Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.     

Kinetic estimation of the base sequence complexity of poly(A)-containing mRNA in Ehrlich-ascites-tumor cells and its abundance in nuclear RNA.

Poly(A)-containing messenger RNA was isolated from polysomes of Ehrlich ascites tumor cells, and analyzed for sequence complexity by hybridization to its complementary DNA. The results indicate the presence of about 27,000 diverse mRNA species in mouse Ehrlich ascites tumor cells. Total nuclear RNA was also hybridized to cDNA transcribed from polysomal poly(A)-containing mRNA up to an rot of 3,000 M . s. It was found that all classes of the polysomal poly(A)-containing mRNA sequences were also present in the nucleus, although the distribution varied. About 2% of the total nuclear RNA sequences were expressed as total polysomal poly(A)-containing mRNA. We also report that the total percentage of the haploid mouse genome transcribed in Ehrlich cells is significantly higher than that found in other mouse cells previously examined for poly(A)-containing mRNA sequence complexity.     

Effects of androgens on the complexity of poly(A) RNA from rat prostate

Poly(A)-containing RNA, isolated from rat ventral prostate, has been analyzed for its base sequence complexity. The kinetics of hybridization of total cellular poly(A)-containing RNA with its complementary DNA (cDNA) from normal and castrated animals are markedly different. RNA from normal animals consists of three abundance classes, about 36% comprises one or two highly abundant RNA sequences, 29% consists of about 24 sequences and the remainder is a scarce class of approximately 8200 sequences. In contrast, the hybridization kinetics of prostatic RNA from castrated animals demonstrate that there is a moderate abundance class of 53 sequences and a scarce class of about 7800 sequences, but that a class of abundant sequences is not present. Using normal prostatic cDNA as a probe, we showed that the abundant sequences were not absent but reduced 10 fold following a 3-day castration period and 100 fold after 7 days. Such heterologous hybridization experiments also suggest that there is significant sequence homology in the RNA sequences present in the prostate irrespective of the hormonal status of the animals. The major effect of testosterone appears to be the regulation of the abundance of specific RNA sequences.     

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.     

Comparison of cytoplasmic and nuclear poly(A)-containing RNA sequences in Xenopus liver cells.

Poly(A)-containing RNAs from cytoplasm and nuclei of adult Xenopus liver cells are compared. After denaturation of the RNA by dimethysulfoxide the average molecule of nuclear poly(A)-containing RNA has a sedimentation value of 28 S whereas the cytoplasmic poly(A)-containing RNA sediments slightly ahead of 18 S. To compare the complexity of cytoplasmic and nuclear poly(A)-containing RNA, complementary DNA (cDNA) transcribed on either cytoplasmic or nuclear RNA is hybridized to the RNA used as a template. The hybridization kinetics suggest a higher complexity of the nuclear RNA compared to the cytoplasmic fraction. Direct evidence of a higher complexity of nuclear poly(A)-containing RNA is shown by the fact that 30% of the nuclear cDNA fails to hybridize with cytoplasmic poly(A)-containing RNA. An attempt to isolate a specific probe for this nucleus-restricted poly(A)-containing RNA reveals that more than 10(4) different nuclear RNA sequences adjacent to the poly(A) do not get into the cytoplasm. We conclude that a poly(A) on a nuclear RNA does not ensure the transport of the adjacent sequence to the cytoplasm.     

Sequence complexity of polyadenylated ribonucleic acid from soybean suspension culture cells

The sequenc complexity of total poly(A) RNA from a higher plant system, soybean cultured cells, was determined. Labeled cDNA synthesized from the poly(A) RNA hybridized exclusively with the unique sequence component of total soybean DNA. Analysis of the hybridization reaction between cDNA and the poly(A) RNA template revealed three abundance classes in the poly(A) RNA. These classes represent 18, 44, and 38% of the poly(A) RNA and contain information for approximately 60, 1900 and 30,000 different 1400-nucleotide RNA molecules. From these results, the total sequence complexity of poly(A) RNA was estimated to be 4.5 X 10(7) nucleotides. Saturation hybridization of labeled unique DNA with RNA showed that the total cell RNA represents 12.4% of the unique DNA sequence complexity, or 6.4 X 10(7) nucleotides, while poly(A) RNA respresent 8.7% of the unique DNA sequence complexity, or 3.3 X 10(7) nucleotides. Thus, it is estimated that 50--70% of total RNA sequence complexity is contained in poly(A) RNA in these cells.     

