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

The effect of polyamines on Qβ and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA $\text{replicase > A}$ protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Qβ RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.

DNA sequence organization in rice genome has been investigated by studying the extent of DNA reassociation as a function of DNA fragment size. The reassociation kinetics curves indicate that there is no sequence interspersion of repeated and single-copy sequences at a DNA length of 6500 nucleotide pairs. The reassociation at lower $\text{C}{}_0\text{t}$ values, however, shows that there is an interspersion of DNA sequences within the repetitive DNA fraction. The size distribution of the repeated sequences is determined by agarose gel electrophoresis of $\text{C}{}_0\text{t}$ 50 DNA fraction of 6500-nucleotide-pair-long rice DNA digested with S₁ nuclease. About 35–40% of the S₁-resistant duplexes consist of 6000–6400 nucleotide pairs and 60–65% have a fragment size less than 150 nucleotide pairs. Among plants, rice is the first genome in which no DNA sequence interspersion is observed.

Three assays have been designed to detect the cleavage of duplex ΦX174 DNA at single-base mis-matches. Studies with S₁ nuclease failed to detect cleavage at mis-matches. S₁ nuclease digestion at 37 and 55°C failed to produce a preferential degradation of a multiply mis-matched heteroduplex when compared to a mis-match-free homoduplex as analyzed by sedimentation on sucrose gradients. Other heteroduplex templates were not cleaved by S₁ nuclease at a defined single-base mis-match when assayed by gel electrophoresis or by marker rescue. In all cases, the amount of S₁ nuclease employed was at least 10-times more than that required to render a single-stranded ΦX174 DNA molecule completely acid soluble. The rate of hydrolysis of single-base mis-matches by S₁ nuclease was estimated to be less than 0.016% of the rate at a base in single-strand ΦX174 DNA. In no instance did we detect activity by S₁ nuclease directed at mis-matched sites in our template molecules. Similarly, the single-strand specific endonuclease from Neurospora crassa does not cleave heteroduplex templates at a defined single-base mismatch when assayed by marker rescue.

A formylmethionyl-tRNA_f deacylase has been purified about 330-fold from a crude initiation factor preparation (1 M NH₄Cl ribosomal wash) from Escherichia coli Q13. The enzyme was nearly homogeneous and had an apparent molecular weight of 24 000. Rat liver methionyl-tRNA_f and E. coli methionyl-tRNA_m were not hydrolyzed significantly by the enzyme under standard conditions. Qβ RNA- and AUG(A)_n-directed polypeptide synthesis was inhibited by the enzyme. The inhibition was at the level of initiation of polypeptide synthesis. The enzymatic activity was inhibited by various factors necessary for polypeptide synthesis. The activity was inhibited more by NH₄Cl and spermidine than by Mg²⁺, GTP and ATP. The complex of formylmethionyl-tRNA_f, initiation factor 2 and GTP was resistant to enzymatic hydrolysis, and the resistance was enhanced by the addition of AUG and ribosomes to the above reaction mixture.

Hybridization of an actin cDNA clone (pSA38) to restriction enzyme digests of Strongylocentrotus purpuratus DNA indicates that the sea urchin genome contains at least five different actin genes. A sea urchin genomic clone library was screened for recombinants which hydridize to pSA38 and four genomic clones were isolated. Restriction maps were generated which indicate that three of these recombinants contain different actin genes, and that the fourth may be an allele to one of these. The restriction maps suggest that one clone contains two linked actin genes. This fact, which was confirmed by heteroduplex analysis, indicates that the actin gene family may be clustered. The linked genes are oriented in the same direction and spaced about 8.0 kilobases apart. In heteroduplexes between genomic clones two intervening sequences were seen. Significant homology is confined to the actin coding region and does not include any flanking sequence. Southern blot analysis reveals that repetitive DNA sequences are found in the region of the actin genes.

