Dominant Lethals Research Articles

Ontogenes in Drosophila Melanogaster and a Model of Speciation

A new type of mutations-dominant lethals with a facultative manifestation -were discovered in D. melanogaster in 2000. These mutations were named conditional mutations. Under restrictive genetic conditions, the mutations manifest themselves as dominant lethals, whereas dominant lethality disappears under permissive conditions, displaying a set of other manifestations. The genes responsible for the emergence of conditional mutations were named ontogenes. The experiments with mutations in ontogenes have revealed the following processes: (1) genome editing in germline cells; (2) induction of high mutagenesis rates in germline cells of the mutants for ontogenes; (3) zygotic selection; (4) isolation of mutants; and (5) alterations in the lethality of mutants with time. The specific features in the manifestation of ontogenes together with the listed processes formed the background for construction of the model of speciation named the regeneration model. The event of speciation is represented as the regeneration of the working state of a genetic system disturbed by the emergence of a mutation in an ontogene. According to the model, it is ontogenes that are in charge of speciation and, eventually, the structure of living matter in the form of individual species. The significance of Mendelian protein-coding genes and Darwinian selection of the fittest according to these genes are doubtless but not paramount.

The development of an adaptive response of <i>Drosophila melanogaster</i> to genotoxicity of alkyl(aryl)sulfurnyl 1,2,4-triazoles

The widespread use of triazoles in agriculture as retardants has led to the need to investigate the possible mutagenic effects of these compounds on natural ecosystems adjacent to agrocoenoses. Model experiments were set up to investigate the ability to induce dominant lethal mutations by synthesized triazoles: 1,2,4-triazole: (1,2,4-triazole (1,2,4-TrH), N-triazolid methanesulfonic acid (CH₃SO₂TrH), N-triazolid benzene sulfonic acid (PhSO₂1,2,4-Tr) and N-triazolid toluene sulfonic acid (4-CH₃ ArSO₂1,2,4-Tr) in Drosophila melanogaster adults. The possibility of developing an adaptive response to these compounds in Drosophila melanogaster was studied. A relationship between the structure and physicochemical properties of the studied alkyl(aryl)sulfonyl 1,2,4-triazoles and their mutagenicity was analyzed. A series was constructed in which mutagenicity for Drosophila adults decreases in the following order: CH₃SO₂TrH 1,2,4-TrH PhSO₂1,2,4-TrH 4-CH₃ArSO₂1,2,4-TrH, both when exposed to non-toxic doses of substances (0,001 mg/ml concentration) and when exposed to compounds at a dose of LD₅₀. It has been established that a short-term (within a day) preliminary action on adults by nontoxic doses (0,0001 mg/ml) followed by action on the same adults by these substances in LD₅₀ dose causes an adaptive response only in females of Drosophila melanogaster. Long-term exposure (the entire larval life period of drosophila) to a nontoxic dose and then repeated exposure of the same Drosophila melanogaster adults to the same compounds at the LD₅₀ dose caused a significant decrease in the number of induced dominant lethalities in females as well as in drosophila males. Consequently, the adaptive response developed only in tissues with an active cell division. It is supposed that the repair mechanisms leading to the development of an adaptive response in adults and larvae are different.

Manipulating Insect Sex Determination Pathways for Genetic Pest Management: Opportunities and Challenges.

Sex determination pathways in insects are generally characterised by an upstream primary signal, which is highly variable across species, and that regulates the splicing of a suite of downstream but highly-conserved genes (transformer, doublesex and fruitless). In turn, these downstream genes then regulate the expression of sex-specific characteristics in males and females. Identification of sex determination pathways has and continues to be, a critical component of insect population suppression technologies. For example, “first-generation” transgenic technologies such as fsRIDL (Female-Specific Release of Insects carrying Dominant Lethals) enabled efficient selective removal of females from a target population as a significant improvement on the sterile insect technique (SIT). Second-generation technologies such as CRISPR/Cas9 homing gene drives and precision-guided SIT (pgSIT) have used gene editing technologies to manipulate sex determination genes in vivo. The development of future, third-generation control technologies, such as Y-linked drives, (female to male) sex-reversal, or X-shredding, will require additional knowledge of aspects of sexual development, including a deeper understanding of the nature of primary signals and dosage compensation. This review shows how knowledge of sex determination in target pest species is fundamental to all phases of the development of control technologies.

Evaluation of Additional Drosophila suzukii Male-Only Strains Generated Through Remobilization of an FL19 Transgene.

