Asymmetric template function of microbial deoxyribonucleic acids: transcription of ribosomal and soluble ribonucleic acids

In Bacillus subtilis and Escherichia coli, 16 and 23S ribosomal ribonucleic acid (rRNA) hybridize exclusively with the heavy (H) strand of methylated albuminkieselguhr (MAK)-fractionated complementary deoxyribonucleic acid (DNA) strands. All the soluble RNA (4S RNA) in B. subtilis and 66 to 75% of the 4S RNA in E. coli also hybridize with the H strand. Interspecific hybridization shows that E. coli 23S rRNA also binds selectively to the DNA H strand of Salmonella typhimurium. The hybridization peak for all three cellular RNA components is specifically located in the late-eluting region of the absorbance profile of the DNA H strand. The early-eluting region of the light (L) strand preferentially inhibits the hybridization between the peak region of the H strand and 23S rRNA. These regions are considered to represent the transcribing sequences and their complements for 23S rRNA in the separated H and L strands of DNA, respectively.     

Evidence for a Single-Stranded Adenovirus-Associated Virus Genome: Isolation and Separation of Complementary Single Strands

Single-stranded adenovirus-associated virus type 2 deoxyribonucleic acid (AAV-2 DNA) has been isolated from the virion after enzymatic pretreatment of the particles by heating at 53 C for 1 hr in 0.015 m NaCl plus 0.0015 m sodium citrate in the presence of 1% sodium dodecyl sulfate. Double-stranded AAV-2 DNA present as a marker is not denatured by this treatment. AAV-2 single-stranded DNA is composed of two complementary species which can be separated in neutral CsCl when 5-bromodeoxyuridine has been substituted for thymidine in the DNA. The present report is the first documented instance of the separation of complementary strands of an animal virus DNA.     

Competent Diplococcus pneumoniae Accept Both Single- and Double-Stranded Deoxyribonucleic Acid

The transforming activity of fractionated complementary strands of Diplococcus pneumoniae deoxyribonucleic acid (DNA) bands at the position of fully denatured DNA in CsCl at pH 11.0, and is completely (> 99.8%) destroyed by digestion with exonuclease-I. These results prove that pure single strands transform the normally prepared competent cells of this species. Their efficiency is about 0.5% that of native DNA of comparable size.     

Asymmetric Transcription of Bacteriophage Mu-1

The deoxyribonucleic acid (DNA) of bacteriophage Mu-1 can be separated into its complementary strands by poly(U,G) binding and equilibrium centrifugation. DNA-ribonucleic acid (RNA) hybridizations in liquid show that more than 98% of "early" phage-specific RNA and over 96% of "late" messenger species bind to the heavy [poly(U,G)-binding] strand of Mu-1 DNA. A small (1.45%) but significant amount of late RNA binds to the light strand. The significance of this RNA fraction is discussed in connection with the peculiar structure of denatured and reannealed Mu-1 DNA.     

Physicochemical properties of kinetoplast DNA from Crithidia acanthocephali. Crithidia luciliae, and Trypanosoma lewisi

The protozoa Crithidia and Trypanosoma contain within a mitochondrion a mass of DNA known as kinetoplast DNA (kDNA) which consists mainly of an association of thousands of small circular molecules of similar size held together by topological interlocking. Using kDNA from Crithidia acanthocephali, Crithidia luciliae, and Trypanosoma lewisi, physicochemical studies have been carried out with intact associations and with fractions of covalently closed single circular molecules, and of open single circular and unit length linear molecules obtained from kDNA associations by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium centrifugation. Buoyant density analyses failed to provide evidence for base composition heterogeneity among kDNA molecules within a species. The complementary nucleotide strands of kDNA molecules of all three species had distinct buoyant densities in both alkaline and neutral cesium chloride. For C. acanthocephali kDNA, these buoyant density differences were shown to be a reflection of differences in base composition between the complementary nucleotide strands. The molar ratios of adenine: thymine:guanine:cytosine, obtained from deoxyribonucleotide analyses were 16.8:41.0:28.1:14.1 for the heavy strand and 41.6:16.6:12.8:29.0 for the light strand. Covalently closed single circular molecules of C. acanthocephali (as well as intact kDNA associations of C. acanthocephali and T. lewisi) formed a single band in alkaline cesium chloride gradients, indicating their component nucleotide strands to be alkaline insensitive. Data from buoyant density, base composition, and thermal melting analyses suggested that minor bases are either rare or absent in Crithidia kDNA. The kinetics of renaturation of 32P labeled C. acanthocephali kDNA measured using hydroxyapatite chromatography were consistent with at least 70% of the circular molecules of this DNA having the same nucleotide sequence. Evidence for sequence homologies among the kDNAs of all three species was obtained from buoyant density analyses of DNA in annealed mixtures containing one component kDNA strand from each of two species.     

