J. Doskočil

Distribution of 5-methylcytosine in pyrimidine sequences of deoxyribonucleic acids.

Abstract Several samples of DNA from different mammalian organs and from wheat germ were degraded with Burton reagent and the location of derivatives of 5-methylcytosine in separate fractions of the hydrolysate was investigated. In mammalian DNAs 5-methylcytosine was found to occur almost exclusively in the fraction of solitary pyrimidine nucleotides and in the terminal groups of polypyrimidine series carrying monoesterified phosphorus on C-3′ of deoxyribose. The ratio of 5-methylcytosine to cytosine was nearly identical in these positions. In the DNA from wheat germ the highest degree of replacement occurred in similar positions, but a fairly high amount of 5-methylcytosine was found in the sequence MpT. The relation of these findings to earlier evidence obtained by enzymic degradation of DNA is discussed. It is concluded that the replacement of cytosine by 5-methylcytosine is determined by the nature of the nucleotide attached to C-3′ of deoxycytidine. The replacement occurs with highest probability in the sequence CpG; somewhat less probable is the replacement in the sequence CpT, whereas in the sequences CpM, CpC and CpA the substitution takes place only rarely, even in preparations with high overall content of 5-methylcytosine.

Pulse-polarographic analysis of double-stranded RNA.

Abstract The reducibility of double-stranded (ds) RNA of phage f2 and its thermally denatured form was studied by the derivative (differential) pulse polarography. It was shown that the denatured RNA produced at neutral and acidic pH values a peak at negative potentials. A peak of dsRNA was much smaller and appeared at more positive potentials. The polarographic behaviour of dsRNA basically agreed with that of DNA; the peaks of dsRNA and its denatured form were however better separated. The lowest amount of the denatured RNA necessay for the determination was about 50 ng. A good sensitivity of pulse polarography for changes in RNA conformation, including damages of the RNA double helix caused by initial attack of an enzyme, low doses of ionizing radiation, etc., was demonstrated. The method was used for testing dsRNA samples prepared for biological experiments; a correlation between the polarographic behaviour of the RNA samples and their antiviral and interferon-inducing activities was found.

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.

PLURIMODAL DISTRIBUTION OF BASE COMPOSITION IN DNA OF SOME HIGHER PLANTS

DNA was isolated from chromatin of Zea mays, Pisum sativum and Solanum tuberosum. Derivative thermal melting curves of unsheared DNA as well as thermal chromatograms of sonicated DNA indicate the presence of discrete cooperatively melting segments with different base composition, consisting of about 103 nucleotide pairs. Maize DNA has a broad and highly asymmetric distribution of base composition, containing a large proportion of components very rich in guanine and cytosine; pea DNA is characterized by an asymmetric distribution with a tail in the region of low content of guanine and cytosine, while potato DNA gives a roughly symmetrical distribution profile.

The effects of 5-azacytidine and 5-azauridine on protein synthesis in Escherichia, coli

In cultures of E., coli deficient in cytidine deaminase 5-azacytidine is a weak inhibitor of total protein synthesis in spite of being extensively incorporated into RNA. Strong inhibition of protein synthesis in wild-type strains is due to 5-azauridine formed from 5-azacytidine by deamination. However, the blocking of replication of phage T4, shown previously to be due to primary inhibition of replication of phage DNA, is a function of 5-azacytidine itself.

Inducible nucleoside permease in escherichia coli

Summary Mutants of E. coli resistant to both 5-azacytidine and showdomycin assume a partially sensitive phenotype when grown in the presence of thymidine. The kinetics of several other types of nucleoside conversion, known to depend on nucleoside-transporting system, is also altered toward the behavior of wild-type cells. It appears that a nucleoside-transporting component, distinct from the constitutive permease of non-induced wild-type cells, and coded by a different gene, is induced concurrently with the enzymes of the deo group.

Temperate and virulent forms of phage theta attacking Bacillus licheniformis.

