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C D Laird

Publications and source records attributed to C D Laird.

At least 19 recordsLinked to original sources
PubMed2000

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

Alleles↗
PubMed2000

Enrichment for submitotic cell populations using flow cytometry.

BACKGROUND: One of the most dramatic events during the course of the mammalian cell cycle is mitosis, when chromosomes condense and segregate, the nuclear envelope breaks down, and the cell divides into two daughter cells. Although cells undergoing mitosis are cytologically distinguishable from nonmitotic cells, few molecular markers are available to specifically identify mitotic cells, especially cells within different stages of mitosis. METHODS: We applied the flow cytometric method of Juan et al. (Cytometry 32:71-77, 1998) to obtain cells with various levels of the molecular markers cyclin B1 and phosphorylated histone H3; fluorescence microscopy was then used to identify sorted cells in different stages of mitosis. RESULTS: We observed the substantial enrichment of submitotic cell populations. CONCLUSIONS: This method represents an effective approach to obtain an enriched population of submitotic cells without the use of drug treatments or prior synchronization.

Biomarkers↗
PubMed1998

Very late DNA replication in the human cell cycle.

G2 was defined originally as the temporal gap between the termination of DNA replication and the beginning of mitosis. In human cells, the G2 period was estimated to be 3-4 h. However, the absence of replicative DNA synthesis during this period designated G2 has never been shown conclusively. In this report, we show that, at some autosomal and X linked loci, programmed DNA replication continues within 90 min of mitosis. Furthermore, the major accumulation of cyclin B1, a cell-cycle marker that is usually ascribed to G2, overlaps extensively with very late DNA replication. We conclude that the G2 period is much shorter than previously thought and may, in some cells, be nonexistent.

Cell Cycle↗
PubMed1998

Fragile-X syndrome and myotonic dystrophy: parallels and paradoxes.

Fragile-X syndrome and myotonic dystrophy are caused by triplet repeat expansions embedded in CpG islands in the transcribed non-coding regions of the FMR1 and the DMPK genes, respectively. Although initial reports emphasized differences in the mechanisms by which the expanded triplet repeats caused these diseases, results published in the past year highlight remarkable parallels in the likely molecular etiologies. At both loci, expansion is associated with altered chromatin, aberrant methylation, and suppressed expression of the adjacent FMR1 and DMAHP genes, implicating epigenetic mediation of these genetic diseases.

Animals↗
PubMed1997

A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication.

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains FMR1, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5' of FMR1 and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3' of FMR1. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.

Cell Cycle↗
PubMed1997

Separation of cells at different times within G2 and mitosis by cyclin B1 flow cytometry.

Multivariate flow cytometry using specific cyclin proteins and DNA content can identify cell populations at different points within the cell cycle. Quantification of cyclin B1 and DNA content reveals that cells with high levels of cyclin B1 predominantly have a 4C DNA content and are therefore in G2 or mitosis. We have examined whether separation of cells by levels of cyclin B1 could be used to discriminate cells at discrete times within these phases. Post-replicative cells progressively enter into fractions with higher levels of cyclin B1, indicating that this protein can be used as a marker of time in G2. Furthermore, cells in particular phases of mitosis can be greatly enriched by separation based on cyclin B1 levels. This method can thus be used to isolate cells at specific times within G2 and mitosis, periods of the cell cycle that have been difficult to study by cell fractionation.

Adult↗
PubMed1997

Sequence analysis of long FMR1 arrays in the Japanese population: insights into the generation of long (CGG)n tracts.

