Specific cleavage of kinetoplast minicircle DNA from Leishmania tarentolae by mung bean nuclease and identification of several additional minicircle sequence classes

Multiple sequence classes of kinetoplast minicircle DNA from Leishmania tarentolae were cleaved by mung bean nuclease in the presence of formamide, yielding unit length linear molecules which retained the anomalous electrophoretic mobility in acrylamide characteristic of minicircle DNA. No specific cleavage site sequence common to all minicircle sequence classes was apparent, although the main region of nuclease cleavage was localized approximately 350 bp from the unique SmaI restriction site of the conserved region found in all minicircle sequence classes. Covalent closure of the minicircle substrate was not a requirement for cleavage, as linearized network-derived or cloned minicircles were also cleaved by mung bean nuclease at similar locations. The partial sequences of several new minicircle sequence classes released from the network by mung bean nuclease are also reported.     

Repeated sequences in the DNA of the eukaryotic genome

Long DNA molecules from a cucumber satellite, the cucumber main band, mung bean, and Chinese hamster ovary (CHO) were digested with mung bean nuclease I, which was used as a probe for high AT regions. The digests were viewed under the electron microscope, and the distribution of sizes for the fragments of nuclease-treated plant DNA showed that the main band cucumber and the mung bean have regions along their genomes spaced at approximately 0.3 to 0.4 μ that are sensitive to the nuclease. The satellite from the cucumber contains these sites at intervals generally of 0.1 μ or less, whereas CHO DNA has these regions at intervals of 0.05 to 1.40 μ in length. The long DNA from the main band of the cucumber and the CHO were also partially melted in formamide at 37°C to denature preferentially the regions along the DNA molecules that are rich in AT. Measurements of the distances from the center of each loop to the center of the adjacent loops showed that these distances for the main band cucumber DNA tended to occur at approximately every 0.4 μ, whereas the corresponding distances for the Chinese hamster DNA were less regular, occurring every 0.1 to 1.0 μ.     

Activity of single-stranded DNA endonucleases in mung bean is associated with cell division

A single-strand-specific endonuclease from mung bean sprouts is widely usedin molecular biology. However, the biological role of this enzyme is unknown. We studied the spatial and temporal activity of single-stranded DNA endonucleases in mung bean seedling by following enzyme activity that linearizes supercoiled plasmid DNA, a characteristic of this type of enzyme. The formation of a linear molecule from supercoiled DNA was found to occur in two distinguishable steps. The first, which involves introducing a nick into the supercoiled DNA and relaxing it, is very rapid and complete within a few seconds. The second step of cleaving the opposite strand to generate a unit-length linear duplex DNA is a relatively slow process. Analysis of the DNA cleavage sites showed the nuclease preferentially cuts supercoiled DNA at an AT-rich region. Varying levels of nuclease activity could be detected in different tissues of the mung bean seedling. The highest activity was in the root tip and was correlated with histone H1 kinase activity. This implies a link between nuclease activity and cell division. Induction of cell division in mung bean hypocotyls with auxin promoted formation of root primordia and considerably increased the activity of single-stranded DNA endonucleases. The nuclease activity and histone H1 kinase activity were reduced in mung bean cuttings treated with hydroxyurea, but not in cuttings treated with oryzalin. The potential function of single-stranded DNA endonucleases is discussed.     

DNA hairpin loops in solution. Correlation between primary structure, thermostability and reactivity with single-strand-specific nuclease from mung bean.

Hairpin structures formed by seven DNA inverted repeats have been studied by PAGE, UV(CD)-spectroscopy and nuclease cleavage. The hairpins consisted of (CG)3 stems and loops of 2, 3 and 4 residues. Thermal stabilities (Tm) have been determined in low and high ionic strength buffers, where the hairpins were structured in the B- and Z-DNA form respectively. The thermodynamic parameters of hairpin formation have been obtained by a two-state analysis of the hairpin-coil transitions. It is found that, on increasing the number of bases in the loop from 2 to 3 and 4, the Tms of the B-hairpins decrease, whereas the Tms of the same hairpins in the Z-form increase. This confirms previous evidence (1,2) that in a hairpin molecule the size and structure of the loop are modulated by the conformation of the helical stem. Moreover, B-hairpins with loops comprising 2, 3 and 4 bases have been digested with the single-strand-specific nuclease from mung bean. In our experimental conditions (0 degrees C) the nuclease preferentially cleaves the unbonded nucleotides of the loops. However, the rates of loop hydrolysis, which roughly follow a first-order kinetics, markedly depend on the size of the loop. At a ratio of 3 enzyme units/micrograms DNA, the half-lives of hairpins which are expected to form loops of 4, 3 and 2 residues are 90, 145 and 440 minutes respectively. Thermostability and enzymatic digestion data suggest that two-membered loops can be formed in B-hairpins but not in Z-hairpins.     

