Fluorometric quantitation of single-stranded DNA: a method applicable to the technique of alkaline elution

The use of Hoechst dye 33258 for the fluorometric quantitation of single-stranded DNA was investigated for the purpose of developing a simple nonradiometric method of quantitating DNA in fractions collected during the analysis of DNA damage by the method of alkaline elution. The sensitivity of the assay allowed amounts of single-stranded DNA as small as 100 ng to be quantitated reliably. The requirement of a near-neutral pH necessitated that alkaline samples be buffered in order to perform DNA quantitation. However, that the addition of a predetermined volume of buffered dye solution to each sample is the only manipulation required prior to fluorescence measurement makes this procedure the simplest yet described for quantitating DNA collected during alkaline elution.     

Structure of the single-stranded polyribonucleotide polycytidylic acid.

The Fourier transformation was obtained from the experimental diffuse X-ray scattering curve of adult human oxyhaemoglobin in an aqueous solution. The correlation function of the form of the molecule was calculated from known coordinates of horse haemoglobin atoms. The distribution function of inhomogeneities was deduced from the above Fourier transformation and the correlation function. This distribution function of inhomogeneities is described by a series of maxima providing evidence for a short and long-range order extending up to 5 nm which is close to the maximum dimension of the haemoglobin molecule.     

Integration of Rous sarcoma virus DNA during transfection

We have investigated the organization and integration sites of Rous sarcoma virus (RSV) DNA in NIH 3T3 mouse cells transformed by transfection with unintegrated and integrated donor RSV DNAs. RSV DNAs of different cell lines transformed by unintegrated donor DNA were flanked by different cellular DNA sequences, indicating that RSV DNA integrates at multiple sites during transfection. The RSV genomes of cells transformed by transfection were colinear with unintegrated RSV DNA, except that deletions within the terminal repeat units of RSV DNA were detected in some cell lines. These results suggested that the terminal repeat sequences of RSV DNA did not necessarily provide a specific integration site for viral DNA during transfection. In addition, cell lines transformed by integrated RSV DNAs contained both the RSV genomes and flanking cellular sequences of the parental cell lines, indicating that integration of integrated viral DNA during transfection occurred by recombinational events within flanking cellular DNA sequences rather than at the terminal of viral DNA. Integration of RSV DNA during transfection thus appears to differ from integration of RSV DNA in virus-infected cells, where the terminal repeat units of viral DNA provide a highly specific integration site. Integration of donor DNA during transfection of NIH 3T3 cells instead appears to proceed by a pathway which is nonspecific for both donor and recipient DNA sequences.     

In vitro synthesis of simian virus 40 DNA. II. Evidence for a repair mechanism.

The technique of density labeling of DNA by BrdU was used to characterize the material synthesized in vitro by cytoplasmic extracts of SV40 infected cells incubated in the presence of simian virus 40 (SV40) DNA component I molecules (Girard et al, Biochimie, this volume). In a first experiment, the template was labeled beforehand in vivo using [14C]-BrdU, and the in vitro incubation was carried out in the presence of [3H]-dGTP and [3H]-dTTP. In a second experiment, the template was labeled in vivo with 32P, and the in vitro incubation was in the presence of [3H]-dGTP and BrdUTP. After digestion with the restriction endonuclease Hind II + III, the fragments from the end products of the reaction were analyzed by density gradient centrifugation, at pH 7 and pH 13. In both experiments the DNA product molecules had the same density as the resepctive DNA templates. Cellular enzymes seem to be responsible for this in vitro synthesis of DNA, since cytoplasmic extracts from uninfected cells were almost as active as those from SV40 infected cells. The system was proved efficient in the conversion of "open circular" molecules (component II DNA molecules) to covalently closed circular DNA molecules (relaxed component I molecules). The use of DNA complexed with histones did not impart viral specificity to the system. It is concluded that the cytoplasmic extract is only capable of supporting the repair synthesis of added viral DNA.     

