A radiometric method for developing the alkaline sucrose gradient sedimentation patterns of DNA from nondividing cells.

A radiometric method for developing the alkaline sucrose gradient sedimentation patterns of DNA from nonlabeled cells is described. The principle of the method is the labeling of the DNA contained in the gradient fractions by means of the binding of a labeled amino acid to DNA in the presence of formaldehyde. The procedure involves incubation of the fractions with the labeling reagent, filtration of the incubation mixtures through nitrocellulose filters, and radiometry of the filters. The relationship between the radioactivity on the filters and the DNA concentration in the sample is linear; the DNA detection sensitivity is sufficient to escape overloading of the gradients. It was shown that the nonlabeled mammalian cell DNA sedimentation patterns developed by the method described and those of DNA from the same cells labeled with [³H]thymidine in vivo are identical.     

Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

The separation of DNA segments attached to proteins.

A simple assay for DNA segments bearing tightly bound proteins is described. This assay depends on the observation that proteins, of any type tested, bind quantitatively to glass fiber filters. When a protein is firmly attached to DNA, this DNA segment is retained while DNA not associated with protein will pass through the filter. Depending on the preparation of DNA, backgrounds as low as 3 × 10^?4 of the input DNA have been obtained. Using this technique it should be possible to specifically recover 1 restriction segment in 3000 that happens to be firmly bound to a protein. The protein or DNA-protein complex can be released by very dilute sodium dodecyl sulfate and after its removal by dialysis, nearly complete rebinding can be achieved. The procedure should find some use in removing traces of protein from DNA solutions as well as for the determination of proteins themselves. Single chain DNA and RNA are not retained but backgrounds are higher, ca. 2 × 10^?2. The procedure should have some application to single chain DNA and RNA-protein complexes.     

Commercial detection methods for biotinylated gene probes: Comparison with32P-labeled DNA probes

This study had two objectives: (a) to determine whether biotinylated DNA probes could be substituted for³²P-labeled DNA probes to detect the presence of the TEM-1 -lactamase gene in crude bacterial preparations, and (b) to evaluate two commercial detection systems for biotinylated probes—an alkaline phosphatase kit produced by Bethesda Research Laboratories (BRL) and an acid phosphatase kit produced by Enzo Biochem. Both the kits produced nonspecific reactions with TEM-1-negative organisms. Treatment with chloroformphenol and proteinase K did not remove these nonspecific reactions. When plasmid DNA was purified by electrophoresis and transferred to nitrocellulose filters by the Southern blot method, there was no qualitative difference between the biotinylated and radioactive probes. However, the³²P-labeled probes were quantitatively 100 times more sensitive than the biotinylated probes. In addition, the Enzo Biochem kit and the³²P-labeled probes could be used with charged nylon membranes, whereas the BRL kit could be used only with nitrocellulose filters.     

High-affinity microtubule protein-higher organism DNA complexes. Many-fold enrichment in repetitive mouse DNA sequences comprised of satellite DNAs

We have examined aspects of the interaction of cycled microtubule protein preparations with ³⁵S-labeled mouse DNA tracer in a competition system with unlabelled competitor E. coli or mouse DNA. The nitrocellulose filter binding assay was used to measure interaction by scintillation counting. DNA molecular weight affected the levels of filter retained ³⁵S-labelled mouse tracer DNA. Filter retention levels increased if ³⁵S-labelled mouse DNA tracer size was increased, and the filter binding level decreased if competitor DNA size was increased. There was a sizeable, reproducible difference in the ³⁵S-labelled mouse DNA tracer binding level of about 1% when E. coli or mouse DNA competitors were compared. Mouse DNA more effectively competed with ³⁵S-labelled mouse DNA for microtubule protein binding than did E. coli DNA, suggesting that a small class of higher-organism DNA sequences interacts very strongly with microtubule protein. From other studies we know this to be the MAP fraction (Marx, K.A. and Denial, T. (1984) in The Molecular Basis of Cancer (Rein, R., ed.), Alan R. Liss, New York, in the press; and Villasante, E., Corces, V.G., Manso-Martinez, R. and Avila, J. (1981) Nucleic Acids Res. 9, 895–908). We find that this difference in competitor DNA strength is qualitatively similar under high-stringency conditions (0.5 M NaCl, high competitor [DNA]) we developed for examining high-affinity complexes. Under high-stringency conditions we isolated 1.2% and 0.6% of ³⁵S-labelled mouse DNA at 4200 and 350 bp respective sizes as nitrocellulose filter bound DNA-protein complexes. At both molecular weights these high-affinity DNA sequences, isolated from the filters, were shown to be significantly enriched in repetitive DNA sequences by S₁ nuclease solution reassociation kinetics. The kinetics are consistent with about a 4-fold mouse satellite DNA enrichment as well as enrichment in other repetitious DNA sequence classes. The high molecular weight filter-bound DNA samples were sedimented to equilibrium in CsCl buoyant density gradients and found to contain primarily mouse satellite DNA density sequences (1.691 g/cm³) with some minor fractions at other density positions (1.670, 1.682, 1.705, 1.740, 1.760 g/cm³) similar to those observed by our laboratory in previous investigations of micrococcal nuclease-resistant chromatin (Marx, K.A. (1977) Biochem. Biophys. Res. Commun. 78, 777–784). That the high-affinity microtubule-bound DNA was some 3–5-fold enriched in mouse satellite sequences was demonstrated by its characteristic BstNI restriction enzyme cleavage pattern     

