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PubMed1995

An A-DNA triplet code: thermodynamic rules for predicting A- and B-DNA.

The ability to predict macromolecular conformations from sequence and thermodynamic principles has long been coveted but generally has not been achieved. We show that differences in the hydration of DNA surfaces can be used to distinguish between sequences that form A- and B-DNA. From this, a "triplet code" of A-DNA propensities was derived as energetic rules for predicting A-DNA formation. This code correctly predicted > 90% of A- and B-DNA sequences in crystals and correlates with A-DNA formation in solution. Thus, with our previous studies on Z-DNA, we now have a single method to predict the relative stability of sequences in the three standard DNA duplex conformations.

Base Sequence↗

PubMedDate not supplied

Molecular cloning and sequencing of a human hepatitis delta (delta) virus RNA.

Human hepatitis delta (delta) virus (HDV) is a form of defective virus, which infects humans only in the presence of a co-infecting hepatitis B virus (HBV). HDV superinfection in a chronic HBV carrier often results in severe chronic hepatitis and cirrhosis, whereas acute HDV and HBV co-infection is frequently associated with fulminant hepatitis. HDV consists of a 36-nm particle, which contains an envelope with HBV surface antigen, and a nucleocapsid containing the hepatitis delta-antigen (HDAg) and an RNA genome of 1.75 kilobases (kb). Recently, the genomic RNA from an HDV serially passaged in chimpanzees has been cloned and sequenced in a study which showed that the HDV RNA is a single-stranded circular molecule with properties similar to those of viroid or virusoid. However, it is not known whether serial passages in chimpanzees had altered the properties of human HDV. Here we report the cloning and sequencing of an HDV RNA isolated directly from a patient with acute delta-hepatitis. The sequence showed considerable divergence (11%) from that of the chimpanzee-adapted HDV. Five open reading frames (ORFs) of more than 100 amino acids in both genomic and anti-genomic sense were found. The largest ORF in antigenomic sense, which can code for 214 amino acids, may correspond to the HDAg.

Amino Acid Sequence↗

PubMed1995

Collagen binding activity of recombinant and N-terminally modified annexin V (anchorin CII).

We have cloned the full coding cDNA sequence of chicken annexin V and of a mutant lacking 8 amino acid residues of the N-terminal tail for prokaryotic expression. Both proteins were synthesized in Escherichia coli upon induction with isopropyl thio-beta-D-galactoside, and were purified following two different protocols: one based on the ability of these proteins to interact reversibly with liposomes in the presence of calcium, and the other based on two sequential ion-exchange chromatographic steps. Spectroscopical analysis of recombinant annexin V revealed that binding of calcium did not change the circular dichroism spectra indicating no significant changes on the secondary structure; however, a conformational change affecting the exposition to the solvent of the tryptophan residue 187 was detected by analysis of fluorescence emission spectra. Recombinant annexin V binds with high affinity to collagen types II and X, and with lower affinity to collagen type I in a calcium-independent manner. Heat denaturing of collagen decreases this interaction while pepsin-treatment of collagen almost completely abolishes annexin V binding. Mutated annexin V interacts with collagen in a similar way as the nonmutated recombinant protein, indicating that the N-terminal tail of annexin V is not essential for collagen binding.

Animals↗

PubMed1979

Poly(2-fluoroadenylic acid). The role of basicity in the stabilization of complementary helices.

The polymerization of 2-fluoroadenosine 5'-diphosphate by polynucleotide phosphorylase to give high molecular weight poly(2-fluoroadenylic acid), poly(fl2A), is described. Both the single-stranded and double-stranded (acid) forms of poly(fl2A) exhibit strikingly similar ultraviolet and circular dichroism spectra to those of poly(A), and the enzymatic polymerization rates and thermal hyperchromicities of the two polymers are also very similar. However, the pKa of poly(fl2A) for protonation at N-1 is 2.9 compared to 5.9 for poly(A) under similar conditions. Poly(fl2A) forms a triple-stranded helix with poly(U) which has ultraviolet and cd spectra very reminiscent of poly(A) . 2 poly(U), but no conditions could be found which permitted the formation of a double helix. In the Escherichia coli ribosome system poly(fl2A) codes for the synthesis of polylysine, as does poly(A), although the rate and extent of incorporation were less in the former case. The role of basicity of adenine N-1 in these interactions is discussed.

