Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies

In vitro studies of muscle mitochondrial metabolism in patients with mitochondrial myopathy have identified a variety of functional defects of the mitochondrial respiratory chain, predominantly affecting complex I (NADH-CoQ reductase) or complex III (ubiquinol-cytochrome c reductase) in adult cases. These two enzymes consist of approximately 36 subunits, eight of which are encoded by mitochondrial DNA (mtDNA). The increased incidence of maternal, as opposed to paternal, transmission in familial mitochondrial myopathy suggests that these disorders may be caused by mutations of mtDNA. Multiple restriction endonuclease analysis of leukocyte mtDNA from patients with the disease, and their relatives, showed no differences in cleavage patterns between affected and unaffected individuals in any single maternal line. When muscle mtDNA was studied, nine of 25 patients were found to have two populations of muscle mtDNA, one of which had deletions of up to 7 kilobases in length. These observations demonstrate that mtDNA heteroplasmy can occur in man and that human disease may be associated with defects of the mitochondrial genome.     

Evidence from mitochondrial DNA that African honey bees spread as continuous maternal lineages

African honey bees have populated much of South and Central America and will soon enter the United States. The mechanism by which they have spread is controversial. Africanization may be largely the result of paternal gene flow into extant European populations or, alternatively, of maternal migration of feral swarms that have maintained an African genetic integrity. We have been using both mitochondrial and nuclear DNA restriction fragment length polymorphisms to follow the population dynamics between European and African bees. In earlier reports, we suggested that if African honey bees had distinctive mitochondrial (mt) DNA, then it could potentially distinguish the relative contributions of swarming and mating to the Africanization process. Because mtDNA is maternally inherited, it would not be transmitted by mating drones and only transported by queens accompanying swarms. Furthermore, the presence of African mtDNA would reflect unbroken maternal lineages from the original bees introduced from Africa. The value of mtDNA for population studies in general has been reviewed recently. Here we report that 19 feral swarms, randomly caught in Mexico, all carried African mtDNA. Thus, the migrating force of the African honey bee in the American tropics consists of continuous African maternal lineages spreading as swarms. The mating of African drones to European queens seems to contribute little to African bee migration.     

Paternal inheritance of mitochondrial DNA in mice.

For nearly 20 years it has been assumed on the basis of low-resolution experiments that mitochondrial (mt)DNA, in contrast to the genes in the nucleus, has an exclusively maternal mode of inheritance in animals. Using the polymerase chain reaction, paternally inherited mtDNA molecules have now been detected in mice at a frequency of 10(-4), relative to the maternal contributions. These mice were hybrids between two inbred strains (C57BL/6J and Mus spretus) whose mtDNAs can be distinguished easily. This new mode of inheritance provides a mechanism for generating heteroplasmy and may explain mitochondrial disorders exhibiting biparental transmission.     

Birth of parthenogenetic mice that can develop to adulthood

Only mammals have relinquished parthenogenesis, a means of producing descendants solely from maternal germ cells. Mouse parthenogenetic embryos die by day 10 of gestation. Bi-parental reproduction is necessary because of parent-specific epigenetic modification of the genome during gametogenesis. This leads to unequal expression of imprinted genes from the maternal and paternal alleles. However, there is no direct evidence that genomic imprinting is the only barrier to parthenogenetic development. Here we show the development of a viable parthenogenetic mouse individual from a reconstructed oocyte containing two haploid sets of maternal genome, derived from non-growing and fully grown oocytes. This development was made possible by the appropriate expression of the Igf2 and H19 genes with other imprinted genes, using mutant mice with a 13-kilobase deletion in the H19 gene as non-growing oocytes donors. This full-term development is associated with a marked reduction in aberrantly expressed genes. The parthenote developed to adulthood with the ability to reproduce offspring. These results suggest that paternal imprinting prevents parthenogenesis, ensuring that the paternal contribution is obligatory for the descendant.     

