Influence of deficiency of the histone gene-containing 38B-40 region on X-chromosome template activity and the white gene position effect variegation in Drosophila melanogaster.

Summary The deficiency of the 38B-40 region containing histone genes in one of the 2nd chromosomes of D. melanogaster triploid intersexes increases the template activity of X-chromosomes both in vivo and in vitro without noticeably affecting autosome activity. This deficiency in the heterozygous state inhibits the variegated position effect of the white gene in the T(1;3)w ^vcotranslocation in diploid females and males, but not affect their rate of development. The variegation suppressor Su(var)hg-1 not only suppress the gene position effect in diploid flies, but also increases the template activity of X-chromosomes in triploid intersexes.The results are discussed with respect to the dependence of gene activity on the structure of chromosomes (density of DNP packing).     

Bestimmung des Heterochromatisierungsstadiums beim white-Positionseffekt mittels r?ntgeninduzierter mitotischer Rekombination in der Augenanlage von Drosophila melanogaster

Summary Position-effect variegation of eye pigmentation in the examined Dp(1;3)N ^264-58 females is due to an insertion of a X-chromosome section including the white-locus into the proximal heterochromatic region of the third chromosome. The light and dark pigmented areas have a cell lineage basis (Fig. 2). Flies bearing the w ⁺-duplication had two X-chromosomes marked with w ^a lz ⁵⁰ e and w ^a rb rux ² respectively (Fig. 3). X-ray induced mitotic recombination in presumptive eye cells of larvae resulted in w ^a lz ^50e /w ^a rb rux ² twin mosaic spots in the adult eyes. After young larvae were treated twin spots appeared, which had one partner light colored and one dark. Such combinations were rarely found after older larvae were treated. Treatment of young larvae in addition produced twin spots with one or both partners variegated (Figs. 5 and 6). Sometime after the stage at which younger larvae were treated and before the stage at which older larvae were treated the translocated w ⁺-gene in each cell was determined for function or no function. As a result the progeny of each of these cells synthesized pigment or not during the pupal stage. At a temperature of 25.5° C the developmental phase during which determination, i.e. heterochromatization of the white gene, takes place, begins not earlier than 39 hours after egg laying and ends about 8 hours later (Fig. 7). In females heterozygous for the short arm of the heterochromatic Y-chromosome linked distally to the X-chromosome (Y ^S X/X) one twin spot partners is homozygous for this arm (Y ^SX/Y^S X), the other lacks it (X/X; Fig.4a). The Y ^SX/Y^S X-partner were more frequently dark pigmented than the X/X-partners (Tables 3 and 4). This shows that heterochromatization of the translocated w ⁺-genes is markedly influenced by the genotype of the single cell. When two genotypes with varied amounts of heterochromatin were compared (Fig. 4) no difference in the phases of heterochromatization could be observed (Table 5). Therefore, when position-effect variegation is modified by varying the amount of heterochromatin in the genome the modification is probably not due to a shift in the phase of heterochromatization.

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Vorgelegt von E. Hadorn     

Studying Trans-Effects of Modifiers on the Position-Effect Variegation in a Set of Euchromatin–Heterochromatin Rearrangements in Drosophila melanogaster

The effects of suppressors of position-effect variegation were studied in a set of euchromatin–heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl.The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin–heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.     

A marked ring-Y-chromosome in Drosophila hydei with a new type of white variegation

A ring-Y chromosome, R(Y)w ^m, of D. hydei is described which carries a complete set of fertility genes, a NOR region and a small X-chromosomal insertion (w ^m), which may be used as a marker. The ring has been characterized by various staining techniques. It was derived from a w ^mCo Y chromosome by X-ray treatment of spermatocytes. Its mode of origin allows to fix the gene order in the distal region of the long arm of the w ^mCoY chromosome. The white ⁺ gene included in the ring shows a new type of position-effect variegation which is described and discussed in the context of an earlier hypothesis on a dual function of the white locus.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila

Variations in compaction of chromosomal material of the rearrangements Dp(1;f) 1337, Dp(1;f) R, Dp(1;1)pn2b, and T(1;4)w ^m258-21, which display an extended position effect, were characterized. Morphological changes found in these rearangements were assigned to two major types: (i) continuous compaction, in which bands and interbands located distal to the eu/heterochromatin junction fuse into one compacted block of chromatin. The extent of compaction is increased by enhancers of position effect (low temperature, removal of the Y or 2R chromosome heterochromatin). In extreme cases compaction extends over dozens of bands. (ii) Discontinuous compaction, in which at least two zones of compaction separated by morphologically normal zones can readily be identified. As a result, some regions located at a greater distance from heterochromatin may be compacted more frequently than others than map nearer to it. A few regions (1D, 2B1-12, 2D) were shown to be most frequently compacted in all rearrangements investigated. The 2B13-18, 2C1-2, 2E, and 2F regions exhibited the lowest frequencies of compaction. Compaction of the zone containing the 2B1-12 bands is always accompanied by inactivation of the ecs locus, which maps in the 2B3-5 puff. At the same time the 2C1-2 and 2E bands located nearer to the breakpoint can retain normal morphology and puffing in response to ecdysterone. The results are interpreted as morphological manifestations of the discontinuity of the spreading effect.by W. Beermann     

Nurse cell polytene chromosomes of Drosophila melanogaster otu mutants: Morphological changes accompanying interallelic complementation and position effect variegation

Combinations of certain mutant alleles of the ovarian tumor gene permit the production of viable eggs. Two alleles that behave in this way are otu⁷ and otu¹. Females homozygous for either allele are sterile, and their ovarian nurse cells (NC) contain giant polytene chromosomes of various morphologies. Fertile flies (otu⁺ / otu⁺, otu⁻ / otu⁷, otu⁺ / otu¹¹) have endopolyploid nurse cells with typical dispersed chromosomes. Fertile hybrids (otu⁷ / otu¹¹) produce large numbers of polytene chromosomes comparable to, and often larger than, classic salivary gland (SG) chromosomes. Therefore, these otu hybrids provide a unique system for studying, at the chromosomal level, the activation and expression of genes functioning during oogenesis. The otu gene encodes a long and a short isoform. The normal long isoform appears to be responsible for the dispersion of chromosomes during the endomitotic DNA replications occurring in ovarian NCs. The genetic inactivation of euchromatic genes placed next to pericentric heterochromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding chromosome regions. A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SG cells and NCs was made using the Dp(1;1)pn2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp (1;1) pn2b rearrangement were 92.6% vs. 15.8%, respectively; for Dp(1;f) 1337, these values were 56.8% vs. 9.7%. Therefore heterochromatin belonging to germ line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterachromatin from somatic chromosomes. Dev. Genet. 20:163–174. 1997. © 1997 Wiley-Liss, Inc.     

Modification of position-effect variegation by competition for binding to Drosophila satellites

white-mottled (w^m4) position-effect variegation (PEV) arises by translocation of the white gene near the pericentric AT-rich 1.688 g/cm³ satellite III (SATIII) repeats of the X chromosome of Drosophila. The natural and artificial A•T-hook proteins D1 and MATH20 modify w^m4 PEV in opposite ways. D1 binds SATIII repeats and enhances PEV, presumably via a recruitment of protein partners, whereas MATH20 suppresses it. We show that D1 and MATH20 compete for binding to identical sites of SATIII repeats in vitro and that conditional MATH20 expression results in a displacement of D1 from pericentric heterochromatin in vivo. In the presence of intermediate levels of MATH20, we show that this displacement becomes selective for SATIII repeats. These results strongly suggest that the suppression of w^m4 PEV by MATH20 is due to a displacement of D1 from its preferred binding sites and provide additional support for a direct role of D1 in the assembly of AT-rich heterochromatin.     

