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Biomedical subjects

T E Wagner

Publications and source records attributed to T E Wagner.

At least 91 records · Page 5Linked to original sources

Expression of the human growth hormone variant gene in cultured fibroblasts and transgenic mice.

The nucleotide sequence of the human growth hormone variant gene, one of the five members of the growth hormone gene family, predicts that it encodes a growth hormone-like protein. As a first step in determining whether this gene is functional in humans, we have expressed a mouse metallothionein I/human growth hormone variant fusion gene in mouse L cells and in transgenic mice. The growth hormone variant protein expressed in transiently transfected L cells is distinct from growth hormone itself with respect to reactivity with anti-growth hormone monoclonal antibodies, behavior during column chromatography, and isoelectric point. Transgenic mice expressing the growth hormone variant protein are 1.4- to 1.9-fold larger than nontransgenic controls, suggesting that the protein has growth-promoting properties.

Animals↗

Enhanced viral resistance in transgenic mice expressing the human beta 1 interferon.

Recombinant plasmids pMTIF-beta 1A and pMTIF-beta 1B were constructed by fusing the metallothionein I promoter-regulatory region to the human beta 1 interferon (HuIFN-beta 1) gene. These linearized fusion genes were then introduced into mouse germ lines by zygote microinjection. The chromosomal integration and the germ line transmission of the injected DNA sequences in the resulting transgenic mice were detected by DNA dot blot and Southern transfer hybridizations. The sera of at least two strains of metallothionein/HuIFN transgenic mice were found to protect human WISH cells against vesicular stomatitis virus infection, and this activity could be neutralized by preincubation with anti-HuIFN-beta 1 antibody. These transgenic mice demonstrated significantly enhanced resistance to pseudorabies virus compared with nontransgenic mice when inoculated with pseudorabies virus. The level of resistance seemed to correlate with the concentrations of HuIFN-beta 1 in serum. These transgenic mice may be used as models to study IFN-induced responses and may serve as prototypes to generate disease-resistant animals.

Animals↗

Endogenous human growth hormone (GH) modulates the effect of gonadotropin-releasing hormone on pituitary function and the gonadotropin response to the negative feedback effect of testosterone in adult male transgenic mice bearing human GH gene.

The consequences of human GH (hGH) secretion on hypothalamic, pituitary, and testicular functions in adult male transgenic mice bearing the hGH gene were evaluated. Two experiments were conducted. In Exp I, transgenic and nontransgenic littermate mice were treated with saline or GnRH in saline (1 ng/g BW). Fifteen minutes after the above treatment, blood samples were obtained for hormone measurements. In Exp II, transgenic and nontransgenic littermate mice were bilaterally castrated, treated with peanut oil or testosterone propionate (TP; 1 microgram/g BW). Blood samples were obtained from half of the animals at 24 h and from the remaining mice 48 h after oil or TP injection. In transgenic mice expressing the hGH gene, plasma PRL levels were significantly lower (P less than 0.001), but circulating LH levels were higher (P less than 0.001) than those in their normal littermates. Administration of GnRH significantly increased (P less than 0.001) plasma LH levels in both groups of mice. However, relative to basal LH levels, the LH response to GnRH treatment was attenuated in transgenic mice. Basal as well as GnRH-stimulated levels of FSH and testosterone were similar in transgenic and nontransgenic littermate mice. In normal castrated mice, plasma LH levels were significantly suppressed (P less than 0.001) 24 h after a single injection of TP. However, the same treatment was ineffective in transgenic mice. The negative feedback effect of TP on plasma FSH levels was also attenuated in transgenic mice. Since it is known that hyperprolactinemia increases LH secretion in male mice, our results demonstrate that transgenic mice are hypoprolactinemic with regard to pituitary PRL secretion, yet due to the lactogenic function of hGH, these animals are physiologically hyperprolactinemic. Thus, the expression of the hGH gene results in a derangement of hypothalamic-pituitary function in adult male transgenic mice.

Animals↗

Genetic engineering of laboratory and livestock mammals.

