Search PubMed⌕ Search

Biomedical subjects

O J Miller

Publications and source records attributed to O J Miller.

At least 37 records · Page 2Linked to original sources
PubMed1991

Isolation of large numbers of chromosome 3-specific cosmids containing clusters of rare restriction-endonuclease sites.

We tested 519 chromosome 3-specific cosmids for the presence of rare restriction-endonuclease sites in a search for cosmids containing HTF islands. We have identified 49 cosmids (9% of those tested) that contain multiple rare restriction-endonuclease sites. The cosmids were digested with several common cutting restriction endonucleases to liberate small fragments which were tested as unique-sequence chromosome 3-specific hybridization probes and for evolutionary sequence conservation. Unique-sequence hybridization probes isolated from the cosmids were hybridized to a somatic cell hybrid deletion mapping panel to subchromosomally localize the cosmids. Fragments from many of these cosmids demonstrated conservation of sequence through evolution, and these fragments hybridize to distinct transcripts. These cosmids should therefore prove a useful resource for the identification of many chromosome 3-specific genes, in addition to having potential use as linking clones for pulsed-field gel mapping studies.

Biological Evolution↗
PubMed1990

Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone.

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.

Animals↗
PubMed1990

Relationship of mouse minor satellite DNA to centromere activity.

Chromosomes from a female mouse cell line were identified by Q-banding prior to in situ hybridization with 3H-labeled mouse minor satellite (satellite II) DNA. No cell was found in which every chromosome was labeled, but grain counts showed that every active centromeric region had minor satellite sequences. In the mouse T (10;13)199H translocation, the breakpoint was within the minor satellite array, leaving clusters of minor satellite at the C-bandless active centromere of the 13(10) chromosome and at the interstitial C-band of the 10(13) chromosome, which is not associated with centromeric activity. In a mouse A9 (L-cell derived) marker chromosome with one terminal and two interstitial C-bands, only the terminal C-band was adjacent to an active centromere, but minor satellite DNA was present at all three sites. Minor satellite DNA was not detected on the Y chromosome, although the presence of a small amount of divergent satellite sequences on this chromosome could not be ruled out.

Animals↗
PubMed1990

A human alpha satellite DNA subset specific for chromosome 12.

We have isolated a DNA clone (pBR12, locus D12Z3) which identifies an alphoid subset specific for chromosome 12. This alphoid subset has an EcoRI periodicity of 680 bp and is characterized by a higher-order repeat of about 1.4 kb (eight basic units of about 170 bp each) as revealed by several restriction enzymes. The sequence analysis confirmed the alphoid nature of pBR12 and the dimeric organization.

Blotting, Southern↗
PubMed1989

The organization of the mouse satellite DNA at centromeres.

The mouse genome contains a major and a minor satellite DNA family of repetitive DNA sequences. The use of 5-azacytidine has allowed us to demonstrate that these satellite DNAs are organized in two separate domains at the centromeres of mouse chromosomes. The minor satellite is closer to the short arms of the acrocentric chromosomes than the major satellite. The major satellite is farther away, flanking the minor satellite and adjacent to the euchromatic long arm of each mouse chromosome. At the level of resolution afforded by the in situ hybridization technique it would appear that the organization of the centromeric domain of the mouse is similar to that in man. That is, both contain two repetitive DNA sequence families arranged in major blocks.

Animals↗
PubMed1989

A human alphoid DNA clone from the EcoRI dimeric family: genomic and internal organization and chromosomal assignment.

We isolated an alpha satellite DNA clone (pC1.8), 17 kb long, which is composed exclusively of tandemly repeated 340-bp EcoRI fragments. Hybridization studies using 37 random EcoRI dimers subcloned from pC1.8 showed that they are heterogeneous. The sequence of 5 dimers, 3 of them adjacent, confirmed this observation and showed that the heterogeneity is more accentuated among the second monomers. The chromosomal assignment under high stringency conditions showed that this alphoid subset is located on chromosomes 1, 5, and 19. No conditions that eliminate the hybridization on any one of those chromosomes were found. This suggests that, in contrast to many other chromosome-specific alpha satellite subsets, the single chromosome subsets of this family are virtually indistinguishable by hybridization techniques.

