Biomedical subjects

W S Ward

Publications and source records attributed to W S Ward.

35 records · Page 2Linked to original sources

PubMed1993

Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures.

The flat, hooked-shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non-random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.

Animals↗

PubMed1993

Deoxyribonucleic acid loop domain tertiary structure in mammalian spermatozoa.

In the four decades since the discovery of the basic structure of the DNA double helix, researchers have been investigating the more dynamic tertiary structures that DNA assumes in the various forms of chromatin. The tertiary structure of DNA is important because it is directly related to the function of the genome: for the cell to access the information that is present in the genome accurately and efficiently, the DNA must be in an organized form. This paper reviews the recent work on one particular enigmatic structural form of eukaryotic DNA, that of the highly condensed spermatozoa. Based on the literature and on recently completed experiments in the field, a new model for DNA packaging in sperm nuclei is presented. In this model, each individual DNA loop domain in the sperm chromatin is condensed into a toroid-shaped structure termed the DNA loop doughnut.

Animals↗

PubMed1992

Diagnosis and staging of bladder cancer.

The need for prompt diagnosis and accurate staging of bladder cancer cannot be over-emphasized, as delays in diagnosing the tumor are clearly associated with a poor prognosis. Accurate staging using the American Joint Committee system is essential if clinicians are to compare the results of different therapies.

Biopsy↗

PubMed1991

Nuclear structure and the three-dimensional organization of DNA.

The organization of DNA within the nucleus has been demonstrated to be both cell and tissue specific and is arranged in a non-random fashion in both sperm and somatic cells. Nuclear structure has a pivotal role in this three-dimensional organization of DNA and RNA and contributes as well to forming fixed organizing sites for nuclear functions, such as DNA replication, transcription, and RNA processing. In sperm, DNA is also organized in a specific fashion by the nuclear matrix and DNA-protamine interactions. Within somatic cells, the nuclear matrix provides a three-dimensional framework for the tissue specific regulation of genes by directed interaction with transcriptional activators. This differential organization of the DNA by the nuclear matrix, in a tissue specific manner, contributes to tissue specific gene expression. The nuclear matrix is the first link from the DNA to the entire tissue matrix system and provides a direct structural linkage to the cytomatrix and extracellular matrix. In summary, the tissue matrix serves as a dynamic structural framework for the cell which interacts to organize and process spatial and temporal information to coordinate cellular functions and gene expression. The tissue matrix provides a structural system for integrating form and function.

Animals↗

PubMed1991

DNA packaging and organization in mammalian spermatozoa: comparison with somatic cells.

Mammalian sperm DNA is the most tightly compacted eukaryotic DNA, being at least sixfold more highly condensed than the DNA in mitotic chromosomes. To achieve this high degree of packaging, sperm DNA interacts with protamines to form linear, side-by-side arrays of chromatin. This differs markedly from the bulkier DNA packaging of somatic cell nuclei and mitotic chromosomes, in which the DNA is coiled around histone octamers to form nucleosomes. The overall organization of mammalian sperm DNA, however, resembles that of somatic cells in that both the linear arrays of sperm chromatin and the 30-nm solenoid filaments of somatic cell chromatin are organized into loop domains attached at their bases to a nuclear matrix. In addition to the sperm nuclear matrix, sperm nuclei contain a unique structure termed the sperm nuclear annulus to which the entire complement of DNA appears to be anchored when the nuclear matrix is disrupted during decondensation. In somatic cells, proper function of DNA is dependent upon the structural organization of the DNA by the nuclear matrix, and the structural organization of sperm DNA is likely to be just as vital to the proper functioning of the spermatozoa.

Animals↗

PubMed1991

DNA attachment to the hamster sperm nuclear matrix and nuclear annulus.

