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A D Riggs

Publications and source records attributed to A D Riggs.

At least 19 recordsLinked to original sources
PubMed1992

Parental imprinting studied by allele-specific primer extension after PCR: paternal X chromosome-linked genes are transcribed prior to preferential paternal X chromosome inactivation.

The preferential inactivation of the paternal X chromosome in extraembryonic cells during early mouse development is an example of parental imprinting, but it has not been studied at the transcriptional level because standard methods of measuring RNA levels do not allow detection of allele-specific RNAs in individual early embryos. We sought to determine whether the paternal allele of the X chromosome-linked gene for 3-phosphoglycerate kinase 1 (Pgk-1), which is located very near the center of X chromosome inactivation, is transcribed prior to differentiation of extraembryonic lineages. Previous reports indicated that in heterozygous embryos there is a delay in the appearance of the phosphoglycerate kinase 1 allozyme encoded by the paternal X chromosome until 2 days after the appearance of the corresponding maternal allozyme. We report results obtained by use of a reverse transcription/PCR-based method which allows the quantitative measurement of allele-specific RNA. The assay is sensitive enough for the quantitative analysis in single embryos of allele-specific transcripts differing by only one nucleotide. We have used this assay to analyze mouse embryos heterozygous at the Pgk-1 and Hprt [hypoxanthine (guanine) phosphoribosyltransferase] loci, and we find that individual 8-cell and blastocyst embryos express both Hprt and Pgk-1 paternal transcripts, as do pooled 2- to 4-cell embryos. These results are discussed in view of the apparent temporal delay in paternal expression of the Pgk-1 gene at the enzyme level.

Alleles↗
PubMed1992

A potentially critical Hpa II site of the X chromosome-linked PGK1 gene is unmethylated prior to the onset of meiosis of human oogenic cells.

Hpa II site H8 is in the CpG-rich 5' untranslated region of the human X chromosome-linked gene for phosphoglycerate kinase 1 (PGK1). It is the only Hpa II site in the CpG "island" whose methylation pattern is perfectly correlated with transcriptional silence of this gene. We measured DNA methylation at site H8 in fetal oogonia and oocytes and found, using a quantitative assay based on the polymerase chain reaction, that purified germ cells isolated by micromanipulation were unmethylated in 47-day to 110-day fetuses, whereas ovaries depleted of germ cells and non-ovary tissues were methylated. We conclude that site H8 is unmethylated in germ cells prior to the onset of meiosis and reactivation of the X chromosome.

Base Sequence↗
PubMed1992

X-chromosome inactivation and cell memory.

Mammalian X-chromosome inactivation is an excellent example of the faithful maintenance of a determined chromosomal state. As such, it may provide insight into the mechanisms for cell memory, defined as the faithful maintenance of a determined state in clonally derived progeny cells. We review here the aspects of X-chromosome inactivation that are relevant to cell memory and discuss the various molecular mechanisms that have been proposed to explain its occurrence, with emphasis on DNA methylation and a recently proposed mechanism that depends on the timing of replication.

Animals↗
PubMed1992

A sensitive, quantitative assay for measurement of allele-specific transcripts differing by a single nucleotide.

We have found that the single nucleotide primer extension assay, a PCR-based assay currently used qualitatively to measure allelic differences in DNA, can be used quantitatively to measure allele-specific transcripts differing by only a single nucleotide. We show that total RNA containing the Pgk-1a transcript can be specifically detected even in a 1000-fold excess of RNA containing the Pgk-1b transcript.

Alleles↗
PubMed1992

Binding of transcription factors creates hot spots for UV photoproducts in vivo.

Cyclobutane dipyrimidines and less than mean value of 6-4 dipyrimidines are the two major classes of mutagenic DNA photoproducts produced by UV irradiation of cells. We developed a method to map cyclobutane dipyrimidines at the DNA sequence level in mammalian cells. The frequency of this class of photoproducts was determined at every dipyrimidine along the human phosphoglycerate kinase-1 (PGK1) promoter sequence and was compared to the UV-induced frequency distribution of mean value of 6-4 dipyrimidines. After irradiation of living cells containing active or inactive PGK1 genes, enzymatic or chemical cleavage at UV photoproducts, and amplification by ligation-mediated polymerase chain reaction, photofootprints were seen in all regions which bind transcription factors and appear as DNase I footprints. Photoproduct frequency within transcription factor binding sites was suppressed or enhanced relative to inactive genes or naked DNA with enhancements of up to 30-fold. Since photoproducts are mutagenic, this indicates that photoproduct (mutation?) hot spots may be tissue specific in mammals.

Animals↗
PubMed1991

Sex chromosome evolution: platypus gene mapping suggests that part of the human X chromosome was originally autosomal.

To investigate the evolution of the mammalian sex chromosomes, we have compared the gene content of the X chromosomes in the mammalian groups most distantly related to man (marsupials and monotremes). Previous work established that genes on the long arm of the human X chromosome are conserved on the X chromosomes in all mammals, revealing that this region was part of an ancient mammalian X chromosome. However, we now report that several genes located on the short arm of the human X chromosome are absent from the platypus X chromosome, as well as from the marsupial X chromosome. Because monotremes and marsupials diverged independently from eutherian mammals, this finding implies that the whole human X short arm region is a relatively recent addition to the X chromosome in eutherian mammals.

