Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis最新文献

This study compares over 70 recognition sites for restriction endonucleases on mtDNAs from various control versus malignant cells, derived from Syrian hamster, chick embryo, viper and human cells, exhibiting a wide spectrum of cellular transformation and tumor histories. Agents for transformation in vitro and in vivo include Rous sarcoma viruses, simian virus 40, polyoma virus and adenovirus. The results show a striking intraspecific sequence homogeneity of different mtDNAs regardless of tissue origin and oncogenic history. mtDNA from human biopsy specimens of tumor versus pathologically normal areas yielded indistinguishable restriction cleavage patterns reflecting either the ‘wild-type’ form (with seven restriction endonucleases) or, in one individual, a variant pattern detected with HpaI. The precise position of the HpaI variant site was determined on the physical map of human mtDNA. Additional cleavage sites in the previously reported restriction map of Syrian hamster mtDNA are also presented. It is concluded that (1) mtDNA sequences in higher animal cells are highly conserved in malignant transformation; (2) no evidence for integration of viral sequences in mtDNA is apparent; (3) variant patterns in mtDNA are likely to be intraspecific polymorphisms that pre-exist neoplastic transformation. The possibility is discussed that altered regulatory interaction with the mitochondrial genome, rather than evident changes in mtDNA primary structure, determine anomalous mitochondrial functions in malignant transformation.

The disappearance of tRNA during the maturation of rabbit reticulocytes produced under the stress of phenylhydrazine-induced hemolysis was studied. The tRNA content of reticulocytes and of erythrocytes derived from them was compared. The results show that tRNA persists longer after reticulocyte maturation than ribosomes and than the ability to incorporate amino acids into protein. Considerable uniformity of tRNA degradation was noted with about 15% of the tRNA for most amino acids remaining after reticulocyte maturation. The half-life of tRNA in the maturing cells is estimated to be 50–60 h. There is little tRNA lacking the 3′-terminal pCpCpA moiety in cells derived from reticulocytes.

A number of parameters were explored to increase the transformation efficiency of E. coli with $\text{pBR}\text{322}\text{eukaryotic}$ DNA chimera, formed via d(A) · d(T) and d(G) · d(C) homopolymer tails. Of the E. coli strains analyzed, E. coli strain RR1 was the most efficient bacterial host. A clear optimum of nucleotide tail length existed for both types of homopolymer. The optimum hybridization temperature for chimera formation was found to be approx. 57°C. In the case of d(A) · d(T)-linked chimeras, 30 min was sufficient for optimum chimera formation. In contrast, d(C) · d(G)-linked chimeras required up to 2 h to give the best yields (as measured by transformation efficiency). Other minor factors affecting the transformation process are also explored and discussed.

The azo dye Congo Red has a high affinity for nucleotide-binding enzymes. We have studied the binding of Congo Red to RNA polymerase by circular dichroism (CD) and difference absorption spectroscopy, steady-state kinetics, and nitrocellulose filter-binding. Induced CD shows that a large number of Congo Red molecules bind to the holoenzyme. CD also demonstrates that the core enzyme at low ionic strengths has a distinctive Congo Red binding site which is not present in the holoenzyme, nor in the core enzyme at higher ionic strengths or in the pressece of poly(dT). CD studies indicate that Congo Red can readily displace double-stranded polynucleotides (T7 DNA or poly[d(A-T)]) from RNA polymerase. Single-stranded DNA (poly(dT) and T7 DNA in open complexes) is not displaced from RNA polymerase except at high Congo Red concentrations. Both kinetics and nitrocellulose filter-binding measurements support this conclusion. Difference spectra indicate that the bound Congo Red molecules undergo stacking. We postulate that RNA polymerase binds Congo Red in a region with which a segment of DNA normally interacts, and that Congo Red is a potent inhibitor because the stacked dye has a polyanionic character.

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent light lethality.