Differential activity of maternally and paternally derived chromosome regions in mice

Although both parental sexes contribute equivalent genetic information to the zygote, in mammals this information is not necessarily functionally equivalent. Diploid parthenotes possessing two maternal genomes are generally inviable, embryos possessing two paternal genomes in man may form hydatidiform moles, and nuclear transplantation experiments in mice have shown that both parental genomes are necessary for complete embryogenesis. Not all of the genome is involved in these parental effects, however, because zygotes with maternal or paternal disomy for chromosomes 1, 4, 5, 9, 13, 14 and 15 of the mouse survive normally. On the other hand, only the maternal X chromosome is active in mouse extraembryonic membranes, maternal disomy 6 is lethal, while non-complementation of maternal duplication/paternal deficiency or its reciprocal for regions of chromosome 2, 8 and 17 has been recognized. We report that animals with maternal duplication/paternal deficiency and its reciprocal for each of two particular chromosome regions show anomalous phenotypes which depart from normal in opposite directions, suggesting a differential functioning of gene loci within these regions. A further example of non-complementation lethality is also reported.     

The genetic basis of family conflict resolution in mice

Asymmetries in the costs and benefits of parental investment for mothers, fathers and offspring result in family conflict over the production and provisioning of young. In species where females provide most resources before and after birth, the resolution of this conflict may be influenced by genes expressed in mothers and by maternally and paternally inherited genes expressed in offspring. Here we disentangle these effects by means of reciprocal mating and cross-fostering of litters between two strains of mice that differ with respect to the typical resolution of family conflict. We find that differences in litter size between these two strains are determined by paternal genotype, whereas differences in provisioning are under maternal control, showing that there is antagonistic coadaptation of maternal and paternal effects on distinct life-history traits. Maternal provisioning is also influenced by the type of foster offspring. Contradictory to theoretical expectations, however, we find no evidence for a negative correlation across strains between maternal provisioning and offspring demand. Instead, we show that there is positive coadaptation such that offspring obtain more resources from foster mothers of the same strain as their natural mother, irrespective of their father's strain.     

Genomic imprinting determines methylation of parental alleles in transgenic mice

Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.     

Parental origin of chromosomes involved in the translocation t(9;22).

Functionally equivalent genetic maternal can be labelled by an epigenetic marking process and used differentially depending on whether its origin is maternal or paternal. This phenomenon is known as genomic imprinting and is manifested at either the chromosomal or gene level. Genomic imprinting seems to play an important role in cancer predisposition syndromes, and phenotypic consequences are evident in constitutional deletion syndromes and uniparental disomies. Moreover, there seems to be a preferential retention of paternal alleles in sporadic tumours such as Wilms' tumour, rhabdomyosarcoma, osteosarcoma and retinoblastoma. To investigate whether chromosomes involved in acquired abnormalities of haematologic neoplasms show a similar 'parent of origin' bias, we studied the inheritance of the translocated chromosomes 9 and 22 in cases of Philadelphia-chromosome-positive leukaemia, using unique specific chromosome band polymorphisms. Here we show that the translocated chromosome 9 was of paternal origin, whereas the translocated chromosomes 22 were derived exclusively from the maternal copy, in 11 cases with reliable polymorphisms. Our data therefore provide evidence that imprinting phenomena may play an important role in acquired tumour-specific chromosome rearrangements.     

Embryological and molecular investigations of parental imprinting on mouse chromosome 7

Mouse embryos with duplications of whole maternal (parthenogenetic and gynogenetic) or paternal (androgenetic) genomes show reciprocal phenotypes and do not develop to term. Genetic complementation has identified the distal region of chromosome 7 (Chr 7) as one of the regions for which both a maternal and paternal chromosome copy are essential for normal development, presumably because of the presence of imprinted genes whose expression is dependent on their parental origin. Embryos with the maternal duplication and paternal deficiency of distal Chr 7 are growth retarded and die around day 16 of gestation; the reciprocal paternal duplication embryos die at an unidentified earlier stage. We report here the incorporation of cells with the paternal duplication into chimaeras, resulting in a striking growth enhancement of the embryos. One gene located on mouse distal Chr 7 (ref. 5) is the insulin-like growth factor 2 (Igf2) gene, an embryonic mitogen. In embryos with the maternal duplication of distal Chr 7, the two maternal alleles of the Igf2 gene are repressed. The presence of two paternal alleles of this gene in many cells is probably responsible for the growth enhancement observed in chimaeras. We propose that there are other imprinted genes in this Chr 7 region. We also compare the imprinting of this subgenomic region with phenotypes resulting from the duplication of the whole parental genome in parthenogenones and androgenones.     