Drug Stability↗

PubMed1976

The relative positions of sea urchin histone genes on the chimeric plasmids pSp2 and pSp17 as studied by electronmicroscopy.

The relative positions of the sea urchin histone genes and the spacer regions on the chimeric plasmids pS p2 and pSp17 have been mapped by hybridizing total histonemessenger RNA to single strands of the plasmid DNAs. The lengths and spacing between the several RNA:DNA duplex regions on the single strands of DNA were measured by the gene 32-ethidium bromide electron microscope mapping method. We find that the genes are interdigitated with spacer sequences of different lengths; that there are three coding sequences on pSp2, all on the same strand, with the relative order H1, H4, and B4; and that there are two coding sequences on pSp17, both on the same strand, corresponding to the messages denoted B1 and B2-B3, where B4, B1, and B2-3 are electrophoretically resolved components of histone mRNA, all of size intermediate between the larger H1 and the smaller H4 message.

Chimera↗

PubMed1998

Engineering, expression and biochemical characterization of the hemoglobin domain of a Erwinia chrysanthemi flavohemoprotein.

An artificial hemoglobin-like domain has been constructed by engineering the gene coding for the multi-domain flavohemoprotein from the bacterium Erwinia chrysanthemi. This domain was designed by molecular modelling, cloned and over-expressed in Escherichia coli. The holo-protein was obtained in large quantities after extraction from inclusion bodies and refolding in presence of alkaline hemin. The purified 140-residue domain was studied and characterized to gain new insights into the biochemical function of the recombinant domain and the biological role of this new flavohemoprotein. The structural and functional features of this domain in solution were studied using far-ultraviolet circular dichroism, resonance Raman, proton-NMR spectroscopy, flash laser photolysis and molecular modelling. The recombinant domain is shown to be folded properly and active. This hemoglobin-like domain is able to bind oxygen and carbon monoxide with very high affinity. It exhibits a rapid auto-oxidation which may explain its tight association with a flavin containing reductase domain. A functional model of this hemoglobin is discussed and compared with the X-ray structures of other hemoproteins.

Circular Dichroism↗

PubMed1998

Characterization of the C-terminal propeptide involved in bacterial wall spanning of alpha-amylase from the psychrophile Alteromonas haloplanctis.

The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at the mid-exponential phase and is cleaved by a nonspecific protease into the mature enzyme and the propeptide. The purified C-terminal propeptide displays several features common to beta-pleated transmembrane proteins. It has no intramolecular chaperone function because active alpha-amylase is expressed by Escherichia coli in the absence of the propeptide coding region. In E. coli, the 70-kDa precursor is directed toward the supernatant. When the alpha-amylase coding region is excised from the gene, the secretion helper can still promote its own membrane spanning. It can also accept a foreign passenger, as shown by the extracellular routing of a beta-lactamase-propeptide fusion protein.

Amino Acid Sequence↗

PubMed2003

Ionization chamber dosimetry of small photon fields: a Monte Carlo study on stopping-power ratios for radiosurgery and IMRT beams.

Absolute dosimetry with ionization chambers of the narrow photon fields used in stereotactic techniques and IMRT beamlets is constrained by lack of electron equilibrium in the radiation field. It is questionable that stopping-power ratio in dosimetry protocols, obtained for broad photon beams and quasi-electron equilibrium conditions, can be used in the dosimetry of narrow fields while keeping the uncertainty at the same level as for the broad beams used in accelerator calibrations. Monte Carlo simulations have been performed for two 6 MV clinical accelerators (Elekta SL-18 and Siemens Mevatron Primus), equipped with radiosurgery applicators and MLC. Narrow circular and Z-shaped on-axis and off-axis fields, as well as broad IMRT configured beams, have been simulated together with reference 10 x 10 cm2 beams. Phase-space data have been used to generate 3D dose distributions which have been compared satisfactorily with experimental profiles (ion chamber, diodes and film). Photon and electron spectra at various depths in water have been calculated, followed by Spencer-Attix (delta = 10 keV) stopping-power ratio calculations which have been compared to those used in the IAEA TRS-398 code of practice. For water/air and PMMA/air stopping-power ratios, agreements within 0.1% have been obtained for the 10 x 10 cm2 fields. For radiosurgery applicators and narrow MLC beams, the calculated s(w,air) values agree with the reference within +/-0.3%, well within the estimated standard uncertainty of the reference stopping-power ratios (0.5%). Ionization chamber dosimetry of narrow beams at the photon qualities used in this work (6 MV) can therefore be based on stopping-power ratios data in dosimetry protocols. For a modulated 6 MV broad beam used in clinical IMRT, s(w,air) agrees within 0.1% with the value for 10 x 10 cm2, confirming that at low energies IMRT absolute dosimetry can also be based on data for open reference fields. At higher energies (24 MV) the difference in s(w,air) was up to 1.1%, indicating that the use of protocol data for narrow beams in such cases is less accurate than at low energies, and detailed calculations of the dosimetry parameters involved should be performed if similar accuracy to that of 6 MV is sought.

