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Chromosome breakage plays an important role in the evolution of karyotypes and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster, we find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of 3 different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all 3 chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the 3 chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment vary significantly between the 3 chromosomes and are correlated with the origin of the centromere and the amount of pericentric heterochromatin. We suggest that different centromere strengths could account for this.

Chromosome breakage plays an important role in the evolution of karyotypes, and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not well understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes inDrosophila melanogasterwe find that breakage under tension also tends to occur in specific hotspots. Our experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. We analyzed the breakage patterns of three different ring-Xchromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all three chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, we found that the hotspot locations are not conserved between the three chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility.. Additionally, the frequency of dicentric breakage and the durability of their spindle attachment varies significantly between the three chromosomes and is correlated with the origin of the centromere and the amount of pericentric heterochromatin they carry. We suggest that different centromere strengths could account for this.

Many insects maintain mutualistic associations with bacterial endosymbionts, but little is known about how they originate in nature. In this study, we describe the establishment and manipulation of a synthetic insect-bacterial symbiosis in a weevil host. Following egg injection, the nascent symbiont colonized many tissues, including prototypical somatic and germinal bacteriomes, yielding maternal transmission over many generations. We then engineered the nascent symbiont to overproduce the aromatic amino acids tyrosine and phenylalanine, which facilitate weevil cuticle strengthening and accelerated larval development, replicating the function of mutualistic symbionts that are widely distributed among weevils and other beetles in nature. Our work provides empirical support for the notion that mutualistic symbioses can be initiated in insects by the acquisition of environmental bacteria. It also shows that certain bacterial genera, including the Sodalis spp. used in our study, are predisposed to develop these associations due to their ability to maintain benign infections and undergo vertical transmission in diverse insect hosts, facilitating the partner-fidelity feedback that is critical for the evolution of obligate mutualism. These experimental advances provide a new platform for laboratory studies focusing on the molecular mechanisms and evolutionary processes underlying insect-bacterial symbiosis.

Site-specific recombinases are widely used tools for analysis of genetics, development, and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because the FRT and lox target sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the outcome of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites in Drosophila melanogaster and in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Site-specific recombinases are widely used tools for analysis of genetics, development and cell biology, and many schemes have been devised to alter gene expression by recombinase-mediated DNA rearrangements. Because theFRTandloxtarget sites for the commonly used FLP and Cre recombinases are asymmetrical, and must pair in the same direction to recombine, construct design must take into account orientation of the target sites. Both direct and inverted configurations have been used. However, the consequence of recombination between target sites on sister chromatids is frequently overlooked. This is especially consequential with inverted target sites, where exchange between oppositely oriented target sites on sisters will produce dicentric and acentric chromosomes. By using constructs that have inverted target sites inDrosophila melanogasterand in mice, we show here that dicentric chromosomes are produced in the presence of recombinase, and that the frequency of this event is quite high. The negative effects on cell viability and behavior can be significant, and should be considered when using such constructs.

Double-strand DNA breaks are repaired by one of several mechanisms that rejoin two broken ends. However, cells are challenged when asked to repair a single broken end and respond by: (1) inducing programmed cell death; (2) healing the broken end by constructing a new telomere; (3) adapting to the broken end and resuming the mitotic cycle without repair; and (4) using information from the sister chromatid or homologous chromosome to restore a normal chromosome terminus. During one form of homolog-dependent repair in yeast, termed break-induced replication (BIR), a template chromosome can be copied for hundreds of kilobases. BIR efficiency depends on Pif1 helicase and Pol32, a nonessential subunit of DNA polymerase δ. To date, there is little evidence that BIR can be used for extensive chromosome repair in higher eukaryotes. We report that a dicentric chromosome broken in mitosis in the male germline of Drosophila melanogaster is usually repaired by healing, but can also be repaired in a homolog-dependent fashion, restoring at least 1.3 Mb of terminal sequence information. This mode of repair is significantly reduced in pif1 and pol32 mutants. Formally, the repaired chromosomes are recombinants. However, the absence of reciprocal recombinants and the dependence on Pif1 and Pol32 strongly support the hypothesis that BIR is the mechanism for restoration of the chromosome terminus. In contrast to yeast, pif1 mutants in Drosophila exhibit a reduced rate of chromosome healing, likely owing to fundamental differences in telomeres between these organisms.

