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

A method is described for estimating rapidly the relative proportion of total DNA that is of mitochondrial origin in small quantities of the yeast, Saccharomyces cerevisiae. This procedure involves the mechanical disruption of cells followed by the addition of small amounts of radioactively labeled yeast nuclear and mitochondrial DNA to the lysate. Both labeled and unlabeled DNAs are then co-extracted from the mixture and separated into nuclear and mitochondrial DNA components by poly(l-lysine) Kieselguhr column chromatography. The resulting specific radioactivities of each species of DNA, when compared to the amount of labeled DNA initially added, are related to the relative proportion of unlabeled nuclear and mitochondrial DNA in the original cell sample. The isotope dilution procedure reported here is shown to be both reproducible and to reflect the true relative concentrations of each species of DNA in this yeast.

Primary hepatocyte monolayers, derived from chick embryos, can be cultured from the onset in a completely chemically defined medium, free of added hormones. The liver cells synthesize and secrete a wide spectrum of plasma proteins for several days in this serum-free environment. Addition of fetal bovine serum elicits a 3–5-fold increase in the production of certain plasma proteins: fibrinogen, albumin, and the $\text{α}{}_1\text{-}\text{globulin}$ M. This effect of serum is selective; transferrin and plasminogen syntheses are enhanced less than 1.5-fold. Significant stimulation is observed with 0.1% fetal bovine serum, and half-maximal values for individual plasma proteins are obtained with concentrations ranging between 0.4 and 1%. The stimulatory activity of serum shows no developmental or species specificity. Plasma is as active as serum derived from the same blood sample. The hepatocytes respond rapidly to serum, significant changes in albumin synthesis occurring less than 1 h after serum addition or removal. The effect of short exposure is fully reversible. These results establish the capacity of low concentrations of serum to stimulate plasma protein synthesis and underscore the importance of studying the effects of hormones and other factors under serum-free conditions. The findings suggest that, in addition to the classical hormones, ubiquitous but as yet uncharacterized serum components play a role in controlling this major hepatic function.

An ATP-dependent deoxyribonuclease has been partially purified from extracts of Caulobacter crescentus cells in a procedure involving ion-exchange and affinity chromatography. The enzyme was purified approximately 350-fold and was free of contaminating nucleolytic and ATPase activity. The nuclease hydrolyzes linear, double-stranded DNA with subsequent release of short oligonucleotides, mostly from one to four bases in length. The release of nucleotides is accompanied by hydrolysis of ATP, 7.6 nmol ATP being consumed for each nmol of acid-soluble products of DNA degradation. The enzyme shows an absolute requirement for divalent cations and is most active at pH 7.6 to 8.8. The molecular weight of the nuclease, estimated by gel filtration and sucrose density gradient centrifugation, is 280 000.

An endonuclease purified from germinating pea (Pisum sativum) seeds has been shown to catalyze the hydrolysis of heat-denatured single-stranded DNA. Since P. sativum endonuclease shows appreciable activity in the presence of DNA destabilizing agents and, unlike many similar endonucleases, significant activity at neutral pH, it is a potentially valuable tool for studies of the secondary structure of nucleic acids. The residual hydrolysis of duplex DNA is directed towards partially denatured, A,T-rich areas in native DNA. The rate of hydrolysis of deoxypoly-nucleotides was in the order $\text{poly(dT) ⪢ denatured DNA > poly(dA) > poly(dA-dT) = native DNA}$. Neither poly(dC), poly(dG) nor poly(dC) · poly(dG) were attacked by the enzyme. Supercoiled, covalently closed circular phage PM2 form I DNA is converted to singly hit nicked circular form II and doubly hit linear form III duplexes. Prolonged treatment with enzyme does not further cleave the linear form III DNA. Addition of increasing concentrations of NaCl in the incubation mixture suppresses the conversion of form I to form II, but not the conversion of form II to form III, which is enhanced with the increasing ionic strength. The enzymatically relaxed circular form, I°, obtained by unwinding of supercoiled DNA with a DNA-relaxing protein, is resistant to the action of the enzyme. Molecules with intermediate superhelix densities do not serve as substrates. The sites of cleavage of P. sativum endonuclease in PM2 DNA occur within regions that are readily denaturable in a topologically constrained superhelical molecule.

The mechanism of inhibition of RNA polymerase-catalyzed synthesis of RNA by actinomycin D and the phenanthridinium derivatives ethidium bromide and 3,8-diamino-6-ethylphenanthridinium bromide (DEMB) is examined. A general kinetic equation describing the dependence of RNA synthesis on DNA template concentration is derived and distinct expressions corresponding to various possible mechanisms of inhibition are subsequently obtained by introducing into the equations assumptions as appropriate for the individual mechanisms. The fitting of the experimental results of inhibition into the resulting equations has suggested that ethidium bromide and DEMB inhibit RNA polymerase by forming an inhibitor-template complex which interferes with enzyme recognition of, and binding to, appropriate sites on the template (binding inhibition). The fitting of the dependence of the rate of RNA synthesis on the bound-inhibitor to DNA ratios to the derived kinetic expressions also allows a tentative distinction to be made as to whether ethidium bromide and DEMB interfere with RNA synthesis by a mechanism of ‘partial’ or ‘complete’ inhibition.