HeLa cell cytoplasmic mRNA contains three classes of sequences: predominantly poly(A)-free, predominantly poly(A)-containing and bimorphic

The mRNA species which exist in the HeLa cell polyribisomes in a form devoid of A sequences longer than 8 nucleotides constitute the poly(A)-free class of mRNA. The rapidly labelled component of this mRNA class shares no measurable sequence homology with poly(A)-containing RNA. If poly(A)-free mRNA larger than 12 S labelled for 2 h in vivo is hybridized with total cellular DNA, it hybridizes primarily with single-copy DNA. When a large excess of steady poly(A)-containing RNA is added before hybridization of labelled poly(A)-free RNA, no inhibition of hybridization occurs. This indicates the existence of a class of poly(A)-free mRNA with no poly(A)-containing counterpart. Some mRNA species can exist solely as poly(A)-containing mRNAs. These mRNAs in HeLa cells are found almost exclusively in the mRNA species present only a few times per cell (scarce sequences). Some mRNA species can exist in two forms, poly(A)containing and lacking, as evidenced by the translation data in vitro of Kaufmann et al. [Proc. Natl Acad. Sci. U.S.A. 74, 4801--4805 (1977)]. In addition, if cDNA to total poly(A)-containing mRNA is fractionated into abundant and scarce classes, 47% of the scarce class cDNA can be readily hybridized with poly(A)-free mRNA. 10% of the abundant cDNA to poly(A)-containing mRNA will hybridize with poly(A)-free sequences very rapidly while the other 90% hybridize 160 times more slowly, indicating two very different frequency distributions. The cytoplasmic metabolism of these three distinct mRNA classes is discussed.     

Comparison of poly(A)-containing RNAs in different cell types of the lower eukaryote Schizophyllum commune

Poly(A)-containing RNAs were isolated from morphologically different cells of the fungus Schizophyllum commune. Using mRNA markers the number-average length of poly(A)-containing RNA in total RNA and in purified poly(A)-containing RNA was estimated as 1100 nucleotides. Number-average length of poly(A)-tracts was 33 nucleotides. 2.5% of total RNA is poly(A)-containing RNA and probably up to 7.5% are non-polyadenylated polydisperse RNA sequences. Saturation hybridization of poly(A)-containing RNA to gap-translated [3H]DNA resulted in 16% of the reactive single-copy DNA to become S1 nuclease resistant. It was found that purified poly(A)-containing RNA represented the entire RNA complexity, i.e. 10 000 different RNA sequences in S. commune. RNA sequences isolated from morphologically different mycelia and from fruiting and non-fruiting mycelia were identical for at least 90%.     

Androgenic regulation of messenger RNA in rat epididymis

1. The regulation by testosterone of mRNA complexity and mRNA activity was investigated in rat caput and cauda epididymidis. 2. The sequence complexity of cytoplasmic poly(A)-containing RNA from normal rats was determined by homologous hybridization with radiolabelled complementary DNA probes by using RNA in excess. Computer analysis of results suggested that hybridization could best be described by curves composed of two components distinguished by their relative abundance. Thus caput-epididymidal RNA consists of approx. 260 moderately abundant and 16400 scarce sequences, whereas cauda-epididymidal RNA consists of approx. 124 moderately abundant and 13400 scarce sequences. Judging by heterologous-hybridization reactions, castration did not result in appreciable alterations in either sequence complexity or the relative abundance of the two classes of poly(A)-containing RNA. 3. To investigate if individual mRNA sequences were regulated by androgens, mRNA was translated in a cell-free system derived from reticulocyte lysate. Since most of the translation products had a different mobility on sodium dodecyl sulphate/polyacrylamide gels from the authentic proteins synthesized in tissue minces, antibodies were used to identify specific translation products. Antibodies to the two related major proteins (mol.wt. 18500 and 19000) secreted by the caput epididymidis and whose synthesis is stimulated by testosterone both precipitated a single translation product of mol.wt. 21000. That this polypeptide was a precursor to the secreted proteins was suggested by the fact that the addition of microsomal membranes isolated from dog pancreas resulted in the appearance of a polypeptide of mol. wt. 19000. 4. Translation of RNA from the caput epididymidis of rats of different hormonal status showed that mRNA activity for the 21000-dalton polypeptide declined after castration, but could be restored by treating rats with testosterone. 5. It is concluded that testosterone stimulates the synthesis of a major protein secreted by the caput epididymidis by regulating its mRNA activity.     