The fluorescent dye 4’-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by Ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10 sec. exposure to UV. In most cases the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images or interpreting images in experiments that use these dyes in combination with other fluorochromes.

The addition of a new telomere onto a chromosome break, a process termed healing, has been studied extensively in organisms that utilize telomerase to maintain their telomeres. In comparison, relatively little is known about how new telomeres are constructed on broken chromosomes in organisms that do not use telomerase. Chromosome healing was studied in somatic and germline cells of Drosophila melanogaster, a nontelomerase species. We observed, for the first time, that broken chromosomes can be healed in somatic cells. In addition, overexpression of the telomere cap component Hiphop increased the survival of somatic cells with broken chromosomes, while the cap component HP1 did not, and overexpression of the cap protein HOAP decreased their survival. In the male germline, Hiphop overexpression greatly increased the transmission of healed chromosomes. These results indicate that Hiphop can stimulate healing of a chromosome break. We suggest that this reflects a unique function of Hiphop: it is capable of seeding formation of a new telomeric cap on a chromosome end that lacks a telomere.

This is the author accepted manuscript. The final version is available from AAAS via http://dx.doi.org/10.1126/science.aac7932

In 1946, H. J. Muller was awarded the Nobel Prize in Medicine for his discovery with Drosophila melanogaster , some 20 years earlier, that exposure to X-rays caused mutation ([Muller 1927][1]). This led to the identification of a large number of Drosophila mutants and chromosome rearrangements, many

Telomere loss was produced during development of Drosophila melanogaster by breakage of an induced dicentric chromosome. The most prominent outcome of this event is cell death through Chk2 and Chk1 controlled p53-dependent apoptotic pathways. A third p53-independent apoptotic pathway is additionally utilized when telomere loss is accompanied by the generation of significant aneuploidy. In spite of these three lines of defense against the proliferation of cells with damaged genomes a small fraction of cells that have lost a telomere escape apoptosis and divide repeatedly. Evasion of apoptosis is accompanied by the accumulation of karyotypic abnormalites that often typify cancer cells, including end-to-end chromosome fusions, anaphase bridges, aneuploidy, and polyploidy. There was clear evidence of bridge–breakage–fusion cycles, and surprisingly, chromosome segments without centromeres could persist and accumulate to high-copy number. Cells manifesting these signs of genomic instability were much more frequent when the apoptotic mechanisms were crippled. We conclude that loss of a single telomere is sufficient to generate at least two phenotypes of early cancer cells: genomic instability that involves multiple chromosomes and aneuploidy. This aneuploidy may facilitate the continued escape of such cells from the normal checkpoint mechanisms.

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.

Following the advent of a gene targeting technique in Drosophila, different methods have been developed to modify the Drosophila genome. The initial demonstration of gene targeting in flies used an ends-in method, which generates a duplication of the target locus. The duplicated locus can then be efficiently reduced to a single copy by generating a double-strand break between the duplicated segments. This method has been used to knock out target genes by introducing point mutations. A derivative of this method is reported here. By using different homologous regions for the targeting and reduction steps, a complete deletion of the target gene can be generated to produce a definitive null allele. The breakpoints of the deletion can be precisely controlled. Unlike ends-out targeting, this method does not leave exogenous sequence at the deleted locus. Three endogenous genes, Sir2, Sirt2, and p53 have been successfully deleted using this method.

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.

Ends-in and ends-out refer to the two arrangements of donor DNA that can be used for gene targeting. Both have been used for targeted mutagenesis, but require donors of differing design. Ends-out targeting is more frequently used in mice and yeast because it gives a straightforward route to replace or delete a target locus. Although ends-in targeting has been successful in Drosophila , an attempt at ends-out targeting failed. To test whether ends-out targeting could be used in Drosophila , we applied two strategies for ends-out gene replacement at the endogenous yellow ( y ) locus in Drosophila . First, a mutant allele was rescued by replacement with an 8-kb y + DNA fragment at a rate of ≈1/800 gametes. Second, a wild-type gene was disrupted by the insertion of a marker gene in exon 1 at a rate of ≈1/380 gametes. The I-SceI endonuclease component alone is not sufficient for targeting: the FLP recombinase is also needed to generate the extrachromosomal donor. When both components are used we find that ends-out targeting can be approximately as efficient as ends-in targeting, and is likely to be generally useful for Drosophila gene targeting.

