pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

HP1 is a small nonhistone chromosomal protein of Drosophila melanogaster predominantly localized to the pericentric heterochromatin. We have shown previously that mutations in the HP1 coding sequences are associated with dominant suppression of heterochromatic position-effect variegation, and with recessive lethality. When fused to an Hsp70 heat shock gene promoter, the cDNA encoding HP1 supports the heat shock-inducible accumulation of HPI protein in transgenic flies; this cDNA construct complements the dominant suppression of position-effect variegation associated with mutations in the HP1 gene. Here, we report experiments demonstrating that the heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations in a heat shock-dependent fashion. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7–8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos.     

The Heterochromatin-Associated Protein Hp-1 Is an Essential Protein in Drosophila with Dosage-Dependent Effects on Position-Effect Variegation

Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.     

Third chromosome suppressor of position-effect variegation loci in Drosophila melanogaster

Summary As a result of a genetic analysis of 63 third chromosome suppressor mutations of position-effect variegation 12 different loci showing dominant suppression have been identified and their map positions determined. A compilcation of the genetic data available for each suppressor locus is given. The strong suppressor effects of the mutations have been quantified by measurements of white variegation inw ^m4h /w ^m4h ,w ^m4h /Y andw ^m4h /O flies. Mutant alleles of three loci were found in these studies to dominate over the strong enhancer effect of complete loss of the Y chromosome. Most of the identified loci suppressing position-effect variegation represent essential genetic funtions; only three loci represent nonessential functions. Mutations of two loci display recessive butyrate sensitivity and lethal interaction with the heterochromatic Y chromosome suggesting that these genes affect chromosomal condensation. Studies with deficiencies and triploids revealed that most of the loci represent haplo-abnormal suppressor functions. The use of the isolated mutant material for genetic, developmental and molecular studies of processes connected with gene inactivation in position-effect variegation is discussed.Dedicated to Prof. H.J. Becker on the occasion of his 6th birthday     

Butyrate sensitive suppressor of position-effect variegation mutations in drosophila melanogaster

Summary Mutations at a locus on chromosome II of D. melanogaster suppressing position-effect variegation mutations have been identified which display recessive butyrate sensitivity. Survival of homozygous mutant flies is significantly reduced on medium containing sodium n-butyrate. The butyrate sensitive suppressor mutations are further characterized by recessive female sterility and reduced survival of homozygotes. Complementation analysis showed their allelism. The locus of these mutations, Su-var (2) 1, has been localized to 40.5±0.2 and, by using interstitial duplications, to region 31CD on the cytogenetic map. Moreover, the mutant alleles of the Su-var (2) 1 locus display a lethal interaction with the heterochromatic Y chromosome. The presence or absence of a Y chromosome in males or females has a strong influence on the viability of homozygous or transheterozygous suppressor flies. All the genetic properties of Su-var (2) 1 mutants suggest strongly that this locus affects chromosome condensation.     

Functional dissection of the Drosophila modifier of variegation Su(var)3-7

An increase in the dose of the heterochromatin-associated Su(var)3-7 protein of Drosophila augments the genomic silencing of position-effect variegation. We have expressed a number of fragments of the protein in flies to assign functions to the different domains. Specific binding to pericentric heterochromatin depends on the C-terminal half of the protein. The N terminus, containing six of the seven widely spaced zinc fingers, is required for binding to bands on euchromatic arms, with no preference for pericentric heterochromatin. In contrast to the enhancing properties of the full-length protein, the N terminus half has no effect on heterochromatin-dependent position-effect variegation. In contrast, the C terminus moiety suppresses variegation. This dominant negative effect on variegation could result from association of the fragment with the wild type endogenous protein. Indeed, we have found and mapped a domain of self-association in this C-terminal half. Furthermore, a small fragment of the C-terminal region actually depletes pericentric heterochromatin from endogenous Su(var)3-7 and has a very strong suppressor effect. This depletion is not followed by a depletion of HP1, a companion of Su(var)3-7. This indicates that Su(var)3-7 does not recruit HP1 to heterochromatin. We propose in conclusion that the association of Su(var)3-7 to heterochromatin depends on protein-protein interaction mediated by the C-terminal half of the sequence, while the silencing function requires also the N-terminal half containing the zinc fingers.     