We have studied the interaction of the fluorescent lanthanide, terbium(III) (Tb³⁺), with polynucleotides and linear and superhelical DNA, through employment of mono- and multivalent cations as competitive inhibitors. Increasingly effective competitive inhibition of the Tb³⁺-nucleic acid interaction was achieved, for the most part, in the cation order $\text{monovalent < divalent < tetravalent}$. The divalent cation Cu²⁺ proved to be an exception to this trend, and was the strongest competitive inhibitor of all cations tested, exhibiting an affinity for Tb³⁺ binding sites over twice that of Tb³⁺ itself. Unexpectedly, a narrow range of low sodium ion concentration (8–20 mM) was found to be effective in inducing localized unwinding or unstacking of linear and supercoiled DNA double helices, a phenomenon detectable through the use of both Tb³⁺ fluorescence enhancement and ultraviolet spectroscopy. Within a similar range of low sodium ion concentration, moreover, histone H1 was substantially more effective in displacing terbium ion from DNA than either histones H2B or H4, but at higher ionic strength, this difference was absent. These results further confirm the sensitivity and specificity of Tb³⁺ as a conformational probe of nucleic acids.

1. The effect of Ni²⁺ on respiration, volume changes and Ca²⁺ movements was investigated in rat liver mitochondria. 2. Ni²⁺ inhibited Ca²⁺ uptake into respiring mitochondria, Ca²⁺-stimulated respiration and swelling in Ca²⁺ salts, whereas it did not inhibit either state 4 and NDP-stimulated respiration, or swelling in K⁺ salt in the presence of valinomycin. 3. The inhibitory concentration of Ni²⁺ depended strongly on the applied Ca²⁺ concentration. As revealed by direct methods, 50% inhibition of Ca²⁺ influx was achieved by approx. 2-fold excess of Ni²⁺. 4. If added to Ca²⁺-loaded mitochondria, Ni²⁺ gave rise to slow Ca²⁺ release and inhibited uncoupler-induced efflux slightly. 5. It is concluded that Ni²⁺ is a potent inhibitor of mitochondrial Ca²⁺ transport. Ca²⁺ influx is far more sensitive to inhibition than Ca²⁺ efflux.

Guinea pig epidermal DNAase I was purified from an epidermal extract by a procedure including DEAE-cellulose chromatography, Sephadex G-100 gel filtration and Con A-Sepharose affinity chromatography. The purified enzyme contained no detectable activities of acid DNAase, alkaline RNAase, phosphodiesterase or acid or alkaline phosphatase, but was contaminated with acid RNAase activity. The molecular weight of the enzyme was estimated to be 33 000 by sucrose density gradient centrifugation and Sephadex G-100 gel filtration. Its isoelectric point is $\text{5.2 ± 0.1}$. The enzyme requires divalent cations and exhibits two pH optima that are dependent on divalent cations: in the presence of Mn²⁺, the optimum pH is about 7.5 in 50 mM Tris-HCI buffer and in the presence of Mn²⁺, the pH optimum is 6.4 in 50 mM cacodylate-HCI buffer. The enzyme hydrolyzes native DNA about 6-times faster than denatured DNA, producing 5′-phosphoryl and 3′-hydroxyl terminated oligonucleotides with an average chain length of about eight nucleotides, and converts double-stranded and circular DNA to relaxed and linear forms. The enzyme is inhibited by G-actin and antiserum against bovine pancreatic DNAase A. Thus this enzyme is classified as DNAase I.

The conversion of single-stranded DNA in S13 intact phage particles to the double-stranded replicative form DNA was observed in cell extracts prepared from Escherichia coli H560 (S13^S, polA, endA) cells lysed with lysozyme and the non-ionic detergent, Brij 58. The DNA product, which associated with a rapidly sedimenting component, was identified as RFII-DNA with a gap by sedimentation analysis. The conversion was inhibited by $\text{N-}\text{ethylmaleimide}$, but not by rifampicin, nicotinamide mononucleotide or polymyxin B. The dnaB gene product was involved in the replicative system. Similar extracts prepared from a S13-resistant E. coli strain K12W6 also catalyzed this synthesis.

Poly(5-methoxycytidylic acid) [poly(mo⁵C)] has been prepared by polymerization of 5-methoxycytidine diphosphate with polynucleotide phosphorylase. The poly(mo⁵C) was of high molecular weight and formed a 1 : 1 hybrid with poly(I). Although the $\text{T}{}_{\mathrm{<mtext>m</mtext>}}$ of this hybrid is higher than that of poly(I) · poly(C) in 0.1 M salt, poly(I) · poly(mo⁵C) is not an inducer of interferon.