Recent advances in recombinant genetics have made possible the transfer of cloned genes from one organism to the genome of another. Research with mice made transgenic by insertion of rat or human genes has provided direct evidence that transferred genes can be incorporated into the germline and expressed in the recipient. Current technology for gene transfer involves microinjection of the recombinant genes into the male pronucleus of the zygote. Resulting transgenic mice, when mated as adults, produced offspring that contained and expressed the transgenes. These observations serve as indications of the possibilities that exist for genetic engineering in livestock species. Although there are some technical problems to be overcome before livestock embryos can be genetically altered by these means, the genes for producing growth hormone transgenic livestock are currently available, and research groups are working toward this objective. In addition to this work with growth hormone genes, there are many other potential applications for genetic engineering livestock to produce more highly efficient production; however, there is considerable research to be done before the full potential of this technology can be achieved. It will be necessary to identify other genes that have potential for improving the production efficiency of livestock, and it will be necessary to gain a more complete understanding of the developmental and molecular biology of livestock. The potential impact of this technology in farm animal production is enormous, but, in the short term, it will be a costly endeavor.

Animal Husbandry↗

Visualization of pronuclei in living bovine zygotes.

Bovine embryos were surgically collected from the oviducts of superovulated crossbred heifers 48 h postonset of estrus. The one-celled ova were treated with 4'-6'-diamidino-2-phenylindole (DAPI) and observed under ultraviolet light by fluorescence microscopy. Both male and female pronuclei were visualized, identified and subjected to micromanipulation.

Animals↗

Microinjection of a rabbit beta-globin gene into zygotes and its subsequent expression in adult mice and their offspring.

We have transferred a gene coding for rabbit beta-globin into the male pronucleus of mouse zygotes by direct microinjection. Some of these zygotes developed into mature mice which contained this gene and appeared to be producing a rabbit globin. Evidence for the presence of the gene in these animals was provided by Southern blot hybridization analysis. Evidence for the expression of the rabbit gene in these transformed mice and their offspring was provided by hemoglobin isoelectric focusing analysis and specific serological reactivity between mouse anti-rabbit hemoglobin antiserum and a hemolysate from the mice that developed from the microinjected zygotes. The use of this zygote transformation may allow the introduction and expression of a broad range of genetic elements in mammals.

Animals↗

Human sperm chromatin organization: isolation of homogeneous (25 K b.p.) DNA fragments from in situ chromatin degradation in human sperm cells.

Autodegradation of the chromatin contents of human sperm cells occurs in the presence of dithiothreotol at pH 8.0. The products of this degradation are chromosomal fragments containing DNA molecules approximately 25 K b.p. in length and showing spheroidal subunit structure. From the length of these fragments the direction of one axis of a crystalline organization of human sperm chromatin is suggested.

Chromatin↗

Human sperm chromatin has a nucleosomal structure.

Digestion of human sperm chromatin with micrococcal nuclease reveals an 160 base pair (b.p.) DNA fragment that is further degraded to a series of DNA fragments remarkably similar to the micrococcal nuclease digestion products of eukaryote somatic cellular chromatin. DNase I digestion of human sperm chromatin also yields an identical pattern of DNA fragments to that observed upon DNase I digestion of somatic chromatin. These data, together with earlier electron microscopic observations, suggest a nucleosomal structure for human sperm chromatin.

Chromatin↗

Analysis of binding interactions between histone core complex and simian virus 40 DNA. A comparison of acetylated versus non-acetylated histone core complexes.

The interactions of acetylated and non-acetylated core complex histones with simian virus 40 (SV40) DNA 1 have been analyzed. A modified filter-binding assay utilizing micrococcal nuclease, which allows quantification of histone octamer binding to DNA has been developed. Using this assay it was determined that both non-acetylated core complex histones ad core complex histones acetylated with acetyl adenylate to levels existing in vivo bind cooperatively to SV40 DNA 1. Although both interactions are cooperative, the magnitude of the cooperativity parameter, omega, is significantly less in the acetylated case. This difference in cooperativity is in contrast to the nearly identical intrinsic association constant, K, observed in both cases.

Acetylation↗

Chemically induced gene expression. Manipulation of the transforming ability of simian virus 40 minichromatin by specific chemical hyperacetylation of histones H3 and H4.

