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Biomedical subjects

W Reik

Publications and source records attributed to W Reik.

At least 73 records · Page 4Linked to original sources
PubMed1996

Instability of long inverted repeats within mouse transgenes.

Various sequences in the mammalian genomes are unstable. One class of sequence arrangement is long inverted repeats, which are known to be unstable in bacteria and yeast. While in mammals some evidence suggests that short inverted repeats (<10 bp long) may show instability, nothing is known about the stability of long inverted repeats. Here we describe two unrelated multicopy transgenes in the mouse (loci 109 and OX1-5), each of which contains a long inverted repeat that shows substantial mitotic instability. This instability also occurs in the germline so that mutant transgenes appear within pedigrees at a high frequency. The mutation processes acting at these two inverted repeats are complex and can involve insertion or deletion, and can result in stabilization of the transgene. At transgene 109 mutational events range from very small rearrangements at the centre of the inverted repeat to complete transgene deletion. In addition we show that the rates of mutation at the inverted repeat of transgene OX1-5 can vary between the male and female germlines and between inbred strains of mice, suggesting the possibility of a genetic analysis to identify loci that modulate inverted repeat instability.

Animals↗
PubMed1996

Imprinting mutation in the Beckwith-Wiedemann syndrome leads to biallelic IGF2 expression through an H19-independent pathway.

The Beckwith-Wiedemann syndrome (BWS) is genetically linked to chromosome 11p15.5, and a variety of observations suggest that deregulation of imprinted genes in this region is causally involved in the pathogenesis of the disease. It has been shown that in some patients without cytogenetic abnormalities the otherwise repressed maternal copy of the insulin-like growth factor 2 (IGF2) gene is expressed, leading to biallelic expression of IGF2. In some of these cases, this is accompanied by repression and DNA methylation of the maternal (otherwise active) copy of the neighbouring H19 gene. Hence, it is attractive to think that mutations may interfere with some aspect of H19 imprinting, thus leading to an inactive maternal allele, and indirectly to activation of the maternal IGF2 allele as reported in mice with an H19 gene deletion. However, no mutations have been identified so far in these patients. The only known mutations associated with BWS are maternally transmitted translocations, which are clustered in two locations centrometric to IGF2. The first cluster is 200-400 kb from IGF2 and the second is several megabases away. Hence, genes located far from the translocation breakpoints are potentially deregulated by them. Here we provide the first evidence of alteration of imprinting in a translocation family, with biallelic expression of IGF2 and altered DNA replication patterns in the IGF2 region. Interestingly, H19 imprinting was normal, suggesting an H19-independent pathway to biallelic IGF2 transcription. DNA methylation in IGF2 remained monoallelic, suggesting that the mutation in this family had uncoupled allele-specific methylation from expression.

Alleles↗
PubMed1996

The Wellcome Prize Lecture. Genetic imprinting: the battle of the sexes rages on.

Genomic imprinting in mammals is an important genetic mechanism by which genes are expressed or repressed depending on which parent they have been inherited from. Some properties of the imprinting mechanism are already established; notably, some of the effects of imprinting on mammalian development can be explained by the phenotypic effects of a number of specific imprinted genes, which include major fetal growth factors. An evolutionary explanation of imprinting has also been suggested. Some of the molecular mechanisms of imprinting are known, and these include the modification of DNA and chromosomes in the form of DNA methylation and possibly heritable chromatin structures. Loss of imprinting or altered imprinting is implicated in a large number of genetic diseases and cancers. Many important issues remain to be resolved; these include the precise molecular mechanisms and, in particular, the nature of the primary imprints that are inherited from the parental gametes, and the genes that control the imprinting process. Isolation of the majority of imprinted genes and the elucidation of their phenotypic effects and physiology are major goals for the future. These studies will provide important insights into human genetics, and will connect evolutionary understanding with physiology, genetic disease and human behaviour.

Animals↗
PubMed1996

Genetic conflict in early development: parental imprinting in normal and abnormal growth.