The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus

In sexually reproducing animals, a crucial step in zygote formation is the decondensation of the fertilizing sperm nucleus into a DNA replication-competent male pronucleus. Genome-wide nucleosome assembly on paternal DNA implies the replacement of sperm chromosomal proteins, such as protamines, by maternally provided histones. This fundamental process is specifically impaired in sésame (ssm), a unique Drosophila maternal effect mutant that prevents male pronucleus formation. Here we show that ssm is a point mutation in the Hira gene, thus demonstrating that the histone chaperone protein HIRA is required for nucleosome assembly during sperm nucleus decondensation. In vertebrates, HIRA has recently been shown to be critical for a nucleosome assembly pathway independent of DNA synthesis that specifically involves the H3.3 histone variant. We also show that nucleosomes containing H3.3, and not H3, are specifically assembled in paternal Drosophila chromatin before the first round of DNA replication. The exclusive marking of paternal chromosomes with H3.3 represents a primary epigenetic distinction between parental genomes in the zygote, and underlines an important consequence of the critical and highly specialized function of HIRA at fertilization.     

Delayed activation of the paternal genome during seed development

Little is known about the timing of the maternal-to-zygotic transition during seed development in flowering plants. Because plant embryos can develop from somatic cells or microspores, maternal contributions are not considered to be crucial in early embryogensis. Early-acting embryo-lethal mutants in Arabidopsis, including emb30/gnom which affects the first zygotic division, have fuelled the perception that both maternal and paternal genomes are active immediately after fertilization. Here we show that none of the paternally inherited alleles of 20 loci that we tested is expressed during early seed development in Arabidopsis. For genes that are expressed at later stages, the paternally inherited allele becomes active three to four days after fertilization. The genes that we tested are involved in various processes and distributed throughout the genome, indicating that most, if not all, of the paternal genome may be initially silenced. Our findings are corroborated by genetic studies showing that emb30/gnom has a maternal-effect phenotype that is paternally rescuable in addition to its zygotic lethality. Thus, contrary to previous interpretations, early embryo and endosperm development are mainly under maternal control.     

Microorganisms associated with chromosome destruction and reproductive isolation between two insect species

Microorganisms have been implicated in causing cytoplasmic incompatibility in a variety of insect species, including mosquitoes, fruitflies, beetles and wasps. The effect is typically unidirectional: incompatible crosses produce no progeny or sterile males, whereas the reciprocal crosses produce normal progeny. The parasitic wasp Nasonia vitripennis is one of the few species in which the cytogenetic mechanism of incompatibility is known. In this species the paternal chromosome set forms a tangled mass in a fertilized egg and is eventually lost. Here we report that cytoplasmic microorganisms are associated with complete bidirectional incompatibility between N. vitripennis and a closely related sympatric species, N. giraulti. Microorganisms can be seen in the eggs of both species. Hybrid offspring are normally not produced in crosses between the two species, but do occur after elimination of the microorganisms by antibiotic treatment. A cytogenetic and genetic study shows that bidirectional interspecific incompatibility is due to improper condensation of the paternal chromosomes. Microorganism-mediated reproductive isolation is of interest because it could provide a rapid mode of speciation. The mechanism of incompatibility in Nasonia is also of interest as a potential tool for studying chromosome imprinting and chromosome condensation.     

CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus

The Insulin-like growth factor 2 (Igf2) and H19 genes are imprinted, resulting in silencing of the maternal and paternal alleles, respectively. This event is dependent upon an imprinted-control region two kilobases upstream of H19 (refs 1, 2). On the paternal chromosome this element is methylated and required for the silencing of H19 (refs 2-4). On the maternal chromosome the region is unmethylated and required for silencing of the Igf2 gene 90 kilobases upstream. We have proposed that the unmethylated imprinted-control region acts as a chromatin boundary that blocks the interaction of Igf2 with enhancers that lie 3' of H19 (refs 5, 6). This enhancer-blocking activity would then be lost when the region was methylated, thereby allowing expression of Igf2 paternally. Here we show, using transgenic mice and tissue culture, that the unmethylated imprinted-control regions from mouse and human H19 exhibit enhancer-blocking activity. Furthermore, we show that CTCF, a zinc finger protein implicated in vertebrate boundary function, binds to several sites in the unmethylated imprinted-control region that are essential for enhancer blocking. Consistent with our model, CTCF binding is abolished by DNA methylation. This is the first example, to our knowledge, of a regulated chromatin boundary in vertebrates.     