Hamster sperm DNA is packaged so tightly that it is the most highly condensed eukaryotic DNA known. Nevertheless, the sperm genome is also organized in a very specific fashion by two nuclear structures, the nuclear matrix and the nuclear annulus. The nuclear matrix organizes sperm DNA into loop domains approximately 46 Kb in length by specific attachment sequences. When the sperm nucleus is induced to decondense, the nuclear matrix almost completely dissipates, leaving only the nuclear annulus, a structure located at the implantation fossa within the nucleus. All the DNA remains anchored to the annulus during decondensation. Rotary shadowing transmission electron microscopy of isolated nuclear annuli retaining only a small portion of this anchored DNA suggests that the DNA is anchored to the annulus in a series of mini-loops, 5-10 Kb in length. These data support our previous suggestions that sperm DNA is organized in a very specific manner.

Animals↗

PubMed1990

Specific organization of genes in relation to the sperm nuclear matrix.

We have recently reported that mammalian sperm DNA, the most highly condensed and functionally inert eukaryotic DNA, is organized into DNA loop domains attached at their bases to a sperm nuclear matrix (Chromosoma, 98: 153, 1989). We report here the specific arrangement of genes within the sperm loop domains by measuring the proximity of six hamster genes to the sperm nuclear matrix. After restriction endonuclease treatment of sperm nuclear matrices, five genes were clearly not associated with the sperm nuclear matrix and only one, alpha A-crystallin, was within 2 Kb of the matrix. This suggests that sperm DNA is organized in a specific manner in relation to the sperm matrix.

Animals↗

PubMed1990

Allelic loss of chromosomes 16q and 10q in human prostate cancer.

Recent advances in understanding the molecular genetics of common adult tumors have indicated that multiple genetic alterations including the activation of oncogenes and the inactivation of tumor suppressor genes are important in the pathogenesis of these tumors. Loss of heterozygosity is a hallmark of tumor suppressor gene inactivation and has been used to identify chromosomal regions that contain these genes. We have examined allelic loss in the most common tumor in men, prostate cancer. Twenty-eight prostate cancer specimens have been examined for loss of heterozygosity at 11 different chromosomal arms including 3p, 7q, 9q, 10p, 10q, 11p, 13q, 16p, 16q, 17p, and 18q. Fifty-four percent (13/24) of clinically localized tumors and 4 of 4 metastatic tumors showed loss of heterozygosity on at least one chromosome. Chromosomes 16q and 10q exhibited the highest frequency of loss of heterozygosity with 30% of tumors showing loss at these chromosomes. These data demonstrate that allelic loss is a common event in prostate cancer and suggest that chromosomes 16q and 10q may contain the sites of tumor suppressor genes important in the pathogenesis of human prostate cancer.

Alleles↗

PubMed1989

DNA loop domains in mammalian spermatozoa.

The highly condensed and tightly packaged DNA of hamster spermatozoa was found to be organized into topologically constrained DNA loop domains attached at their bases to a nuclear matrix. The loop domains of the sperm nuclei differed from somatic cell loop domains from the same animal in two aspects. Sperm loop domains were 60% smaller than somatic cell loop domains, with an average DNA length of 46 +/- 7 kb in sperm as compared with 76 +/- 11 kb in brain. Secondly, unlike virtually all somatic cell DNA known which is negatively supercoiled, sperm DNA was devoid of detectable supercoiling. The presence of the loop domain structure in the highly condensed DNA of mammalian spermatozoa suggests that this motif is a fundamental aspect of eukaryotic DNA organization.

Animals↗

PubMed1989

Identification of a sperm nuclear annulus: a sperm DNA anchor.

We report the isolation and characterization of a new nuclear structure from spermatozoa of the golden Syrian hamster which we term the nuclear annulus. The nuclear annulus was located at the implantation fossa, the point at which the tail is joined to the sperm head, inside the nucleus adjacent to the inner nuclear envelope. Extraction of sperm nuclei with 2 M NaCl and 10 mM dithiothreitol caused the solubilization of the protamines and DNA decondensation. This released the DNA from its structural constraints except that the DNA remained anchored to the nuclear annulus, which survived the extraction procedure. Nuclear annuli were isolated and examined by scanning electron microscopy. The nuclear annulus was a double-crescent, ring-shaped structure, 2.10 +/- 0.16 micron long and 1.36 +/- 0.13 micron wide. We believe that the nuclear annulus may play an important role in the organization of sperm DNA.