Parental (genomic) imprinting is the process by which the differential expression of maternal and paternal alleles at certain genetic loci in mammalian embryos occurs. Such loci are implicated in the control of fetal, placental and neonatal growth, and, more generally, in diverse aspects of fetal nutrient acquisition and maternal-fetal interactions. Not surprisingly, the aberrant expression of imprinted genes is implicated in a range of embryonic and fetal abnormalities. We outline how an evolutionary theory, based on classic parent-offspring conflict theory, relates to certain fetal growth abnormalities. In particular, we suggest that growth abnormalities resulting from the manipulation of preimplantation mammalian embryos in vitro (for example large calf syndrome) may reflect the occurrence of genetic conflict over the fetal growth programme in the early preimplantation period.

Cell Nucleus↗
PubMed1996

Analysis of parent-specific gene expression in early mouse embryos and embryonic stem cells using high-resolution two-dimensional electrophoresis of proteins.

Genomic imprinting is an important genetic mechanism in mammals whereby certain genes are epigenetically modified and their expression altered according to their parental origin. The most important consequence of this is the requirement for both a maternal and a paternal genome for normal development to proceed to term. Although there are many instances of specific phenotypes (in the mouse) and diseases (in humans) resulting from imbalances in the parental chromosomes, it is only in the past few years that some of the imprinted genes responsible have been identified. It is however unclear what proportion of the genome is imprinted, particularly in the early embryo. To address the question to what extent parent-specific gene expression occurs in the early embryo and with a possible view to identifying new imprinted genes, the protein profiles of parthenogenetic and normal blastocysts were compared using the technique of high-resolution two-dimensional electrophoresis. The protein profiles of parthenogenetic, androgenetic and normal embryonic stem cells were also compared. Hence parent-specific gene expression was examined in embryonic and extraembryonic lineages of the early embryo. Approximately 1000 polypeptides were examined in each of the analyses, however no parent-specific differences were observed for any of these polypeptides. From this result, it is concluded that expression of genes encoding these polypeptides is identical from the parental chromosomes. These findings have important implications for estimates of the number of imprinted genes in the genome and for the interpretation of phenotypes of parthenogenetic and androgenetic embryos.

Animals↗
PubMed1995

Genetic conflict and evolution of mammalian X-chromosome inactivation.

The existence of parentally imprinted gene expression in the somatic tissues of mammals and plants can be explained by a theory of intragenomic genetic conflict, which is a logical extension of classical parent-offspring conflict theory. This theory unites conceptually the phenomena of autosomal imprinting and X-chromosome inactivation. We argue that recent experimental studies of X-chromosome inactivation and androgenetic development address previously published predictions of the conflict theory, and we discuss possible explanations for the occurrence of random X-inactivation in the somatic tissues of eutherians.

Animals↗
PubMed1995

Chromatin structure and imprinting: developmental control of DNase-I sensitivity in the mouse insulin-like growth factor 2 gene.

The insulin-like growth factor 2 (Igf2) gene on distal mouse chromosome 7 is expressed predominantly from the paternal allele. In previous studies we identified two regions of paternal allele-specific methylation; one at approximately 3 kb upstream of promoter 1, and a second in the 3', coding portion of the gene. The 3' region is methylated in an expressing tissue (fetal liver), whereas in a non-expressing tissue (fetal brain), it is not methylated. By contrast, in the 5' region, the paternal allele is highly methylated in all tissues. Here, we have studied another characteristic of chromatin, namely, sensitivity to DNase-I and have focused our developmental analysis on the two differentially methylated regions of Igf2. In the upstream region, four clustered DNase-I hypersensitive sites (HSS) were detected in embryonic stem (ES) cells and in midgestation embryos, but not in neonatal liver or brain. In promoter 1 (P1), at approximately 0.3 kb upstream of exon 1, we detected a tissue-specific HSS that was present in neonatal liver, in which P1 is active, but was absent in ES cells, the embryo, and in neonatal brain. No DNase-I HSS were detected in the 3' differentially methylated region of Igf2. In all these regions, we did not detect differences in DNase-I sensitivity between the parental chromosomes. These results establish major developmental and tissue-specific control of chromatin in the Igf2 locus. The presence of the HSS upstream of Igf2 precedes transcriptional activation of the Igf2 gene and may be indicative of a promoter for another transcript that is transcribed in the opposite direction. The HSS in P1 is largely liver-specific; this promoter therefore is differently regulated than the more general fetal promoters P2 and P3. Whereas methylation can be allele-specific, presumably reflecting the gene imprint, the nuclease sensitivity, as detected by our assay, is not. These results, taken together with previous observations, reveal developmental and tissue-specific complexity in the expression of the parental imprint at the level of chromatin and transcription. We propose that epigenetic features of tissue-specific control and of the control of allelic expression are intricately linked.