Degree of methylation of transgenes is dependent on gamete of origin

Data derived from both pronuclear transplantation experiments and classical genetic experiments indicate that the maternal and paternal genetic contributions to the mammalian zygote nucleus do not function equivalently during subsequent development. These observations have been interpreted as resulting from differential 'genome imprinting' during male and female gametogenesis. The molecular mechanism responsible for genome imprinting is unknown, but data gathered to date require that the mechanism fulfill at least four criteria: (1) the imprint must be physically linked to the pronucleus; (2) the imprint must persist through DNA replication and cell division; (3) the mechanism must be capable of affecting gene expression; and (4) the mechanism must be capable of switching the identity of the imprint from one sex to the other in successive generations. One molecular mechanism which could satisfy the first three criteria is differential DNA methylation during gametogenesis itself, or before formation of the zygote nucleus during embryogenesis. We present data indicating that the methylation patterns of exogenous DNA sequences in transgenic mice can be changed by switching their gamete of origin in successive generations. These data suggest that DNA methylation can also satisfy the fourth criterion for an imprinting mechanism.     

Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting

Imprinted genes are epigenetically marked during gametogenesis so that they are exclusively expressed from either the paternal or the maternal allele in offspring. Imprinting prevents parthenogenesis in mammals and is often disrupted in congenital malformation syndromes, tumours and cloned animals. Although de novo DNA methyltransferases of the Dnmt3 family are implicated in maternal imprinting, the lethality of Dnmt3a and Dnmt3b knockout mice has precluded further studies. We here report the disruption of Dnmt3a and Dnmt3b in germ cells, with their preservation in somatic cells, by conditional knockout technology. Offspring from Dnmt3a conditional mutant females die in utero and lack methylation and allele-specific expression at all maternally imprinted loci examined. Dnmt3a conditional mutant males show impaired spermatogenesis and lack methylation at two of three paternally imprinted loci examined in spermatogonia. By contrast, Dnmt3b conditional mutants and their offspring show no apparent phenotype. The phenotype of Dnmt3a conditional mutants is indistinguishable from that of Dnmt3L knockout mice, except for the discrepancy in methylation at one locus. These results indicate that both Dnmt3a and Dnmt3L are required for methylation of most imprinted loci in germ cells, but also suggest the involvement of other factors.     

Role of paternal and maternal genomes in mouse development

There has been much speculation on whether mammalian eggs with two male pronuclei can develop normally. Eggs with two female pronuclei can sometimes develop as far as the 25-somite stage but with only very meagre extraembryonic tissues. We suggested that the genome undergoes specific imprinting during gametogenesis and that some paternal genes may be necessary for normal development of the extraembryonic tissues, in which only the maternal X chromosome remains active. However, the need for the maternal genome for development to term is not yet unequivocally established. The detailed study described here demonstrates that while between 40 and 50% of heterozygous reconstituted eggs with a male and a female pronucleus develop to term, none of the eggs with two male pronuclei does so. Furthermore, embryos in the latter case are very retarded, even though the trophoblast develops relatively well compared with embryos having two female pronuclei. Our combined results indicate that while the paternal genome is essential for the normal development of extraembryonic tissues, the maternal genome may be essential for some stages of embryogenesis.     

A global disorder of imprinting in the human female germ line

Imprinted genes are expressed differently depending on whether they are carried by a chromosome of maternal or paternal origin. Correct imprinting is established by germline-specific modifications; failure of this process underlies several inherited human syndromes. All these imprinting control defects are cis-acting, disrupting establishment or maintenance of allele-specific epigenetic modifications across one contiguous segment of the genome. In contrast, we report here an inherited global imprinting defect. This recessive maternal-effect mutation disrupts the specification of imprints at multiple, non-contiguous loci, with the result that genes normally carrying a maternal methylation imprint assume a paternal epigenetic pattern on the maternal allele. The resulting conception is phenotypically indistinguishable from an androgenetic complete hydatidiform mole, in which abnormal extra-embryonic tissue proliferates while development of the embryo is absent or nearly so. This disorder offers a genetic route to the identification of trans-acting oocyte factors that mediate maternal imprint establishment.