Characterization of gastric mucosal membranes. VI. The presence of channel-forming substances

Highly purified gastric membranes were extracted by ionic or non-ionic detergents. Further fractionation was carried out on acrylamide gel electrophoresis. Some defined bands, incorporated into artificial bilayer membranes, produced discrete conductance changes characteristics of channel activity. A low molecular weight peak, purified through two cycles of electrophoresis was characterized as an anion-selective, positively voltage dependent channel, and a model to account for the action of the transport ATPase incoporating this channel concept is suggested.     

The vasoactive intestinal peptide (VIP): VI. The 17-norleucine analog of the sequence 14–28

A pentadecapeptide amide with the C-terminal sequence (14–28) of the vasoactive intestinal peptide (VIP), but with the methionine residue in position 17 replaced by l-norleucine, was synthesized. The synthesis was carried out through stepwise chain lengthening, by the in situ technique. The norleucine-containing pentadecapeptide, l-arginyl-l-lysyl-l-glutaminyl-l-norleucyl-l-alanyl-l-valyl-l-lysyl-l-lysyl-l-tyrosyl- l-leucyl-l-asparaginyl-l-seryl-l-isoleucyl-l-leucyl-l-asparaginamide, was as active in relaxing different smooth-muscle preparations as the methionine-containing parent sequence.     

In vitro synthesis of simian virus 40 DNA. I. Synthesis by a soluble extract from infected CV1 cells.

Simian Virus 40 (SV40) DNA replication was studied in vitro using cell free extracts prepared from SV40 infected CV1 cells. The cells were fractionated into a soluble cytoplasmic fraction and nuclei. The nuclei were lysed with high salt and used to prepare a soluble nuclear fraction. Both fractions displayed DNA polymerase activity as measured with activated calf thymus DNA. However, only the cytoplasmic fraction was active when SV40 DNA comonent I molecules were used as template. Under these conditions, the cytoplasmic extract was shown to catalyse the SV40 DNA dependent, in vitro incorporation of the four deoxyribonucleotides into DNA molecules which had, at both neutral and alkaline pH, the same sedimentation behavior as authentic SV40 DNA component I and component II molecules. Optimal Mg++ concentration was 5-8 mM. Incorporation of label into DNA component I molecules showed an initial lag of about 15 min., after which it was linear with time for up to 5 hrs at 32 degrees. Incorporation into DNA component II molecules proceeded without obvious lag and reached a plateau after approximately 2 hrs of incubation. It is concluded that the cytoplasmic extract supports the in vitro synthesis of SV40 DNA and that DNA component II molecules appear to be a precursor to DNA component I molecules in the reaction. Labeling of viral DNA molecules was highly dependent on ATP and on an ATP generating system. In the absence of ATP and of the energy generating system, incorporation occurred but both template and newly synthesized DNA molecules were extensively degraded.     

Infection of macrophages with Friend virus: relationship to the spontaneous regression of viral erythroleukemia.

Resident peritoneal macrophages from normal mice were refractory to infection with the RFV or conventional strains of Friend virus (FV). Stimulation of DNA synthesis in the macrophage population by induction of an exudate in vivo or treatment in vitro with macrophage colony-stimulating factor resulted in productive infection following exposure to virus. Similarly, normal resident macrophages did not become infected in vivo following transfer to leukemic mice, while exudate macrophages did become infected. Bone marrow macrophage stem cells were stimulated to replicate and mature in clonal agar cultures in the presence of colony-stimulating factor. These replicating stem cells could be infected with RFV, as shown by virus production in the resultant progeny macrophages. Transfer of normal resident peritoneal macrophages to leukemic progressor mice caused regression of the disease. In contrast, transfer of normal bone marrow cells was ineffective in causing leukemia regression. During erythroleukemogenesis induced by RFV, macrophage precursor cells in all of the mice became infected with virus. In mice with a progressive and lethal leukemia, mature end-stage macrophages were produced which were also infected with virus. In mice in which the leukemia would later spontaneously regress, the infected stem cells were eliminated and the marrow became repopulated with uninfected cells. The resultant progeny macrophages which appeared in the peritoneal cavity were uninfected and thus capable of participating in or causing leukemia regression.     

