Purification of Albumin mRNA from Rat Liver

Abstract

Albumin mRNA was purified from rat liver by indirect immunoprecipitation of albumin‐synthesizing polysomes. Monospecific antibodies against albumin, raised in rabbits, were added to rat liver polysomes and the antibody‐polysome complex formed was adsorbed to goat antibodies against rabbit immunoglobulins, coupled to p‐aminobenzyl‐cellulose according to Schütz et al. [Nucleic Acid Res. 4, 71–84 (1979)]. Approximately 8–10% of the polysomes were bound to the matrix. They were then eluted from the column, RNA was isolated and mRNA recovered by chromatography on oligo(dT)‐cellulose. By translation of the mRNA in vitro in a wheat germ system, it was estimated that approximately 90% of the albumin‐synthesizing mRNA was precipitated and that the albumin mRNA fraction obtained was enriched 8–10‐fold for albumin‐coding sequences. Complementary DNA was transcribed from this mRNA. Albumin‐specific cDNA was purified by hybridization of the cDNA to polysomal rat liver mRNA up to a limited rot value of 2 × 10−2 mol · 1−1· s, isolation of the double strands by hydroxyapatite chromatography and recovery of the hybridized cDNA after alkali digestion of RNA. Kinetic data obtained by hybridization of this cDNA against total cytoplasmic and purified mRNA demonstrate that more than 90% of this cDNA comprises only one sequence component. An r0t1/2 value of 2.2 × 10−3 mol · l−1· s was estimated for the immunoprecipitated mRNA fraction. This indicates already a high degree of purity of the mRNA component of this fraction, as this RNA still contains approximately 50% ribosomal RNA. By electrophoresis of glyoxal‐denatured RNA in agarose gels, a molecular weight of 780000 was found for albumin mRNA, a value which corresponds well with a sequence complexity of 880000, estimated from the kinetics of the mRNA‐cDNA hybridization. Titrations of albumin cDNA with total polysomal poly(A)‐containing RNA indicated a content of approximately 10% for albumin mRNA in this fraction. Total nuclear RNA was shown to contain approximately 0.1% albumin‐specific sequences, whereas poly(A)‐containing nuclear RNA was about 4‐fold enriched for albumin sequences.

Isolation and Characterization of Xenopus laevis Albumin mRNA

“…It is possible that either these albumin mRNA molecules had not been polyadenylated during the processing of the initial mRNA transcripts [38] or they reflect aging of the mRNA molecules [39]. Similar findings have been reported for other mRNA species such as ovalbumin [21,40,41], globin [22,42], casein [43], vitellogenin [25] and rat liver albumin [23,24,44] mRNAs.…”

“…As vitellogenin mRNA has already been isolated and fully characterized [25] the next step in the analysis of such a system was to isolate, purify and characterize the mRNA coding for albumin. Various methods have been described forthe isolation ofalbumin mRNAfrom rat liver [23,24,30,44] and mouse liver [31]. The purification of albumin mRNA from the total nucleic acids of frog liver was achieved by the use of oligo(dT)-cellulose chromatography and fractionation of the resulting poly(A)-rich RNA on sucrose gradients containing 50% MeZSO.…”

Δομη Χρωματινης Και Ρυθμιση Της Γονιδιακης Εκφρασης

“…"Επίσης συμφωνεί μέ τά ευρήμα τα άλλων ομάδων γιά τό mRNA της άλβουμίνης άπό συκώτι έπίμυος (Taylor καί Tse, 1976 e Strair et al, 1977" Sala-Trepat et al, 1979" Hofer et al, 1979.…”