Chromosomal structure is altered by mutations that suppress or enhance position effect variegation

We examined the genetic, morphological, and molecular effects of position effect variegation inDrosophila, and the effects of mutations that either suppress [Su(var)] or enhance [E(var)] this phenomenon. All eightSu(var) mutations examined strongly suppress the inactivation of variegating alleles of the genes white [In(l) w ^m4 ], brown [In (2R)bw ^VDe2 ] and Stubble [T(2;3)Sb ^V ]. TheE(var) mutation enhances variegation of these loci. The chromosomal region 3C-E (26 bands) which includes the white locus is usually packaged as heterochromatin in salivary glands of the variegating strainw ^m4 . Addition of any of theSu(var) mutations restores a more euchromatic morphology to this region. In situ hybridization to polytene chromosomes and DNA blot analyses of gene copy number demonstrate that the DNA of thew ⁺ gene is less accessible to its probe in the variegatingw ^m4 strain than it is in the wildtype or variegation-suppressed strains. Blot analysis of larval salivary gland DNA indicates that the white gene copy number does not vary among the strains. Hence, the differences in binding of thew ⁺ gene probe in the variegating and variegation-suppressed strains reflect differences in chromosomal packaging rather than alterations in gene number. The effects of variegation and theSu(var) mutations on chromatin structure were analyzed further by DNAse I digestion and DNA blot hybridization. In contrast to their dramatic effects on chromosomal morphology and gene expression, theSu(var) mutations had negligible effects on nuclease sensitivity of the white gene chromatin. We suggest that the changes in gene expression resulting from position effect variegation and the action of theSu(var) mutations involve alterations in chromosomal packaging.     

Mutants affecting position-effect heterochromatinization in Drosophila melanogaster

The dominant suppressor Su(var)b ¹⁰¹ and the dominant enhancer En(var)c ¹⁰¹ were found to affect significantly white variegation in a strongly variegating line of the w ^m4 chromosome (w ^m4h ) which has been used as standard rearrangement for a genetic dissection of position-effect variegation (Reuter and Wolff, 1981). Both mutations were also shown to affect position-effect heterochromatisation in T(1;4)w ^m258-21 and variegation in all the rearrangements tested (white, brown, scute and bobbed variegation). These results suggest that the genes identified encode functions essential for the manifestation of gene inactivation in position-effect rearrangements. It seems also reasonable to assume that in all the rearrangements tested identical heterochromatisation processes lead to inactivation of the genes whose phenotype is variegated.     

A rapid decrease in number of the complete <Emphasis Type="Italic">ninja</Emphasis> element and concomitant increase of the defective element in a strain of <Emphasis Type="Italic">Drosophila simulans</Emphasis>

The ninja element, originally isolated from an unstable white mutant strain white-milky (w^mky) of Drosophila simulans, is a member of the retrotransposon family with long terminal repeats (LTRs). We show that ninja is present in high copy numbers in the w^mky-derivative sublines white-chocolate (w^cho) and white-persimmon1 (w^psm1), in a low copy number in another derivative subline white-milky 3 (w^mky3), and in only a few copies in a wild type strain. We have cloned the ninja elements from these sublines and examined their structures. Most of the elements cloned (38 out of 41 independent clones) from w^cho were full length. In contrast, only 9 of 23 independent clones from w^mky3 were full length. We hypothesize that ninja elements were integrated and lost frequently in the w^mky strain and its derivative genomes, and that a rapid decrease in numbers of the ninja element was caused not by an increased rate of loss but by a reduction of integration of full length ninja elements in w^mky3. Each defective element had a unique deletion and/or an insertion except for the three from w^mky3, which had exactly the same 81-bp deletion in each of the 5 and 3 LTRs. The 5 and 3 ends of the deletion appeared to represent sequences similar to those of Drosophila consensus splicing sites. Ectopic splicing may have produced these defective ninja elements.     

Isolation of dominant suppressor mutations for position-effect variegation in Drosophila melanogaster

Summary Dominant suppressor mutations for position-effect variegation have been isolated by using a strongly variegated line carrying the w ^m4 chromosome (w ^m4h) and the dominant enhancer mutant En(var)c ¹⁰¹. The use of an effective genetic test system made it possible to isolate more than 100 strongly dominant suppressor mutations for position-effect variegation. This suggests that the phenomenon of position-effect variegation is characterised by a complex genetic basis. The significance of the isolated mutants to genetic dissection of structural and regulatory functions of the eukaryotic chromosome is discussed.     

