The inhibition of bovine and rat parotid deoxyribonuclease I by skeletal muscle actin. A biochemical and immunocytochemical study.

Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.     

Respiration-stimulated activation of DNAase I associated with mitochondria]

The influence of respiration and Ca++ transport in the liver mitochondria on the activation of DNAase I, associated with these organelles, was studied. It was shown that 96% of the total activity of this enzyme in mitochondria is in the latent state. Aeration of the mitochondrial suspension leads to a sharp increase in the enzyme activity. The activation of DNAase I is inhibited by EGTA addition (optimal pH 8.0), and stimulated in mitochondria, releasing Ca++. It is concluded that the activation of DNAase I is dependent on the state of cellular energetics. Participation of mitochondrial phospholypase A, activated by the Ca++ release from mitochondria during DNAase I activation is suggested.     

DNAase I sensitivity of genes expressed during myogenesis.

Cultures of a rat myogenic cell line were used to examine the question of whether in proliferating precursor cells genes which are programmed to be expressed later in development, in the same cell lineage, differ in DNAase I sensitivity from genes which are never expressed in these cells. Nuclei isolated from proliferating mononucleated myoblasts, differentiated cultures containing multinucleaged fibers, and rat brain, were treated with DNAase I. The sensitivity of the genes coding for the muscle-specific alpha-actin, myosin light chain 2 and the nonmuscle beta-actin was measured by blot hybridization of nuclear DNA with the corresponding cloned cDNA and genomic DNA probes. The sensitivity of these genes was compared to that of a gene not expressed in the muscle tissue. The results showed that in the muscle precursor cells, the potentiality of tissue-specific genes to be expressed is not reflected in DNAase I sensitivity. The changes which render these genes preferentially sensitive to DNAase I take place during the transition to terminal differentiation. The results showed also that the region of DNAase I sensitivity of the alpha-actin gene in the differentiated cells ends between 40 to 700 bp 5' to the structural gene. No DNAase I hypersensitive site was detected 5' to the alpha-actin gene.     

Effect of X-ray induced DNA damage on DNAase I hypersensitivity of SV40 chromatin: relation to elastic torsional strain in DNA.

The effect of X-irradiation on DNAase I hypersensitivity of SV40 minichromosomes within nuclei or free in solution was investigated. The susceptibility of the specific DNA sites in the control region of minichromosomes to DNAase I decreased in a dose dependent manner after irradiation of isolated nuclei. On the other hand, the irradiation of minichromosomes extracted from nuclei in 0.1 M NaCl-containing buffer almost did not affect the level of their hypersensitivity to DNAase I. This suggests that DNAase I hypersensitivity may be determined by two different mechanisms. One of them may be connected with elastic torsional strain within a fraction of minichromosomes and another seems to be determined by nucleosome free region. The first mechanism may be primarily responsible for the hypersensitivity of minichromosomes within nuclei. After irradiation of the intact cells, DNAase I hypersensitivity tested in nuclei substantially increased. This was connected with activation of endogeneous nucleases by X-irradiation which led to accumulation of single- and double-strand breaks superimposed to DNAase I induced breaks in the control region of SV40 DNA.     

In situ lymphoid cells of mouse mammary tumors. I. Development and evaluation of a method for the separation of lymphoid cells from mouse mammary tumors

A method of separating lymphoid cells from solid mouse mammary tumors was developed and evaluated. In this method the tumors are digested with 0.01% collagenase, 0.01% DNAase, and 0.025% trypsin in Dulbecco's PBS into suspensions of cells with a viability of 90%. The suspensions are fractionated on a continuous gradient of Ficoll in tissue culture medium. In model experiments this gradient was found to separate, cleanly, admixed cells of an established mammary tumor cell line and dissociated thymus glands. Recovery rates were 50% for the tumor cells and 80% for the thymocytes. The preparation of the cell suspensions and the gradient separation procedure are not harmful to the cells as indicated by trypan blue exclusion and the ability to grow in cell culture.     

Subcellular localization of gamma-glutamyltransferase in calf thymocytes.

The subcellular localization of gamma-glutamyltransferase in calf thymocytes was investigated and compared with that of alkaline phosphodiesterase I, alkaline nitrophenyl phosphatase, succinate-tetrazolium oxidoreductase (succinate-INT reductase) and lactate dehydrogenase after two different methods of cell disruption and differential centrifugation. Most of the activity was recovered in the crude membrane fractions (43.0%), but significant amounts co-pelleted with the large-granule (mitochondria) fractions (31%). The specific activity of the gamma-glutamyltransferase in the purified plasma membrane was 30-50 times that of the enzyme in the cell homogenate and had a similar subcellular distribution to the plasma-membrane markers, alkaline phosphodiesterase I and alkaline nitrophenyl phosphatase. It was concluded that gamma-glutamyltransferase was primary a plasma-membrane-bound enzyme, and that its location in other subcellular fractions was probably due to their contamination with plasma-membrane vesicles.     

