Fine-Structure Analysis and Genetic Organization at the Base of the X Chromosome in DROSOPHILA MELANOGASTER

Genetic organization at the base of the X chromosome was studied through the analysis of X-ray-induced deficiencies. Deficiencies were recovered so as to have a preselected right end "anchored" in the centric heterochromatin to the right of the su(f) locus. "Free" ends of deficiencies occurred at any of 22 intervals in Section 20 and in the proximal portion of Section 19 of Bridges' (1938) polytene chromosome map. The distribution of 130 such free ends of deficiencies induced in normal, In(1)sc ⁸, and In(1)w^m4 chromosomes suggests that on the single section level, genes are flanked by "hot" or "cold" sites for X-ray-induced breaks, and that occurrence of the hot spots is dependent on their interaction with the fixed-end sites in the centric heterochromatin. In the light of these results, it is argued that long heterochromatic sequences separate the relatively few genes in Section 20, and thus endow it with several characteristics typical of heterochromatic regions. Section 20 is considered to be a transition region between the mostly heterochromatic and mostly euchromatic regions of the X chromosome; the differences between them are suggested as being merely quantitative.     

Sex Chromosome Translocations in the Evolution of Reproductive Isolation

Haldane's rule states that in organisms with differentiated sex chromosomes, hybrid sterility or inviability is generally expressed more frequently in the heterogametic sex. This observation has been variously explained as due to either genic or chromosomal imbalance. The fixation probabilities and mean times to fixation of sex-chromosome translocations of the type necessary to explain Haldane's rule on the basis of chromosomal imbalance have been estimated in small populations of Drosophila melanogaster. The fixation probability of an X chromosome carrying the long arm of the Y(X·Y^L) is approximately 30% greater than expected under the assumption of no selection. No fitness differences associated with the attached Y^L segment were detected. The fixation probability of a deficient Y chromosome is 300% greater than expected when the X chromosome contains the deleted portion of the Y. It is suggested that sex-chromosome translocations may play a role in the establishment of reproductive isolation.     

Male-Sterilizing Interactions between Duplications and Deficiencies for Proximal X-Chromosome Material in DROSOPHILA MELANOGASTER

The genetic limits of sixty-four deficiencies in the vicinity of the euchromatic-heterochromatic junction of the X chromosome were mapped with respect to a number of proximal recessive lethal mutations. They were also tested for male fertility in combination with three Y chromosomes carrying different amounts of proximal X-chromosome-derived material (B^SYy⁺, y⁺Ymal¹²⁶ and y⁺Ymal⁺). All deficiencies that did not include the locus of bb and a few that did were male-fertile in all male-viable Df(1)/Dp(1;Y) combinations. Nineteen bb deficiencies fell into six different classes by virtue of their male-fertility phenotypes when combined with the duplicated Y chromosomes. The six categories of deficiencies are consistent with a formalism that invokes three factors or regions at the base of the X, one distal and two proximal to bb, which bind a substance critical for precocious inactivation of the X chromosome in the primary spermatocyte. Free duplications carrying these regions or factors compete for the substance in such a way that, in the presence of such duplications, proximally deficient X chromosomes are unable to command sufficient substance for proper control of X-chromosome gene activity preparatory to spermatogenesis. We conclude that there is no single factor at the base of the X that is required for the fertility of males whose genotype is otherwise normal.     

