Molecular biologist in fields of DNA replication and repair, plasmids and bacteriophage, amoeboid cell signalling, early embryonic development, X-chromosome activity in development, late origin of germ line, epigenetic programming and deprogramming, embryonic stem cells, single cell molecular biology to detect gene mutation, gene transcription and cDNA libraries, and gene modification, preimplantation diagnosis of genetic disease, embryonic genes re-expressed in cancers.
We have constructed amplified cDNA preparations from replicate samples of human oocytes and indiv... more We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription-polymerase chain reaction (RT-PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.
A highly sensitive biochemical microassay has been developed for adenosine deaminase (ADA; EC 3.5... more A highly sensitive biochemical microassay has been developed for adenosine deaminase (ADA; EC 3.5.4.4), the enzyme deficient in approximately 20% of cases of severe combined immuno-deficiency disease (SCID). The microassay is capable of detecting femtomolar amounts of reaction product in a single blastomere from a mouse 8-cell embryo and thus is sensitive enough to be considered for the possible preimplantation diagnosis of SCID in human embryos.
Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) c... more Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) coded for by a gene on the X chromosome, compared with embryos from XX females. Groups of unfertilized eggs and individual embryos at the 8-cell, morula and blastocyst stages were assayed for HPRT activity. An autosomally coded enzyme (APRT) was assayed simultaneously in the same reaction mix as a control. There is a substantial increase in HPRT activity by the 8-cell stage. However, the mean activity of HPRT in embryos of XO mothers remains half that in embryos of XX mothers. This suggests a significant maternally inherited component of HPRT activity in 8-cell embryos. By the 9- to 16-cell morula stage the HPRT activities in the two groups of embryos become similar due, presumably, to a transition to embryo-coded activity; HPRT activities in individual morulae from XX mothers show a bimodal distribution consistent with the hypothesis that both X-chromosomes are active in XX embryos at th...
ABSTRACT Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of... more ABSTRACT Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt 220 to 13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endog- enous Xist gene). The testis-specific expression is corre- lated with hypomethylation of the transgene, although to different extents in different lines. Following paternal trans- mission, expression of the Xist promoter-luciferase con- struct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypometh- ylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development. Mol. Reprod. Dev. 49:356–367, 1998.
A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ line is show... more A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ line is shown by analysis of gene dosage effects on activity of an X-linked enzyme. The ratio of activities of the X-linked enzyme HPRT and an autosomal enzyme APRT are determined in XX and XY germ cells from embryonic gonads from the 12th to the 17th day of pregnancy. Mitotic stages of XX and XY germ cells on the 12th day have similar HPRT: APRT ratios, but on the 13th day the ratios are significantly higher in XX than XY germ cells. As the XX germ cells enter meiosis they show a marked increase in HPRT:APRT ratio which is primarily due to a rise in X-linked HPRT activity. Comparisons are made with XO germ cells on the 12th and 14th day. On the 12th day, XO do not differ from XX and XY germ cells, suggesting that only one X chromosome is active in XX germ cells at this stage. On the 14th day, on the other hand, the HPRT:APRT ratios in XO and XY germ cells are similar but in XX germ cells the ratio ...
International Journal of Developmental Biology, 2001
This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and... more This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and spatial aspects of X-chromosome inactivation and imprinting and the role of methylation in X-inactivation in these processes during female mouse embryo development. To enable molecular studies of embryos, we developed a plethora of single cell assays for specific enzyme activity, gene mutation and methylation, and RNA transcription. While in Anne's Unit, I used these same single cell assays to pioneer the procedures for preimplantation diagnosis of genetic disease, now an established clinical approach to prevention of the birth of children with severe genetic disease. At the Institute of Child Health in London, we continue to develop new highly sensitive molecular procedures--currently for the creation of cDNA libraries from human preimplantation embryos, primordial germ cells and embryonal stem cells. We are using these cDNA preparations to isolate human developmental genes and emb...
Journal of embryology and experimental morphology, 1978
Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) c... more Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) coded for by a gene on the X chromosome, compared with embryos from XX females. Groups of unfertilized eggs and individual embryos at the 8-cell, morula and blastocyst stages were assayed for HPRT activity. An autosomally coded enzyme (APRT) was assayed simultaneously in the same reaction mix as a control. There is a substantial increase in HPRT activity by the 8-cell stage. However, the mean activity of HPRT in embryos of XO mothers remains half that in embryos of XX mothers. This suggests a significant maternally inherited component of HPRT activity in 8-cell embryos. By the 9- to 16-cell morula stage the HPRT activities in the two groups of embryos become similar due, presumably, to a transition to embryo-coded activity; HPRT activities in individual morulae from XX mothers show a bimodal distribution consistent with the hypothesis that both X-chromosomes are active in XX embryos at th...
