Daltons Of DNA Research Articles

Radiosensitivities of DNA molecules in lymphocytes from the circulating blood of man

The radiosensitivities of DNA molecules from lymphocytes of human circulating blood were examined by alkaline sucrose gradient centrifugation. The single-strand breaks of DNA per rad, including the breaks formed under the present alkaline condition, were 1.2 plus or minus 0.1 breaks per 10-12 daltons DNA per rad. When the cells were transformed, the number of breaks was found to increase to 1.8 plus or minus 0.2 breaks per 10-12 daltons DNA per rad. The lymphocytes are capable of rejoining radiation-induced single-strand breaks of DNA. The rate of rejoining was dependent upon types of the suspending medium. The rate increased to ten times of that of the non-transformed cells upon transformation.

Radiosensitivities of DNA Molecules in Lymphocytes From the Circulating Blood of Man

Abstract The radiosensitivities of DNA molecules from lymphocytes of human circulating blood were examined by alkaline sucrose gradient centrifugation. The single-strand breaks of DNA per rad, including the breaks formed under the present alkaline condition, were 1.2 plus or minus 0.1 breaks per 10–12 daltons DNA per rad. When the cells were transformed, the number of breaks was found to increase to 1.8 plus or minus 0.2 breaks per 10–12 daltons DNA per rad. The lymphocytes are capable of rejoining radiation-induced single-strand breaks of DNA. The rate of rejoining was dependent upon types of the suspending medium. The rate increased to ten times of that of the non-transformed cells upon transformation.

Investigation of methods for measurement of radioactivity in tritiated DNA and applications to assays for DNA polymerase activity

Methods for collection and counting of 3H-labeled DNA on nitrocellulose and glass fiber filters have been investigated. The findings are of potential importance in determining the amount of radioactivity in DNA as well as other macromolecules. The highest counting efficiencies were observed using glass fiber filters, NCS for dissolving DNA, and a toluene seintillation mixture for counting. However, glass fiber filters, even with large amounts of co-precipitant albumin, failed to collect all of low molecular weight (approx 185,000 daltons) DNA. Thus, in many applications nitrocellulose filters proved to be more advantageous.

The nature of single-strand breaks in DNA following treatment of L-cells with methylating agents

DNA from untreated L-cells had a weight average molecular weight (Mw) of 5.7 ± 0.58·10 8 daltons as measured by sedimentation in an alkaline sucrose gradient. This value was reduced by one half after the cells were treated for 1 h with 8 μg/ml of N-methyl- N-nitrosourea (MNUA), 34 μg/ml of methyl methanesulfonate (MMS) or 0.16 μg/ml of N-methyl- N′-nitro- N-nitrosoguanidine (MNNG). That dose of MNUA produced 52 methylations per 5.7·10 8 daltons DNA. 20% of these were not purine derivatives and were assumed to contain some phosphotriesters. That dose of MMS (above) produced 290 methylations per 5.7·10 8 daltons DNA and about 14% of these were not purine derivatives. The rates of loss of methylated purines from DNA were 2.3% per hour for 7-methylguanine (7-MeG), 7.4% per hour for 3-methyladenine (3-MeA) and no detectable loss of O 6-methylguanine (O 6-MeG) over a 12 h period. Since phosphotriesters are alkali-labile the single-strand breaks probably arose from this structure and did not form within the cell. This conclusion is supported by the following considerations. MNUA was more effective than MMS at reducing the molecular weight of DNA, as measured in alkaline medium. The greater SN 1 character of MNUA would cause a greater formation of phosphotriesters than would MMS.

Clustering of transfer RNA genes of Xenopus laevis

The arrangement of the reiterated DNA sequences complementary to transfer RNA has been studied in Xenopus laevis. Prehybridization of denatured DNA with an excess of unfractionated tRNA results in a small but well-defined increase in the buoyant density of fragments which contain sequences homologous to tRNA. The density increase is smaller than that found for 5 S DNA, but is the same or nearly so for all tRNA coding sequences examined. These results indicate that the majority of tRNA genes are clustered together with spacer DNA, the average size of which is estimated to be approximately 0.5 × 10 6 daltons (native) DNA. In high molecular weight native DNA preparations, the sequences homologous to unfractionated tRNA, tRNA Val, tRNA 1 Met and tRNA 2 Met band in CsCl at 1.707, 1.702, 1.708 and 1.711 g cm −3, respectively. The mean buoyant densities are constant at all molecular weights examined but they do not correspond to the base compositions of the complementary tRNA species. These results indicate that isocoding genes are linked to spacer DNA in separate and extensive gene clusters, and that the different clusters contain different spacer DNA sequences. These clusters form well-defined cryptic DNA satellites which are potentially separable from each other as well as from other chromosomal DNA.

Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis

Euglena chloroplast DNA has a buoyant density in CsCI of 1.686. Shearing this DNA produces a satellite band at density 1.700. The satellite, easily lost during preparative CsCI gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 × 10 6 daltons of DNA.

Endonuclease-sensitive sites in the DNA of irradiated bacteria: A rapid and sensitive assay

The endonucleolytic activity from an extract of Micrococcus luteus is used to detect small amounts of damage in the DNA of bacteria exposed to ultraviolet light or γ rays. The extract is added directly to a lysate of the bacteria, and after a short period of incubation a sample of the bacterial DNA is assayed for endonuclease-sensitive sites by sedimentation through alkaline sucrose gradients. As few as one or two sites per 10 8 daltons of DNA can be detected by this method.

An improved method for measuring cross-links in the DNA of mammalian cells: The effect of nitrogen mustard

This paper describes a technique for quantitatively measuring both the number of interstrand cross-links and the number of strand breaks introduced into the DNA of cells by a drug. The method has been illustrated by studying the effect of nitrogen mustard on the DNA of a murine lymphoma cell (L5178Y). The results have been compared with measurements of the molecular weight of DNA in alkaline sucrose gradients. It was found that treatment of L5178Y cells for one hour at 37 °C with 1 μM nitrogen mustard introduced two cross-links and one strand-break per 10 9 daltons of DNA. The method is also suitable for observing low levels of cross-linking. It is estimated that one cross-link in 2 · 10 9 daltons of DNA can be detected.

Letter: DNA repair: a simple enzymatic assay for human cells.

An assay is described based on the ability of certain endonucleases in Micrococcus luteus extracts to react specifically at sites of DNA damage, and produce single-strand breaks. The breaks were detected by sedimenting the DNA through alkali. DNA repair was assumed to occur if the number of endonuclease sensitive sites decreased during post-treatment incubation of cells. It is claimed that improvements have been obtained in an established technique by adding M. luteus extract to partially disintegrated cells, instead of to isolated purified DNA, enabling samples to be processed in 30 min with most of the manipulations at 0 deg C. As few as one or two endonuclease-sensitive sites per 100 million daltons of DNA could be detected. The potential of the method is demonstrated with results obtained from uv-irradiated human diploid fibroblasts. (UK)

Repeated DNA sequences in a Balbiani ring

Newly synthesized RNA from a short region of chromosome IV of Chironomus tentans salivary gland cells was studied by RNA-DNA hybridization. The region is dominated by a puff, Balbiani ring 2, likely to be involved in the genetic determination of secretory proteins. Hybridization of filter-bound Chironomus tentans DNA by BR-2 † † Abbreviations used: BR, Balbiani ring. RNA was studied in a micro-assay for RNA-DNA hybrids. The proportion of DNA hybridized at saturation was 0.17%, corresponding to 200 × 10 6 daltons of DNA per haploid genome. Cytological hybridization showed that this DNA is mainly located in the BR-2 region. Since the size of BR-2 RNA is about one order of magnitude smaller than the size of BR-2 DNA, several transcription units may be contained in the BR-2 region. The rate of hybrid formation was determined with DNA in excess to measure the number of binding sites for BR-2 RNA. Nucleolar 38 s RNA, known to have about 100 binding sites in the haploid genome, was used for comparison. BR-2 RNA hybridized twice as fast as 38 s RNA, and retained an almost linear reaction rate until 15% of the input RNA had hybridized. This result suggests that the average multiplicity of sequences within BR-2 DNA is about 200, and that a considerable part of BR-2 RNA hybridizes with repeated DNA sequences. Furthermore, the results indicate that BR-2 RNA and its template DNA are internally repeated, because the multiplicity of BR-2 DNA is about one order of magnitude greater than the maximal number of transcription units. Cytological hybridization experiments with RNA from two additional Balbiani rings (BR-1 and BR-3) as well as from BR-2 indicated that the three BR's differ with regard to information content, and that BR-1 like BR-2 contains repeated nucleotide sequences. Thus it is likely that the three BR's code for different secretory proteins.

