William Meinke

Rapid isolation of mouse DNA from cells in tissue culture.

A rapid method for the isolation of DNA from mouse cells grown in tissue culture is described. Cells are lysed in 0.24 m sodium phosphate, pH 6.8 buffer, containing 1% sodium dodecyl sulfate, 8 m urea, and 10−3m ethylene-diaminetetraacetic acid, and the crude lysate applied to a column of hydroxyapatite. RNA and proteins are removed from the column with 0.24 m sodium phosphate buffer containing 8 m urea while DNA is selectively eluted with 0.48 m sodium phosphate buffer. There is almost total recovery of cellular DNA from the column and the DNA is virtually free from proteins and RNA. This extraction procedure indicates that a mouse cell contains about 8 × 10−6 μg of DNA.

Physical properties of cytoplasmic membrane-associated DNA

Abstract Some of the physical properties of a cytoplasmic membrane-associated DNA isolated from a diploid human lymphocyte cell line have been examined. Cytoplasmic membrane-associated DNA extracted from lymphocytes labeled with either [3H]or [14C]thymidine had a specific activity lower than nuclear DNA extracted from the same cells. Analysis of cytoplasmic membrane-associated DNA in the electron microscope shows that the molecules are linear and have a mean length of 1·75 μm; the average sedimentation coefficient of this DNA is 16·6 S, which corresponds to a molecular weight of 4·2×106. Cytoplasmic membrane-associated and nuclear DNA band at identical positions in both neutral and alkaline CsCl gradients with buoyant densities of 1·699 g/ml and 1·752 g/ml, respectively. Native cytoplasmic membrane-associated DNA is double-stranded and has a mole fraction of guanine plus cytosine of 40± l %. Sheared, denatured cytoplasmic membrane-associated DNA reassociates as two distinct fractions whose rates of reassociation differ by about four decades: the complexity of the reassociation of this DNA tends to rule out the possibility that it arises from either mycoplasmal or viral contamination of our cell cultures. The slowly reassociating fraction of cytoplasmic membrane-associated DNA reassociates about ten times faster than the unique sequences of nuclear DNA. This could represent potential genetic information for about 100,000 diverse genes of 1000 nucleotide pairs each. At present the function of cytoplasmic membrane-associated DNA in these cells is unknown.

Simian virus 40‐related DNA sequences in a human brain tumor

Papovaviruses can induce experimental brain neoplasms in animals, and some papovaviruses have been implicated in the formation of various human tumors. We examined a series of seven human brain tumors removed at craniotomy for the presence of viral DNA sequences by the technique of DNA-DNA hybridization. Simian virus 40 (SV40) DNA was labeled in vitro and used as a “probe” for detecting related DNA sequences in cellular DNA extracted from brain tumors. SV40-related DNA sequences were found in DNA extracted from one tumor, a glioblastoma multiforme. It was calculated that approximately 1.2 viral genome equivalents per diploid cell were present in the tumor. Since the rate of reassociation of the probe deviated from ideal second-order kinetics, it is surmised that either only a portion of the SV40 genome was present in tumor cells or, alternatively, that the probe detected a related human papovavirus.

Studies on the structure and formation of polyoma DNA replicative intermediates.

Abstract Circular replicative forms of polyoma DNA with two branch points and three branches have been found in electron microscope studies. These replicative forms were isolated from material sedimenting between 18 s and 25 s in neutral CsCl density gradients. Experiments employing short pulse-labeling periods with [ 3 H]thymidine indicated that a large portion of the polyoma DNA replicative intermediates which labeled rapidly, sedimented as a heterogeneous 25 s component. The 25 s component had a buoyant density identical to DNA extracted from polyoma virus and hybridized to polyoma viral DNA with the same efficiency as did the reference supercoiled polyoma DNA. Kinetic analysis of polyoma DNA replication and pulse-chase experiments revealed that a fraction of the 25 s DNA consisted of stable forms. The position of banding of the 25 s DNA in CsCl-ethidium bromide equilibrium density gradients and electron microscope studies showed that some 25 s DNA molecules were catenated dimers. Most of the catenated dimers were composed of two linked open circular monomer units. Some of the catenated dimers were open circular monomers linked to supercoiled monomers. Additionally, open circular and supercoiled dimers of polyoma DNA were seen. A model is discussed for the formation of progeny supercoiled polyoma DNA molecules and for the formation of catenated and open circular dimers of polyoma DNA by a replication mechanism.