The human fragile-X syndrome is associated with expansions of a (CGG)n triplet repeat within the FMR1 gene. Whilst normal FMR1 arrays consist of variable numbers of (CGG)7-13 blocks punctuated with single AGG triplets, unstable arrays contain longer blocks of uninterrupted (CGG)n. The degree of instability, and subsequent risk of expansion to the fragile-X mutation, is dependent upon the length of this uninterrupted repeat. Detailed analyses of normal FMR1 array structures suggest that longer uninterrupted blocks of repeat could arise either through a process of gradual slippage or a more dramatic loss of an intervening AGG triplet. Up to 15% of Japanese and Chinese individuals have FMR1 triplet arrays centred on 36 repeats in length, a modal group not found in Caucasians. As longer FMR1 arrays have been associated with high-risk fragile-X haplotypes in some populations, we investigated the nature of these larger arrays. Sequence analysis revealed that the unusual length is due to the presence of a novel (CGG)6 block within the array. Several haplotypically related arrays contain blocks of (CGG)16 or (CGG)15, consistent with the fusion of adjacent (CGG)9 and (CGG)6 blocks after loss of the intervening AGG triplet. This is compatible with inferences from the Caucasian population that AGG loss is a mechanism by which long blocks of identical repeats are generated.

Dinucleotide Repeats↗
PubMed1997

Epigenetic variation illustrated by DNA methylation patterns of the fragile-X gene FMR1.

Genomic methylation patterns of mammals can vary among individuals and are subject to dynamic changes during development. In order to gain a better understanding of this variation, we have analyzed patterns of cytosine methylation within a 200 bp region at the CpG island of the human FMR1 gene from leukocyte DNA. FMR1 is normally methylated during inactivation of the X chromosome in females and it is also methylated and inactivated upon expansion of CGG repeats in fragile-X syndrome. Patterns of methylation (epigenotypes) were determined by the sequencing of bisulfite-treated alleles from normal males and females and alleles from a family of five brothers who are methylation mosaics and are affected to various degrees by the fragile-X syndrome. Our data indicate that: (i) methylation of individual CpG cytosines is strikingly variable in hypermethylated epigenotypes obtained from a single individual, suggesting that maintenance of cytosine methylation is a dynamic process; (ii) methylation of non-CpG cytosines in the region studied may occur but is rare; (iii) mosaicism of methylation in the analyzed fragile-X males is remarkably similar to that found for the active X and inactive X alleles in normal females, suggesting that the methylation mosaicism of some fragile-X males reflects similar on and off states of FMR1 expression that exist in normal females; (iv) hypermethylation is slightly more pronounced on fragile-X alleles than on normal inactive X alleles of females; (v) the general dichotomy of hypo- and hypermethylated alleles persisted over the 5 year period that separated samplings of the fragile-X males; (vi) methylation variability was most pronounced at a consensus binding sequence for the alpha-PAL transcription factor, a sequence that may play a role in regulating expression of FMR1.

Alleles↗
PubMed1996

In vitro expansion of GGC:GCC repeats: identification of the preferred strand of expansion.

The human fragile-X syndrome, a major cause of inherited mental retardation, is associated with expansion of the trinucleotide repeat GGC:GCC. Repetitive sequences in DNA are subject to slippage during catalysis by DNA polymerases. We characterized the extent of slippage of synthetic GGC:GCC repeats by various DNA polymerases: Taq DNA polymerase, Klenow fragment of DNA polymerase I, DNA Sequence, DNA polymerase-alpha and polymerase-beta, as well as HIV reverse transcriptase. All of these enzymes were found to expand GGC:GCC repeats, with the most extensive expansion exhibited by Taq DNA polymerase. Starting with a template and primer, each 15 nucleotides (nt) in length, the product of one round of synthesis by Taq polymerase is as long as 250 nt. Sequence analysis of cloned DNA fragments expanded by Taq polymerase indicates that expansion involves multiple triplet additions and that it is asymmetric. The asymmetric distribution of terminal nucleotides in the expanded product is consistent with active expansion of the GCC strand and passive additions onto the GGC strand. The preferential elongation and expansion of the GCC strand was confirmed in studies utilizing longer repeats within a single-stranded M-13 template.