Unscheduled DNA synthesis at low X-ray doses

Studies on the kinetics of unscheduled DNA synthesis in human lymphocytes following high radiation doses ( Radiation Res., 1969, 39, pp. 400–412) have been extended to the dose range of 600–2400 rad. As in the case of the high doses, the data can be modeled in terms of a single enzyme system. There are, however, differences; at high doses the ratio of radiation-induced substrate (lesions) to enzyme is much greater than 1 and a Michaelis Menten plot of the data is possible. Below 1000 rad, this ratio appears to be less than 1 and consequently the Michaelis Menten plot breaks down. In the dose range of 600–2400 rad, the data are represented in terms of a dose-dependent rate constant. The rate constant increases as the dose decreases from 2400 to about 1000 rad and then reaches a constant value of 3 h −1 which corresponds to a half-life of about 14 min. Under the assumption of a single enzyme system, the high-dose data and low-dose data can be used together to obtain an estimate of the average number of enzymes per lymphocyte. A value of 10–20 thousand enzyme units per cell is obtained. This is equivalent to 1 enzyme unit per 2 to 4·10 8 dalton of DNA. At low doses, the amount of exogenous thymidine (TdR) incorporated as a result of radiation-induced unscheduled DNA synthesis amounts to 6 molecules of TdR/rad/cell at saturated concentration of exogenous TdR (about 5 μM). Coupled with the enzyme model, this amounts to approximately 10–20 lesions per rad or that 1 or 2 nucleotides are incorporated per lesion.

Plus and minus single-stranded DNA separately encapsidated in adeno-associated satellite virions.

Based on physical and chemical determinations, the mnolecular weight of the type 4 adeno-satellite virus is 5.4 X 10(6) daltons, and the virion contains 1.4 X 10(6) daltons of DNA. Denaturation and renaturation studies indicate that the viral genome is a single-stranded DNA molecule and that each virion contains either a minus or a plus strand. Upon extraction, the minus and plus strands unite to form double-stranded DNA molecules with no obvious excess of unpaired strands.

Physical and mapping properties of distant linkages between genetic markers in transformation of Bacillus subtilis.

DNA purified by a procedure based on phenol extraction contained many intact linkages, i.e. between genetic markers showing less than 20% cotransfer. The molecular weight of this DNA, as revealed by zone centrifugation, varied between 4.5×107 and 2.5×108. Linkages with cotransfer frequencies greater than 10% were found to consist of not more than 6.0×107 daltons of DNA, while one linkage showing only 2% cotransfer was provisionally estimated to be about 1.5×108 daltons. With the aim of extending the transformation map of B. subtilis, 16 mutations for nutritional requirements, not suspected—on the basis of the phenotypes involved—to be linked to any other markers, were examined for linkage with each other and to markers on the existing map. Three new pairs of distantly linked markers were found, but no linkage to any location on the known map.

The binding of RNA polymerase to DNA

The amount of RNA polymerase that can bind to DNA is measured by separating the enzyme bound to DNA from unbound enzyme by zone centrifugation. In a solution containing 0.05 m-KCl, 5 m m-MgCl 2 and 0.01 m-Tris buffer (pH 7.9), the maximum amount of Escherichia coli RNA polymerase that can bind to T7 DNA is 1.8 μg of enzyme per μg of DNA, or one polymerase molecule (molecular weight of 8.8 × 10 5) per 5 × 10 5 daltons of DNA. The maximum amount of RNA polymerase that can bind to f1 DNA is 11 μg per μg of DNA. The binding reaction is reversible, does not require magnesium ions, and is sensitive to the ionic strength and pH of the solution. Dinitrophenol (3 × 10 −2 m) and proflavine sulfate (10 μg/ml.) inhibit the reaction, but actinomycin (1 mμmole/ml.) does not. sRNA both charged and uncharged competes directly with DNA for the binding site on the enzyme.