Animals↗

PubMed1986

Cross-linking of cytokeratins to DNA in vivo by chromium salt and cis-diamminedichloroplatinum(II).

The in vivo cross-linking of cytokeratins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 and cis-diamminedichloroplatinum(II) (cis-DDP) was studied. Cytokeratin-DNA complexes were obtained by high-speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate. The cytokeratins were identified electrophoretically and immunologically by use of polyclonal and monoclonal antibodies. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to K2CrO4 for at least 4 h, and the amount of keratin-DNA complexes increased with the incubation time. Each of the three Novikoff ascites hepatoma cytokeratins (p39, p49, and p56) showed a different apparent rate of cross-link formation with DNA. Cytokeratin-DNA complexes were detectable in our system only with K2CrO4 concentrations of 200 microM or greater, and saturation in cross-linking was effected at approximately 2 mM. Higher K2CrO4 concentrations (up to 5 mM) did not produce further significant increases in the amount of cross-linked cytokeratins. Chromium and cis-DDP cross-linked the same cytokeratins at approximately the same ratios; however, both agents cross-linked the major cytokeratins selectively, since not all cytokeratins present in Novikoff hepatoma cell lysates could be cross-linked to DNA. Further evidence of DNA-cytokeratin complexes was obtained by CsCl gradient centrifugation. Our results document the ability of chromate and cis-DDP to produce DNA-cytokeratin cross-links in vivo and show that in live Novikoff hepatoma cells some, but not all, of the components of intermediate filaments are within cross-linking distance of DNA.

Animals↗

PubMed1985

Cross-linking of Novikoff ascites hepatoma cytokeratin filaments.

We have investigated the structure of solubilized cytokeratins from Novikoff ascites hepatoma using the cleavable cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) in the presence of 6 M urea to effect partial complex melting. By two-dimensional gel electrophoresis, in which the protein cross-links were broken in the second dimension, we have identified two major complexes as a p39-p56 dimer and a (p39-p56)2 tetramer, p39 and p56 being two of the major cytokeratins in Novikoff ascites hepatoma. Experiments investigating possible relationships between the dimer and tetramer employed immunoblots and two monoclonal antibodies which recognized either p56 or p39 cytokeratins. When very low protein concentrations were cross-linked, the dimer was the predominant product. As protein concentration increased, we noted a decrease in dimers and a corresponding increase in tetramers, suggesting that the dimer may be a precursor to the tetramer. In support of the cross-linking experiments, two-dimensional gel electrophoresis using 4 M urea in the first dimension indicated a predominant association of p56 and p39 in the Novikoff ascites hepatoma cytokeratin complexes.

Animals↗

PubMed1985

Chromium-induced cross-linking of nuclear proteins and DNA.

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.

Animals↗

PubMed1984

Association of cytokeratin p39 with DNA in intact Novikoff hepatoma cells.

The relationship between the rat tumor-associated cytokeratin p39 (Mr 39,000) and cellular DNA has been studied in intact cells. Using a DNA-protein crosslinking technique, incubation with cis-dichlorodiammineplatinum, we present evidence for the association of p39 with DNA in intact Novikoff ascites hepatoma cells. The cells were treated with cis-dichlorodiammineplatinum and solubilized in NaDodSO4-containing buffer, and the DNA was pelleted by high-speed centrifugation. By immunotransfer analysis, the cytokeratin was found in the DNA pellet of the crosslinked samples while absent from the controls. This result was further substantiated by CsCl density-gradient centrifugation. Collectively, these results suggest a cytokeratin-DNA association at the filament binding sites on or near the nuclear lamina.

