Ph.D. - Biomedical Sciences (Biochemistry)

Permanent URI for this collection


Recent Submissions

Now showing 1 - 10 of 36
  • Item
  • Item
  • Item
  • Item
    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.
  • Item
    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.
  • Item
    Investigation of manganese binding potential in neoplastic and non-neoplastic tissue
    ([Honolulu], 1971) Zimmerman, Norman
    In an attempt to discover methods of controlling cancer, two basic philosophies of research prevail. The first involves testing vast multitudes of drugs in the hope that one may be found which destroys the malignancy without killing the patient. The second espouses the finding of differences between neoplastic and non-neoplastic tissue in the hope that these differences may elucidate the nature of the carcinogenic process. Once this is known, one would hope to be able, teleologically, to select possible methods of cure. Clearly, a great number of differences between neoplastic tissue and the tissue of origin of the neoplasm simply reflect cellular differences and in no way relate to the carcinogenic process. The dissertation will be concerned with the differences in manganese binding potential in neoplastic and non-neoplastic tissue that has been observed by the use of Electron Paramagnetic Resonance (EPR). A general discussion of EPR and the role of manganese in normal tissue will be followed by a proposed theory of carcinogenesis. Evidence will then be presented for differences in manganese binding potential between neoplastic tissue and the tissue of origin for six different systems. Competition studies will be utilized to show that the effect observed with manganese cannot be seen for a variety of other ions (i.e., it is not a common ion effect). A brief discussion on endogenous manganese levels will be included. A discussion of purification of the binding system will follow. In conclusion, data will be presented regarding manganese binding potential with respect to the carcinogenic process.
  • Item
    The pyrimidine tracts of bacteriophage øX174
    ([Honolulu], 1969) Bickel, Riva Pearl Wenig
    The bacteriophage ϕX174t containing single stranded, circular DNA as its genetic material, has been studied in some detail with respect to its chemistry and biological function. The frequency of the pyrimidine tracts in the wild type bacteriophage has been determined t and the preponderance of the amount of longer tracts of seven to ten pyrimidine nucleotides, over that which would be expected from a random distribution noted. Further studies of the longer pyrimidine tracts were carried out using the ρ- mutant bacteriophage, which is lysis defective, showing delayed lysis in dilute cultures of the host, E. coli, and lysing only about ten per cent of the cells in dense culture. The DNA of the phage contains a discontinuity, a small region which is not single stranded, and therefore not susceptible to the action of E. coli exonuclease I. This enzyme digests only single stranded DNA and works only on free 5' OH ends. Therefore, digesting ϕX174 chromosomes with pancreatic DNA'ase, an endonuclease, to a limited extent, followed by digestion with E. coli exonuclease I, leaves a population of DNA of varying lengths, all having the same 5' end at the discontinuity. Within this population, then, the frequency of an individual tract's survival, depends on its distance from the discontinuity, and can be used to determine its position on the ϕX174 chromosome. Methods for preparation of pyrimidine tracts, their separation by length into isopliths, and their subfractionation according to their relative cytosine and thymine content have already been developed. When more than one tract of the same length and base composition existed, further separations were not possible. Only the unique tracts could be mapped. This, in the ρ- mutant, consisted of one decanucleotide, two each of nona and octanucleotides and one each of hepta and hexanucleotides. All of these tracts were located in the region of about 30 to 80 per cent of the distance around the chromosome from the discontinuity. The pyrimidine tracts of the ρ- mutant and wild type bacteriophage were compared. There is a decanucleotide of composition C3T7 present in the wild type phage, but not in the ρ- mutant. Some evidence indicates that this mutation, that of a pyrimidine to a purine, occurs at a cytosine residue, leaving an octanucleotide, CIT7 present in the ρ- mutant, but not in the wild type phage. The nature of the ρ- mutation is discussed, along with the possibility that the longer pyrimidine tracts may have a role in initiation of the DNA to messenger RNA transcription process.
  • Item
    A study of the biosynthesis of growth hormone and prolactin in bovine pituitary slices and cell-free systems
    ([Honolulu], 1969) Robertson, Mary Chalmers
    The biosynthesis of growth hormone and prolactin was studied in both slice and cell-free systems from bovine anterior pituitary tissue. Slice preparations were highly active in incorporating radioactive amino acids into prolactin t growth hormone and protein. Modification of existing procedures were employed for the isolation of the labeled hormones in a relatively purified state. A number of physical t chemical, and biological criteria. were used to demonstrate that the radioactive proteins were indistinguishable from reference standards. These included amino acid composition, N- and C-terminal analysis, gel filtration, ion-exchange chromatography, polyacrylamide gel electrophoresis, and Ouchterlony immunodiffusion. Using the same procedure, the relationship of growth hormone releasing factor (GRF) to the synthesis and release of growth hormone in vitro with both bovine and