Studies on Bacillus subtilis bacteriophage φ15

Abstract The growth characteristics and physicochemical properties of Bacillus subtilis bacteriophage φ15 have been investigated and compared with φ29. Differences in growth characteristics of these phages were found in various stages of spore germination and outgrowth which may account for the absence of plaque formation by φ15 on wild-type cells. Both φ15 and φ29 genomes were incorporated into spores when cells were infected at a certain critical time during host cell development. φ15 is morphologically indistinguishable from φ29. The DNA of φ15 is a linear duplex molecule having a mean length of 6.1 μm. The S value of this DNA is 23 ± 0.4 S. These values correspond to a molecular weight of approximately 12 × 106 daltons. At least seven structural proteins of φ15 were recognized in SDS-acrylamide gel electrophoresis. The sum of the molecular weights of φ15 proteins is approximately 354,000 daltons. This corresponds to about 59% of the coding capacity of double-stranded DNA of φ15. φ15 DNA was found to hybridize with φ29 DNA.

Reassociation and dissociation of cytoplasmic membrane-associated DNA.

Abstract The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA ( C 0 t −3 ) appears to consist of sequences derived from a single “family”. About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a C 0 t 1 2 value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.

Nucleotide Sequence of Bovine Papillomavirus Type 2 Late Region

The late region of bovine papillomavirus type 2 (BPV-2) DNA has been identified. The complete nucleotide sequence of the region was determined and revealed two large open reading frames. The DNA sequence results and the predicted amino acid sequence of putative polypeptides encoded by this region are presented. Comparative analysis of the BPV-2 late region and the corresponding area of BPV-1 was performed. This study demonstrates that identical genetic organization and considerable nucleotide sequence conservation exists between these two serotypes.

Further Characterization of Herpes Virus Persistence

Infection of cell cultures with herpes simples virus type 1 (HSV-1) under standard culture conditions yielded persistently infected cells capable of continued growth in the presence of virus and of forming colonies in agarose. The ability of an infected culture to yield cells able to survive HSV-1 infection was directly related to the presence of S phase cells (cells actively engaged in DNA synthesis) at the time of infection. Only when very high multiplicities of infecting virus (greater than 10) were used did cultures fail to yield persistently infected cells capable of colonial growth in agarose. Cell clones derived from colonies grown in agarose established cell cultures which possessed all the characteristics of HSV-1 persistently infected cultures. When cultures were cloned a second time in agarose, as a rare event, cell cultures which did not immediately liberate infectious virus could be isolated. These cell cultures, however, possessed an increased resistance to superinfection by HSV-1. On continued cultivation they reverted to persistence as exhibited by the sudden onset of virus cytopathic effects and release of infectious virus.

Differential effects of metabolic inhibitors on cytoplasmic membrane associated and nuclear DNA

Abstract The cellular functions necessary for transport of cytoplasmic membrane associated DNA from nucleus to cytoplasm have been investigated utilizing inhibitors of macromolecular synthesis. Hydroxyurea, fluorodeoxyuridine, cytosine arabinoside, and ethidium bromide did not prevent transport of cytoplasmic membrane associated DNA to the cytoplasm. In contrast, rifampicin and N-demethyl rifampicin totally inhibited the appearance of newly synthesized DNA on cytoplasmic membranes, while dimethyl-benzyl-demethyl rifampicin was partially inhibitory.