Cloning, characterization, and tissue distribution of messenger RNA species expressed at moderate and scarce frequencies within the lactating guinea pig mammary gland.
In the lactating guinea pig mammary gland, the most abundant mRNA species encoding the major milk proteins, alpha-lactalbumin and caseins A, B, and C, have been extensively studied. Here we describe the isolation and characterization of cloned cDNA sequences representative of moderately abundant and scarce mammary gland mRNA species present at estimated concentrations of 1,400 (pgpO5), 540 (pgpKE6), 36 (pgpK1), and 2 (pgpJF4) copies per sequence per cell. RNA blotting showed these to represent mRNA species of 1,150, 1,900, 1,250, and 3,300 nucleotides in size, respectively. Hybrid selection cell-free synthesis showed that the mRNAs encoded proteins of Mr 33,000 (pgpO5), 58,000 (pgpKE6), and 36,000 (pgpK1). Studies on the tissue distribution of mammary gland mRNAs showed that the mRNA species of lower abundance, but not milk protein mRNAs, were expressed in other tissues but at concentrations differing from those in the mammary gland. None were expressed in all tissues, and so were not typical "housekeeping" proteins. We have used these cloned cDNA species to reinvestigate the apparent differential accumulation of moderately abundant poly(A)-containing mRNA species in polyadenylated and nonpolyadenylated cytoplasmic RNA populations of the mammary gland. Unlike previous observations, based on RNA excess hybridization using fractionated cDNA probes, the use of sequence-specific cloned cDNA probes showed that little intact mRNA was present in the nonpolyadenylated fraction. Thus previous observations were a reflection of the preferential accumulation of fragments of moderately abundant mRNA species, possibly a result of enhanced turnover. The significance of our results in terms of future investigations into factors which determine mRNA accumulation and tissue-specific expression is discussed.
D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002474:Cell-Free System; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification; D004262:DNA Restriction Enzymes; D005260:Female; D006168:Guinea Pigs; D007774:Lactation; D008099:Liver / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000378:metabolism*; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011247:Pregnancy; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
D012333:RNA, Messenger; D004247:DNA; D004262:DNA Restriction Enzymes