Agarose Gel Electrophoretic Analysis Research Articles

Plasma thiols inhibit hemin-dependent oxidation of human low-density lipoprotein

Oxidative modification of human low-density lipoprotein (LDL) renders it atherogenic. Previous studies demonstrated that plasma thiols promote oxidation of LDL by free ferric iron (Fe 3+). The current study investigated effects of plasma thiols on oxidation of LDL by hemin, a physiological Fe 3+–protoporphyrin IX complex thought to be capable of initiating LDL oxidation in vivo. In contrast to free Fe 3+ which is incapable of oxidizing LDL in the absence of an exogenous reductant, hemin readily promoted LDL oxidation. During incubation of LDL (0.2 mg of protein/ml) with hemin (10 μM) at 37°C for 6 h, thiobarbituric acid-reactive substances (TBARS), a marker of lipid oxidation, increased from 0.3 (±0.1) nmol/mg of LDL protein to a maximal concentration of 45.8 (±5.2) nmol/mg of LDL protein. Under the same experimental conditions, lipid-conjugated dienes, another marker of lipid oxidation, increased from non-detectable to near-maximal levels of 78–187 nmol/mg of LDL protein, and lipoprotein polyunsaturated fatty acyl-containing cholesteryl ester content decreased to 15–36% of that present in native (i.e. unoxidized) LDL. Continued incubation of LDL with hemin for up to 24 h resulted in no further significant alterations in lipoprotein levels of TBARS, lipid-conjugated dienes, and cholesteryl esters. In addition to these chemical modifications indicative of lipoprotein oxidation, agarose gel electrophoretic analysis indicated that exposure of LDL to hemin resulted in conversion of the lipoprotein to an atherogenic form as evidenced by its increased anodic electrophoretic mobility. Addition of physiological concentrations of plasma thiols (either cysteine, homocysteine or reduced glutathione; 1–100 μM, each) inhibited hemin-mediated oxidation of LDL. Thus, whereas the maximal TBARS concentration was achieved following 6 h of incubation of LDL with hemin alone, addition of thiol extended the time required to attain maximal TBARS concentration to ≥12 h. Similar antioxidant effects of thiols on formation of lipid-conjugated dienes, loss of cholesteryl esters, and lipoprotein anodic electrophoretic mobility were also observed. However, all thiols were not equally effective at inhibiting hemin-dependent LDL oxidation. Thus, whereas reduced glutathione was most effective at inhibiting hemin-dependent LDL oxidation, an intermediate effect was observed for homocysteine, and cysteine was least effective. The inhibition of hemin-mediated LDL oxidation by plasma thiols reported here confirms a previous observation that, under certain conditions, thiols can function as antioxidants, but contrasts with the previously documented pro-oxidant effect of the same thiols on oxidation of LDL by free Fe 3+. These contrasting effects of plasma thiols on hemin- and free Fe 3+-mediated LDL oxidation indicate that, in vivo, the ability of thiols to function as either anti- or pro-oxidants during LDL oxidation may, at least in part, be determined by the type of oxidant stress to which the lipoprotein is exposed.

Preliminary study pointing out a significant alteration in the biochemical composition of MUC2 in colorectal mucinous carcinoma

Objectives: In this study, we characterized colonic MUC2 mucin from a mucinous carcinoma cell line and tried to find out carcinoma-associated alterations by comparing the results with those obtained from its benign phenotype previously. Design and methods: The molecular size distribution of the extracted molecules and their reactivity with two different MUC2 polypeptide antibodies indicated the presence of precursor and mature forms of the mucin in both cell lines. Isopycnic density gradient centrifugation gave good resolution of mature and precursor forms of MUC2 as assessed by agarose gel electrophoresis. Using this approach, we compared the different forms of MUC2 between benign and malign colonic cells. Results: In the comparison, we detected some aberrant glycosylated MUC2 molecules in mucinous carcinoma cell line. Agarose gel electrophoretic analysis of the low-density fractions indicated that these molecules are more charged than precursors, however, they are smaller and/or less glycosylated than mature MUC2 molecules. Conclusion: The identification of unusual partially glycosylated forms of the major colonic mucin MUC2 is novel and unexpected. Implication of defective processes in the post translational modification/ processing of MUC2 opens a new field in the cancer mucin biology.