When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage., Author Summary Using the fruitfly as an experimental model system, we produced chromosomes that were broken at one end, and thus lacked the normal telomeric cap. The occurrence of such chromosomes is thought to promote carcinogenesis. A cell's response to such chromosomes is therefore of great interest. In somatic cells the tumor suppressors Chk2 and P53 can induce suicide of cells with such a chromosome and eliminate the danger. In the male germline, though, such chromosomes can be healed by the addition of a new telomere cap, and may then be transmitted to the next generation. We find that Chk2 and P53 regulate healing in the germline, but in seemingly opposite directions. Chk2 functions independently of P53 to eliminate cells with a damaged chromosome, while P53 is required to repopulate the germline after this Chk2-mediated elimination. Cells that carry a broken chromosome continue to divide in Chk2 mutants. We observed that the broken ends of sister chromatids may fuse in meiosis and that the fused chromatids produce a bridge spanning two cells at the second meiotic division. This structure elicits a previously undiscovered mechanism to eliminate sperm derived from such cells, providing an added safeguard to maintain genome integrity through the germline.

We previously described a method for targeted homologous recombination at the yellow gene of Drosophila melanogaster. Because only a single gene was targeted, further work was required to show whether the method could be extended to become generally useful for gene modification in Drosophila. We have now used this method to produce a knockout of the autosomal pugilist gene by homologous recombination between the endogenous locus and a 2.5-kb DNA fragment. This was accomplished solely by tracking the altered genetic linkage of an arbitrary marker gene as the targeting DNA moved from chromosome X or 2 to chromosome 3. The results indicate that this method of homologous recombination is likely to be generally useful for Drosophila gene targeting.

Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence.

Genetic imprinting is defined as a reversible, differential marking of genes or chromosomes that is determined by the sex of the parent from whom the genetic material is inherited [1]. Imprinting was first observed in insects where, in some species, most notably among the coccoids (scale insects and allies), the differential marking of paternally and maternally transmitted chromosome sets leads to inactivation or elimination of paternal chromosomes [2]. Imprinting is also widespread in plants and mammals [3,4], in which paternally and maternally inherited alleles may be differentially expressed. Despite imprinting having been discovered in insects, clear examples of parental imprinting are scarce in the model insect species Drosophila melanogaster. We describe a case of imprint-mediated control of gene expression in Drosophila. The imprinted gene – the white+ eye-color gene – is expressed at a low level when transmitted by males, and at a high level when transmitted by females. Thus, in common with coccoids, Drosophila is capable of generating an imprint, and can respond to that imprint by silencing the paternal allele.

The ability to place a series of gene constructs at a specific site in the genome opens new possibilities for the experimental examination of gene expression and chromosomal position effects. We report that the FLP- FRT site-specific recombination system of the yeast 2mu plasmid can be used to integrate DNA at a chromosomal FRT target site in Drosophila. The technique we used was to first integrate an FRT- flanked gene by standard P element-mediated transformation. FLP was then used to excise the FRT- flanked donor DNA and screen for FLP-mediated re-integration at an FRT target at a different chromosome location. Such events were recovered from up to 5% of the crosses used to screen for mobilization and are easily detectable by altered linkage of a white reporter gene or by the generation of a white + gene upon integration.

We show that site-specific recombination can be used to engineer chromosome rearrangements in Drosophila melanogaster. The FLP site-specific recombinase acts on chromosomal target sites located within specially constructed P elements to provide an easy screen for the recovery of rearrangements with breakpoints that can be chosen in advance. Paracentric and pericentric inversions are easily recovered when two elements lie in the same chromosome in opposite orientation. These inversions are readily reversible. Duplications and deficiencies can be recovered by recombination between two elements that lie in the same orientation on the same chromosome or on homologues. We observe that the frequency of recombination between FRTs at ectopic locations decreases as the distance that separates those FRTs increases. We also describe methods to determine the absolute orientation of these P elements within the chromosome. The ability to produce chromosome rearrangements precisely between preselected sites provides a powerful new tool for investigations into the relationships between chromosome arrangement, structure, and function.