This review covers data on changing patterns of DNA methylation and the regulation of gene expres... more This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight-cell stage to the blastocyst stage in preimplantation embryos, and global de novo methylation begins at implantation. We have used X-chromosome inactivation in female embryos as a model system to study specific CpG sites in the X-linked Pgk-1 and G6pd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methylation of the X-linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X-chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra-embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by "adaptive epigenetic/genetic inheritance" is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become "fixed" as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are "acquired characteristics." An extension of this hypothesis is "foreign gene acquisition and sorting" (selection or silencing of gene function according to use). "Kidnapping" and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more "junk" DNA. Adaptive epigenetic/genetic inheritance challenges the "central dogma" that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution.
We have constructed amplified cDNA preparations from replicate samples of human oocytes and indiv... more We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription-polymerase chain reaction (RT-PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.
A highly sensitive biochemical microassay has been developed for adenosine deaminase (ADA; EC 3.5... more A highly sensitive biochemical microassay has been developed for adenosine deaminase (ADA; EC 3.5.4.4), the enzyme deficient in approximately 20% of cases of severe combined immuno-deficiency disease (SCID). The microassay is capable of detecting femtomolar amounts of reaction product in a single blastomere from a mouse 8-cell embryo and thus is sensitive enough to be considered for the possible preimplantation diagnosis of SCID in human embryos.
Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) c... more Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) coded for by a gene on the X chromosome, compared with embryos from XX females. Groups of unfertilized eggs and individual embryos at the 8-cell, morula and blastocyst stages were assayed for HPRT activity. An autosomally coded enzyme (APRT) was assayed simultaneously in the same reaction mix as a control. There is a substantial increase in HPRT activity by the 8-cell stage. However, the mean activity of HPRT in embryos of XO mothers remains half that in embryos of XX mothers. This suggests a significant maternally inherited component of HPRT activity in 8-cell embryos. By the 9- to 16-cell morula stage the HPRT activities in the two groups of embryos become similar due, presumably, to a transition to embryo-coded activity; HPRT activities in individual morulae from XX mothers show a bimodal distribution consistent with the hypothesis that both X-chromosomes are active in XX embryos at th...
ABSTRACT Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of... more ABSTRACT Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females, a process involving a regulatory gene, Xist (X-inactive specific transcript). Xist maps to the X-inactivation centre and is expressed from the inactive X chromosome in female somatic cells and at the time of X inactivation during spermatogenesis in the male. In female preimplantation embryos, Xist demonstrates imprinting in that the paternal allele inherited from the sperm is preferentially expressed. This preferential paternal Xist expression is correlated with paternal X inactivation in the extraembryonic lineages at the blastocyst stage. We have analysed a 233-bp Xist promoter fragment (nt 220 to 13) for its ability to direct appropriate expression and its regulation by DNA methylation. This minimal promoter sequence directs expression of the luciferase reporter gene following injection of the construct into one-cell embryos. In vitro methylation of the construct before injection represses transcription. In six different transgenic lines, expression of the Xist promoter-luciferase transgene occurs only in the testis of the males (as for the endog- enous Xist gene). The testis-specific expression is corre- lated with hypomethylation of the transgene, although to different extents in different lines. Following paternal trans- mission, expression of the Xist promoter-luciferase con- struct in preimplantation embryos is correlated with degree of hypomethylation in the testis and the degree of hypometh- ylation of the transgene in embryos at the morula stage. It is concluded that the patterns of methylation of the transgene in sperm (and in microinjected transgenes) can regulate the activity of the Xist promoter in the preimplantation embryo and thus support the hypothesis that gametic methylation patterns govern imprinted expression of the endogenous Xist gene in development. Mol. Reprod. Dev. 49:356–367, 1998.
