Conformational study on ketamine by circular dichroism and ultraviolet spectroscopy

Since the enantiomers of the N‐methyl‐D ‐aspartate (NMDA) receptors antagonist ketamine have different pharmacological profiles, CD and UV spectroscopy were applied for the study of conformer equilibrium and pH dependence in ketamine solutions. The assignment of the configurations and conformations was performed on the basis of the “octant rule” and UV spectra. In accord with published data, it was established that, on protonation, the phenyl group of the ketamine molecule occupies an axial position, while for the base form, the ratio of conformers containing axial/equatorial aryl moieties is strongly solvent‐dependent. The CD and UV spectra indicate the presence of an intramolecular H‐bond C=O····H—N in the conformer with axial aryl moiety. Chirality 11:280–285, 1999. © 1999 Wiley‐Liss, Inc.     

肝硬化患者幽门螺杆菌感染与血氨的关系

目的 :探讨幽门螺杆菌 (Hp)感染对肝硬化患者血氨浓度的影响。 方法 :35例肝硬化患者及 2 5例消化性溃疡患者分别测定Hp感染情况及空腹血氨浓度 ,对于Hp阳性病人口服质子泵抑制剂三联方案1周后测定空腹血氨。结果 :Hp阳性的肝硬化患者空腹血氨较Hp阴性的肝硬化患者空腹血氨显著升高(P <0 0 1)。根除Hp后 ,肝硬化患者空腹血氨浓度显著下降 (P <0 0 5 )。 结论 :Hp感染可引起肝硬化患者血氨浓度升高 ,对此类患者采用Hp根除治疗 ,可显著降低血氨浓度。抗Hp治疗可能有助于预防及治疗肝硬化患者的高氨血症及由此诱发的肝性脑病及亚临床肝性脑病。     

Insulin Resistance,Hyperleptinemia, and Obstructive Sleep Apnea in Launois‐Bensaude Syndrome

Objective: Launois‐Bensaude Syndrome (LBS) is a very rare cause of obesity, characterized by a symmetrical accumulation of a very large number of lipomata in different regions of the body, excluding the face, the forearms, and the shanks. Obesity is known to be closely associated with insulin resistance, hyperleptinemia, and obstructive sleep apnea (OSA). We were interested in studying whether these conditions are also present in patients with obesity due to LBS with a similar frequency as in patients with “simple” truncal obesity. Research Methods and Procedures: We performed polysomnography and hyperinsulinemic euglycemic clamp studies and measured serum leptin in three patients with LBS and in six patients with “simple” truncal obesity, matched for sex and body mass index (LBS group, 36.39 kg/m²; controls, 35.82 kg/m²). Results: Polysomnography revealed severe OSA in one LBS patient with marked “horsecollar lipomata.” In the other LBS patients, no OSA could be demonstrated. The leptin levels of the two groups were comparable (LBS group, 36.39 μg/liter; controls, 37.18 μg/liter) and the insulin responsiveness index was also comparable in the two groups (LBS group, 3.47 μmol/kg · minute; controls, 3.79 μmol/kg · minute). Discussion: Patients with LBS demonstrated similar metabolic features in terms of insulin sensitivity and hyperleptinemia as patients with “simple” truncal obesity. LBS is not strictly associated with OSA.     

Serological responses of FldA and small-molecular-weight proteins of Helicobacter pylori: correlation with the presence of the gastric MALT tissue