Alleles↗
PubMed1995

A novel strategy to identify maternal and paternal inheritance in the mouse.

A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two-dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N-terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: alpha-enolase, cyclophilin and beta-group hemoglobins. The parental effects observed for alpha-enolase and cyclophilin were found to be due to parent-specific post-translational modifications of these proteins. For the beta-group hemoglobins our results suggested parental effects on the transcriptional level.

Amino Acid Isomerases↗
PubMed1995

Imprinting mutations in the Beckwith-Wiedemann syndrome suggested by altered imprinting pattern in the IGF2-H19 domain.

Regional regulations of parental imprinting in the IGF2-H19 domain of imprinted genes was studied in the Beckwith-Wiedemann syndrome (BWS). We identified BWS patients who had inherited a normal biparental chromosome complement of the chromosome 11p15.5 region (where IGF2 and H19 reside), but had an altered pattern of allelic methylation of both genes, with the maternal chromosome carrying a parental imprinting pattern. In fibroblasts, IGF2 was expressed from both parental alleles and H19 was not expressed, precisely as predicted from the altered pattern of allelic methylation. Interestingly, DNA replication patterns of the 11p15.5 region remained asynchronous as in controls. Our results therefore provide the first example of the dissociation of regional control of DNA replication from regional control of allelic methylation and expression in imprinting. We suggest that the altered pattern of allelic methylation and expression arises in the germline or in the early embryo from defects in resetting or setting of imprinting in maternal germline. Potential candidate regions for mutations include the previously identified translocation breakpoint clusters and the H19 gene itself. The finding of possible 'imprinting mutations' in BWS raises the prospect of identifying genetic factors that control imprinting in this region.

Base Sequence↗
PubMed1995

Analysis of polysulfate-binding domains in porcine proacrosin, a putative zona adhesion protein from mammalian spermatozoa.

Proacrosin is one of the major proteins found within the acrosomal vesicle of mammalian spermatozoa. Previous work has shown that it binds non-enzymatically and with high affinity to polysulfate groups on zona pellucida glycoproteins (ZPGPs) thereby leading to the hypothesis that at fertilization it functions as a secondary ligand molecule to retain acrosome-reacted spermatozoa on the surface of the egg. In the present work we have investigated the nature and extent of the polysulfate binding domain on boar sperm proacrosin using a combination of group-specific modifying reagents, fragmentation analysis, peptide synthesis and expression of deletion recombinants in E. coli bacteria. Taken overall, our results show that arginine, lysine and histidine residues located between Gly 93 and Ala 275, together with the participation of His 47 and Arg 50, are necessary for maximum polysulfate binding activity. The secondary and tertiary structure of this central peptide domain is also important to ensure correct alignment of basic residues with complementary sulfate groups on ZPGPs. Proacrosin, therefore, has many properties in common with other polysulfate binding proteins, such as antithrombin III and sea urchin sperm binding, in having a conformation-dependent domain containing basic amino acids that mediates specific protein-protein interactions. These observations strengthen the hypothesis that proacrosin is a multifunctional protein with a major role as a ligand molecule at fertilization.