Cytogenetic Analysis of a Segment of the Y Chromosome of DROSOPHILA MELANOGASTER

Males carrying a large deficiency in the long arm of the Y chromosome known to delete the fertility gene kl-2 are sterile and exhibit a complex phenotype: (1) First metaphase chromosomes are irregular in outline and appear sticky; (2) spermatids contain micronuclei; (3) the nebenkerns of the spermatids are nonuniform in size; (4) a high molecular weight protein ordinarily present in sperm is absent; and (5) crystals appear in the nucleus and cytoplasm of spermatocytes and spermatids. In such males that carry Ste⁺ on their X chromosome the crystals appear long and needle shaped; in Ste males the needles are much shorter and assemble into star-shaped aggregates. The large deficiency may be subdivided into two shorter component deficiencies. The more distal is male sterile and lacks the high molecular weight polypeptide; the more proximal is responsible for the remainder of the phenotype. Ste males carrying the more proximal component deficiency are sterile, but Ste ⁺ males are fertile. Genetic studies of chromosome segregation in such males reveal that (1) both the sex chromosomes and the large autosomes undergo nondisjunction, (2) the fourth chromosomes disjoin regularly, (3) sex chromosome nondisjunction is more frequent in cells in which the second or third chromosomes nondisjoin than in cells in which autosomal disjunction is regular, (4) in doubly exceptional cells, the sex chromosomes tend to segregate to the opposite pole from the autosomes and (5) there is meiotic drive; i.e., reciprocal meiotic products are not recovered with equal frequencies, complements with fewer chromosomes being recovered more frequently than those with more chromosomes. The proximal component deficiency can itself be further subdivided into two smaller component deficiencies, both of which have nearly normal spermatogenic phenotypes as observed in the light microscope. Meiosis in Ste ⁺ males carrying either of these small Y deficiencies is normal; Ste males, however, exhibit low levels of sex chromosome nondisjunction with either deficient Y. The meiotic phenotype is apparently sensitive to the amount of Y chromosome missing and to the Ste constitution of the X chromosome.     

An antagonism between the telomeric and polycomb-dependent chromatin at the end of terminally deleted <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> chromosomes

The results obtained at our laboratory during several last years suggest that a specific telomeric chromatin is formed at the terminal regions of Drosophila melanogaster DNA of 4–5 kb in length. In this work, we have studied how the telomeric chromatin influences the Polycomb-dependent repression. Many Polycomb proteins are involved in the formation of subtelomeric chromatin and inhibit the expression of a transgene inserted into it. However, the question on a positive or negative effect of telomeric chromatin on the assembly of subtelomeric protein complexes is yet open. Using a model system, in which a P element inserted upstream of the yellow gene promoter and located at the end of a terminally deleted chromosome can serve as a binding site for Pc-G proteins in the presence of ph ^p1 mutant allele, we have shown that a specific structure of the telomeric chromatin negatively influences the formation of Polycomb-dependent repression complex. These results suggest an antagonism between the telomeric and subtelomeric (Pc-G-dependent) chromatins.     

Chromosomal segregational mechanisms in ant-lions (Myrmeleontidae,Neuroptera)

In a single male specimen of Myrmeleon mexicanum Banks the sex chromosomes, normally X and Y, were replaced by what appeared to be X¹X² and Y. These segregated as expected on that interpretation in only half of the spermatocytes — in the other half, one X and the Y segregated from the other X. This atypical segregation is explicable on the assumption that one of the supposed Xs is a supernumerary, not a sex chromosome, and the diploid complement of the male comprises six pairs of autosomes plus a supernumerary and the X and Y sex chromosomes. The orientation of the X chromosomes at first metaphase was variable: kinetochoric activity may be localized midway the length of the chromosome, as in gonial mitosis, or terminally. Comparative study of three congeneric species, seven of Brachynemurus, one of Psammoleon, and one of Vella showed normal segregation in all, and no evidence for secondary kinetochoric activity. In nine of the species studied one pair of autosomes was unconjoined at first metaphase in 0.3%–1.2% of primary spermatocytes. These autosomes segregated precociously with the sex chromosomes in the central unit of the spindle. In one exceptional male of Brachynemurus hubbardi Currie all first meiotic metaphases showed this behavior, and a compound X¹X²/Y¹Y² system was thus simulated. Bivalent formation replaced distance segregation of sex chromosomes in 0.4%–3.2% of the spermatocytes in seven of the thirteen species studied. These sex-bivalents frequently displayed partial or complete failure in congression.     