New Instruments for High Throughput Receptor Binding Assays

Abstract

The TopCount^(R) Microplate Scintillation Counter and the Matrix 9600^(R) Direct Beta Counter are microplate compatible instruments developed to meet the needs of investigators using radioisotope assays adapted for very high throughput. This paper describes these instruments and their application to receptor binding assays. When combined with the appropriate sample handling equipment and filter media, use of these multi-detector instruments improves sample handling efficiency and shortens overall counting time. The assay protocols including filtration through glass fiber mats and membrane filters have been investigated. Results obtained from these new instruments are compared to standard techniques using conventional liquid scintillation and gamma counting.     

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.     

An improved method for detecting foreign DNA in plasmids of Escherichia coli.

A new procedure has been developed for lysing bacterial colonies on nitrocellulose filters and immobilizing the released DNA on the filters. The procedure involves the use of lysozyme and Triton X-100. When used in conjunction with in situ hybridization, this method has proven effective in detecting DNA recombinants, while eliminating the problems of false positives and variation between duplicate filters that are seen with other methods.     

Recovery of DNA segments from agarose gels

After electrophoresis, DNA can be efficiently recovered by solubilization of agarose gels with NaClO₄, followed by retention of DNA on glass fiber filters. After removal of the NaClO₄ by ethanol, the DNA can be extracted with a low salt buffer.     

Isolation and visualisation of alkali stable protein/DNA complexes

Distinct polypeptides, 54,000–68,000 daltons in size, are alkali-stably bound to eukaryotic DNA. DNA fragments several hundred base pairs in length associated with these polypeptides are preferentially retained on glass fibre filters from solutions containing 1 M sodium chloride. About 50 percent of the protein/DNA complexes present in total DNA are retained on filters together with about 2 percent of the DNA. This preferential binding is demonstrated (a) by the ratio of ³H and ³⁵S radioactivity retained on filters after filtration of DNA from [³H]thymidine and L-[³⁵S]methionine labelled cells, (b) radioiodination of the material retained on filters and passing filters respectively and (c) by electron microscopical visualisation of the polypeptide component in the complexes after chemical modification with dinitrofluorobenzene (DNFB) followed by incubation with dinitrophenyl (DNP) specific antibodies.     

Identification of human satellite DNA sequences associated with chemically resistant nonhistone polypeptide adducts

A fraction of DNA fragments of highly purified and completely unfolded eukaryotic DNA inevitably remains associated with chemically resistant nonhistone DNA-polypeptide complexes. This fraction can be isolated by nitrocellulose filtration because the polypeptide-associated DNA fragments are retained on nitrocellulose filters while bulk DNA passes through the filters. The fraction of AluI-fragmented DNA from human placenta retained on filters as a result of the binding factors (R-DNA, 12%) represents a subset of genomic sequences with a sequence complexity different from unfractionated DNA and DNA recovered in the filtrate (F-DNA). DNA sequences prevalent in the retained fraction were detected by differential plaque hybridization of a recombinant gt10 library with radiolabeled F- and R-DNA fractions. Several recombinant phages showing much stronger hybridization signals with the R-DNA probe than with the F-DNA probe were selected, plaque-purified and analyzed. Analysis of the inserts of such clones showed that repetitive DNA sequences of the alphoid dimeric and tetrameric family, satellite III and satellite III-like sequences are highly enriched in the retained fraction, which indicates that these sequences specifically attract the polypeptides involved in the tightly bound and resistant complexes. This property of repetitive sequences is of interest since tandemly repetitive sequences have been suggested to code for locus-specific fixation and stabilization of the chromatin fiber in the cell nucleus.by L. ManuelidisThis work contains parts of the Ph.D. thesis of M.P. (University of Giessen).     

Hybridization method for quantitation of replicating polyoma DNA sequences.

Polyoma DNA was cleaved with restriction endonuclease HpaII, the fragments were separated by gel electrophoresis and transferred in good yield to separate nitrocellulose filters by a modification of the procedure of E. M. Southern (1975, J. Mol. Biol.98, 503–517). The filters were then used in hybridization experiments to localize the isotope in different parts of the polyoma genome after in vitro incorporation of labeled deoxyribonucleoside triphosphates into the DNA.     

High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM^–1 E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.     

Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of ³H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated ³H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA.     

