Some effects of triton x-100 on pea etioplasts

When pea etioplast preparations were treated with Triton X-100, the membranes disappeared, the pigments were solubilized, and the organelles appeared to disintegrate. Low speed centrifugation (2000g) of the preparations following treatment with Triton X-100 resulted in a pellet which contained considerable quantities of plastid material. This included RNA polymerase and DNA polymerase activity, much of the DNA, about 30% of the RNA, and 50% of the protein of the washed plastid. The amount of RNA polymerase and DNA polymerase activity associated with the low speed pellet was dependent on the pH during Triton treatment. Significant quantities of the RNA polymerase activity of chloroplasts from spinach, peas, and tobacco were also recovered in the pellet after Triton treatment.     

DNA-hydrolyzing IgG antibodies from the blood of patients with acquired immune deficiency syndrome

DNase activity was analyzed in 110 IgG preparations from the blood of AIDS patients. The relative activity of the preparations varied markedly among patients, being reliably detectable in 96% of the preparations. It was shown with several rigid criteria that DNAase activity is an intrinsic property of antibodies (Abs) from AIDS patients. Not only intact IgG, but also isolated light chains of polyclonal Abs were shown to possess catalytic activity. The abzymes efficiently catalyzed DNA hydrolysis in a wide range of pH (5.0–9.5). The K _M and V _max values were evaluated for Ab-dependent hydrolysis of DNA.     

Genetic Expression in Heterozygous Replicative Form Molecules of phiX174

Heterozygous replicative form molecules of bacteriophage X174 deoxyribonucleic acid (DNA) have been constructed in vitro. These are composed of viral strands extracted from purified preparations of phage bearing ts mutations and complementary strands of either half length or full length synthesized with purified DNA polymerase, in vitro, on DNA from am3 phage. In infections with such heterozygous DNA, involving mutations in each of four different cistrons, phage with the genotype of the complementary strand comprised 1 to 20% of the total phage produced by a spheroplast population. From single-burst analysis of the progeny from DNA heterozygous in one cistron (B), it appears that those phage with the genotype of the complementary strand arise as major components in a small proportion of the infected cells rather than comprising a minor component in most cells. The implications of such a pattern of expression are discussed with respect to mechanisms of phage DNA synthesis.     

Construction of viable and lethal mutations in the origin of bacteriophage 'phi' X174 using synthetic oligodeoxyribonucleotides

Six different synthetic deoxyhexadecamers complementary to the origin of bacteriophage φX174, corresponding to nucleotides 4299 to 4314, except for one preselected nucleotide change were used as primers for DNA synthesis on wild-type φX² DNA as a template. DNA synthesis was performed with Escherichia coli DNA polymerase I (Klenow fragment) in the presence of DNA ligase. Heteroduplex RFIV DNA was isolated and, after limited digestion with DNAase I, complementary strands containing the mutant primers were isolated. The biological activity of these complementary strands was assayed in spheroplasts. Spheroplasts were made from E. coli K58 ung^? (uracil N-glycosylase) to prevent degradation of the complementary strands caused by uracil incorporation (Baas et al., 1980a).Using (5′-³²P) end-labeled primers, it was shown that all tested DNA polymerase preparations, including phage T4 DNA polymerase, contained variable amounts of 5′ → 3′ exonuclease activity. This nick translation activity may result in removal of the mutation in the primers, and therefore in isolation of wild-type complementary DNA instead of mutant complementary DNA.Restriction enzyme analysis of completed RFIV DNA showed that the primers can initiate DNA synthesis at more than one place on the φX174 genome. These complications result in a mixed population of complementary strand DNAs synthesized in vitro. Nevertheless, the desired mutants were picked up with high frequency using a selection test that is based on the difference in ultraviolet light sensitivity of homoduplex and heteroduplex φX174 RF DNA. Heteroduplex φX174 RF DNA is two to three times more sensitive to ultraviolet light irradiation than is homoduplex φX174 RF DNA (Baas &; Jansz, 1971,1972). Phage DNA derived from single plaque lysates of two of the six mutant complementary strand DNA preparations yielded, after annealing with wild-type complementary strand DNA, heteroduplex DNA with high frequency. DNA sequence analysis in the origin region of RF DNA obtained from these two phage preparations revealed the presence of the expected mutation. RFI DNA of these two origin mutants was nicked by φX174 gene A protein in the same way as wild-type φX174 RFI DNA.Phage DNA derived from single plaque lysates of the other four mutant complementary strand DNA preparations yielded exclusively homoduplex DNA after annealing with wild-type complementary strand DNA. It is concluded that priming with these deoxyhexadecamers resulted in the synthesis of complementary φX174 DNA with lethal mutations. The implications of these results, the construction of two silent, viable φX174 origin mutants and the failure to detect four others, for the initiation mechanism of φX174 RF DNA replication are discussed.     

