Patterns of DNA synthesis and mitosis in the coenobial alga Pediastrum boryanum (Turp.) Meneghini were analyzed by cytophotometric measurements of individual, Feulgen‐stained nuclei from swarming zoospores aggregating into colonies, and cells in colonies varying in age from 12 to 96 h after their initial transfer to fresh culture medium. A haploid genome size of 0.2 pg DNA (corresponding to roughly 11 × 1012 daltons, or 1.64 × 105 kb) was estimated by comparative measurements of nuclei from zoospores or young colonies and chicken erythrocyte (RBC) nuclei which were included with each set of Pediastrum slides as an internal reference standard of 2.5 pg DNA/cell. Although nuclear morphology and extent of chromatin condensation vary with different stages of colony development, nuclear division in P. boryanum appears to follow each cycle of DNA replication with no accumulation of DNA beyond the 2C level. Cytoplasmic cleavage resulting in the formation of individual zoospores is delayed until completion of mitosis, as is the demise of the pyrenoid. After 96 h of culture, 40% of all colonies have cells that are 8‐ or 16‐nucleate and some colonies have 32 nuclei/cell. Release of zoospores within vesicles occurs at this time to complete a cycle of asexual reproduction.

The mechanism of action of insulin and epidermal growth factor was studied by genetic and biochemical means. Particular emphasis was placed on the ability of these factors to induce DNA synthesis and the relationship of endocytosis to that ability. Insulin was crosslinked to the active fragment A of diphtheria toxin. This conjugate specifically killed cultured mouse cells through an insulin receptor-mediated process. The conjugate was used to select genetic variants resistant to its cytotoxic effect. Six resistant variants were isolated, 2 of which retained very low insulin receptor activity. When these two variants were further analyzed both displayed altered cell shape and growth properties. The CI-3 variant also was shown to have a deficient lysosomal system and failed to efficiently degrade epidermal growth factor. This variant was, however, fully responsive to the mitogenic action of EGF. This suggested that lysosomal processing is unimportant in the production of a mitogenic stimulus by EGF. EGF was found to be endocytosed by fibroblasts through 2 separate pathways. One pathway involves an unidentified organelle and correlated with increased degradation of the ligand. The other pathway involves a Golgi-like component and is correlated with a lack of degradation and uptake into a dense, non-lysosomal organelle. Uptake of EGF into this non-lysosomal component, which we named mitosomes, correlated with the ability of EGF to induce DNA synthesis. From these results, a model was constructed for the coupling of endocytosis, uptake into mitosomes and the stimulation of DNA synthesis.

Bibliography: leaves 127-166. This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes.

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

Vaccinia virus infected monkey kidney cells had been previously shown to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA from mutant virus resistant to hydroxyurea were digested with restriction endonucleases and were shown to have substoichiometric amounts of the Hind III F fragment. Additional information from Southern blotting experiments localized the putative small subunit (R2) gene to the left end of the Hind III F fragment of the vaccinia virus genome. The entire open reading frame of the R2 gene and the flanking regions was sequenced and the translated sequence found to be 80% homologous to the mouse R2 polypeptide. A combination of in situ and in vitro experiments addressed the question of macromolecular interactions involving vaccinia ribonucleotide reductase (FIR). Replication of double stranded viral DNA occurs in very discrete loci in infected cells and these DNA factories can be isolated from gently lysed cell in sucrose gradients. RR was detected at low levels (less than 5% of the total R2) with the rapidly sedimenting DNA by using antibodies against FIR. In situ crosslinking experiments were attempted with no specific interaction determined at this time. Immunolocalization experiments have given evidence for localization of large subunit (R1) polypeptide to the viral inclusion bodies. The most conclusive results utilized anti-idiotypic antibodies against the antibodies to R2 protein. lmmunolocalization experiments have shown the putative R2 binding protein to be localized at the sites of viral DNA synthesis. lmmunoprecipitations show a single predominant viral polypeptide which also has proven to be a DNA binding (phospho)protein. Screening a lambda phale expression library of vaccinia with the anti-idiotypic antibody localized the binding site to the carboxy terminal 81 amino acids in open reading frame 1-3 of the vaccinia genome. The open reading frame was cloned into a pET11c expression vector and the partially purified recombinant protein was shown to have specificity for single-stranded DNA as well as stimulate vaccinia RR activity. / Graduation date: 1993

