Studies on the isolation and characterization of hair follicle messenger RNA

Abstract

The work described in this thesis was directed towards the isolation, purification and fractionation of mRNA from guinea pig hair follicles, and also towards the charaterization of this follicle mRNA by translation in cell-free protein-synthesizing systems. The following results were obtained:- (1) Procedures for the preparation of hair fo1lic1e tissue homogenate were thoroughly investigated with the aims of maximizing RNA yields while minimizing contamination by active ribonuclease and the shearing of large RNA molecules. Procedures for the isolation of deproteinized RNA from the fo1lic1e homogenate were also investigated with the aim of maximizing yields of undegraded, biologically-active mRNA. This work led to the first isolation of follicle RNA fractions containing mRNA activity. This activity was assayed by stimulation of amino acid incorporation in a heterologous (wheat embryo) cell-free protein-synthesizing system. (2) Follicle RNA was fractionated, and the various fractions obtained were partially characterized with respect to their protein translation products in the wheat embryo cell-free system. Parameters affecting the efficiency of the follicle mRNA -primed wheat embryo system for the synthesis of large follicle proteins were investigated. (3) The activity of the hair follicle cell-free protein-synthesizing system was improved, and a modified cell-free system prepared from follic1e tissue was tested as a possible assay system for follicle mRNA fractions. As part of continuing studies on the hair follicle honogenate system, the follicle arginine transfer enzyme, whose existence was first postulated by Lock (1973), was further characterized.