Aggregate formation from short fragments of plant DNA

Large aggregates have been observed after partial reassociation of pea (Pisum sativum L.) DNA preparations sheared to mean single strand fragment lengths as short as 350 nucleotides. At high DNA concentrations and conditions of salt and temperature which require only moderate precision of base pairing, aggregates pelletable by brief centrifugation account for 30 to 40% of the total DNA from peas, while calf thymus DNA reassociated under similar conditions forms less than 10% pelletable structures. In contrast to networks formed during the reassociation of long DNA fragments containing interspersed repetitive sequences, these aggregates contain a high percentage of double-stranded DNA and are enriched in repetitive sequences.     

Preparation of labeled nucleic acids (nick translation and iodination) for DNA homology and rRNA hybridization experiments

Nick-translated DNA preparations may contain very short fragments, causing inconsistent DNA homology results. This appears to be due to extensive fragmentation of the high molecular weight DNA used in the labeling procedure. DNA preparations from organisms containing DNA of low mol% G+C content appear to be the most fragmented. The problem was circumvented by labeling these DNA preparations with¹²⁵I. Sample preparation protocols have been developed for the routine¹²⁵I labeling of DNA and rRNA from a wide range of organisms.     

Methods for Detection of Anticarsia gemmatalis Nucleopolyhedrovirus DNA in Soil

Two methods, phenol-ether and magnetic capture-hybridization (MCH), were developed and compared with regard to their sensitivities and abilities to extract the DNA of the insect baculovirus Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) from soil and to produce DNA amplifiable by PCR. Laboratory experiments were performed with 0.25 g of autoclaved soil inoculated with different viral concentrations to optimize both methods of baculovirus DNA extraction and to determine their sensitivities. Both procedures produced amplifiable DNA; however, the MCH method was 100-fold more sensitive than the phenol-ether procedure. The removal of PCR inhibitors from the soil appeared to be complete when MCH was used as the viral DNA isolation method, because undiluted aliquots of the DNA preparations could be amplified by PCR. The phenol-ether procedure probably did not completely remove PCR inhibitors from the soil, since PCR products were observed only when the AgMNPV DNA preparations were diluted 10- or 100-fold. AgMNPV DNA was detected in field-collected soil samples from 15 to 180 days after virus application when the MCH procedure to isolate DNA was coupled with PCR amplification of the polyhedrin region.     

A comparative study of the protein components of ribonucleoprotein particles isolated from rat liver and hepatoma nuclei