Base Sequence↗
PubMed1989

Genetic correction of hereditary disease.

Several hereditary disorders may be potentially correctable by the introduction and incorporation of the normal gene into human tissues using a variety of systems. Although technical issues surrounding integration, stable expression and potential insertional mutagenesis to the treated cells has not yet been fully resolved, enough scientific progress has already been made to consider somatic cell gene therapy acceptable from both the scientific and ethical viewpoints. Tissue-specific stem cell and embryonic stem cell transplantation will allow therapy earlier in the developing embryo. As technical problems are eliminated, these procedures will become morally permissible, as they will allow the correction of devastating hereditary disease.

Ethics, Medical↗
PubMed1988

Relationship between the number and function of human ribosomal genes.

The relative number of ribosomal RNA genes of the acrocentric chromosomes in one individual was measured by counting grains after in situ hybridization of 3H-labeled human 18S rDNA to fixed metaphase chromosomes. The relative amount of ribosomal RNA gene activity of each of the same chromosomes was estimated by determining the frequency with which the chromosome's nucleolus organizer region (NOR) was silver stained, the size of the silver-stained region, and how often the chromosome was found in satellite association. Results were similar in phytohemagglutinin-stimulated T-lymphocytes, Epstein-Barr virus transformed lymphoblasts, and fibroblasts. One chromosome 21 had few gene copies and low activity. One chromosome 22 had many gene copies but low activity. Both chromosomes 14 had few gene copies but high activity. The level of expression that can be achieved by rRNA gene clusters can, therefore, be determined by factors other than the number of gene copies.

B-Lymphocytes↗
PubMed1987

Overabundance of rare-cutting restriction endonuclease sites in the human genome.

A human chromosome 3-specific cosmid library was constructed from a somatic cell hybrid containing human chromosome 3 as its only human component. This library was screened to identify 230 human recombinants which contained an average insert size of 37 kilobases. DNA prepared from 54 of these cosmids, representing 2000 kilobases of human DNA, was then tested for restriction endonuclease sites for EcoRI, HindIII, KpnI, XhoI, and DraI, as well as those of the rare-cutting restriction endonucleases NotI, SfiI, NruI, MluI, SacII, and BssHII. Sites for the latter enzymes were much more abundant than would be expected from theoretical calculations, reflecting non-random clustering of these sites. This has important implications for the use of these enzymes in the construction of physical maps of chromosomes. Some individual cosmids contained large numbers of rare sites, offering an alternative means of physically mapping chromosomes based upon identifying clusters of rare restriction sites. These clusters appear to be spaced an average of 1000 kb apart.

Animals↗
PubMed1987

Generation of human B-cell hybridomas secreting monoclonal anti-myelin-associated glycoprotein antibodies from a patient with neuropathy.

Human hybridomas that secrete monoclonal IgM anti-myelin-associated glycoprotein antibodies were generated by fusion with cells from a patient with peripheral neuropathy and IgM monoclonal gammopathy. Karyotypic analysis of the hybridoma cells revealed no chromosomal abnormalities. The cells were positive for cell-surface idiotype HLA-DR and the plasma cell antigen PCA-1, and negative for the B-cell determinant B4 and for Leu-1, which has been postulated to distinguish a subpopulation of B cells that secrete IgM with autoantibody activity.

Antibodies, Anti-Idiotypic↗
PubMed1987

p82H identifies sequences at every human centromere.

A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.

Base Sequence↗
PubMed1987

Human ribosomal DNA fragments amplified in hamster cells are transcribed only by RNA polymerase II and are not silver stained.

Cloned human rRNA gene fragments that included the promoter region were introduced into Chinese hamster dihydrofolate reductase-deficient (dhfr-) cells by cotransformation with a dhfr minigene and amplified by selection for methotrexate resistance. The human ribosomal DNA was transcribed by RNA polymerase II, not RNA polymerase I or III. The metaphase chromosome regions containing the transcriptionally active human ribosomal DNA failed to show silver staining.