Polyamines stimulate natural RNA-directed DNA synthesis by Rauscher murine leukemia virus DNA polymerase

In the presence of optimal concentrations of Mg²⁺, spermine and spermidine were found to stimulate rabbit globin mRNA-directed cDNA synthesis by Rauscher murine leukemia virus (R-MuLV) DNA polymerase. Stimulation of DNA synthesis did not occur with the polyamines putrescine or cadaverine, nor could exogenously provided salt or ammonium ions duplicate the stimulation. Analysis of the mechanism of stimulation showed that inclusion of spermine in reaction mixtures a) increased Vmax and decreased apparent Km with respect to the globin mRNA-oligo(dT) tem?late-primer complex, and b) decreased the quantity of oligo (dT) required for optimal rates of cDNA synthesis on a fixed quantity of mRNA template. Genomic 70S RNA-directed cDNA synthesis was also stimulated by spermine addition to reaction mixtures, but only at supra-optimal RNA concentrations. Our results suggest that stimulation of R-MuLV DNA polymerase activity by polyamines is primarily due to stabilization of the enzyme-templateprimer initiation complex resulting in increased efficiency of initiation of cDNA synthesis.     

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.     

Identification of a protein linked to the ends of adenovirus DNA.

A DNA-protein complex from human adenoviruses has been further characterized by electron microscopy, radiochemical labeling and analytical ultracentrifugation. Preparations of the complex contain a large percentage of forms of DNA which are either circular or oligomeric and are readily distinguishable from preparations of pronase-treated adenovirus DNA by analytical ultracentrifugation in cesium chloride gradients containing 4 M guanidinium chloride. The protein component has been iodinated in vitro with ¹²⁵I using Bolton and Hunter reagent, and SDS-polyacrylamide gel analysis of the labeled protein indicates that it has an apparent molecular weight of 55,000 daltons. DNAase I digestion of the DNA-protein complex labeled with ³²PO₄ results in release of a ³²PO₄-labeled protein which remains labeled even after boiling in 1% SDS and 1% mercaptoethanol. Subsequent digestion of this entity with snake venom phosphodiesterase leads to release of ³²P₄-labeled 5′-phosphate deoxynucleotides. Digestion of the DNA-protein complex with Eco R1 and analysis of the isolated restriction fragments indicates that the protein is present on each terminal fragment. We conclude that there is a protein of 55,000 daltons directly attached to each 5′ end of molecule probably via a covalent linkage. We propose that the protein functions during DNA replication by facilitating priming of the progeny strands, thus allowing the 5′ ends of the DNA to be replicated.     

Primary structure of the southern bean mosaic virus coat protein: First results

Studies on the southern bean mosaic virus coat protein have established the molecular weight of this protein, its amino acid composition, the nature of its C-terminal amino acid, and the blockage of the N-terminal residue by an acetyl group. After hydrolysis of the protein by trypsin, the hydrolysate was fractionated by ion-exchange chromatography. Among the purified tryptic peptides were isolated the N- and the C-terminal peptides where sequences were determined, principally by mass spectrometry.     

DNA recognition and cleavage by the EcoP15 restriction endonuclease.

EcoP15 is a restriction-modification enzyme coded by the P15 plasmid of Escherichia coli. We have determined the sites recognized by this enzyme on pBR322 and simian virus 40 DNA. The enzyme recognizes the sequence:

In restriction, the enzyme cleaves the DNA 25 to 26 base-pairs 3′ to this sequence to leave single-stranded 5′ protrusions two bases long.