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Biomedical subjects

M Monk

Publications and source records attributed to M Monk.

At least 55 records · Page 3Linked to original sources
PubMed1992

Methylation of CpG sites of two X-linked genes coincides with X-inactivation in the female mouse embryo but not in the germ line.

To further our understanding of initiation and imprinting of X-chromosome inactivation, we have examined methylation of specific CpG sites of X-linked Pgk-1 and G6pd genes throughout female mouse development. Methylation occurs around the time of inactivation and earlier for Pgk-1, which is closer to the X-inactivation centre. In female primordial germ cells, the inactive X chromosome escapes methylation; this may underly the reversibility of inactivation at meiosis. Similarly, the genes are unmethylated on the inactive X chromosome in sperm; hence, the imprint specifying preferential X-inactivation in extra-embryonic tissues must reside elsewhere.

Animals↗
PubMed1991

Decrease in DNA methylase activity during preimplantation development in the mouse.

During early mouse development, there are large-scale changes in DNA methylation. These changes may be due to the availability or stability of the enzyme, DNA methyltransferase (methylase), which is responsible for maintenance of DNA methylation. A microassay for methylase activity in preimplantation embryos shows that the level of maternally inherited enzyme is extremely high in the egg and that this activity is stable for the first three cleavage divisions. However, from the 8-cell to the blastocyst stage, there is a marked and absolute decrease in enzyme activity.

Animals↗
PubMed1990

Amplification of a beta-haemoglobin sequence in individual human oocytes and polar bodies.

A 680 base-pair sequence of the human beta-haemoglobin gene was reproducibly amplified in individual unfertilised human oocytes and in first polar bodies isolated from them. Specificity and sensitivity of amplification were achieved by two sequential reactions with two sets of primers, amplifying first a 725 base-pair sequence and secondly a 680 base-pair sequence from within the first amplified fragment. A restriction enzyme digest of the DNA amplified from a single oocyte with the endonuclease Dde I confirmed the identity of the amplified beta-haemoglobin fragment; this technique provides a diagnostic test for the genetic defect responsible for sickle cell anaemia. Analysis of the DNA from the first polar body may enable detection of such defects in unfertilised eggs from carrier women. Selection of eggs without the defect for fertilisation may therefore obviate the need for diagnostic procedures on embryos.

Amino Acid Sequence↗
PubMed1990

Changes in DNA methylation during mouse embryonic development in relation to X-chromosome activity and imprinting.

Changing DNA methylation patterns during embryonic development are discussed in relation to differential gene expression, changes in X-chromosome activity and genomic imprinting. Sperm DNA is more methylated than oocyte DNA, both overall and for specific sequences. The methylation difference between the gametes could be one of the mechanisms (along with chromatin structure) regulating initial differences in expression of parental alleles in early development. There is a loss of methylation during development from the morula to the blastocyst and a marked decrease in methylase activity. De novo methylation becomes apparent around the time of implantation and occurs to a lesser extent in extra-embryonic tissue DNA. In embryonic DNA, de novo methylation begins at the time of random X-chromosome inactivation but it continues to occur after X-chromosome inactivation and may be a mechanism that irreversibly fixes specific patterns of gene expression and X-chromosome inactivity in the female. The germ line is probably delineated before extensive de novo methylation and hence escapes this process. The marked undermethylation of the germ line DNA may be a prerequisite for X-chromosome reactivation. The process underlying reactivation and removal of parent-specific patterns of gene expression may be changes in chromatin configuration associated with meiosis and a general reprogramming of the germ line to developmental totipotency.

Animals↗
PubMed1990

Preimplantation sexing and diagnosis of hypoxanthine phosphoribosyl transferase deficiency in mice by biochemical microassay.

Hypoxanthine phosphoribosyl transferase (HPRT)-deficient male embryos derived from heterozygous (carrier) female mice were diagnosed by biochemical microassay of X-chromosome-coded HPRT activity in a single cell taken from the 8-cell embryo or in 5-10 cells sampled from the blastocyst. In the latter procedure, carrier female blastocysts could also be distinguished from affected males, and normal males and females, as having intermediate HPRT activity in the sampled trophectoderm cells. During the assay procedures, the operated preimplantation embryos were cultured. They were then transferred, in batches as diagnosed, to recipient females. The resulting fetuses were sexed by gonad morphology and assayed for HPRT activity. All those identified as HPRT-negative embryos by biopsy at the 8-cell or blastocyst stages were indeed HPRT-negative males. The heterozygous females were also correctly identified by the trophectoderm biopsy procedure. The sex of an embryo can also be diagnosed by HPRT activity dosage in a single blastomere taken from 8-cell embryos from a normal mating and cultured for 12 hours before assay. Both X chromosomes are active in female morulae and the blastomeres sampled from female preimplantation embryos have twice the X-coded HPRT activity compared to those from the male embryos. The accuracy of this procedure for sexing was again verified by transfer of the putative male and putative female embryos into recipient females.