Changes in site specificity of single-strand-specific endonucleases on supercoiled PM2 DNA with temperature and ionic environment.

Mung bean nuclease sites in supercoiled PM2 DNA at neutral pH were located by linearizing the singly-nicked circular DNA product with venom phosphodiesterase followed by restriction endonuclease mapping. The locations of the sites varied with small changes in temperature and in concentration of NaC1 or magnesium ion. Different environmental changes which affect duplex stability in the same direction showed similar effects on the number of sites and in some cases resulted in identical cleavage patterns. Venom phosphodiesterase and P1 nuclease showed cleavage patterns similar to mung bean nuclease under the same environmental conditions and showed similar variations in cleavage patterns when environmental conditions were changed. Relaxed, closed-circular DNA was slowly cleaved at numerous sites whose locations did not vary with environment. Changes in site specificity are likely the result of environmental effects on the conformation of supercoiled DNA as opposed to effects on the single-strand-specific endonucleases themselves.     

Action of single-strand specific nucleases on model DNA heteroduplexes of defined size and sequence.

The sensitivity of the model DNAs containing dA-dG and dtg-dG heteroduplex regions of defined length to S1 and mung bean single-strand specific nucleases was tested by polyacrylamide gel electrophoretic analysis of the distribution of product oligonucleotides. Single-base mismatch heteroduplexes were extremely resistant to these nucleases, although low levels of cleavage at the heteroduplex nucleotide were observed at high nuclease concentrations. The nuclease sensitivity of dA-dtg heteroduplex regions increased gradually as the length of the heteroduplex region increased frome one to six nucleotides. The sensitivity of dG-dG heteroduplexes three to five nucleotides long was considerably greater than that of the single dtg-dG mismatch.     

Binding of DNA hairpins to an assembler-strand as part of a primordial translation device

A crucial event in the process leading to the origin of life is the emergence of a simple translation device. To approach experimental realization of this device the binding ability of short DNA hairpins to complementary oligonucleotides fixed on a solid support was investigated. The binding is achieved by base pairing between the loop nucleotides of the hairpins containing different numbers of adenosine residues and oligothymidylates covalently linked to cellulose. The loop has to consist of at least five nucleotides to achieve binding. The exact number of established base pairs was determined in two ways. First, the elution temperatures of hairpins and those of oligoadenylates which had the length of the loop were compared. Secondly, the architecture of the loop was analyzed by means of the single-strand-specific nuclease from mung bean acting as structural probe. Onlyn-2 of n loop nucleotides of a hairpin are able to form base pairs. Therefore, a strong evidence for the formation of a triplet of base pairs between primeval tRNA and mRNA sufficient to stabilize the complex enzyme-free is given.     

Mung bean nuclease exhibits a generalized gene-excision activity upon purified Plasmodium falciparum genomic DNA.

A novel set of reaction conditions for mung bean nuclease has been described in which Plasmodium genes were specifically excised as intact fragments from purified DNA. We have now determined that under the new conditions mung bean nuclease cleaves precisely at sites outside of the coding region of every P. falciparum gene for which the extent of the protein coding region in genomic DNA is known. We conclude that this enzyme activity is probably a general one for P. falciparum genes. Introns are not specifically cleaved, although one gene contained a cleavage site within an intron. There is no direct relationship between dA.dT-richness and sites of cleavage under these conditions. Also contrary to the expectations of a model based on cleavage at denaturation bubbles, there was no general relationship between the concentration of the DNA denaturant, formamide, and the size of the resulting gene-containing fragments. Thus, the data strongly suggest the involvement of an altered DNA structure near gene boundaries in determining the recognition sites for this enzyme activity.     