Organized packaging of kinetoplast DNA networks

The kinetoplast DNA (kDNA) of Trypanosoma equiperdum is organized as a complex structure of catenated circular DNA molecules. The major component of the kDNA network is the one kilobase minicircle that is present at about 10,000 copies per network. We have developed two assays to examine the structure of kDNA networks compacted in vitro with spermidine. Our results suggest that minicircles are arranged into a regular structure with an exposed domain which is DNAase I- and restriction-sensitive and a protected domain which is resistant to restriction endonucleases and DNAase I. This regularly packaged structure is dependent upon spermidine compaction and the circularity of the kDNA, but does not require supercoiled minicircles or catenated networks.     

Features of the structure of replicating and non-replicating chromatin in chicken erythroblasts.

The digestion by DNAase I of DNA synthesised by isolated chicken erythroblasts was examined in isolated nuclei. It was found that newly synthesised DNA was susceptible to DNAase I but matured to a relatively resistant form with increasing time after replication as observed in mammalian systems. The presence of trypsin in the digestion exposed all of the DNA to DNAase I action. Examination of the digestion products showed that the newly replicated DNA differed little from the more mature form in the structure of the DNA-protein complex but that the difference in susceptibility was probably a result of a differential rate of access of the DNAase to the new and old DNA.     

Netropsin, distamycin A, bis-netropsins as selective inhibitors of restrictases and DNAse I

The simultaneous analysis of DNAase I "footprinting" data and restriction endonucleases inhibition data was performed on the same DNA end-labelled fragment. The inhibition induced by netropsin, a number of bis-netropsins and distamycin A was investigated. These experiments led us to the following conclusions. The restriction endonucleases inhibition by the ligands is caused by the ligand molecules binding in the close vicinity to the restriction endonuclease recognition sequence. The zone of +/- 4 bp from the center of the restriction endonuclease recognition sequence can be defined as the zone of the influence of the bounded ligand on the restriction endonuclease. But in this case the intersection of recognition sequence and the binding site occupied by a single ligand molecule is not sufficient for the inhibition to occur. Restriction endonuclease cutting sites protected by netropsin can be predicted basing upon known nucleotide sequence specificity of netropsin. Netropsin and bis-netropsins show different nucleotide sequence specificity. This fact can be used for selective inhibition of restriction endonucleases.     

Spectrophotometric method of studying the cleavage of DNA-duplexes by restriction endonucleases]

A spectrophotometric method for continuous monitoring the cleavage of DNA duplexes by type II restriction endonucleases was proposed. The time course of cleavage of a 14-membered DNA duplex by MvaI endonuclease was obtained. The spectrophotometric method is characterized by rapidity and high precision in determining the kinetic parameters of the reaction. It can be recommended for testing the preparations for the presence of restriction endonucleases, rapid determination of the activity of any restriction endonucleases, highly precise quantitative analysis of the restriction enzyme catalysed reactions.     

Extracellular manganese-stimulated deoxyribonuclease as a marker event in sporulation of Bacillus subtilis.

A considerable amount of Mn2+-stimulated DNAase (deoxyribonuclease) activity is released by Bacillus subtilis 168 during sporulation in a glucose-deficient medium; much smaller amounts are released during starvation for phosphate or nitrogen. Protein synthesis is required. Two forms of evidence are presented that production of the DNAase is associated with events late in stage II of sporulation. 19 Thymidine starvation, which inhibits the biochemical events associated with sporulation, also inhibits release of the DNAase. 2. Several asporogenous mutants blocked at stage II or earlier and unable to produce alkaline phosphatase (a stage-II event) do not produce the enzyme. Mutants blocked towards the end of stage II or later produce both enzymes. During sporulation of the wild-type strain, the DNAase appears about 1 h after alkaline phosphatase. The results suggest that production of the DNAase is controlled by a still-undiscovered stage-II genetic locus.     

Correlation between DNase I hypersensitive sites and putative regulatory sequences in human immunoglobulin genes of the kappa light chain type.

The human lymphoid cell lines Walker and Daudi constitute a particularly suitable system for studies on the chromatin structure of K light chain genes (see preceding paper). The rearranged and non-rearranged alleles of Walker cells were found to be about equally sensitive towards digestion with DNAase I. A DNAase I hypersensitive site was mapped 0.13 kb upstream of the leader segment of the rearranged VK genes; it comprises a region in which promoter-like regulatory elements were discovered recently. Additional hypersensitive sites are located further upstream. A hypersensitive site in the JK-CK intron coincides with a putative tissue specific enhancer element. A hypersensitive region down-stream of CK overlaps with the cleavage/polyadenylation recognition signal which is flanked by sequences related to the above mentioned putative regulatory sequences. The coincidence between DNAase I hypersensitive sites and those sequences may be functionally significant.     

