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D J Wolgemuth

Publications and source records attributed to D J Wolgemuth.

107 records · Page 6Linked to original sources
PubMed1985

Separation of mouse testis cells on a Celsep (TM) apparatus and their usefulness as a source of high molecular weight DNA or RNA.

The use of a self-contained unit-gravity cell separation apparatus for separation of populations of mouse testicular cells is described. The apparatus, a Celsep (TM), maximizes the unit area over which sedimentation occurs, reduces the amount of separation medium employed, and is quite reproducible. Cells thus isolated have been good sources for isolation of DNA, and notably, high molecular weight RNA.

Animals↗
PubMed1985

Localization of mRNAs in mouse testes by in situ hybridization: distribution of alpha-tubulin and developmental stage specificity of pro-opiomelanocortin transcripts.

Gene expression of mouse testicular germ cells and surrounding somatic cells during spermatogenesis was examined by RNA:cDNA hybridization in situ and concomitant Northern and dot blot analysis. Particular attention was paid to obtaining fixation and hybridization procedures for use with mouse testes to accomplish sensitive and precise localization with the in situ technique. alpha-Tubulin mRNAs were localized in virtually all testis cell types. In elongating spermatids, a unique labeling pattern was visualized; possibly corresponding to the position of the asymmetrically displaced cytoplasm. Pro-opiomelanocortin (POMC) transcripts in the adult mouse testis detected by Northern blot analysis were shown to be of a smaller size (approximately 600-800 nt) than the pituitary POMC transcripts, similar to what has been reported recently for POMC expression in the rat testis and mouse Leydig cell lines. Localization of POMC mRNAs by in situ hybridization was primarily to Leydig cells, although some labeling of spermatogonia and spermatocytes within the seminiferous epithelium was detected. A cellular or developmental specificity of the pattern of POMC localization was observed: obvious labeling of Leydig cells was limited to interstitial regions which were surrounded completely or in part by tubules in stages IX to XII of the cycle of the seminiferous epithelium.

Animals↗
PubMed1985

Haploid expression of a unique c-abl transcript in the mouse male germ line.

RNA from immature mouse testes was shown to lack a low-molecular-weight c-abl transcript previously noted to be the predominant species in adult testes. The developmental pattern of appearance of this c-abl variant was determined by analyzing RNA obtained from purified populations of testicular cells in different stages of spermatogenesis. The appearance of the c-abl testicular variant was coincident with the entry of the germ cells into their haploid state and suggested that the regulated expression of this proto-oncogene may be important in the normal differentiation of the male germ line.

Animals↗
PubMed1984

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.

Animals↗
PubMed1984

Formation of the rabbit zona pellucida and its relationship to ovarian follicular development.

The zona pellucida is the unique extracellular glycoprotein matrix which is assembled during growth of the mammalian oocyte. The present studies were carried out to examine the formation of this structure in relation to the differentiation of ovarian cell types during follicular development. Specific antibodies were developed against total rabbit ZP proteins as well as against ZP proteins electrophoretically purified by high-resolution two-dimensional polyacrylamide electrophoresis gels (2D-PAGE). Antibodies were characterized by (a) immunoelectrophoresis, (b) a Staphylococcus aureus protein A binding assay, and (c) immunoblotting following 2D-PAGE separation of ZP proteins. Immunoperoxidase localization with these antibodies was used to determine the stage of ovarian follicular development at which ZP antigens first appear as well as to evaluate the cellular and extracellular distribution of these proteins throughout folliculogenesis. The ZP proteins were first observed in the cytoplasm and at the periphery of the oocytes surrounded by a thin squamous follicular cell layer. No staining was observed in the cytoplasm of follicle cells during early folliculogenesis. As the ZP matrix was assembled extracellularly, the intensity of staining of the outer and inner regions could be distinguished. This differentiation of the matrix coincided with the differentiation of the follicular cells into a multilayer cell complex. At this stage, specific ZP proteins are localized within the cytoplasm of the inner layers of these follicular cells. The staining is then diminished in cells of preantral follicles. These studies demonstrate that the formation of the ZP is an excellent model system to study the early stages of follicular development and cell differentiation.

Animals↗
PubMed1984

Effects of clinostat rotation on mouse meiotic maturation in vitro.

The effects of microgravity on meiosis, fertilization, and early embryonic development in mammals are being examined by using a clinostat to reorient the cells with respect to the gravity vector. A clinostat capable of supporting mammalian cells in tissue culture has been developed. Initial studies have focused on examining the effects of clinostat rotation on meiotic maturation in mouse oocytes. Oocytes recovered from ovarian follicles were subjected to clinostat rotation on a horizontal or vertical axis or to static conditions for a 16 hr period. No gross morphological changes and no effects on germinal vesicle breakdown were observed under any rotation conditions (1/4, 1, 10, 30, 100 RPM). Success of meiotic progression to Metaphase II was comparable among experimental and control groups except at 100 RPM, where a slight inhibition was observed.