To solve the mechanism for the complete cessation of DNA synthesis in Tetrahymena cells involved in the amino acid starvation, the nature of DNA polymerase activity was investigated in crude enzyme preparations or in toluene-permeabilized specimens. In crude enzyme preparations from growing cells, ³H-TTP incorporation into acid-insoluble products showed little dependency on exogenous DNA template, while incorporation increased markedly in the presence of ATP. These characteristics were very similar to those of replicative DNA synthesis in permeabilized Escherichia coli.Variations of DNA and RNA polymerase activities following transfer of exponentially growing Tetrahymena cells to amino acid-deprived medium showed that in the crude enzyme preparations DNA polymerase activity dropped sharply within 3 h after the transfer and practically no activity was detected thereafter, whereas RNA polymerase activity did not disappear in the same preparations. Such enzyme kinetics coincided well with the kinetics of in vivo synthesis of the corresponding nucleic acid.The cessation of DNA synthesis in the amino acid-starved cells may be due not to the activation of DNase or a soluble polymerase inhibitor, nor to the deficiency of each kind of deoxyribonucleoside triphosphate or magnesium ion or ATP generation system. It follows from this that the cessation of DNA polymerase activity in the starved cells may be due to the deficiency of DNA polymerase or its associated factor(s) as a reflection of short life-span of such a protein.     

Purification and Properties of Deoxyribonucleic Acid Binding Factor Isolated from the Surface of Streptococcus sanguis Cells

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S₁ action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.     

Electroporation-mediated transfection of mammalian cells with crude plasmid DNA preparations

We designed a simple and reproducible electroporation-mediated transfection procedure with which to screen mammalian expression vector-constructed cDNA libraries. Using a specific chamber composed of five parallel electrodes, the recipient cells can be electroporated separately with 40 plasmid DNA preparations in a single experiment. Over 300 crude plasmids prepared from E. coli (DH-5) carrying a pcD2neo-vector-derived cDNA library were tested. The efficiency of stable transfection by electroporation with crude plasmid DNA preparations was 10-times higher than with the CsCl-purified plasmid DNA. When the crude plasmids were digested with RNase, the efficiency of stable transfection markedly decreased, indicating that the contaminating bacterial RNA in the crude plasmid preparations has a strong carrier effect during the electroporation. Even when salmon sperm DNA or genomic DNA from the recipient cells was used as the carrier of the purified plasmid, the efficiency was not higher than that using the crude preparations. This procedure is useful not only for screening a number of cDNAs but also for routinely introducing biologically active foreign genes into cultured mammalian cells.     

Inhibition of a nuclease contaminant in the commercial preparations of Escherichia coli alkaline phosphatase.

Escherichia coli alkaline phosphatase is a valuable reagent for removal of terminal phosphate from both ribo-and deoxyribo-oligonucleotides or from restriction enzyme fragments of DNA. Some commercial preparations of this enzyme were found to be contaminated with nucleases which could degrade both DNA and RNA. These contaminating nucleases can be completely eliminated by carrying out the enzymic reaction in the presence of 0.1-1% sodium dodecyl sulfate without any loss of phosphatase activity. This report has immediate application in the sequence analysis of DNA or RNA.     

Plasmid recombination in Haemophilus influenzae

DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded square with one double-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.     

Isolation and structure of phage lambda head-mutant DNA

High molecular weight DNA accumulates in bacteria in which λ is multiplying but cannot complete the formation of new phage particles due to a defect in head assembly. Accumulated λ DNA has been isolated from induced mitomycin C-treated lysogens by means of a shift in buoyant density labels from heavy to light and fractionation by density-gradient sedimentation for completely light DNA. Head formation was blocked in these lysogens by amber mutations in genes D or E, which specify the two major head proteins. The purified DNA is at least 80% λ by DNA-DNA hybridization and some preparations are close to 100% λ by this test.     

Formation and subsequent removal of O6-methylguanine from deoxyribonucleic acid in rat liver and kidney after small doses of dimethylnitrosamine