Animals↗
PubMed1990

Variation in epigenetic inheritance.

Changing patterns of DNA methylation may underlie differential gene expression in development. Additional sources of variation in allelic methylation may be introduced by parental differences as well as by gamete of origin.

Alleles↗
PubMed1990

Use of a HpaII-polymerase chain reaction assay to study DNA methylation in the Pgk-1 CpG island of mouse embryos at the time of X-chromosome inactivation.

A HpaII-PCR assay was used to study DNA methylation in individual mouse embryos. It was found that HpaII site H-7 in the CpG island of the X-chromosome-linked Pgk-1 gene is less than or equal to 10% methylated in oocytes and male embryos but becomes 40% methylated in female embryos at 6.5 days; about the time of X-chromosome inactivation of the inner cell mass.

Animals↗
PubMed1990

Preferential X-chromosome inactivation, DNA methylation and imprinting.

Non-random X-chromosome inactivation in mammals was one of the first observed examples of differential expression dependent on the gamete of origin of the genetic material. The paternally-inherited X chromosome is preferentially inactive in all cells of female marsupials and in the extra-embryonic tissues of developing female rodents. Some form of parental imprinting during male and female gametogenesis must provide a recognition signal that determines the nonrandomness of X-inactivation but its nature is thus far unknown. In the mouse, the imprint distinguishing the X chromosomes in the extra-embryonic tissues must be erased early in development since X-inactivation is random in the embryonic cells. Random X-chromosome inactivation leads to cellular mosaicism in expression and differential methylation of active and inactive X-linked genes. Transgene imprinting shares many features with X-inactivation, including differential DNA methylation. In this paper we consider when methylation differences in early development affecting X-chromosome activity and imprinting are established. There are processes of methylation and demethylation occurring in early development, as well as changes in the activity of the DNA methylase itself. Methylation of a specific CpG site associated with activity of the X-linked PGK-1 gene has been studied. This site is already methylated on the inactive X chromosome by 6.5 days' gestation, close to the time of X-inactivation. However, differential methylation of this site is not the primary imprint marking the paternal X chromosome for preferential inactivation in the extra-embryonic membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Diagnosis of beta-thalassaemia by DNA amplification in single blastomeres from mouse preimplantation embryos.

Mouse preimplantation embryos were accurately diagnosed as normal or mutant at the beta-major haemoglobin locus by amplification of specific DNA sequences in a single cell. A DNA sequence containing the whole of exon 3 and some 3' untranslated sequences within the beta-major haemoglobin gene was amplified in single blastomeres by means of the polymerase chain reaction (PCR). Blastomeres were removed from embryos of four to eight cells from normal BALB/c mice and from mutant (thalassaemic) BALB/c mice homozygous for a deletion of the whole beta-major haemoglobin gene. The sensitivity of the amplification procedure was enhanced by the sequential use of two sets of oligonucleotide primers for 30 cycles of amplification each, the second pair being located within the segment amplified by the first pair. The product (204 base-pairs) could be easily visualised in ethidium bromide-stained agarose gels. Stringent precautions to prevent contamination were taken, and with these precautions the PCR amplification procedure could be carried out under normal laboratory conditions. These procedures for diagnosis of genetic disease before implantation should be applicable to preimplantation diagnosis of any monogenic disorder in man for which the affected DNA sequence is known.

Alleles↗
PubMed1989

Measurement of HPRT activity in the human unfertilized oocyte and pre-embryo.

The hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase (APRT) activities in individual non-fertilized human eggs and in human pre-embryos (4-cell to blastocyst stage) have been analysed. A wide spread of activities was observed, the mean values of which decline with time post-ovulation for both eggs and advancing pre-embryonic stages. The variation in activities was less in groups of eggs or pre-embryos recovered from a single ovulatory cycle. The activity of HPRT, but not of APRT, was readily detectable in single 4-cell and 8-cell blastomeres. When pre-embryos at various preimplantation stages were exposed to alpha-amanitin, to block transcription of mRNA from the pre-embryonic genome, no clear effect on HPRT activity was observed. It is concluded that the HPRT and APRT activities measured in the pre-embryos studied here are likely to be maternally inherited, and that use of a direct assay for HPRT activity for the pre-implantation diagnosis of Lesch-Nyhan disease would be premature.

Adenine Phosphoribosyltransferase↗
PubMed1989

Til 1--a human lymphoblastoid cell line with minimal DNA methylation.

