DNA filter elution: A window on DNA damage in mammalian cells

This personal account traces a series of studies that led from DNA physical chemistry to anticancer drug mechanisms. Chemical crosslinking as a basis for anticancer drug actions had been suspected since the time of the first clinical reports of the effectiveness of nitrogen mustard in 1946. After the elucidation of the DNA helix-coil transition, several nearly concurrent findings in the early 1960s established the paradigm of DNA interstrand crosslinking. The DNA filter elution phenomenon was discovered in the early 1970s, and lent itself to the development of practical assays for DNA crosslinks and other DNA lesions in mammalian cells. The assays allowed studies of the effects of DNA damaging agents at pharmacologically or toxicologically relevant doses, and have been widely applied in studies of mutagenic and chemotherapeutic agents. During the period 1979–1986, DNA filter elution studies led to the paradigm of DNA topoisomerases as targets of anticancer drug action, and this has become one of the most active areas of anticancer drug development.     

A simple method for detecting drug effects on the DNA of mammalian cells

A simple method for detecting drug or X-ray effects on the DNA of intact cells is described. This method uses an easily constructed viscometer to measure changes in the viscosity of alkaline cell lysates. The method has been tested by comparing the effects of 4′-[(9-acridinyl)amino]methanesulfon-o-anisidide (oAMSA), 4′-[(9-acridinyl)amino]methanesulfon-m-anisidide (mAMSA), daunorubicin, and adriamycin on cellular DNA.     

A method for enucleating cultured mammalian cells

A method is described for enucleating cells which normally could not be enucleated due to their poor adhesion to the growth surface. The technique consists of linking ConA to the surface and then applying the cells. This results in cell adhesion firm enough to withstand the centrifugal forces necessary to enucleate. The method has been applied to fibroblastic, epithelioid and lymphoid cell lines.     

A simplified in situ solubilization procedure for the determination of DNA and cell number in tissue cultured mammalian cells

A rapid and simple procedure for the direct solubilization of DNA from adherent cultured cells, or sedimented cells, has been developed and employed in the measurement of DNA in small numbers of endothelial cells (1 X 10(3).     

A model for the spatio-temporal organization of DNA replication in mammalian cells

The spatio-temporal organization of chromosomal DNA replication was analyzed using a model based on a "DNA unit" (or decondensation unit) hypothesis. The model is an extension of the fork movement theory of Huberman & Riggs (1968) and can account for a partially deterministic and partially stochastic order of DNA replication in chromosomes. It presumes that each chromosome is composed of DNA units that are arranged in sequence and that are replicated in parallel. A deterministic wave of chromatin decondensation propagates along the DNA unit continuously and progressively providing a field for the random activation of replication origin. Assignment of replication times to DNA compartments by a Monte Carlo method was programmed based on the model and the program was used to stimulate DNA synthesis rate curves that can be measured by the method of Dolbeare et al. (1983, 1985). The shape of the curve is shown to constrain possible parameter values of the model, which include the rate of fork movement, the fraction of chromatin that is decondensed at the start of S-phase, the initial number of origins activated, the rate at which new origins are activated, etc. The chromosomal organization that controls the molecular level of DNA replication is briefly reviewed and its relevance to the model is also discussed.     

A new method for the in vitro determination of the bile tolerance of potentially probiotic lactobacilli

Applied Microbiology and Biotechnology - A new in vitro method was developed to determine the bile tolerance of potentially probiotic lactobacilli. The overnight culture of various lactobacilli...     

Improved method to prepare RNA-free DNA from mammalian cells

To isolate DNA for nucleoside analog incorporation studies, many investigators use RNase A to remove RNA from total cellular nucleic acid. We observed persistence of ribonucleotides from RNA in nucleic acid samples treated with RNase A alone. Although incubation of [5-³H]uridine-labeled nucleic acid with 50 μg/ml RNase A decreased tritium by 97%, HPLC analysis of the resulting DNA preparation digested to nucleosides revealed high levels of ribonucleosides. Increasing RNase A 10-fold (500 μg/ml) effected only a 1.7-fold reduction in ribonucleosides. Overall, the level of ribonucleosides was one-fourth that of the deoxynucleosides, primarily due to the high levels of guanosine. It was hypothesized that the ribonucleosides originated from guanosine-rich tracts of RNA since RNase A cuts preferentially 3′ to pyrimidine monophosphates and to some extent after AMP. The addition of 0.05 μg/ml RNase T1, which preferentially cleaves RNA 3′ to GMP, decreased total ribonucleosides by nearly 20-fold. In conclusion, we have developed a rapid method which removes greater then 99% of cellular RNA from nucleic acid extracts and a reversed-phase HPLC procedure that detects RNA contamination more sensitively than [5-³H]uridine labeling. These methods are useful for the determination of analog incorporation into DNA, especially for agents which incorporate into both DNA and RNA.     

A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei.