Salinity induced nuclear and DNA degradation in meristematic cells of soybean (Glycine max (L.)) roots

Changes in the nuclei of meristematic root cells of soybean (Glycine max (L.) Merr. cv. Acme) in response to severe salinity were studied. Root growth was inhibited by 200 mM NaCl, when 1 mM CaCl_2 was present in the culture media. Increasing CaCl_2 up to 5 mM partially prevented this inhibition. However, inhibition also occurred with 100~mM NaCl without CaCl_2. We examined the meristematic cells under a series of NaCl treatments. Nuclear deformation of the cells occurred with 24 h of 150 mM or higher NaCl, and was followed by degradation of nuclei in the apical region of the root. TEM observation and agarose gel electrophoretic analysis confirmed that root tip nuclear DNA deformed or degraded with 150 mM or higher NaCl concentrations.

Investigation of the positive response of ethyl acrylate in the mouse lymphoma genotoxicity assay.

To develop a better understanding of the relationship between ethyl acrylate (EA)-induced cytotoxicity and mutation frequency in the mouse lymphoma assay (MLA) we measured the effects of EA treatment to ML cells on: (1) nonprotein sulfhydryl (NPS) levels; (2) mitochondrial rhodamine 123 (Rh123) uptake; (3) the DNA elution slope (single-strand breakage) and Y intercept of fitted curves (cytotoxicity and double-strand breakage) in the alkaline elution assay; (4) the appearance of apoptosis; and (5) the pulsed-field gel electrophoretic resolution of high-molecular-weight DNA. EA reduced NPS in both a time- and concentration-dependent manner. By 30 min, > or = 20 micrograms/ml EA reduced NPS by 50% or greater. By 4 h, > or = 10 micrograms/ml markedly decreased both NPS cell content (> or = 71.5% reduction) and mitochondrial Rh123 uptake (10-50 micrograms/ml; 9-62%), the latter effect being further enhanced by washing and incubation for an additional 2 h (12-85%). EA did not induce single-strand breaks in the alkaline elution assay. Only highly cytotoxic EA concentrations (80-87% reduction in RCG at 40-50 micrograms/ml) caused both increases in the elution slope and parallel drops (Y intercept) in the elution curve in the alkaline elution assay. Conventional agarose gel electrophoretic analysis of the DNA neutral fraction of these high dose preparations showed evidence for both apoptosis (180-bp oligonucleosomal DNA laddering effect) and random smearing of DNA (necrosis). Pulsed-field gel electrophoresis of directly loaded high dose cell preparations revealed both high- and low-molecular-weight DNA double-strand breaks, but only at the highest concentrations. These observations indicated that the EA-induced mutagenic response correlated best with cellular cytotoxicity mediated by NPS depletion and mitochondrial membrane impairment.

PCR‐Based Detection of Artificial Latent Infections of Geranium by Xanthomonas campestris pv. pelargonii

AbstractA procedure entailing biological enrichment and PCR amplification was developed to detect small populations of Xanthomonas campestris pv. pelargonii (X.c, pv. pelargonii) in tissues of geranium. Known numbers of colony forming units (CFU) of X.c. pv. pelargonii were introduced into ‘Red Elite’ geraniums through wounding of petioles and stems. Immediately after inoculation, sections of the petioles and stems were harvested and incubated for 24 or 48 h in nutrient broth (biological enrichment). After enrichment, bacterial cells were collected by centrifugation, followed by rapid extraction of total nucleic acid from the cells with GeneReleaser™, PCR amplification of DNA with pathovar‐specific primers, and ethidium bromide‐stained agarose gel electrophoretic analysis of the PCR products. After 48 h biological enrichment, it was possible to detect as few as 1 CFU of Xc. pv. pelargonii in stems and petioles collected immediately after inoculation, with the detection limit ranging between 1 and 120 CFU during multiple experiments. It also was possible to detect systemic movement of the bacterium in intact plants sampled 24 h after inoculation with a minimal inoculum (4 CFU). This procedure may have application in geranium certification programs concerned with the detection of latent infections associated with low levels of X.c.pv. pelargonii.