PURPOSE: We tested whether the serological response to Flavodoxin-A (FldA) protein and anti-Helicobacter pylori immunoblots correlated to the degree of mucosaassociated lymphoid tissue (MALT) in the stomach. METHODS: Eighty H. pyloni-infected patients with different degrees of MALT in the stomach were investigated with serum sampling and endoscopy on enrolment, the 2nd and the 12th months after anti-H. pylori therapy. All sera were tested for the anti-FldA protein and anti-H. pylon immunoblots, including 19.5, 26.5, 30, 35, 89, and 116 KDa (CagA). Regression of follicular gastritis was assessed by histology. RESULTS: Patients with dense lymphoid follicles had higher prevalence rates of anti-FldA protein, 19.5, 26.5, and 30 KDa antibodies of H. pylori (p < .05). Histologic downgrade of MALT was observed in 25% (10/40) of patients in the 2nd month and in 60% (23/38) in the 12th month after H. pylori therapy. After H. pylori eradication, the patients with MALT and dense lymphoid follicles had significantly negative seroconversions of 19.5, 26.5, 30, and 35 KDa antibodies (p < .05), but not of CagA and FldA. CONCLUSION: Patients with gastric MALT and dense lymphoid follicles had different anti-H. pylori serological responses to those with scanty or an absence of lymphoid follicles. The negative seroconversion of the smaller-molecular-weight proteins, but not CagA and FldA, may correlate with the regression of MALT by H. pylori eradication.     

Conformational Peculiarities of Polynucleotides with a Nonrandom Base Sequence According to the 1H→ 3H Exchange Rate in C8H Groups of Purinic Residues

Abstract

We have determined the ¹H→³H exchange rate constants between water and C8H groups of purinic residues of alternating polynucleotides poly(dA-dT)·poly(dA-dT), poly(dG-dC)·poly(dG- dC) and poly(dA-dC)·poly(dG-dT) as well as homopolynucleotides poly(dA)·poly(dT) and poly(dG)·poly(dC) in aqueous solutions with high-salt concentrations (3 M NaCl and 4–6 M CsF), in water-ethanol (60%) solution and in 0.15 M NaCl at 25°C. The rate constants for adenine (k_A) and guanine (k_G) of polynucleotides were compared with corresponding constants for E.coli DNA, dGMP nd dAMP at the same conditions. The relation between exchange rates and conformations of polynucleotides permits the study of their conformational peculiarities in solution.

Of three alternating polynucleotides examined in 0.15 M NaCl the exchange retardation was observed only for poly(dA-dT)·poly(dA-dT) as compared with that in B-DNA, which is in good agreement with the B-alternating “wrinkled” DNA model. The conformations of poly(dG-dC)·poly(dG-dC) and poly(dA-dC)·poly(dG-dT), according to the exchange data obtained, are within the B form. For homopolynucleotides in 0.15 M NaCl, the k_A value for poly(dA)·poly(dT) is nearly the same as k_A for B-DNA, which indicates the similarity of their conformations, whereas the k_G value for poly(dG)·poly(dC) is 1.7-fold lower in comparison with the k_G value in B-DNA. This seems to be connected with the existence of B? A conformation equilibrium for poly(dG)·poly(dC) in solution.

The increase of NaCl concentration to 3 M results in a B→Z transition in the case of poly(dG-dC)·poly(dG-dC) and in the shift of B?A equilibrium towards the A-form in the case of poly(dG)·poly(dC), as is evidenced by alterations of their K_G values. Poly(dA-dT)·poly(dA-dT) in 6 M CsF and poly(dA-dC)·poly(dG-dT) in 4.3 M CsF maintain their inherent conformations in 0.15 M NaCl in spite of the fact that they are characterised by the “X-type” CD-spectrum at these conditions. According to the exchange data the conformation of poly(dA)·poly(dT) in 6 M CsF corresponds to the “heteronomous” DNA model or some other structure with lower accessibility of C8H groups of adenylic residues.     

Immunological Studies in Kaposi's Sarcoma in Uganda

An evaluation of humoral and cellular immune mechanisms was performed on patients with Kaposi''s sarcoma in Uganda. Antibody responses and immunoglobulin levels were normal in all patients studied. Nevertheless, a striking impairment in the delayed hypersensitivity response to dinitrochlorobenzene was noted in patients with the “malignant” type of tumour.     

Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization

In the article by Bandyopadhyay et al. entitled “Spermidinyl-CoA-based HAT inhibitors block DNA repair and provide cancer-specific chemo- and radiosensitization,” which appeared in the September 1, 2009 issue of Cell Cycle (pp.2779-88), the first sentence in part (D) of the legend to Figure 3 appeared incorrectly as “(D) Inhibition of g-H2A.X acetylation by 1a: Top row – SDS-PAGE/Western of g-H2A.X in H358 cells treated with 10 µM CPT (1 hour) followed by incubation for an additional hour in the presence (right) or absence (left) of 50 µM 1a (60 µg/lane).” The correct sentence reads as follows: “(D) Inhibition of g-H2A.X acetylation by 1a: Top row – SDS-PAGE/Western of g-H2A.X in H358 cells treated with 5nM CPT (18 hours) in the presence (right lane) or absence (left lane) of 50 µM 1a (60 µg/lane).”     

Interactions between k-Casein and β-Lactoglobulin: Effects of Anions on Covalent Stabilization of the Complex

Structure breaking anions, trichloroacetate ( — TCA~) and thiocyanate ( — SCN~), significantly increased the interaction between 7c-casein (K-C) and /Mactoglobulin (B-Lg) to give the covalently stabilized K-C/B-Lg tetrameric (A4) complex, whereas “structure making” anions, chloride ( — Cl^-) and especially sulfate ( —SO4), reduced it. The reactivity of K-C with B-Lg in the presence of these anions followed the order, — TCA~ > — SCN~ > — Cl” > — SO4 (100mm). The percentile distribution of the covalent bonded K-C/B-Lg A4 complex after 720 s of heating at 70°C in — TCA~ (lOOmm) was one order greater than it was in — SO^- During storage of the heated K- C/B-Lg mixture, the B-Lg A3/K-C interaction in — TCA~ was rapid and uneffected by the holding temperature, whereas in — SO4, the reaction rate was inversely related to the temperature, being apparently controlled by the relative concentration of monomeric K-C.     

Interplay between halogen bonds and hydrogen bonds in OH/SH···HOX···HY (X = Cl, Br; Y = F, Cl, Br) complexes

The character of the cooperativity between the HOX···OH/SH halogen bond (XB) and the Y―H···(H)OX hydrogen bond (HB) in OH/SH···HOX···HY (X = Cl, Br; Y = F, Cl, Br) complexes has been investigated by means of second-order Møller?Plesset perturbation theory (MP2) calculations and “quantum theory of atoms in molecules” (QTAIM) studies. The geometries of the complexes have been determined from the most negative electrostatic potentials (V _S,min) and the most positive electrostatic potentials (V _S,max) on the electron density contours of the individual species. The greater the V _S,max values of HY, the larger the interaction energies of halogen-bonded HOX···OH/SH in the termolecular complexes, indicating that the ability of cooperative effect of hydrogen bond on halogen bond are determined by V _S,max of HY. The interaction energies, binding distances, infrared vibrational frequencies, and electron densities ρ at the BCPs of the hydrogen bonds and halogen bonds prove that there is positive cooperativity between these bonds. The potentiation of hydrogen bonds on halogen bonds is greater than that of halogen bonds on hydrogen bonds. QTAIM studies have shown that the halogen bonds and hydrogen bonds are closed-shell noncovalent interactions, and both have greater electrostatic character in the termolecular species compared with the bimolecular species.

Electron Microscopy of Supercoiled pEJ4 DNA Containing Homopurine · Homopyrimidine Sequences

Abstract

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine · homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine · homopyridine tract assumes an unusual conformation) a single thick “stem” or a “denaturation bubble” in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC 19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The “denaturation bubbles” were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only “stems”. The “stems” were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine · homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.     

Cell wall glycoproteins and polysaccharides of parenchyma of Phaseolus coccineus

Fractionation of the cell wall material of parenchyma of mature runner beans with and without chlorite-HOAc treatment, clearly showed that at least two main types of wall proteins were present. One relatively rich in hydroxyproline (HP) associated with α′-cellulose, from which most (90%) of it could be readily liberated by chlorite-HOAc treatment and the other relatively poor in HP associated with hemicellulose A. The chlorite HOAC solubilized “glycoprotein” contained a high proportion of arabinose and galactose. It was purified by PhOH-H₂O fractionation and the molar ratios of HP, arabinose, galactose, xylose, rhamnose, glucose and uronic acid in the purified glycoprotein (“glycoprotein X”) were 1:2·6:2·4:0·2:0·2:0·1:0·3. The principal amino acids of glycoprotein X were HP (43·5 mol%), serine and proline which together comprised 66 mol% of the total. These results suggest that the HP-rich wall glycoprotein is associated with cellulose microfibrils and approximates in conformation to polyhydroxyproline carrying arabinose and galactose oligosaccharide side chains.     