Base Sequence↗
PubMed1993

Association of fragile X syndrome with delayed replication of the FMR1 gene.

The fragile X syndrome is commonly associated with mutant alleles of the FMR1 gene that are hypermethylated and have large expansions of CGG repeats. We present data here on the replication timing of FMR1 that confirm predictions of delayed replication of alleles from affected males. The normal FMR1 allele replicates late in S phase, while alleles from affected males replicate later, the major peak of replication occurring in the flow cytometry fraction usually referred to as G2/M. The delayed timing of replication is not the direct result of a single replication fork stalling at the expanded CGG repeat, because delayed replication was observed for regions on both sides of the repeat. The domain of altered replication timing includes sites at least 150 kb 5' and 34 kb 3' of the repeat, indicating that genes in addition to FMR1 may be affected.

Animals↗
PubMed1992

Estimating the stability of the proposed imprinted state of the fragile-X mutation when transmitted by females.

Fragile-X syndrome is a major cause of mental retardation in humans. The X-inactivation imprinting model accounts for the unusual pattern of inheritance and expression of this syndrome. According to this model, the fragile-X mutation creates a local block to the attempted reactivation of the mutant X chromosome prior to oogenesis. This local block results in an "imprinted" fragile-X chromosome that is deleterious in males and in females for whom this chromosome is predominantly the active X chromosome. The imprinted state of the fragile-X mutation is inferred to be stable when transmitted by an imprinted female because the penetrance of the syndrome in sons of affected females is estimated to be 1.0. To provide a more precise estimate of the stability of the proposed fragile-X imprint, we have analyzed published pedigrees that include restriction fragment length polymorphism and cytogenetic data from sibships with mothers who are interpreted as having an imprinted fragile-X allele. We conclude that the fragile-X imprint was stable in 46 out of 48 female meioses. This analysis leads to a preliminary estimate of about 96% for the stability of the imprint through female meiosis. Two imprinted females had progeny who appeared to be carriers of a nonimprinted fragile-X allele. If this interpretation is correct, then reversion from the imprinted to the nonimprinted state, or "erasure," can occasionally occur when the mutant fragile-X allele is transmitted by an imprinted female. We discuss the genetic and epigenetic significance of possible female erasure. We request DNA and cytogenetic information from unpublished pedigrees to quantify further the stability, during female meiosis, of the proposed imprinted state of the mutant fragile-X allele.

Female↗
PubMed1992

Methylation analysis of CGG sites in the CpG island of the human FMR1 gene.

The fragile-X syndrome of mental retardation is associated with an expansion in the number of CGG repeats present in the FMR1 gene. The repeat region is within sequences characteristic of a CpG island. Methylation of CpG dinucleotides that are 5' to the CGG repeat has been shown to occur on the inactive X chromosome of normal females and on the X chromosome of affected fragile-X males, and is correlated with silencing of the FMR1 gene. The methylation status of CpG sites 3' to the repeat and within the repeat itself has not previously been reported. We have used two methylation-sensitive restriction enzymes, AciI and Fnu4HI, to further characterize the methylation pattern of the FMR1 CpG island in normal individuals and in those carrying fragile-X mutations. Our results indicate that: (i) CpG dinucleotides on the 3' side of the CGG repeat are part of the CpG island that is methylated during inactivation of a normal X chromosome in females; (ii) the CGG repeats are also part of the CpG island and are extensively methylated as a result of normal X-chromosome inactivation; (iii) similar to normal males, unaffected fragile-X males with small CGG expansions are unmethylated in the CpG island; for affected males, the patterns of methylation are similar to those of a normal, inactive X chromosome; (iv) in contrast to the partial methylation observed for certain sites in lymphocyte DNA, complete methylation was observed in DNA from cell lines containing either a normal inactive X chromosome or a fragile-X chromosome from an affected male.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Epigene conversion: a proposal with implications for gene mapping in humans.