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.     

Carnitine suppression of position-effect variegation in Drosophila melanogaster

Carnitine is a well-known naturally occurring compound, very similar to butyrate, with an essential role in intermediary metabolism mainly at the mitochondrial level. Since butyrate inhibits the enzyme histone deacetylase and is capable of suppressing position-effect variegation in Drosophila melanogaster, we tested a further possible function of carnitine in the nucleus, using an assay for the suppression of position-effect variegation. We tested three physiological forms of carnitine (l-carnitine, l-propionylcarnitine, l-acetylcarnitine) for the ability to suppress two different chromosomal rearrangements, inducing variegation of the white ⁺ and brown ⁺ genes. The results show that the carnitine derivatives are capable of suppressing the position-effect variegation, albeit with different efficiencies. The carnitine derivatives interact lethally with Su-var(2)1 ⁰¹, a mutation that induces hyperacetylation of histones, whilst hyperacetylated histories accumulated in both the nuclei of HeLa cells and Drosophila polytene chromosomes treated with the same compounds. These results strongly suggest that the carnitine derivatives suppress position-effect variegation by a mechanism similar to that of butyrate. It is suggested that carnitines may have a functional role in the nucleus, probably at the chromatin level.     

Varied expression of a Y-linked P[w+] insert due to imprinting in Drosophila melanogaster.

During gametogenesis, a gene can become imprinted affecting its expression in progeny. We have used the expression of a Y-linked P[w+]YAL transposable DNA element as a reporter system to investigate the effect of parental origination on the expression of the w+ insert. Expression of w+ was greater in male progeny when the Y chromosome, harboring the insert, was inherited from the parental male rather than from the parental female. Imprinting was not due to a genetic background influence in the males, since the only difference among the males was the parental origin of the Y chromosome. It was also observed that the genetic background can affect imprinting, since w+ expression was also higher in males when the Y was derived from C(1)DX attached-X parental females rather than from C(1)RM attached-X parental females. Though the heterochromatic imprinting mechanism is unknown, a mutated Heterochromatin Protein 1 (HP1) gene, which is associated with suppression of position-effect variegation, increases expression of the w+ locus in the P[w+]YAL insert, indicating that HP1 may play a role in Y chromosome 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.     

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.     

Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila.

The Su(var)205 gene of Drosophila melanogaster encodes heterochromatin protein 1 (HP1), a protein located preferentially within beta-heterochromatin. Mutation of this gene has been associated with dominant suppression of position-effect variegation. We have cloned and sequenced the gene encoding HP1 from Drosophila virilis, a distantly related species. Comparison of the predicted amino acid sequence with Drosophila melanogaster HP1 shows two regions of strong homology, one near the N-terminus (57/61 amino acids identical) and the other near the C-terminus (62/68 amino acids identical) of the protein. Little homology is seen in the 5' and 3' untranslated portions of the gene, as well as in the intronic sequences, although intron/exon boundaries are generally conserved. A comparison of the deduced amino acid sequences of HP1-like proteins from other species shows that the cores of the N-terminal and C-terminal domains have been conserved from insects to mammals. The high degree of conservation suggests that these N- and C-terminal domains could interact with other macromolecules in the formation of the condensed structure of heterochromatin.     

