Related base sequences in the DNA of simple and complex organisms. IV. Evolutionary divergence of base sequence in mouse L-cell cytoplasmic and nucleus-restricted RNA

Nuclear and cytoplasmic RNAs of mouse L-cells were compared for base sequence relatedness between and within them. The relationship of each of these RNAs to the DNA of other rodents and the rabbit was determined. RNA restricted to the mouse L-cell nucleus is unrelated in base sequence to cytoplasmic RNA. There is less diversity of base sequence within the gene families transcribing nucleus-restricted RNA than within those transcribing cytoplasmic RNA, suggesting more recent evolutionary origin. The gene families which code for mouse L-cell unique nuclear RNA are far more divergent from those of other rodents than are cytoplasmic RNA gene families. They are unrelated in base sequence to any gene families in rabbit DNA, suggesting that these gene families evolved after the separation of the ancestors of the orders Rodentia and Lagomorpha. The known characteristics of the RNA restricted to the nucleus in eukaryotic cells are summarized.This investigation was supported by U.S. Public Health Service Training Grant GM 00182 and Research Grant GM 12449 from the Institute of General Medical Sciences.     

Discrete and heterogeneous high molecular weight RNAs complementary to a long dispersed repeat family (a possible transposon) of human DNA

Approximately 1% of heterogeneous nuclear RNA and approximately 0.035% of cytoplasmic RNA from a cultured line of human lymphoblastoid cells is complementary to a long dispersed repetitious sequence that comprises at least 6% of human DNA. The complementary nuclear RNA is both heterogeneously and discretely sized and is present in both poly(A)-terminated and non-poly(A)-terminated molecules. The complementary cytoplasmic RNA is mainly in discretely sized molecules ranging in size from approximately 600 to 8200 bases, some of which are most abundantly represented in poly(A)-terminated molecules, whereas others are most abundantly represented in non-poly(A)-terminated molecules. Few, if any, of the complementary cytoplasmic RNAs can be found associated with polyribosomes. The dispersed repeat sequence exhibits substantial restriction enzyme fragment length polymorphisms in human DNA and is also present in mouse DNA, although some regions of the human repeat appear to be more abundantly represented in mouse DNA than are other regions.     

Virus-Specific Ribonucleic Acid in Cells Producing Rous Sarcoma Virus: Detection and Characterization

Cells producing Rous sarcoma virus contain virus-specific ribonucleic acid (RNA) which can be identified by hybridization to single-stranded deoxyribonucleic acid (DNA) synthesized with RNA-directed DNA polymerase. Hybridization was detected by either fractionation on hydroxyapatite or hydrolysis with single strand-specific nucleases. Similar results were obtained with both procedures. The hybrids formed between enzymatically synthesized DNA and viral RNA have a high order of thermal stability, with only minor evidence of mismatched nucleotide sequences. Virus-specific RNA is present in both nuclei and cytoplasm of infected cells. This RNA is remarkably heterogeneous in size, including molecules which are probably restricted to the nucleus and which sediment in their native state more rapidly than the viral genome. The nature of the RNA found in cytoplasmic fractions varies from preparation to preparation, but heterogeneous RNA (ca. 4-50S), smaller than the viral genome, is always present in substantial amounts.     

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.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

THE CYTOPLASMIC CONTROL OF NUCLEAR ACTIVITY IN ANIMAL DEVELOPMENT

1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.     