End-to-End Association of <Emphasis Type="Italic">X</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> Chromosomes in Mouse Meiosis

ACCORDING to the hypothesis of Crew and Koller¹ and Koller and Darlington², there are homologous segments in the X and Y chromosomes of the mouse and other mammals. The homologous regions in the mouse were believed to be localized in the extremely short arms proximal to the kinetochores. The end-to-end association at meiosis was thought to be the result of the formation of a chiasma between these homologous regions³. Electron microscopy revealed a short synaptonemal complex in mouse meiotic cells⁴. However, partial sex linkage has never been demonstrated in the mouse⁵ and other authors^6–10 believe that the X and Y chromosomes associate only by connexion between the chromosome ends furthest from the centromeres.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

Cytological identification of two X-chromosome types in the wood lemming (Myopus schisticolor)

In the wood lemming (Myopus schisticolor) three genetic types of sex chromosome constitution in females are postulated: XX, X^*X and X^*Y (X^*=X with a mutation inactivating the male determining effect of the Y chromosome). Males are all XY. It is shown in the present paper that the two types of X chromosomes, X and X^*, exhibit differences in the G-band patterns of their short arms. In addition, it was demonstrated in unbanded chromosomes that the short arm in X^* is shorter than in X. The origin of these differences is still obscure; but they allow to identify and to distinguish the individual types of sex chromosome constitution, as of XX versus X^*X females and of X^*Y females versus XY males, on the basis of G-banded chromosome preparations from somatic cells.     

X-Y Exchange and the Coevolution of the X and Y Rdna Arrays in Drosophila melanogaster

The nucleolus organizers on the X and Y chromosomes of Drosophila melanogaster are the sites of 200-250 tandemly repeated genes for ribosomal RNA. As there is no meiotic crossing over in male Drosophila, the X and Y chromosomal rDNA arrays should be evolutionarily independent, and therefore divergent. The rRNAs produced by X and Y are, however, very similar, if not identical. Molecular, genetic and cytological analyses of a series of X chromosome rDNA deletions (bb alleles) showed that they arose by unequal exchange through the nucleolus organizers of the X and Y chromosomes. Three separate exchange events generated compound X·Y ^L chromosomes carrying mainly Y-specific rDNA. This led to the hypothesis that X-Y exchange is responsible for the coevolution of X and Y chromosomal rDNA. We have tested and confirmed several of the predictions of this hypothesis: First, X· Y^L chromosomes must be found in wild populations. We have found such a chromosome. Second, the X·Y^L chromosome must lose the Y^L arm, and/or be at a selective disadvantage to normal X⁺ chromosomes, to retain the normal morphology of the X chromosome. Six of seventeen sublines founded from homozygous X·Y^Lbb stocks have become fixed for chromosomes with spontaneous loss of part or all of the appended Y^L. Third, rDNA variants on the X chromosome are expected to be clustered within the X⁺ nucleolus organizer, recently donated (" Y") forms being proximal, and X-specific forms distal. We present evidence for clustering of rRNA genes containing Type 1 insertions. Consequently, X-Y exchange is probably responsible for the coevolution of X and Y rDNA arrays.     

Mapping a mutator,mu2, which increases the frequency of terminal deletions in Drosophila melanogaster

A mutator, mu2, in Drosophila melanogaster has been identified recently that potentiates the recovery of terminal deficiencies. The deleted chromosomes behave as if they had been capped; that is, they are protected from degradation and from fusion with other chromosome fragments. The mutator maps near the telomere on the left arm of chromosome 3. Using the selectable marker Aprt, 150 deficiencies for region 62 of the cytological map have been recovered. These deficiencies identify the map position of mu2 as 62B11-C1. A yeast artificial chromosome spanning this region has been subcloned into lambda phage, and the positions of deficiency breakpoints on either side of the mu2 gene have been identified within the subclones. These positions limit the location of the left end of the gene to a 23 kb region. In the course of these experiments, three additional, presumptive mutant alleles were identified, suggesting that other mutator alleles remain undiscovered in many standard laboratory stocks.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

