Methylmercuric Hydroxide Research Articles

Methylmercuric hydroxide

Methylmercuric hydroxide

The use of reverse transcriptase for efficient first- and second-strand cDNA synthesis from single- and double-stranded RNA templates

Molecular characterization of eight distinct, difficult-to-clone RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry and several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates, combined with random primers, were denatured with methyl mercuric hydroxide. Reverse transcriptase was added followed by the addition of RNase H. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently into a T-tailed vector after adding an A-overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel-purified dsRNA and with single-stranded RNA templates.

RNA-dependent RNA polymerase activity associated with virus-like dsRNA in Eimeria maxima and E. necatrix of the domestic fowl.

RNA-dependent RNA polymerase (RDRP) activity was identified in lysates of Eimeria maxima sporozoites and E. necatrix sporozoites and merozoites. Pretreatment of cell lysates with DNase I, RNase A, proteinase K and actinomycin D prior to RDRP assay was employed to characterize RDRP activity. DNase I and actinomycin D had little effect, while proteinase K abolished RDRP activity in both species. RNase A at a concentration of 1 mg/ml also reduced the polymerase activity in E. maxima and E. necatrix sporozoite lysates to 2% and 0%, respectively. Gel electrophoresis of RDRP products revealed that while most migrated at sizes less than 3 kb, a proportion of labelled products of E. necatrix and E. maxima also migrated to the sizes of their respective putative viral genomes. The RDRP products of E. necatrix were shown to be single-stranded by digestion with RNase in both low- and high-salt solutions and by methylmercuric hydroxide treatment. Moreover, the RDRP products of E. necatrix only hybridized to the 5.6-kb dsRNA of E. necatrix but not to the 4.5-kb dsRNAs of E. necatrix or E. maxima.

Northern analysis of highly folded goat α s1 Casein mRNA

Northern blotting using glyoxal to denature a highly folded mRNA, such as goat α s1-Casein E, can lead to the detection of multiple incompletely denatured forms. Formaldehyde appears to be the most suitable agent for Northern blotting due to its effective denaturing capacity and lower toxicity than methylmercuric hydroxide.

Complementation of xeroderma pigmentosum cells by microinjection of mRNA fractionated under denaturing conditions: an estimation of sizes of XP-E and XP-G mRNA

Excision repair deficiencies in groups A and G xeroderma pigmentosum (XP) cells are transiently complemented after microinjection of HeLa poly(A)+RNA, but those in groups D and F are not complemented (Legerski et al., 1984). We tested XP cells belonging to the seven complementation groups, A-G, and Cockayne's syndrome (CS) cells belonging to the two complementation groups, A and B, for transient correction by microinjection of total poly(A)+RNA from HeLa cells. Among the XP cells, unscheduled DNA synthesis (UDS) was increased only in XP-A cells by microinjection of total poly(A)+RNA. However, UDS was increased in XP-E and XP-G cells as well as in XP-A cells by microinjection of concentrated poly(A)+RNA fractionated on a 5–25% sucrose density gradient containing methylmercuric hydroxide. The sizes of XP-E and XP-G mRNA were estimated to be 1.5–2.7 kb and 2.0–3.8 kb, respectively, by comparison to internal marker RNAs including 18S rRNA, 28S rRNA, HPRT mRNA and XPAC mRNA. RNA synthesis recovery after UV exposure in CS cells was not increased by microinjection of either total poly(A)+RNA or fractionated RNA. These results provide estimates of the sizes of XP-E and XP-G proteins and will facilitate molecular cloning of DNA repair genes, especially of XP-E and XP-G genes.

Separation of translationally active mRNAs by reversed-phase ion-pair high-performance liquid chromatography

An ion-pair high-performance liquid chromatographic method on C4 columns was developed for the separation of mRNAs. The addition of methylmercuric hydroxide markedly influenced the separation according to length of these molecules. A method is given to recover minute amounts of translatable mRNA from the organic phase. The resolution of mRNAs improved with increasing pore size of the column support.

The octanol/water partition coefficient of methylmercuric chloride and methylmercuric hydroxide in pure water and salt solutions

The I-octanol/water partition coefficient (K,.) of methylmercury was determined at pH 7 in water and in aqueous solutions ranging from 0.0003 to 0.6000 m in sodium chloride. It was also determined over a pH range of 2 to 10 with a fixed sodium chloride concentration of 0.0045 M. In these experiments, K,, was seen to increase with increasing chloride concentrations in the range 0.0 to 0.075 m and to decrease with increasing pH above 7. Results were similar whether low levels of methylmercuric chloride or methylmercuric hydroxide were used as the starting materials. Partition- ing wai not affected by changes in ionic strength. Keywords-Octanol/.ater partition coefficient Methylmercury K.,,,

Analysis of polyadenylated RNA from brain synaptosomes and mitochondria

We have isolated RNA from sheep brain synaptosomes and mitochondria separated by an aqueous two-phase system composed of dextran and poly(ethylene glycol). RNA was fractionated through oligo(dT)-cellulose columns and analyzed by electrophoresis through agarose slab gels containing methylmercuric hydroxide and stained with ethidium bromide. The electrophoretic patterns of the poly(A)-containing RNA fraction from synaptosomes and mitochondria are very similar although some high molecular weight RNA species, clearly visible in the synaptosomal fraction, are scarcely detected in the mitochondrial preparations. The electrophoretic analysis of a cleaner RNA preparation from digitonin-treated free mitochondria (mitoplasts) showed that all the poly (A)-RNA species of the synaptosomal preparation are also present in mitoplast. These results strongly suggest that all the discrete poly(A)-RNA species identified in brain synaptosomes are of mitochondrial origin.