In Drosophila there exist several examples of gene expression that can be modified by an interaction between alleles; this effect is known as transvection. The inference that alleles interact comes from the observations that homologous chromosomes pair in mitotically dividing cells, and that chromosome rearrangements can alter the phenotype produced by a pair of alleles. It is thought that heterozygous rearrangements impede the ability of alleles to pair and interact. However, because the existing data are inconsistent, this issue is not fully settled. By measuring the frequency of site-specific recombination between homologous chromosomes, we show that structural heterozygosity inhibits the pairing of alleles that lie distal to a rearrangement breakpoint. We suggest that some of the apparent conflicts may owe to variations in cell-cycle lengths in the tissues where the relevant allelic interactions occur. Cells with a longer cell cycle have more time to establish the normal pairing relationships that have been disturbed by rearrangements. In support, we show that Minute mutations, which slow the rate of cell division, partially restore a transvection effect that is disrupted by inversion heterozygosity.

The pugilist-Dominant mutation results from fusion of a portion of the gene encoding the tri-functional Methylene Tetrahydrofolate Dehydrogenase (E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3) to approximately one kb of a heterochromatic satellite repeat. Expression of this fusion gene results in an unusual ring pattern of pigmentation around the eye. We carried out experiments to determine the mechanism for this pattern. By using FLP-mediated DNA mobilization to place different pugD transgenes at pre-selected sites we found that variation in repeat length makes a strong contribution to variability of the pug phenotype. This variation is manifest primarily as differences in the thickness of the pigmented ring. We show that similar phenotypic variation can also be achieved by changing gene copy number. We found that the pugD pattern is not controlled by wingless, which is normally expressed in a similar ring pattern. Finally, we found that physical injury to a pugD eye can lead to pigment deposition in parts of the eye that would not have been pigmented in the absence of injury. Our results are consistent with a model in which a metabolite vital for pigment formation is imported from the periphery of the eye, and pugD limits the extent of its transport towards the center of the eye, thus revealing the existence of a hitherto unknown mechanism of localized transport in the eye.

Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryABR120G missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cell's primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryABR120G pathology, confirming the link between NADP/H metabolism and CryAB., Author Summary Cardiomyopathy is a specific form of heart disease that involves progressive restructuring of the heart muscle, resulting in reduced function and increased chance of sudden heart failure. Several mutations have been identified that cause inherited cardiomyopathy, including mutations in a gene called alpha B-crystallin. The work we report here puts this mutant gene into the fruit fly Drosophila melanogaster, where it causes heart defects that are similar to the human condition. Like cataracts reported in humans, this mutant gene also causes defective development of the fly eye. By examining defective fly hearts and eyes, and asking how genes involved in oxidation/reduction (redox) reactions influence these defects, we find that genes which control the redox environment of the cell also control the heart and eye defects. Oxidative stress, defined as an excess of harmful oxygen radicals, is a common concept in disease. Our work shows that in some circumstances, the genes that generate a more reduced cellular environment may also contribute to disease.

The transposase source delta 2-3(99B) was used to mobilize a P element located at sites on chromosomes X, 2 and 3. The transposition event most frequently recovered was a chromosome with two copies of the P element at or near the original site of insertion. These were easily recognized because the P element carried a hypomorphic white gene with a dosage dependent phenotype; flies with two copies of the gene have darker eyes than flies with one copy. The P element also carried direct repeats of the recombination target (FRT) for the FLP site-specific recombinase. The synthesis of FLP in these flies caused excision of the FRT-flanked white gene. Because the two white copies excised independently, patches of eye tissue with different levels of pigmentation were produced. Thus, the presence of two copies of the FRT-flanked white gene could be verified. When the P elements lay in the same orientation, FLP-mediated recombination between the FRTs on separated elements produced deficiencies and duplications of the flanked region. When P elements were inverted, the predominant consequence of FLP-catalyzed recombination between the inverted elements was the formation of dicentric chromosomes and acentric fragments as a result of unequal sister chromatid exchange.