Animals↗
PubMed1987

Gene therapy.

Severe genetic disorders are potentially correctable by the addition of a normal gene into tissues. Although the technical problems involving integration, stable expression, and insertional damage to the treated cell are not yet fully solved, enough scientific progress has already been made to consider somatic cell gene therapy acceptable from both the ethical and scientific viewpoints. The resolutions to problems evolving from somatic cell gene therapy will help to overcome the technical difficulties encountered presently with germ line gene manipulation. This procedure would then become morally permissible as it will cause, in time, a reduction in the pool of abnormal genes in the population. Enhancement genetic engineering is technically feasible but morally unacceptable. Eugenic genetic engineering is not technically possible or ethically permissible in the foreseeable future.

Ethics, Medical↗
PubMed1985

Cellular uptake and localization of fluorescent derivatives of phorbol ester tumor promoters.

The synthetic fluorescent derivatives of 12-O-tetradecanoylphorbol-13-acetate (TPA), dansyl-TPA, dansyl-TPA-20-acetate and dansyl-TPA-13-desacetate, have ID50 values in the [3H]PDBu binding assay of 2nM, 30nM and 1000nM respectively; the ID50 value of TPA is 4nM. Dansyl-TPA is also equipotent with TPA as an activator of protein kinase C(PKC) producing half maximum stimulation at 2nM. Dansyl-TPA-13-desacetate is almost as potent as dansyl-TPA, while dansyl-TPA-20-acetate is completely inactive as an activator of PKC. The cellular uptake of these fluorescent TPA derivatives tends to parallel their activity in the [3H]PDBu binding assay. Treatment of C3H 10T1/2 cells with 100nM dansyl-TPA results in intense fluorescence of the entire cytoplasm, while the nucleus is virtually devoid of fluorescence. The uptake of fluorescence is quenched by an excess of TPA. Thus, dansyl-TPA rapidly enters cells and binds to specific sites distributed throughout the cytoplasm. Presumably these sites reflect the cellular localization of phorbol ester receptors and protein kinase C.

Animals↗
PubMed1985

Pattern of undermethylation of the major satellite DNA of mouse sperm.

Enzymatic hydrolysis and base analysis by high performance liquid chromatography showed that mouse satellite DNA had 30-50% less 5-methylcytosine in sperm than in somatic tissue (1.59 mols % vs 2.40-3.11 mols %). Maxam-Gilbert sequencing and analysis of the intensity of the cytosine bands indicated that the level of methylation of the eight CpGs of the consensus sequence in sperm satellite DNA ranged from 0 to about 50%, considerably lower than the levels reported in somatic tissues. The Mn1I site containing one of these CpGs was cut much more extensively in satellite DNA from sperm than from liver, confirming the undermethylation of this site in sperm DNA.

5-Methylcytosine↗
PubMed1985

DNA methylation is not increased in mouse-human somatic cell hybrids.

The level of DNA methylation in three mouse-human cell lines that retained different human chromosomes and in the parental mouse and human lines has been determined by high-pressure liquid chromatography (HPLC). The level of methylation is similar in the hybrid and parental cells, indicating that interspecific somatic cell hybridization followed by preferential chromosome segregation can occur without an increase in overall DNA methylation.

Animals↗
PubMed1985

Dosage compensation in mammals: why does a gene on the inactive X yield less product than one on the active X?

An expressed gene on the inactive mammalian X chromosome yields less product than the same gene on the active X. Characteristics of the inactive X which might be responsible for this are late replication, chromatin clumping, and altered patterns of DNA methylation. If an expressed gene on the inactive X is not replicated until late in S, it will be present in two copies for a shorter fraction of the cell cycle than its early replicating homologue and therefore yield less product. Alternatively, transcription may be slowed by a microenvironment of highly condensed chromatin or by an abnormal pattern of methylation of the DNA template. Experiments are proposed by which to test these and related hypotheses.

Animals↗