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.     

Sequence complexity of heterogeneous nuclear RNA in sea urchin embryos.

The sequence complexity of heterogeneous nuclear RNA is sea urchin gastrulas was measured by RNA-driven hybridization reactions with nonrepetitive sea urchin DNA. 28.5% of the sequence complexity of the genome is represented in the nuclear RNA. This amounts to 1.74 X 10(8) nucleotides of diverse sequence, more than 10 times the nucleotide complexity of the polysomal messenger RNA extracted from sea urchin embryos at the same stage. The complex set of nuclear RNA sequences driving this hybridization reaction was shown to be the same as the rapidly labeled hnRNA, using pulse-labeled nuclear RNA as driver.     

Sequence diversity of polysomal mRNAs in roots and shoots of etiolated and greened pea seedlings

The sequence complexity and abundance of polysomal mRNA populations of pea seedlings were measured using RNA excess hybridization to both single-copy DNA and complementary DNA. The estimated sequence complexity of the polysomal mRNA populations was 2.5·10⁷ nucleotides or 19,400 different mRNAs of average size. Since the haploid genome size of pea was found to be 4.0·10⁹ nucleotide pairs, only 0.62% of the total haploid genome of pea was transcribed into polysomal mRNA. The roots and shoots of 4-d etiolated and light-grown seedlings contained similar numbers of diverse mRNAs. The RNA excess hybridizations, using single-copy DNA enriched for sequences transcribed in either light-grown shoots or etiolated roots and single-copy DNA depleted of such sequences, indicated that at least 92% of the sequence complexity of polysomal mRNAs was identical in roots and shoots irrespective of the presence of a functional photosynthetic system. In contrast, RNA excess hybridization to complementary DNA revealed that 21% of the polysomal polyadenylated mRNA mass found in light-grown shoots was absent in etiolated roots. The kinetics of these hybridizations indicated that this was due to the appearance of a limited number of abundant mRNAs under conditions of illumination.     

Sequential Sepharose chromatographic isolation of polysomes and polysomal RNAs depleted in nuclear RNA from Xenopus.

Analyses of sequence complexities, stage specific gene expression, and mRNA sequence divergence require polysomal RNA preparations relatively free of nuclear RNA contamination. Conventional procedures for the isolation of uncontaminated polysomal RNAs which rely on sucrose density centrifugations are laborious and unsuitable for large scale isolations. We describe here a method using sequential Sepharose chromatography for isolating polysomes and polysomal RNAs depleted for nuclear RNA. Polysomes and polysomal RNAs isolated from livers of Xenopus stimulated to produce vitellogenin were capable of directing protein synthesis in vitro and showed little evidence of degradation. The polysomal RNAs contained less than 0.5% of nuclear RNA.     

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.     

Preferential expression of unique sequences adjacent to middle repetitive sequences in mouse cytoplasmic RNA.

Total single-copy DNA and single-copy DNA contiguous to middle repetitive sequences were isolated from mouse brain by successive hydroxylapatite column chromatographies. These DNAs, termed repeat-contiguous single-copy DNA, were found to constitute 48% of the total single-copy DNA. The saturation hybridization values of these two DNA probes to nuclear RNA and cytoplasmic RNA containing polyA of mouse brain and liver were measured. The saturation hybridization levels of total single-copy DNA to brain and liver nuclear RNA were 13.5% and 8.8%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 13.3% and 8.5%, respectively. On the contrary, the saturation hybridization levels of single-copy DNA to cytoplasmic RNA containing polyA of brain and liver were 3.8% and 2.0%, respectively, and those of repeat-contiguous single-copy DNA to the same RNA samples were 5.8% and 4.0%, respectively. Similar results were obtained with total cytoplasmic RNA. These results indicate that about half the steady state nuclear RNA is transcribed from repeat-contiguous single-copy DNA, and that cytoplasmic RNA containing polyA is mainly derived from repeat-contiguous single-copy DNA.     