The mechanisms that cells use to monitor telomere integrity, and the array of responses that may be induced, are not fully defined. To date there have been no studies in animals describing the ability of cells to survive and contribute to adult organs following telomere loss. We developed assays to monitor the ability of somatic cells to proliferate and differentiate after telomere loss. Here we show that p53 and Chk2 limit the growth and differentiation of cells that lose a telomere. Furthermore, our results show that two copies of the genes encoding p53 and Chk2 are required for the cell to mount a rapid wildtype response to a missing telomere. Finally, our results show that, while Chk2 functions by activating the p53-dependent apoptotic cascade, Chk2 also functions independently of p53 to limit survival. In spite of these mechanisms to eliminate cells that have lost a telomere, we find that such cells can make a substantial contribution to differentiated adult tissues., Author Summary In this work, we describe two simple assays for examining the fate of cells that lose a telomere. We applied these assays to examine the role of DNA damage response genes in controlling the fate of such cells. The checkpoint kinase Chk2 is known to activate the p53 tumor suppressor to promote apoptosis of cells with DNA damage, including the loss of a telomere. In work described here, we discovered that Chk2 can also act independently of p53 to eliminate cells that have lost a telomere. We also show for the first time in Drosophila that the genes encoding Chk2 and p53 are haplo-insufficient, as they are in humans. These critical discoveries demonstrate that the response to DNA damage, in the form of telomere loss, has an unexpectedly high degree of functional conservation from Drosophila to humans. This greatly strengthens the utility of Drosophila as a model to characterize the mechanisms that cells use to respond to telomere loss and, most critically, the mechanisms by which such cells can escape apoptosis. The original assay we describe in this work provides a basis for high-throughput genome-wide genetic screens to identify these mechanisms.

Ring chromosomes are of basic interest to the geneticist and cell biologist who study their behavior in meiotic and mitotic divisions. In addition, the mitotic instability associated with some ring-X chromosomes has proven useful in Drosophila as a means to produce gynandromorphs for developmental studies. We describe a method to construct ring-X chromosomes in Drosophila via I-CreI-mediated exchange in rDNA, and then rapidly diagnose the recovery of ring chromosomes via FLP-mediated sister chromatid exchange within the ring. The method we describe provides a ready means to tailor the genetic content of ring-X chromosomes, making it suited to produce ring-X chromosomes for a variety of experimental purposes.

Previously, we observed that heterochromatic 4 and Y chromosomes that had experienced breakage in the male germline were frequently transmitted to progeny. Their behavior suggested that they carried functional telomeres. Here we show that efficient healing by de novo telomere addition is not unique to heterochromatic breaks.

We present detailed protocols for two methods of gene targeting in Drosophila. The first, ends-out targeting, is identical in concept to gene replacement techniques used routinely in mammalian and yeast cells. In Drosophila, the targeted gene is replaced by the marker gene white + (although options exist to generate unmarked targeted alleles). This approach is simple in both the molecular cloning and the genetic manipulations. Ends-out will likely serve most investigators' purposes to generate simple gene deletions or reporter gene "knock-ins." The second method, ends-in targeting, targets a wild-type gene with an engineered mutated copy and generates a duplication structure at the target locus. This duplication can subsequently be reduced to one copy, removing the wild-type gene and leaving only the introduced mutation. Although more complicated in the cloning and genetic manipulations (see Note 1), this approach has the benefit that the mutations may be introduced with no other remnant of the targeting procedure. This "surgical" approach will appeal to investigators who desire minimal perturbation to the genome, such as single nucleotide mutation. Although both approaches appear to be approximately equally efficient (see Note 2), each method has separate strengths and drawbacks. The choice of which approach is best depends on the researcher's goal.

We present detailed protocols for two methods of gene targeting in Drosophila. The first, ends-out targeting, is identical in concept to gene replacement techniques used routinely in mammalian and yeast cells. In Drosophila, the targeted gene is replaced by the marker gene white + (although options exist to generate unmarked targeted alleles). This approach is simple in both the molecular cloning and the genetic manipulations. Ends-out will likely serve most investigators' purposes to generate simple gene deletions or reporter gene "knock-ins." The second method, ends-in targeting, targets a wild-type gene with an engineered mutated copy and generates a duplication structure at the target locus. This duplication can subsequently be reduced to one copy, removing the wild-type gene and leaving only the introduced mutation. Although more complicated in the cloning and genetic manipulations (see Note 1), this approach has the benefit that the mutations may be introduced with no other remnant of the targeting procedure. This "surgical" approach will appeal to investigators who desire minimal perturbation to the genome, such as single nucleotide mutation. Although both approaches appear to be approximately equally efficient (see Note 2), each method has separate strengths and drawbacks. The choice of which approach is best depends on the researcher's goal.

