Polysomal Polyadenylated RNA Research Articles

Gene expression in friend erythroleukemia cells following the induction of hemoglobin synthesis

When treated with dimethylsulphoxide (DMSO), Friend cells accumulate the messenger RNAs (mRNA) coding for α- and β-globins, and the ratio of α- to β-globin mRNA changes. The concentration of globin mRNA sequences in the nuclear polyadenylated RNA of the DMSO-treated Friend cell is significantly (50–100-fold) lower than that in the polysomal polyadenylated RNA, showing that post-transcriptional controls must modulate the concentration of globin sequences in the polysomal RNA. Both the untreated and DMSO-treated Friend cells are expressing several thousand other polyadenylated mRNAs. No differences, other than the increase in globin mRNA sequences, can be detected when the mRNA populations of the two cells are compared by heterologous hybridization reactions involving the RNA from one cell and a complementary DNA (cDNA) copy of the RNA from the other cell. A similar result is obtained when nuclear polyadenylated RNAs are compared, indicating that no extensive restriction in gene expression occurs while globin mRNA is accumulating. The vast majority of the mRNAs expressed in Friend cells are not specific to erythroid cells, since there is a high degree of homology between the polyadenylated mRNAs of DMSO-treated Friend cells and those of a transformed fibroblast cell line.

Complexity of nuclear and polysomal polyadenylated RNA in a pluripotent embryonal carcinoma cell line.

The base-sequence complexities and relative abundance of polysomal and nuclear polyadenylated [poly(A+)] RNA sequences have been analyzed in a pluripotent embryonal carcinoma cell line. Polysomal RNA and nuclear poly(A+) RNA have a complexity representing respectively 0.5% and 2.5% of the single copy component of haploid mouse DNA (1.8 X 10(6) K base pairs). By hybridization with specific cDNAs, three abundance classes were found in polysomal poly(A+) RNA, representing respectively 31%, 33%, and 36% of the RNA, with base sequence complexities of 0.1 X 10(3), 0.9 X 10(3), and 14.5 X 10(3) kilobases. This corresponds to 7000-8000 different mRNA species of an average length of 2000 nucleotides, present on an average of 5 to 600 copies per cell. In nuclear RNA, a major class of abundance was found with a complexity of 100 X 10(3) kilobases, each sequence being present in 1 copy per nucleus. The majority of the polysomal poly(A+) RNA sequences are represented in the nuclear poly(A+) RNA but are present in a more restricted range of relative abundance implying posttranscriptional mechanisms of quantitative modulation: polysomal RNA sequences appear to be preferentially transcribed into nuclear cDNA suggesting a preferential location of these sequences close to poly(A) sequences. The presence of a specialized gene product, globin specific RNA, could not be detected either in the nuclear or polysomal compartments of embryonal carcinoma cells, even at levels that would have detected one sequence per 50 cells.

Comparison of polysomal polyadenylated RNA from embryonal carcinoma and committed myogenic and erythropoietic cell lines

Using the technique of mRNA-cDNA hybridization, we have examined the polysomal poly(A) + mRNA base-sequence complexity in three different mouse cell lines: mouse embryonal carcinoma cells, myoblast cells and Friend erythroleukemic cells. These cells express 7700, 13,200 and 6200 mRNA sequences, respectively, distributed in three frequency classes. Reciprocal heterologous hybridization experiments revealed that there is a large degree of homology, a subset of 6000 common sequences being present on the polysomes of all three cell types. Myoblast mRNA is capable of hybridizing all reactive embryonal carcinoma cell cDNA, with kinetics close to the homologous embryonal carcinoma cell curve, thus indicating that all embryonal carcinoma cell sequences are present on myoblast polysomes, the majority at similar abundance. Conversely, embryonal carcinoma cell mRNA fails to hybridize 12% of myoblast cDNA, apparently arising primarily from the complex frequency class. This was confirmed by using myoblast fractions partially enriched in abundant and rare sequences. As a proportion of the rare class, this 12% fraction represents about 4500 sequences close to the difference in base-sequence complexity between myoblast and embryonal carcinoma cells. Homologous and heterologous hybridization with total and fractionated Friend cell cDNA probes revealed that all Friend cell polysomal poly(A) + RNA sequences are common to embryonal carcinoma cell polysomes—apart from a small group of sequences drawn from the abundant class, corresponding to about 10% of Friend cell cDNA. This represents about 12 sequences from the abundant class. In addition, certain common sequences in the abundant Friend cell frequency class are present at lower frequency in embryonal carcinoma cell polysomes. Friend cell polysomal poly(A) + RNA fails to hybridize 7–10% embryonal carcinoma cell cDNA apparently derived from the rare frequency class. As a fraction of the rare class, this corresponds approximately to the difference (about 1500 sequences) in complexity between the Friend and embryonal carcinoma cell lines.

Distribution of polyadenylated RNA sequences in the cytoplasm of Drosophila cells

Complementary DNA (cDNA) was synthesized by reverse transcriptase, on a template of total cytoplasmic polyadenylated RNA from Drosophila cells. This cDNA was hybridized to an excess of total cytoplasmic polyadenylated RNA, polysomal polyadenylated RNA and non-polysomal cytoplasmic polyadenylated RNA. Our results show that a significant fraction of the total cytoplasmic polyadenylated RNA sequences are not bound to polysomes. However, the majority of these sequences are present in the population of non-polysomal polyadenylated cytoplasmic RNA molecules.

Homology relationship between the messenger RNA and heterogenous nuclear RNA of mouse L cells. A DNA excess hybridization study

Methods were developed for the isolation and use of large quantities of unique DNA sequences complementary to polysomal polyadenylated messenger RNA (poly(A)+mRNA). The complementary DNA was prepared by annealing poly-(A)+mRNA to total non-repetitive genomic DNA and subsequent purification of the DNA-RNA hybrids away from the bulk of the DNA. The mDNA ‡ was tested for specificity in a series of DNA excess hybridization reactions involving mRNA and various subfractions of heterogeneous nuclear RNA. The results suggest that the mRNA is not grossly contaminated with non-mRNA sequences and thus the method may be used to obtain mDNA for use in experiments where mDNA excess hybridizations are required. Furthermore, use of the mDNA probe indicated that more than about 12% and possibly as much as 26%±5% homology between large (>45 S) hnRNA (either polyadenylated or non-adenylated) and poly(A)+ mRNA. Small (≤28 S) polyadenylated hnRNA was found to be more than twofold enriched in mRNA sequences as compared to large hnRNA or small non-adenylated hnRNA. A comparison of the percentage homology and the size difference between the large hnRNA and mRNA molecules suggests that a substantial proportion of these hnRNA molecules are potential precursors of mRNA.

A kinetic estimation of base sequence complexity of nuclear poly(A)-containing RNA in mouse friend cells

Complementary DNA (cDNA) has been transcribed by viral reverse transcriptase from poly(A)-containing nuclear RNA prepared from growing mouse Friend cells (clone M2). Annealing experiments with this cDNA have demonstrated that a large proportion of the poly(A) tracts in M2 cell nuclear RNA are adjacent to RNA sequences which are transcribed from nonrepetitive DNA in the mouse genome. The kinetics of hybridization of cDNA to template RNA indicate that nuclear poly(A)-containing RNA consists of at least two abundance classes, the more complex of which is transcribed from approximately 3% of the genome. Thus there are at least 5 times more unique DNA sequences represented in nuclear polyadenylated RNA than in polysomal polyadenylated RNA. Moreover, there is evidence for posttranscriptional mechanisms which alter the relative concentrations of some (at least) gene transcripts between nucleus and cytoplasm.