Drosophila suzukii (D. suzukii) (Matsumura, 1931; Diptera: Drosophilidae), also known as spotted wing Drosophila, is a worldwide pest of fruits with soft skins such as blueberries and cherries. Originally from Asia, D. suzukii is now present in the Americas and Europe and has become a significant economic pest. Growers largely rely on insecticides for the control of D. suzukii. Genetic strategies offer a species-specific environmentally friendly way for suppression of D. suzukii populations. We previously developed a transgenic strain of D. suzukii that produced only males on a diet that did not contain tetracycline. The strain carried a single copy of the FL19 construct on chromosome 3. Repeated releases of an excess of FL19 males led to suppression of D. suzukii populations in laboratory cage trials. Females died as a consequence of overexpression of the tetracycline transactivator (tTA) and tTA-activated expression of the head involution defective proapoptotic gene. The aim of this study was to generate additional male-only strains that carried two copies of the FL19 transgene through crossing the original line with a piggyBac jumpstarter strain. Males that carried either two chromosome 3 or a singleX-linked transgene were identified through stronger expression of the red fluorescent protein marker gene. The brighter fluorescence of the X-linked lines was likely due to dosage compensation of the red fluorescent protein gene. In total, four X-linked lines and eleven lines with two copies on chromosome 3 were obtained, of which five were further examined. All but one of the strains produced only males on a diet without tetracycline. When crossed with wild type virgin females, all of the five two copy autosomal strains examined produced only males. However, the single copy X-linked lines did not show dominant female lethality. Five of the autosomal lines were further evaluated for productivity (egg to adult) and male competition. Based on these results, the most promising lines have been selected for future population suppression experiments with strains from different geographical locations.

Two User-Friendly Molecular Markers Developed for the Identification of Hybrid Lethality Genes in Brassica oleracea

Cabbage (Brassica oleracea) is an important vegetable crop that is cultivated worldwide. Previously, we reported the identification of two dominant complementary hybrid lethality (HL) genes in cabbage that could result in the death of hybrids. To avoid such losses in the breeding process, we attempted to develop molecular markers to identify HL lines. Among 54 previous mapping markers closely linked to BoHL1 or BoHL2, only six markers for BoHL2 were available in eight cabbage lines (two BoHL1 lines; three BoHL2 lines; three lines without BoHL); however, they were neither universal nor user-friendly in more inbred lines. To develop more accurate markers, these cabbage lines were resequenced at an ~20× depth to obtain more nucleotide variations in the mapping regions. Then, an InDel in BoHL1 and a single-nucleotide polymorphism (SNP) in BoHL2 were identified, and the corresponding InDel marker MBoHL1 and the competitive allele-specific PCR (KASP) marker KBoHL2 were developed and showed 100% accuracy in eight inbred lines. Moreover, we identified 138 cabbage lines using the two markers, among which one inbred line carried BoHL1 and 11 inbred lines carried BoHL2. All of the lethal line genotypes obtained with the two markers matched the phenotype. Two markers were highly reliable for the rapid identification of HL genes in cabbage.

The haplolethality paradox of the wupA gene in Drosophila.

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3’ end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

Functional Consequences of the Evolution of Matrimony, a Meiosis-Specific Inhibitor of Polo Kinase.

Meiosis is a defining characteristic of eukaryotes, believed to have evolved only once, over one billion years ago. While the general progression of meiotic events is conserved across multiple diverse organisms, the specific pathways and proteins involved can be highly divergent, even within species from the same genus. Here we investigate the rapid evolution of Matrimony (Mtrm), a female meiosis-specific regulator of Polo kinase (Polo) in Drosophila. Mtrm physically interacts with Polo and is required to restrict the activity of Polo during meiosis. Despite Mtrm’s critical role in meiosis, sequence conservation within the genus Drosophila is poor. To explore the functional significance of this rapid divergence, we expressed Mtrm proteins from 12 different Drosophila species in the Drosophila melanogaster female germline. Distantly related Mtrm homologs are able to both physically interact with D. melanogaster Polo and rescue the meiotic defects seen in mtrm mutants. However, these distant homologs are not properly degraded after the completion of meiosis. Rather, they continue to inhibit Polo function in the early embryo, resulting in dominant maternal-effect lethality. We show that the ability of Mtrm to be properly degraded, and thus release Polo, is partially due to residues or motifs found within Mtrm’s least-conserved regions. We hypothesize that, while Mtrm regions critical for its meiotic function are under strong purifying selection, changes that occurred in its unconserved regions may have been advantageous, potentially by affecting the timing or duration of meiosis and/or the early embryonic divisions.