Comparison of photosystem II complexes isolated from tobacco and two chlorophyll deficient tobacco mutants

A comparative study of photosystem II complexes isolated from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contains normal stacked thylakoid membranes, and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked grana or essentially unstacked thylakoids with occasional membrane doublings, has been carried out. The corresponding photosystem II complexes had an O₂ evolving activity ranging from 290 (for the wild type) to 1100 mol O₂ x mg chlorophyll^-1 x h^-1 (for the mutant Su/su var. Aurea). The reduced photosynthetic unit size was also obvious in the mangenese and cytochrome_b559 content. The photosystem II complex from the wild type contained 4 Mn and 1 cytochrome_b559 per 200 to 280 chlorophylls, while the corresponding value for the mutant Su/su var. Aurea was 4 Mn and 1 cytochrome_b559 per 35 to 60 chlorophylls. We have also examined the polypeptide composition and show that the photosystem II complex from the wild type consisted of polypeptides of 48, 42, 33, 32, 30, 28, 23, 21, 18, 16 and 10 kDa, while the mutant complex mainly contained the polypeptides of 48, 42, 33, 32, 30, 28 and 10 kDa. In the mutant photosystem II complex the light-harvesting chlorophyll protein (peptide of 28 kDa) was reduced by a factor of 5 to 6 as compared to the wild type. With respect to the peptide composition and the photosynthetic unit size, the Triton-solubilized photosystem II complex from the mutant Su/su var. Aurea was very similar to O₂ evolving photosystem II reaction center core complexes.Abbreviations PS photosystem - chl chlorophyll - LHCP light-harvesting chlorophyll a/b protein complex     

Mitomycin C induces genomic rearrangements involving transposable elements in Drosophila melanogaster

Summary Mitomycin C was injected into the abdomen of male flies of the y ² sc¹ w^aG strain of Drosophila melanogaster. They were mated with females bearing attached-X chromosomes, and the male offspring (F₁) were analysed for the appearance of mutations in the X chromosome. We observed y ¹ and sc ⁺ reversions induced either by excision of mdg4 (gypsy) with retention of one long terminal repeat (LTR) or by insertion of a foreign sequence into mdg4, partial reversion of the w ^aG mutation, w ^aGw ^aGd, and unstable f mutations. The overall mutation frequency was considerably higher than in control flies of the y ² sc¹ w^aG strain. Possible mechanisms of genomic rearrangements induced by Mitomycin C, in particular the role of homologous recombination, are discussed.     

Genetic instability in Drosophila melanogaster. Evidence for insertion mutations

Summary An X chromosome in Drosophila melanogaster is described which is mutationally unstable. Mutational events were identified through phenotypic changes associated with a tandem duplication of the X chromosome in which the white locus is present in duplicate. The left segment of the tandem duplication was marked with the mutant w ^sp, the right segment with mutant w ^17G. Some of the phenotypic changes were identified as deletions involving the w ^17G marked segment of the duplication. Other phenotypic changes involved the left segment in which phenotypically w ^sp mutated to w. Experimental evidence is presented which attributes these latter mutations to insertions of foreign DNA into the w locus equivalent to the insertion mutations of E. coli.     

Species-specific DNA sequences for identification of somatic hybrids between Lycopersicon esculentum and Solanum acaule

Summary Species-specific highly repeated DNA sequences can be used to screen the progeny of protoplast fusions combining different species. Such probes are easy to clone and can be detected by fast methods, e.g., hybridization to total genomic DNA. Furthermore, due to their high copy number, hybridization signals are strong and represent more than one locus, unlike isozymes or resistance markers. After cloning and screening for species-specific DNA sequences we characterized the highly repeated DNA sequences of the solanaceous species Solanum acaule and Lycopersicon esculentum var. gilva. DNA sequencing and hy ridization revealed a prominent, tandemly arranged satellite DNA repeat of 162 bp in Lycopersicon esculentum and a different satellite repeat of 183 bp, also tandemly organized, in Solanum acaule. Each repeat is absent in the respective other species. Therefore, we have used these DNA repeats as markers to distinguish regenerated interspecific somatic hybrids from the respective fusion partners. These hybrids were clearly identified by Southern hybridization and dot-blot assays to the respective ³²P-labelled satellite DNA.     