A procedure originally developed to isolate transcriptionally active from less active rat ventral prostate chromatin, employing minimal shear and centrifugation through a dense sucrose gradient, was used to separate prostate chromatin actively synthesizing DNA from total chromatin. It was verified that maximum DNA synthesis in ventral prostates of rats 6 days after castration occurred after administration of testosterone propionate daily for 3 days. When minced ventral prostates from such animals were incubated with [³H]thymidine, and the ‘heavy’ and ‘light’ chromatin fractions were separated by sucrose gradient centrifugation, most radioactive DNA was present in the ‘light’ fraction at the top of the gradient. Incorporation was due to DNA synthesis and not to repair, as judged by inhibition with N-ethylmaleimide. Results of in vitro and in vivo thymidine pulse-chase experiments were consistent with initial labelling of DNA-in a replication complex and subsequent sequestration of radioactive DNA in forms resistant to release by the preparative procedure. Although about half the estimtated total endogenous DNA polymerase activity detected in vitro was present in the heavy fraction, the apparent specific activity of the enzyme in the ‘light’ fraction was 5-times as high. Lastly, when equal concentrations of DNA from the separated chromatin fractions were shadowed with platinum-palladium and examined by electron microscopy, 5-times as many Y-shaped structures were seen in the light fraction. This procedure facilitates the isolation of enzymatically active DNA structures undergoing semiconservative replication and study of their subsequent molecular ‘processing’ into forms no longer susceptible to separation by this comparatively gentle method of chromatin preparation. Since the method also yields transcriptionally active chromatin fractions from rat liver and Chinese hamster ovary cell nuclei, it should be applicable to the study of DNA synthesis in these and many other cells.

Cyclic AMP derivatives increase the rate of synthesis of tyrosine aminotransferase in Reuber H35 hepatoma cells. Various studies lend support to the hypothesis that cyclic AMP increases the synthesis of tyrosine aminotransferase by acting at a posttranscriptional site. The presence of a limited non-translatable pool of tyrosine aminotransferase mRNA prior to the formation of the translatable tyrosine aminotransferase mRNA implicates a possible site of action of cyclic AMP. We compared the capacity of $\text{N}{}^6\text{,O}{}^2\text{′-}\text{dibutyryl}$ cyclic AMP to induce tyrosine aminotransferase synthesis when untranslatable tyrosine aminotransferase mRNA sequences are present or absent. The transition of a condition in which non-translatable tyrosine aminotransferase mRNA sequences were present to a condition in which they were absent was established by preinduction of Reuber H35 cells with dexamethasone, followed by addition of actinomycin D. In the time period thereafter, the amount of non-translatable mRNA decreased and 1.5–2 h after addition of actinomycin D, only translatable tyrosine aminotransferase mRNA was present. It can be seen that the induction of tyrosine aminotransferase synthesis by dibutyryl cyclic AMP follows the normal decrease of tyrosine aminotransferase mRNA. We present evidence that dibutyryl cyclic AMP in Reuber H35 hepatoma cells regulates tyrosine aminotransferase synthesis at a posttranscriptional site independent of the pool of nontranslatable tyrosine aminotransferase mRNA sequences, but influencing the efficiency of translation of active tyrosine aminotransferase mRNA.

Chromatin isolated from herpes simplex virus type 1-infected baby hamster kidney cells contains a number of tightly associated virus-induced polypeptides. A subset of these proteins bind to immobilized DNA in vitro (Vmw 175, 155, 130, 63, 43, $\text{38}\text{39}$). Virus-induced polypeptides extractable with acid from infected cell chromatin include Vmw 155, the major capsid protein of herpes simplex virus type 1 virions, and Vmw 63 and $\text{38}\text{39}$ which are heterogeneous with respect to charge and are phosphorylated. These chromatin preparations, in the presence of deoxynucleoside triphosphates and MgCl₂ were capable of synthesizing viral and cell DNA in a reaction which was stimulated by the addition of ATP, riboNTPs and potassium acetate. In vitro synthesized viral DNA cosedimented with prelabelled parental DNA but the single-stranded product was smaller than parental DNA. Density labelling indicated that extensive synthesis was taking place and all BamHI fragments of viral DNA were represented by the DNA synthesized in vitro.

The optimum monovalent cation concentration (Na⁺ or K⁺) for the relaxation of superhelical DNA by the rat liver nicking-closing enzyme under conditions of DNA excess was found to be 150–200 mM. The detection of a nicked DNA species after stopping a reaction with alkali depends on having a high molar ratio of enzyme to DNA and is maximal between 50 and 100 mM monovalen cation. Varying the salt concentration from 15 to 200 mM appears to have no effect on the catalysis of the nicking-closing reaction by the enzyme. Instead different salt optima in these two assays can be explained by the observation that the nicking-closing enzyme acts by a processive mechanism below 100 mM salt and becomes nonprocessive above 150 mM. The salt elution of the nicking-closing enzyme from resting cell chromatin appears to be similar to that which one would expect for the elution of the enzyme from naked DNA. However, greater than 70% of the chromatin associated enzyme activity remained bound to chromatin from growing cells at 300 mM salt, a concentration at which there is no significant binding to naked DNA in vitro.