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.     

Mitotic Recombination in the Heterochromatin of the Sex Chromosomes of DROSOPHILA MELANOGASTER

The frequency of spontaneous and X-ray-induced mitotic recombination involving the Y chromosome has been studied in individuals with a marked Y chromosome arm and different XY compound chromosomes. The genotypes used include X chromosomes with different amounts of X heterochromatin and either or both arms of the Y chromosome attached to either side of the centromere. Individuals with two Y chromosomes have also been studied. The results show that the bulk of mitotic recombination takes place between homologous regions.     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25°?C and β-heterochromatic in X0 males at 14°?C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin.     

Differential Uptake of Tritiated Thymidine into Hetero- and Euchromatin in Melanoplus and Secale

Grasshoppers of the species Melanoplus differentialis were injected with tritium-labelled thymidine. At intervals thereafter autoradiographic stripping film was applied over Feulgen squashes and sections. In this species during early prophase of meiosis the sex chromosome forms a heterochromatic block large enough to be resolved in tritium autoradiographs. A study of the squash preparations reveals that the sex chromosome is synthesizing DNA at a different period of time from the euchromatic autosomes. Since there is a developmental sequence of spermatocyte cysts along the testicular tubes it is possible from the sections to show that the heterochromatin synthesizes DNA later than does the euchromatin. To find out whether the results obtained in Melanoplus were characteristic of heterochromatin in general, young seedlings of rye were grown in a tritiated thymidine solution and Feulgen squashes were made as for Melanoplus. In rye leaf nuclei there is a large block of heterochromatin constituted by the proximal regions of the chromosomes and a euchromatic one formed by the median and distal regions of the same chromosomes. Here also the heterochromatin synthesizes DNA at a different period of time from the euchromatin. It is concluded that in rye the asynchrony of synthesis occurs within each chromosome. Counts of silver grains over the two types of chromatin in nuclei of Melanoplus and Secale disclosed that the number of grains per unit area was two to three times higher over the heterochromatin. To check the DNA content, Feulgen photometric measurements were made of Melanoplus nuclei at the same stage. The Feulgen and grain counts agree in showing that the heterochromatin contains two to three times more DNA per unit area than the euchromatin.     

Parameters of Male and Female Recombination Influenced by the T-007 Second Chromosome in DROSOPHILA MELANOGASTER

The T-007 second chromosome line of Drosophila melanogaster, previously shown to contain genetic elements responsible for male recombination induction, appears to affect several parameters of recombination in females. In T-007 heterozygous females, the distribution of recombination (but not the total frequency) is changed from that observed in control females; relative increases are observed in the more proximal regions of the second, third and X chromosomes, while relative decreases are observed more distally. These changes are paralleled by altered coefficient of coincidence values and in an increased nondisjunction frequency of second chromosomes. The distribution of recombination in females is strikingly similar to that observed in males as measured along the second and third chromosomes, and the frequency of nondisjunction of the X and Y chromosomes is increased in T-007 heterozygous males. Based upon these results and responses to the effect of structurally rearranged heterologues (the "interchromosomal effect"), it is suggested that T-007 affects the preconditions for meiotic exchange in females. It is not yet known if elements responsible for these effects are the same elements responsible for the numerous other traits associated with the T-007 second chromosome.     

An Analysis of Regional Constraints on Exchange in DROSOPHILA MELANOGASTER Using Recombination-Defective Meiotic Mutants

The frequency of crossing over per unit of physical distance varies systematically along the chromosomes of Drosophila melanogaster . The regional distribution of crossovers in a series of X chromosomes of the same genetic constitution, but having different sequences, was compared in the presence and absence of normal genetically mediated regional constraints on exchange. Recombination was examined in Drosophila melanogaster females homozygous for either normal sequence X chromosomes or any of a series of X chromosome inversions. Autosomally, these females were either (1) wild type, (2) homozygous for one of several recombination-defective meiotic mutations that attenuate the normal regional constraints on exchange or (3) heterozygous for the multiply inverted chromosome TM2. The results show that the centromere, the telomeres, the heterochromatin and the euchromatic-heterochromatic junction do not serve as elements that respond to genic determinants of the regional distribution of exchanges. Instead, the results suggest that there are several elements sparsely distributed in the X chromosome euchromatin. Together with the controlling system affected by recombination-defective meiotic mutations, these elements specify the regional distribution of exchanges. The results also demonstrate that the alteration in the distribution of crossovers caused by inversion heterozygosity (the interchromosomal effect) results from the response of a normal controlling system to an overall increase in the frequency of crossing over, rather than from a disruption of the system of regional constraints on exchange that is disrupted by meiotic mutations. The mechanisms by which regional constraints on exchange might be established are discussed, as is the possible evolutionary significance of this system.     

