Mung Bean Nuclease Digestion Research Articles

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA

Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 5' → 3' exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo- and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 5' → 3' exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabidopsis and Nicotiana benthamiana, leading to chlorosis but surprisingly few disruptions in the cleavage of polycistronic rRNA and mRNA precursors. In contrast, antisense RNAs accumulated massively, suggesting that the failure of chloroplast RNA polymerase to terminate effectively leads to extensive symmetric transcription products that are normally eliminated by RNase J. Mung bean nuclease digestion and polysome analysis revealed that this antisense RNA forms duplexes with sense strand transcripts and prevents their translation. We conclude that a major role of chloroplast RNase J is RNA surveillance to prevent overaccumulation of antisense RNA, which would otherwise exert deleterious effects on chloroplast gene expression.

Hepatitis C Virus Is Primed by CD81 Protein for Low pH-dependent Fusion

Hepatitis C virus (HCV) entry into permissive cells is a complex process that involves interactions with at least four co-factors followed by endocytosis and low pH-dependent fusion with endosomes. The precise sequence of receptor engagement and their roles in promoting HCV E1E2 glycoprotein-mediated fusion are poorly characterized. Because cell-free HCV tolerates an acidic environment, we hypothesized that binding to one or more receptors on the cell surface renders E1E2 competent to undergo low pH-induced conformational changes and promote fusion with endosomes. To test this hypothesis, we examined the effects of low pH and of the second extracellular loop (ECL2) of CD81, one of the four entry factors, on HCV infectivity. Pretreatment with an acidic buffer or with ECL2 enhanced infection through changing the E1E2 conformation, as evidenced by the altered reactivity of these proteins with conformation-specific antibodies and stable association with liposomes. However, neither of the two treatments alone permitted direct fusion with the cell plasma membrane. Sequential HCV preincubation with ECL2 and acidic buffer in the absence of target cells resulted in a marked loss of infectivity, implying that the receptor-bound HCV is primed for low pH-dependent conformational changes. Indeed, soluble receptor-pretreated HCV fused with the cell plasma membrane at low pH under conditions blocking an endocytic entry pathway. These findings suggest that CD81 primes HCV for low pH-dependent fusion early in the entry process. The simple triggering paradigm and intermediate conformations of E1E2 identified in this study could help guide future vaccine and therapeutic efforts to block HCV infection.

The Archaeon Haloarcula marismortui has Few Modifications in the Central Parts of its 23 S Ribosomal RNA

Post-transcriptional modifications were mapped in domains II, IV and V of 23S RNA from the archaeon Haloarcula marismortui. The RNA was investigated by two primer extension techniques using reverse transcriptase and three mass spectrometry techniques. One primer extension technique utilized decreasing concentrations of deoxynucleotide triphosphates to detect 2'-O-ribose methylations and other polymerase blocking modifications. In the other, the rRNA was chemically modified, followed by mild alkaline hydrolysis to map pseudo-uridine groups (Psis). RNA fragments for mass spectrometry were isolated from 23S rRNA by site-directed RNase H or mung bean nuclease digestion followed by gel purification. Modified RNase digestion fragments were identified with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and the modifications were further studied by tandem MS. Psis suggested by the primer extension technique were verified by specific cyanoethylation and mass spectrometric detection. A total of only five post-transcriptionally methylated nucleotides and three Psis were detected in the three 23S rRNA domains. One of the methylated nucleotides has not been reported while a dispute about the number of Psis is solved. The limited number of modified nucleotides suggests that H. marismortui does not have special needs for extensive rRNA modifications. We have performed detailed investigations on the three-dimensional location and molecular interactions of the modified nucleotides by computer analysis. Our results show that all the modified positions are at regions with RNA-RNA contacts and all except one are at the surface of the subunit and in functionally important regions.

Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of human colonic microbiota

Various molecular–biological approaches using the 16S rRNA gene sequence have been used for the analysis of human colonic microbiota. Terminal- restriction fragment length polymorphism (T-RFLP) analysis is suitable for a rapid comparison of complex bacterial communities. Terminal-restriction fragment (T-RF) length can be calculated from a known sequence, thus one can predict bacterial species on the basis of their T-RF length by this analysis. The aim of this study was to build a phylogenetic assignment database for T-RFLP analysis of human colonic microbiota (PAD-HCM), and to demonstrate the effectiveness of PAD-HCM compared with the results of 16S rRNA gene clone library analysis. PAD-HCM was completed to include 342 sequence data obtained using four restriction enzymes. Approximately 80% of the total clones detected by 16S rRNA gene clone library analysis were the same bacterial species or phylotypes as those assigned from T-RF using PAD-HCM. Moreover, large T-RFs consisted of common species or phylotypes detected by both analytical methods. All pseudo-T-RFs identified by mung bean nuclease digestion could not be assigned to a bacterial species or phylotype, and this finding shows that pseudo-T-RFs can also be predicted using PAD-HCM. We conclude that PAD-HCM built in this study enables the prediction of T-RFs at the species level including difficult-to-culture bacteria, and that it is very useful for the T-RFLP analysis of human colonic microbiota.