Characterization of DOPA decarboxylase mRNA in rat pheochromocytoma

Total poly (A +) RNA has been extracted from rat pheochromocytoma and translated in vitro by means of a reticulocyte lysate system. We show that two antisera, prepared against pig kidney DOPA decarboxylase (DDC) or rat pheochromocytoma DDC, immunoprecipitate an in vitro synthetized 50 kDa polypeptide identified as DDC by competition experiments with pure DDC. The proportion of specific mRNA has been calculated and represents 0.05% of total poly A + mRNA. Its size has been established by electrophoresis in methylmercuric hydroxide containing agarose gel, corresponding to a 2.2 kb length mRNA.

Association of three RNA molecules with potato mop-top virus

Potato mop-top virus particles, purified from systemically infectedNicotiana benthamiana plants and then disrupted by heating with sodium dodecyl sulphate and 2-mercaptoethanol, contained only a single polypeptide of Mr 19 100 detectable by polyacrylamide gel electrophoresis. Single-stranded RNA preparations from virus particles, when subjected to electrophoresis in an agarose gel containing methylmercuric hydroxide as a denaturant, were shown to contain approximately equal proportions of three RNAs of 6.5, 3.2 and 2.5 kb. Double-stranded RNA preparations extracted from systemically infectedN. benthamiana leaves or from locally infectedN. debneyi leaves, was shown by polyacrylamide gel electrophoresis to contain a major species of 3.2 kbp and two minor species of 6.5 and 2.4 kbp.

Ribosomal RNA-based diagnosis of Plasmodium falciparum malaria

We have identified useful target sites for the diagnosis of malaria infections by oligonucleotide hybridization on the small subunit RNA of Plasmodium falciparum. Acetic acid works as effectively as formaldehyde or methyl mercuric hydroxide in procedures designed to apply RNA to filters. We have confirmed the findings of others that the stability of ribosomal RNA suffices for its use as a target for diagnosis. We have achieved a detection level of at least 0.00046% parasitemia and suggest that detection of a single parasite is well within reach of this technology.

Translation of the human C3b/C4b receptor mRNA in a cell-free system and by Xenopus oocytes.

The C3b/C4b complement receptor (CR1) is a large, single-chain integral membrane glycoprotein present on erythrocytes, leukocytes, glomerular podocytes, and splenic dendritic-reticular cells that mediates the binding of complement-coated particles and immune complexes. CR1 is unusual in that it is polymorphic in size with the four allelic variants having molecular weights of 190,000, 220,000, 250,000, and 280,000 (SDS-PAGE, reducing conditions). The in vitro translation of the common (Mr 220,000) allelic variant CR1 has been achieved by using mRNA in lysates of rabbit reticulocytes and in Xenopus oocytes. HL-60, a promyelocytic human leukemic cell line, was treated with DMSO to induce differentiation and synthesis of CR1. Poly(A+) RNA was purified from these cells by column chromatography on oligo(dT)-cellulose. In the rabbit reticulocyte system, no CR1 was detected unless the translation mixture was denatured. In the presence of methylmercuric hydroxide, the CR1 translation product, unlike most translation products, had the same molecular weight in gel electrophoresis as the high-mannose-containing pro-CR1 and was 15-20K larger than nonglycosylated CR1. This suggests that a cotranslational modification of CR1 structure occurs, probably involving a proteolytic cleavage event. When poly(A+) RNA was translated in Xenopus oocytes, CR1 could be detected by treatment of oocytes with anti-CR1 monoclonal antibody followed by fluorescein-conjugated goat anti-mouse IgG. CR1 was diffusely distributed but preferentially localized to the vegetal surface. The molecular weight of this product, identified in immunoprecipitates of lysates of [35S]methionine-labeled oocytes, was identical with that of CR1 of HL-60.

Biological function of the rotavirus protein VP4: Observations on porcine isolates from China

Rotaviruses isolated from pigs in China were grown in MA104 cells. One tissue-culture-adapted isolate consisted of two subpopulations (variants), the RNA profiles of which differed in the relative migration of RNA segment 4 only. The variants were separated by plaque purification and by recovery from limiting dilutions and remained genetically stable. The variant possessing the slower migrating RNA segment 4, called 4S, grew faster and formed large plaques after 4-6 days incubation, whereas the variant possessing the faster migrating RNA segment 4, called 4F, grew more slowly and formed only microscopic plaques after 10-14 days incubation. The protein product of the 4F RNA occurred in much lower concentration in infected cells than the product of the 4S RNA. The RNA segments 4 of the two variants were found to be closely related when tested by dot hybridization under stringent conditions. The 4S RNA is more resistant to denaturation with methyl mercuric hydroxide than is the 4F RNA. The relevance of these findings to the biological functions of rotaviruses is discussed.