We have developed a non-enzymatic acetylating procedure, closely resembling the situation in vivo, utilizing acetyl adenylate, an acetylating agent in vivo, that mimics the enzymatic hyperacetylation of specific histone species. Analysis of the acetylated species of calf thymus histones produced from reaction with soluble chromatin yielded the same species generated in vivo and observed during active gene transcription. Four species of histone H4 and three of histone H3 occur with no alteration in histones H2A or H2B. This procedure has been utilized to hyperacetylate simian virus 40 (SV40) minichromatin in vitro in order to study the effect of acetylated compared to non-acetylated minichromatin in cellular transformation of cultured Balb/3T3 cells. Transformed cell foci appeared only in the cultures infected with hyperacetylated SV40 minichromatin. To select for cellular transformation, foci were transferred to agar-lined culture flasks and grown in the suspension of 1% methylcellulose. The selected cells were plated on slides and analyzed for the presence of T-antigen by indirect immunofluorescence. The hyperacetylated-minichromatin-infected cells exhibited T-antigen-specific fluorescence, while non-acetylated-minichromatin-treated cells and normal cells showed no specific fluorescence. These results suggest a major role for histone hyperacetylation in the mechanism of SV40 viral transformation.

Animals↗

Fine structure of human sperm chromatin.

The fine structure of human sperm chromatin is revealed by electron microscopy. Human sperm chromatin is characterized by fibers composed of discrete spherical organizational units not unlike somatic nucleosomes. During the decondensation process fibers composed of joined spherical units of both 400-A and 150-A diameters were observed.

Chromatin↗

Subunit structure of Eutherian sperm chromatin.

The beta-mercaptoethanol induced decondensation of spermatozoon cell nuclei from several Eutherian species has been followed from the intact spermatazoon cell to the solubilized linear unit sperm chromosomal fiber using fluorescence and electron microscopy. Data from nuclease digestion studies in conjunction with electron microscopic evidence indicate that the gross structure of the unit Eutherian sperm chromosomal fiber consists of DNA folded around sperm specific histone multimers spaced regularly along the fiber generating a linear array of sperm nucleosomes connected by short stretches of uncomplexed DNA. The sperm nucleosomes, 80 A in diameter are separated by 20 A filaments of DNA. This structure is remarkably similar to the structure of somatic chromatin although the protein components of the two chromosomes are markedly different. It seems likely that chromosomal fibers, similar to those described herein, may be present in the male pronucleus following fertilization.

Animals↗

Phosphorylation and dephosphorylation of histone (V (H5): controlled condensation of avian erythrocyte chromatin. Appendix: Phosphorylation and dephosphorylation of histone H5. II. Circular dichroic studies.

During avian erythropoiesis, the blast cells of the bone marrow mature into polychromatic erythrocytes (late stages knwon as reticulocytes) and then into mature red blood cells. When chickens are made anemic, the proportion of immature cells in the anemic bone marrow increases dramatically. The level of the lysine-rich histones. H1 and H5, has been found to be constant in the blood and bone marrow of normal and anemic chickens. This implies that H5 replaces H1 quantitatively. Urea-aluminum-lactate starch gel electrophoresis of H5 from these sources show that the degree of phosphorylation of H5 is proportional to the number of immature cells. About 70% of the H5 from the most immature bone marrow is phosphorylated, while 50% of the H5 from anemic blood is phosphorylated and H5 in normal blood is almost completely devoid of phosphate. When immature cells of the anemia bone marrow are incubated in the presence of inorganic 32P and [3H]lysine and [3H]arginine, extensive 32P incorporation is found in the phospho species. A minimum of nine phosphorylated components have been demonstrated by starch gel electrophoresis. The incorporation of 3H is time dependent. After 1.5 h of labeling, 3H is found in H5 containing 0, 1, 2, and 3 phosphates. tthe combined data suggest that newly synthesized H5 becomes progressively phosphorylated and that at the terminal stage of development, the phosphorylated H5 is completely dephosphorylated. These events may be important in controlling the timing of chromatin condensation.

Anemia↗