Skeletal muscle protein content of lipolytic inhibitor G(0)/G(1) switch gene-2 protein: the effect of endurance training

The first and rate-limiting step of lipolysis is the removal of the first fatty acid from a triglyceride molecule; it is catalyzed by adipose triglyceride lipase (ATGL). ATGL is co-activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 protein (G0S2). G0S2 has also recently been identified as a positive regulator of oxidative phosphorylation within the mitochondria. Previous research has demonstrated in cell culture, a dose dependent mechanism for inhibition by G0S2 on ATGL. However our data is not consistent with this hypothesis. There was no change in G0S2 protein content during an acute lipolytic inducing set of contractions in both whole muscle, and isolated mitochondria yet both ATGL and G0S2 increase following endurance training, in spite of the fact that there should be increased reliance on intramuscular lipolysis. Therefore, inhibition of ATGL by G0S2 appears to be regulated through more complicated intracellular or post-translation regulation.

Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.