Tissue-specific patterns of methylated deoxycytidine residues in the mammalian genome are preserved by postreplicative methylation of newly synthesized DNA. DNA methyltransferase (MTase) is here shown to associate with replication foci during S phase but to display a diffuse nucleoplasmic distribution in non-S phase cells. Analysis of DNA MTase-beta-galactosidase fusion proteins has shown that association with replication foci is mediated by a novel targeting sequence located near the N-terminus of DNA MTase. This sequence has the properties expected of a targeting sequence in that it is not required for enzymatic activity, prevents proper targeting when deleted, and, when fused to beta-galactosidase, causes the fusion protein to associate with replication foci in a cell cycle-dependent manner.     

A non-radioactive automated method for DNA sequence determination

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.     

A single-reaction method for DNA sequence determination.

A chemical method for the determination of DNA sequence is presented. Heating of DNA, labeled at a single 3' extremity, in the presence of formamide results in efficient cleavage of phosphodiester bonds 3' of A, G, and C residues. The relative efficiency is A = G greater than C. The bias between A and G is solved by a simple pretreatment (photoreaction in methylene blue) followed by heating in formamide. The entire procedure does not require any intermediate purification step or handling of hazardous chemicals and allows determination of the sequence on only two electrophoretic lanes. Its simplicity and rapidity favor automatization.     

A digitonin-based procedure for the isolation of mitochondrial DNA from mammalian cells

A procedure for the isolation of closed circular mitochondrial DNA (mtDNA) from cells permeabilized with digitonin is described. Compared to the standard procedure in which cells are broken after osmotic swelling, the digitonin-based procedure is more consistent and results in higher mtDNA yields.     

A specific method of high sensitivity for the determination of adenosine in the incubation medium of fat tissue

A suitable method for the measurement of adenosine in the incubation medium of fat tissue (200-500 mg) has been developed. The method is based on the specificity of the adenosine deaminase reaction and on the high sensitivity of a fluorescent method for adenine derivatives. The decrease of fluorescence in a sample after treatment with this enzyme is used for measuring adenosine in the range of 50-500 pmoles/tube. This method is highly specific and is not affected by other adenine derivatives present in the sample. Instead of acetic acid, perchloric acid was used in the fluorescent reaction, thus increasing the amount of adenosine dependent fluorescence. With this modification of the original fluorescent method, perchloric acid extracts can be used without further processing after deproteinization of the samples. Using this method, we could measure the adenosine release of fat pads of Wistar rats incubated in Krebs-Ringer-albumin buffer without concentration or purification procedures.     

Multiple sites for the initiation of microtubule assembly in mammalian cells.

The pattern of microtubule regrowth in mammalian fibroblast and epithelial cells has been examined by immunofluorescence of cytoskeletal preparations with antibody to tubulin. After reversal of treatment with colcemid, vinblastine or low temperature, microtubules appear to grow simultaneously from several distinct initiation sites located within 5 microns of the nucleus of mouse and human fibroblasts. Each site initiates the growth of 10-30 microtubules. More than 70% of the mouse fibroblasts have between 5 and 10 initiation sites with an average of 8. The human fibroblasts have an average of 5 sites per cell. The average number and numerical distribution of sites per fibroblast cell are not affected by time of exposure to colcemid or the concentration of colcemid applied to the cells. Multiple microtubule initiation sites are also observed during the process of microtubule depolymerization. In addition to growth from these complex initiation sites, microtubules appear to grow singly from the perinuclear region of human fibroblasts. The regrowth of individual microtubules from the perinuclear growth is especially prominent in epithelial cell lines from rat kangaroo and pig. These epithelial lines have only a single complex initiation site per cell. Two classes of complex initiation sites can be distinguished in microtubule regrowth experiments in human and mouse fibroblasts after exposure to griseofulvin. Microtubules first grow extensively from a single distinct site, which has approximately 20 microtubules growing from it and may be the centriole or centriolar pair. Subsequently, microtubules regrow from other perinuclear complex initiation sites. It thus appears that at least three distinct classes of initiation sites can be observed in mammalian cells: primary sites, which regrow microtubules first after griseofulvin treatment; secondary sites, which are distinct perinuclear sites and recover from griseofulvin treatment more slowly than the primary sites; and tertiary sites or sites of growth of single microtubules, also located near the cell nucleus.     

A preparative method for obtaining enucleated mammalian cells.

Analysis of the 2D gel electrophoretic pattern of ribosomal proteins from both the small and large subunit of rat liver were made at various times following partial hepatectomy. No changes were observed in the electrophoretic mobility of proteins from the 60S subunit during periods of 2 hr. to 72 hr. of liver regeneration. Changes were observed, however, in two proteins of the 40S subunit a short time after partial hepatectomy. Protein S₆ disappeared from its normal position and a new spot appeared as a more negative form as early as 2 hr. post regeneration. This modification persisted for at least 18 hr. At 72 hr., S₆ returned to its normal position. Protein S₂, on the other hand, underwent a different pattern of change during the early stages of liver regeneration. S₂ was observed to migrate as 2 spots at 2 hr. after partial hepatectomy and this pattern was preserved at the 4 hr. period. At 8 hr., the pattern was further modified to 2 spots which was distinct from the earlier change. This pattern was similar at 12 hr. At 18 hr. only the normal S₂ protein was observed. No further change in S₂ migration was observed at the 72 hr. period of liver regeneration.