Retrotransposon-Induced Ectopic Expression of Cut Causes the Om(1a) Mutant in Drosophila Ananassae

Optic morphology (Om) mutations in Drosophila ananassae map to at least 22 loci scattered throughout the genome. They are semidominant, neomorphic, nonpleiotropic, and are associated with the insertion of a retrotransposon, tom. The Om(1A) gene, which is cytogenetically linked to the cut locus, was cloned using a DNA fragment of the cut locus of Drosophila melanogaster as a probe. Three of the eight alleles of Om(1A) examined have insertion of the tom element within a putative cut region. The γ-ray-induced revertants of Om(1A) are accompanied with cut lethal mutations and rearrangements within the cut coding region. In the eye imaginal discs of the Om(1A) mutants, differentiation of photoreceptor clusters is suppressed, abnormal cell death occurs in the center and the cut protein is expressed ectopically. D. melanogaster flies transformed with a chimeric cut gene under the control of a heat-inducible promoter show excessive cell death in the region anterior to the morphogenetic furrow, suppressed differentiation to photoreceptor clusters and defect in the imaginal eye morphology when subjected to temperature elevation. These findings suggest that the tom element inserted within the Om(1A) region induces ectopic cut expression in the eye imaginal discs, thus resulting in the Om(1A) mutant phenotype.     

Analysis of the Om(1d) Locus in Drosophila Ananassae

From the ca;px stock, which is the progenitor of Om mutants caused by insertions of the tom retrotransposon, 50 kb of genomic DNA including the Om(1D) locus was cloned by tom tagging and chromosome walking. Southern blot analyses of six Om(1D) mutants exposed one or two tom elements inserted at five nonrandom sites within an 18-kb distal segment of the restriction map; the phenotypic uniformity between these mutants was not affected by variations in the position, number or orientation of their inserts. Spontaneous revertants or more extreme derivatives of Om(1D) alleles were nonlinearly associated with losses or gains of tom inserts. Seven of eight radiation induced derivatives of Om(1D) mutants had one breakpoint of a chromosome rearrangement in polytene section 13A which includes the Om(1D) locus. Two Om(1D) derivatives, a spontaneous revertant and an induced extreme allele, were associated with overlapping deficiencies which define a region that is likely to contain the Om(1D) coding seguences proximal to the tom insertion sites. Incidental results confirm the previously indicated homology of the Om(1D) locus with the Bar locus of Drosophila melanogaster.     

A 2-Mb YAC/BAC-based physical map of the ovum mutant (Om) locus region on mouse chromosome 11

The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization. We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11. Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus. This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region. We determined the maximum physical interval for Om to be 1400 kb. In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region. Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region. This integrated map provides support for the identification of additional candidate genes for the DDK syndrome.     

Genetic analysis of the Om(2D) locus in Drosophila ananassae.

The Om(2D)63 mutants were mutagenized by gamma-ray irradiation and DEB feeding. A total of nine revertants were recovered and characterized; eight revertants were homozygous-lethal expressing no appreciable abnormality in cuticular pattern and central nervous system, and all failed to complement the lethality with each other. Two of the eight expressed embryonic lethality and were associated with cytologically detectable deletions including the putative Om(2D) locus, while four were associated with rearrangements in a region distal to the insertion sites of the tom elements. No rearrangement was detected in the remaining two by Southern blot analysis. One of the nine revertants was homozygous-viable with wild-type eyes and was associated with a reciprocal translocation with the break points at 48B in 2R (Om(2D) locus) and 96A in 3R. Based on these data, it is concluded that interaction between the region comprised of a single complementation group of the recessive lethal and the inserted tom elements seems to be responsible for the Om(2D) mutant phenotype. In addition, two induced dominant enhancers specific to Om(2D)63 were identified; both mapped on chromosome 2.     

Maternal transmission ratio distortion at the mouse Om locus results from meiotic drive at the second meiotic division

We have observed maternal transmission ratio distortion (TRD) in favor of DDK alleles at the Ovum mutant (Om) locus on mouse chromosome 11 among the offspring of (C57BL/6 x DDK) F(1) females and C57BL/6 males. Although significant lethality occurs in this backcross ( approximately 50%), differences in the level of TRD found in recombinant vs. nonrecombinant chromosomes among offspring argue that TRD is due to nonrandom segregation of chromatids at the second meiotic division, i.e., true meiotic drive. We tested this hypothesis directly, by determining the centromere and Om genotypes of individual chromatids in zygote stage embryos. We found similar levels of TRD in favor of DDK alleles at Om in the female pronucleus and TRD in favor of C57BL/6 alleles at Om in the second polar body. In those embryos for which complete dyads have been reconstructed, TRD was present only in those inheriting heteromorphic dyads. These results demonstrate that meiotic drive occurs at MII and that preferential death of one genotypic class of embryo does not play a large role in the TRD.     

