Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland

Abstract

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87--93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

Casein kinase in cytosol of rat and mouse mammary epithelial cells isolated from histone kinase by MgCl2 treatment and influence of pregnancy and lactation on the enzyme activity.

“…Therefore, the existence of casein kinase in the Golgi apparatus (Bingham & Farrell, 1974) seems to corroborate the current view on the biosynthesis of casein and the majority of casein may be synthesized on membrane-bound polysomes, although Devinoy et al (1979) and Houdebine (1977) suggested that free polysomes are responsible for a substantial part of casein synthesis and Craig et al (1979) supported this hypothesis, they suggested that the greater part of this casein may be hydrolysed by intracellular proteases. Regarding the secretion of the casein synthesized on free polysomes, this may occur along the line of fat droplet secretion, since Kurosumi et al (1968) observed electron microscopically that the lumina of lipid droplets contain granules, i. e. casein, when the lumina are removed from the cell membrane.…”

Identification of Limulus polyphemus haemocyanin messenger RNA

“…Glassware used in the extraction of RNA was heat-sterilized, and all solutions and polyware were autoclaved as described by Craig et al (1979). Limulus haemocyanin was purified as described previously (Lamy et al, 1979), and the native haemocyanin dissociated by pH change and separ-ated into five components or zones by chromatography on DEAE-Sepharose CL-6B (Sullivan et al, 1976).…”

“…The RNA was applied to the column equilibrated in 0.5% (w/v) SDS/0.5 M-NaCl/I mM-EDTA/lOmM-Tris/HCl buffer, pH 7.4. After a wash in the same buffer lacking SDS, the column temperature was raised to 600C and the poly(A)+ RNA eluted with lOmM-Tris/HCl buffer, pH 7.4, and precipitated with ethanol and recovered by centrifugation (see Craig et al, 1979).…”

Construction and characterization of a plasmid containing complementary DNA to mRNA encoding the N-terminal amino acid sequence of the rat glutathione transferase Ya subunit

“…Isolation of poly(A)-containing RNA from free polyribosomal RNA was then performed as previously described in Craig et al (1976). Cell-free protein synthesis and product analysis Cell-free protein synthesis was carried out in a nuclease-treated reticulocyte lysate as described by Pelham & Jackson (1976) and SDS/polyacrylamide-gel electrophoretic analysis of [35S]methionine-labelled peptides was performed by using slab gels as described previously (Craig et al, 1979). Fluorography followed the procedure of Bonner & Laskey (1974), except that dimethyl sulphoxide was replaced by glacial acetic acid throughout (Burckhardt et al, 1979).…”