Graduation date: 1993

The ability to faithfully replicate DNA is dependent upon the maintenance and regulation of its precursors, the deoxyribonucleoside triphosphates. Enzymes encoded by the bacteriophage T4 have been widely used as models of biochemical processes. A body of evidence supports the concept that the bacteriophage T4 enzymes involved in deoxyribonucleotide biosynthesis are associated as a complex within the infected Escherichia coli. This dissertation describes the continued examination of the protein-protein interactions involved in deoxynucleotide biosynthesis of bacteriophage T4. My studies on the protein-protein interactions involved in deoxyribonucleotide biosynthesis focused on two unique phage proteins, the dCMP hydroxymethylase enzyme and the translational regulator RegA. An initial study was undertaken to determine if the generation of anti-idiotypic antibodies would prove useful in the identification of direct interactions between dCMP hydroxymethylase and other proteins of the deoxyribonucleotide synthetase complex. For the initial generation of anti-idiotypic antibodies, polyclonal rabbit antibodies were generated to affinity purified anti-dCMP hydroxymethylase polyclonal rabbit IgG. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the bacteriophage T4 dTMP synthase. A second method to generate anti-idiotypic antibodies was attempted with the translational regulator RegA. The generation of anti-idiotypic antibodies to the RegA protein involved the purification of anti-RegA rabbit Fab fragments and the generation of anti-anti-RegA polyclonal antibodies within mice. This alternative method was determined to be inferior to the initial method for generating anti-idiotypic antibodies. Additional studies involved the examination of RegA protein-protein interactions using affinity chromatography. A number of bacteriophage T4 early proteins were determined to associate with an immobilized RegA column. / Graduation date: 1992

The effects of conjugated linoleic acid (CLA) on lipogenesis and preadipocyte proliferation in young pigs were evaluated in two separate experiments. The first compared dietary effects of linoleic acid, beef tallow, and CLA on composition, lipogenesis, and DNA synthesis. Eighteen pigs weaned at 17 d of age were allotted randomly to corn-based diets supplemented with 1.5% corn oil, 1.5% tallow, or 1.5% CLA. The second experiment evaluated the effects of CLA included with diets high in polyunsaturated fat or beef tallow. Twenty-four pigs weaned at 17 d of age were allotted randomly to one of four corn-based diets supplemented with: 15% corn oil, 12% corn oil + 3% CLA, 15% tallow, and 12% tallow + 3% CLA. The piglets in both trials were fed a basal diet for 7 d and their respective diet for 35 d. [U-14C]Glucose incorporation into total lipids was (experiment 1): 10.64, 11.04, 13.64; (experiment 2): 21.15, 17.54, 21.34, and 19.52 nmol/(105 cells per h) for subcutaneous (s.c.) adipose tissue from corn oil, tallow, CLA; corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively. Tritiated thymidine incorporation into DNA was not different in s.c. adipocytes across treatment groups, but was 5,581, 2,794, 6,573, and 3,760 dpm/(105 cells per h) in s.c. stromal vascular cells from corn oil, corn oil + CLA, tallow, and tallow + CLA-fed piglets, respectively (CLA main effect p<0.034). Additionally, there was a greater proportion of s.c. adipocytes in the smaller, 180-pL cell fraction from the corn oil + CLA-fed pigs (p<0.0074). CLA in the diet increased the s.c. adipose tissue concentration of 18:0 and decreased 16:1 and 18:1 (p<0.05), suggesting depression of stearoyl-coenzyme A desaturase (SCD) enzyme activity in the CLA-fed pigs. The concentration of CLA isomers was raised only slightly in s.c. adipose tissue with the addition of CLA to the diets even though the CLA oil contained 62% CLA isomers. No effects on the growth of young pigs were observed. However, CLA caused a more saturated fatty acid composition and may suppress preadipocyte proliferation, apparent SCD activity, and lipid filling of smaller cells.

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997

Bibliography: leaves 127-166. / xvi, 166, [64] leaves, [11] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study describes the isolation, sequencing and characterization of two cDNAs encoding barley (1-4)-B-xylanase isoenzymes X-I and X-II and the gene corresponding to isoenzyme X. The results of genomic Southern blot analyses indicate that the barley (1-4)-B-xylanase gene family consists of at least 3 genes which are mapped to a single locus on the long arm of chromosome 7(5H). The cDNA is used to monitor tissue-specific expression, developmental regulation and hormonal control of the (1-4)-B-xylanase genes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 1997