Genetic test for myophosphorylase deficiency in Charolais cattle

Abstract Objective To develop a simple test for the determination of genetic susceptibility to myophosphorylase deficiency in Charolais cattle. Animals 48 adult Charolais cattle and 233 calves from one herd and 3 Charolais cattle from 2 other herds. Sixty Piedmontese and 34 Saler cattle provided negative-control samples. Procedure Cattle were from a Charolais herd in which myophosphorylase deficiency was identified and 2 other herds in which cattle had signs compatible with the disease. Genomic DNA was isolated from heparinized blood samples. A segment of the myophosphorylase gene containing the mutation site was amplified by polymerase chain reaction assays, and the genotype (normal vs affected allele) was determined by using restriction enzyme and agarose gel electrophoretic analysis. Results The 3 myophosphorylase genotypes (homozygous normal, homozygous affected, and heterozygous) could be readily identified. Segregation of the affected allele could be determined in an extended pedigree, and all clinically affected cattle were homozygous for this allele. Determination of the distribution of normal and affected alleles in a large population did not indicate a strong selective advantage for heterozygous carriers in this herd. Heterozygotes were also identified in Charolais cattle from the 2 other herds. Conclusions Breeders of Charolais cattle can use this genetic test to perform marker-assisted selection and remove cattle with the mutant myophosphorylase allele from the breeding population. Alternatively, they could more accurately determine selective advantages and disadvantages for cattle with the affected allele. Clinical Relevance Development of this test enables rapid genetic screening of Charolais and related breeds of cattle for detection of the mutation responsible for myophosphorylase deficiency. (Am J Vet Res 1998;59:267–270)

Evaluation of DNA analysis for evidence of apoptosis in megaloblastic anaemia.

This study involved DNA analysis of bone marrow cells of 15 patients with megaloblastic anaemia. The diagnosis was based on the morphological changes seen in the bone marrow, associated with either a low red cell folate or serum vitamin B12 level and an adequate response to appropriate therapy as confirmation of the diagnosis. Flow cytometric DNA analysis showed an increase in the S and G2 phases of the cell cycle, but conventional agarose gel electrophoretic DNA analysis did not confirm the characteristic 'ladder pattern' which might have been expected in classic apoptosis. In addition, cells showing morphological changes suggestive of apoptosis, such as nuclear condensation and fragmentation, did not show evidence of DNA fragmentation using the ApopTag in situ digoxigenin nucleotide labelled, peroxidase detection system. Further studies using annexin V flow cytometric analysis and pulsed field gel electrophoresis were also unable to detect evidence of apoptosis as a significant cause of cell death in megaloblastic anaemia.

Role of c-myc in Tamoxifen-Induced Apoptosis in Estrogen-Independent Breast Cancer Cells

The antiestrogen tamoxifen (TAM) is effective in the treatment of estrogen receptor (ER)-positive as well as some ER-negative breast cancers. However, the precise mechanism of action of TAM, especially in estrogen-independent cells, remains unclear. Previous work by our laboratory has demonstrated that TAM induces the morphologic and biochemical changes that are characteristic of apoptosis in both ER-positive and ER-negative cells. We compared the effect of TAM at a clinically achievable concentration on cell growth and apoptosis with the effect of TAM on c-myc (also known as C-MYC) messenger RNA (mRNA) and protein expression in ER-negative MDA-231 cells. MDA-231 cells were treated for up to 72 hours with 1.0 microM TAM alone or in the presence of 50 microM c-myc antisense or nonsense oligonucleotides. c-myc mRNA expression was determined by northern blot analysis, protein expression by western blot analysis, cell growth inhibition counts, and DNA cleavage by agarose gel electrophoretic analysis. Differences between the mean values from different treatment groups were compared with the use of the two-sided Wilcoxon Ranksum test. TAM treatment for 72 hours increased c-myc mRNA five-fold (from a relative radiolabeled hybridization signal intensity of 17 +/- 4 up to 93 +/- 10; P<.05) and c-MYC protein threefold (from a relative immunofluorescence signal intensity of 28 +/- 7 up to 83+/-21; P< .05). The induction of c-myc by TAM was accompanied by internucleosomal DNA cleavage characteristic of apoptotic cell death. Addition of c-myc antisense oligonucleotide (5'CACGTTGAGGGGCAT-3') to MDA-231 cells resulted in a nearly twofold decrease of basal c-myc mRNA (P< .05) and a sevenfold decrease of basal c-Myc protein (P< .05) expression. Addition of c-myc antisense oligomer also antagonized the TAM-induced increase in c-myc mRNA (P< .05) and protein expression (P< .05) and inhibited TAM-induced cytostasis (P< .01) and apoptosis. In parallel experiments, addition of the nonsense oligomer had no effect on any of the measured parameters. These results indicate that the effects of TAM on ER-negative MDA-231 cells may be mediated through c-myc overexpression. c-myc may play a critical role in the growth and progression of MDA-231 breast cancer cells.