Heritability of the maternal meiotic drive system linked to Om and high-resolution mapping of the Responder locus in mouse

Matings between (C57BL/6 x DDK)F(1) females and C57BL/6 males result in a significant excess of offspring inheriting maternal DDK alleles in the central region of mouse chromosome 11 due to meiotic drive at the second meiotic division. We have shown previously that the locus subject to selection is in the vicinity of D11Mit66, a marker closely linked to the Om locus that controls the preimplantation embryo-lethal phenotype known as the "DDK syndrome." We have also shown that observation of meiotic drive in this system depends upon the genotype of the sire. Here we show that females that are heterozygous at Om retain the meiotic drive phenotype and define a 0.32-cM candidate interval for the Responder locus in this drive system. In addition, analysis of the inheritance of alleles at Om among the offspring of F(1) intercrosses indicates that the effect of the sire is determined by the sperm genotype at Om or a locus linked to Om.     

Formal Relations between Om Mutants and Their Suppressors in Drosophila Ananassae

Optic morphology (Om) mutants associated with insertions of the tom transposable element at each of three tested loci are neomorphs as defined by the phenotypic equivalence of +/+/Om with +/Om and of +/Om/Om with Om/Om. Mutants behaving as suppressors of Om mutants and mapping to at least six loci are recovered from the same source and in similar frequency as Om mutants. The semidominant and nonpleiotropic suppressors at four of the six loci display defective eye phenes themselves, and the phenotypically normal mutants at a fifth locus are suspected alleles of a gene represented by recessive furrowed eye mutants. These and other properties imply that the suppressors, like suppressible Om mutants, are neomorphic due to insertion of the tom element into a hypothetical sequence they share with other members of a set of genes involved in development of the eye. Concurrently premature expression of both the suppressor and suppressed mutants would allow interaction of their products just as in normal development.     

A genetic test to determine the origin of maternal transmission ratio distortion. Meiotic drive at the mouse Om locus

We have shown previously that the progeny of crosses between heterozygous females and C57BL/6 males show transmission ratio distortion at the Om locus on mouse chromosome 11. This result has been replicated in several independent experiments. Here we show that the distortion maps to a single locus on chromosome 11, closely linked to Om, and that gene conversion is not implicated in the origin of this phenomenon. To further investigate the origin of the transmission ratio distortion we generated a test using the well-known effect of recombination on maternal meiotic drive. The genetic test presented here discriminates between unequal segregation of alleles during meiosis and lethality, based on the analysis of genotype at both the distorted locus and the centromere of the same chromosome. We used this test to determine the cause of the transmission ratio distortion observed at the Om locus. Our results indicate that transmission ratio distortion at Om is due to unequal segregation of alleles to the polar body at the second meiotic division. Because the presence of segregation distortion at Om also depends on the genotype of the sire, our results confirm that the sperm can influence segregation of maternal chromosomes to the second polar body.     

Molecular and histological characterizations of the Om(2D) mutants in Drosophila ananassae.

A series of transposon-induced optic morphology (Om) mutants found in a hypermutable marker stock of Drosophila ananassae provides a useful system for analyzing the molecular mechanism of eye morphogenesis. In the present study, one of the 25 Om loci so far reported, Om(2D), has been subjected to histological and molecular analyses as a first step toward understanding the role of Om genes in eye morphogenesis. Histological abnormalities observed during eye morphogenesis of the mutant, i.e. cell death within the eye-antennal discs of third instar larvae, and loss of the lamina, disorganized ommatidia and atrophied optic lobes in adults, were all comparable to those reported with various eye morphology mutants of D. melanogaster. Approximately 25 kb of genomic DNA including the Om (2D) locus was cloned by tom tagging. Southern blot and cloning analyses of two alleles of the Om (2D) locus revealed that insertions of the tom element occurred at three sites within 359 bp; two tandemly arrayed toms sharing one long terminal repeat at the junction and an internally deleted tom were present 359 bp apart from each other in Om (2D) 63, while a single tom in reverse orientation was present within the 359 bp in Om (2D) 10a. Host DNA sequences at the three insertion sites were TATAT or AATAT, and ATAT was duplicated upon the tom insertion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The maternal DDK syndrome phenotype is determined by modifier genes that are not linked to Om

