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.     

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.     

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.     

Translocations used to generate chromosome segment duplications in <Emphasis Type="Italic">Neurospora</Emphasis> can disrupt genes and create novel open reading frames

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.     

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.     

Escape from repeat-induced point mutation of a gene-sized duplication in Neurospora crassa crosses that are heterozygous for a larger chromosome segment duplication

In Neurospora crassa the ability of an ectopic gene-sized duplication to induce repeat-induced point mutation (RIP) in its target gene was suppressed in crosses that were heterozygous for another larger chromosome segment duplication. Specifically, the frequency of RIP in the erg-3 gene due to a 1.3-kb duplication was reduced if the chromosome segment duplications Dp(IIIR > [I;II]) AR17, Dp(VIR > IIIR) OY329, or Dp(IVR > VII) S1229 were present in either the same or the other parental nucleus of the premeiotic dikaryon. We suggest that the larger duplications act as sinks to titrate the RIP machinery away from the smaller duplication. In contrast, RIP efficiency was relatively unaffected in comparably unproductive interspecies crosses with N. intermedia and N. tetrasperma. These findings offer a novel explanation for the observed persistence of the transposable element Tad in only a subset of Neurospora strains.     

Collateral damage: Spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene inNeurospora crassa

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered inNeurospora 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 theerg-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 theerg-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 oferg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into theerg-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 bacterialhph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.     

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.     

Quantitative determination of Der p I allergen levels in allergenic extracts and house-dust samples

Summary House-dust mites are responsible for serious respiratory diseases in humans. An ELISA assay to detectDermatophagoides pteronyssinus (Dp) mites has been set up. This assay, based on quantitative determination of the majorDp allergen, called Der p I, has been applied for the analysis of growing mite cultures and house-dust samples.Der p I allergen levels in mite cultures are well correlated with the number of mites present as well as with the biological activity of the corresponding extracts. Different methods for detectingDp mites in house-dust samples were compared. The ELISA method shows good sensitivity and specificity, and is particularly suitable for routine analyses.     

Tandem duplications in Drosophila melanogaster

In the tandem duplication Dp(1;1)Gr approximately one quarter of the euchromatic part of the X-chromosome is duplicated. Dp(1;1)Gr itself has no phenotypic effect, but it can be made visible by combining different alleles within the tandem duplication and the homologous X-chromosome. In heterozygous females crossing-over between the two X-chromosomes is strongly reduced while at the same time crossing-over in the distal regions of the autosomes is increased.     

<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> heat shock protein 70 is able to suppress the thermosensitivity of an <Emphasis Type="Italic">Escherichia coli</Emphasis> DnaK mutant strain

Heat shock protein 70 (Hsp70) and heat shock protein 40 (Hsp40) are molecular chaperones that ensure that the proteins of the cell are properly folded and functional under both normal and stressful conditions. The malaria parasite Plasmodium falciparum is known to overproduce a heat shock protein 70 (PfHsp70) in response to thermal stress; however, the in vivo function of this protein still needs to be explored. Using in vivo complementation assays, we found that PfHsp70 was able to suppress the thermosensitivity of an Escherichia coli dnaK756 strain, but not that of the corresponding deletion strain (dnaK52) or dnaK103 strain, which produces a truncated DnaK. Constructs were generated that encoded the ATPase domain of PfHsp70 fused to the substrate-binding domain (SBD) of E. coli DnaK (referred to as PfK), and the ATPase domain of E. coli DnaK coupled to the SBD of PfHsp70 (KPf). PfK was unable to suppress the thermosensitivity of any of the E. coli strains. In contrast, KPf was able to suppress the thermosensitivity in the E. coli dnaK756 strain. We also identified two key amino acid residues (V401 and Q402) in the linker region between the ATPase domain and SBD that are essential for the in vivo function of PfHsp70. This is the first example of an Hsp70 from a eukaryotic parasite that can suppress thermosensitivity in a prokaryotic system. In addition, our results also suggest that interdomain communication is critical for the function of the PfHsp70 and PfHsp70-DnaK chimeras. We discuss the implications of these data for the mechanism of action of the Hsp70-Hsp40 chaperone machinery.     