rat pituitaries was studied. Although no effect on synthesis and only a slight effect on release were noted, these results must be considered inconclusive because of the small number of samples studied. With a cell-free system, consisting of ribosomes plus pH 5 enzyme fraction, and fortified with ATP, GTP, and Mg++, the optimal conditions for biosynthesis of growth hormone and general protein were established. When a polysome-enriched preparation was resolved into fractions of discrete particle size on sucrose density gradients, incorporation of radioactive amino acids into the hormone was most active with polysomes containing six to seven ribosomal units. The isolated radioactive product was indistinguishable from the authentic growth hormone standard by four physical criteria; ion-exchange chromatography, Sephadex gel filtration, polyacrylamide disc gel electrophoresis and sucrose density gradient centrifugation. In addition, it displayed specific antibody binding. Examination by similar physical criteria, of the radioactive polypeptide associated with the prolactin fraction isolated from the pituitary ribosomal system, revealed no evidence of incorporation of labeled amino acids into the monomer form of prolactin. Several explanations have been offered for the failure to demonstrate synthesis of prolactin with this cell-free system.
  • Item
    The effect of steroids on RNA synthesis in various tissues of the rat
    ([Honolulu], 1969) Philleo, William Wallace
    Single doses of aldosterone were administered to normal rats, and the effect of RNA synthesis was studied. The maximal stimulation of the incorporation of l4c-ATP into RNA by nuclei isolated from rat kidneys was observed 30 minutes after an intravenous injection of aldosterone. Thereafter, the RNA synthesis decreased to a level less than that of the control, reaching a minimum at 2.5 hours. The return to control level was followed by further oscillations in RNA synthesis. Likewise, in isolated brain nuclei, an oscillation in RNA synthesis was observed following an intravenous injection of aldosterone. The effect of single injections of aldosterone, cortisol, deoxycorticosterone acetate, testosterone or progesterone was studied on RNA synthesis by isolated rat spleen nuclei. It was found that the l4C-ATP incorporation was initially inhibited following the injection of aldosterone, cortisol or deoxycorticosterone acetate. The initial response to an injection of testosterone or progesterone was a stimulation in the spleen RNA synthesis. The response to the administration of aldosterone was similar to that of cortisol. Subsequent to the initial inhibition, a stimulation followed by a return to the level of the control, was observed and secondary inhibitions were found. RNA synthesis in the thymus was initially inhibited following the administration of aldosterone. RNA synthesis was studied in the nuclei isolated from the kidney and spleen of adrenalectomized or hypophysectomized rats injected with aldosterone. Oscillations were observed in the RNA synthesis of both tissues of the endocrinectomized rats. It was concluded that the oscillations in RNA synthesis were not a function of some form of homeostatic control involving the pituitary and adrenal glands. Aldactone (SC 9420) was found to modify the RNA synthesis of nuclei isolated from the kidney, spleen and thymus of intact, adrenalectomized or hypophysectomized rats. Again, oscillations in RNA synthesis were observed, however there was the suggestion that the adrenals or the pituitary might be involved in the response as observed in the kidney and the thymus. Experiments were carried out in which both aldosterone and Aldactone were injected, and their action on RNA synthesis was observed in the kidney, spleen and thymus. The results indicated that these two steroids alter RNA synthesis independently from each other. Analysis of the RNA synthesized in vitro by the isolated nuclei of the kidney and spleen indicated that only a single species of RNA was made. This 5 s RNA was identical to that species of RNA present in nuclei, prior to incubation for the incorporation of l4C-ATP.
  • Item
    The behavior of horse liver alcohol dehydrogenase in guanidine hydrochloride solutions
    ([Honolulu], 1969) Green, Robert William
    The Z average molecular weight of horse liver alcohol dehydrogenase (LADH) in dilute neutral buffer was found to be 78,200 by sedimentation equilibrium. One molar guanidine hydrochloride (GuHCl) reversibly inhibited LADH activity. The inhibition was competitive with limiting nicotinamide adenine dinucleotide and mixed with limiting ethanol. When EDTA was added to LADH in 1 M GuHCl, zinc was removed followed by aggregation. Sedimentation velocity experiments indicated that no subunit was formed under these conditions. Higher concentrations of GuHCl resulted in dissociation of LADH. In 3 M GuHCl, the sedimentation equilibrium data indicated the presence of a reversible association-dissociation system involving subunit, dimer, and trimer. EDTA removed zinc but had no effect on the molecular weight of LADH in 3 M GuHCl. Reduction and alkylation of LADH in 3 M GuHCl resulted in almost complete dissociation into two subunits. Apparently, sulfhydryl residues are involved in subunit association, while zinc plays little or no role. The Z average molecular weight of LADH in 5 M GuHCl containing mercaptoethanol was found to be 45,700, a value higher than expected. The discrepancy may be attributed to incomplete dissociation, a small error in apparent partial specific volume, or solvent binding. Measurements of molecular weight, sedimentation coefficient, and intrinsic viscosity of LADH in 5 M GuHCl indicate that the structure of LADH is composed of two subunits.