Algorithms↗

PubMed2005

Monte Carlo techniques for scattering foil design and dosimetry in total skin electron irradiations.

Total skin electron irradiation (TSEI) with single fields requires large electron beams having good dose uniformity, dmax at the skin surface, and low bremsstrahlung contamination. To satisfy these requirements, energy degraders and scattering foils have to be specially designed for the given accelerator and treatment room. We used Monte Carlo (MC) techniques based on EGS4 user codes (BEAM, DOSXYZ, and DOSRZ) as a guide in the beam modifier design of our TSEI system. The dosimetric characteristics at the treatment distance of 382 cm source-to-surface distance (SSD) were verified experimentally using a linear array of 47 ion chambers, a parallel plate chamber, and radiochromic film. By matching MC simulations to standard beam measurements at 100 cm SSD, the parameters of the electron beam incident on the vacuum window were determined. Best match was achieved assuming that electrons were monoenergetic at 6.72 MeV, parallel, and distributed in a circular pattern having a Gaussian radial distribution with full width at half maximum = 0.13 cm. These parameters were then used to simulate our TSEI unit with various scattering foils. Two of the foils were fabricated and experimentally evaluated by measuring off-axis dose uniformity and depth doses. A scattering foil, consisting of a 12 x 12 cm2 aluminum plate of 0.6 cm thickness and placed at isocenter perpendicular to the beam direction, was considered optimal. It produced a beam that was flat within +/-3% up to 60 cm off-axis distance, dropped by not more than 8% at a distance of 90 cm, and had an x-ray contamination of <3%. For stationary beams, MC-computed dmax, Rp, and R50 agreed with measurements within 0.5 mm. The MC-predicted surface dose of the rotating phantom was 41% of the dose rate at dmax of the stationary phantom, whereas our calculations based on a semiempirical formula in the literature yielded a drop to 42%. The MC simulations provided the guideline of beam modifier design for TSEI and estimated the dosimetric performance for stationary and rotational irradiations.

Calibration↗

PubMed2005

First sequenced mitochondrial genome from the phylum Acanthocephala (Leptorhynchoides thecatus) and its phylogenetic position within Metazoa.

The complete sequence of the mitochondrial genome of Leptorhynchoides thecatus (Acanthocephala) was determined, and a phylogenetic analysis was carried out to determine its placement within Metazoa. The genome is circular, 13,888 bp, and contains at least 36 of the 37 genes typically found in animal mitochondrial genomes. The genes for the large and small ribosomal RNA subunits are shorter than those of most metazoans, and the structures of most of the tRNA genes are atypical. There are two significant noncoding regions (377 and 294 bp), which are the best candidates for a control region; however, these regions do not appear similar to any of the control regions of other animals studied to date. The amino acid and nucleotide sequences of the protein coding genes of L. thecatus and 25 other metazoan taxa were used in both maximum likelihood and maximum parsimony phylogenetic analyses. Results indicate that among taxa with available mitochondrial genome sequences, Platyhelminthes is the closest relative to L. thecatus, which together are the sister taxon of Nematoda; however, long branches and/or base composition bias could be responsible for this result. The monophyly of Ecdysozoa, molting organisms, was not supported by any of the analyses. This study represents the first mitochondrial genome of an acanthocephalan to be sequenced and will allow further studies of systematics, population genetics, and genome evolution.

Acanthocephala↗

PubMed1991

Immunohistochemical identification of cholinergic neurons in the myenteric plexus of guinea-pig small intestine.

It is well established that acetylcholine is a neurotransmitter at several distinct sites in the mammalian enteric nervous system. However, identification of the cholinergic neurons has not been possible due to an inability to selectively label enteric cholinergic neurons. In the present study an immunohistochemical method has been developed to localize choline acetyltransferase, the synthetic enzyme for acetylcholine, in order that cholinergic neurons can be visualized. The morphology, neurochemical coding and projections of cholinergic neurons in the guinea-pig small intestine were determined using double-labelling immunohistochemistry. These experiments have revealed that many myenteric neurons are cholinergic and that they can be distinguished by their specific combinations of immunoreactivity for neurochemicals such as calretinin, neurofilament protein triplet, substance P, enkephalin, somatostatin, 5-hydroxytryptamine, vasoactive intestinal peptide and calbindin. On the basis of their previously described projections, functional roles could be attributed to each of these populations. The identified cholinergic neurons are: motorneurons to the longitudinal muscle (choline acetyltransferase/calretinin); motorneurons to the circular muscle (choline acetyltransferase/neurofilament triplet protein/substance P, choline acetyltransferase/substance P and choline acetyltransferase alone); orally directed interneurons in the myenteric plexus (choline acetyltransferase/calretinin/enkephalin); anally directed interneurons in the myenteric plexus (choline acetyltransferase/somatostatin, choline acetyltransferase/5-hydroxytryptamine, choline acetyltransferase/vasoactive intestinal peptide); secretomotor neurons to the mucosa (choline acetyltransferase/somatostatin); and sensory neurons mediating myenteric reflexes (choline acetyltransferase/calbindin). This information provides a unique opportunity to identify functionally distinct populations of cholinergic neurons and will be of value in the interpretation of physiological and pharmacological studies of enteric neuronal circuitry.

Animals↗

PubMed1976

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.

Chromosome Mapping↗

PubMed2005

Hepatitis B virus DNA integration in hepatocellular carcinoma after interferon-induced disappearance of hepatitis C virus.

OBJECTIVES: Hepatocellular carcinoma (HCC) has been reported in patients in whom hepatitis C virus (HCV) was eliminated by interferon (IFN) therapy. We examined the pathogenesis of HCC in patients with sustained viral response. METHODS: Operable HCC developed in 7 of 342 patients cured of HCV infection by IFN monotherapy. No patient abused alcohol or had diabetes mellitus or obesity. Resected specimens of HCC were histologically evaluated. DNA extracted from HCC was examined by polymerase chain reaction (PCR) to locate hepatitis B virus (HBV) DNA. HBV integration sites in human genome were identified by cassette-ligation-mediated PCR. RESULTS: HBV DNA was not amplified in serum samples from any of the seven patients with HCC and was found in liver in four patients. In the latter four patients, HBV DNA was integrated into the human genome of HCC. In two of these patients, covalently closed circular HBV (cccHBV) was also detected. The patients with HBV DNA integration were free of HCV for more than 3 yr. In two of the three patients without HBV DNA integration, the surrounding liver showed cirrhosis. The liver of HCC with HBV DNA integration had not progressed to cirrhosis. Three of the four tumors with HBV integration had one integration site each, located at chromosomes 11q12, 11q22-23, and 22q11, respectively. The other tumor had two integration sites, situated at chromosomes 11q13 and 14q32. At chromosome 11q12, HBV DNA was integrated into protein-coding genome, the function of which remains unclear. CONCLUSION: Integrated HBV DNA may play a role in hepatocarcinogenesis after the clearance of HCV by IFN treatment.

Aged↗

PubMed1987

Stress-behavior interactions in hamster tumor growth.

The aim of this study was to investigate the effects of a mechanical stressor and individual behavior differences (separately and in combination) on tumor development in the female Syrian hamster. Studies by other investigators have documented the tumor-enhancing effects of such mechanical stressors as rotational stress. Previous studies by our group found that both size of tumor and time to tumor detection were significantly related to a dimension we call "activation." Eighty 100-day old female Syrian hamsters were placed in circular plexiglas environments in groups of 10. Nineteen days after introduction to the cages, a stress condition was imposed on half the animals (four cages). This consisted of shaking each cage of animals three times a week for three 10-minute intervals. Each group's behavior was videotaped in multiple samples to document pre- and poststress behaviors. Twelve days after the stress condition was initiated, each animal was injected subcutaneously midback with one melanoma tumor fraction. Animals were palpated every three days to determine time to detection of tumor. The videotaped behavior samples were coded for behaviors associated with "activation," inactivity, and interaction. Factor analysis resulted in basically the same first factor of activation found in our previous studies. Hamsters in the nonstressed groups had a significantly longer time to tumor development than those in the stressed groups (22.5 days vs. 12.6 days, p less than 0.005). While no prestress behaviors were associated significantly with time to tumor detection, the poststress activation factor was significantly correlated with longer time to tumor development in the stressed group (r = .61, p less than 0.0001). These results suggest that while the stress condition is more powerful than prestress individual behaviors in affecting the outcome variable, stress appears to interact with the individual behaviors related to "activation" to mitigate the negative effects of stress on tumor growth.