<Emphasis Type="Italic">X</Emphasis> Chromosome Inactivation in Cells from an Individual heterozygous for Two <Emphasis Type="Italic">X</Emphasis>-Linked Genes

THE Lyon hypothesis of X chromosome inactivation predicts that in mammalian females, somatic cells are mosaic with respect to whether the active X chromosome is of maternal or paternal origin and that this chromosomal mosaicism is heritable somatically¹. Studies of cell clones derived from females who were heterozygous for genes at one of several X-linked loci^2–6 have provided good evidence for such mosaicism. Proof that only one of the two X chromosomes, however, is active in any given cell rests on the demonstration that the cell or its clone expresses all of the X-linked genes from one parent and none from the other parent. For this purpose it is useful to examine cloned cells from female subjects for genetic markers representing allelic genes at two or more of the parental loci. This study was undertaken to determine whether genes at the X-linked loci for glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase (PGK) are consistently expressed in the eis position in cloned cells as would be expected from a single parental contribution.     

Healing of Broken Linear Dicentric Chromosomes in Yeast

In yeast, meiotic recombination between a linear chromosome III and a haploid-viable circular chromosome will yield a dicentric, tandemly duplicated chromosome. Spores containing apparently intact dicentric chromosomes were recovered from tetrads with three viable spores. The spore containing the dicentric inherited URA3 (part of the recombinant DNA used to join regions near the ends of the chromosome into a circle) as well as HML, HMR and MAL2 (located near the two ends of a linear but deleted from the circle). The Ura⁺ Mal⁺ colonies were highly variegated, giving rise to as many as seven distinctly different stable ("healed") derivatives, some of which were Ura⁺ Mal ⁺, others Ura⁺ Mal^- and others Ura ^- Mal⁺. The colonies were also sectored for five markers (HIS4, LEU2, CRY1, MAT and THR4) initially heterozygous in the tandemly duplicated dicentric chromosome.—Southern blot and genetic analyses have demonstrated that these stable derivatives arose from mitotic break-age of the dicentric chromosome, followed by one of several different healing events. The majority of the stable derivatives contained circular or linear chromosomes apparently resulting from homologous recombination between a broken chromosome end and a homologous region on the other end of the original dicentric duplicated chromosome. A smaller proportion of events resulted in apparently uniquely healed linear chromosomes in which the broken chromosome acquired a new telomere. In two instances we recovered chromosome III partially duplicated with a novel right end. We have also found one derivative that had also experienced rearrangement of repeated DNA sequences found adjacent to yeast telomeres.     

Cytogenetic Analysis and Chromosomal Characteristics of the Polymorphic 18S rDNA of Haliotis discus hannai from Fujian,China

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)₃ displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.     

X-4 Translocations and Meiotic Drive in Drosophila melanogaster Males: Role of Sex Chromosome Pairing

Males carrying certain X-4 translocations exhibit strongly skewed sperm recovery ratios. The X^P4^D half of the translocation disjoins regularly from the Y chromosome and the 4^PX^D half disjoins regularly from the normal 4. Yet the smaller member of each bivalent is recovered in excess of its pairing partner, apparently due to differential gametic lethality. Chromosome recovery probabilities are multiplicative; the viability of each genotype is the product of the recovery probability of its component chromosomes. Meiotic drive can also be caused by deficiency for X heterochromatin. In(1)sc^4Lsc^8R males show the same size dependent chromosome recoveries and multiplicative recovery probabilities found in T(1;4)B^S males. Meiotic drive in In(1)sc^4Lsc^8R males has been shown to be due to X-Y pairing failure. Although pairing is regular in the T(X;4) males, the striking phenotypic parallels suggest a common explanation. The experiments described below show that the two phenomena are, in fact, one and the same. X-4 translocations are shown to have the same effect on recovery of independently assorting chromosomes as does In(1)sc^4Lsc^8R. Addition of pairing sites to the 4^PX^D half of the translocation eliminates drive. A common explanation—failure of the distal euchromatic portion of the X chromosome to participate in X:Y meiotic pairing—is suggested as the cause for drive. The effect of X chromosome breakpoint on X-4 translocation induced meiotic drive is investigated. It is found that translocations with breakpoints distal to 13C on the salivary map do not cause drive while translocations broken proximal to 13C cause drive. The level of drive is related to the position of the breakpoint—the more proximal the breakpoint the greater the drive.     