A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ line is show... more A cycle of inactivation and reactivation of one X chromosome in the female (XX) germ line is shown by analysis of gene dosage effects on activity of an X-linked enzyme. The ratio of activities of the X-linked enzyme HPRT and an autosomal enzyme APRT are determined in XX and XY germ cells from embryonic gonads from the 12th to the 17th day of pregnancy. Mitotic stages of XX and XY germ cells on the 12th day have similar HPRT: APRT ratios, but on the 13th day the ratios are significantly higher in XX than XY germ cells. As the XX germ cells enter meiosis they show a marked increase in HPRT:APRT ratio which is primarily due to a rise in X-linked HPRT activity. Comparisons are made with XO germ cells on the 12th and 14th day. On the 12th day, XO do not differ from XX and XY germ cells, suggesting that only one X chromosome is active in XX germ cells at this stage. On the 14th day, on the other hand, the HPRT:APRT ratios in XO and XY germ cells are similar but in XX germ cells the ratio ...
International Journal of Developmental Biology, 2001
This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and... more This chapter reviews my 18 years of research in Anne's Unit including studies on temporal and spatial aspects of X-chromosome inactivation and imprinting and the role of methylation in X-inactivation in these processes during female mouse embryo development. To enable molecular studies of embryos, we developed a plethora of single cell assays for specific enzyme activity, gene mutation and methylation, and RNA transcription. While in Anne's Unit, I used these same single cell assays to pioneer the procedures for preimplantation diagnosis of genetic disease, now an established clinical approach to prevention of the birth of children with severe genetic disease. At the Institute of Child Health in London, we continue to develop new highly sensitive molecular procedures--currently for the creation of cDNA libraries from human preimplantation embryos, primordial germ cells and embryonal stem cells. We are using these cDNA preparations to isolate human developmental genes and emb...
Journal of embryology and experimental morphology, 1978
Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) c... more Embryos from XO female mice begin development with half the activity levels of an enzyme (HPRT) coded for by a gene on the X chromosome, compared with embryos from XX females. Groups of unfertilized eggs and individual embryos at the 8-cell, morula and blastocyst stages were assayed for HPRT activity. An autosomally coded enzyme (APRT) was assayed simultaneously in the same reaction mix as a control. There is a substantial increase in HPRT activity by the 8-cell stage. However, the mean activity of HPRT in embryos of XO mothers remains half that in embryos of XX mothers. This suggests a significant maternally inherited component of HPRT activity in 8-cell embryos. By the 9- to 16-cell morula stage the HPRT activities in the two groups of embryos become similar due, presumably, to a transition to embryo-coded activity; HPRT activities in individual morulae from XX mothers show a bimodal distribution consistent with the hypothesis that both X-chromosomes are active in XX embryos at th...
This review covers data on changing patterns of DNA methylation and the regulation of gene expres... more This review covers data on changing patterns of DNA methylation and the regulation of gene expression in mouse embryonic development. Global demethylation occurs from the eight-cell stage to the blastocyst stage in preimplantation embryos, and global de novo methylation begins at implantation. We have used X-chromosome inactivation in female embryos as a model system to study specific CpG sites in the X-linked Pgk-1 and G6pd housekeeping genes and in the imprinted regulatory Xist gene to elucidate the role of methylation in the initiation and maintenance of differential gene activity. Methylation of the X-linked housekeeping genes occurs very close in time to their inactivation, thus raising the question as to whether methylation could be causal to inactivation, as well as being involved in its maintenance. A methylation difference between sperm and eggs in the promoter region of the Xist gene, located at the X-chromosome inactivation centre, is correlated with imprinted preferential inactivation of the paternal X chromosome in extra-embryonic tissues. Based on our data, a picture of the inheritance of methylation imprints and speculation on the significance of the Xist imprint in development is presented. On a more general level, an hypothesis of evolution by "adaptive epigenetic/genetic inheritance" is considered. This proposes modification of germ line DNA in response to a change in environment and mutation at the site of modification (e.g., of methylated cytosine to thymine). Epigenetic inheritance could function to shift patterns of gene expression to buffer the evolving system against changes in environment. If the altered patterns of gene activity and inactivity persist, the modifications may become "fixed" as mutations; alternatively, previously silenced gene networks might be recruited into function, thus appearing as if they are "acquired characteristics." An extension of this hypothesis is "foreign gene acquisition and sorting" (selection or silencing of gene function according to use). "Kidnapping" and sorting of foreign genes in this way could explain the observation that increased complexity in evolution is associated with more "junk" DNA. Adaptive epigenetic/genetic inheritance challenges the "central dogma" that information is unidirectional from the DNA to protein and the idea that Darwinian random mutation and selection are the sole mechanisms of evolution.
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