The DDK syndrome is a polar, early embryonic lethal phenotype caused by incompatibility between a maternal factor of DDK origin and a paternal gene of non-DDK origin. Both maternal factor and paternal gene have been mapped to the Om locus on mouse Chromosome (Chr) 11. The paternal contribution to the syndrome has been shown to segregate as a single locus. Although the inheritance of the maternal contribution has not been characterized in depth, it as been assumed to segregate as a single locus. We have now characterized the segregation of the DDK fertility phenotype in over 240 females. Our results demonstrate that females require at least one DDK allele at Om to manifest the syndrome. However, the DDK syndrome inter-strain cross-fertility phenotype of heterozygous females is highly variable and spans the gamut from completely infertile to completely fertile. Our results indicate that this phenotypic variability has a genetic basis and that the modifiers of the DDK syndrome segregate independently of Om. Received: 24 November 1998 / Accepted: 19 January 1999     

The Om(1e) Mutation in Drosophila Ananassae Causes Compound Eye Overgrowth Due to Tom Retrotransposon-Driven Overexpression of a Novel Gene

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, we have carried out molecular analyses. A putative Om(1E) locus cloned by tom tagging and chromosome walking contained two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrated that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affected morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes.     

Isolation of Drosophila clones encoding maternally restricted RNAs

We have isolated molecular clones encoding RNAs which appear to be transcribed predominantly or exclusively during oogenesis in Drosophila melanogaster. These clones were isolated by virtue of their homology to polyadenylated RNAs present in very early embryos and absent from older embryos. An analysis of the developmental expression of three cloned genes is presented. The genomic position of one of these clones coincides closely with the position of the maternal effect lethal gene swallow, providing strong evidence that we have cloned this locus.     

Deletion mapping of the T/t complex: evidence for a second region of critical embryonic genes

The developmental effects of three different deletion mutations of the T/t complex of the mouse have been studied. The three mutations, TOak Ridge (OR), TOrleans (TOrl), and THair pin (THp), each produce a unique homozygous lethal phenotype: THp homozygotes fail to develop normally past the morula stage, TOrl homozygotes past the blastocyst stage, and TOR homozygotes past the egg cylinder stage. In compound embryos (TX/TY), the lethal phenotype observed corresponds to the shared length of deleted chromosome. This interaction allows the regions of chromosome 17, containing genetic information critical to early mammalian development, to be mapped.     

Saccharomyces cerevisiae porin pore forms complexes with mitochondrial outer membrane proteins Om14p and Om45p

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.     

Transmission-Ratio Distortion through F(1) Females at Chromosome 11 Loci Linked to Om in the Mouse Ddk Syndrome

We determined the genotypes of >200 offspring that are survivors of matings between female reciprocal F(1) hybrids (between the DDK and C57BL/6J inbred mouse strains) and C57BL/6J males at markers linked to the Ovum mutant (Om) locus on chromosome 11. In contrast to the expectations of our previous genetic model to explain the ``DDK syndrome,' the genotypes of these offspring do not reflect preferential survival of individuals that receive C57BL/6J alleles from the F(1) females in the region of chromosome 11 to which the Om locus has been mapped. In fact, we observe significant transmission-ratio distortion in favor of DDK alleles in this region. These results are also in contrast to the expectations of Wakasugi's genetic model for the inheritance of Om, in which he proposed equal transmission of DDK and non-DDK alleles from F(1) females. We propose that the results of these experiments may be explained by reduced expression of the maternal DDK Om allele or expression of the maternal DDK Om allele in only a portion of the ova of F(1) females.     

Cytological mapping of Om mutants of Drosophila ananassae.

Semidominant, optic morphology (Om) mutants in Drosophila ananassae have been genetically mapped to at least 25 loci throughout the genome (Hinton, 1984; 1988). Among them, four X-linked Om mutants were proved to be associated with the insertion of a transposable element, tom (Shrimpton et al., 1986; Tanda et al., 1988). In the present study, cytological mapping of autosomal Om mutants was carried out by in situ hybridization to polytene chromosomes using a cloned tom element as a probe. The cytological site for each autosomal Om mutant has been determined to a single band of the salivary gland chromosomes.     

