A series of mutable alleles of the white locus in Drosophila melanogaster

Summary The origin and phenotypes of a number of zeste mutant stocks with mutable white loci are described. Each newly arising form was lighter in eye color than the mutant it originated from. In each case the lighter pigmentation is believed to be due to an increase in genetic material in the proximal region of the white locus, the increase supposedly being the result of unequal crossing over. Some of the mutations which arose in the mutable stocks are reversions. They occurred in males as well as in homo- and heterozygous females. The reversions are believed to be due to a decrease in genetic material in the proximal region of the white locus. The decrease is assumed to be the result of intrachromosomal recombination. At least some of these events took place premeiotically. New mutants which originate frequently from mutable stocks are stable. In addition to the structure of the mutable white locus there is probably at least one still unknown factor which affects its mutability since the frequency of mutations arising in the mutable stocks decreases over the years.     

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.     

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.     

The genetics of a new mutable allele at the white locus in Drosophila melanogaster

Summary The genetics of a third case of high mutation frequency at the white locus in Drosophila melanogaster has been analyzed. The new mutable allele, w ^+u, mutates from a wild-type to a white-eyed phenotype in both males and females. The mutational event is 1) premeiotic, 2) not associated with crossingover, 3) sensitive to genetic modification, and 4) restricted to germinal tissue. The only mutants produced by w ^+u are deletions of the white locus. These deficiencies include subsites 4 and 5 of the white locus, but are cytologically unobservable. The mutable allele itself maps to subsite 4.The mutational properties of w ^+u are unlike those of the other highly mutable white alleles which have been interpreted in terms of phage-like controlling elements. Rather, the properties of w ^+u favor a model based on the premature termination of chromosome replication near the terminus of a replicon which leads to a chromosome deficient for the material between the point of premature termination and the end of the replicon.Supported by NIH predoctoral traineeship GM-150 and by NIH research grant GM-07428 to Dr. W. K. Baker.From a dissertation submitted to the Division of Biological Sciences of The University of Chicago in partial fulfillment of the requirements for the degree of Doctor of Philosophy.     

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.     

The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

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.     

Position Effects and Variegation Enhancers in an Autosomal Region of DROSOPHILA MELANOGASTER

A dominant eye color mutation was found associated with a third chromosome inversion broken distally at or near the karmoisin (kar) locus in 87C and proximally within centric heterochromatin. Suppressibility of the mutant phenotype by an extra Y chromosome indicated that this was an example of dominant position-effect variegation. When heterozygous with deficiencies uncovering the kar locus, this inversion chromosome was found to be lethal unless a region in 87EF was also deleted. Extra Y chromosomes rescued inversion/deletion heterozygotes, while removal of the Y chromosome from heterozygous males deficient for the region in 87EF was lethal. Thus, a variegating lethal lies near the breakpoint in 87C, and a wild-type gene that enhances its variegation lies in 87EF. Furthermore, deletion of the region in 87EF was found to strongly suppress white-mottled-4 (w^m4) variegation, while deletion of another region in 87BC suppressed less strongly. These results indicate that essential genes on autosomes are sensitive to position effects, and loci that enhance variegation, as defined by deficiency mapping, are very common.     

Maternal influence upon the V-type gene position effect in Drosophila melanogaster.