Polyadenylate-deficient analogues of poly(A)-containing mRNA sequences in cultured AKR mouse embryo cells

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30–40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.     

The expression of a plant genome in hnRNA and mRNA.

Representation of genomic kinetic sequence classes and sequence complexities were investigated in nuclear and polysomal RNA of the higher plant Petroselinum sativum (parsley). Two different methods indicated that most if not all polysomal poly(A) -RNA is transcribed from unique sequences. As measured by saturation hybridization in root callus and young leaves 8.7% and 6.2%, respectively, of unique DNA were transcribed in mRNA corresponding to 13.700 and 10.000 average sized genes. Unique nuclear DNA hybridized with an excess of polysomal poly(A)mRNA to the same extent as with total polysomal RNA. 3H-cDNA - poly(A)mRNA hybridization kinetics revealed the presence of two abundance classes with 9.200 and about 30 different mRNAs in leaves and two abundance classes with 10.500 and 960 different mRNAs in callus cells. The existence of plant poly(A)hnRNA was proven both by its fast kinetics of appearance, its length distribution larger than mRNA, and its sequence complexity a few times that of polysomal RNA.     

Comparative analysis of polyadenylated RNA complexity in soybean hypocotyl tissue and cultured suspension cells

Growth parameters of suspension culture cells of soybean (Glycine max L.) were compared between cells grown in medium with (+) auxin and without (−) auxin. Growth rates were greater for (+) auxin cells. Cells transferred to (−) auxin medium primarily expanded in size while (+) auxin cells initially divided and then expanded. Two methods were used to estimate polyadenylated RNA sequence complexity. Kinetic analysis gave a sum of component complexity values of 36,000 and 64,000 diverse poly(A) RNA sequences of about 1,400 nucleotides in (+) and (−) auxin grown cells, respectively. The most striking difference between these cell populations was the increase in the poly(A) RNA sequence complexity in cells grown in medium without auxin. RNA complexities were also determined by the saturation of `single' copy DNA by poly(A) RNAs from (+) and (−) auxin suspension cells. These saturation studies estimated the total complexity of (+) and (−) auxin suspension cells as 41,000 and 57,000 diverse sequences, respectively. Suspension cells in auxin-depleted medium produced about 20,000 more diverse sequences than (+) auxin cells. Comparisons of poly(A) complexities were also made among auxin-treated and untreated hypocotyl cells from the intact plant relative to suspension culture cells. Mixed populations of poly(A) RNA from these tissues and cells allowed the determination of shared sequences among them. When all combinations of poly(A) RNA were mixed, the percentage of `single' copy DNA that saturated was equivalent to diverse sequence complexity estimates of about 60,000. When mixed poly(A) RNA from suspension cells from (+) and (−) auxin medium were compared, they shared about 40,000 sequences and (−) auxin cells contained an additional 20,000. Both (+) and (−) tissue culture cells shared a subset of about 20,000 sequences with cells from (+) and (−) auxin treated hypocotyl. A third subset of about 20,000 sequences was shared by (−) auxin suspension cells and hypocotyl treated with or without auxin, a subset most of which were not shared by (+) auxin suspension cells. Kinetic and saturation data estimates of poly(A) RNA complexity compared favorably and indicated that exogenous auxin treatment can dramatically alter the complexity of all classes of poly(A) RNAs in cultured cells.