BALB/c alleles at modifier loci increase the severity of the maternal effect of the "DDK syndrome"

The Om locus was first described in the DDK inbred mouse strain: DDK mice carry a mutation at Om resulting in a parental effect lethality of F(1) embryos. When DDK females are mated with males of other (non-DDK) inbred strains, e.g., BALB/c, they exhibit a low fertility, whereas the reciprocal cross, non-DDK females x DDK males, is fertile (as is the DDK intrastrain cross). The low fertility is due to the death of (DDK x non-DDK)F(1) embryos at the late-morula to blastocyst stage, which is referred to as the "DDK syndrome." The death of these F(1) embryos is caused by an incompatibility between a DDK maternal factor and the non-DDK paternal pronucleus. Previous genetic studies showed that F(1) mice have an intermediate phenotype compared to parental strains: crosses between F(1) females and non-DDK males are semisterile, as are crosses between DDK females and F(1) males. In the present studies, we have examined the properties of mice heterozygous for BALB/c and DDK Om alleles on an essentially BALB/c genetic background. Surprisingly, we found that the females are quasi-sterile when mated with BALB/c males and, thus, present a phenotype similar to DDK females. These results indicate that BALB/c alleles at modifier loci increase the severity of the DDK syndrome.     

Co-transfer of parthenogenotes and single porcine embryos leads to full-term development of the embryos

It has long been known that several embryos are needed to establish and maintain pregnancy during early gestation in pigs. In this study, we assessed whether co-transfer of parthenogenotes with a single embryo was sufficient to maintain development of the embryo. Embryos were recovered at morula and early blastocyst stages from gilts that had been artificially inseminated. Parthenogenotes were produced from oocytes matured in vitro, activated by electrical stimulation, and then cultured for 110h. Those that had developed to morula- or blastocyst-like stages at 110h post-activation were transferred to recipient pigs either with or without morula or blastocyst stage embryos. Parthenogenotes that were transferred to recipients in the absence of embryos were viable up to 30 days post-activation and formed limb-buds; at 40 days, however, all were dead or degenerate. Among pigs that received both parthenogenotes and a single embryo, seven of nine (77.8%) delivered a normal piglet at full-term. This study therefore demonstrates that parthenogenotes can be used in place of embryos to establish pregnancy and promote development of a single co-transferred embryo. This method may be applied to rescue high value porcine embryos that are difficult to produce, such as transgenic cloned embryos, or for embryos frozen as a genetic resource.     

Irreversible barrier to the reprogramming of donor cells in cloning with mouse embryos and embryonic stem cells

Somatic cloning does not always result in ontogeny in mammals, and development is often associated with various abnormalities and embryo loss with a high frequency. This is considered to be due to aberrant gene expression resulting from epigenetic reprogramming errors. However, a fundamental question in this context is whether the developmental abnormalities reported to date are specific to somatic cloning. The aim of this study was to determine the stage of nuclear differentiation during development that leads to developmental abnormalities associated with embryo cloning. In order to address this issue, we reconstructed cloned embryos using four- and eight-cell embryos, morula embryos, inner cell mass (ICM) cells, and embryonic stem cells as donor nuclei and determined the occurrence of abnormalities such as developmental arrest and placentomegaly, which are common characteristics of all mouse somatic cell clones. The present analysis revealed that an acute decline in the full-term developmental competence of cloned embryos occurred with the use of four- and eight-cell donor nuclei (22.7% vs. 1.8%) in cases of standard embryo cloning and with morula and ICM donor nuclei (11.4% vs. 6.6%) in serial nuclear transfer. Histological observation showed abnormal differentiation and proliferation of trophoblastic giant cells in the placentae of cloned concepti derived from four-cell to ICM cell donor nuclei. Enlargement of placenta along with excessive proliferation of the spongiotrophoblast layer and glycogen cells was observed in the clones derived from morula embryos and ICM cells. These results revealed that irreversible epigenetic events had already started to occur at the four-cell stage. In addition, the expression of genes involved in placentomegaly is regulated at the blastocyst stage by irreversible epigenetic events, and it could not be reprogrammed by the fusion of nuclei with unfertilized oocytes. Hence, developmental abnormalities such as placentomegaly as well as embryo loss during development may occur even in cloned embryos reconstructed with nuclei from preimplantation-stage embryos, and these abnormalities are not specific to somatic cloning.