Complexity of RNA in Eggs of DROSOPHILA MELANOGASTER and MUSCA DOMESTICA

Comparative measurements are presented of the sequence complexity of the RNA stored in the eggs of two dipteran flies, Musca domestica and Drosophila melanogaster. The genome of Musca is about five times the size of the Drosophila genome and contains about 3.6 times as much single-copy sequence. As shown earlier, the interspersion of repetitive and single-copy sequence is of the short-period form in Musca, and is of the long-period form in Drosophila. The egg RNA complexities were determined by hybridization of excess RNA with radioactively labeled single-copy DNA. Complexity is expressed as the length (in nucleotides) of diverse single-copy sequence represented in the RNA. The complexity of the RNA of the Musca egg is about 2.4 x 10⁷ nucleotides, and that of the Drosophila egg is about 1.2 x 10⁷ nucleotides. The RNA of the Musca egg is similar to or very slightly lower in complexity than that of other egg RNAs, e.g., those of Xenopus and sea urchin. Compared to all previously measured egg RNAs, Drosophila egg RNA is low in sequence complexity.     

Complexity and characterization of polyadenylated RNA in the mouse brain

The complexity of nuclear RNA, poly(A)hnRNA, poly(A)mRNA, and total poly(A)RNA from mouse brain has been measured by saturation hybridization with nonrepeated DNA. These DNA populations were complementary, respectively, to 21, 13.5, 3.8, and 13.3% of the DNA. From the RNA Cot required to achieve half-sturation, it was estimated that about 2.5–3% of the mass of total nuclear RNA constituted most of the complexity. Similarly, complexity driver molecules constituted 6–7% of the mass of the poly(A)hnRNA. 75–80% of the poly(A)mRNA diversity is contained in an estimated 4–5% of the mass of this mRNA. Poly(A)hnRNA constituted about 20% of the mass of nuclear RNA and was comprised of molecules which sedimented in DMSO-sucrose gradients largely between 16S and 60S. The number average size of poly(A)hnRNA determined by sedimentation, electron microscopy, or poly(A) content was 4200–4800 nucleotides. Poly(A)mRNA constituted about 2% of the total polysomal RNA, and the number average size was 1100–1400 nucleotides. The complexity of whole cell poly(A)RNA, which contains both poly(A)hnRNA and poly(A)mRNA populations, was the same as poly(A)hnRNA. This implies that cytoplasmic polyadenylation does not occur to any apparent qualitative extent and that poly(A)mRNA is a subset of the poly(A)hnRNA population. The complexity of poly(A)hnRNA and poly(A)mRNA in kilobases was 5 × 10⁵ and 1.4 × 10⁵, respectively. DNA which hybridized with poly(A)mRNA renatures in the presence of excess total DNA at the same rate as nonrepetitive tracer DNA. Hence saturation values are due to hybridization with nonrepeated DNA and are therefore a direct measure of the sequence complexity of poly(A)mRNA. These results indicate that the nonrepeated sequence complexity of the poly(A)mRNA population is equal to about one fourth that observed for poly(A)hnRNA.     

Similarity of hnRNA sequences in blastula and pluteus stage sea urchin embryos

We have compared the total single-copy sequences transcribed as nuclear RNA in blastula and pluteus stage embryos of the sea urchin Tripneustes gratilla by hybridization of excess nuclear RNA with purified radioactive single-copy DNA. The kinetics of hybridization of either blastula or pluteus nuclear RNA with single-copy DNA show a single pseudo-first-order reaction with 34% of the single-copy genome. From the rate of the reaction and the purity of the nuclear RNA, it can be estimated that the reacting RNAs are present on the average at a concentration of one molecule per 14 nuclei. A mixture of blastula and pluteus RNA also hybridizes with 34% of the single-copy genome, indicating that the total complexity of RNAs transcribed at both stages is no greater than transcribed at each stage alone. The identity of the sequences transcribed by blastula and pluteus embryos was further examined by fractionation of the labeled DNA into sequences complementary and not complementary to pluteus RNA. This was achieved by hybridization of single-copy DNA to high pluteus RNA Cot, and separation of the hybridized and nonhybridized DNA on hydroxylapatite. Using either the DNA complementary or noncomplementary with pluteus RNA, essentially identical amounts of RNA:DNA hybrids are formed at high RNA Cot with blastula or pluteus RNA. Gross changes in the total RNA sequences transcribed do not appear to be involved in the developmental changes between blastula and pluteus, even though 45% of the mRNA sequences change between these two stages (Galau et al., 1976).