Modeling, using nucleases, of the effect of radiation on Chinese hamster fibroblasts. On the role of single-stranded DNA breaks, induced by DNAase I, in the initiation of chromosome aberrations

DNAase I injected to Chinese hamster fibroblast cells resulted in the chromosome aberration induction at all stages of the cell cycle and death of cells. Comparison of the effects of DNAase I and gamma-radiation on Chinese hamster cells showed that with close values of the induced DNA breaks there were close values of the cytogenetic damage and the number of DNA damages per aberrant cell.     

The chromatin structure of specific genes: I. Evidence for higher order domains of defined DNA sequence.

When the chromatin of Drosophila is examined by digestion with DNAase I or micrococcal nuclease, no general structural organization above the level of the nucleosome is revealed by the cleavage pattern. In contrast, the DNAase I cleavage pattern of specific regions of the Drosophila chromosome shows discrete bands with sizes ranging from a few kilobase pairs (kb) to more than 20 kb. Visualization of such higher order bands was achieved by the use of the Southern blotting technique. The DNAase I-cleaved fragments were transferred onto a nitrocellulose sheet after size fractionation by gel electrophoresis. Hybridization was then carried out with radioactively labeled cloned fragments of DNA from D. melanogaster. For the five different chromosomal regions examined, each gives a unique pattern of higher order bands on the autoradiogram; the patterns are different for different regions. Restriction enzyme cleavage of the fragments generated indicates that the preferential DNAase I cleavage sites in chromatin are position-specific. The chromosomal regions bounded by preferential DNAase I cleavage sites are referred to as supranucleosomal or higher order domains for purposes of discussion and analysis. The micrococcal nuclease cleavage pattern of chromatin at specific loci was also examined. In the one case studied in detail, this nuclease also cleaves at position-specific sites.     

DNAaseI-hypersensitive minichromosomes of SV40 possess an elastic torsional strain in DNA.

Previously, we have shown that DNA in a small fraction (2-5%) of SV40 minichromosomes was torsionally strained and could be relaxed by treating minichromosomes with topoisomerase I. This fraction was enriched with endogeneous RNA polymerase II (Luchnik et al., 1982, EMBO J., 1, 1353). Here we show that one and the same fraction of SV40 minichromosomes is hypersensitive to DNAase I and is relaxable by topoisomerase I. Moreover, this fraction completely loses its hypersensitivity to DNAase I upon relaxation. The possibility that this fraction of minichromosomes can be represented by naked DNA is ruled out by the results of studying the kinetics of minichromosome digestion by DNAase I in comparison to digestion of pure SV40 DNA and by measuring the buoyant density of SV40 chromatin in equilibrium CsCl gradient. Our data obtained with SV40 minichromosomes may be relevant to the mechanism responsible for DNAase I hypersensitivity in the loops or domains of cellular chromatin.     

Some aspects of the desoxyribonuclease activities of animal tissues

It has been found that many animal tissues contain "acid" desoxyribonucleases with pH optima near 5.2. A chemical method for the determination of this activity is described. The pancreatic desoxyribonuclease crystallized by Kunitz and shown to have a neutral pH optimum occurs in the pancreas together with the "acid" enzyme, but only the "neutral" enzyme occurs in the pancreatic juice. The ratio of "neutral" to "acid" DNAase activities in the pancreas is greater than 200, but in all other tissues examined there is no appreciable concentration of the neutral enzyme. It is concluded that neutral DNAase, like trypsin or lipase, has a digestive function. Some problems in the activation of the secretory enzyme in neutral pancreatic extracts are described. This activation can be interpreted in terms of a specific inhibitor or an inactive form of the enzyme. A comparison of the "acid" DNAase activities of different organs of the calf, horse, chicken, mouse, and rat indicates a possible connection between the DNAase concentration of a tissue and its capacity for proliferation or regeneration. However, the comparative DNAase activities of fetal and adult tissues do not support the view that DNAase function is limited to some simple role in the mechanics of cell division. Studies on the incorporation of glycine-N¹⁵ into the desoxypentose nucleic acids of avian red cells, and mouse liver, pancreas, and kidney show that the N¹⁵ uptake into the DNA of the chromosome is most rapid in tissues with high DNAase concentrations. No N¹⁵ incorporation is observed in the DNA of avian red cells, which have negligible concentrations of the enzyme. The analyses of tissues and nuclei isolated in non-aqueous media show that the bulk of the enzyme occurs in the cytoplasm of the cell, and that nuclear concentrations vary from tissue to tissue. A theory relating the DNAase activity of the cell to its over-all desoxypentose nucleotide metabolism is discussed. No evidence has been found for the presence of inhibitors of the "acid" DNAase in animal tissues.