Animals↗
PubMed1982

Transcription and accurate polyadenylation in vitro of RNA from the major late adenovirus 2 transcription unit.

Nucleoprotein complexes with in vitro transcription activity were isolated from HeLa cells late in lytic infection with Adenovirus type 2 (Ad2). Both polymerase II and polymerase III were active in these extracts, and greater than 85% of the labeled RNA was Ad2-specific. Electrophoretic analyses and Southern blot analyses demonstrated that RNA complementary to the entire 30 kb late transcription unit including RNA near the presumed termination site was synthesized. The addition of DRB (5,6,dichloro-1-beta-D-ribofuranosyl-benzimidazole) in vivo prior to the isolation of the complexes resulted in accumulation of polymerase II at the promoter proximal sites, but nascent chains started in vivo in DRB were successfully elongated in vitro. Approximately 10% of the RNA labeled in vitro contained poly(A), and the length of poly(A) was very similar to that of nuclear RNA isolated from Ad2-infected cells. The in vitro sites of poly(A) addition were specific--labeled poly(A)-terminated RNA molecules ended at a point on the genome coincident with previously mapped poly(A) sites of mRNAs produced in vivo. In addition, the polyadenylation enzyme (or enzymes) cosediment with the nucleoprotein complexes during sucrose gradient centrifugation since gradient purified complexes synthesize poly(A) containing RNA in vitro in the absence of any added nuclear extract.

Adenoviruses, Human↗
PubMed1981

DNA replication-mediated recombination of molecules of adenovirus 2 DNA.

Molecules of adenovirus 2 DNA were isolated as nucleoprotein complexes late in infection of HeLa cells and examined by electron microscopy. Some were interpreted as representing intermediates in recombination events. The structures observed were consistent with predicted intermediates according to the model for genetic recombination proposed by Meselson and Radding [Meselson, M. S. & Radding, C. M. (1975) Proc. Natl. Acad. Sci. USA 72, 358-361] and constitute physical evidence from animal cells in support of this model.

Adenoviruses, Human↗
PubMed1980

Visualization of genetic recombination intermediates of human adenovirus type 2 DNA from infected HeLa cells.

The study of recombination in prokaryotes has been facilitated by the availability of recombinational mutants and simple genetic elements such as phages and plasmids. These small but defined molecules of DNA have been especially useful for electron microscopic analysis of structural detail of molecules undergoing recombination both in vivo and in vitro. A limitation in the structural analysis of plasmid recombination is the absolute number of recombining molecules which can be identified and analysed amidst a background of nonrecombining molecules. This limitation would be of even greater consequence in studies of genetic recombination in animal cells. We therefore chose virus-infected animal cells as a model system for the study of the molecular mechanism of genetic recombination in higher organisms. HeLa cells infected with adenovirus serotype 2 (Ad-2) offer several advantages for studying recombination: (1) the virus contains a small and well characterized genome of about 35 kilobases; (2) a large amount of Ad-2 DNA is accumulated during lytic infection and host DNA synthesis is suppressed; (3) Ad-2 recombines at a very high frequency; and (4) similar to phages, animal viruses and Ad-2 in particular are believed to use many of the host cell's enzymes in necessary metabolic processes, presumably including recombination. In this study we used electron microscopic techniques to visualize the structures of in vivo Ad-2 DNA recombination intermediates. Molecules were observed with structures at putative cross-over points which were consistent with the molecular mechanism of recombination proposed by Holliday. In addition, we observed Ad-2 DNA molecules engaged in recombination which were simultaneously serving as templates for replication and/or transcription. To the best of our knowledge, this is the first visualization of in vivo recombination intermediates of discrete DNA molecules isolated from eukaryotic cells.

Adenoviruses, Human↗
PubMed1980

Repetitive DNA sequences in methotrexate- and methasquin-sensitive and -resistant Chinese hamster cell lines.

DNA purified from a Chinese hamster cell line of lung fibroblast origin (DC83F) was analyzed by density gradient centrifugation and by gel electrophoresis after restriction endonuclease digestion in order to fractionate discrete repetitive fractions within the total DNA. No obvious satellite DNAs were resolved using the CsCl or Ag-Cs2SO4 density gradient conditions described herein. However, analysis of the digestion products of a battery of restriction endonucleases indicated that three of these enzymes, EcoR1, HaeIII, and XhoI, yielded discrete fragments which could be visualized with EtBr staining or identified by scintillation counting of [3H]DNA. DNAs from several highly (greater than or equal to hundredfold increased resistance) antifolate-resistant sublines of DC-3F, characterized by a large homogeneously staining region (HSR) in the chromosome complement, were examined with both techniques and compared to the parental, antifolate-sensitive cell line DNA. The density gradient profiles and electrophoretic patterns of restriction endonuclease digests were identical among all the cell lines examined and were indistinguishable from those of the parental DC-3F DNA.

Animals↗