Functional properties of the heterochromatic sequences inducing w m4 position-effect variegation in Drosophila melanogaster

In position-effect variegation euchromatic genes are brought into the vicinity of heterochromatic sequences as a result of chromosomal rearrangements. This results in the inactivation of these genes in a proportion of cells causing a variegated phenotype. Tartof et al. (1984) have shown that the flanking heterochromatin in the w ^m4 variegating rearrangement in Drosophila melanogaster is homologous to the Type I inserts found in some portions of the rDNA repeats. We have studied the functional properties of these sequences using 51 revertant chromosomes, several variant lines of w ^m4 , strong enhancer mutations of position-effect variegation and X heterochromatin deletions. Our results suggest an array of tandemly repeated sequences showing additive effects and probably subject to magnification and reduction in number. Since only 3 of the 51 revertants isolated do not show variegation if strong enhancer mutations are introduced, only a very short sequence must be essential for the induction of white gene inactivation in w ^m4 . This suggests that the heterochromatic junction itself is sufficient to initiate the variegation of an adjacent gene. Parental source as well as paternal effects on the activity of these sequences have been detected. Revertant chromosomes of w ^m4 can be found after P-directed mutagenesis in hybrid dysgenic crosses suggesting mobile genetic elements at the breakpoints of inversion w ^m4 . These results are discussed with respect to the structural basis of positioneffect variegation as well as the function of certain heterochromatic sequences.     

Euchromatic inversions causing mosaic gene expression in Drosophila hydei: A study of forked and Zebra

In D. hydei two new mutants, In(1)f³ and IN(5)Z, show obvious mosaic gene expression. Their phenotypic expression is susceptible to the breeding temperature and to the addition of a supernumerary Y chromosome to the chromosome set. In this respect the mutants resemble standard cases of position-effect variegation based on the action of heterochromatin. However, since neither centromeric nor sex chromosomal heterochromatin apparently are involved, the mutations point to a new type of variegation provoked by euchromatic sections. The mosaic patterns of these mutants, in particular those of In(1)f³, will be described.     

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 mutable white-inversion in Drosophila melanogaster

Summary From a zeste mutant stock with a mutable white locus a new mutant (z w ^w ) was isolated. It has a white-eyed phenotype and a short X-chromosome inversion (In(1)w ^w ) which extends from salivary chromosome bands 3B2-C1 to 4B4-C1. In giant chromosomes of heterozygotes the inversion is unusually tightly paired. Probably because of this intimate pairing the recombination frequencies for regions near the inversion are not decreased in comparison to those for structurally normal chromosomes. The inversion chromosome is mutable. The mutations which arise have pigmented eyes and can be subdivided into two groups. One group is characterized by a re-inversion to normal chromosome structure. The mutability of the white locus appears to be independent of the inversion and reinversion. The process of reinversion is discussed.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor ^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann     

An analysis of white-mottled mutants inDrosophila hydei,with observations on X-Y exchanges in the male

Chromosomes and phenotypes of four different sex-linkedwhite-mottled mutants of the position-effect variogation type were studied. Three mutants (w ^m1,w ^m2,w ^m3) are X-chromosomal rearrangements which shift the w⁺ locus into a position close to heterochromatin, but which have different ouchromatic and heterochromatic breaks. The fourth, a spontaneous derivative ofw ^m1, is an insertional duplication of part of the X chromosome, including thew ⁺ andN ⁺loci. The duplicated segment is inserted into the distal part of the long arm of the heterochromatic Y chromosome. It is designated,w ^m CoY, orXw ^m Co when transferred to the X chromosome.Three chromosomal types (w ^m1,w ^m CoY) and (Xw ^m Co) having the same cuchromatic break near thew ⁺ locus, cause large-spotted eyes whereas two others (w ^m2,w ^m3) produce a popper-and-salt type of mottling. From the position of the various eu- and heterochromatic breaks, it appears that the distance of thew ⁺ locus to the point of reunion with heterochromatin, rather than the amount or type of adjoining heterochromatin, dietates the phenotypic action of the displacedw ⁺ locus, in the sense of a spreading effect on two proposed functional subunits within thew ⁺ locus.The pigmentation background against which the mottling effect is produced, i.e., a givenw-allele with its characteristic colour, or other eye colour mutations, does not seem to affect the type of mottling. Drosopterins and ommochromes react in the same way to modifing factors like temperature and supernumerary Y chromosomes. Two mutants (w ^m2 andw ^m CoY) while reacting in the same manner to Y chromosomes showed an opposite temperature response.By exchange between the heterochromatin of the Y and X chromosome inw/w ^m CoY males thew ^m Co duplication was transferred between the sex chromosomes with a certain regularity. It is not yet known wether the exchanges are mitotic or meiotic in origin but their heterochromatic nature has been demonstrated cytologically.     