ChemInform Abstract: Methyl(2‐mercapto‐4‐methylpyrimidinato)mercury(II): A Methylmercury(II) Derivative with an Unusual Metal‐Metal Interaction.

AbstractThe mercury‐mercury distance in crystals of (III), obtained by reaction of (I) with methyl mercuric hydroxide (II), is determined by structural analysis to be 3.1 Å.

Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs.

The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.

Proteins expressed by Mill Door/79 virus, a Kemerovo serogroup orbivirus transmitted by the ticks Ixodes uriae.

Mill Door/79 virus, an orbivirus of the Kemerovo serogroup, Great Island Complex, was shown to induce in infected cells 10 segments of double-stranded RNA with a total molecular weight of 11.46 X 10(6) daltons. Using methyl mercuric hydroxide as a denaturing agent the double-stranded RNA was translated in a cell-free system producing 11 polypeptides, 10 of which co-migrated with those produced in Mill Door/79 virus infected Vero cells. The segments were separated and individually translated in a cell-free system allowing their coding assignments to be made. These assignments were confirmed by partial proteolysis.

Isolation and characterization of milk protein nuclear RNAs in rat mammary gland

Methods have been developed to isolate high-molecular-weight pre-mRNAs from lactating mammary gland, a tissue high in RNase levels. These methods involved isolation of nuclei at −20°C in 50% glycerol, and nucleic acid extraction using a guanidine thiocyanate-CsCl protocol. Specific RNAs were detected using α-, β-, and γ-casein and whey acidic protein nick-translated cDNA and genomic DNA probes by hybridization in situ to pre-mRNAs fractionated on agarose gels containing 10 m m methylmercuric hydroxide. Using these techniques it was possible to isolate poly(A)-containing gene-sized primary transcripts in the case of the two smaller genes, β-casein and whey acidic protein. A very complex pattern of pre-mRNAs was observed for the β-casein transcripts, including detection of a species which may represent an excised intron. Probes for the α- and γ-casein genes revealed much lower abundance and complexity of RNA precursors. These methods have proven useful in the initial analysis of RNA processing of these hormonally regulated milk protein gene transcripts.

Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.

Isolation and partial characterization of the messenger RNA encoding the B880 holochrome protein of Rhodospirillum rubrum

The B880 holochrome messenger RNA was extracted from cultures of the photosynthetic bacterium Rhodospirillum rubrum. It was purified by chromatography on Sepharose 4B followed by sucrose density gradient centrifugation. The purified fractions were shown to program an Escherichia coli cell-free system into synthesizing both the alpha and the beta polypeptides of the holochrome. The translation products were identified by immunoprecipitation with specific antibodies raised against these polypeptides. The latter are effective competitors with the translation products for antigen-antibody complex formation. The purest mRNA preparations contained approximately 33% holochrome messenger RNA activity. Its most probable size, as determined by agarose gel electrophoresis in the presence of 6 M urea or methylmercuric hydroxide, is approximately 620 nucleotides. Since the combined sizes of the alpha and beta polypeptides add up to only 106 amino acid residues, we conclude that the holochrome mRNA is most probably polycistronic.

Molecular cloning of a cDNA complementary to a UDP-glucose pyrophosphorylase mRNA of Dictyostelium discoideum

Uridine diphosphoglucose pyrophosphorylase (UTP: -α- d-glucose-1-phosphate uridyltransferase, EC 2.7.7.9) is an essential enzyme for normal development of Dictyostelium discoideum and its specific activity increases 3- to 10-fold by the later stages of development. Previous experiments have shown that additional forms of the enzyme appear concomitantly with this increase and that two uridine diphosphoglucose pyrophosphorylase (UDPGP) polypeptides are immunoprecipitated from the in vitro translation products of total cellular RNA at any stage of development ( B. F. Fishel, R. E. Manrow and R. P. Dottin, 1982, Dev. Biol. 92, 175–187). Using an in vitro translation-immunoprecipitation assay of UDPGP mRNA, we show that an increase in the amount of translatable mRNA is correlated with the accumulation of enzyme during development. A cDNA bank was constructed from a mRNA population that had been enriched for UDPGP mRNA by size fractionation on sucrose gradients containing methylmercuric hydroxide ( C. W. Schweinfest, R. W. Kwiatkowski, and R. P. Dottin, 1982, Proc. Natl. Acad. Sci. USA 79, 4997–5000). A 1.8-Kb cDNA complementary to a UDPGP mRNA was identified after screening the bank by hybridization selection and translation. Only the mRNA encoding the higher molecular weight in vitro translation product is hybrid selected by this cDNA. In hybrid-arrested translation experiments, the coding strand of this cDNA selectively inhibits the translation of only one of the two in vitro translation products. Therefore, there are two distinct UDPGP mRNAs.