New insights into sex chromosome evolution in anole lizards (Reptilia,Dactyloidae)

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X₁X₁X₂X₂/X₁X₂Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n?=?36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.     

A New Resource for Characterizing X-Linked Genes in Drosophila melanogaster: Systematic Coverage and Subdivision of the X Chromosome With Nested,Y-Linked Duplications

Interchromosomal duplications are especially important for the study of X-linked genes. Males inheriting a mutation in a vital X-linked gene cannot survive unless there is a wild-type copy of the gene duplicated elsewhere in the genome. Rescuing the lethality of an X-linked mutation with a duplication allows the mutation to be used experimentally in complementation tests and other genetic crosses and it maps the mutated gene to a defined chromosomal region. Duplications can also be used to screen for dosage-dependent enhancers and suppressors of mutant phenotypes as a way to identify genes involved in the same biological process. We describe an ongoing project in Drosophila melanogaster to generate comprehensive coverage and extensive breakpoint subdivision of the X chromosome with megabase-scale X segments borne on Y chromosomes. The in vivo method involves the creation of X inversions on attached-XY chromosomes by FLP-FRT site-specific recombination technology followed by irradiation to induce large internal X deletions. The resulting chromosomes consist of the X tip, a medial X segment placed near the tip by an inversion, and a full Y. A nested set of medial duplicated segments is derived from each inversion precursor. We have constructed a set of inversions on attached-XY chromosomes that enable us to isolate nested duplicated segments from all X regions. To date, our screens have provided a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. These duplication chromosomes will be valuable resources for rescuing and mapping X-linked mutations and identifying dosage-dependent modifiers of mutant phenotypes.MANY eukaryotes of biomedical and agricultural importance—including humans, other mammals, birds, and Drosophila—are heterogametic. Their sex chromosomes differ drastically in size and genetic composition. In species with X and Y chromosomes, males carry only one copy of each X-linked gene. This poses a serious challenge for experimental geneticists, because males inheriting a mutation in a vital X-linked gene die before they can be used in genetic crosses. In fact, the hemizygosity of X-linked genes in males has been a significant barrier to the functional analysis of many X-linked genes and is largely responsible for the poor genetic characterization of X chromosomes relative to autosomes in most organisms.The lethality of X-linked mutations can be rescued by providing a wild-type copy of the mutated gene elsewhere in the genome. This can be accomplished with a transgenic construct if the molecular identity of the mutated gene is known. In many cases, however, the mutated gene has not been identified and it is necessary to provide wild-type function with a multigene interchromosomal duplication, i.e., a segment of the X inserted in another chromosome. If the proximal and distal extents of the duplicated segment are known, phenotypic rescue maps the mutated gene to the defined X chromosome region.Multigene deletions can also be used to map X-linked mutations by complementation, but crosses between individuals carrying deletions and X-linked lethal mutations are impossible without rescuing the lethality of either the deletion or the lethal mutation in males. Projects at the Bloomington Drosophila Stock Center and elsewhere (Parks et al. 2004; Ryder et al. 2007) have generated large collections of deletions with molecularly defined breakpoints in Drosophila melanogaster, but the utility of the X deletions is limited without duplications of the corresponding chromosomal regions.Duplications are potentially important for gene discovery. Identifying sets of genes involved in the same cellular process is a major focus of functional genomics research and this can be accomplished genetically by identifying dosage-sensitive modifiers of mutant phenotypes. Often, increasing or decreasing the copy number of a gene will enhance or suppress the phenotype associated with mutating another gene involved in the same process. Screening collections of deletions is a popular way to identify interacting genes in Drosophila (for examples, see Seher et al. 2007; Zhao et al. 2008; Aerts et al. 2009; Salzer et al. 2010) and was a major impetus for the assembly of the Bloomington Stock Center “Deficiency Kit,” which provides maximal coverage of the genome with the fewest deletions. Though dosage-sensitive modifiers