An association between a lymphocyte antigen in sheep and the response to vaccination against the parasite Trichostrongylus colubriformis

Lymphocyte antigens were tested in sheep which had been selected for responsiveness to vaccination against the intestinal nematode Trichostrongylus colubriformis. These sheep had been bred in an assortative mating programme which produced offspring designated as either “high responders” or “low responders”, with highly heritable resistance or susceptibility.Ovine lymphocyte antigen (OLA) typing antisera were obtained from parous ewes in the course of matings which produced the high and low responder flocks. A particular antigen (SY1) was found to be present in high frequency on the lymphocytes of high responder (72·2%) and in lower frequency (21·9%) on the lymphocytes of low responder rams. In ewes, the frequency for high responders was 65·7% and for low responders it was 33·5%. A similar association between the SY1 antigen and low faecal egg count was found in random-bred sheep which had been vaccinated with irradiated larvae and challenged with normal larvae. The conclusion was drawn that this lymphocyte antigen was likely to be part of the sheep major histocompatibility complex which influenced the immune response of sheep to vaccination against the parasite.     

Quantifying greenhouse gases from the production,transportation and utilization of charcoal in developing countries: a case study of Kampala,Uganda

Purpose

This study aims to quantify greenhouse gases (GHGs) from the production, transportation and utilization of charcoal and to assess the possibilities of decreasing greenhouse gases (GHGs) from the charcoal industry in general in Uganda. It also aims to assess the emission intensity of the Ugandan “charcoal production” sector compared to that of some other major charcoal producing nations.

Methods

This work was done in accordance with ISO 14040 methodology for life-cycle assessment (LCA), using GABi 4.0—a software for life-cycle assessment. A cradle-to-grave study was conducted, excluding emissions arising from machinery use during biomass cultivation and harvesting. The distance from charcoal production locations to Kampala was estimated using ArcGIS 10.0 software and a GPS tool. Emission data from a modern charcoal production process (PYREG methane-free charcoal production equipment), which complies with the German air quality standards (TA-Luft), was compared with emissions from a traditional charcoal production process. Four coupled scenarios were modelled to account for differences in the quantity of greenhouse gases emitted from the “traditional charcoal production phase”, “improved charcoal production phase (biomass feedstock sourced sustainably and unsustainably)”, “transportation phase” and “utilization phase”. Data for this study was obtained via literature review and onsite measurements.

Results and discussion

The results showed that greenhouse gases emitted due to charcoal supply and use of traditional production technique in Kampala was 1,554,699 tCO₂eq, with the transportation phase accounting for approximately 0.15 % of total greenhouse gases emitted. The utilization phase (charcoal cookstoves) emitted 723,985 tCO₂eq (46.6 %), while the charcoal production phase emitted 828,316 tCO₂eq (53.3 %). Changing the charcoal production technology from a traditional method to an improved production method (PYREG charcoal process) resulted in greenhouse gases reductions for the city of 230,747 tCO₂eq; however, by using sustainably sourced biomass, this resulted in reductions of 801,817 tCO₂eq.

Conclusions

This study showcased and quantified possible GHG emission reduction scenarios for the charcoal industry in Uganda. The result of 3 tCO₂eq emitted per tonne of charcoal produced, using earth mound method, can be applied to other countries in Eastern Africa where similar charcoal production methods are used; this will allow for somewhat better regional estimates of the inventory of greenhouse gas emissions from the production of charcoal. The results of this study also suggests that the primary use of charcoal for cooking will lead to increases in GHG emissions and increases in deforestation on the long term, if legal frameworks are not made to ensure that biomass used for charcoal production is obtained via sustainable sources or if alternative cheap energy-generating technologies for cooking are not developed and deployed to the masses.     