Contrasting response of euchromatin and heterochromatin to translocation in polytene chromosomes of Drosophila melanogaster

Studies on Feulgen-DNA content in the polytene chromosomes of D. melanogaster T(14)w ^m258-21 heterozygotes showed that when the euchromatic region 3D₁-E₂ is located next to the heterochromatic breakpoint it contains less DNA than in the non-translocated homologue (Hartmann-Goldstein and Cowell, 1976). In contrast to the region adjacent to the breakpoint, region 3C_1–10, which contains intercalary heterochromatin, shows more DNA in the translocated than in the non-translocated chromosome. Transposition may induce morphologically euchromatic regions containing putatively underreplicated sequences to undergo additional replication cycles. Region 2E₁-3A₄, distal to 3C₁ and at some distance from the heterochromatic breakpoint is apparently unaffected. Extended replication and reduced DNA content in regions which have undergone chromosomal rearrangement could be accounted for by varying degrees of blockage of replication in individual strands of the polytene chromosome.     

Characterization of mutations that enhance position-effect variegation in Drosophila melanogaster

Summary Several mutants that enhance the gene inactivation associated with position-effect variegation [E(var) mutants] have been characterized. These include three ethyl methanesulfonate (EMS)-induced lesions and a second chromosome duplication. Each of the EMS mutations maps to a discrete euchromatic site on the third chromosome. One is located within the chromosomal region occupied by a cluster of Su(var) mutations. All four E(var) mutants enhance the inactivation of several different variegators and therefore they appear to influence position-effect variegation generally. However, the enhancement caused by the single site E(var) mutations is less striking than that caused by the duplication or by loss of the Y chromosome. The interaction between the E(var) mutants and selected Su(var) mutations, as well as the effects of extra Y heterochromatin on E(var) expression, have also been investigated. Based on the results of these studies, various hypothetical functions of the E(var) ⁺ products are suggested.     

JIL-1 and Su(var)3-7 Interact Genetically and Counteract Each Other's Effect on Position-Effect Variegation in Drosophila

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms was unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity.SU(VAR)3-9, a histone methyltransferase, Su(var)2-5, HP1a, and Su(var)3-7, a 1250-residue zinc-finger protein are all inherent components of pericentric heterochromatin (Rea et al. 2000; Eissenberg and Elgin 2000; Schotta et al. 2002; Delattre et al. 2004; Ebert et al. 2004) and are important factors for silencing of reporter genes by heterochromatic spreading in Drosophila (for review see Weiler and Wakimoto 1995; Girton and Johansen 2008). Su(var)3-9 has been shown to catalyze most of the dimethylation of the histone H3K9 residue which in turn can promote HP1a and Su(var)3-7 recruitment (Schotta et al. 2002; Jaquet et al. 2006). In addition, Su(var)3-9, HP1a, and Su(var)3-7 can directly interact with each other, suggesting a model where interdependent interactions between Su(var)3-9, HP1a, and Su(var)3-7 lead to heterochromatin assembly at pericentric sites (Lachner et al. 2001; Schotta et al. 2002; Elgin and Grewal 2003; Jaquet et al. 2006). Heterochromatin formation in Drosophila is initiated early in development through active removal of H3K4 methylation by the LSD1 demethylase homolog Su(var)3-3 (Rudolph et al. 2007). Subsequently, a developmentally regulated balance between Su(var)3-3 H3K4 demethylase, Su(var)3-9 H3K9 methyltransferase, and RPD3 H3K9 deacetylase activity contribute to conserve the distinction between euchromatic and heterochromatic domains (Rudolph et al. 2007). Thus, highly complex interactions between multiple heterochromatic and euchromatic factors are likely to contribute to the regulation of a dynamic balance between the distinct chromatin environments promoting gene activity and gene silencing.It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase (Jin et al. 1999; Wang et al. 2001) is a major regulator of chromatin structure (Deng et al. 2005; 2008) and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing (Ebert et al. 2004; Zhang et al. 2006; Lerach et al. 2006; Bao et al. 2007). In the absence of the JIL-1 kinase, the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms with the most pronounced increase on the X chromosomes (Zhang et al. 2006; Deng et al. 2007). However, overall levels of the H3K9me2 mark and HP1a were unchanged, suggesting that the spreading was accompanied by a redistribution that reduces the levels in pericentromeric heterochromatin. Genetic interaction assays demonstrated that the lethality as well as some of the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-9 gene (Zhang et al. 2006; Deng et al. 2007). This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene where no genetic interactions were detectable between JIL-1 and Su(var)2-5 (Deng et al. 2007) Thus, these findings indicate that while Su(var)3-9 histone methyltransferase activity may be a factor in the lethality and chromatin structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1a. However, the potential role of the third major heterochromatin component, Su(var)3-7, was not addressed in these studies. Here we show that Su(var)3-7, like Su(var)3-9, genetically interacts with JIL-1, that reducing the dose of Su(var)3-7 significantly reduces the lethality of JIL-1 null mutants, and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalacing effect on position-effect variegation (PEV).     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