Development of a genetic system for a purple sulfur bacterium: Conjugative plasmid transfer inChromatium vinosum

Establishment of a system for manipulative genetics in phototrophic sulfur bacteria of the family Chromatiaceae has mainly been hampered by the lack of reliable methods for growth of these organisms on agar surfaces, techniques for streaking, growth on selective media, screening for antibiotic resistance markers, and most importantly by the lack of a system for DNA transfer. We, therefore, developed minimal and complex agar media forChromatium vinosum strain D (DSM 180T), a representative of the purple sulfur bacteria. Sensitivity ofC. vinosum towards a broad range of antibiotics was tested in liquid cultures and solidified media, allowing us to select appropriate antibiotic resistance markers. Furthermore, a system for conjugative transfer of IncP-mobilizable plasmids fromEscherichia coli toC. vinosum was established. Broad-host-range IncQ vectors were mobilized toC. vinosum with the aid of plasmid RP4 either present extrachromosomally or integrated in the chromosome ofE. coli S17-1. Conjugation efficiencies of up to 1 were observed. Agarose gel electrophoretic analysis showed that transconjugants contained the transferred plasmids in addition to the two detectable plasmids of wild-typeC. vinosum. All genetic markers tested (kanamycin, gentamicin, ampicillin, amikacin, tetracycline) were expressed inC. vinosum. Furthermore, high-frequency transfer of plasmid RP4 fromC. vinosum toE. coli and toRhodospirillum rubrum K100 was demonstrated.

Effects of plasmid DNA sizes and several other factors on transformation of Bacillus subtilis ISW1214 with plasmid DNA by electroporation.

Plasmid DNAs in the range from 2.9 to 12.6 kbp were transferred into Bacillus subtilis ISW1214 intact cells by the use of electroporation. The transformation efficiency (transformants per microgram plasmid DNA) decreased with increases of size of the DNA. However that of 2.0 x 10(3) transformants per microgram of DNA were done routinely, by using a plasmid with a large molecular size of 12.6 kbp. The size of plasmid DNA in the range of 3.7 to 12.6 kbp did not affect the molecular efficiency (transformants per molecule input DNA). The transformation efficiency as high as 9.3 x 10(4) transformants per microgram of purified plasmid pUB110 was obtained, using a cell concentration of 7.6 x 10(10) cells/ml and DNA concentration of 4 micrograms/ml in buffer containing 0.3 M sucrose, 1 mM CaCl2, and 1 mM sodium citrate (pH 6.0) under optimal pulse conditions of an electric field strength of 7 kV/cm and a duration of 500 mus with a single squared pulse at 0 degrees C. The gene expression for antibiotic resistance after electroporation was completed within 1.5 h. The transformants were confirmed to harbor the same intact plasmid by agarose gel electrophoretic analysis.

A new genetic assay for rifampicin susceptibility of Mycobacterium tuberculosis.

A new method for testing rifampicin (RFP) susceptibility of Mycobacterium tuberculosis was developed. This method is based on detection of the internal sequence derived from 71-kDa heat shock protein mRNA in tubercle bacilli heat-treated in the presence of RFP. The target sequence was amplified by reverse transcription and PCR, followed by agarose gel electrophoretic analysis. No amplification occurred in one RFP-susceptible strain by exposure to 45 degrees C for 45 min in Middlebrook 7H9 broth containing RFP (10 micrograms/ml) after overnight incubation at 37 degrees C. On the other hand, an amplified 275-bp product was obtained from the RFP-resistant strain MY-129. In a subsequent trial using 65 clinical isolates, this method defined their RFP susceptibility levels as well as the verification of the MICs obtained by the conventional agar dilution method, with the exception of one RFP-susceptible strain. Thus, this method provides a rapid and practical system to determine RFP susceptibility in M. tuberculosis.

Differential diagnosis of infectious laryngotracheitis from other avian respiratory diseases by a simplified PCR procedure

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of avian infectious laryngotracheitis virus (ILTV) in chicken trachea, chorio-allantoic membrane (CAM), infected hepatoma cells and infectious cell culture supernatant. Samples were prepared by dilution in distilled water. After boiling and low speed centrifugation, samples were used for PCR analysis with two primers without special labeling. The PCR analysis for ILT virus could be completed in less than 8 h. Standard agarose gel electrophoretic analysis of the PCR products revealed a prominent band of 300 base-pairs in samples from ILTV-infected specimens, but not from specimens containing Newcastle disease virus, infectious bronchitis virus, avian adenovirus, fowlpox virus, Pachecoz or Marek's disease virus. One single ILTV infected cell or 10 plaque forming units of ILTV could be detected with this procedure. The procedure can be used for the identification of ILTV and the differentiation of ILTV from other avian respiratory tract infectants.