Acrosin↗
PubMed1994

Allelic methylation of H19 and IGF2 in the Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome with associated embryonal tumours. Most BWS cases are sporadic but familial cases occur in 15% of patients and in these there is linkage to chromosome 11p15. In addition, a small number of patients have cytogenetic abnormalities involving chromosome 11p15. Approximately 20% of sporadic BWS patients have uniparental paternal disomy (UPD) of chromosome 11p15. This finding together with the observation that penetrance in familial cases depends on parental transmission, suggests that the gene(s) for BWS are imprinted. The recent demonstration of biallelic expression of the otherwise maternally imprinted IGF2 gene in some BWS patients implicates excess IGF2 expression in the disease. Here we have analysed the allele-specific methylation patterns in the IGF2 gene and in the neighbouring and reciprocally imprinted H19 gene in a group of 42 BWS patients, 10 of which were mosaic UPD cases. We found that allelic methylation of both genes was normal in all non-UPD cases, with the paternal allele being methylated, and was increased in UPD cases in proportion with the disomic lineage. These findings suggest that sporadic BWS is not associated with a general alteration of methylation imprinting of the IGF2 and H19 genes. The methylation assay used in this study thus also offers a simple and reliable diagnostic test of UPD for 11p15.5. An unexpected finding was a distortion of the frequency of AvaII alleles at the IGF2 locus exclusively in UPD BWS cases (P < 0.001). This further implicates the IGF2 gene in aspects of the BWS phenotype.

Alleles↗
PubMed1994

Developmental control of allelic methylation in the imprinted mouse Igf2 and H19 genes.

The Insulin-like growth factor 2 (Igf2) and H19 genes are reciprocally imprinted and closely linked. Igf2 encodes a fetal growth-factor and is predominantly expressed from the paternal allele, while H19 is expressed from the maternal allele and encodes a transcript which may downregulate cellular proliferation. One of the epigenetic modifications thought to be involved in parental imprinting is DNA methylation. Here we analyse methylation in two regions of the Igf2 gene, one approx. 3 kb upstream of the gene and one in the 3' part of the gene. Both regions are more methylated on the expressed paternal chromosome. Genomic sequencing of individual chromosomes in the first region shows this parent-specific methylation to be highly mosaic; interestingly, individual sperm chromosomes carry different methylation patterns into the egg. In the more 3' region, which is fully methylated in sperm, the level of methylation on the paternal allele is highly tissue-specific and is correlated with expression of the gene in fetal tissues. Hence, the paternal allele is highly methylated in fetal liver (high expression) but is undermethylated in fetal brain (virtually no expression). Adult choroid plexus, a brain tissue in which Igf2 is expressed from both alleles and H19 is not expressed, represents an apparent loss of imprinting. Here, both Igf2 and H19 adopt a paternal type methylation pattern on both parental chromosomes. Analysis of early-passage androgenetic and parthenogenetic embryonic stem (ES) cells shows that the methylation patterns of Igf2 and H19 on maternal and paternal chromosomes are very similar. Androgenetic and parthenogenetic teratomas derived from these ES cells show the appropriate paternal and maternal patterns, respectively, of allelic methylation in both genes. Our results suggest that allelic methylation patterns in Igf2 and H19 arise early in embryogenesis and change progressively during development. Some of these developmental changes are apparently under tissue-specific control.

Alleles↗
PubMed1993

The inheritance of germline-specific epigenetic modifications during development.

Parental genomes in mammals are programmed in the germline with heritable epigenetic modifications that exert control on the expression of specific (imprinted) genes. DNA methylation is one form of epigenetic modification which shows marked genome-wide variations in the germline and during early development. Certain transgene loci also demonstrate (reversible) germline-specific methylation imprints that are heritable in somatic tissues during development. Recently, four endogenous genes have been identified whose expression is dependent on their parental origin. The mechanism of genomic imprinting and the role of imprinted genes during development is beginning to be analysed. Three of these genes map to the mouse chromosome 7. Human chromosomes 11p13, 11p15, and 15q11-13 are associated with disorders exhibiting parental origin effects in their patterns of inheritance. These regions share syntenic homology with mouse chromosome 7. The relationship between parental imprints, germline-dependent epigenetic inheritance and totipotency is also under investigation using embryonic stem cells derived from the epiblast. These cells are pluripotent or totipotent and evidence indicates the presence of at least the primary parental imprints. However, imprints inherited from the paternal germline in androgenetic cells are apparently more stable than those from the female germline in parthenogenetic cells.

Animals↗