Properties of condensed chromatin in barley nuclei

A method for isolation and purification of intact nuclei from barley leaves was developed and several properties of the chromatin were studied. The dense structure of the main part of the chromatin does not alter the accessibility of the DNA to nucleases. 60% of the nuclear DNA can be degraded by micrococcal endonuclease. Nevertheless the solubility of the chromatin fragments depends on the extent of nuclease digestion; solubilisation occurring only when the major part of the internucleosomal DNA was degraded (30% of digestion). Electron microscopic observations suggest that this was due to particularly dense organization of the chromatin in situ. The possible physiological meaning of some of these properties are discussed.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Mutational specificity of ethyl methanesulfonate in excision-repair-proficient and -deficient strains of Drosophila melanogaster

Summary The vermilion gene was used as a target to determine the mutational specificity of ethyl methanesulfonate (EMS) in germ cells of Drosophila melanogaster. To study the impact of DNA repair on the type of mutations induced, both excision-repair-proficient (exr ⁺) and excision-repair-deficient (exr ^–) strains were used for the isolation of mutant flies. In all, 28 mutants from the exr ⁺ strain and 24 from the exr ^– strain, were characterized by sequence analysis. In two mutants obtained from the exr ⁺ strain, small deletions were observed. All other mutations were caused by single base-pair changes. In two mutants double base-pair substitutions had occurred. Of the mutations induced in the exr ⁺ strain, 22 (76%) were GCAT transitions, 3 (10%) ATTA transversions, 2 (6%) GCTA transversions and 2 (6%) were deletions. As in other systems, the mutation spectrum of EMS in Drosophila is dominated by GCAT transitions. Of the mutations in an exr ^– background, 12 (48%) were GCAT transitions, 7 (28%) ATTA transversions, 5 (20%) GCTA transversions and 1 (4%) was a ATGC transition. The significant increase in the contribution of transversion mutations obtained in the absence of an active maternal excision-repair mechanism, clearly indicates efficient repair of N-alkyl adducts (7-ethyl guanine and 3-ethyl adenine) by the excision-repair system in Drosophila germ cells.     

aubergine mutations in Drosophila melanogaster impair P cytotype determination by telomeric P elements inserted in heterochromatin

Transposable P elements inserted in the heterochromatic Telomeric Associated Sequences on the X chromosome (1A site) of Drosophila melanogaster have a very strong capacity to elicit the P cytotype, a maternally transmitted condition which represses P element transposition and P-induced hybrid dysgenesis. This repressive capacity has previously been shown to be sensitive to mutant alleles of the gene Su(var)205, which encodes HP1 (Heterochromatin Protein 1), thus suggesting a role for chromatin structure in repression. Since an interaction between heterochromatin formation and RNA interference has been reported in various organisms, we tested the effect of mutant alleles of aubergine, a gene that has been shown to play a role in RNA interference in Drosophila, on the repressive properties of telomeric P elements. Seven out of the eight mutant alleles tested clearly impaired the repressive capacities of the two independent telomeric P insertions at 1A analyzed. P repression by P strains whose repressive capacities are not linked to the presence of P copies at 1A were previously found to be insensitive to Su(var)205; here, we show that they are also insensitive to aubergine mutations. These results strongly suggest that both RNA interference and heterochromatin structure are involved in the establishment of the P cytotype elicited by telomeric P elements, and reinforce the hypothesis that different mechanisms for repression of P elements exist which depend on the chromosomal location of the regulatory copies of P.Communicated by G. Reuter