Animals↗

PubMed1984

Characterization of monoclonal antibodies to novikoff hepatoma cytokeratin antigen p39.

Three stable monoclonal antibody-producing mouse hybridoma lines have been developed which produce high-titer, immunoglobulin M antibodies specific for the Novikoff ascites hepatoma (NAH) Mr 39,000 cytokeratin antigen (p39). Immunotransfer assays of cytoskeletal protein-enriched fractions indicated p39 to be present in a range of rat tissues, including colon, breast, lung, and uterus. Two-dimensional gel immunoblots confirmed that immunoreactivity in the latter tissues was for polypeptides with similar isoelectric points to those of NAH p39; however, reactivity in the colon contained a wide range of additional isomeric forms. Immunohistochemical localization studies with these antibodies revealed enrichment of p39 in the simple epithelia or ductular structures of organs containing this antigen. In all sections examined, glandular epithelia were observed to be only weakly immunoreactive. Additional immunoblot and immunocytochemical analyses with the monoclonal antibodies and polyspecific antisera to NAH cytokeratin suggest the human Mr 40,000 cytokeratin to be similar to but not identical to NAH p39.

Animals↗

PubMedDate not supplied

DNA organization in human spermatozoa.

Previous studies from this laboratory on hamster spermatozoa have demonstrated that rodent sperm DNA is packaged into the sperm nucleus in a specific manner by nuclear structures. The entire genome is organized into DNA loop domains attached at their bases to a sperm nuclear matrix, the skeletal structure of the nucleus. When nuclei are completely decondensed, the nuclear matrix dissipates, and the entire genome remains anchored to a single structure located at the base of the tail, termed the nuclear annulus. Here, we have extended these studies to human sperm nuclei, which were found to be similar to hamster. Human sperm DNA was found to be organized into loop domains attached at their bases to a nuclear matrix. The average size of the human sperm halo of DNA surrounding the extracted sperm nucleus (made up of DNA loop domains) was about 50% smaller than those that have been reported for somatic cells (this corresponds to an approximate loop domain size of 26.8 +/- 2.1 kb). Human sperm DNA also remained anchored to the base of the tail when completely decondensed, indicating the existence of a nuclear annulus-like structure in human spermatozoa; but, unlike the hamster nuclear annulus, the human annulus could not be isolated because of its structural instability when separated from the tail. Using human centromere repeats as a probe for in situ hybridization, we examined the packaging of individual DNA sequences within the sperm nucleus. These studies demonstrate that human sperm DNA is highly organized by nuclear structures.

Adult↗

PubMedDate not supplied

Structure of the rRNA genes in the hamster sperm nucleus.

We have examined the structure of the major ribosomal RNA (rRNA) genes in the hamster sperm nucleus, using fluorescent in situ hybridization (FISH). The rRNA genes are present as tandemly repeated clusters located at the telomeric ends of the short arms of five pairs of acrocentric chromosomes in the Syrian golden hamster (as they are in humans). In somatic cells, these five chromosome pairs come together to form the nucleolus, the site of rRNA synthesis. The nucleolus remains intact through S phase of the cell cycle, breaking apart only during late G2 and mitosis when the chromosomes condense. Mammalian sperm nuclei are the final products of meiotic division and morphological differentiation that includes a dramatic chromatin condensation. Consequently, it was not immediately obvious whether the rRNA genes would be condensed into a nucleolus-like structure in the mature spermatozoa, or separated, as they are in mitotic chromosomes. We found that of 117 sperm nuclei examined, 91.5% contained between two and five FISH signals for the rRNA gene clusters, and 64.0% contained four (29%) or five (35%) signals. In decondensed hamster sperm nuclei, the rRNA hybridized signals were separated into independent strands. These data collectively indicate that the chromosomes containing the rRNA genes are not bound together into a pre-nucleolar structure in fully condensed mammalian sperm nuclei.

Animals↗