Evolutionary forces acting on the repetitive DNA of heterochromatin are not constrained by the same considerations that apply to protein-coding genes. Consequently, such sequences are subject to rapid evolutionary change. By examining the Troponin C gene family of Drosophila melanogaster, which has euchromatic and heterochromatic members, we find that protein-coding genes also evolve in response to their chromosomal location. The heterochromatic members of the family show a reduced CG content and increased variation in DNA sequence. We show that the CG reduction applies broadly to the protein-coding sequences of genes located at the heterochromatin:euchromatin interface, with a very strong correlation between CG content and the distance from centric heterochromatin. We also observe a similar trend in the transition from telomeric heterochromatin to euchromatin. We propose that the methylation of DNA is one of the forces driving this sequence evolution.

To investigate structure-function relationships in gamma-glutamyl carboxylases, the enzyme from Drosophila melanogaster was characterized. Four cysteine residues were shown to be important determinants for enzymatic activity. Native Drosophila substrates have not yet been identified, but propeptides of human prothrombin and factor IX are recognized by the Drosophila enzyme. The presence of the propeptide region increased apparent affinity by approximately 200-fold, and mutation of a hydrophobic residue of factor IX propeptide (F-16A) decreased carboxylation by 90%, as in the human enzyme. Substrate recognition appears to be highly conserved between the human and Drosophila gamma-glutamyl carboxylases. Inactivation of Drosophila gamma-glutamyl carboxylase by non-sense mutations or insertional mutagenesis by P-element insertion have no apparent effects on growth and fertility under laboratory conditions.

The sequence and the structure of DNA methyltransferase-2 (Dnmt2) bear close affinities to authentic DNA cytosine methyltransferases. A combined genetic and biochemical approach revealed that human DNMT2 did not methylate DNA but instead methylated a small RNA; mass spectrometry showed that this RNA is aspartic acid transfer RNA (tRNA Asp ) and that DNMT2 specifically methylated cytosine 38 in the anticodon loop. The function of DNMT2 is highly conserved, and human DNMT2 protein restored methylation in vitro to tRNA Asp from Dnmt2-deficient strains of mouse, Arabidopsis thaliana, and Drosophila melanogaster in a manner that was dependent on preexisting patterns of modified nucleosides. Indirect sequence recognition is also a feature of eukaryotic DNA methyltransferases, which may have arisen from a Dnmt2-like RNA methyltransferase.

The heat-shock response is a programmed change in gene expression carried out by cells in response to environmental stress, such as heat. This response is universal and is characterized by the synthesis of a small group of conserved protein chaperones. In Drosophila melanogaster the Hsp70 chaperone dominates the profile of protein synthesis during the heat-shock response. We recently generated precise deletion alleles of the Hsp70 genes of D. melanogaster and have used those alleles to characterize the phenotypes of Hsp70-deficient flies. Flies with Hsp70 deletions have reduced thermotolerance. We find that Hsp70 is essential to survive a severe heat shock, but is not required to survive a milder heat shock, indicating that a significant degree of thermotolerance remains in the absence of Hsp70. However, flies without Hsp70 have a lengthened heat-shock response and an extended developmental delay after a non-lethal heat shock, indicating Hsp70 has an important role in recovery from stress, even at lower temperatures. Lack of Hsp70 also confers enhanced sensitivity to a temperature-sensitive lethal mutation and to the neurodegenerative effects produced by expression of a human polyglutamine disease protein.