Conditional Mutations in Drosophila: Concept of Genes That Control Individual Development

The conditional mutations in D. melanogaster are produced by gamma-irradiation, maintained in laboratory cultures, and inherited as gene mutations. However, their manifestation differs from the conventional mutations by several specific features. The most noticeable specific feature is their conditional nature, i.e., a conditional mutation manifests itself in the individuals of a certain genotype being silent in the individuals with another genotype. A particular procedure for mutation recovery determines what these genotypes will be. An overwhelming number of mutations are conditional dominant lethals. The viable mutation carriers display a drastically decreased fertility. Early zygotic lethality is inherited according to parental type (maternal or paternal). The carriers of conditional mutations give the offspring with a high rate of monstrosities. The possibility for the offspring to form monstrosities is inherited according to a parental (maternal or paternal) type. The level of fertility of conditional mutants is altered by chromosomal rearrangements. The chromosomal rearrangements themselves cause a decrease in fertility. Lethality of the progenies produced by the parents carrying rearrangements is inherited according to a parental (maternal or paternal) type. The results allow for a set of logical arguments in favor of that 1) the genome has a specialized system of genes (ontogenes) that control the course of individual development; 2) unlike a classical gene, acting according to the scheme DNA à RNA à protein, the ontogene implements the regulation according to the scheme DNA à RNA; and 3) the course of individual development is programmed by double-strand RNAs produced by ontogenes in germline cells.

Epididymal CYP2E1 plays a critical role in acrylamide-induced DNA damage in spermatozoa and paternally mediated embryonic resorptions†.

Acrylamide is a ubiquitous toxicant in human lives, due to its formation in many food products. Acrylamide induces dominant lethal mutations with administration of 25 mg/kg bw/day for 5 days in male mice. Cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) is responsible for this dominant lethality. CYP2E1 is the only enzyme responsible for the conversion of acrylamide to the highly reactive metabolite glycidamide, which forms adducts with DNA. CYP2E1 is present predominantly in the liver, as well as the brain, kidney, intestines, and spleen. Within the male mouse reproductive tract, CYP2E1 localizes to spermatocytes. However, embryo resorptions have been demonstrated to occur only with exposure of the late stages of spermatogenesis and spermatozoa. It was determined that CYP2E1 is additionally expressed within the mouse epididymal epithelium, and this localization is responsible for acrylamide-induced dominant lethality. Further, an equivalent profile of CYP2E1 expression was identified in the human reproductive tract. While spermatozoa of both species were also established to possess CYP2E1, this did not contribute to acrylamide-induced DNA damage. In vitro studies strengthened these findings further, revealing that acrylamide exposure only induces DNA damage in human and mouse spermatozoa following metabolism by the mouse epididymal epithelial cell line (mECap18) to glycidamide. These findings emphasize, for the first time, the vital role of the epididymis in the reproductive toxicity associated with acute acrylamide exposure.

Chronic Acrylamide Exposure in Male Mice Results in Elevated DNA Damage in the Germline and Heritable Induction of CYP2E1 in the Testes.

Acute acrylamide exposure in male rodents results in reduced reproductive performance and dominant lethality. However, the reproductive effects of low dose chronic exposure, which better reflects the nature of human exposure, remain far less certain. Human dietary consumption of acrylamide has been estimated at an average of 1-4 µg/kg bw/day. In order to simulate this exposure, male mice were provided with acrylamide (1 µg/ml) via their drinking water continuously for six months, which was equivalent to a human dose of 10.5 µg/ kg bw/day. This exposure regime increased DNA damage in the spermatozoa, without affecting a concomitant reduction in overall fertility. The offspring of acrylamide treated mice did not have an increased incidence of skin papilloma formation following the two-stage tumor induction protocol. However, the male offspring of acrylamide treated fathers had significantly increased levels of DNA damage in their spermatozoa, despite having had no direct toxicant exposure. It was also found that the F0, and most crucially, F1 mice had increased levels of CYP2E1 protein in their germ cells. This is significant as CYP2E1 is the sole enzyme responsible for conversion of acrylamide to its harmful metabolite glycidamide. This altered expression may be due to epigenetic alterations. Additionally, the F0 and F1 mice had increased oxidative adducts in the DNA of their germ cells, which was hypothesized to arise as a byproduct of increased CYP2E1 activity. Therefore, chronic paternal acrylamide exposure in mice has consequences for their offspring, and raises concerns for the effects of acrylamide exposure in the human population and the accumulated effects with multiple generations of exposure.