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25μF, a transformation efficiency as high as 1.4×107 transformants per μg of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.

Selective Inhibitory Effect of Bufalin on Growth of Human Tumor Cells in vitro: Association with the Induction of Apoptosis in Leukemia HL‐60 Cells

We found that bufalin, an active principle of the Chinese medicine chan'su, has selective inhibitory effects on the growth of various human cancer cells. In order to examine whether the growth‐inhibitory effect of bufalin on human cancer cells is associated with apoptosis, human leukemia cells were treated with bufalin. HL‐60, ML1, and U937 leukemia cells treated with bufalin at 10−8M and above had condensed and fragmented nuclei. Flow cytometric analysis of these cells treated with bufalin showed fragmented DNA smaller than that of the G1 phase. DNA of HL‐60 cells treated with bufalin showed a ladder pattern characteristic of apoptosis, as analyzed by agarose gel electrophoretic analysis. DNA synthesis and topoisomerase II activity of HL‐60 cells were markedly inhibited as the concentration of bufalin was increased. The concentration needed for inducing apoptosis of HL‐60 cells was 10−8M, which is comparable to that of camptothecin, but lower than those of other antitumor drugs such as cisplatin, VP16 and all‐trans retinoic acid. Apoptosis was not observed when human mononuclear and polymorphonuclear cells were treated with 10−6M bufalin for 24 h. These results indicate the association of the growth‐inhibitory effect of bufalin with the induction of apoptosis, at least in HL‐60 cells, and suggest the usefulness of bufalin for differentiation‐apoptosis‐inducing therapy for cancer.

A variant insulin promoter in non-insulin-dependent diabetes mellitus.

To test the hypothesis that alterations in regulatory regions of the insulin gene occur in a subset of patients with non-insulin-dependent diabetes mellitus (NIDDM), the promoter region was studied by polymerase chain reaction (PCR) amplification directly from genomic DNA, followed by high-resolution polyacrylamide gel electrophoresis under nondenaturing conditions. By using this method a previously identified HincII polymorphism (GTTGAC to GTTGAG at position-56) in American Blacks was readily detected, indicating that single base changes could be observed. In the course of screening the insulin promoter from 40 American Black subjects with NIDDM, an apparent larger allele was found in two individuals. Both patients were shown to have in addition to a normal allele, a larger allele containing an 8-bp repeat, TGGTCTAA from positions -322 to -315 of the insulin promoter. To facilitate rapid screening for the 8-bp repeat, a high-resolution agarose gel electrophoretic analysis was adopted. DNA from American Black NIDDM subjects (n = 100) and nondiabetic subjects (n = 100) was PCR amplified and analyzed. The 8-bp repeat was present in five NIDDM subjects, and one nondiabetic subject. DNA from Mauritius Creoles, also of African ancestry, was analyzed, and the 8-bp repeat was present in 3 of 41 NIDDM subjects, and 0 of 41 nondiabetic subjects. Analysis of glucose metabolism in three presumed normal sibs of an NIDDM patient with an 8-bp repeat revealed that one sib had overt diabetes, and two sibs were glucose intolerant, but there was no consistent segregation of the insulin promoter variant with the diabetes phenotype. The variant promoter was not present in 35 Caucasian NIDDM patients or in 40 Pima Indians. To test the biological consequences of the 8-bp repeat sequence in the insulin promoter, a normal and variant promoter were subcloned into a luciferase plasmid, and reporter gene activity assessed by transient transfection into mouse insulinoma (beta TC1) and hamster insulinoma (HIT) cells. The promoter activity of the variant allele was found to be reduced to 37.9 +/- 10.3% of the activity of the normal promoter in HIT cells (P less than 0.01, n = 4), and 49.1 +/- 6.4% in beta TC1 cells (P less than 0.01, n = 6). These data thus suggest that a naturally occurring variant of the insulin promoter may contribute to the diabetes phenotype in 5-6% of Black NIDDM patients.