Base composition of deoxyribonucleic acid of the temperature-sensitive kanamycin-resistant R factor, Rts1

The buoyant density of deoxyribonucleic acid (DNA) from the temperature-sensitive R factor, Rts1, was determined by CsCl density-gradient centrifugation. Rts1 was found to consist of a single species of DNA of density 1.705 g/cm(3), which corresponds to a base composition of 45% guanine plus cytosine. This value is distinct from the densities previously reported for other R factors, suggesting that Rts1 represents a new molecular class of R factors.     

Asymmetric Distribution of Guanine Plus Thymine Between Complementary Strands of Deoxyribonucleic Acid of Members of the Enterobacteriaceae

Analysis of the deoxyribonucleic acid prepared from Proteus mirabilis, Escherichia coli, and Serratia marcescens in an alkaline CsCl gradient has shown that there is an asymmetric distribution of guanine plus thymine residues between the complementary strands of the deoxyribonucleic acid.     

Fractionated Strands of Bacterial Deoxyribonucleic Acid III. Transformation Efficiencies and Rates of Phenotypic Expression

Preferential binding of guanine-rich ribopolymers to one of the complementary strands of denatured deoxyribonucleic acid (DNA) of Diplococcus pneumoniae permitted the fractionation of the complements in CsCl density gradients. Phenotypic expression of the newly acquired genes for four drug resistances was more rapid in cells transformed by the heavy fractions than in those transformed either by light fractions or by unfractionated DNA. The efficiencies of transformation with the two complements were nearly equal for the four markers tested. Both efficiency and expression results were the same whether we assayed the residual activity or the activity obtained by annealing the fractions with excess recipient DNA.     

Analysis of Euglena gracilis chloroplast deoxyribonucleic acid with a restriction endonuclease, EcoRI.

Cleavage of chloroplast deoxyribonucleic acid (DNA) of Euglena gracilis Z with restriction endonuclease RI from Escherichia coli (EcoRI) yielded 23 bands upon electrophoresis in gels of agarose. Four of the bands contained twice the stoichiometric amount of DNA. One of these bands contained two similarly sized fragments. The sum of the molecular weight of the 24 different fragments equaled the molecular weight of the circular molecule. The restriction fragments had different buoyant densities, with four having distinctly heavy densities in CsCl. Restriction fragments with a high buoyant density were preferentially lost when broken chloroplast DNA was purified by equilibrium density gradient centrifugation. Hybridization of chloroplast ribosomal ribonucleic acid to intact chloroplast DNA determined that there are two cistrons for 16S and 23S ribosomal ribonucleic acid. These two cistrons are located on six restriction fragments, all of which have buoyant densities greater than the intact molecule of chloroplast DNA.     

Relationship of mRNA from Productively Infected Cells to the Complementary Strands of Adenovirus Type 2 DNA

The complementary strands of adenovirus type 2 (Ad2) DNA were separated by buoyant density gradient centrifugation with poly (U, G). The complementary strand DNA was shown to remain intact through the course of strand separation. The l-strand of Ad2 DNA, appearing in the less dense complex with poly (U, G) in neutral CsCl density gradients, was shown to have a buoyant density in alkaline (pH 12.5) CsCl density gradients which is 2 to 3 mg per ml greater than that of its complement (h-strand). Renaturation of purified complementary strand DNA was observed only in mixtures of h- and l-strand DNA, and then with the second-order reaction rate expected for Ad2 DNA. Hybridization of the complementary strands of Ad2 DNA with cytoplasmic mRNA isolated from infected HeLa cells was performed in liquid phase and analyzed by hydroxylapatite chromatography. Before viral DNA synthesis (6 h after infection), 13 to 18% of the h-strand and 30 to 35% of the l-strand were represented in viral mRNA. Late (18 h) after infection the mRNA represented 20 to 25% and 63 to 68% of the h- and l-strands, respectively. Most, if not all sequences present in viral mRNA before viral DNA synthesis were also present in the cytoplasm late in infection.     

