Formamide Gels Research Articles

New Insights into the Mechanism of Initial Transcription

RNA polymerases undergo substantial structural and functional changes in transitioning from sequence-specific initial transcription to stable and relatively sequence-independent elongation. Initially, transcribing complexes are characteristically unstable, yielding short abortive products on the path to elongation. However, protein mutations have been isolated in RNA polymerases that dramatically reduce abortive instability. Understanding these mutations is essential to understanding the energetics of initial transcription and promoter clearance. We demonstrate here that the P266L point mutation in T7 RNA polymerase, which shows dramatically reduced abortive cycling, also transitions to elongation later, i.e. at longer lengths of RNA. These two properties of the mutant are not necessarily coupled, but rather we propose that they both derive from a weakening of the barrier to RNA-DNA hybrid-driven rotation of the promoter binding N-terminal platform, a motion necessary to achieve programmatically timed release of promoter contacts in the transition to elongation. Parallels in the multisubunit RNA polymerases are discussed.

Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11

In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His(44), Met(90), His(97), and His(127), and CuB, a second degenerate octahedral geometry with the addition of Glu(46). The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

(286) Identification of Genes in Tomato Roots Induced by Aluminum Stress using Fluorescent cDNA Differential Display

The goal of this study was isolate genes that are regulated by Al treatments of tomato roots growing in vitro. For Al treatment, germinating tomato seeds were plated on a MS agar medium supplemented with 0, 350, and 1200 μM AlCl3 for 30 days. Total RNA was extracted from root tissues and separated on denature formamide gel to check their quantity and quality. Equal amount of total RNA from treatment and control was treated with DNAse I (Genhunter, TN) to remove genomic DNA contamination. cDNA was obtained by reverse transcription using all the regents in RNA Image Kit (Genhunter, TN). The cDNA was amplified using the fluorescently labeled anchor primers (Oligo dT-A, C, G) and 16 random primers. Amplification products were separated by electrophoresis in 6% nondenaturing polyacrylamide gels and DNA bands were observed by scanning the gel on a FMBIOIII scanner. After comparing the band profile on the gel image, fragments of gene that showed changes in intensity compared to control (0 μM AlCl3) were isolated from the gel manually. These bands were re-amplified with the same pair of primers as the original amplification and cloned onto PCR-Trap cloning vector (Genhunter, TN). After DNA sequence analysis and homology comparison with NCBI database, we have identified clone # C01HBa0256E08 on L. esculentum chromosome 01, clone # C10HBa0111D09 on chromosome 10 and clone # LE_HBa-31H5 on chromosome 4.

Identification of a Ubiquinone-binding Site That Affects Autophosphorylation of the Sensor Kinase RegB

Rhodobacter capsulatus regulates many metabolic processes in response to the level of environmental oxygen and the energy state of the cell. One of the key global redox regulators of the cell's metabolic physiology is the sensor kinase RegB that controls the synthesis of numerous energy generation and utilization processes. In this study, we have succeeded in purifying full-length RegB containing six transmembrane-spanning elements. Exogenous addition of excess oxidized coenzyme Q1 is capable of inhibiting RegB autophosphorylation approximately 6-fold. However, the addition of reduced coenzyme Q1 exhibits no inhibitory effect on kinase activity. A ubiquinone-binding site, as defined by azidoquinone photo affinity cross-linking, was determined to lie within a periplasmic loop between transmembrane helices 3 and 4 that contains a fully conserved heptapeptide sequence of GGXXNPF. Mutation of the phenylalanine in this heptapeptide renders RegB constitutively active in vivo, indicating that this domain is responsible for sensing the redox state of the ubiquinone pool and subsequently controlling RegB autophosphorylation.

Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos.

Retinoic acid (RA) is a physiological agent that has a wide range of biological activity and appears to regulate developmental programs of vertebrates. However, little is known about the molecular basis of its metabolism. Here we have identified a novel cytochrome P450 (P450RA) that specifically metabolizes RA. In vitro, P450RA converts all-trans RA into 5,8-epoxy all-trans RA. P450RA metabolizes other biologically active RAs such as 9-cis RA and 13-cis RA, but fails to metabolize their precursors, retinol and retinal. Overexpression of P450RA in cell culture renders the cells hyposensitive to all-trans RA. These functional tests in vitro and in vivo indicate that P450RA inactivates RA. The P450RA gene is not expressed uniformly but in a stage- and region-specific fashion during mouse development. The major expression domains in developing embryos include the posterior neural plate and neural crest cells for cranial ganglia. The expression of P450RA, however, is not necessarily inducible by excess RA. These results suggest that P450RA regulates the intracellular level of RA and may be involved in setting up the uneven distribution of active RA in mammalian embryos.

Reevaluation of the exact CAG repeat length in hereditary cerebellar ataxias using highly denaturing conditions and long PCR

Hereditary cerebellar ataxias, including spinocerebellar ataxia type I (SCA1), dentato-rubro-pallidoluysian atrophy (DRPLA), and Machado-Joseph disease (MJD), have been associated with unstable CAG repeats. The length of the CAG repeat is a major factor in determining the age of onset of these diseases. In electrophoresis through acrylamide gels with formamide, the CAG repeat length following the polymerase chain reaction (PCR) coincides with the sequence-determined repeat length after subcloning. However, without formamide, PCR products with long CAG repeats appear 1-4 repeats shorter than when electrophoresed with formamide, and the repeat lengths are variable. In addition, the larger the CAG repeats are, the more difficult are the PCR reactions. A mixture containing thermostable Taq and Pwo DNA polymerases (so-called "long PCR") is much more sensitive than that with Taq polymerase alone in detecting- expanded CAG repeats. Therefore, highly denaturing conditions, especially formamide gel electrophoresis, and the "long PCR" protocol should be used to evaluate the exact CAG repeat length. We have used these principles to detect unstable CAG repeats. The normal ranges are 14-34 repeats for MJD, 6-31 repeats for DRPLA, and 21-32 repeats for SCA1.

Ultra-low expansion glass from gels

Pyrolysis of mixed titanium and silicon metal halides produces a commercial glass (7.4% TiO2) with ultra-low thermal expansion that is essentially zero over the temperature range of 0 to 300°C. A colloidal particulate gel process involving potassium silicate, titania sol and formamide gel reagent was found to produce glass compositions with similar low expansion behavior. Due to the strongly basic nature of the precursor solutions, special titania sols had to be prepared that were stable in these alkali silicate solutions. The preferred TiO2 sols were those containing quaternary ammonium stabilizing counter-ions. These sols served not only as the source of homogeneously distributed titania, but they may also serve as nucleating species that contribute to particle growth and pore size control of the gel network. The large pore (∼0.3 µm) TiO2/SiO2 gel structures were easily dealkalized, dried and sintered to uncracked glass shapes. Plates up to 9.5 cm×6.6 cm× 0.5 cm thick and some intricate cast shapes were produced and their glass properties evaluated.

Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47°C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.

The effect of essential amino acid deprivation on poly(A) metabolism in Neurospora crassa

Poly(A) messenger RNA was isolated from a methionine auxotroph of Neurospora crassa grown to exponential and early stationary phases, and from mycelia deprived of the essential amino acid. The polyadenylate regions were isolated from the mRNA preparations by enzymatic digestion and affinity chromatography, and their lengths determined by electrophoresis on 10% acrylamide 90% formamide gels. There was no significant difference in the lengths of the poly(A) regions between exponential and early stationary phase cultures. However, the poly(A) regions of mRNA isolated from cells undergoing amino acid deprivation were larger and more heterogeneous than the poly(A) regions isolated from cells grown in amino acid-supplemented medium. Whole cell extracts prepared from both amino acid-supplemented and amino acid-deprived cultures were assayed for their ability to synthesize and degrade poly(A). No significant difference between the two extracts was detected in either poly(A) polymerase or poly(A)-degrading activities. It was concluded that continuing translational activity is required for the shortening of poly(A) tracts in N. crassa.

Tissue- and stage-specific RNA patterns in Drosophila as revealed by gradient formamide gel electrophoresis.

The in vitro labeled RNAs in the accessory glands from male adults, and in the salivary glands and fat body from developing larvae of Drosophila melanogaster were separated on linear gradient acrylamide gels in formamide. The patterns appear to be tissue- and stage-specific, and several lines of evidence indicate that the visualized radioactive bands include both hnRNA and messages.

Transcription and processing of rRNA in higher plant cells (Daucus carota, Petroselinum crispum, Acer pseudoplatanus)

Actively dividing cells from parsley (Petroselinum crispum) and carrot (Daucus carota) (bothApiaceae) andAcer pseudoplatanus (Aceraceae) were used to detect the primary gene product of the rDNA in plant cells. Parsley and carrot cells were labelled with [32P] orthophosphate. In both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels under non-denaturing conditions. Its molecular weight did not exceed 2.5 × 106 daltons. The component emerged from the heterogenous material after a labelling period of 5–10 min. In parsley cells 45 min after onset of incubation labelled mature rRNA (25S and 18S) arrived in the cytoplasm. InAcer pseudoplatanus (incubation period 60 min) two rapidly labelled components did emerge from polyacrylamide gels; their molecular weights were 2.3 and 3.2− 3.4 × 106 daltons. After electrophoresis under denaturing conditions the larger component was no longer present, thus indicating that it was an aggregate of different RNA molecules. The molecular weights of the rRNA precursors ofD. carota andP. crispum determined after electrophoresis in formamide gels were about 2.1 × 106 daltons. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 × 106 daltons.

Alternate Capping Mechanisms for Transcription of Spring Viremia of Carp Virus: Evidence for Independent mRNA Initiation

Two alternate mechanisms of mRNA capping for spring viremia of carp virus have been observed. Under normal reaction conditions, a ppG residue of the capping GTP is transferred to a pA moiety of the 5' termini of mRNA transcripts. However, in reaction conditions where GppNHp is used instead of GTP, an alternate capping mechanism occurs whereby a pG residue of the capping GTP is transferred to a ppA moiety of the transcripts. The first mechanism is identical to that described previously for vesicular stomatitis virus (G. Abraham, D. P. Rhodes, and A. K. Banerjee, Nature [London] 255:37-40, 1975; A. K. Banerjee, S. A. Moyer, and D. P. Rhodes, Virology 61:547-558, 1974), and thus appears to be a conserved function during the evolution of rhabdoviruses. The alternate mechanism of capping indicates not only that capping can take place by two procedures, but also that the substrate termini have di- or triphosphate 5' ends, indicating that they are probably independently initiated. An analog of ATP, AppNHp, has been found to completely inhibit the initiation of transcription by spring viremia of carp virus, suggesting that a cleavage between the beta and gamma phosphates of ATP is essential for the initiation of transcription. However, in the presence of GppNHp, uncapped (ppAp and pppAp), capped (GpppAp), and capped methylated (m7GpppAmpAp and GpppAmpAp) transcripts are detected. Size analyses of oligodeoxythymidylic acid-cellulose-bound transcripts resolved by formamide gel electrophoresis demonstrated that full-size mRNA transcripts are synthesized as well as larger RNA species. The presence of GppNHp and S-adenosylhomocysteine in reaction mixtures did not have any effect on the type of unmethylated transcription products. Our results favor a transcription model postulated previously (D. H. L. Bishop, in H. Fraenkel-Conrat and R. R. Wagner, ed., Comprehensive Virology, vol. 10, Plenum Press, New York, 1977; D. H. L. Bishop and A. Flamand, in D. C. Burke and W. C. Russell, ed., Control Processes in Virus Multiplication, Cambridge University Press, Cambridge, 1975; D. H. L. Bishop and M. S. Smith, in D. Nayak, ed., The Molecular Biology of Animal Viruses, Marcel Dekker, New York, 1977; P. Roy and D. H. L. Bishop, J. Virol. 11:487-501, 1973) in which mRNA synthesis is initiated independently; they do not support a model for transcripts being synthesized by plus-strand cleavage (A. K. Banerjee, G. Abraham, and R. J. Colonno, J. Gen. Virol. 34:1-8, 1977; A. K. Banerjee, R. J. Colonno, D. Testa, and M. T. Franze-Fernandez, in B. M. J. Mahy and R. D. Barry, ed., Negative Strand Viruses and the Host Cells, Academic Press, London, 1978).

Aminoacylation and messenger functions of eggplant mosaic virus RNA

The predominant RNA of eggplant mosaic virus (EMV) was found to have a molecular weight of 1.9 × 10 6 by formamide gel electrophoresis. Electrophoretic analysis of virion protein under dissociating conditions revealed two polypeptides, a major component of 21,000 daltons, and a minor component of 22,000 daltons. Both peptides were present in translation products coded by RNA isolated from virions, but the proportion of the 22,000-dalton peptide was higher in products synthesized using RNA isolated from EMV-infected Datura leaves as messenger. Since the viral RNA showed a marked tendency to aggregate, it is possible that these polypeptides were translated from trace amounts of small EMV RNAs present as contaminants of the 1.9 × 10 5-dalton genomic RNA. Although the addition of tRNA from infected or healthy Datura leaves, or from wheat germ, stimulated amino acid incorporation, no changes were discerned in the profile of cell-free translation products after electrophoretic separation. Nonaminoacylated and valylated EMV RNA stimulated similar levels of amino acid incorporation, and the translation products appeared identical. Valine bound to genomic EMV RNA was not donated during protein synthesis.

The Molecular Weight of rRNA Precursor Molecules and Their Processing in Higher Plant Cells

Actively dividing callus cells of higher plants (Petroselinum crispum, Daucus carota, Acer pseudoplatanus) were used to detect the primary gene product of rDNA in vivo. Parsley and carrot cells were labelled with [32P]orthophosphate. Under non-denaturing conditions, in both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels. Its molecular weight did not exceed 2.5 x 10(6) dalton. Under denaturing conditions, 2.0--2.1 x 10(6) dalton were determined on formamide gels. This rRNA precursor was already present after a labelling period of 5--10 min. In parsley cells labelled mature rRNA (25S and 18S) arrived in the cytoplasm 45 min after onset of incubation. In Acer pseudoplatanus incubated with [3H]uridine two rapidly labelled components did emerge from polyacrylamide gels without formamide; their molecular weights were 2.3 and 3.2--3.4 x 10(6) dalton. After electrophoresis in formamide, the larger component disappeared, thus indicating that it would be an intermolecular aggregate of different RNAs. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 x 10(6) dalton.

Precursors of alpha and beta globin messenger RNAs

Several structural properties of the pulse-labeled globin messenger RNA precursor molecules that sediment faster than steady-state (10 S) globin mRNA (Curtis & Weissmann, 1976; Ross, 1976; Kwan et al., 1977; Bastos & Aviv, 1977) are investigated. The radioactive, globin-specific RNA resolves into two peaks during electrophoresis in polyacrylamide gels prepared in 98% formamide. The molecular weights of the larger and the smaller RNAs are 600,000 and 280,000, respectively, as compared with the average molecule weight of cytoplasmic globin mRNA, 200,000. When RNA from cells labeled for 10 minutes is analyzed by formamide gel electrophoresis, a precursor larger than 600,000 M r is not detected. Most, if not all, of the molecules in the 600,000 and 280,000 M r RNA fractions are polyadenylated. The 600,000 M r RNA contains β globin mRNA sequences, but little, if any, a globin sequences; the 280,000 M r RNA contains both α and β sequences. At steady-state the 14-day-old mouse fetal liver erythroblast contains approximately 50 molecules of the 600,000 M r RNA, 1000 molecules of the 280,000 M r RNAs, and 60,000 molecules of mature globin mRNA. The half-lives of the 600,000 and 280,000 M r RNAs calculated using steady-state levels are 2 minutes and 17 minutes, respectively. These data demonstrate that β globin gene sequences are transcribed into a molecule (β-precursor) that is threefold larger than mature β globin mRNA. In contrast, the largest transcript of the α globin genes detected in these experiments is only 1.4-fold larger than mature α globin mRNA. The possibility that the 280,000 M r, β-specific RNA is a processing intermediate generated by cleavage of the 600,000 M r β-precursor is discussed, along with alternative hypotheses.

Preparation of a complementary DNA for leghaemoglobin and direct demonstration that leghaemoglobin is encoded by the soybean genome.

In soybean root nodules, leghaemoglobin (Lb) accounts for 25--30% of the total soluble protein but is not detected in other tissues. In order to determine whether the Lb genes are plant or bacterial in origin a cDNA probe for Lb was prepared from 9S poly (A) containing mRNA of root nodules. Although this 9S mRNA directed synthesis of predominantly three forms of Lb in vitro, the kinetics of hybridisation of cDNA and the 9S mRNA showed a transition at about 30% hybridisation which suggested that the 9S-cDNA was not pure Lb-cDNA. The abundant, Lb-cDNA was prepared by two cycles of hybridising 9S mRNA and cDNA to a Rot of 3 X 10(-3) and isolation of the hybridised cDNA on hydroxyapatite. The Lb-cDNA was homogeneous in hybridisation analysis with 9S mRNA and electrophoresis in 98% formamide gels. This cDNA hybridised with soybean DNA and not with Rhizobium DNA, thus directly demonstrating that Lb genes are of plant origin. Titration of Lb-cDNA with soybean DNA showed that Lb genes are reiterated about forty-fold per haploid genome.

Species identification and genome mapping of cytoplasmic adenovirus type 2 RNAs synthesized late in infection.

Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R.EcoRI and endo R.SmaI. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The SmaI-D region (0.38 to 0.51 on the unit genome) and the EcoRI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by SmaI-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity of the respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experiments. The multiple transcripts encoded by the EcoRI-D fragment were found to contain sequences that are present in early cytoplasmic RNA. These studies enabled preparation of a map which accounts for transcription of approximately 67% of the r strand of the adenovirus 2 genome.

Synthesis of RNA from cellulose-bound complementary DNA.

Radioactively labeled RNAs were synthesized from cellulose-bound cDNA templates using Escherichia coli RNA polymerase. Hybridization of this RNA to excess unlabeled cDNA approached 100%, indicating the complementarity of product and template. The average length of the RNA product, as determined by formamide gels, was approximately 40% of the template length. Hybridization of unlabeled globin RNA produced by this technique to labeled globin cDNA indicated the population of RNA sequences represented at least 80% of the template sequences. Approximately 30% of the RNA product by mass contains poly(A) tails as determined by binding to oligo(dT)-cellulose. The template can be reused for several cycles of synthesis with little loss of synthetic capability and therefore, can amplify the amount of mRNA initially used to produce the template.

Phylogenetic Relationships Between Mycoplasmas and Other Procaryotes Based upon the Electrophoretic Behavior of Their Ribosomal Ribonucleic Acids

The migration of ribosomal ribonucleic acids (rRNA's) from bacteria, bacterial L forms, mycoplasmas, and acholeplasmas in nondenaturing and formamide gels has been examined. Mycoplasmal and acholeplasmal RNAs migrate in a pattern distinct from each other and from bacteria in nondenaturing gels. The differences observed appear to be mainly due to conformational differences in the molecules. When examined under denaturing conditions in formamide gels, the 23S RNAs of all organisms examined comigrated. The 16S RNAs of mycoplasmas and acholeplasmas comigrated and appear to be of a smaller molecular weight than bacterial 16S RNA. It is suggested that the differences observed are indicative of significant phylogenetic divergence between mycoplasmas and bacteria or bacterial L forms.