The quantitation and repair of double-strand DNA breaks in human fibroblasts has been determined using a method involving the nondenaturing elution of DNA from a filter. DNA from cells from two human fibroblast lines exposed to γ-radiation from 0 to 10 000 rad showed increasing retention on a filter with decreasing radiation dose, and the data suggest a linear relationship between double-strand breaks induced and radiation dose. The ability of normal human fibroblasts to repair double-strand breaks with various doses of radiation was demonstrated, with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 10 min for repair of 5000 rad exposure and 39 min for repair of 10 000 rad damage. The kinetics of the DNA rejoining were not linear and suggest that, as in the repair of single-strand breaks, both an initial ‘fast’ and a later ‘slow’ mechanism may be involved.

DNA (N-DNA) prepared under conditions eliminating the exposure of chromatin to cytoplasmic components exhibits some special properties not observed for DNA prepared by standard methods (S-DNA). N-DNA, having a sedimentation coefficient of 24.7 S and a firmly bound protein content of 0.7%, can be cleaved (in contrast to S-DNA) by treatment with chelating agents, into stable subunits having a mean molecular weight of about 500 000. This cleavage was shown to be an ordered process which involved no enzymatic or shear degradation. It was accompanied by the release of phosphopeptides. The analyses of these phosphopeptides revealed the presence of two main fractions. One contained phosphoserine and glycine ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ about 1 400), and the other contained phosphoserine, glycine, alanine, glutamic and aspartic acids ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ about 900). The amount of released phosphopeptides could be correlated to the extent of cleavage.

The widespread occurrence of physical binding between biological macromolecules and small molecules has prompted us to hypothesize that physical binding contributes to DNA alkylation specificity. The preferred physical binding sites for a CH⁺₃-like test probe were predicted for several sequences of DNA using molecular mechanics free space calculation methods. Sequences containing $\text{A = T}$ basepairs direct physical binding to the minor groove, whereas sequences containing $\text{G ≡ C}$ basepairs direct physical binding to the major groove. Physical binding calculations were also performed for model ‘unwound’ DNA conformations. The results of the test probe studies were subsequently employed as starting points to predict the preferred physical binding sites for the more complicated case of an actual alkylating agent, the dimethylaziridinium ion. These studies demonstrate that physical binding specificity is highly dependent upon DNA sequence and conformation, and correlates well with the DNA alkylation site specificity observed for alkylating agents in the dimethylaziridine class.

Mouse myeloma DNA polymerase γ was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)_n · (dT)_12–18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of $\text{M}{}_{\mathrm{r}}\text{= 47 000}$ changed proportionally with DNA polymerase γ activity. A minor polypeptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 140 000}$ also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by ¹²⁵I-labeled peptide mapping indicates that the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide in the mouse myeloma DNA polymerase γ preparation is structurally related to the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide of chick embryo DNA polymerase γ (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002–7009). An antibody against chick embryo DNA polymerase γ cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the “expected” specific activity with respect to the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide and the molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{= 188000}$ on the assumption of a tetrameric structure of the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide). Results indicate that the $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 47 000 polypeptide is a component of the mouse myeloma DNA polymerase γ.

The three-dimensional structure of blasticidin S hydrochloride pentahydrate, a member of the cytosine amino nucleoside antibiotics, has been solved using diffractometer data and refined to an R value of 0.115. The crystal data are a = 13.500(5), b = 20.387(7), $\text{c = 4.824(2)}\text{A}\text{̊}$, β = 98.66(3)°, Z = 2, Dc = 1.389 g · cm⁻³, space group P2₁. The nucleoside base conformation is anti ($\text{ч = 86°}$) and the 2′,3′-unsaturated pyranosyl sugar exhibits a half-chair (°H₅) conformation. The amide plane is twisted from the trans position by about 10°. The guanidium group and the amino group of the amino acid chain are positively charged, while the carboxyl group of the sugar is ionized. The chloride ion is surrounded by water molecules only, in a trigonal prismatic arrangement. The molecule has an extended conformation and there is an intramolecular hydrogen bond between the ammonium group and the carboxyl group. A striking feature of blasticidin is that all the hydrophilic groups lie on one side of the molecule and the hydrophobic groups on the other. Amicetin also shows a similar feature and this might be linked to the commonality of their antibiotic functions. Hydrogen bonds link the hydrophilic sides of adjacent molecules forming double chains parallel to the b-axis. The hydrophobic sides of adjacent double chains are separated by a water layer.