1. The amounts of 7-methylguanine and O⁶-methylguanine present in the DNA of liver and kidney of rats 4h and 24h after administration of low doses of dimethylnitrosamine were measured. 2. O⁶-Methylguanine was rapidly removed from liver DNA so that less than 15% of the expected amount (on the basis of 7-methylguanine found) was present within 4h after doses of 0.25mg/kg body wt. or less. Within 24h of administration of dimethylnitrosamine at doses of 1mg/kg or below, more than 85% of the expected amount of O⁶-methylguanine was removed. Removal was most efficient (defined in terms of the percentage of the O⁶-methylguanine formed that was subsequently lost within 24h) after doses of 0.25–0.5mg/kg body wt. At doses greater or less than this the removal was less efficient, even though the absolute amount of O⁶-methylguanine lost during 24h increased with the dose of dimethylnitrosamine over the entire range of doses from 0.001 to 20mg/kg body wt. 3. Alkylation of kidney DNA after intraperitoneal injections of 1–50μg of dimethylnitrosamine/kg body wt. occurred at about one-tenth the extent of alkylation of liver DNA. Removal of O⁶-methylguanine from the DNA also took place in the kidney, but was slower than in the liver. 4. After oral administration of these doses of dimethylnitrosamine, the alkylation of kidney DNA was much less than after intraperitoneal administration and represented only 1–2% of that found in the liver. 5. Alkylation of liver and kidney DNA was readily detectable when measured 24h after the final injection in rats that received daily injections of 1μg of [³H]dimethylnitrosamine/kg for 2 or 3 weeks. After 3 weeks, O⁶-methylguanine contents in the liver DNA were about 1% of the 7-methylguanine contents. The amount of 7-methylguanine in the liver DNA was 10 times that in the kidney DNA, but liver O⁶-methylguanine contents were only twice those in the kidney. 6. Extracts able to catalyse the removal of O⁶-methylguanine from alkylated DNA in vitro were isolated from liver and kidney. These extracts did not lead to the loss of 7-methylguanine from DNA. 7. The possible relevance of the formation and removal of O⁶-methylguanine in DNA to the risk of tumour induction by exposure to low concentrations of dimethylnitrosamine is discussed.     

Nucleotide sequences from messenger RNA transcribed from the operator-proximal portion of the tryptophan operon of Escherichia coli

³²P-labeled messenger RNA transcribed in vivo from the operator-proximal portion of the tryptophan operon of Escherichia coli was purified by DNA/RNA hybridization. The mRNA preparations obtained were subjected to polyaerylaamide gel electrophoresis, and a number of discrete labeled bands were detected. Characterization of the labeled bands and of purified, unbanded mRNA preparations, by partial sequence analysis of the oligonucleotides obtained following T₁ and pancreatic RNase digestion, revealed that the bands represented discrete segments of the trp mRNA molecule. This observation suggests that endonucleolytic cleavage occurs in vivo at specific sites in the mRNA molecule.     

The characterization of the proteins which are secreted by the mucocysts of Tetrahymena thermophila

The ‘mucus’ which is discharged by the ciliated protozoan Tetrahymena thermophila has been isolated. All preparations have been shown to consist of a highly consistent mixture of at least 30 proteins varying in molecular weight between 200 000 and 10 000. In marked contrast to mammalian mucins these preparations have a very low (3% w/v) carbohydrate content with glucosamine being the major sugar. The amino acid analyses show that aspartic and glutamic acids and their amides together with serine and glycine account for 45% of the amino acids.     

KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.     

The ribosomal cistrons of the water mold Achlya bisexualis

Total DNA was extracted from vegatative mycelia of the water mold Achlya bisexualis. Fractionation of the DNA in CsCl gradients resulted in two components: a major component with a buoyant density of 1.697 g cm^?3 and a minor component with a density of 1.685 g cm^?3. The minor component has been identified as mitochondrial DNA based on extractions from isolated mitochondria and Triton X-100 washed nuclei. Detergent washing of the nuclei yielded DNA in which the mitochondrial DNA component was absent, while the isolated mitochondrial preparations contained DNA enriched in the 1.685 g cm^?3 component. Hybridization studies of A. bisexualis DNA to rRNA show that the ribosomal cistrons have a buoyant density coincident with that obtained with the nuclear DNA. In addition, preliminary evidence indicates that the mitochondrial DNA does not hybridize to the cytoplasmic RNA under the conditions used for this study. Ribosomal RNA hybridized to about 0.65% of the total DNA.