The Effect of Modifiers of Position-Effect Variegation on the Variegation of Heterochromatic Genes of Drosophila Melanogaster

Dominant modifiers of position-effect variegation of Drosophila melanogaster were tested for their effects on the variegation of genes normally located in heterochromatin. These modifiers were previously isolated as strong suppressors of the variegation of euchromatic genes and have been postulated to encode structural components of heterochromatin or other products that influence chromosome condensation. While eight of the modifiers had weak or no detectable effects, six acted as enhancers of light (lt) variegation. The two modifiers with the strongest effects on lt were shown to also enhance the variegation of neighboring heterochromatic genes. These results suggest that the wild-type gene products of some modifiers of position-effect variegation are required for proper expression of genes normally located within or near the heterochromatin of chromosome 2. We conclude that these heterochromatic genes have fundamentally different regulatory requirements compared to those typical of euchromatic genes.     

Reduced DNA polytenization of a minichromosome region undergoing position-effect variegation in Drosophila

Molecular analysis of a Drosophila minichromosome, Dp(1;f)1187, revealed a relationship between position-effect variegation and the copy number reductions of heterochromatic sequences that occur in polytene cells. Heterochromatin adjacent to a defined junction with euchromatin underpolytenized at least 60-fold. Lesser reductions were observed in euchromatic sequences up to 103 kb from the breakpoint. The copy number changes behaved in all respects like the expression of yellow, a gene located within the affected region. Both copy number and yellow expression displayed a cell-by-cell mosaic pattern of reduction, and adding a Y chromosome, a known suppressor of variegation, increased both substantially. We discuss the possibility that changes in replication alter copy number locally and also propose an alternative model of position-effect variegation based on the somatic elimination of heterochromatic sequences.     

Ectopic HP1 promotes chromosome loops and variegated silencing in Drosophila

A transgene inserted in euchromatin exhibits mosaic expression when targeted by a fusion protein made of the DNA-binding domain of GAL4 and the heterochromatin-associated protein HP1. The silencing responds to the loss of a dose of the dominant modifiers of position-effect variegation Su(var)3-7 and Su(var)2-5, the locus encoding HP1. The genomic environs of the insertion site at 87C1 comprise the dispersed repetitive elements micropia and alphagamma. In the presence of the GAL4-HP1 chimera, the polytene chromosomes of this line form loops between the insertion site of the transgene and six other sections of chromosome 3R, as well as, rarely, with pericentric and telomeric heterochromatin. In contrast to the insertion site of the transgene at 87C, the six loop-forming sites in the euchromatic arm were each previously described as intercalary heterochromatin. Moreover, GAL4-HP1 tethering on one homologue trans-inactivates the reporter on the other. HP1, probably together with other partners, could thus facilitate the coalescence of dispersed middle repetitive sequences, and organize the heterochromatic structure responsible for the variegated silencing of nearby euchromatic genes.     

A Cytogenetic and Genetic Characterization of a Group of Closely Linked Second Chromosome Mutations That Suppress Position-Effect Variegation in Drosophila Melanogaster

Characterization of a group of dominant second chromosome suppressor of position-effect variegation (PEV) (Su(var)) mutants has revealed a variety of interesting properties, including: maternal-effect suppression of PEV, homozygous lethality or semilethality and male-specific hemizygous lethality, female infecundity, acute sensitivity to the amount of heterochromatin in the cell and sensitivity to sodium butyrate. Deficiency/duplication mapping and complementation tests have revealed that eight of the mutants define at least two genes in section 31 of the left arm of chromosome 2 and they suggest that a ninth corresponds to an additional nonessential Su(var) gene within or near this region. The effects of specific deficiencies and a duplication on PEV indicate that the expression of one or more of the Su(var) genes in this region of the chromosome is dose-dependent, i.e., capable of haplo-abnormal suppression and triplo-abnormal enhancement. Interestingly, the appearance of certain visible phenotypes among a subset of the mutants suggests that they may possess antimorphic properties. Our results are consistent with the hypothesis that two of these Su(var) genes encode structural components of heterochromatin. We also report that two previously isolated mutants located in 31E and 31F-32A act as recessive suppressors of PEV.