Epigenetic modification of DNA is now recognized as a potentially important factor in the inheritance and expression of some mutations; its ability to complicate human genetic analysis is concurrently becoming apparent. One unusual form of epigenetic modification, dominant position-effect variegation (PEV), has been used as a model for Huntington disease. In dominant PEV, a fully dominant mutant phenotype results from stable epigenetic inactivation of an allele adjacent to the structural alteration (cis-inactivation) combined with a complementary inactivation of the homologous normal allele (trans-inactivation). We now propose that trans-inactivation of the normal allele may occasionally persist through meiosis. Such "epigene conversion" occurring at the Huntington disease locus in a few percent of meioses would largely account for the published anomalies in that region's genetic map. This concept could also explain anomalous linkage map data for other disease-causing alleles in humans.

Chromosome Mapping↗
PubMed1991

Hormonal control of gene expression: interactions between two trans-acting regulators in Drosophila.

The steroid hormone 20-hydroxyecdysone (20HE) and the Broad-Complex locus (BRC) are involved in regulating developmental changes in gene expression around the time of metamorphosis in Drosophila. We have investigated the regulatory interactions between 20HE, BRC, and a set of genes expressed in the fat body of third-instar Drosophila larvae. RNA levels for two hormone-inducible genes, Larval Serum Protein-2 and P1, accumulate to normal levels in BRC-mutant larvae. In contrast, RNA levels for the P6 gene were affected by mutations at BRC. On the basis of the results of experiments in which hormone concentrations were varied in BRC-mutant or wild-type larvae, we conclude that 20HE can both increase and decrease P6 RNA levels in the absence of BRC product(s). BRC appears to be a trans-acting modulator of the response of P6 to the hormone. We propose that BRC attenuates the repressive effect of the hormone, expanding the range of hormone concentrations that induce the gene, thus allowing P6 RNA to reach high levels during the third larval instar. The results are discussed in relation to other genes that are regulated by the same two trans-acting factors. A model is presented that refines the model of Ashburner et al. (1974, Cold Spring Harbor Symp. Quant. Biol. 38: 655-662) for the hormonal regulation of gene activity.

Age Factors↗
PubMed1991

The X-inactivation imprinting model of the fragile-X syndrome: annotated references, 1990.

The X-inactivation imprinting model of the fragile-X syndrome (1), the inference from this model and from pedigree data that human oogonia are derived from two progenitor cells present after the initial event that leads to chromosome imprinting (13), and the general model of fragile sites (8), provide a theoretical framework that is consistent with data on the fragile-X syndrome. This framework has led to novel predictions, some of which have been tested. Results of most tests have been consistent with predictions (10, 13, 17, 18, 27, 28). More direct tests are necessary to explore the underlying mechanisms of chromosome change, here termed imprinting, of the molecular basis of the change, and of the mutation itself.

Dosage Compensation, Genetic↗
PubMed1990

Proposed genetic basis of Huntington's disease.

I propose that Huntington's disease (HD) is caused by dominant position-effect variegation, a phenomenon for which new information is available in Drosophila melanogaster. The essential features of this proposal are that (1) the HD mutation is the result of a chromosome alteration that inactivates transcription of a nearby structural gene or genes (cis-inactivation); the combination of this proposed chromosome alteration and the structural gene(s) is termed the HD allele; (2) there is pairing in some somatic cells between the HD and HD+ alleles on homologous chromosomes; (3) as a result of this somatic pairing, the HD mutation also inactivates transcription of the HD+ structural gene on the normal homologue (trans-inactivation), resulting in complete dominance of the mutation; (4) polymorphism for an X-linked recessive modifier of position-effect variegation means that the age of onset of symptoms of HD will depend on which parent the HD mutation was inherited from. The fully dominant nature of HD and the parental-source effect on the age of onset are thus both understandable within the genetic and epigenetic paradigm of position-effect variegation.

Adolescent↗