Yeast telomere length varies in response to changes in the amount of polyC1-3A in the cell.

Yeast chromosomes terminate in a GC-rich tail of DNA. Previous investigations have shown that the length of this tail can change in response to genetic variation. Here we present data that show that the length can also alter in response to changes in the amount of the GC-rich DNA found elsewhere in the nucleus.

[Calorimetric study of the polyG-polyC complex]. / Kalorimetricheskoe issledovanie kompleksa poliG-poliTs.

Heat effects of polyG-polyC melting in neutral aqueous solutions have been measured using differential scanning microcalorimeter with an extended temperature range. The limiting value of melting enthalpy is 53 +/- 4 kJ per mole of base pairs and melting temperature dependence on the sodium concentration can be expressed by the empiric relation Tm = 13.2 log(Na+) + 420 K.

Implications for the formation of abasic sites following modification of polydeoxycytidylic acid by acrolein in vitro.

Polydeoxycytidylic acid (poly dC) was incubated with excess acrolein. A Nensorb 20 nucleic acid purification cartridge was used to bind the polymeric material in the poly dC/acrolein reaction mixture. The non-polymeric material eluted from this column had a UV absorbance four times higher than that of the control. The fluorescence spectrum of the eluted material did not correspond to that of unmodified cytosine. Separate aliquots of the reaction mixture were digested to deoxynucleotide 3'-monophosphates by incubation with micrococcal nuclease and spleen phosphodiesterase. The products were converted to 32P-labeled deoxynucleotide 3',5'-bisphosphates by incubation with T4 polynucleotide kinase and excess [gamma-32P]ATP. The 3'-monophosphate was selectively removed by incubation with nuclease P1. Two-dimensional thin-layer chromatography (TLC) on polyethyleneimine cellulose (PEI)-cellulose and detection of 32P-labeled deoxynucleotide 5'-monophosphates by autoradiography failed to provide evidence for the formation of an acrolein adduct of deoxycytidine 5'-monophosphate. When acrolein-modified deoxycytidine 3'-monophosphate was 32P post-labeled, a new product, which co-chromatographed with UV markers synthesized by reaction of acrolein with deoxycytidine 5'-monophosphate, was detected. These data show that acrolein-modified deoxycytidine 3'-monophosphates are substrates for 32P labeling by T4 polynucleotide kinase and are stable under the assay conditions employed. The inability to detect the acrolein-modified nucleotides after reaction with poly dC in vitro suggests that the modified bases are lost from poly dC by cleavage of the N-glycosyl bond resulting in the formation of an abasic site.

Cyclic voltammetry of DNA at a mercury electrode: an anodic peak specific for guanine.

Synthetic homopolyribonucleotides poly(A), poly(U), poly(C), and poly(G), poly(A, G, U), apurinic acid and native and denatured DNA from calf thymus were analyzed by means of cyclic voltammetry (CV) using a hanging mercury drop electrode. It was shown that guanine containing polynucleotides, i.e. poly(G), poly(A, G, U) and DNA yield an anodic peak of guanine in the vicinity of a potential of -0.3 V (against a saturated calomel electrode). The guanine peak appeared only at a sufficiently negative switching potential (about -2 V). The appearance of the guanine peak was conditioned by a reduction of guanine residues in the region of the switching potential and reoxidation of the reduction product in the vicinity of -0.3 V. Native and thermally denatured DNAs were investigated under the conditions of both complete and incomplete coverage of the electrode in various background electrolytes. Both DNA forms yielded anodic CV peaks of guanine with the peak of denatured DNA being always higher than that of native DNA. Irradiation of native DNA with relatively small doses of gamma radiation (5-120 Gy) resulted in an increase of the anodic peak. A comparison of changes induced by gamma radiation in the anodic (guanine) and cathodic (reduction of adenine and cytosine) peaks showed a steeper increase of the cathodic peak as compared to that of the anodic one. It has been concluded that in the given dose range the DNA double-helical structure is mainly damaged in the adenine-thymine rich regions.

Preparation and sequencing of the cloacin fragment of Streptomyces aureofaciens 16S RNA.

A fast method for isolation of a 3'-terminal fragment of Streptomyces aureofaciens 16S RNA was developed. The procedure involves reaction of 70S ribosomes with cloacin DF13 and subsequent fractionation of the reaction mixture by polyacrylamide gel electrophoresis. The cloacin fragment was eluted from the gel and used directly for 3'-end labeling with cytidine-3',5'-[5'-32P]bisphosphate. The labeled RNA fragment was sequenced by the enzymatic method. It consists of 50 nucleotides and has the sequence 5'-GUCGUAACAAGGUAACCGUACCGGA-AGGUGCGGUUGGAUCACCUCCUUUCOH. The differences from the E. coli and Bacillus sequences and their possible influence on the rate and specificity of polypeptide synthesis are discussed.

