Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell.     

Mouse ubiquitous B2 repeat in polysomal and cytoplasmic poly(A)+RNAs: uniderectional orientation and 3''-end localization.

A cDNA library in pBR322 was prepared with cytoplasmic poly(A)+RNA from mouse liver cells. From 1 to 1.5% of clones hybridized to either B1 or B2 ubiquitous repetitive sequences. Several clones hybridizing to a B2 repeat were partially sequenced. The full-length B2 sequence was found at the 3'-end of abundant 20S poly(A)+RNA (designated as B2+mRNAx) within the non-coding part of it. B2+mRNAx is concentrated in mouse liver polysomes and absent from cytoplasm of Ehrlich carcinoma cells. The B2 sequence seems to be located at the 3'-end of some other mRNAs as well. To determine the orientation of the B2 sequence in different RNAs, its two strands were labeled, electrophoretically separated, and used for hybridization with Northern blotts containing nuclear, cytoplasmic and polysomal RNAs. In nuclear RNA, the B2 sequence is present in both orientations; in polysomal and cytoplasmic poly(A)+RNAs, only one ("canonical") strand of it can be detected. Low molecular weight poly(A)+B2+RNA [1] also contains the same strand of the B2 element. The conclusion has been drawn that only one its strand can survive the processing. This strand contains promoter-like sequences and AATAAA blocks. The latter can be used in some cases by the cell as mRNA polyadenylation signals.     

Low-molecular RNA subcellular localization and its binding strength with cellular structures

Some fractions of low molecular weight (LMW) nuclear RNAs were shown to be present in the cytoplasm of rat liver cells. In addition to known 4S tRNA, 5S and 5,8S rRNAs U3 and 8S1 LMW nuclear RNAs, 8SII and 8SIII LMW RNAs have been detected in RNA preparations of free total and membrane-bound polysomes. The U3 and 8SI polysoma I RNAs seem to be associated with high molecular weight polysomal RNA. Using thermal phenol fractionation, that some LMW RNAs were shown to be slightly bound to the cellular structures whereas some others are bound more tightly. Considerable amounts of LMW RNAs are tightly bound to the chromosome-nucleolar apparatus. They can be extracted only at 85 degrees C. The data presented are discussed with regard to LMW nuclear and polysomal RNAs functions.     

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.     

Diversity of gene expression in goldfish brain

The sequence complexity of nuclear and polysomal RNA from goldfish brain and kidney was measured by RNA-driven hybridization reactions with single-copy [3H]DNA. At saturation, brain nuclear and polysomal RNA were complementary to 23.2 and 6.7% of the DNA probe, respectively. In contrast to these findings, nuclear and polysomal RNA from kidney hybridized to 16.1 and 3.1% of the single-copy DNA, values that were significantly lower than that obtained in the CNS. Taken together, the results focus attention on the striking diversity of gene expression in goldfish brain and extend to lower vertebrates the observation that nervous tissue expresses significantly more genetic information than other somatic tissues or organs.     

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.     

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.