Characterization of the deoxyribonucleic acid of various strains of halophilic bacteria

Bacteria classified as extreme halophiles, in the genera Halobacterium and Halococcus, contain deoxyribonucleic acid (DNA) which displays two components in a CsCl equilibrium density gradient. The base composition of the major DNA component ranges from 66 to 68% guanine plus cytosine (GC), whereas that of the satellite DNA comprising some 11 to 36% of the total, is between 57 and 60% GC. Purification of the bacterial cells in a CsCl density gradient and other more conventional strain purification procedures both indicated that the presence of the satellite DNA component is not a result of mixed cultures.     

Nuclear Fraction of Bacillus subtilis as a Template for Ribonucleic Acid Synthesis

A "nuclear fraction" prepared from Bacillus subtilis was a more efficient template than purified deoxyribonucleic acid for the synthesis of ribonucleic acid by exogenously added ribonucleic acid polymerase isolated from B. subtilis. The initial rate of synthesis with the nuclear fraction was higher and synthesis continued for several hours, yielding an amount of ribonucleic acid greater than the amount of deoxyribonucleic acid used as the template. The product was heterogenous in size, with a large portion exceeding 23S. When purified deoxyribonucleic acid was the template, a more limited synthesis was observed with a predominantly 7S product. However, the ribonucleic acids produced in vitro from these templates were very similar to each other and to in vivo synthesized ribonucleic acid as determined by the competition of ribonucleic acid from whole cells in the annealing of in vitro synthesized ribonucleic acids to deoxyribonucleic acid. Treatment of the nuclear fraction with heat (60 C for 15 min) or trypsin reduced the capacity of the nuclear fraction to synthesize ribonucleic acid to the level observed with purified deoxyribonucleic acid.     

Cellular Origin of a Mouse Leukemia Viral Ribonucleic Acid

Mouse erythroblastosis virus, a member of the mouse leukemia virus group, was obtained from chronically infected C(3)H mouse embryo cells and purified on sucrose gradients. The ribonucleic acid (RNA) extracted from ribonuclease-treated virus consisted of a rapidly sedimenting (72S) species and a more slowly sedimenting component (4 to 30S). The 72S RNA did not contain base sequences homologous to deoxyribonucleic acid (DNA) from infected cells as determined by hybridization studies. In contrast, the slowly sedimenting RNA enclosed within the virus had base sequences homologous to DNA from infected and uninfected C(3)H mouse embryo cells.     

Chromatographically Fractionated Complementary Strands of Bacillus subtilis Deoxyribonucleic Acid: Biological Properties

Biological, physical, and chromatographic properties of methylated albuminkieselguhr (MAK)-fractionated complementary strands, designated as light (L) and heavy (H), of Bacillus subtilis deoxyribonucleic acid (DNA) are presented. The pattern of transforming activity along the MAK elution profile of alkilidenatured DNA shows that the residually active molecules selectively fractionated ahead of the L strand fraction, whereas the most active self-annealed molecules fractionated preferentially at the trailing end of the H strand fraction. The restoration rate of transforming activity in the late-eluting H molecules was rapid and independent of concentration during the annealing reaction. The data suggest that the self-annealing activity in the H strand is due in part to the formation of intrastrand secondary structures. Hydroxyapatite chromatography of self-annealed L and H strands yielded a major fraction (I) of highly purified strand preparations devoid of transforming activity and hypochromicity, and a minor "nativelike" fraction (II). Sedimentation velocity measurements show that, in addition to the mutual complementary nature of the L and H fractions, they differ in molecular size and possibly configuration.