A transgenic male-only strain of the New World screwworm for an improved control program using the sterile insect technique.

BackgroundThe New World screwworm, Cochliomyia hominivorax, is a devastating pest of livestock endemic to subtropical and tropical regions of the Western hemisphere. The larvae of this species feed on the tissue of living animals, including man, and can cause death if untreated. Over 60 years ago, the sterile insect technique (SIT) was developed with the aim of eradicating this pest, initially from Florida but subsequently from all of North and Central America. From the outset it was appreciated that SIT would be more efficient if only sterile males were released in the field, but this was not possible until now.ResultsHere, we report on the development and evaluation of the first sexing strains of C. hominivorax that produce only males when raised on diet without tetracycline. Transgenic lines have been developed that possess a tetracycline repressible female-lethal genetic system. Ten of these lines show high female lethality at the late larval/pupal stages and three of them present dominant female lethality. Most of the lines were comparable to the wild type parental strain in several fitness parameters that are relevant to mass rearing in a production facility. Further, three lines performed well in male mating success and male competition assays, suggesting they would be sexually competitive in the field. Consequently, one transgenic line has been selected by the New World Screwworm Program for evaluation under mass rearing conditions.ConclusionsWe conclude that the promising characteristics of the selected sexing strains may contribute to reduce production costs for the existing eradication program and provide more efficient population suppression, which should make a genetic control program more economical in regions were C. hominivorax remains endemic.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0296-8) contains supplementary material, which is available to authorized users.

Time to Empower Release of Insects Carrying a Dominant Lethal and Wolbachia Against Zika

RIDL (release of insects with dominant lethality) and Wolbachia are 2 potentially powerful tools in the fight against Zika, but their technological advancement is being hampered by policy barriers. In this study, we discuss what could be done to overcome these regulatory deadlocks.

Genotoxicity Assessment of an Organophosphate Insecticide, Ethion Employing Mosquito Genome (Diptera:<i>Culicidae</i>)

The preferential motives of the present exploration are attributed to the genotoxicity evaluation of an organophosphate pesticide, ethion on the heredity material of Culex quinquefasciatus , exposed to sublethal aliquot, LC 20 , for the continuation of 24 h, implementing two in vivo approaches viz., the testicular chromosomal aberrations analysis and dominant lethal test (DLT). Mutagenic, as well as clastogenic consequences of ethion on germinal chromosomes were observed, specifically through relatively elevated incidences of nondisjunctions during pole-wise movement of chromosomes, excessively condensed heterochromatinizations regions, chromosomal fusions due to stickiness, aneuploidy eventually by expressed by hypoploidy, lags of whole or chromosomal fragments and furthermore, pole to pole extended chromosomal bridges, in comparison to control population, with statistical corresponding values 26.03 ± 15.5 in treated stock as against 12.67 ± 3.8 in control groups. For DLT, treated male imagoes were allowed to crossmate with normal females to procure egg rafts and enhanced percentage frequency of unhatched eggs corresponds to value 12.39 ± 3.00 in treated stocks as correlated to 4.23 ± 1.12 in control stocks. Subsequently, statical examination of procured data, from both executed parameters, demonstrated significant outcomes, when scrutinized through Student's t-test (P < 0.05). The overall conclusion indicated that the induced genotoxicity of ethion in germinal chromosomes could be the probable reason for dominant lethality observed during DLT.

Mutation analysis of four Chinese families with pure hereditary spastic paraplegia: pseudo- X-linked dominant inheritance and male lethality due to a novel ATL1 mutation.