The conformation of single-stranded nucleic acids tDNA versus tRNA

Conformational analyses using the single-strand-specific nuclease from mung bean and restriction endonucleases have been performed on a series of DNA fragments related to the sequence of the yeast initiator tRNA(Met). Mung bean nuclease cleaves DNA fragments exclusively in some, but not all, single-stranded regions as predicted by RNA secondary structural rules. Comparison of cleavage patterns of yeast initiator tRNA(Met), tDNA(Met) (a DNA oligomer having the sequence of tRNA(Met] and the anti-tDNA(Met) (the complement of tDNA(Met] suggests that the conformation of the three molecules is very similar. Furthermore, both tDNA and anti-tDNA are cleaved by HhaI and CfoI restriction endonucleases at two GCG/C sites which would be in double-stranded regions (the acceptor and dihydrouridine stem), if the two molecules adopt the tRNA cloverleaf structure. On the other hand, minor cleavage products show that the core region, i.e. the extra loop area, is slightly more exposed in tDNA and in anti-tDNA than in tRNA. Therefore, we submit that the global conformation of nucleic acids is primarily dictated by the interaction of purine and pyrimidine bases with atoms and functional groups common to both RNA and DNA. In this view the 2'-hydroxyl group, in tRNA at least, is an auxiliary structural feature whose role is limited to fostering local interactions, which increase the stability of a given conformation.     

Complexes of DNA hairpins and a single-stranded oligonucleotide detected by affinity chromatography and mung bean nuclease cleavage

The emergence of a simple translation device consisting of an assembler strand (primordial mRNA) and RNA hairpins (primordial tRNA) is presumed to be an important step leading to the origin of life. The assumption of a non-enzymatic interaction of primordial tRNA and mRNA is experimentally approached. DNA hairpins containing five or more adenosine residues in the loop are able to bind to complementary oligonucleotides covalently bound to cellulose. The exact number of base pairs formed between the hairpins and the assembler strand is determined by two methods applied to DNA hairpin/assembler complexes. The melting temperature of a complex is measured and the cleavage pattern by nuclease from mung bean is determined. The loop of the smallest hairpin able to bind consists of five adenosine residues and only three base pairs are formed. This supports the idea of a primordial recognition similar to the contemporary codon-anticodon interaction.     

Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively.     

Specific hydrolysis of the cohesive ends of bacteriophage lambda DNA by three single strand-specific nucleases.

Procedures have been worked out for Aspergillus nuclease S1 and mung been nuclease to quantitatively cleave off both of the 12-nucleotide long, single-stranded cohesive ends of lambdaDNA. This cleavage is indicated by the almost complete elimination of the repair incorporation of radioactive nucleotides by DNA polymerase into the digested DNA. With S1 nuclease, cleavage was complete at 10 degrees as well as at 30 degrees. Under the conditions for quantitative cleavage of the single-stranded regions there was no digestion of the double-stranded lambdaDNA. The mung bean nuclease cleaved off the cohesive ends completely at 30 degrees but at 5 degrees, the cleavage was not complete even at high enzyme concentration. The nearest neighbor analysis of the repaired DNA indicates that at 5 degrees about four nucleotides remained undigested. The mung bean nuclease also introduced, under the conditions used, some nicks into double-stranded DNA as determined by the repair incorporation. The Escherichia coli exonuclease VII cleaved off part of the cohesive ends of lambdaDNA, leaving two nucleotides on each end as single-stranded tails.     

Mung bean nuclease cleavage of a dA + dT-rich sequence or an inverted repeat sequence in supercoiled PM2 DNA depends on ionic environment

We have determined the nucleotide sequences around two alternative sites cleaved in supercoiled PM2 DNA by single-strand-specific mung bean nuclease in different ionic environments. In 10 mM Tris-HC1 (pH 7.0, 37 degrees C), the major site is a dA+dT-rich sequence which maps with a known early denaturation region at 0.75 map units. About 30 cleavages occurred in a 135 bp region. Cleavages were largely excluded at (dA)n . (dT)n (n = 3-7) sequences. Cleavage patterns of this type have not been previously observed in dA+dT-rich sequences. With the addition of 0.1 M NaC1 the major alternative site occurred in a hyphenated inverted repeat sequence 500 bp away (0.70 map units) and did not map to an early denaturation region. One major and 4 minor cleavages occurred in the region between the repeats, suggesting that a hairpin containing at most a 12 bp stem and 10 base loop is recognized. The basis for nuclease recognition of the dA+dT-rich sequence is not clear. The differences in the sequences and cleavage patterns at the alternative sites indicate that their secondary structures differ.     

Phytotoxicity of selected herbicides to mung bean (Phaseolus aureus Roxb.)