[Study of poly(C) and poly(I)-poly(C) complexes with model phospholipid membranes by infrared spectroscopy]. / Izuchenie kompleksov poly(C) i poly(I)-poly(C) s model'nymi fosfolipidnymi membranami metodom IK-spektroskopii.

The temperature dependence of poly(C) is shown by the infrared spectroscopy to be different for the free polynucleotide and for the polynucleotide in complexes with membranes. The intensity of stretching vibrations of C = 0 bond of poly(C) in the complex appears to be sensitive to the temperature. The intensity of this band is sharply decreased by increasing the temperature. This effect depends upon concentration of Mg2+-cations. Adsorption of poly(I)-poly(C) on the surface of vesicles from phosphatidylcholine results in the increase of the double helix.

[Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex]. / Otsenka razmera nepreryvnogo uchastka poli(G), neobkhodimogo dlia biologicheskoi aktivnosti kompleksa (G).poli(Ts).

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.

Genetic control of chromosome length in yeast.

The chromosomes of the yeast Saccharomyces cerevisiae terminate with sequences that have the form poly(C1-3-A). In this paper, we show that within an individual yeast strain all chromosomes end with tracts of poly(C1-3-A) of similar lengths; however, different strains can have tracts that vary in length by a factor of two. By a genetic analysis, we demonstrate that yeast cells have a mechanism that allows them to change rapidly the length of their chromosomes by altering the length of the poly(C1-3-A) tract.

Heterogeneity of the polyribocytidylic acid tract in aphthovirus: biochemical and biological studies of viruses carrying polyribocytidylic acid tracts of different lengths.

In this paper we report a study of a sample of foot-and-mouth disease virus carrying two polyribocytidylic acid [poly(C)] tracts of different lengths. By plaque purification in tissue culture, we isolated two populations of particles, one carrying the long poly(C) tract and the other carrying only the short homopolymer. The fingerprints of both viruses were indistinguishable from each other and from that of the virus present in the original sample, suggesting that the main difference between the two types of particles is limited to the poly(C) tracts of their genomic RNAs, to the flanking sequences of the poly(C) tract, or to both. In addition, some biological properties of these viruses are reported, such as stability upon serial passages in different cell lines, plaque size, and pathogenicity for cattle. The results indicate that the size of the poly(C) tract is not directly related to the virulence of these viruses. However, the size of the homopolymer could play a role in determining their efficiency of replication, and it appears that the particles with the short poly(C) tract might have some replicative advantage over those carrying the long one.

Electrochemistry of double-stranded complexes of synthetic polyribonucleotides having interferonogenic and antiviral activity.

Double-stranded (ds) complexes of poly(C) with poly(G) and poly(G,I) were studied using differential pulse polarography (DPP) and differential pulse voltammetry at a pyrolytic graphite electrode (DPV). The complex formed by copolymer was found to be DPP inactive. On the other hand, poly(G).poly(C) yielded a small DPP peak corresponding to single-stranded (ss) poly(C). It was suggested that ss poly(C) present in the solutions of poly(G).poly(C) appeared due to the existence of segments in poly(G) during the complex-forming process in which guanine residues were unable to be hydrogen-bonded with bases in poly(C). Polynucleotide complexes investigated in this report yielded a DPV peak corresponding to electrooxidation of guanine residues, which was markedly lower than that yielded by ss polymers. Moreover, this DPV peak yielded by the complex prepared from an equimolar mixture of poly(G) and poly(C) was still markedly higher than that yielded by poly(G,I).poly(C), or by poly(G).poly(C) prepared in the excess of poly(C). The lowering of the DPV peak was explained as being particularly due to the presence of the polynucleotide segments with an intact and regular secondary structure. The results of our electrochemical analysis of the complexes investigated were compared with their biological activity reported earlier. This comparison calls attention to the fact that biological effectiveness of these biopolymers may be dependent on details of their secondary structure which may be monitored using the methods of electrochemical analysis.

[Heterogeneity of the poly(C) tract of encephalomyocarditis virus RNA]. / Neodnorodnost' poly(C)-trakta RNK virusa éntsefalomiokardita.

The nucleotide sequence of the RNase T1-resistant fragment of encephalomyocarditis virus RNA that includes the poly(C) tract was determined by gel sequencing and mobility shift methods. This sequence is (5') AC126(127) UCUCUCUC9UAACG (3'). The results show that the poly(C) tract is discontinuous, i.e., it is interrupted by the UCUCUCU-sequence. The tract displays anomalously high mobility in polyacrylamide gels as compared to random polynucleotides, indicating that electrophoretic determination of its length gives underestimated values.