could also be identified using increased gene dosage, the use of duplications in enhancer and suppressor screens remains largely unexplored. Assembling sets of duplications providing efficient genomic coverage would likely popularize this experimental approach.The size of duplicated segments determines how duplication chromosomes are used experimentally. Small duplicated segments allow high resolution gene mapping, but they are not suitable for other purposes. Only large duplicated segments are capable of rescuing the lethality of sizable multigene X deletions. Likewise, large duplicated segments provide efficiency in initially localizing mutations and identifying dosage-dependent modifiers. Despite their usefulness, interchromosomal duplications of large segments are among the hardest chromosomal rearrangements to isolate. In Drosophila, many existing duplications were recovered fortuitously as three-breakpoint aberrations following irradiation, but such rearrangements are rare and difficult to identify in screens. Other duplications were methodically constructed from preexisting rearranged chromosomes. This approach works well when it is possible, but it can be used only when progenitor aberrations with appropriate breakpoints are available. Because of these difficulties, the selection of duplication strains generated by Drosophila workers over the past several decades is not satisfactory for many purposes. The duplications are often difficult to use experimentally, their breakpoints are sparsely distributed along the X chromosome and only roughly mapped, and substantial gaps in coverage exist. Obviously, improved duplication resources are needed.Here we present the methodology and progress of a project at the Bloomington Drosophila Stock Center to construct interchromosomal duplications of large, megabase-scale X segments. Our approach builds on the long history of manipulating Drosophila chromosomes in vivo (Novitski and Childress 1976; Ashburner et al. 2005), but we have eliminated the need for preexisting aberrations by generating progenitor chromosomes using the FLP-FRT system. Indeed, this site-specific recombination system has had an enormous impact on the ability of fly geneticists to engineer many kinds of novel chromosomes (Golic and Golic 1996; Parks et al. 2004; Ryder et al. 2007). We will demonstrate how we have combined FLP-mediated recombination and other chromosome manipulation techniques to produce Y-linked duplications of large X segments. As we will show, appending X segments to Y chromosomes rather than autosomes has advantages both for the synthesis and experimental use of X duplications.To date, we have generated a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. We anticipate completion of the project within the coming year. Using these duplications, mutations and genetic modifiers can be mapped first to large X intervals using a tiling set of the largest duplicated segments and then to small chromosome intervals using subsets of the duplications. These duplications will also facilitate deletion mapping. The creation of a set of stocks providing complete duplication coverage and extensive breakpoint subdivision of the X chromosome in a consistent genetic background will remove an impediment to investigating the functions of X-linked genes that has frustrated generations of Drosophila geneticists.     

Homozygous and Hemizygous Viability Variation on the X Chromosome of DROSOPHILA MELANOGASTER

We report here a study of viability inbreeding depression associated with the X chromosome of Drosophila melanogaster. Fifty wild chromosomes from Mt. Sinai, New York, and 90 wild chromosomes from Death Valley, California, were extracted using the marked FM6 balancer chromosome and viabilities measured for homozygous and heterozygous females, and for hemizygous males, relative to FM6 males as a standard genotype. No statistically significant female genetic load was observed for either chromosome set, although a 95% confidence limit estimated the total load <0.046 for the samples pooled. About 10% of the Death Valley chromosomes appear to be "supervital" as homozygotes. There is little evidence for a pervasive sex-limited detrimental load on the X chromosome; the evidence indicates nearly identical viability effects in males and homozygous females excluding the supervital chromosomes. The average degree of dominance for viability polygenes is estimated between 0.23 to 0.36, which is consistent with autosomal variation and implies near additivity. We conclude that there is little genetic load associated with viability variation on the X chromosome and that the substantial reduction in total fitness observed for chromosome homozygosity in an earlier study may be due largely to sex-limited fertility in females.