Factors Affecting Recognition and Disjunction of Chromosomes at Distributive Pairing in Female DROSOPHILA MELANOGASTER. I. Total Length VS. Arm Length

The behavior of a compound metacentric fourth chromosome (see PDF) has been examined to determine whether arm length or total length is the basis for recognition in distributive pairing. Recognition was judged by the frequency with which the (see PDF) nondisjoined from a series of X duplications (Dp), ranging in size from ≤ 0.3 to > 4 times the size of a single fourth chromosome. Dp, (see PDF) nondisjunction was measured in the absence and in the presence of a competitor, a compound metacentric X. In both situations, total length and not arm length, was found to confer the characteristic recognition property to the (see PDF). A comparison of Dp, (see PDF) nondisjunction curves for both the noncompetitive and competitive situations with analogous Dp, 4 curves previously obtained, show the Dp, (see PDF) curves to be similar in shape to those obtained earlier but displaced one unit to the right, corresponding precisely to the difference in size between the (see PDF) and the 4. Rules governing chromosome recognition for acrocentrics were found completely applicable to metacentrics; disjunctive behavior of metacentrics differed from that of acrocentrics in that two arms conferred on a chromosome the capacity to act as the intermediate of a trivalent when size no longer warranted this attribute. This capacity, itself, is size-dependent.     

<Emphasis Type="Italic">Daphnia</Emphasis> growth is hindered by chemical information on predation risk at high but not at low food levels

Daphnia hyalina × galeata (Dhg) and D. pulicaria (Dp) are ready to pay greater costs in terms of predation risk avoidance at high rather than at low food levels. Such costs are easier to assess in Daphnia than in large long-lived and difficult-to-handle herbivores, since they can be precisely determined in a few-day experiment as the reduced growth (P=A−R) resulting from diminished assimilation (A) and/or increased respiration (R). In experiments with Daphnia grown for six days from the neonate to the first clutch of eggs, which were given different levels of algal food (Scenedesmus at concentrations from 0.05 to 1.60 mg C l⁻¹), individual growth was lower in the presence of fish kairomone (chemical information on fish predation; present at a concentration that induces antipredator defensive behavior and life histories) than in the absence of kairomone (control). The difference from the control was negligible at the lowest food levels, and gradually increased with increasing food concentration. At a food concentration of 1.6 mg C l⁻¹, growth was reduced by 9–32 and 0–8% in Dhg and Dp, respectively, compared to the controls. A similar reduction was observed in the body length of six-day-old animals (Dhg 6–19%, Dp 0–14%), but not in the first clutch reproductive effort (clutch volume). Daphnia had a greater number of eggs per clutch in the presence of the kairomone, but smaller eggs, so that the total volume of eggs in a clutch was the same with and without kairomone. The amplification of the effect of the kairomone due to high food levels was weaker in Dp, a species that rarely coexists with planktivorous fish in natural habitats.     

Metformin mediates cardioprotection against aging‐induced ischemic necroptosis

Necroptosis is crucially involved in severe cardiac pathological conditions. However, whether necroptosis contributes to age‐related intolerance to ischemia/reperfusion (I/R) injury remains elusive. In addition, metformin as a potential anti‐aging related injury drug, how it interacts with myocardial necroptosis is not yet clear. Male C57BL/6 mice at 3–4‐ (young) and 22–24 months of age (aged) and RIPK3‐deficient (Ripk3^?/?) mice were used to investigate aging‐related I/R injury in vivo. Metformin (125 μg/kg, i.p.), necrostatin‐1 (3.5 mg/kg), and adenovirus vector encoding p62‐shRNAs (Ad‐sh‐p62) were used to treat aging mice. I/R‐induced myocardial necroptosis was exaggerated in aged mice, which correlated with autophagy defects characterized by p62 accumulation in aged hearts or aged human myocardium. Functionally, blocking autophagic flux promoted H/R‐evoked cardiomyocyte necroptosis in vitro. We further revealed that p62 forms a complex with RIP1‐RIP3 (necrosome) and promotes the binding of RIP1 and RIP3. In mice, necrostatin‐1 treatment (a RIP1 inhibitor), RIP3 deficiency, and cardiac p62 knockdown in vivo demonstrated that p62‐RIP1‐RIP3‐dependent myocardial necroptosis contributes to aging‐related myocardial vulnerability to I/R injury. Notably, metformin treatment disrupted p62‐RIP1‐RIP3 complexes and effectively repressed I/R‐induced necroptosis in aged hearts, ultimately reducing mortality in this model. These findings highlight previously unknown mechanisms of aging‐related myocardial ischemic vulnerability: p62‐necrosome‐dependent necroptosis. Metformin acts as a cardioprotective agent that inhibits this unfavorable chain mechanism of aging‐related I/R susceptibility.     