Cytochemical hybridization with fluorochrome-labeled RNA. I. Development of a method using nucleic acids bound to agarose beads as a model.

A new procedure to label RNA at the 3'-terminus with a fluorochrome molecule is described. The thiosemicarbazides derived from tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) were prepared by reacting these compounds with hydrazine in dimethylsulfoxide (DMSO):pyridine, 99:1 (v/v). They coupled efficiently to the aldehydes generated by periodate oxidation of RNAs. We determined, using Sepharose to which different nucleic acids and proteins had been bound, that the label added no specific binding properties to the RNA, and did not interfere with duplex formation of labeled poly(U) and poly(A). The stability of the fluorochrome-RNA bond under conditions generally used for hybridization was investigated. The bond was found to be unstable at 66 degrees C in 3 x SSC, 0.1% SDS (50% loss within 45 min) but stable for at least 40 hr at 23 degrees C in 70% formamide/3 x SSC. The hybridization characteristics of complementary RNA, both fluorochrome- and 3H-labeled, were investigated using DNA-Sepharose beads as a cytochemical model. Hybridization was measured by scintillation counting of microliter quantities of beads and quantitative fluorescence microscopy of individual Sepharose beads. No influence of the label on the specificity and stability of the hybrids was found. Maximum specific fluorescence was found after hybridization at 23 degrees C in 70% formamide/3 x SSC. These results made possible the successful use of fluorochrome-labeled RNA to perform cytochemical hybridization followed by detection of the hybrids with fluorescence microscopy. This will be described in an accompanying article.

Sequence and location of the poly C tract in aphtho- and cardiovirus RNA.

The poly C tract in the RNA of the aphtho- and cardio viruses has been examined in several isolates of foot-and-mouth disease virus (FMDV) and encephalomyocarditis (EMC) virus. The length of the tract is variable, containing 100 to 170 bases in the FMDV isolates and 80 to 250 bases in the EMC virus isolates. Each poly C tract contains c. 10% A and U residues, located at the 5' end, i.e. most of the tract is a continuous run of C residues. The position of the tract on the genome was the same in each of the FMDV isolates, about 400 bases from the 5' end, whereas in the EMC virus isolates it was about 150 bases from the 5' end.

More precise location of the polycytidylic acid tract in foot and mouth disease virus RNA.

The polycytidylic acid [poly(C)] tract in foot and mouth disease virus RNA has been located about 400 nucleotides from the 5' end of the RNA by analysis of the products from the digestion of the RNA with RNase H in the presence of oligodeoxyguanylic acid [oligo(dG)]. This treatment produces a small fragment (S) containing the small protein covalently linked to the RNA and a large fragment (L) that migrates faster than untreated RNA on low-percentage polyacrylamide gels, lacks the poly(C) tract as shown by RNase T1 digestion and oligo(dG)-cellulose binding, and is no longer infective. Polyacrylamide gel electrophoresis of fragment S suggests that it is about 400 nucleotides long, in agreement with the size estimated from the proportion of radioactivity in the fragment. Analysis of the RNase T1 digestion products of S shows that it contains only those oligonucleotides mapping close to the poly(C) tract that is situated near the 5' end of the virus RNA.

Terbium as a solid-state probe for RNA.

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.

Studies on the chemical composition and biological properties of transfer factor.

Dialyzable transfer factor (dTF) was fractionated on Sephadex G-10 and G-25 fine columns, and biological activity was found in 3 fractions. One of these, designated VIa, and having a tendency to adsorb to the Sephadex G-10 gel, was shown to have a therapeutic effect on certain immunological diseases. Analysis of this fraction on thin-layer and gas chromatography and with infrared and mass spectroscopy indicated that about half of this fraction was composed of uracil; additional unidentified heterocyclic and aromatic substances were present in this fraction. Adjacent fraction V contained tyrosine and a small polyribonucleotide, and fraction VII hypoxanthine and additional unidentified components. Our results suggest that the therapeutic activity of dTF is not mediated through an immunologically specific informational molecule, but is rather based on non-specific stimulation of the expression of the immune response.

The location of the ploy(C) tract in the RNA of foot-and-mouth disease virus.

Fragments of foot-and-mouth disease virus RNA of decreasing size, containing the 3' poly(A) sequence have been prepared by alkali treatment and sucrose gradient centrifugation followed by oligo(dT)-cellulose affinity chromatography. Polyacrylamide gel electrophoresis of the ribonuclease T1 resistant oligonucleotides from these polyadenylated fragments has enabled us to locate the position of some of the longer oligonucleotides on the RNA. In particular the poly C tract has been shown to be near the 5' end of the RNA; The possible function of the poly(C) tract is discussed in the light of these findings.