over in the middle of each zone. We suppose that the type of chiasma interference, in Podospora, is a typical one as it is in Drosophila. It seems that both these phenomena are under common genetical control.In the LG6, we observe a weaker positive chiasma interference without crossing-over localization.Laboratoire associé au C.N.R.S.n^o 086     

Chromosome segment duplications in Neurospora crassa and their effects on repeat-induced point mutation and meiotic silencing by unpaired DNA

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.     

The complete karyotype of Aspergilius niger: the use of introduced electrophoretic mobility variation of chromosomes for gene assignment studies

A method is described for unambiguous assignment of cloned genes to Aspergillus niger chromosomes by CHEF gel electrophoresis and Southern analysis. All of the eight linkage groups (LGs), with the exception of LG VII, have previously been assigned to specific chromosomal bands in the electrophoretic karyotype of A. niger. Using a LG VII-specific probe (nicB gene of A. niger) we have shown that LG VII corresponds to a chromosome of about 4.1 Mb. Furthermore, genetic localization of three unassigned genes (glaA, agIA and pepA) in strains in which these genes had been replaced by a selectable marker gene led to a revised karyotype for the chromosomes corresponding to LGs VIII and VI. The revised electrophoretic karyotype reveals only 5 distinct bands. The presence of three pairs of equally sized chromosomes precluded assignment of genes to one specific chromosome in the wild-type strain. However, unambiguous chromosome assignment of cloned genes using CHEF-Southern analysis was demonstrated using a set of A. niger strains with introduced chromosomal size variation. The availability of these tester strains obviates the need to isolate or construct mutant. strains for the purpose of chromosome assignment.     

Genetic analysis of wild-isolated Neurospora crassa strains identified as dominant suppressors of repeat-induced point mutation

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.     

Isolation and characterization of a new gene, sre, which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ^? strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ^? mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells.     

Gene inactivation in Arabidopsis thaliana is not accompanied by an accumulation of repeat-induced point mutations

Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase (hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism.