Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)⁺ RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.     

Molecular cloning of lupin leghemoglobin cDNA

Poly(A)⁺RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences specific for nodules were selected by differential colony hybridization using³²P-labeled cDNA synthesized either from nodule poly(A)⁺RNA or from poly(A)⁺RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)⁺RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules.     

A model for regulation of ColE1-like plasmid replication by uncharged tRNAs in amino acid-starved Escherichia coli cells

It has been previously observed that various ColE1-like plasmids replicate differentially in Escherichia coli cells during the relaxed response to amino acid starvation. Here we develop a kinetic model to explain these observations based on the possibility of interaction of the 3' CCA-OH sequence with the UGG triplets in loops of RNA I and RNA II encoded by ColE1-like plasmids. According to our model, when the interaction of uncharged CCA with RNA I is possible, the replication of the ColE1-like plasmid is affected by differences in the concentration of various tRNAs in the starved cell, but it is not affected by the tRNA concentration if the hypothetical pairing occurs between the CCA-OH and RNA II. Using the previously determined parameters for the pBR322 plasmid, the concentration of uncharged tRNAs in the amino acid starved relaxed strains and the assumed efficiency of binding of tRNA and RNA I, we show that our model explains the differences in pBR322 copy number in the relaxed strain starved for several amino acids.     

The ribosomal DNA of Drosophila melanogaster is organized differently from that of Drosophila hydei

On the X chromosome of Drosophila melanogaster there is a single tandem array of 240 ribosomal RNA genes. The majority of these contain an insertion, known as type I, in the 28 S coding region. Previous genetic and electron microscopic studies indicated that genes bearing the type I insertion (ins⁺) are interspersed at random with those lacking it (ins^?). In contrast, Renkawitz-Pohl et al. (1981) have analyzed the restriction pattern of X chromosomal ribosomal DNA in Drosophila hydei and demonstrated that in this case ins⁺ genes are segregated from ins^?. This suggests either that the rDNA is organized differently in these two species or that the restriction enzyme technique reveals significant clustering not detected by previous methods. By using an appropriate restriction enzyme, we demonstrate that ins⁺ and ins^? genes are intermingled at random in D. melanogaster. These experiments also indicate that genes containing the short form of the insertion are flanked by a larger spacer upstream than downstream.     

Restoration of mutability in non-mutable Escherichia coli carrying different plasmids.

Summary N and I group plasmids, which increase methylmethane sulfonate (MMS) mutagenesis in lexA ⁺ strains of E. coli WP2 may be divided into two classes: those restoring part of the mutability of lexA ^- strains (class I) and those leaving lexA ^- strains non-mutable (class II). Almost complete restoration of MMS mutability is obtained by class I plasmids in a partially suppressed lexA rnm strain, while class II plasmids cause far fewer MMS revertants in this strain than in lexA ⁺. A pair of class I and II plasmids in lexA ^- shows a synergistic effect on mutability. These two classes do not coincide with plasmid division into incompatibility groups.     

Physical characterization of plasmids from Streptomyces kasugaensis MB273

Plasmid DNA of molecular weight 6.8 × 10⁶ was isolated from Streptomyces kasugaensis MB273. The plasmid DNA showed a single CsCl-ethidium bromide density gradient centrifugation, in neutral sucrose gradient centrifugation, and in agarose gel electrophoresis. When this DNA was digested with BamHI or SalI endonucleases, an unexpected number of fragments were found on agarose gel electrophoresis. Molecular weight summation of fragments obtained from double restriction enzyme digestions suggested that the plasmid DNA was a mixture of two different plasmids. This was confirmed by constructing recombinant plasmids between S. kasugaensis plasmid DNA and pBR322, and then by isolating two plasmids after SalI endonuclease treatment followed by sucrose gradient centrifugation. One of the plasmids (pSK1) had a single recognition site for BamHI, EcoRI, and SalI, and three sites for BglII. The other plasmid (pSK2) had a single recognition site for EcoRI and BglII, two recognition sites for BamHI, and no cleavage site for SalI. The cleavage maps of these plasmids were constructed using several restriction endonucleases.     

DNA-dependent RNA polymerases from Drosophila melanogaster adults: Isolation and partial characterization

In preparation for the isolation and biochemical characterization of putative RNA polymerase mutants, DNA-dependent RNA polymerases of Drosophila melanogaster adults were isolated and partially characterized. Approximately 70% of the female adult RNA polymerase is located in ovaries. Multiple forms of ovarian RNA polymerases I and II are separable by DEAE-Sephadex chromatography. The two forms of RNA polymerase II differ in ammonium sulfate optima. RNA polymerase IIA is more active with double-stranded DNA as template, whereas RNA polymerase IIB transcribes single-stranded DNA most efficiently. Rechromatography of RNA polymerase IIA on DEAE-Sephadex results in the loss of ability of this form to transcribed double-stranded DNA most efficiently. Ovariectomized carcasses have two forms of RNA polymerase I and one form of RNA polymerase II and each transcribes single-stranded DNA most efficiently. As judged by gel filtration chromatography, female adult extracts have forms of RNA polymerase II that differ in molecular weight and template preference.Supported by Grants GM23456 from the NIH and 11259 from the City University Research Foundation.     

Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

DNA-dependent RNA polymerases from murine testis. Properties of two activities.

Multiple forms of DNA-dependent RNA polymerase activities have been isolated from nuclei of mouse testis. Using highly purified nuclei, two activities can be solubilized and are separable by DEAE-Sephadex chromatography; peak I eluting at 0.11–0.14 M and peak II eluting at 0.24–0.27 M (NH₄)₂SO₄. A third form of RNA polymerase activity is observed eluting at 0.31–0.33 M (NH₄)₂SO₄ when an extract from a less highly purified nuclear preparation is analysed. At concentrations of 0.125 μg/ml, peak I is insensitive to the toxin α-amanitin, peak II is totally inhibited, and peak III is partially inhibited. Peak I activity is optimal at pH 8.4 in the presence of Mg²⁺ (2–6 mM) or Mn²⁺ (1 mM) and uses native and heat-denatured DNA template equally well. Peak II has optimal activity at pH 7.9 in the presence of Mn²⁺ (2 mM) and heat-denatured DNA. Mg²⁺ has little effect on the activity of peak II.     

A Novel Type II NAD+-Specific Isocitrate Dehydrogenase from the Marine Bacterium Congregibacter litoralis KT71

In most living organisms, isocitrate dehydrogenases (IDHs) convert isocitrate into ɑ-ketoglutarate (ɑ-KG). Phylogenetic analyses divide the IDH protein family into two subgroups: types I and II. Based on cofactor usage, IDHs are either NAD⁺-specific (NAD-IDH) or NADP⁺-specific (NADP-IDH); NADP-IDH evolved from NAD-IDH. Type I IDHs include NAD-IDHs and NADP-IDHs; however, no type II NAD-IDHs have been reported to date. This study reports a novel type II NAD-IDH from the marine bacterium Congregibacter litoralis KT71 (ClIDH, GenBank accession no. EAQ96042). His-tagged recombinant ClIDH was produced in Escherichia coli and purified; the recombinant enzyme was NAD⁺-specific and showed no detectable activity with NADP⁺. The K _m values of the enzyme for NAD⁺ were 262.6±7.4 μM or 309.1±11.2 μM with Mg²⁺ or Mn²⁺ as the divalent cation, respectively. The coenzyme specificity of a ClIDH Asp487Arg/Leu488His mutant was altered, and the preference of the mutant for NADP⁺ was approximately 24-fold higher than that for NAD⁺, suggesting that ClIDH is an NAD⁺-specific ancestral enzyme in the type II IDH subgroup. Gel filtration and analytical ultracentrifugation analyses revealed the homohexameric structure of ClIDH, which is the first IDH hexamer discovered thus far. A 163-amino acid segment of CIIDH is essential to maintain its polymerization structure and activity, as a truncated version lacking this region forms a non-functional monomer. ClIDH was dependent on divalent cations, the most effective being Mn²⁺. The maximal activity of purified recombinant ClIDH was achieved at 35°C and pH 7.5, and a heat inactivation experiment showed that a 20-min incubation at 33°C caused a 50% loss of ClIDH activity. The discovery of a NAD⁺-specific, type II IDH fills a gap in the current classification of IDHs, and sheds light on the evolution of type II IDHs.     

Solvent effects on ouabain binding to the (Na+,K+)-ATPase of rat brain

The effects of the solvents deuterated water (²H₂O) and dimethyl sulfoxide (Me₂SO) on [³H]ouabain binding to (Na⁺,K⁺)-ATPase under different ligand conditions were examined. These solvents inhibited the type I ouabain binding to the enzyme (i.e., in the presence of Mg²⁺+ATP+Na⁺). In contrast, both solvents stimulated type II (i.e., Mg²⁺+P_i-, or Mn²⁺-dependent) binding of the drug. The solvent effects were not due to pH changes in the reaction. However, pH did influence ouabain binding in a differential manner, depending on the ligands present. For example, changes in pH from 7.05 to 7.86 caused a drop in the rate of binding by about 15% in the presence of Mg²⁺+Na⁺+ATP, 75% in the Mg²⁺+P_i system, and in the presence of Mn²⁺ an increase by 24% under similar conditions. Inhibitory or stimulatory effects of solvents were modified as various ligands, and their order of addition, were altered. Thus, ²H₂O inhibition of type I ouabain binding was dependent on Na⁺ concentration in the reaction and was reduced as Na⁺ was elevated. Contact of the enzyme with Me₂SO, prior to ligands for type I binding, resulted in a greater inhibition of ouabain binding than that when enzyme was exposed to Na⁺+ATP first and then to Me₂SO. Likewise, the stimulation of type II binding was greater when appropriate ligands acted on enzyme prior to addition of the solvent. Since Me₂SO and ²H₂O inhibit type I ouabain binding, it is proposed that this reaction is favored under conditions which promote loss of H₂O, and E₁ enzyme conformation; the stimulation of type II ouabain binding in the presence of the solvents suggests that this type of binding is favored under conditions which promote the presence of H₂O at the active enzyme center and E₂ enzyme conformation. This postulation of a role of H₂O in modulating enzyme conformations and ouabain interaction with them is in concordance with previous observations.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Incompatibility properties of Col E1 and pMB1 derivative plasmids: Random replication of multicopy replicons

The incompatibility properties of Col E1-like plasmids have been examined in Rec⁺ and RecA^? bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.     

Effect of altered efficiency of the RNA I and RNA II promoters on in vivo replication of ColE1-like plasmids in Escherichia coli

Summary Thermal inactivation of the dnaA gene product leads to a considerable decrease in the rate of replication of ColE1-like plasmids. To test the possiblity that the dnaA protein may affect synthesis of RNA I, which is an inhibitor of primer formation, or synthesis of RNA II, which is the primer precursor for replication of ColE1 (Tomizawa and Itoh 1982), the effect of the dnaA46 mutation on the efficiency of the RNA I and the RNA II promoters was examined. It appears that thermal inactivation of the dnaA protein results in a considerable increase in the activity of the RNA I promoter. We suggest that overproduction of RNA I in dnaA mutants grown at the restrictive temperature is responsible for the reduced replication of ColE1-like plasmids.It has been found that addition of rifampicin to cultures of the dnaA46 or the dna ⁺ strain grown at 42°C results in a dramatic increase in the rate of replication of ColE1-like plasmids. We show that the activity of the RNA II promoter at 42°C is exceptionally resistant to rifampicin. In the presence of the drug, this leads, to an altered ratio of RNA I to RNA II, in favor of the latter RNA species.     

Form II DNA-dependent RNA polymerase from Drosophila melanogaster: General in vitro catalytic properties and template interactions

Several in vitro properties of partially purified form II RNA polymerase from Drosophila melanogaster embryo nuclei are described. The enzyme preparation is free from contaminating RNase, protein kinase, and polyphosphate kinase activities and can be used to study the incorporation of -³²P-labeled nucleoside triphosphates. The enzyme exhibits a biphasic heat inactivation pattern which is probably related to differential lability of its two subforms. However, a considerable protection against heat inactivation is provided by the nucleoside triphosphates present in the in vitro reaction system such that the enzyme catalyzes RNA synthesis in a nearly linear mode for over 2 hr at 30 C. Two initiation inhibitors, rifamycin AF/013 and polyriboinosinic acid (poly[I]), were tested against this enzyme. Rifamycin AF/013 was found unsuitable for critical studies because of the high concentrations necessary for total inhibition (200 µg/ml) and particularly because of the obligate use of solvents which secondarily have a destabilizing effect on native DNA. Poly[I] was found to effectively block initiation at very low concentrations (1 µg/ml). The enzyme rapidly forms poly[I]-resistant preinitiation complexes on both double- and single-stranded DNA. These complexes decay with a half-life of 2.5–3 min. RNA synthesis from poly[I]-resistant complexes amounts to 10% of the total potential synthesis on both double- and single-stranded DNA. Enzyme-DNA saturation experiments indicate that the form II enzyme discriminates two types of sites on Drosophila DNA, tight binding and weak binding, from which RNA synthesis proceeds slowly and rapidly, respectively. The tight-binding sites appear to be analogous to those sites with which the enzyme is able to form poly[I]-resistant complexes.This investigation was supported by funds from The National Research Council of Canada (NRC A9722).