Mitochondrial DNA copy number in bovine oocytes and somatic cells

Restriction endonuclease analysis and direct nucleotide sequencing of bovine mitochondrial DNA have revealed a high apparent rate of sequence divergence between maternally related individuals. One possible mechanism that would account for the high rate involves nonuniform amplification and/or segregation of mitochondrial DNA during development of the oocyte. We report here experiments which quantitate the amount of mitochondrial DNA in the bovine oocyte as compared to bovine somatic cells. Total DNA was isolated from purified oocytes, separated by agarose gel electrophoresis, and immobilized on nitrocellulose filters. Hybridization with the complete mitochondrial DNA genome or cloned mitochondrial DNA restriction fragments revealed a 100-fold increase in oocyte mitochondrial DNA as compared to somatic cells. Developing oocytes contained about 4.5 pg or 2.6 × 10⁵ copies per cell, whereas primary bovine tissue culture cells contained 0.045 pg or 2.6 × 10³ copies per cell. These experiments demonstrate directly the amplification of mitochondrial DNA in mammalian oocytes and are consistent with models which could generate mitochondrial DNA polymorphisms by unequal amplification of mitochondrial genomes within an animal.     

Organization of human δ- and β-globin genes in cellular DNA and the presence of intragenic inserts

We have analyzed human cellular DNA for its δ- and β-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with ³²P-β-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3′ end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the δ and β genes and those containing sequences flanking either 5′ to the δ gene or 3′ to the β gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, δ- and β-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.     

Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays

A several-fold greater counting efficiency is observed for protein labeled with [³H]leucine than for free [³H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for ¹⁴C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.     

Hydroxyapatite-mediated transfer of DNA from diluted solutions to nitrocellulose or nylon membranes

Transfer of DNA (from 0.1 to 10 micrograms) from diluted solutions of variable volumes (1-10 ml) and various composition (2 M NaCl; 4 M LiCl, 8 M urea; 4 M CsCl; 20% sucrose) to nitrocellulose or nylon membranes was achieved with the use of hydroxyapatite. This absorbent that binds nucleic acids effectively and independently of ionic strength and composition of solution (except for chelators and phosphate ions) easily dissolves in small volumes of acids (for example, in 10% TCA). This phenomenon provides the opportunity to deliver the acid-insoluble precipitates to membrane filters. After alkaline denaturation on the filter followed by a fixation step (baking or UV irradiation for nitrocellulose or nylon filters, respectively), DNA hybridizes effectively with nick-translated DNA probes. The method is simple, reproducible, sensitive, and useful for working with diluted DNA solutions containing interfering substances.     

In situ DNA-RNA hybridization using in vivo bromodeoxyuridine-labeled DNA probe

Summary An in vivo 5-bromodeoxyuridine (BrdUrd) labeled DNA probe was used for in situ DNA-RNA hybridization. BrdUrd was incorporated into plasmid DNA by inoculating E. coli with Luria-Bertani (LB) culture medium containing 500 mg/L of BrdUrd. After purification of the plasmid DNA, specific probes of the defined DNA fragments, which contained the cloned insert and short stretches of the vector DNA, were generated by restriction endonuclease. The enzymatic digestion pattern of the BrdUrd-labeled plasmid DNA was the same as that of the non-labeled one. BrdUrd was incorporated in 15%–20% of the total DNA, that is, about 80% of the thymidine was replaced by BrdUrd. Picogram amounts of the BrdUrd-labeled DNA probe itself and the target DNA were detectable on nitrocellulose filters in dot-blot spot and hybridization experiments using a peroxidase/diaminobenzidine combination. The BrdUrd-labeled DNA probe was efficiently hybridized with both single stranded DNA on nitrocellulose filters and cellular mRNA in in situ hybridization experiments. Through the reaction with BrdUrd in single stranded tails, hybridized probes were clearly detectable with fluorescent microscopy using a FITC-conjugated monoclonal anti-BrdUrd antibody. The in vivo labeling method did not require nick translation steps or in vitro DNA polymerase reactions. Sensitive, stable and efficient DNA probes were easily obtainable with this method.     

Using different sorbents for the concentration of enteroviruses

Comparative investigations were carried out to evaluate the efficiency of concentration (EC) of enteroviruses (poliovirus type 1 and simian rotavirus SA-11) using macroporous glass (brands MPG-1000 VGKh, USSR, and SO1, Czechoslovakia) and membrane filters (MF): (nitrocellulose PNTs 0.45, USSR, Millipore HAWP 0.45, USA, Synpor 0.45, Czechoslovakia as well as polycapromide MF Pall 0.2, FRG, and FMPA 0.55, USSR). To assess the sorption properties of filters, poliovirus preparations and rabbit serum gamma-globulin were labelled with radioactive isotopes. Nitrocellulose filters (Millipore and PNTs) proved to be superior in providing for 64-90% virus sorption and 20-24% protein sorption. Elution experiments using solutions of different chemical nature--protein solutions (triptosophosphate broth and beef extract), amino acid mixture (glycine + arginine), chaotropic salt (sodium trichloroacetate mixed with lysine)--showed that protein solutions better eluted rotavirus SA-11 from nitrocellulose filters and macroporous glass (MPG). The utilization of nitrocellulose filters and MPG as sorbents enables a 10-40-fold concentration of enteroviruses depending on the chosen eluent. Comparisons of EC values for rotavirus SA-11 obtained using porous glass MPS-1000 VGKh and SO1 indicated that MPS-1000 VGKh was superior both with respect to recovery efficiency (R) and concentration factor (CF).