The action of the Notch locus in Drosophila hydei

The phenotypic effects of different doses of the dominant, sex-linked mutant Notch (N) and its wildtype allele (N ⁺) were studied in Drosophila hydei, N being lethal in homozygous or hemizygous condition. Various dosage combinations were made by using N ⁺ N and N ⁺ N ⁺ attached-X chromosomes as well as X and Y N ⁺-duplication chromosomes (w ^mCoY, Xw^mCo,and DpCo ^Nt). The N mutant used, N ⁶⁸, is associated with a small inversion: In (I) N ⁶⁸.The wing phenotype was found to depend solely on the number of functional (N ⁺) alleles present, irrespective of the dose of N. Females with a single dose of N ⁺ are phenotypically Notch, females with three or four doses of N ⁺ show a Confluens wing phenotype. The latter occurs in varying degrees of expression which seem to be correlated with the relative amounts of sex-chromosomal heterochromatin present. In males the N ⁺ locus behaves as a dosage compensated locus either on the X or the Y chromosome.In the w ^mCo (w⁺N⁺) duplication, the w ⁺ locus shows variegation when placed over white, whereas N ⁺ placed over N ⁶⁸ does not. The former being situated closer to the heterochromatin in this aberration, this is consistent with the idea of gene inhibition by heterochromatin but at the same time would imply a very limited spreading effect.     

Pleiotropic effects associated with the deletion of heterochromatin surrounding rDNA on the X chromosome of Drosophila

In Drosophila melanogaster X chromosome heterochromatin (Xh) constitutes the proximal 40% of the X chromosome DNA and contains a number of genetic elements with homologous sites on the Y chromosome, one of which is well defined, namely, the bobbed locus, the repetitive structural locus for the 18S and 28S rRNAs. This report presents the localisation of specific repeated DNA sequences within Xh and the employment of this sequence map in constructing new chromosomes to analyse the nature of the heterochromatin surrounding the rDNA region. Repeated sequences were located relative to inversion breakpoints which differentiate Xh cytogenetically. When the rDNA region was manipulated to be in a position in the chromosome so that it was without the Xh which normally surrounds it, the following obser-vations were made, (i) The rDNA region of Xh is intrinsically hetero-chromatic, remaining genetically active and yet possessing major heterochromatic properties even in the absence of the flanking heterochromatin regions, (ii) The size of the deletion removing the portion of Xh normally located distal to the rDNA region affected the dominance relationship between the X and Y nucleolar organizers (activity/endoreduplication assayed in male salivary glands). The X rDNA without any flanking heterochromatin was dominant over Y rDNA while the presence of some Xh allowed both the X and Y rDNA to be utilized, (iii) Enhancement of the position effect variegation on the white locus was demonstrated to occur as a result of the Xh deletions generated. EMS mutagenesis studies argue that the regions of Xh flanking the rDNA region contain no vital loci despite the fact that they strongly effect gene expression in some genotypes. This is consistent with early studies using X-ray mutagenesis (Lindsley et al., 1960). The pleiotropic effects of deleting specific regions of Xh is discussed in relation to the possible influence of heterochromatin on the organisation of the functional interphase nucleus.     

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.