Methylation has been shown to be correlated with several fundamental cellular processes, including changes in gene expression, alterations in chromatin structure and inactivation of the mammalian X chromosome. It is possible, therefore, that the methylation status of a particular sequence may reflect involvement in a number of processes. Given this potentially confused situation, it is clear that many studies would be facilitated if unmethylated or minimally methylated DNA from a mammalian source were available. A major use of such DNA would be in the construction of long-range physical maps. In many cases, long-range physical maps are a prerequisite for the eventual isolation of disease genes that have been localised to a particular chromosomal region by other means (e.g. genetic linkage studies). Many of the enzymes used in such long-range mapping experiments are methylation sensitive, which makes it difficult to determine how many sites for a particular enzyme are present in any DNA sequence. Here we report the finding of a minimally methylated DNA in the human lymphoblastoid cell line, Til 1. The methylation level of Til 1 DNA was analysed in several ways and compared with that in human lymphocytes, placental tissue and other lymphoblastoid cell lines. The results showed clear and reproducible differences in methylation among the cell types, both at a global level and in the vicinities of specific DNA sequences. Lymphocyte DNA had the highest level of methylation while placental DNA and cell line DNA had lower but similar levels. Til 1 had abnormally low levels of 5-methylcytosine when measured directly, and no detectable methylation at any of the restriction sites examined.

5-Methylcytosine↗
PubMed1988

Pre-implantation diagnosis of HPRT-deficient male and carrier female mouse embryos by trophectoderm biopsy.

In an animal model for Lesch-Nyhan syndrome, the affected male embryos, as well as the carrier female embryos, have been successfully identified by biochemical microassay of a sample of trophectoderm cells taken from the mouse embryos at the blastocyst stage. The embryos were removed from the uterus, diagnosed and returned to the uterus within 2 days without the need for cryopreservation. The diagnosis was confirmed at 14 days gestation by analysis of the hypoxanthine phosphoribosyl transferase (HPRT) status of the fetuses. Live young were obtained from biopsied embryos after transfer.

Animals↗
PubMed1988

Microassay for adenosine deaminase, the enzyme lacking in some forms of immunodeficiency, in mouse preimplantation embryos.

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.

Adenosine Deaminase↗
PubMed1988

DNA methylation in the developing marsupial embryo.

Marsupial development differs from early development of placental mammals in that the blastocyst is unilaminar, so that both embryonic and extraembryonic cells are derived from a single layer of cells (protoderm) which faces the blastocyst cavity. Also, all cells in female marsupial conceptuses so far examined show preferential paternal X-inactivation. To test for a possible correlation between cell position, paternal X-inactivation and DNA hypomethylation, marsupial DNA preparations from three regions, embryo, vascular yolk sac and avascular yolk sac, were digested with methyl-specific restriction endonucleases, separated on agarose gels and end-labelled with 32P-dCTP. The size distribution of the fragments obtained indicated three levels of methylation: high methylation of embryonic DNA, intermediate levels of methylation of vascular yolk sac DNA and hypomethylation of avascular yolk sac DNA. The degree of methylation of repeat sequences, observed as discrete bands in end-labelled HpaII digests, was correlated with the overall methylation of tissue DNA. Thus, the difference in methylation in embryonic and extraembryonic DNA was similar to that described for the mouse conceptus, and the outside cell position of marsupial fetal precursor cells did not correlate with hypomethylation. HpaII tiny fragments, which indicate the presence of CpG-rich islands of DNA, were evident in the marsupial digests. In the mouse DNA, these islands are associated with gene transcription and provide one route to cloning of unique gene sequences.

Animals↗
PubMed1988

Expression of injected HPRT minigene DNA in mouse embryos and its inhibition by antisense DNA.

We have used a highly sensitive biochemical microassay to monitor the expression of a cloned minigene for hypoxanthine phosphoribosyl transferase (HPRT, EC.2.4.2.8) in preimplantation mouse embryos. The mouse HPRT promoter and the mouse metallothionein promoter (MT-I) function equally well in embryos at the 2-cell stage whereas the viral SV40 promoter does not allow HPRT expression. Induced HPRT activity from the MT-I HPRT minigene construct occurs in cleavage embryos cultured in the presence of cadmium. In contrast, negation of enzyme expression from the injected minigene DNA is mediated by simultaneous injection of HPRT antisense DNA.

Adenine Phosphoribosyltransferase↗
PubMed1988

Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere.

Single blastomeres were isolated from zona-free 8-cell mouse embryos and assayed for X-linked hypoxanthine phosphoribosyl transferase (HPRT) activity and autosome-linked adenine phosphoribosyl transferase (APRT) activity. At this stage of development both X chromosomes are active in female embryos. Hence, a bimodal distribution of HPRT: APRT ratios, corresponding to male (XY) and female (XX) biopsied samples, was observed due to the 2-fold difference in gene dosage for HPRT activity. Batches of putative male and female embryos identified in this way were transferred to pseudopregnant recipient females. Development of the seven-eighths embryos was equivalent to that of control zona-free intact embryos. Sex determination by measurement of X-linked gene dosage was accurate and rapid enough to allow transfer of embryos of known sex without the need for cryopreservation.

Adenine Phosphoribosyltransferase↗