We studied four Chinese families with pure hereditary spastic paraplegia (HSP) to investigate the clinical features and associated genetic mutations. Linkage analysis was performed for all families to map the disease locus onto autosomal chromosomes, and related loci involved in HSP on the X chromosome were also examined. Polymerase chain reaction (PCR) sequencing was used to detect gene mutations. To confirm the influence of a splice-site mutation on mRNA, we used reverse transcription-PCR and direct sequencing. Linkage analysis and ATL1 gene sequencing of amniocytes were performed for prenatal genetic diagnosis. One missense variant (c.1517T>A) and a splice-site mutation (c.1245+1G>A) in SPAST, and two missense variants (c.715C>T, c.1204T>G) in ATL1 were identified. The c.1245+1G>A mutation caused a deletion of exon 9 in the SPAST gene. Prenatal genetic diagnosis showed that fetus did not carry the ALT1 c.1204T>G mutation. Follow-up was maintained for 5 years, and the negative result was confirmed by evidence of a healthy growing boy. We identified two novel mutations and two previously reported mutations in SPAST and ATL1, respectively. The family with the ATL1 c.1204T>G mutation exhibited male-lethality, female infancy-onset, and pseudo- X-linked dominant transmission, which had never been previously reported for HSP. Characteristic facial features were also noticed. The boy on whom prenatal gene diagnosis was performed is healthy and without unusual facies, suggesting that the c.1204T>G mutation might be related to these features. The results extend the genetic spectrum of HSP and suggest that linkage analysis remains a powerful tool in gene discovery studies.

The molecular biology of the olive fly comes of age.

BackgroundOlive cultivation blends with the history of the Mediterranean countries since ancient times. Even today, activities around the olive tree constitute major engagements of several people in the countryside of both sides of the Mediterranean basin. The olive fly is, beyond doubt, the most destructive pest of cultivated olives. The female fly leaves its eggs in the olive fruit. Upon emergence, the larvae feed on the olive sap, thus destroying the fruit. If untreated, practically all olives get infected. The use of chemical insecticides constitutes the principal olive fly control approach. The Sterile Insect Technique (SIT), an environmentally friendly alternative control method, had been tried in pilot field applications in the 1970's, albeit with no practical success. This was mainly attributed to the low, non-antagonistic quality of the mixed-sex released insects. Many years of experience from successful SIT applications in related species, primarily the Mediterranean fruit fly, Ceratitis capitata, demonstrated that efficient SIT protocols require the availability of fundamental genetic and molecular information.ResultsAmong the primary systems whose understanding can contribute towards novel SIT approaches (or its recently developed alternative RIDL: Release of Insects carrying a Dominant Lethal) is the reproductive, since the ability to manipulate the reproductive system would directly affect the insect's fertility. In addition, the analysis of early embryonic promoters and apoptotic genes would provide tools that confer dominant early-embryonic lethality during mass-rearing. Here we report the identification of several genes involved in these systems through whole transcriptome analysis of female accessory glands (FAGs) and spermathecae, as well as male testes. Indeed, analysis of differentially expressed genes in these tissues revealed higher metabolic activity in testes than in FAGs/spermathecae. Furthermore, at least five olfactory-related genes were shown to be differentially expressed in the female and male reproductive systems analyzed. Finally, the expression profile of the embryonic serendipity-α locus and the pre-apoptotic head involution defective gene were analyzed during embryonic developmental stages.ConclusionsSeveral years of molecular studies on the olive fly can now be combined with new information from whole transcriptome analyses and lead to a deep understanding of the biology of this notorious insect pest. This is a prerequisite for the development of novel embryonic lethality female sexing strains for successful SIT efforts which, combined with improved mass-reared conditions, give new hope for efficient SIT applications for the olive fly.

Transgenic expression of the N525S-tuberin variant in Tsc2 mutant (Eker) rats causes dominant embryonic lethality.

The Tsc2 product, tuberin, negatively regulates the mTOR pathway. We have exploited the Eker (Tsc2-mutant) rat system to analyse various Tsc2 mutations. Here, we focus on the N525S-Tsc2 variant (NSM), which is known to cause distinct symptoms in patients even though normal suppression of mTOR is observed. Unexpectedly, we were repeatedly unable to generate viable rats carrying the NSM transgene. Genotypic analysis revealed that most of the embryos carrying the transgene died around embryonic day after 14.5—similar to the stage of lethality observed for Eker homozygotes. Thus, the NSM transgene appeared to have a dominant lethal effect in our rat model. Further, no significant differences were observed for various signal transduction molecules in transiently expressed NSM cells compared to WT. These results indicate that a non-mTOR pathway, critical for embryogenesis, is being regulated by tuberin, providing a link between tuberin expression and the severity of Tsc2 mutation-related pathogenesis.