The homing endonuclease I-CreI recognizes a site in the gene encoding the 23S rRNA of Chlamydomonas reinhardtii. A very similar sequence is present in the 28S rRNA genes that are located on the X and Y chromosomes of Drosophila melanogaster. In this work we show that I-CreI expression in Drosophila is capable of causing induced DNA damage and eliciting cell cycle arrest. Expression also caused recombination between the X and Y chromosomes in the heterochromatic regions where the rDNA is located, presumably as a result of a high frequency of double-strand breaks in these regions. Approximately 20% of the offspring of males expressing I-CreI showed exceptional inheritance of X- and Y-linked markers, consistent with chromosome exchange at rDNA loci. Cytogenetic analysis confirmed the structures of many of these products. Exchange between the X and Y chromosomes can be induced in males and females to produce derivative-altered Y chromosomes, attached-XY, and attached-X chromosomes. This method has advantages over the traditional use of X rays for generating X-Y interchanges because it is very frequent and it generates predictable products.

Homologous recombination can produce directed mutations in the genomes of a number of model organisms, including Drosophila melanogaster. One of the most useful applications has been to delete target genes to generate null alleles. In Drosophila, specific gene deletions have not yet been produced by this method. To test whether such deletions could be produced by homologous recombination in D. melanogaster we set out to delete the Hsp70 genes. Six nearly identical copies of this gene, encoding the major heat-shock protein in Drosophila, are found at two separate but closely linked loci. This arrangement has thwarted standard genetic approaches to generate an Hsp70-null fly, making this an ideal test of gene targeting. In this study, ends-out targeting was used to generate specific deletions of all Hsp70 genes, including one deletion that spanned ∼47 kb. The Hsp70-null flies are viable and fertile. The results show that genomic deletions of varied sizes can be readily generated by homologous recombination in Drosophila.

In recombinational DNA double-strand break repair a homologous template for gene conversion may be located at several different genomic positions: on the homologous chromosome in diploid organisms, on the sister chromatid after DNA replication, or at an ectopic position. The use of the homologous chromosome in mitotic gene conversion is thought to be limited in the yeast Saccharomyces cerevisiae and mammalian cells. In contrast, by studying the repair of double-strand breaks generated by the I-SceI rare-cutting endonuclease, we find that the homologous chromosome is frequently used in Drosophila melanogaster, which we suggest is attributable to somatic pairing of homologous chromosomes in mitotic cells of Drosophila. We also find that Drosophila mitotic cells of the germ line, like yeast, employ the homologous recombinational repair pathway more often than imperfect nonhomologous end joining.

Genomic imprinting is well known as a regulatory property of a few specific chromosomal regions and leads to differential behavior of maternally and paternally inherited alleles. We surveyed the activity of two reporter genes in 23 independent P-element insertions on the heterochromatic Y chromosome of Drosophila melanogaster and found that all but one location showed differential expression of one or both genes according to the parental source of the chromosome. In contrast, genes inserted in autosomal heterochromatin generally did not show imprint-regulated expression. The imprints were established on Y-linked transgenes inserted into many different sequences and locations. We conclude that genomic imprinting affecting gene expression is a general property of the Drosophila Y chromosome and distinguishes the Y from the autosomal complement.

Zinc-finger nucleases (ZFNs) are hybrids between a nonspecific DNA-cleavage domain and a DNA-binding domain composed of Cys2His2 zinc fingers. Because zinc fingers can be manipulated to recognize a broad range of sequences, these enzymes have the potential to direct cleavage to arbitrarily chosen targets. We have tested this idea by designing a pair of ZFNs that recognize a unique site in the yellow (y) gene of Drosophila. When these nucleases were expressed in developing larvae, they led to somatic mutations specifically in the y gene. These somatic mosaics were observed in approximately one-half of the males expressing both nucleases. Germline y mutations were recovered from 5.7% of males, but from none of the females, tested. DNA sequences were determined and showed that all of the mutations were small deletions and/or insertions located precisely at the designed target. These are exactly the types of alterations expected from nonhomologous end joining (NHEJ) following double-strand cleavage of the target. This approach promises to permit generation of directed mutations in many types of cells and organisms.

We used a recently developed method to produce mutant alleles of five endogenous Drosophila genes, including the homolog of thep53 tumor suppressor. Transgenic expression of the FLP site-specific recombinase and the I-SceI endonuclease generates extrachromosomal linear DNA molecules in vivo. These molecules undergo homologous recombination with the corresponding chromosomal locus to generate targeted alterations of the host genome. The results address several questions about the general utility of this technique. We show that genes not near telomeres can be efficiently targeted; that no knowledge of the mutant phenotype is needed for targeting; and that insertional mutations and allelic substitutions can be easily produced.