Summary We have studied the influence of various factors on the V-type position effect of the white gene transposed to heterochromatin as a result of different chromosome rearraugements in D. melanogaster. Variegation due to the white gene position effect is much weaker if the flies have received Dp(1;3)w^vco from parental males, and not females. The origin of the chromosome rearrangement does not have this influence in the case of T(1;4)w^m5 or has it to insignificant extent in the case of In(1)w^m4. The Y-chromosome in maternal genome strongly suppresses Dp(1;3)w^vco-induced variegation even in the progeny which has not received an extra Y-chromosome but only if this progeny gets Dp(1;3)w^vco from the same female. The low temperature (16° C) at which parental females have developed, considerably affects the position effect in the progeny with Dp(1;3)w^vco, whereas the temperature of males' development has no influence at all. The maternal temperature effect occurs also when Dp(1;3)w^vco has come down from the father, though it is stronger if the mother subjected to low temperature treatment bore the rearrangement. The influence of temperature seems to take effect at the final stages of oogenesis.The data obtained lead one to suppose that the influence of genotypic and external factors on variegation is passed to the next generation of flies in different ways. The direction of crosses and additional Y-chromosome heterochromatin in the maternal genome seem to affect variegation in the progeny through changes in the properties (structure) of the chromosome rearrangement expressing the position effect. As to the temperature of the mothers development, only a small part of its influence may be accounted for by the same mechanism, whereas most of the temperature influence seems to be passed on through other components of the nucleus or through the cytoplasm.     

The influence of heterochromatin, inversion-heterozygosity and somatic pairing on x-ray induced mitotic recombination in Drosophila melanogaster

Summary Mitotic recombination has been induced with X-rays in Drosophila melanogaster larvae and assayed later as twin mosaic spots in the adult eyes. When the X-chromosomes are marked with zeste and white and the third chromosomes with roughoid and sepia, the frequency of twin spots was about 20 times higher for the X-chromosome than for the third chromosome. The greater amount of heterochromatin in the X-chromosome was considered responsible for the difference.Experiments with different inversion heterozygotes support this interpretation. Euchromatic inversions of different lengths have, when heterozygous, little or no influence on the twin spot frequency. The shorter the heterochromatic segment between the kinetochore and the proxomal break point of the inversion the stronger is the reduction of the twin spot frequency.The heterozygotes for the long sc ⁸ and sc ^S1 inversions gave exceptionally low twin spot frequencies. It seems possible that potential twin spot daughter cells die after recombination because of genetic imbalance and/or lack of proper cell separation resulting from the persistence of the dikinetic chromosome elements.To test whether inaccurate somatic pairing in inversion heterozygotes could help explain the low twin spot frequencies in those of sc ⁸ and sc ^S1, neuroblast chromosomes were investigated. They show that chromosomal arrangement during metaphase is determined exclusively by the location of the kinetochore, which always points, irrespective of earlier somatic pairing, toward the center of the metaphase plate. It is possible that there is a lack of proper chromosome alignment at the X-ray sensitive stage for mitotic recombination.     

X-Y exchanges in males of Drosophila hydei carrying the w m Co duplication

Males carrying, inserted on their Y chromosome, a small fragment of X including the w ⁺ (and N ⁺) locus (white-mottled Confluens, w ^m Co), were crossed with the purpose of scoring exceptional progeny. Some of the male and female exceptions were progeny tested and further analysed. Among the various mechanisms which may lead to exceptional offspring, X-Y exchanges proved to occur with a not negligible frequency. The rate was 3%. Nondisjunction accounts for the bulk of the remaining exceptions and appears to be increased considerably in the presence of rearrangements on one or the other of the sex chromosomes.The w ^m Co fragment after having been switched from Y to X by some mechanism other than regular crossing over, may become retransferred to a normal Y chromosome, but at a rate below 3%.     

Cytogenetic analysis of chromosome region 89A of Drosophila melanogaster: isolation of deficiencies and mapping of Po, Aldox-1 and transposon insertions