A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector.

In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (approximately 100 kbp). Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D). No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates. Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin. Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion. This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both Escherichia coli and P. putida.

The structure of mitochondrial DNA from the liverwort, Marchantia polymorpha

The structure of mitochondrial DNA (mtDNA) from cultured cells of the liverwort, Marchantia polymorpha, was analyzed by pulsed-field gel electrophoresis (PFGE) and moving pictures of the fluorescently labeled molecules. Previous electron microscopic analysis with this liverwort revealed a unique property among land plants: mtDNA circles of only one size, that of the 186 kb genome, with no subgenomic circles. Most of the mtDNA was immobile in PFGE and contained complex structures, larger than the genome size with a bright fluorescent node and multiple attached fibers. The mobile mtDNA was mostly linear molecules in monomeric to pentameric lengths of the unit genome that increased following mung bean nuclease digestion, with a corresponding decrease in the immobile fraction. From 0 to 5% of the mtDNA was found as circular molecules the size of the genome and its oligomers; no subgenome-sized circles were present. Radiolabeling revealed that mtDNA synthesis began soon after transfer of cells to fresh medium and most newly replicated mtDNA was immobile; the circular form of the genome was not rapidly labeled.

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.

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.

Cloning and expression of genomic DNA sequences coding for putative erythrocyte membrane-associated antigens of Plasmodium falciparum

Genomic DNA fragments of Plasmodium falciparum generated by mung bean nuclease digestion were cloned in the lambda expression vector λJK2. The resulting library was screened with a rabbit antiserum raised against purified membranes of P. falciparum-infected erythrocytes and with a serum pool from immune humans from an endemic area of Liberia. Positive clones were rescreened with a series of human and monkey sera. Twelve selected clones were analysed in detail. Four of them corresponded to already described membrane-associated P. falciparum antigens. The other positive clones contained inserts which, according to the nucleotide sequence, Southern blot analysis and immunological characteristics, correspond to so far unknown antigens.

Cloning of Four Plant Viruses from Small Quantities of Double-Stranded RNA

The first strand and second strand synthesis were basically that of Gubler and Hoffman with the omission of mung bean nuclease digestion. After second strand synthesis, the DNA was size-fractionated on a Sepharose CL-4B column, C-tailed, annealed to G-tailed pBR322/pUC9, and Escherichia coli DH1/DH5a cells transformed. No differences in length of cDNA and size of inserts was observed when heat- and DMSO-denaturation were used. However, use of MeHg yielded greater incorporation of 32 P-dATP into first strand when compared to the boiling method of denaturation

Nucleotide sequence and characterization of a gene encoding the phytochrome polypeptide from Avena

We have isolated and characterized a gene encoding the phytochrome polypeptide of Avena. Based on nucleotide sequence identity with previously sequenced cDNA clones this gene is designated as type 3 ( phy3). The gene is about 5.9 kb long with six exons and five introns, one each of the latter in the 5 ' and 3 ' -untranslated regions. The largest exon encodes the entire 74-kDa, chromophore-bearing, N-terminal domain of the photoreceptor postulated to be directly involved in its mechanism of action. The transcription start point, identified by mung-bean nuclease digestion, is located 24 to 35 bp downstream from a tandem TATA box. Sequence elements homologous to a number of motifs implicated as upstream regulatory elements in other genes are present in the 5'-flanking DNA of phy3. Particularly intriguing are three elements at positions -140, -470 and -650. These elements share homology with the ‘GT’ motif postulated to be a component of the light-regulatory element of genes encoding the small subunit of ribulose bisphosphate carboxylase.

33] Expression and secretion vectors for yeast

The chapter focuses on methods used to efficiently express and secrete biologically active proteins from Saccharomyces cereoisiae . The chapter describes yeast expression vectors utilizing episomal vectors. The expression cassettes from these vectors can also be integrated into the yeast chromosome, where they are presented at controlled copy number, exhibiting a high degree of mitotic stability. The chapter discusses extrachromosomal replication vectors, centromere plasmids, and the assembly of expression cassettes. One of the advantages of a secretion system is the production of heterologous proteins with authentic NH 2 termini. The methods utilized to analyze heterologous protein secretion are the same as those used for proteins expressed directly in the cytoplasm (direct expression vectors) The chapter describes several related procedures including the Mung Bean nuclease digestion, the S1 nuclease digestion of cohesive termini, and the analysis of carbohydrate additions to heterologous proteins.

Isolation of a gene segment expressed by mature schizonts of Plasmodium knowlesi

Fragments of Plasmodium knowlesi DNA, generated by mung bean nuclease digestion, were ligated into the λgt11 vector. This expression library was immunoscreened with a serum which inhibits invasion of erythrocytes by merozoites in vitro and whose primary specificity is directed against a M r 140 000 merozoite surface antigen. One of the isolated clones contained a 125 base pair insert which hybridized to a 1.8 kilobase species of schizont RNA, indicating that this insert is part of a gene expressed during schizogony.

Physical characterization of a kinetoplast DNA fragment with unusual properties.

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.