In 1931, Mungomery stated "whitegrubs have been for years past, and still are, the worst insect problem confronting the sugar industry". This statement remains true to this day, with canegrubs costing the Australian sugar industry A$7.22 million in lost production and in use of insecticides. The development of a sugarcane cultivar with resistance to canegrub attack would be a valuable addition to the recently implemented canegrub management program. This thesis examined the possibility that natural inhibitors derived from canegrubs could be incorporated in sugarcane to reduce or prevent its destruction by canegrubs. The research described here demonstrated that canegrub haemolymph contains inhibitors with activity against commercially purified enzymes and serine proteases found in crude midgut extracts. A cDNA encoding a potential canegrub protease inhibitor (DA10 12) belonging to the Ascaris family was cloned, but it did not have activity against the major canegrub midgut proteases. This protein does, however, still have potential for modification into a serine protease inhibitor suitable for use as a novel insect resistance transgene. The possibility of using haemolymph derived inhibitors as novel antimetabolites in a canegrub management strategy based on transgenic plants was also explored. The findings suggest that proteins with properties similar to those of DA10 12 will require the presence of a signal peptide and/or codon optimisation for successful expression in sugarcane. The research outlined in this thesis is the first investigation of protease inhibitors in the haemolymph of scarab larvae, and is the first report of an Ascaris family inhibitor that does not inhibit a serine protease.

The epigenetic contribution to phenotype is now well established. Studies over the past decade have shown that proteins that are able to establish and propagate epigenetic modifications are essential for mammalian development. Some of the genes involved in these processes have been identified, but the roles of many remain unknown. The mutagenesis screens for modifiers of position effect variegation in Drosophila suggest that there are over 200 genes that are able to modify epigenetic variegation. We emulated this screen in the mouse to identify mammalian modifiers of a variegating transgene. The screen aimed to identify novel genes involved in epigenetic reprogramming, and to generate mouse models to study the impact of disruption to the epigenome. Inbred male mice carrying a variegating GFP transgene expressed in erythrocytes were mutagenised with ENU. Offspring were screened by flow cytometry and in the initial rounds of mutagenesis, 11 dominant mutant lines were identified. These lines were called MommeDs (Modifiers of murine metastable epialleles, dominant). This thesis describes the mapping and phenotypic characterisation of three Momme lines: MommeD7, MommeD8 and MommeD9. The MommeD9 mutation enhances variegation and was mapped to a 3.4 Mb interval on Chromosome 7. A mutation in a 5? splice site was found in the Trim28 gene. Analysis of Trim28 mRNA and protein in heterozygotes showed that the mutant allele was null. Homozygotes die before mid-gestation. Heterozygotes are viable but display variable and complex phenotypes, including infertility, obesity, behavioural abnormalities and premature death. Obese MommeD9 mice have liver steatosis, impaired glucose tolerance and other indicators of metabolic syndrome. This phenotype has not previously been reported for mice haploinsufficient for Trim28. There is considerable variability of phenotypes among inbred MommeD9 heterozygotes, which suggests a role for epigenetics in phenotypic noise or “intangible variation”. MommeD8 is a semi-dominant enhancer of variegation. Some homozygotes are viable but some die around birth. Viable homozygotes weigh less than wildtype littermates and have increased CpG methylation at the GFP transgene enhancer element. The mutation was mapped to a 4 Mb interval on chromosome 4. Extensive candidate gene sequencing failed to find a mutation and so DNA from mutant and wildtype individuals were sequenced across the entire linked interval by 454 Sequencing technology. MommeD8 individuals carry two point mutations, one is intergenic and the other lies in an intron of the Ppie gene. Analysis of Ppie mRNA in heterozygotes and homozygotes shows that mutants have reduced transcript levels, suggesting that a deficiency in Ppie causes the increased silencing of GFP. The Ppie gene has not been reported to be involved in epigenetic reprogramming and little is known about its function. Mice heterozygous for MommeD7 have a marked increase in expression of GFP. Heterozygotes have a range of hematopoietic abnormalities including splenomegaly, anaemia and reticulocytosis. Homozygotes die at birth and appear pale. The increased GFP in the peripheral blood appears to be the consequence of an increase in reticulocytes. The mutation is linked to a 1.5 Mb interval on Chromosome 7. MommeD7 mice appear to have hematopoietic abnormalities that affect the expression of the erythroid-specific GFP reporter transgene. MommeD7 mice serve as a reminder that, as well as discovering bona fide modifiers of epigenetic reprogramming, the ENU screen can also identify hematopoietic mutants.

Freie Univ., Diss.--Berlin, 1997.

Techn. Universiẗat, Diss., 2005--München. / Enth. 5 Sonderabdr. aus verschiedenen Zeitschr.

University, Diss., 2005--Würzburg. / Erscheinungsjahr an der Haupttitelstelle: 2005.

München, Techn. Univ., Diss., 2004.

München, Techn. Univ., Diss., 2001.

Techn. Hochsch., Diss., 2002--Aachen.

Techn. Hochsch., Diss., 2003--Aachen.

Techn. Hochsch., Diss., 2004--Aachen.