Summary We have initiated a cytogenetic analysis of chromosome region 89A of Drosophila melanogaster by isolating a set of radiation-induced mutations causing loss of function of P[(w)B]1-1, a transposon bearing the white locus inserted in 89A. Complementation tests and cytological examination of these chromosomes identified four new deficiencies (Df(3R)Po ², Df(3R)Po ³, Df(3R)Po ⁴ and Df(3R)c(3)G ² ). The new deficiencies and three previously identified deficiencies (Df(3R)sbd ²⁶, Df(3R)sbd ⁴⁵ and Df(3R)sbd ¹⁰⁵) were tested for the ability to complement mutations in the enzyme loci Po and Aldox-1, the indirect flight muscle genes Tm2 and act88F, the morphological mutations jvl, sbd ² and Sb, the vital loci srp, pnr and mor, and a newly described vital locus l(3)89Aa. We also used linkage analysis to determine the order and relative positions of P[(w)B]1-1 and an independent transposon insertion, P[w⁺]21, with respect to cv-c, Po, Aldox-1 and sbd ². Cytological examination of the deficiencies and analysis of the transformed lines by in situ hybridization permits the correlation of genetically defined regions with specific polytene chromosome bands. A revised cytogenetic map of the 8817–8913 region is presented.     

H3S10 phosphorylation by the JIL-1 kinase regulates H3K9 dimethylation and gene expression at the white locus in Drosophila

The JIL-1 kinase is a multidomain protein that localizes specifically to euchromatin interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase. Genetic interaction assays have suggested that the function of the epigenetic histone H3S10ph mark is to antagonize heterochromatization by participating in a dynamic balance between factors promoting repression and activation of gene expression as measured by position-effect variegation (PEV) assays. Interestingly, JIL-1 loss-of-function alleles can act either as an enhancer or indirectly as a suppressor of w^m4 PEV depending on the precise levels of JIL-1 kinase activity. In this study, we have explored the relationship between PEV and the relative levels of the H3S10ph and H3K9me2 marks at the white gene in both wild-type and w^m4 backgrounds by ChIP analysis. Our results indicate that H3K9me2 levels at the white gene directly correlate with its level of expression and that H3K9me2 levels in turn are regulated by H3S10 phosphorylation.     

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w ^67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w ^67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. P. Georgiev     

Molecular analysis of large transposable elements carrying the white locus of Drosophila melanogaster

A large transposable element (TE) comprising the white-apricot and roughest genes has been found to transpose to well over a hundred sites scattered over the Drosophila genome. We report the cloning of the essential parts of several TEs. TE98 and TE28 sequences were cloned by `walking' along the chromosome from the previously cloned heatshock genes. The ends of the TEs are characterized by dispersed repetitive elements belonging to the foldback (FB) family. FB elements are also associated with two independently isolated transposable elements originating from the white locus, Tp w^c-1 and Tp w^+IV. The strong correlation between FB elements and large composite transposons suggests that a pair of these elements can mobilize large intermediary DNA segments. One particular FB family member, FB-NOF, is associated with TE28, the white-crimson (w^c) mutant, the w^c-derived Tp w^c-1 and probably also with Tp w^+IV. A unique sequence located close to the white end of TE28 was used to clone the borders of TE77 and the surrounding sequences in the bithorax region, indicating that the TE can be used as a probe for gene isolation. Some evolutionary implications of the large composite transposons are discussed.     

Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus

Summary The circadian rhythm phenotypes of eight chromosome aberrations with a breakpoint in the region of the per locus (3B1-2) were analyzed. Two duplications and five deficiencies with a 3B1-2 breakpoint produce either a wild-type or an arrhythmic clock phenotype while one translocation with a 3B1-2 breakpoint, T(1;4)JC43, produces locomotor-activity rhythms with either very-long period (31–39 h), rhythms that grade into arrhythmicity, or completely arrhythmic phenotypes. This is a unique phenotype that had not previously been observed for mutants at the per locus. An extensive complementation analysis of 3B1-2 chromosome aberrations and per mutant alleles provided no compelling evidence for genetic complexity at the per locus. This is in contrast to the report of Young and Judd (1978). Analysis of both the locomotor-activity and eclosion phenotypes of 3B1-2 chromosome aberrations did not uncover differences in the genetic control of these two rhythms. The clock phenotypes of 3B1-2 chromosome aberrations, the three per mutant alleles, and per ⁺ duplications suggest that mutations at the per locus shorten, lengthen, or eliminate periodicity by respectively increasing, decreasing, or eliminating per activity.