Studies on the kinetics of the RNA metabolism in the prostate of normal and castrated rats (author's transl)]

In the prostate of adult Wistar rats the RNA/DNA quotient of the whole organ as well as the amount of RNA and DNA in the nucleus was measured at different times after castration. Furthermore the half-life time for the turnover of the RNA in the nucleus and the cytoplasm was determined for normal and castrated rats with the aid of pulse labelling using [5(-3)H]uridine. A mathematical model was developed to analyze the experimental results. This model enabled us to make differentiated statements on the heterogeneous nuclear RNA (hmRNA) and the remaining RNA in the nucleus. The evaluation of the experimental values gave the following results: 1. By deprivation of androgens the uptake of [3H]uridine into the prostate is lowered. 2. The amount of DNA in the morphologically intact nucleus remains constant at least up to the 12th day after castration. 3. 6 days after castration the amount of hmRNA decreases to 1/10 and that of cytoplasmic RNA to 1/4. 4. The half-life time for the decrease of the whole nuclear RNA is 3.7 d and that of the cytoplasmic RNA 1.7 d. 5. The half-life time for the turnover of hmRNA is 16 min and that of cytoplasmic RNA about 2 days. 6 days after castration the half-life times are unchanged. The experimental results suggest that the observed decrease of nuclear RNA following castration can mainly be attributed to a reduced synthesis of hnRNA, while the decrease of cytoplasmic RNA is first of all caused by an increase in RNA degradation.     

THE SYNTHESIS OF DNA, RNA, AND NUCLEAR PROTEIN IN NORMAL AND TUMOR STRAIN CELLS : IV. HeLa Tumor Strain Cells

Interferometric and photometric measurements have been made on HeLa cells, a strain of cells originally derived from a human carcinoma. From a study of the relations between successive physical measurements on individual cells, it was confirmed that, whereas the net syntheses of nuclear RNA and nuclear protein are closely associated during interphase, they are dissociated from DNA replication to a significant extent. These results on nuclear metabolism agree with others previously reported in cell strains derived from tumors; they contrast with results from freshly prepared normal cells, where the net syntheses of DNA, nuclear RNA, and protein are closely associated during interphase. Cytoplasmic measurements on HeLa cells showed that much of the net synthesis of cytoplasmic RNA is associated with DNA replication as in normal cells, and they failed to detect transfer from the nucleus of a stable RNA component synthesized independently from DNA replication. In auxiliary experiments, an inhibition of the onset of DNA synthesis was produced by a dose of X-rays; under these conditions it was shown that the major part of the accumulation of nuclear protein was independent of DNA replication and that the accumulation of nuclear RNA was equivalent to or slightly less than that of nuclear protein. About half the accumulation of cytoplasmic RNA was inhibited when DNA synthesis was blocked.     

Intracellular Amounts of Nucleic Acids and Protein During Pollen Grain Growth in Tradescantia

The rapid growth, large organelles, and synchronous development of T. paludosa pollen grains make them ideal subjects for cytochemical analysis. A microphotometric study of the nucleoli, chromosomes, and cytoplasm fixed at daily intervals during pollen grain maturation indicated that: 1. DNA (Feulgen) synthesis in the generative nucleus occurred during the first third of interphase, while the DNA content of the vegetative nucleus remained unchanged. 2. Throughout development, changes in RNA (azure B) content, in general, paralleled changes in protein (NYS¹, Millon) content in each organelle of the vegetative cell. Initially, the RNA and protein of all organelles increased up to mid interphase, when chromosomal and nucleolar fractions began to decline despite a continued increase in cytoplasmic RNA and protein. At least 24 hours before anthesis, the vegetative nucleolus had disappeared and chromosomal protein and RNA of the vegetative nucleus were apparently in rapid decline. Such a system offered an opportunity to study the role of the nucleus, especially the nucleolus, in RNA and protein metabolism in the cytoplasm, by noting what cytoplasmic processes could and could not continue at a time when nuclear mechanisms were absent or minimal. It was found that at least 2 fundamental processes continued during this period: both RNA and protein accumulated in the cytoplasm at a rapid rate. It was concluded that the nucleus is not the sole source of cytoplasmic RNA, for the data suggest that there are at least 2 separate and independent, or remotely dependent synthesizing systems, one nuclear and the other cytoplasmic. It is evident that nuclear influence on cytoplasmic synthesis need be neither direct nor immediate.     

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse satellite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.