Transgenic sexing system for genetic control of the Australian sheep blow fly Lucilia cuprina

The New World screwworm and the Australian sheep blowfly Lucilia cuprina are devastating pests of livestock. The larvae of these species feed on the tissue of the living animal and can cause death if untreated. The sterile insect technique or SIT was used to eradicate screwworm from North and Central America. This inspired efforts to develop strains containing complex chromosomal rearrangements for genetic control of L. cuprina in Australia. Although one field trial was promising, the approach was abandoned due to costs and difficulties in mass rearing the strain. As the efficiency of SIT can be significantly increased if only sterile males are released, we have developed transgenic strains of L. cuprina that carry a dominant tetracycline repressible female lethal genetic system. Lethality is due to overexpression of an auto-regulated tetracycline repressible transactivator (tTA) gene and occurs mostly at the pupal stage. Dominant female lethality was achieved by replacing the Drosophila hsp70 core promoter with a Lucilia hsp70 core promoter-5′UTR for tTA overexpression. The strains carry a dominant strongly expressed marker that will facilitate identification in the field. Interestingly, the sexes could be reliably sorted by fluorescence or color from the early first instar larval stage as females that overexpress tTA also overexpress the linked marker gene. Male-only strains of L. cuprina developed in this study could form the basis for a future genetic control program. Moreover, the system developed for L. cuprina should be readily transferrable to other major calliphorid livestock pests including the New and Old World screwworm.

Pediatric Genetics in the Clinic

Abstract Introduction This resource is a case-based learning module on pediatric genetics intended to introduce students to the genetic concepts of X-linked inheritance, dominant lethality, X-chrom...

The ORC/Cdc6/MCM2-7 complex facilitates MCM2-7 dimerization during prereplicative complex formation

The replicative mini-chromosome-maintenance 2–7 (MCM2-7) helicase is loaded in Saccharomyces cerevisiae and other eukaryotes as a head-to-head double-hexamer around origin DNA. At first, ORC/Cdc6 recruits with the help of Cdt1 a single MCM2-7 hexamer to form an ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex. Then, on ATP hydrolysis and Cdt1 release, the ‘initial’ complex is transformed into an ORC/Cdc6/MCM2-7 (OCM) complex. However, it remains unclear how the OCM is subsequently converted into a MCM2-7 double-hexamer. Through analysis of MCM2-7 hexamer-interface mutants we discovered a complex competent for MCM2-7 dimerization. We demonstrate that these MCM2-7 mutants arrest during prereplicative complex (pre-RC) assembly after OCM formation, but before MCM2-7 double-hexamer assembly. Remarkably, only the OCM complex, but not the ‘initial’ ORC/Cdc6/Cdt1/MCM2-7 complex, is competent for MCM2-7 dimerization. The MCM2-7 dimer, in contrast to the MCM2-7 double-hexamer, interacts with ORC/Cdc6 and is salt-sensitive, classifying the arrested complex as a helicase-loading intermediate. Accordingly, we found that overexpression of the mutants cause cell-cycle arrest and dominant lethality. Our work identifies the OCM complex as competent for MCM2-7 dimerization, reveals MCM2-7 dimerization as a limiting step during pre-RC formation and defines critical mechanisms that explain how origins are licensed.

SEC14 and Spectrin Domains 1 (Sestd1), Dishevelled 2 (Dvl2) and Dapper Antagonist of Catenin-1 (Dact1) co-regulate the Wnt/Planar Cell Polarity (PCP) pathway during mammalian development

We previously reported that Sestd1 KO phenocopies Dact1 KO in mice, consistent with a model in which Sestd1 and Dact1 act together to form a crucial functional complex that regulates Vangl2 in the Wnt/Planar Cell Polarity (PCP) pathway. Here, we show that Dvl2, a binding partner of Dact1, also forms complexes with Sestd1, and does so independently of both Dact1 and Vangl2. In cell-based assays, whereas Sestd1 does not alter Dvl2 activation of the Wnt/β-catenin signaling pathway, Dvl2 enhances activation of Rho family GTPases by Dact1 and Sestd1, consistent with a role in the PCP pathway. In mice, although Dvl2 KO is recessive in an otherwise wild type background, it leads to dominant embryonic lethality in either the Sestd1 or Dact1 KO background. This genetic synergy stands in contrast to the epistasis we have previously reported between Sestd1 and Dact1 KO, and suggests independent or semi-independent functions for Dvl2 vs. Sestd1/Dact1 in the regulation of the PCP pathway during development. In conclusion, biochemical and genetic interactions between Dvl2, Sestd1, and Dact1, in addition to prior reported interactions between these same molecules and Vangl2, suggest that all these gene products can form complexes together and regulate the PCP pathway during mammalian development. However, Sestd1 and Dact1 have a closely allied function in the post-translational regulation of Vangl2 that is at least partially distinct from the functions of Dvl2 in this pathway.