On the Control of the Distribution of Meiotic Exchange in DROSOPHILA MELANOGASTER

The effects of eight recombination-defective meiotic mutants on crossing over within the X heterochromatin were examined. Since none permit substantial frequencies of exchange within heterochromatin although six lessen or abolish constraints on the location of exchanges within euchromatin, the systems that prohibit exchange within heterochromatin and that govern where exchanges will occur in euchromatin are under separate genetic control.—A minor component of the effects of mei-218 is the production of nonhomologous exchanges; of mei-9 is the recovery of deleted chromatids; and of mei-41 is the recovery of deleted chromatids and/or a low frequency of heterochromatic exchanges.     

Interchromosomal Effects of Heterochromatic Deletions on Recombination in DROSOPHILA MELANOGASTER

It is now known that partial deletions of the satellite sequences in X-chromosome heterochromatin result in a significant decrease in intrachromosomal recombination in the proximal region of the X chromosome of D. melanogaster (YAMAMOTO and MIKLOS 1978). It is important to ask then if the loss or gain of heterochromatin on the X also alters recombination in other chromosomes of the genome (interchromosomal effects). I have looked for such alterations by measuring recombination in chromosome 3. The results clearly indicate that the partial loss of X-chromosome heterochromatin not only decreases crossing over in the proximal region of the X chromosome itself, but also increases the frequency in chromosome 3, especially in the euchromatic regions around the centromere. Furthermore, the greater the deficiency of X heterochromatin, the higher is recombination in chromosome 3. This finding not only provides further evidence in support of the hypothesis that heterochromatin, in this case mainly composed of satellite DNA, regulates the recombination system, but it demonstrates that when the satellite content of one chromosome of the D. melanogaster genome is altered, there is an alteration in the crossover characteristics of other chromosomes in the same complement. If the amount of satellite DNA in a genome is being continuously altered, then one can predict that the recombination system is also being continually perturbed. Thus, the changing gene combinations produced indirectly by increases or decreases of heterochromatin are among the components available to organisms to break up or form new gene combinations upon which selection can act.     

Studies on the Ribosomal RNA Cistrons in Interspecific Drosophila Hybrids. II. Heterochromatic Regions Mediating Nucleolar Dominance

Interspecific hybrids of D. melanogaster and D. simulans normally exhibit a secondary constriction only at the D. melanogaster nucleolus organizer (NO). This phenomenon, termed nucleolar dominance, occurs only when the NO-bearing sex chromosomes of both species are present in conjunction. Experiments were initiated to localize regions on the sex chromosomes of D. melanogaster involved in mediating this suppression. Sex chromosome heterochromatic rearrangements and deficiencies were introduced into F₁ hybrids and their corresponding effect on simulans NO constriction formation was examined in hybrid mitotic neuroblast tissue. Sex chromosomes deficient for both the D. melanogaster NO and adjacent heterochromatin were unable to restrict the formation of a constriction at the D. simulans NO. The presence of a D. melanogaster NO, however, was not sufficient for the establishment of nucleolar dominance. Results from an array of NO-bearing X and Y chromosome rearrangements and deficiencies indicate that at least one heterochromatic region, proximal to the NO on the D. melanogaster X and distal to the NO on the D. melanogaster Y, affects the induction of this interchromosomal phenomenon.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Dynamic organization of the β-heterochromatin in the Drosophila melanogaster polytene X chromosome

Region 20 of the polytene X chromosome of Drosophila melanogaster was studied in salivary glands (SG) and pseudonurse cells (PNC) of otu mutants. In SG chromosomes the morphology of the region strongly depends on two modifiers of position effect variegation: temperature and amount of heterochromatin. It is banded in XYY males at 25° C and β-heterochromatic in X0 males at 14° C, i.e. it shows dynamic transitions. In PNC chromosomes region 20 is not heterochromatic, but demonstrates a clear banding pattern. Some molecular markers of mitotic heterochromatin were localized by means of in situ hybridization on PNC chromosomes: DNA of the gene su(f) in section 20C, the nucleolar organizer and 359-bp satellite in 20F. The 359-bp satellite, which has been considered to be specific for heterochromatin of the mitotic X chromosome, was found at two additional sites on chromosome 3L, proximally to 80C. The right arm of the X chromosome in SG chromosomes was localized in the inversion In(1LR)pn2b: the telomeric HeT-A DNA and AAGAG satellite from the right arm are polytenized, having been relocated from heterochromatin to euchromatin. Received: 1 July 1998 / Accepted: 7 September 1998     

On the Roles of Heterochromatin and Euchromatin in Meiosis in Drosophila: Mapping Chromosomal Pairing Sites and Testing Candidate Mutations for Effects on X–Y Nondisjunction and Meiotic Drive in Male Meiosis

