The synthesis and enzymatic polymerization of 5-thio- and 5-methylmercurithio-pyrimidine nucleotides.

The 5-thio and 5-methylmercurithio derivatives of UTP, dUTP and dCTP have been synthesized and tested as substrates for nucleic acid polymerases. The 5-thio-nucleotides were polymerized inefficiently by both RNA polymerase and DNA polymerase I of Escherichia coli. The 5-methylmercurithio derivatives of dUTP and dCTP were, however, utilized by DNA polymerase I, an enzyme insensitive to mercurial compounds, although they were potent inhibitors of all other polymerases tested. While polymers containing the 5-thio substituent possess structural abnormalities, most likely interstrand disulfide bridges, polymers containing 5-methylmercurithio groups appear normal. The latter polynucleotides are readily separated from non-sulfated polymers by chromatography on mercuriagarose.

Phytohemagglutinin isolectin subunit composition.

The subunit compositions of individual phytohemagglutinin isolectins from red kidney bean Phaseolus vulgaris were examined by isoelectric focusing and sodium dodecyl sulfate electrophoresis on polyacrylamide gels. Isoelectric focusing reveals heterogeneous but unique and non-overlapping protein band patterns for each of the homotetrameric isolectins, E4 and L4. Isoelectric focusing of the intermediate isolectins which contain both subunits (E3L1, E2L2, and E1L3) show all the protein bands common to isolectins E4 or L4 in proportions relative to their suggested subunit compositions. Polyacrylamide gel electrophoresis in a continuous sodium dodecyl sulfate buffer system gives a single protein band for all of the isolectins. In contrast, a discontinuous sodium dodecyl sulfate buffer procedure resolves isolectins E4 and L4 into single major protein bands of apparent molecular weights 31 700 (+/-600) and 29 900 (+/-200), respectively. Each of the intermediate isolectins contained both protein bands and their relative proportion, as determined by absorbance scanning, confirms the phytohemagglutinin isolectin subunit compositions as E4, E3L1, E2L2, E1L3, and L4.

Nucleic acid-based antiviral drugs against seasonal and avian influenza viruses.

Influenza viruses are etiological agents of deadly flu that continue to pose global health threats, and have caused global pandemics that killed millions of people worldwide. The availability of neuraminidase inhibitors and attenuated vaccines improves our ability to defend against influenza, but their benefits can be significantly limited by drug-resistance and virus mutations. Nucleic acid-based drugs may represent a promising class of antiviral agents that could play a role in the prevention and treatment of influenza. Efficacy studies in animals have shown that ds RNA, such as poly ICLC can provide effective and broad-spectrum prophylaxis against lethal challenges against various strains of influenza A virus. Furthermore, similar level of antiviral protection in mice can be provided by using short fragments of oligonucleotides that induce antiviral immunity. Finally, influenza virus expression can also be specifically inhibited or suppressed using antisense oligonucleotides that bind to viral mRNA encoding key viral proteins. The versatility and potency of nucleic acid-based drugs make them potential drug candidates for used in seasonal or pandemic influenza situations.

Sequence homology among different size classes of plant mtDNAs.

Supercoiled mtDNAs were isolated from tissue culture cells of tobacco, bean, and corn, and the smallest size classes were used to study the relationships among the different size classes of each species through restriction digests and hybridizations. Three of the smallest tobacco mtDNAs [10.1, 20.2, and 28.8 kilobases (kb)], the two smallest bean mtDNAs (1.9 and 3.8 kb), and the two smallest corn mtDNAs (1.5 and 1.8 kb) were extracted from the gels and nick translated. The 10.1-kb tobacco mtDNA hybridizes to all the other tobacco mtDNA size classes and a large percentage of the tobacco mtRNAs. Restriction digests indicate that the 20.2-kb size class is a dimer of the 10.1-kb size class. The 1.9-kb bean mtDNA hybridizes to all but three of the bean mtDNA size classes and hybridizes to two mtRNAs. Restriction digests indicate that the 3.8-kb size class is a dimer of the 1.9-kb size class. The 1.5- and 1.8-kb corn mtDNAs, which do not have any Hha I restriction fragments in common, both hybridize to many of the same size classes of the corn mtDNA profile and, in addition, each hybridizes to a few size classes not recognized by the other. The 1.5- and 1.8-kb size classes both hybridize to two RNAs, one of which they appear to have in common. However, with both the 1.9-kb bean mtDNA and the two corn mtDNAs, the molecular sizes of the two RNAs exceed those of the respective DNAs. The possible role and origin of the many size classes are discussed.