Activation of chromium(VI) by thiols results in chromium(V) formation, chromium binding to DNA and altered DNA conformation

The ability of the thiols glutathione, cysteine, beta-mercaptoethanol and dithiothreitol to effect chromium(VI)-induced DNA damage in vitro has been investigated. Reaction of pBR322 DNA with chromium(VI) in the presence of the thiols led to formation of chromium(V) and chromium--DNA adducts. The extent of chromium binding to DNA differed by several orders of magnitude among the thiols tested, in the order dithiothreitol greater than beta-mercaptoethanol much greater than cysteine greater than or equal to glutathione. The maximal level of chromium(V) formed also differed among the thiols tested, in the order beta-mercaptoethanol greater than dithiothreitol much greater than glutathione greater than or equal to cysteine. Electronic spectral studies of these reactions indicated that the rate of reduction of chromium(VI) is dependent on the thiol tested, in the order cysteine greater than dithiothreitol greater than glutathione greater than beta-mercaptoethanol. Electron paramagnetic resonance studies of these reactions indicate that a significant level of chromium(III) is detected only with cysteine. Chromium--DNA adducts formed by reaction of chromium(VI) in the presence of glutathione or cysteine did not lead to DNA conformational changes detectable upon agarose gel electrophoretic analysis. Changes in DNA conformation were detected as altered electrophoretic mobility of pBR322 DNA on agarose gels after reaction with chromium(VI) in the presence of dithiothreitol or beta-mercaptoethanol. Effects on DNA electrophoretic mobility, which depended on whether the initial conformation of the plasmid was linear or supercoiled, included altered and heterogeneous mobility, as well as complete inhibition of migration of the plasmid. Transmission electron microscopy of chromium--DNA complexes revealed aggregates of several plasmids, as well as condensation of individual plasmids into compact kinked forms. These effects may be due to cross-linking of DNA induced by chromium metabolites. These studies indicate that the levels of chromium bound to DNA are related to the levels and stabilities of the chromium(V) species formed upon reaction of chromium(VI) with the various thiols. Chromium--thiol interactions may play an important role in chromium(VI) genotoxicity.

Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes.

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.

N-ethylmaleimide inhibition of the DNA-binding activity of the herpes simplex virus type 1 major DNA-binding protein.

The major herpes simplex virus DNA-binding protein, designated ICP8, binds tightly to single-stranded DNA and is required for replication of viral DNA. The sensitivity of the DNA-binding activity of ICP8 to the action of the sulfhydryl reagent N-ethylmaleimide has been examined by using nitrocellulose filter-binding and agarose gel electrophoresis assays. Incubation of ICP8 with N-ethylmaleimide results in a rapid loss of DNA-binding activity. Preincubation of ICP8 with single-stranded DNA markedly inhibits this loss of binding activity. These results imply that a free sulfhydryl group is involved in the interaction of ICP8 with single-stranded DNA and that this sulfhydryl group becomes less accessible to the environment upon binding. Agarose gel electrophoretic analysis of the binding interaction in the presence and absence of N-ethylmaleimide indicates that the cooperative binding exhibited by ICP8 is lost upon treatment with this reagent but that some residual noncooperative binding may remain. This last result was confirmed by equilibrium dialysis experiments with the 32P-labeled oligonucleotide dT10 and native and N-ethylmaleimide-treated ICP8.

DNA breakage by hydrolyzable tannins in the presence of cupric ion.

DNA breakage by hydrolyzable tannins of known structures was investigated by agarose or polyacrylamide gel electrophoretic analysis. Hydrolyzable tannins could cause both double-strand and single-strand breakages in λDNA in the presence of Cu2+. The breaking reaction was strongly suppressed at concentrations higher than 100 μm of hydrolyzable tannins. DNA breakage by various tannins was dependent upon their sorts, numbers, and binding sites of the constituent acids and polyalcohols. Metallic ions other than Cu2+ had little effect on the breaking reaction.

Polyacrylamide–agarose gel electrophoretic analysis of tobacco mosaic virus disassembly intermediates

The intermediates of disassembly of the U1, U4, U6, and U7 strains of tobacco mosaic virus (TMV) induced by alkali and urea were directly analysed by electrophoresis in composite polyacrylamide (2.0 to 2.5% (w/v)) – agarose (0.5% (w/v)) gels, using a discontinuous buffer system with Tris, borate, EDTA, and sodium chloride (pH 8.3). The results show no difference between the disassembly patterns of the four strains. In the case of alkaline stripping, two new major partially stripped virus (PSV) particles (PSV 5a and PSV 5c) and several minor intermediates were characterized. The overall results indicate that polyacrylamide–agarose gel electrophoresis is superior to agarose gel electrophoresis and to sucrose density gradient ccntrifugation for the analytical separation of PSVs. Polyacrylamide–agarose gel electrophoresis is thus a rather simple, rapid, and efficient method of studying in vitro disassembly of plant viruses.