Jacques Harel

Persistance d'une synthèse de D-RNA dans le foie de rat traité par l'actinomycine

Abstract Actinomycin D-treated rats were injected with 32 P-labelled orthophosphate, 3 h later the animals were sacrificed and both RNA and DNA were extracted from their livers using phenol plus 0.5% or 1.0% sodium dodecylsulfate. RNA was fractionated by centrifugation in sucrose gradients. Separation of ribosomal and DNA-like RNA from s-RNA was achieved using 1.5 M NaCl or 0.2 M MgCl 2 . Complete inhibition of the synthesis of ribosomal RNA and s-RNA was found in the livers of actinomycin-treated rats, since, whether or not the RNA was treated with snake-venom phosphodiesterase (EC 3.14.1) before analysing the labelled 2′,3′-nucleotides, residual labelling of s-RNA could be attributed exclusively to the end groups pC-pC-pA. Synthesis of another fraction of RNA (8–10 S) was only partially inhibited (50–70%) by actinomycin. The (A+U)/(C+G) ratio of this fraction was identical to the (A+T)/(C+G) ratio of DNA, but generally the proportion of A was much larger (32–33%) than the proportion of U (20–22%). This DNA-like RNA could be recovered in a form closely associated with DNA itself. A similar DNA-like RNA was found in normal rat liver. These findings suggest that synthesis of all cellular RNA fractions is DNA dependent, but synthesis of DNA-like RNA is much less sensitive to actinomycin than synthesis of ribosomal and s-RNA. These facts pose certain questions about the manner in which ‘messenger’ RNA is replicated in mammalian cells.

FRACTIONS WITH DIFFERING BASE COMPOSITION IN RNA FROM MALIGNANT CELLS OF MOUSE.

RNA labeled with 32 P was extracted from mouse ascites tumor cells, and centri-fuged in a linear sucrose gradient. After a pulse of 30 minutes most of the label was in a “heavy” (>30 s) and a “light” (4 to 10 s) fractions. The base composition of the fractions differed depending on whether or not sodium dodecyl-sulfate was included in the phenol extraction procedure. Without sodium dodecylsulfate the radioactivity of the “light” fraction predominated. This fraction contained a high proportion of cytosine which disappeared after treatment with phosphodiesterase. After the enzymic treatment, the base-ratios resembled those of transfer RNA. The “heavy” fraction differed from ribosomal RNA by a low proportion of adenine and a high proportion of uracil. More radioactivity was recovered after a short pulse when sodium dodecylsulfate and phenol were used. The “heavy” fraction differed from ribosomal RNA (28 8) only by slightly higher adenine and uracil contents. A DNA-like RNA was separated from transfer RNA contained in the light fraction (4 to 10 s). DNA-like RNA was also directly separated from DNA fibers. After labeling for 4·5 to 7 hr RNA was uniformly labeled, but there were still differences in the base ratios of the fractions. Ribosomal 26 to 28 s RNA had lower adenine and uracil contents than ribosomal 16 to 18 s RNA.

Possible role of c-fos, c-N-ras and c-mos proto-oncogenes in muscular development.

Time course analyses of various proto-oncogene transcripts compared with cytoskeleton-specific and muscle-specific messenger RNAs (mRNAs) were carried out during growth and differentiation of a clonal line of rat myoblasts that retain the capacity to form non-contractile fibres in vitro. Throughout their growth phase, these cells express consistent levels of c-fos, c-myc, c-Ki-ras and c-N-ras RNA and no c-mos RNA. When the cultures approach confluency the level of c-fos RNA rises sharply 3-4-fold, peaks, and rapidly declines when muscle-specific transcripts start accumulating, to become negligible in myotube-forming cells. These changes occur whatever the concentration in seric factors. By contrast, the level of c-N-ras RNA rises up to 3-fold and both c-myc and c-Ki-ras RNAs are slowly eliminated during the myogenic process, whereas no c-mos RNA is detectable. However, skeletal muscles from prenatal fetuses and adult animals were reproducibly found to contain both low and high levels of c-mos RNA respectively. These data and the demonstration that inactivation of the c-fos gene correlates with the loss of myogenic capability in six lines of neoplastic myoblasts, including four lines transformed by the v-fos oncogene, suggest a physiological function for this proto-oncogene during early stages of myogenesis and for the c-N-ras and c-mos genes in later stages of muscular development.

The small chromatin fragments released by micrococcal nuclease from hepatoma tissue cultured cell nuclei are strongly enriched in coding DNA sequences and are related to an actively transcribed single-stranded DNA fraction

It was shown with the use of specific probes that mild micrococcal nuclease digestion released from chromatin actively-transcribed genes as small nucleosome oligomers. In the present work we demonstrate that most if not all of the active genes are accessible to the nuclease. It was found that the short released fragments are greatly enriched in transcribed DNA sequences, the most enriched being the dimers of nucleosomes since 35% of their DNA could be hybridized to cytoplasmic RNA. The results of cDNA-DNA hybridizations indicate that the monomers and dimers of nucleosomes contain most of the DNA sequences which encode poly(A+) RNAs, however larger released fragments include some transcribed sequences, while the nuclease resistant chromatin is considerably impoverished in coding sites. These evidences are the finding that about 25% of the DNA from the dimers of nucleosomes are exclusively located in this class of fragments, tend to prove that the active chromatin regions are attacked in a non-random way by micrococcal nuclease. We have previously isolated, without using exogenous nuclease, an actively transcribed genomic fraction amounting to 1.5-2% of the total nuclear DNA, formed of single-stranded DNA. In the present study we show that all or nearly all the single-stranded DNA sequences could be reassociated with the DNA fragments present in the released monomers and dimers of nucleosomes. Our observations confirmed our previous finding that the greatest part of single-stranded DNA selectively originates from the coding strand of genomic DNA.