Animals↗

PubMed1987

Primary and secondary structure of bovine retinal S antigen (48-kDa protein).

The complete amino acid sequence of bovine S antigen (48-kDa protein) has been determined by cDNA and partial amino acid sequencing. A 1623-base-pair (bp) cDNA contains an open reading frame coding for a protein of 404 amino acids (45,275 Da). Tryptic peptides and cyanogen bromide peptides of native bovine S antigen were purified and partially sequenced. All of these peptides were accounted for in the long open reading frame. Searching of the National Biomedical Research Foundation data bank revealed no extensive sequence homology between S antigen and other proteins. However, there are local regions of sequence similarity with alpha transducin, including the sites subject to ADP-ribosylation by Bordetella pertussis and cholera toxins and the phosphoryl binding-sites. Secondary structure prediction and circular dichroic spectroscopy show that S antigen is composed predominantly of beta-sheet conformation. Acid-catalyzed methanolysis suggests the presence of low levels of carbohydrate in the molecule.

Amino Acid Sequence↗

PubMed1988

Genetic analysis of tomato golden mosaic virus: the coat protein is not required for systemic spread or symptom development.

The geminiviruses are a unique group of higher plant viruses that are composed of twin isometric particles which contain circular, single-stranded DNA. Tomato golden mosaic virus (TGMV), a whitefly-transmitted agent, belongs to the subgroup of geminiviruses whose members possess a bipartite genome. The TGMV A genome component has the capacity to encode at least four proteins. One of these is the viral coat protein, as inferred by homology with coat-protein, genes of other geminiviruses and by the observation of typical geminate particles in transgenic plants that contain inserts of TGMV A DNA. We have investigated the role of the coat protein in TGMV replication and report here that its coding sequence may be interrupted or substantially deleted without loss of infectivity. However, certain coat-protein mutants showed reproducible delays in time of symptom appearance as well as reduced symptom development, when inoculated onto transgenic Nicotiana benthamiana plants containing the TGMV B component. The most attenuated symptoms were seen with a mutant in which the coat-protein coding sequence was almost entirely deleted. The significance of these findings for the development of plant vectors from TGMV DNA is discussed.

Journal Article↗

PubMed2001

Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.

Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region. Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E. coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes.

Amino Acid Substitution↗

PubMed2004

Targeted DNA integration within different functional gene domains in yeast reveals ORF sequences as recombinational cold-spots.

The efficiency of gene targeting within different segments of genes in yeast was estimated by transforming yeast cells with double-stranded integrative plasmids, bearing functional gene domains [promoter (P), ORF (O) and terminator (T)] derived from the common genetic markers HIS3, LEU2, TRP1 and URA3. Transformation experiments with circular plasmids carrying a single gene domain demonstrated that the 5' and 3' flanking DNA regions (P and T) of the HIS3 and URA3 genes are preferred as sites for plasmid integration by several fold over the corresponding ORFs. Moreover, when plasmids bearing combinations of two or three regions were linearized to target them to a specific site of integration, three of the ORFs were found to be less preferred as sites for plasmid integration than their corresponding flanking regions. Surprisingly, in up to 50% of the transformants obtained with plasmids that had been linearized within coding sequences, the DNA actually integrated into neighbouring regions. Almost the same frequencies of ORF mis-targeting were obtained with plasmid vectors containing only two functional domains ("PO" or "OT") of the gene URA3, demonstrating that this event is not the consequence of competition between homologous DNA regions distal to the ORF. Therefore, we suggest that coding sequences could be considered to be "cold spots" for plasmid integration in yeast.

Aldose-Ketose Isomerases↗