Ph.D. - Biomedical Sciences (Biochemistry)

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    Correlation between intramolecular base composition heterogeneity of DNA and control of transcriptional expression in E. coli temperate phage P2
    ([Honolulu], 1972) Geisselsoder, Janet
    Intramolecular base compositional heterogeneity has been demonstrated in the DNA of the phage P2 by following the optical density of solutions at 260 nm as a function of increasing temperature. First derivative curves of the functions thus generated have been obtained for P2 DNA and the DNA of two closely related phages. Comparison of these curves reveals similarities at the high temperature (high GC) end and differences at the low temperature (low GC) end. All three of these phages can, by supplying the genes concerned with phage particle maturation, serve as helper for the defective phage P4. The strands of P2 DNA have been separated on the basis of their buoyant densities in CsCl solutions when complexed with poly UG. In vivo transcription patterns from these strands in cells infected with various P2 mutants have demonstrated that the "heavy" strand is the one predominantly transcribed, that some "light" strand transcription originates from an operon coding for proteins involved in phage head assembly (thus fuling out a "read-through" mechanism for late gene activation), and that early "light" strand transcription does not originate from DNA deleted in two mutants which is in the right half of the physical map. There is, however, some "light" strand transcription early in infection. P2 DNA has been sheared in: half and the halves separated on the basis of Hg++ binding in Cs2SO4 density gradients. Electron microscopic analysis of partially denatured molecules in these preparations have fixed them with respect to the physical map. In ~ transcription originates primarily from the right, or low GC half early in infection and then shifts to the left, or high GC half. Mutants in genes A and B, which are located in the right half of the genetic map, are defective in both DNA replication and in effecting this shift. Infection with one polar amber mutant under non-permissive conditions has demonstrated that this operon lies :in the right half of the DNA and thus helps to fix the physical map with respect to the genetic map. Two of those regions of the phage DNA which are known to be transcribed from a repressed genome, namely the prophage, appear to be of quite low GC content.
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    The synthesis and release of hormones from human adenohypophyses in vitro
    ([Honolulu], 1971) Siler, Theresa Marie
    Isolation of human prolactin (HP) from fetal and tumor culture medium which was distinct from human growth hormone was achieved by column chromatography. These preparations were studied by chromatoelectrophoresis, radioimmunoassay and polyacrylamide gel electrophoresis (PAGE). The isolated fractions were then compared with two preparations of Pasteels' human prolactin which were further purified by Dr. Lyons (Lyons' PHP and Lyons' cPHP) and the other HP preparations by radioimmunoassay, PAGE and NH2-terminal studies. The HP preparation from fetal culture medium was found to be the most highly purified and immunopotent preparation and most suitable for a radioimmunoassay for human prolactin in plasma. Forty-four human fetal pituitaries and two human pituitary adenomas were studied in organ culture for up to 11 months. Radioimmunoassays for human growth hormone (HGH), human prolactin (HP), luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), adrenocorticotropin hormone (ACTH) and melanocyte-stimulating hormone (MSH) were performed on the culture medium from the pituitary organ cultures. The hormones released into the medium were studied throughout the time in culture and related to the age of the fetal pituitary when first cultured and to its sex. The release of HGH, FSH, LH and ACTH gradually decreased during the first 20 days in vitro to basal or undetectable levels. The release of HP and MSH initially decreased and then increased with the time in culture to a sustained elevated level of release. The total release of each hormone was studied and could be related to various events in fetal development. Pituitaries from six fetuses which had an abnormal total release of pituitary hormones could be related to a fetal abnormality in three cases. Studies of pituitary anlage tissue from embryos as early as five weeks of gestation released hormones in vitro. That these hormones were synthesized in vitro was shown by incorporation of Cl4-leucine into these hormones as determined by PAGE and specific antibody precipitation of each hormone prior to PAGE. Some factors controlling the release of hormones in vitro were studied by the use of modified media. An increased release was obtained with an increased Ca++ and K+ in the medium whereas EDTA caused a decrease in FSH and LH and an increase in HGH and HP. With increased Mg++ concentrations in the medium HGH, ACTH, and HP release decreased. The removal of estradiol from the medium results in increased release of FSH, LH, HGH and ACTH while the HP release was greatly decreased. Various doses of TRH decreased HP release and altered the release pattern of HGH, FSH, and LH. Fetal hypothalamic tissue added to the medium increased HGH, FSH and LH release and extended the duration of release for these hormones while HP release was inhibited. Chick embryo extract was found to have a stimulating effect on the release of each hormones in vitro.