Mung bean (Phaseolus aureus Roxb.) is grown after harvest of wheat during the fallow period. Herbicides such as metsulfuron, atrazine and isoxaflutole are recommended to control weeds in wheat–rice cropping system including weeds of fallow crop. The effects of three herbicides with different modes of action—atrazine, photosystem II inhibitor; metsulfuron, acetolactate synthase inhibitor; and isoxaflutole, 4-hydroxyphenylpyruvatedioxygenase inhibitor—on shoot height, chlorophyll concentrations and cellular damage in herbicide-treated mung bean were studied. While isoxaflutole inhibited shoot growth and chlorophyll concentration of mung bean, atrazine and metsulfuron did not cause reduction in the shoot growth of mung bean. Metsulfuron (226, 452, 1356 and 2260 μg/kg soil) and isoxaflutole (452, 1356 and 2260 μg/kg soil) in soil reduced the concentration of leaf chlorophyll of mung bean compared to the control. Atrazine in soil did not affect the total chlorophyll concentration of mung bean leaves. Electron micrographs showed that untreated mung bean had elongated chloroplasts, thylakoids organized as intact grana, distinct starch grains and a small number of plastoglubuli. Mesophyll cells of atrazine-treated mung bean leaves had swollen chloroplasts and thylakoids with disorganized grana. Leaves of metsulfuron-treated mung bean had swollen chloroplasts with a large number of starch grains. Starch grains were not observed in leaves of mung bean treated with either atrazine or isoxaflutole. Complete disruption of thylakoids was observed in isoxaflutole-treated mung bean leaves. Leaves of atrazine-treated mung bean showed detached microfibrils along with distorted and degenerated secondary walls. Metsulfuron-treated mung bean leaves showed aggregated microfibrils with completely dissolved secondary walls, while isoxaflutole-treated leaves had completely degenerated secondary walls with complete loss of microfibrils. We conclude that isoxaflutole at higher doses, influence mung bean at the morphological, physiological and cellular levels.     

Mung bean nuclease cleaves preferentially at the boundaries of variant surface glycoprotein gene transpositions in trypanosome DNA

Telomere-linked genes coding for the variant surface glycoproteins (VSGs) of African trypanosomes have been difficult to clone because their flanking regions frequently lack restriction sites. Therefore, we constructed a genomic DNA library of fragments generated by digestion of purified trypanosome DNA with mung bean nuclease, an enzyme that cleaves before and after genes in Plasmodium falciparum DNA (McCutchan, T. F., Hansen, J. L., Dame, J. B., and Mullins, J. A. (1984) Science 225, 625-628). Southern hybridizations with several gene probes showed that under the appropriate conditions mung bean nuclease produces discrete trypanosome DNA fragments that are as clearly resolved on an agarose gel as restriction fragments. The majority of VSG genes are on fragments of about 1.7 kilobase pairs. To examine the sites of mung bean nuclease cleavage, the insert boundary sequences of eight recombinant clones in the library containing VSG genes were determined. In general, mung bean nuclease cleaved 300-800 base pairs in front of the VSG start codon and within 50 base pairs on either side of the termination codon. These regions also form the boundaries of VSG gene conversion events indicating that the enzyme recognizes, in part, a conformational structure rather than a specific sequence. The analyzed clones included both telomere-linked and interior basic copy VSG genes indicating that the library potentially contains all of the telomere-linked VSG genes in the genome.     

Ferulic acid impairs rhizogenesis and root growth,and alters associated biochemical changes in mung bean (Vigna radiata) hypocotyls

The present study investigated the effect of ferulic acid (FA; 0–1000 µM) on early growth, and rhizogenesis in mung bean (Vigna radiata) hypocotyls and associated biochemical changes. FA severely affected the radicle elongation and number of secondary roots after 72 h. The root and shoot length, number and length of secondary roots, and seedling dry weight of one-week-old seedlings of mung bean were decreased by 64%. The rooting potential (percent rooting, number and length of adventitious roots) of mung bean hypocotyls under in vitro conditions was significantly inhibited in response to 1–100 µM FA. At 1000 µM there was complete cessation of rooting. FA caused a reduction in the contents of water-soluble proteins and endogenous total phenolics, whereas the activities of proteases, peroxidases, and polyphenol peroxidases increased. The study concludes that FA inhibits root growth and development, and in vitro rooting process in mung bean by interfering with biochemical processes that are crucial for root formation.