The poly(A)(+)RNA sequence complexity is also represented in poly(A)(-)RNA in sea-urchin embryos

The extent to which the poly(A)(+)RNA sequence complexity from sea-urchin embryos is also represented in poly(A)(-)RNA was determined by cDNA cross-hybridization. Eighty percent or more of both the cytoplasmic poly(A)(+)RNA and polysomal poly(A)(+)RNA sequences appeared in a poly(A)(-) form. In both cases, the cellular concentrations of the poly(A)(-)RNA molecules that reacted with the cDNA were similar to the concentrations of the homologous poly(A)(+) sequences. Additionally, few, if any, abundant poly(A)(+)mRNA molecules were quantitatively discriminated by polyadenylation, since the abundant poly(A)(+)sequences were also abundant in poly(A)(-)RNA. Neither degradation nor inefficient binding to oligo (dT)-cellulose can account for the observed cross-reactivity. These data indicate that, in sea-urchin embryos, the poly(A) does not regulate the utilization of mRNA by demarcating an mRNA subset that is specifically and completely polyadenylated.     

Poly(A) and synthesis of polyadenylated RNA in the preimplantation mouse embryo

Investigations were conducted to quantitate polyadenylic acid and estimate the synthesis of polyadenylated RNA in mouse embryos at several stages of preimplantation development. Poly(A) was assayed by molecular hybridization of total embryonic RNA with [³H]polyuridylic acid. The mean values of poly(A) in the ovulated oocytes and in the one-cell, two-cell, and blastocyst stages of the embryo were 1.9, 1.6, 0.68, and 3.8 pg, respectively. Synthesis of polyadenylated RNA was estimated by affinity chromatography of [³H]uridine-labeled embryo RNA on oligo(dT)-cellulose. The proportions of newly synthesized RNA bound by oligo(dT)-cellulose at the 2-cell, 8- to 16-cell, and blastocyst stages were 6.7, 3.5, and 3.3%, respectively. These results suggest that significant quantities of maternal mRNA are present during early development of the mouse, but that polyadenylation of RNA transcribed from the embryonic genome occurs as early as the two-cell stage.     

The identification of cDNA clones that include the 3′ end of mRNA

A method is presented that facilitates the identification of cDNA clones corresponding to the polyadenylated 3′ end of mRNA. It is based on the use of a poly dT probe that is synthesized by homopolymer extension of commercially available oligo dT. The method is shown to work in Southern blot analysis of plasmid preparations and in situ with colonies.     

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.