Isolation of single stranded transcription sites from human nuclear DNA

Single stranded DNA (ss-DNA) isolated from nuclear DNA of human rhabdomyosarcoma (RD line) cells amounts to 2% of total DNA. As compared with native double-stranded DNA (ds-DNA), ss-DNA has a lower mean sedimentation coefficient, higher buoyant density, contains fewer repeated DNA sequences and a greater proportion of it can be hybridized to human RNAs (up to 35% and less than 8% for ds-DNA). The hybridization kinetics determined by S1 nuclease digestion and verified by other methods (hydroxylapatite chromatography, density gradient centrifugation and thermal melting) indicate that ss-DNA corresponds to a great number and variety of transcripts. These data are discussed as evidence for DNA strand dissociation in the course of gene activity.

Nuclear penetration and stimulation of nucleic acids synthesis by poly(A)-poly(U) in mammalian cells☆

Abstract The rapid penetration of poly(A)-poly(U) into cell nuclei is shown by radioautography, by recovery of acid-precipitable material from isolated nuclei and by sucrose gradient centrifugation of nuclear lysates. The majority of poly(A)-poly(U) remains intact in the nuclei for at least 2 1 2 h. This penetration is increased 20-fold by pretreatment of the cells with DEAE Dextran. In cells treated with DEAE Dextran, DNA and RNA syntheses are stimulated by poly(A)-poly(U) from the time the polymer complex is added and for at least 2 1 2 h.

Distribution of repetitious DNA in randomly growing and synchronized Chinese hamster cells

Abstract Diploid Chinese hamster cells were labelled with 32 P for 2 days or synchronized and pulse-labelled with 3 H-thymidine throughout the DNA synthetic (S) period. Total nuclear DNA and DNA fractions having different G + C contents were analysed, using an improved method of hydroxylapatite chromatography. Both uniformly labelled DNA (random DNA) and DNA pulse-labelled throughout the S period, contained a similar proportion (3–4%) of single-stranded sequences (SS-DNA). The amount and G + C content of SS-DNA were very much increased in the highest buoyant density DNA fraction analysed. After denaturing and renaturing at several C 0 t values, the amounts of reassociated DNA were determined. Using random DNA, the G + C content of the reassociated sequences did not differ from that of total DNA regardless of the C 0 t value. Using pulse-labelled DNA, the proportion of highly repetitious sequences (renatured at C 0 t 2) isolated from total DNA as well as from both high and low buoyant density fractions, did not vary throughout the S period, as compared with random DNA. However, both the heavy and light fractions of late S DNA contained a greater proportion of moderately repetitious sequences (renatured at C 0 t 10 and 50) than early S or random DNA. These results are compared with those in other animal species.

Replication of repetitious DNA in synchronized chick fibroblast cells

Abstract Chick embryo fibroblasts in monolayer culture were synchronized by contact inhibition and serum starvation. Nuclear DNA isolated from the [ 3 H] thymidine pulse-labelled cells throughout the period of DNA synthesis (S phase) was analysed by hydroxylapatite chromatography after renaturation at different C 0 t values. It is shown that repeated sequences having different frequencies of reassociation, replicate differently throughout the S period. In order to study the distribution of the repeated sequences, DNA isolated during the S period was fractionated according to its buoyant density. It is shown that only some of the highly reiterated sequences which are included in the high buoyant density DNA fractions, replicate equally well during the early and the late S periods. By contrast, reiterated sequences of the low buoyant density DNA fractions replicate mainly during the late S period.

DNA copies of viral RNA in rat cells transformed by rous sarcoma virus (RSV)

Abstract Using a modified hybridization method at least 50 p cent of the input 70 S RNA from avian tumor virus (RSV) could be annealed with DNA from RSV transformed rat cells, and only 2–3 p cent with DNA from normal cells. The mean number of viral genome copies does not seem to exeed 1 or 2 per tumor cell genome.

Expression of extracellular matrix genes in relation to myogenesis and neoplastic transformation

Fibronectin and alpha 1(I) and alpha 2(I) collagen proteins and RNAs are highly expressed during the growth phase of the non-transformed L6 alpha 1 rat myoblasts. When L6 alpha 1 cells from myotubes following transfer to low serum medium, the levels of fibronectin RNA decrease 8-fold, those of both alpha 2(I) transcripts decrease only 2-fold, while those of both alpha 1(I) transcripts remain stable. The L6 alpha 1 cell-derived non-differentiable low-malignant M4 cell and high-malignant RMS4 cell display only one size of alpha 1(I) and alpha 2(I) transcripts. Compared with L6 alpha 1 myoblasts, the levels of fibronectin and alpha 1(I) RNAs are reduced by factors of 4-5 and 9-10 respectively in both M4 and RMS4 and those of alpha 2(I) RNAs by factors of 10-11 and 20-22 in M4 and RMS4, respectively. Transcription rates are similarly decreased for fibronectin RNA, but are decreased less for collagen RNAs.