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Graduation date: 1970

Call number: LD2668 .T4 1951 U4 / Master of Science

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The A mating type factor of the hymenomycete fungus Coprinus cinereus is a multi-allelic gene complex that controls mating compatibility and sexual development. It contains up to four pairs of specificity genes, the a, b, c, and d gene-pairs. Each gene-pair codes for two homeodomain transcription factors with distinct classes of homeodomain motifs. Mating compatibility between the A42 and A6 factors depends solely on the different alleles of the b gene-pair, b1-1 and b2-1 in A42 and b1-3 and b2-3 in A6. The b1-3 and b2-3 genes of A6 were isolated and the complete DNA sequences of genomic and cDNA clones were determined. Construction of chimeric genes using the A42 and A6 b genes identified the N-terminal regions of the A proteins as being responsible for allele specificity. Analysis of protein-protein interactions showed that b1 and b2 proteins from different alleles of the same gene-pair can dimerise, whereas proteins from the same allele pair can not. It was shown that a region of 90 amino acids at the N-terminus of the b2-3 protein is sufficient for dimerisation with b1-1. This region is predicted to contain an amphipathic helix. A comparison with the equivalent region in the b2-1 protein identifies a similar helix. This suggests that a compatible A mating type reaction and thus allele specificity is recognised by the ability to dimerise through this domain. Polyclonal antibodies were raised against the b1-1 protein and a heterologous yeast expression system was established for testing potential DNA target sites of the b1-1 and b2-3 proteins, both techniques offering potentially useful tools for further molecular analysis of the A mating type proteins.

The purposes for which the fractionation of proteins are carried out are quite varied and manyfold. However, one of the more important reasons is that of determining the nature and the extent of autolysis or proteolysis on the bovine muscle proteins during post mortem aging. Hence, the development of a procedure for the adequate fractionation of muscle proteins would greatly stimulate the interest and research progress in this difficult field of study. The research reported herein pertains to a study of the fractionation of fresh bovine muscle proteins by ion exchange chromatography. A KCl-phosphate buffer, pH 7.5 and an ionic strength of 0.55, was used to extract the proteins from the muscle. This extract was then diluted to specific ionic strengths in order to separate the gross fractions (actomyosin, myosin, sarcoplasmic + actin) from the total KCl-phosphate soluble proteins. The major protein fractions were then separated by diethylaminoethyl-cellulose (DEAE-cellulose) ion exchange chromatographic procedure. A non-linear gradient elution schedule was used throughout the chromatographic procedure. The disc electrophoresis technique was used to determine the homogeneity of the various protein fractions and sub-fractions. The total KCl-phosphate soluble proteins were fractionated into 9-12 fractions by DEAE-cellulose ion exchange chromatography. The number of fractions differed from one sample to another. The disc electrophoresis results paralleled those of the chromatographic procedure. Some of the fractions appeared to be homogeneous while others were not. The KCl-phosphate soluble proteins minus actomyosin were fractionated by column chromatography into 9-10 different protein components. The disc electrophoresis results also indicated that this fraction contained 9-10 components. The sarcoplasmic + actin proteins were fractionated into six fractions by the chromatographic procedure. These fractions appeared to be electrophoretically homogeneous. Although some success was attained in the separation of the actomyosin proteins, the myosin fraction was quite resistant to chromatographic separation. In spite of the ineffectiveness of the DEAE-cellulose column chromatographic technique to fractionate the myosin and actomyosin proteins, the procedure appears to have some value for studying the other protein fractions during autolysis. Moreover, further work on the adoption of this procedure might provide the necessary information to perfect the technique for muscle protein fractionation. / Graduation date: 1963

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