SummaryClear-plaque phage ϑc, attacking bacitracin-producing strains of B. licheniformis, yields spontaneous temperate mutants at high frequency; the temperate mutants fall into several classes phenotypically different in plaque morphology and properties of lysogenised bacteria. The most common phenotype ϑ3 has DNA restriction fragment patterns identical with those of the parent ϑc; some less common temperate forms, i.e. ϑ1 and ϑ2, produce different restriction fragment patterns, sugesting that a part of the original ϑc DNA has been reorganized or replaced by some foreign genetic material. The changed fragment pattern remains stable upon subsequent passaging of the phage or of the lysogenic bacteria. Neither class of temperate phage mutants gives clearplaque revertants at measurable frequency. Lysogenisation of bacteria with any class of temperate phage confers immunity to all temperate forms and to ϑc; virulent mutants ϑvir, which plate with 100% efficiency on lysogens for ϑ1 and ϑ2 but not for ϑ3, occur in stocks of ϑc at a frequency of 10−7. The mutation from ϑc to ϑvir is not accompanied by any change of the restriction fragment patterns of DNA.

Sequence homology and recombination between the genomes of morphologically dissimilar bacteriophages LP 52 and theta

SummaryA restriction fragment map of Bacillus licheniformis temperate phage LP 52 DNA (molecular weight 38.5×106) was established, using restriction endonucleases BamHI (8 target sites), BglI (10 sites), BglII (13 sites) and EcoRI (22 sites). The map is linear, with well-defined ends, without any signs of circular permutation. The DNA of a related phage, LP 51, produced identical restriction fragments. At least 62% DNA of LP 52 has been found homologous to the DNA of the recently discovered, morphologically quite dissimilar, phage ϱ, as demonstrated by hybridization of electrophoretically separated restriction fragments of DNA. Under the same conditions, the DNAs of LP 52 and of the morphologically similar Bacillus subtilis phage ϕ105 did not cross-hybridize. The homologous regions in the genomes of phages LP 52 and ϱ have been shown to be colinear. Comparison of the cleavage maps of phages LP 52 and ϱ has shown that, within the regions of homology, not a single restriction fragment and few restriction sites have been conserved during divergent evolution. Three major regions of heterology were defined; the longest one, covering the right-hand end of the map (73±2.75% up to 100% LP 52 genome length) appeared to contain genes coding for structural proteins of the virions; a shorter region at the left-hand end of the map (coordinates zero to 10.3±3.3% LP 52 genome length) and a very short central region (coordinates 41.8–43.9%) could be identified, the latter apparently containing a regulatory locus responsible for the heteroimmune behavior of the two phages. Recombinants between phages LP 52 and ϱ were isolated. Mapping of recombinant genomes has indicated mutual substitution of allelic pieces of LP 52 and ϱ DNAs upon strict conservation of overall genome length.

Physical mapping of the genomes of lytic and temperate forms of phage theta

SummaryRestriction maps of genomes of the lytic form and diverse temperate mutants of phage ϱ of Bacillus licheniformis were constructed. Most temperate mutants produced fragmentation patterns identical to that of the parent lytic form, ϱc: in other mutants the only detectable change in the map was the deletion of a BglII restriction endonuclease site at 46.5% genome length. In the genomes of two other temperate mutants, ϱ1 and ϱ2, the central part of the genome was replaced by a piece of DNA of equal length, but with a different distribution of restriction sites; the maps of the two mutants failed to reveal any similarity in the location of restriction sites in the inserted DNA. It seems that any alteration comprising the locus around the coordinate 46.5% of the ϱc genome, brings about a transition from the lytic to temperate phenotype, indicating the position of a regulatory gene responsible for positive control of phage replication.

The components of the nucleoside-transporting system in Escherichia coli

A mutant of Escherichia coli resistant to 5-azacytidine was selected and shown to be deficient in the high-affinity component of the nucleoside-transporting system which participates in several types of conversion of nucleosides, such as incorporation and deamination of 5-azacytidine and cytidine as well as phosphorolysis of thymidine. All these reactions are affected in a similar manner when wild-type cells are infected with T4-phage or treated with osmotically disrupted phage, while the treatment of mutant bacteria with this phage does not significantly alter the value of the Michaelis constant for the phosphorolysis of thymidine. Since the metabolic conversion of nucleosides in mutant cells remains susceptible to competitive inhibition with heterologous nucleosides, it seems that another, low-affinity component participates in the transport, which is still active in mutant or phage-infected bacteria. Cytidine is transported predominantly by the low-affinity component, provided its concentration in the medium is sufficiently high: 5-azacytidine, however, requires the high-affinity component for efficient transport at any concentration.