Mapping of pairing sites involved in meiotic homolog disjunction in Drosophilahas led to conflicting hypotheses about the nature of such sites and the role of heterochromatin in meiotic pairing. In the female-specific distributive system, pairing regions appear to be exclusively heterochromatic and map to broad regions encompassing many different sequences. In male meiosis, autosomal pairing sites appear to be distributed broadly within euchromatin but to be absent from heterochromatin, whereas the X-pairing site maps in the centric heterochromatin. The X site has been shown to coincide with the intergenic spacer (IGS) repeats within the rDNA arrays shared between the X and Y. It has not been clear whether the heterochromatic location of this pairing site has any significance. A novel assay for genic modifiers of X–Y chromosome pairing was developed based on the intermediate nondisjunction levels observed in males whose X chromosome lacks the native pairing site but contains two transgenic insertions of single rDNA genes. This assay was used to test several mutations in Su(var)(Suppressor of position effect variegation), PcG(Polycomb-Group) recombination defective, and repair-defective genes. No strong effects on disjunction were seen. However, the tests did uncover several mutations that suppress or enhance the meiotic drive (distorted X-Y recovery ratio) that accompanies X–Y pairing failure. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Centromere-Independent Accumulation of Cohesin at Ectopic Heterochromatin Sites Induces Chromosome Stretching during Anaphase

Pericentric heterochromatin, while often considered as “junk” DNA, plays important functions in chromosome biology. It contributes to sister chromatid cohesion, a process mediated by the cohesin complex that ensures proper genome segregation during nuclear division. Long stretches of heterochromatin are almost exclusively placed at centromere-proximal regions but it remains unclear if there is functional (or mechanistic) importance in linking the sites of sister chromatid cohesion to the chromosomal regions that mediate spindle attachment (the centromere). Using engineered chromosomes in Drosophila melanogaster, we demonstrate that cohesin enrichment is dictated by the presence of heterochromatin rather than centromere proximity. This preferential accumulation is caused by an enrichment of the cohesin-loading factor (Nipped-B/NIPBL/Scc2) at dense heterochromatic regions. As a result, chromosome translocations containing ectopic pericentric heterochromatin embedded in euchromatin display additional cohesin-dependent constrictions. These ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit a significant increase in length during anaphase (termed chromatin stretching). These results provide evidence that long stretches of heterochromatin distant from the centromere, as often found in many cancers, are sufficient to induce abnormal accumulation of cohesin at these sites and thereby compromise the fidelity of chromosome segregation.     

Physical distribution of recombination in B-genome chromosomes of tetraploid wheat

Summary Several studies have indicated a noncorrespondence between genetic and physical distances in wheat chromosomes. To study the physical distribution of recombination, polymorphism for C-banding patterns was used to monitor recombination in 67 segments in 11 B-genome chromosome arms of Triticum turgidum. Recombination was absent in proximal regions of all chromosome arms; its frequency increased exponentially with distance from the centromere. A significant difference was observed between the distribution of recombination in physically short and physically long arms. In physically short arms, recombination was almost exclusively concentrated in distal segments and only those regions were represented in their genetic maps. In physically long arms, while a majority of the genetic distance was again based upon recombination in distal chromosome segments, some interstitial recombination was observed. Consequently, these regions also contributed to the genetic maps. Such a pattern of recombination, skewed toward terminal segments of chromosomes, is probably a result of telomeric pairing initiation and strong positive chiasma interference. Interference averaged 0.81 in 35 pairs of adjacent segments and 0.57 across the entire recombining portions of chromosome arms. The total genetic map lengths of the arms corresponded closely to those expected on the basis of their metaphase-I chiasma frequencies. As a consequence of this uneven distribution of recombination there can be a 153-fold difference (or more) in the number of DNA base pairs per unit (centiMorgan) of genetic length.     

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.     

Structure and replication of Phaseolus polytene chromosomes

The normal morphology of the polytene chromosomes of the embryo suspensor of Phaseolus coccineus is that of a tightly condensed cord with heavily Feulgen staining centromeric heterochromatic regions (α-heterochromatin) and other accessory heterochromatic regions (β-heterochromatin). The replication pattern of the chromosomes has been determined by autoradiographic analysis of material pulsed with ³H-thymidine for various lengths of time. The DNA replication cycle reqires 4–6 hours for completion. During replication chromosome structure becomes diffuse and the β-heterochromatic regions are indistinguishable from the euchromatic regions. The euchromatin is the first to replicate, and replication begins simultaneously at numerous sites in the euchromatin. The β-heterochromatin replicates next, and finally the centromeric heterochromatin. Replication is essentially complete in each of these parts of the chromosome before DNA synthesis begins in the next. The chromosomes are composed of numerous longitudinally running Feulgen positive strands, the equivalent portions of which replicate simultaneously. This indicates that there must be close control of the replication cycle in sister strands.