We have isolated a dominant mutation, pugilistDominant (pug D), that causes variegated reductions in pteridine and ommochrome pigmentation of the Drosophila eye. The effect of pugD on pteridine pigmentation is most dramatic: the only remaining pigment consists of a thin ring of pigment around the periphery of the eye with a few scattered spots in the center. The pugD mutation disrupts a gene that encodes a Drosophila homolog of the trifunctional enzyme methylenetetrahydrofolate dehydrogenase (MTHFD; E.C.1.5.1.5, E.C.3.5.4.9, E.C.6.3.4.3). This enzyme produces a cofactor that is utilized in purine biosynthesis. Because pteridines are derived from GTP, the pigment defect may result from an impairment in the production of purines. The mutant allele consists of a portion of the MTHFD coding region fused to ∼1 kb of highly repetitive DNA. Transcription and translation of both parts are required for the phenotype. The repetitive DNA consists of ∼140 nearly perfect repeats of the sequence AGAGAGA, a significant component of centric heterochromatin. The unusual nature of the protein produced by this gene may be responsible for its dominance. The repetitive DNA may also account for the variegated aspect of the phenotype. It may promote occasional association of the pugD locus with centric heterochromatin, accompanied by inactivation of pugD, in a manner similar to the proposed mode of action for brownDominant.

We investigated the fate of dicentric chromosomes in the mitotic divisions of Drosophila melanogaster. We constructed chromosomes that were not required for viability and that carried P elements with inverted repeats of the target sites (FRTs) for the FLP site-specific recombinase. FLP-mediated unequal sister-chromatid exchange between inverted FRTs produced dicentric chromosomes at a high rate. The fate of the dicentric chromosome was evaluated in the mitotic cells of the male germline. We found that dicentric chromosomes break in mitosis, and the broken fragments can be transmitted. Some of these chromosome fragments exhibit dominant semilethality. Nonlethal fragments were broken at many sites along the chromosome, but the semilethal fragments were all broken near the original site of sister-chromatid fusion, and retained P element sequences near their termini. We discuss the implications of the recovery and behavior of broken chromosomes for checkpoints that detect double-strand break damage and the functions of telomeres in Drosophila.

A transgene was inserted at several different chromosomal sites in Drosophila melanogaster, where its expression was subject to genomic position effects. Quantitative position effects and variegated and constant patterned position effects were observed. We investigated the status of the affected gene in the somatic cells where it normally functions. The FLP site-specific recombinase was used to remove the gene from the chromosome and its expression was then evaluated. We show that the FLP recombinase functions in cells that have finished their developmental program of mitoses. When FLP acts on directly repeated copies of its target site (FRT), the DNA flanked by those FRTs is excised from the chromosome as a closed circle. The extrachromosomal circle is maintained in nondividing cells, and a gene located on such a circle can be expressed. We then demonstrate that a gene subject to either variegated or constant position effect can be relieved of that effect by excision of the gene from the chromosome in cells where it would otherwise be inactive. We also observed a strong inhibition of FLP-mediated recombination for target sites located near centric heterochromatin.

The ability to mark a cell and its descendants genetically so that the resulting cell clone can be distinguished from neighboring cells facilitates studies in animal biology and development. A method of generating clones by inducing homologous mitotic recombination in Drosophila with a site-specific yeast recombinase is described. This method allows for frequent mosaicism after mitotic exchange is induced at predefined sites in the genome.

We have transferred the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination targets (FRTs), into the genome of Drosophila. Flies were transformed with an FLP gene under the control of hsp70 regulatory sequences and with a white gene flanked by FRTs. The heat-induced recombinase catalyzes recombination between FRTs, causing loss of white (seen somatically as white patches in the eye) and, less frequently, gain of white (seen as dark-red patches). Loss and gain frequencies vary with the severity of the heat shock, and patterns of mosaicism vary with the developmental stage at which the heat shock is applied. The recombinase is also active in the germline, producing white-eyed and dark-red-eyed progeny.