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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.

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.

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.

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.

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.

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.

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 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.