Major transcripts containing B1 and B2 repetitive sequences in cytoplasmic poly(A)+RNA from mouse tissues

The cytoplasmic poly(A)+RNAs containing ubiquitous B1 and B2 repeats of the mouse genome in normal tissues and tumors have been studied. Only one strand of the repeats is represented in cytoplasmic RNA in all the cases. Some tumor cells were found to be enriched in 1.4 kb B1+mRNA, 1.6 kb B2+mRNA and small (0.2-04 kb) B1+ and B2+ poly(A)+RNAs. On the other hand, mouse liver and kidney contained high amounts of 2 kb B2+mRNA. Its content increased noticeably in the regenerating liver, but in hepatoma it dropped to a zero level. Thus, the switching on (or off) of B1- and B2-containing mRNAs occurred noncoordinately. At the same time, the activation of the synthesis of small B2+RNA and small B1+RNA was simultaneous.     

mRNA usage during Drosophila melanogaster embryonic development. Analysis of nine cloned DNA segments

A set of nine phage lambda clones containing inserts from Drosophila melanogaster which are complementary to cDNA made from oocyte poly(A)+ RNA were selected from a larger group. These cloned elements code for a range of middle abundant RNA sequences which show no appreciable change in abundance during Drosophila embryogenesis. Seven of the nine clones are complementary to two oocyte RNAs, one to three RNAs and one to four RNAs. This study describes the changes that occur in these RNAs during embryonic development in the polysomal and non-polysomal fraction, and in the poly(A)+ RNA and poly(A)- RNA fraction. In all nine of these clones, greater than 70% of the complementary RNA is found in the polysomal region of a sucrose gradient. This proportion increases somewhat during development. Specific changes have been found during development in the proportion of RNA that is poly(A)+. Depending to the cloned sequence, this proportion may increase, decrease, or remain unchanged. For those clones that show a change, most of this change occurs between 8 and 19 h of development. Our data suggest, furthermore, the presence of a class of non-adenylated RNA being utilized during embryogenesis.     

Stored Messenger Ribonucleoprotein Particles in Differentiated Sclerotia of Physarum polycephalum

Starvation induces vegetative microplasmodia of Physarum polycephalum to differentiate into translationally-dormant sclerotia. The existence and the biochemical nature of stored mRNA in sclerotia is examined in this report. The sclerotia contain about 50% of the poly(A)-containing RNA [poly(A)+RNA] complement of microplasmodia as determined by [³H]-poly(U) hybridization. The sclerotial poly(A)+RNA sequences are associated with proteins in a ribonucleoprotein complex [poly(A)+mRNP] which sediments more slowly than the polysomes. Sclerotial poly(A)+RNP sediments more rapidly than poly(A)+RNP derived from the polysomes of microplasmodia despite the occurrence of poly(A)+RNA molecules of a similar size in both particles suggesting the existence of differences in protein composition. Isolation of poly(A)+RNP by oligo (dT)-cellulose chromatography and the analysis of its associated proteins by polyacrylamide gel electrophoresis show that sclerotial poly(A)+RNP contains at least 14 major polypeptides, 11 of which are different in electrophoretic mobility from the polypeptides found in polysomal poly(A)+RNP. Three of the sclerotial poly(A)+RNP polypeptides are associated with the poly(A) sequence (18, 46, and 52 × 10³ mol. wt. components), while the remaining eight are presumably bound to non-poly(A) portions of the poly(A)+RNA. Although distinct from polysomal poly(A)+RNP, the sclerotial poly(A)+RNP is similar in sedimentation behavior and protein composition (with two exceptions) to the microplasmodial free cytoplasmic poly(A)+RNP. The results suggest that dormant sclerotia store mRNA sequences in association with a distinct set of proteins and that these proteins are similar to those associated with the free cytoplasmic poly(A)+RNP of vegetative plasmodia.     

Cloning and cytogenetic localization of evolutionarily conserved genomic sequences which are expressed in the head of the Drosophila melanogaster imago

Screening of Drosophila melanogaster genomic library was carried out using mouse brain polysomal poly(A)+RNA. As a result, 100 clones were selected, among which 14 clones were picked up after hybridization with fly head poly(A)+RNA. It follows therefore, that these clones contain evolutionary conserved sequences which are expressed in Drosophila fly heads. Analysis of these 14 clones revealed RNA-coding fragments. Comparison of their expression in heads and bodies of Drosophila was carried out. Using in situ hybridization we determined the localization of selected 14 sequences on polytene chromosomes. The possibility of further analysis of some clones to study developmental and functional processes in neural system of Drosophila is discussed.     

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.     

Structure and function of rat liver polysome populations. I. Complexity, frequency distribution, and degree of uniqueness of free and membrane-bound polysomal polyadenylate-containing RNA populations

Free and membrane-bound polysomes were isolated from rat liver in high yields with minimal degradation, cross-contamination, or contamination by nuclear or nonpolysomal cytoplasmic ribonucleoprotein. Poly(A)+ RNA fractions isolated from free and bound polysomal RNA (poly(A)+ RNAfree and poly(A)+ RNAbound) by oligo(dT) cellulose chromatography exhibited number-average lengths of 1,600 and 1,200 nucleotides, respectively, on formamide sucrose gradients. Poly(A)+ RNAfree and poly(A)+ RNAbound contain 9.1 +/- 0.55 and 10.7 +/- 0.50% poly(A) as measured by hybridization to [3H]poly(U) and comprise 2.37 and 1.22% of their respective polysomal RNA populations. Homologous poly(A)+ RNA-cDNA hybridizations revealed that greater than 95% of the mass of poly(A)+ RNAfree and poly(A)+ RNAbound contain nucleotide complexities of about 3.4 x 10(7) and 6.0 x 10(6), respectively. This represents about 20,000 and 5,000 poly(A)+ RNA species of average sizes. Heterologous hybridizations suggested that considerable overlap exists between poly(A)+ RNAfree and poly(A)+ RNAbound sequences that cannot be attributed to cross-contamination. This was confirmed by conducting heterologous reactions using kinetically enriched cDNA populations. Heterologous hybridizations involving poly(A)+ RNA derived from tightly bound polysomes and cDNAfree indicated tha most of the overlapping sequences are not contributed by loosely bound (high-salt releasable) polysomes. The ramifications of these findings are discussed.