Animals↗
PubMed1991

In vivo mapping of a DNA adduct at nucleotide resolution: detection of pyrimidine (6-4) pyrimidone photoproducts by ligation-mediated polymerase chain reaction.

DNA adducts in unique sequences along the mammalian genome are mapped in vivo at single-nucleotide resolution. Pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts] represent one of the two major adduct classes found after UV irradiation of DNA and were shown to play an important role in UV-induced mutagenesis. After UV light treatment of cells, DNA is prepared and chemically cleaved at (6-4) photoproducts with piperidine. Gene-specific fragments are then amplified from total genomic DNA by use of a ligation-mediated polymerase chain reaction. Analysis of the human chromosome X-linked phosphoglycerate kinase (PGK1) gene's promoter has shown that the frequency of (6-4) photoproducts expressed as piperidine-labile sites is (i) high at TpC and CpC dinucleotides, (ii) dependent on the nearest-neighbor bases, (iii) inhibited by the binding of a transcription factor, and (iv) different for DNA derived from the active and inactive X chromosome. This latter difference is mainly a consequence of the presence of 5-methylcytosine (m5C) in CpG dinucleotides on the inactive X chromosome. 5-Methylcytosine in the sequences Tm5CG and Cm5CG inhibits the formation of (6-4) photoproducts. Thus, in addition to in vivo mapping of a DNA adduct at nucleotide resolution, we also report another method for methylation analysis and photofootprinting.

Animals↗
PubMed1991

Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR.

Ligation-mediated polymerase chain reaction (LMPCR) provides adequate sensitivity for nucleotide-level analysis of single-copy genes. Here, we report that chromatin structure can be studied by enzyme treatment of permeabilized cells followed by LMPCR. DNase I treatment of lysolecithin-permeabilized cells was found to give very clear footprints and to show differences between active and inactive X chromosomes (Xa and Xi, respectively) at the human X-linked phosphoglycerate kinase (PGK-1) locus. Beginning 380 bp upstream and continuing 70 bp downstream of the major transcription start site of PGK-1, we analyzed both strands of this promoter and CpG island and discovered the following: (1) The transcriptionally active Xa in permeabilized cells has several upstream regions that are almost completely protected on both strands from DNase I nicking. (2) Nuclei isolated in polyamine-containing buffers lack these footprints, suggesting that data from isolated nuclei can be flawed; other buffers are less disruptive. (3) The Xa has no detectable footprints at the transcription start and HIP1 consensus sequence. (4) The heterochromatic and transcriptionally inactive Xi has no footprints but has two regions showing increased DNase I sensitivity at 10-bp intervals, suggesting that the DNA is wrapped on the surface of a particle; one nucleosome-sized particle seems to be positioned over the transcription start site and another is centered approximately 260 bp upstream. (5) Potassium permanganate and micrococcal nuclease (MNase) studies indicate no melted or otherwise unusual DNA structures in the region analyzed, and MNase, unlike restriction endonuclease MspI, does cut within the positioned particles on the Xi. Results are discussed in the context of X chromosome inactivation and the maintenance of protein and DNA methylation differences between euchromatin and facultative heterochromatin at CpG islands.

Animals↗
PubMed1990

Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells.

Carcinoembryonic antigen (CEA) is one of the best characterized tumor-associated antigens and is extensively used in the in vitro immunodiagnosis of human colon adenocarcinomas. Among a number of anti-CEA monoclonal antibodies, the murine monoclonal antibody T84.66 shows the highest specificity and affinity for CEA and has been used successfully for in vivo tumor imaging in mice and humans. We report here the cloning and sequencing of the genes coding for monoclonal antibody T84.66 and the amino acid sequence of the variable regions for the heavy and light chains. We also report the construction of mouse/human chimeric IgG1 antibody genes using T84.66 variable region genes and human constant region genes. The resulting chimeric gene constructs were transfected into murine myeloma cells (Sp2/0) by electroporation and into Chinese hamster ovary cells by lipofection. The chimeric antibodies obtained exhibited the same specificity and affinity for CEA as that of the T84.66 immunoglobulin produced by the murine hybridoma cell line. Antibody concentrations in culture medium supernatants were clonally variable but similar (15-480 ng/ml) for both Sp2/0 and Chinese hamster ovary transfectants; the average production by Chinese hamster ovary transfectants was only 3-5-fold less than Sp2/0 transfectants. Ascites production of Sp2/0 transfectants is sufficiently high (900 micrograms/ml) for initial in vivo studies with humanized T84.66.

Amino Acid Sequence↗
PubMed1990

Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks.

DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases and Southern blotting. We report here that the sensitivity of this method can be increased several-hundred-fold by applying a ligation-mediated polymerase chain reaction (LM-PCR) procedure following enzyme treatment. DNA is cleaved simultaneously with two restriction enzymes, one sensitive and one insensitive to methylation. After cleavage, a gene-specific oligonucleotide primer is used for primer extension, followed by linker ligation and then conventional PCR. Using this technique, we demonstrate that DNA from 100 cells (about 0.6 ng) can be prepared and qualitatively analyzed for methylation at sites in an X-linked CpG island, and 50 ng of DNA can be analyzed quantitatively. A site 23 bp downstream of the major transcription start site of human phosphoglycerate kinase-1 (PGK-1) is 52 +/- 7 percent methylated in DNA from female blood and greater than 98 percent unmethylated in DNA from male blood or HeLa cells. This method detects quantitatively specific breaks in either double stranded or single stranded DNA. Thus new assays for enzymes and DNA structure can be devised.

Animals↗
PubMed1990

Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis.

A reverse transcriptase-polymerase chain reaction assay (RT-PCR) was used quantitatively to measure accumulated levels of RNA transcripts in total mouse RNAs derived from male germ cells at various spermatogenic stages. RNA levels for two X-linked enzymes, phosphoglycerate kinase (PGK-1) and hypoxanthine phosphoribosyl transferase (HPRT), both decrease during spermatogenesis, although the transcript levels decrease much more rapidly for PGK-1. RNA for the Y-linked ZFY (zinc finger protein) is elevated in all spermatogenic cell fractions tested, being particularly high in leptotene/zygotene spermatocytes and round spermatids. RNA for adenine phosphoribosyltransferase (APRT) increases 5-fold to a peak during late pachynema. RNA for PGK-2, undetectable in spermatogonial cells, increases at least 50-fold by the round spermatid stage. DNA (cytosine-5-)-methyltransferase (MTase) transcript levels are over an order of magnitude higher throughout spermatogenesis than in non-dividing liver cells.

Adenine Phosphoribosyltransferase↗
PubMed1990

DNA methylation and late replication probably aid cell memory, and type I DNA reeling could aid chromosome folding and enhancer function.

DNA methylation in mammals is reviewed, and it is concluded that one role of methylation is to aid cell memory, which is defined as the ability of mitotically derived progeny cells to remember and re-establish their proper cellular identity. Methylation of X-linked CpG-rich islands probably stabilizes X-chromosome inactivation, but other mechanisms appear to be involved. Late replication is discussed as a key ancestral mechanism for X inactivation, and it is emphasized that early and late replication domains may each be self perpetuating. Therefore, early-late replication timing becomes another strong candidate mechanism for cell memory. A chromosome-loop folding enigma is discussed, and it is concluded that special mechanisms are needed to explain the formation and maintenance of specific looped domains. DNA reeling, such as done by type I restriction-modification enzymes, is proposed to provide this special mechanism for folding. DNA reeling mechanisms can help to explain the cis-spreading of X-chromosome inactivation as well as long-range action by enhancers.

Animals↗
PubMed1990

The X chromosome of monotremes shares a highly conserved region with the eutherian and marsupial X chromosomes despite the absence of X chromosome inactivation.

Eight genes, located on the long arm of the human X chromosome and present on the marsupial X chromosome, were mapped by in situ hybridization to the chromosomes of the platypus Ornithorhynchus anatinus, one of the three species of monotreme mammals. All were located on the X chromosome. We conclude that the long arm of the human X chromosome represents a highly conserved region that formed part of the X chromosome in a mammalian ancestor at least 150 million years ago. Since three of these genes are located on the long arm of the platypus X chromosome, which is G-band homologous to the Y chromosome and apparently exempt from X chromosome inactivation, the conservation of this region has evidently not depended on isolation by X-Y chromosome differentiation and X chromosome inactivation.

Animals↗
PubMed1990

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

The 5' region of the gene encoding human X chromosome-linked phosphoglycerate kinase 1 (PGK1) is a promoter-containing CpG island known to be methylated at 119 of 121 CpG dinucleotides in a 450-base-pair region on the inactive human X chromosome in the hamster-human cell line X8-6T2. Here we report the use of polymerase chain reaction-aided genomic sequencing to determine the complete methylation pattern of this region in clones derived from X8-6T2 cells after treatment with the methylation inhibitor 5-azacytidine. We find (i) a clone showing full expression of human phosphoglycerate kinase is fully unmethylated in this region; (ii) clones not expressing human phosphoglycerate kinase remain methylated at approximately 50% of CpG sites, with a pattern of interspersed methylated (M) and unmethylated (U) sites different for each clone; (iii) singles, defined as M-U-M or U-M-U, are common; and (iv) a few CpG sites are partially methylated. The data are interpreted according to a model of multiple, autonomous CpG sites, and estimates are made for two key parameters, maintenance efficiency (Em approximately 99.9% per site per generation) and de novo methylation efficiency (Ed approximately 5%). These parameter values and the hypothesis that several independent sites must be unmethylated for transcription can explain the stable maintenance of X chromosome inactivation. We also consider how the active region is kept free of methylation and suggest that transcription inhibits methylation by decreasing Em so that methylation cannot be maintained. Thus, multiple CpG sites, independent with respect to a dynamic methylation system, can stabilize two alternative states of methylation and transcription.

Animals↗