No description available.

Using a novel purification scheme and a new assay for detection of DNA kinase activity, a Polymin P-precipitable DNA kinase has been identified and characterized from calf thymus extracts. The DNA kinase activity was able to phosphorylate RNA as well as single-stranded and double-stranded DNA, therefore it has been termed Polymin P-precipitable polynucleotide kinase (PP-PNK). The enzyme had a neutral to alkaline, broad pH optimum that distinguished it from the previously described mammalian DNA kinases that have an acidic pH optimum. The sedimentation coefficient of the enzyme was 3.4-3.8 S, indicating a molecular weight of about 50 kDa. Estimates for the K$ sb{ rm M}$ for ATP were 52 $ mu$M and for the oligonucleotide substrate 8 $ mu$M. The activity was inhibited by pyrophosphate anions and to a lesser extent by sulfate anions. These results differentiate PP-PNK from other mammalian polynucleotide kinases.

Whole cell extracts from fresh calf thymus glands were subjected to Polymin P fractionation and Q Sepharose chromatography. Three peaks of DNA kinase activity, designated SNQI, SNQII and SNQIII, were found in the supernatant fraction. Studies of SNQI have revealed an estimated molecular mass of 50-90 kDa by Superose 12 chromatography, and activity gel analysis following SDS-PAGE identified an active polypeptide of approximately 55 kDa. This enzyme preparation, purified 10,000-fold, phosphorylated 5$ sp prime$-OH-terminated oligodeoxyribonucleotides and double stranded DNA, yet was inactive on an oligoriboadenosine ladder. SNQI functions optimally at an acidic pH in 10 mM MgCl$ sb2$, but is inhibited by both sulfate and pyrophosphate anions. The estimated K$ sb{ rm M}$ values were 2.3 $ mu$M for the oligonucleotide substrate and 11.8 $ mu$M for ATP. Similar to an enzymatic activity previously isolated from rat liver, SNQI is the first bovine preparation to display both 5$ sp prime$ kinase and 3$ sp prime$ phosphatase activities. / Partial purification and characterization of SNQII revealed similarities to SNQI, such as an acidic pH optimum and the presence of 3$ sp prime$ phosphatase activity. DNA kinase activity was also demonstrated in two mammalian cell lines.

In cell cycle checkpoint control, the Chk2 family protein kinases play a central role in mediating the cellular responses to DNA damage or DNA replication block. However, at the beginning of this project, there was no evidence for a Drosophila homologue of Chk2. loki was identified in a screen for serine/threonine protein kinases that are expressed in the ovary. Using a phylogenetic analysis, I showed that loki is a Drosophila chk2 orthologue. To characterize the checkpoint function of loki in Drosophila development, we created a loki null mutant and generated anti-Loki antibodies. Under normal laboratory conditions, loki null mutants display no apparent defect during the whole life span. Further functional analysis revealed that loki is not required for the meiotic pachytene checkpoint, the essential DNA replication checkpoint control in syncytial embryos and the DNA damage/replication checkpoint during the larval stage. However, in postblastoderm embryos, loki is required for the DNA damage checkpoint activated by gamma irradiation. Embryos lacking loki are not able to arrest the cell cycle in response to gamma irradiation.

Protein phosphorylation is now recognized to be one of the most important means of regulating protein activity. An approach was taken that was aimed at identifying new protein Serine/Threonine kinase genes in the fruit fly Drosophila melanogaster. Three of the kinases identified were chosen for molecular characterization: a Map kinase-activated protein kinase-2 homolog (DmMAPKAPK-2); a novel female germline specific kinase ( loki); and the homolog of the vertebrate cdk7 genes (Dmcdk7). Among those, Dmcdk7 was chosen for in depth molecular and genetic characterization. Cdk7 has previously been shown in vertebrate systems to phosphorylate and activate many different Cyclin-dependent kinases (Cdks) in vitro. However, conclusive evidence that Cdk7 could act as a Cdk-activating kinase (CAK) in vivo had remained elusive, and became even controversial. Adding to the controversy was the fact that in the budding yeast S. cerevisiae, CAK activity is provided by the CAK1/Civ1 protein which is unrelated to Cdk7. It was therefore proposed that the CAK activity of Cdk7 may be an in vitro artefact. In an attempt to resolve this issue null and temperature sensitive mutations of the Dmcdk7 gene have been created. The results obtained using these mutant alleles of Dmcdk7 demonstrate that cdk7 is necessary for CAK activity in vivo in a multicellular organism. It is shown that cdk7 activity is required for the activation of both Cdc2/Cyclin A and Cdc2/Cyclin B complexes, and for cell division. In addition to validate the function of Cdk7 as a bona fide regulator of the cell cycle, these results suggest that there may be a fundamental difference in the way metazoans and budding yeast effect a key modification of Cdks. Phosphorylation events at different sites (including the T-loop) are also known to be involved in stabilizing the Cdk7/Cyclin H dimer in vitro, and have been shown to occur in vivo. Surprisingly, the in vivo analysis of different phosphorylation mutant forms of DmCdk7 f

Insulin stimulates glucose transport into muscle cells and adipocytes via a process that involves the translocation of GLUT4 proteins from intracellular stores to the cell membrane. The pathway by which this translocation takes place has not been fully elucidated. The purpose of this study was to determine the effect of the calciumdependent calmodulin protein kinase II (CAMKII) inhibitor KN-62 on insulin stimulated 3-0-methylglucose transport in isolated rat epitrochlearis muscles. The primary finding of this investigation was that KN-62 decreased insulin stimulated glucose transport by -35%. KN-04, a structural analogue of KN-62, did not affect insulin stimulated glucose transport. Additional experiments showed that the L-type calcium (Ca 2+) channel inhibitor nifedipine inhibited glucose transport to a similar extent as KN-62 (-29%). Furthermore, no additive inhibitory effect was seen when KN-62 and nifedipine were used in combination. The results of this investigation suggest that CAMKII has a critical role in insulin stimulated glucose transport, and this role may be dependent upon L-type Cat- channel activation. / School of Physical Education

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: G.D. Wright. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.) -- McMaster University, 1999. / Includes bibliographical references (leaves 214-223). Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: G.D. Wright. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 89-108).