Mercurated polynucleotides: new probes for hybridization and selective polymer fractionation.

Polynucleotides containing covalently bound mercury atoms have been prepared by chemical or enzymatic syntheses and some of their physical and biochemical properties studied. The mercury substituents do not appear to alter significantly normal polynucleotide structure. Mercurated polymers function efficiently as templates for nucleic acid polymerases, they are fully susceptible to degradation by standard nucleases, and their denaturation and reannealing properties resemble those of the corresponding nonmercurated polymers. While the Tm's of DNA duplexes are lowered by extensive mercuration, the Tm's of DNA-RNA hybrids and RNA duplexes are either unaffected or elevated. Mercuration, as would be expected, greatly increases the buoyant density of both DNA and RNA. The introduction of as few as one mercury atom per 200 bases permits the selective and quantitative retention of the mercurated polymer probe (and associated nucleotide sequences) on columns of sulfhydryl-agarose. The use of mercurated nucleotides (as polymerase substrates) and oligonucleotides (as primers) in conjunction with sulfhydryl-agarose chromatography provides a simple and efficient method for the isolation of selected polynucleotide sequences, such as specific in vitro transcription products or terminal fragments of duplex DNA. Products absorbed to the affinity resin are readily recovered for further analysis by eluting with buffers containing mercaptoethanol. Although the mercury-carbon bond is somewhat thermolabile, mercurated polynucleotides are suitable as probes in low temperature hybridization studies.

Maturation and germination of Phaseolus vulgaris embryonic axes in culture.

In this study of embryo development in Phaseolus vulgaris L., we found that immature embryonic axes placed in culture show a growth lag before germinating. The length of this lag phase varies according to axis age at excision, but is not affected by transfer to fresh medium, alteration of sucrose concentration between 0.5 and 2%, or whether the culture medium is liquid or agar-solidified. The lag phase was shortened by both actinomycin D and cordycepin treatment, and by treatment with 10(-5) to 10(-6) M benzyladenine. The effect of abscisic acid (ABA) varied with concentration: below a certain level, it had no effect on the lag phase, but above that level it inhibited, germination. This threshold concentration was 10(-7) M for 20-d-old axes but increased to 10(-5) M by the time the axes were 32 to 34 d old. To determine whether the axes were continuing their embryonic development during the lag phase, we tested them for desiccation-tolerance and for synthesis of phaseolin, a seed storage protein which is specific for embryos of P. vulgaris. The ability to germinate after rapid desiccation was acquired by axes at 26 d past anthesis; when axes younger than this were placed in culture, they developed desiccation-tolerance during the lag phase of growth, indicating that they were continuing embryonic maturation. Phaseolin was present in isolated axes, although at lower levels than in cotyledons. It accumulated during axis development in parallel with total protein, staying at about 1% of total protein content. When isolated immature axes were pulsed with (3)H-or (14)C-amino acids, they incorporated label into phaseolin, shown by precipitation with anti-phaseolin antibody. Isolated axes from mature seeds, however, did not synthesize detectable amounts of phaseolin. Immature axes cultured in vitro for a period of one to several days continued synthesizing phaseolin until the day prior to visible germination. Treatment of cultured axes with ABA increased the amount of precursor amino acids incorporated into protein, but had a small or no effect on the relative proportion of phaseolin synthesized. We conclude that P. vulgaris axes in culture continue to develop embryonically for a period of time which seems to be under intrinisc control by the axis. This contrasts with "precocious germanation", a pattern of embryo behavior seen in many other species. When such embryos are excised from seeds while immature and placed in culture, they switch promptly from embryo development into germination. If ABA or water stress is responsible for preventing precocious germination, it may be that a high level of ABA is maintained or synthesized internally by embryonic axes of Phaseolus, while in other embryos the maternal environment supplies ABA and/or causes water stress.

Analysis of four tobacco mitochondrial DNA size classes.