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH:ubiquinone reductase ofNeurospora mitochondria

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH:ubiquinone reductase (Complex I) fromNeurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain ofN. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functionalnuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH:ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.     

Stability of domain 4 of the anthrax toxin protective antigen and the effect of the VWA domain of CMG2 on stability

The major immunogenic component of the current anthrax vaccine, anthrax vaccine adsorbed (AVA) is protective antigen (PA). We have shown recently that the thermodynamic stability of PA can be significantly improved by binding to the Von‐Willebrand factor A (VWA) domain of capillary morphogenesis protein 2 (CMG2), and improvements in thermodynamic stability may improve storage and long‐term stability of PA for use as a vaccine. In order to understand the origin of this increase in stability, we have isolated the receptor binding domain of PA, domain 4 (D4), and have studied the effect of the addition of CMG2 on thermodynamic stability. We are able to determine a binding affinity between D4 and CMG2 (～300 nM), which is significantly weaker than that between full‐length PA and CMG2 (170–300 pM). Unlike full‐length PA, we observe very little change in stability of D4 on binding to CMG2, using either fluorescence or ¹⁹F‐NMR experiments. Because in previous experiments we could observe a stabilization of both domain 4 and domain 2, the mechanism of stabilization of PA by CMG2 is likely to involve a mutual stabilization of these two domains.     

An integrative plasmid and multiple-sized plasmids of Pseudomonas syringae pv. phaseolicola have extensive homology

Summary Plasmids isolated from five strains of the bean pathogen Pseudomonas syringae pv. phaseolicola were characterized by restriction endonuclease and filter hybridization analyses. BamHI and EcoRI restriction patterns revealed that total plasmid DNA from each strain had a high level of sequence homology with pMC7105, a 148 kbp integrative plasmid found in a sixth strain. Only six BamHI fragments from the eight plasmids in these strains failed to hybridize with pMC7105 probe. Four of these fragments, three from pPP6520 and one from pPP6525 of strain PP652, hybridized strongly to plasmid DNA from a closely-related pathovar, P. syringae pv. glycinea. BamHI fragment 8, which is involved in the integration of pMC7105 into the host chromosome, contains a repeat sequence that was present on all the plasmids except pPP6120 (6.8 kbp), pPP6310 (40 kbp) and pPP6520 (45 kbp). Every plasmid but pPP6520 had fragments that showed weak hybridization to the small plasmid, pPP6120. This homology suggests that a second repetitive sequence is common to these plasmids. The large plasmids (148 to 151 kbp) were essentially identical to pMC7105. The intermediate plasmids (122 to 128 kbp) appeared to be derived mainly from pMC7105 or a related plasmid, whereas the smaller plasmids (6.8 to 45 kbp) appear to have been derived in part from sequences not present in pMC7105.     

Synthesis of Conceptually New,Potentially Anti-viral,Low-Toxity,Acid- and Enzyme-stable Furanoid and Pyranoid Nucleosides

A large (80 kbp) plasmid designated as pSLL was isolated from Streptomyces laurentii ATCC 31255 (strain P0). Another covalently closed circular plasmid, pSLS (16 kbp), was found in pSLL-cured PO cells (named PI), which spontaneously formed pocks on a solid medium. The frequency of the generation of pSLL-loss PI cells coordinated positively with the frequency of pock appearance. pSLS was also found as an integrated form in the chromosome of strains PO and Pl. The results indicated that strain PO harbored two plasmids, pSLL and integrated form pSLS, and pSLS seemed to be generated as a free-form plasmid with the loss of pSLL. It was also suggested that pSLL may suppress the excision of the integrated pSLS from the chromosome and that the free form of pSLS may participate in pock formation.