Supercoiled mtDNAs were isolated from tobacco suspension culture cells and three of the smallest size classes (10.1, 20.2 and 30.3 kb) were characterized through denaturation, heteroduplex and restriction mapping. The 20.2 molecule was found to be a head-to-tail dimer of the 10.1 or X size class, while the 30.3 kb size class was found to contain two kinds of molecules, a head-to-tail trimer of X (X3) and a second molecule, ABC. X and ABC had a 118 +/- 35 bp region of homology, and both size classes shared a degree of homology with at least one other size class. Restriction maps of both the X and ABC molecules are presented and the possible origin and role of the many plant mtDNA size classes are discussed.

Supercoiled mitochondrial DNAs from plant tissue culture cells.

Supercoiled mitochondrial DNA (mtDNA) has been isolated in preparative amounts from five different plant species and compared on agarose gels. The gels reveal considerable mtDNA size heterogeneity within each species as well as substantial differences in the range and frequency of the mtDNA size classes among the different plants. The two lowest size classes of three of the plants have been extracted and their molecular weights determined. Comparison of two N. tabacum W38 culture lines has revealed significant differences in their mtDNA size classes. However, despite the totally different supercoil patterns, these two lines have almost identical restriction digests. The availability of preparative amounts of separated mtDNA size classes will make it possible to carry out a detailed analysis of plant mitochondrial genomes.

The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.

A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA.

A simple new procedure was described for producing a sequential series of overlapping clones for use in DNA sequencing. The technique used single-stranded M13 DNA and complementary DNA oligomers to form specific cleavage and ligation substrates. It was, therefore, independent of the sequence of the DNA cloned into the vector. Deletions of varying sizes were generated from one end of the insert through the 3' to 5' exonuclease activity of T4 DNA polymerase. The approximate size of the deletion and therefore the starting point for DNA sequencing could be estimated by electrophoresis of the subcloned phage DNA on a agarose gel. This greatly reduced the number of templates that must be sequenced to obtain a complete sequence. The entire procedure could be carried out in one tube in less than a day. The procedure was used to subclone and sequence the maize mitochondrial 18 S rDNA and 5' flanking region (2622 bases) in less than a week. Other applications of oligomers and single-stranded DNA in the construction of insertions, deletions, and cDNAs are discussed.

Conversion of covalently mercurated nucleic acids to tritiated and halogenated derivatives.

Mercurated nucleic acids are converted to the corresponding tritiated, brominated, and iodinated derivatives by treatment with sodium borotritiide, N-bromosuccinimide, and elemental iodine, respectively. All three reactions occur under mild conditions in neutral aqueous solutions. Mercury-halogen conversions are essentially quantitative at both the mono- and polynucleotide levels. Tritiation reactions also proceed efficiently with mononucleotides, although polymers undergo incomplete demercuration. In spite of the latter limitation , these reactions provide novel and efficient synthetic routes to radiolabeled nucleic acid derivatives.

Direct covalent mercuration of nucleotides and polynucleotides.

Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.

Sequence analysis of the maize mitochondrial 26 S rRNA gene and flanking regions.

A single copy of the large ribosomal 26 S rRNA gene is found in the maize mitochondrial genome. The sequence of this gene and the flanking regions has been determined using the M13 dideoxy sequencing method. The maize mt 26 S rDNA shares a high degree of homology with the Escherichia coli 23 S rDNA, and the approximate 5' and 3' ends of the maize 26 S rDNA have been located by comparison with the E. coli sequence. The maize mt 26 S rDNA has also been compared with the sequences of the maize chloroplast 23 S rDNA, the human mitochondrial 16 S rDNA, part of the yeast mitochondrial 21 S rDNA, and the yeast cytoplasmic 25 S rDNA. In all cases, there are numerous regions of 70% or higher homology.

DNA sequence analysis of a 5.27-kb direct repeat occurring adjacent to the regions of S-episome homology in maize mitochondria.

The DNA sequence of the 5270-bp repeated DNA element from the mitochondrial genome of the fertile cytoplasm of maize has been determined. The repeat is a major site of recombination within the mitochondrial genome and sequences related to the R1(S1) and R2(S2) linear episomes reside immediately adjacent to the repeat. The terminal inverted repeats of the R1 and R2 homologous sequences form one of the two boundaries of the repeat. Frame-shift mutations have introduced 11 translation termination codons into the transcribed S2/R2 URFI gene. The repeated sequence, though recombinantly active, appears to serve no biological function.