The physicochemical essence of the purine·pyrimidine transition mismatches with Watson-Crick geometry in DNA: A·C* versa A*·C. A QM and QTAIM atomistic understanding

It was established for the first time by DFT and MP2 quantum-mechanical (QM) methods either in vacuum, so in the continuum with a low dielectric constant (ε = 4), typical for hydrophobic interfaces of specific protein-nucleic acid interactions, that the repertoire for the tautomerisation of the biologically important adenine·cytosine* (A·C*) mismatched DNA base pair, formed by the amino tautomer of the A and the imino mutagenic tautomer of the C, into the A*·C base mispair (?G = 2.72 kcal?mol^?1 obtained at the MP2 level of QM theory in the continuum with ε = 4), formed by the imino mutagenic tautomer of the A and the amino tautomer of the C, proceeds via the asynchronous concerted double proton transfer along two antiparallel H-bonds through the transition state (TS_A·C*?A*·C). The limiting stage of the A·C*→A*·C tautomerisation is the final proton transfer along the intermolecular N6H···N4 H-bond. It was found that the A·C*/A*·C DNA base mispairs with Watson–Crick geometry are associated by the N6H?N4/N4H?N6, N3H?N1/N1H?N3 and C2H?O2 H-bonds, respectively, while the TS_A·C*?A*·C is joined by the N6–H–N4 covalent bridge and the N1H?N3 and C2H?O2 H-bonds. It was revealed that the A·C*?A*·C tautomerisation is assisted by the true C2H?O2 H-bond, that in contrast to the two others conventional H-bonds exists along the entire intrinsic reaction coordinate (IRC) range herewith becoming stronger at the transition from vacuum to the continuum with ε = 4. To better understand the behavior of the intermolecular H-bonds and base mispairs along the IRC of the A·C*?A*·C tautomerisation, the profiles of their electron-topological, energetical, geometrical, polar and charge characteristics are reported in this study. It was established based on the profiles of the H-bond energies that all three H-bonds are cooperative, mutually strengthening each other. The nine key points, providing a detailed physicochemical picture of the A·C*?A*·C tautomerisation, were revealed and thoroughly examined along the IRC. It was shown that the A*·C base mispair with the population ~1 % obtained at the MP2 level of QM theory in the continuum with ε = 4 is thermodynamically and dynamically stable structure. Its lifetime was calculated to be 5.76·10^?10 s at the MP2 level of QM theory in the continuum with ε = 4. This lifetime, from the one side, enables all six low-frequency intermolecular vibrations to develop, but, from the other side, it is by order less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. This means that the A*·C base mispair “slips away from the hands” of the replication machinery into the A·C* mismatched base pair. Consequently, the authors came to the conclusion that exactly the A·C* base mispair is an active player of the point mutational events and is effectively dissociated by the replication machinery into the A and C* monomers in contrast to the A*·C base mispair, playing the mediated role of a provider of the A·C* base mispair in DNA that is synthesised.     

Organ-specific autoantibodies in children with Helicobacter pylori infection

BACKGROUND: We compared the prevalence of organ-specific autoantibodies in a group of Helicobacter pylori infected children and a group of uninfected children and investigated the relationship between the presence of relevant autoantibodies and the status of the target organs. PATIENTS AND METHODS: One hundred and twenty-four children with dyspepsia (54 boys, 70 girls; mean age 10.5 years; range 4-19) underwent gastroscopy: 56 had H. pylori infection (31 girls, 25 boys), while 68 (37 girls and 31 boys), were H. pylori-negative. All sera were tested for the presence of: parietal cell autoantibodies (PCA), intrinsic factor autoantibodies (IFA), microsomial autoantibodies, thyroglobulin autoantibodies, islet cell autoantibodies, glutamic acid decarboxylase autoantibodies, adrenal cortex autoantibodies, steroid-producing cell autoantibodies; gastrin, pepsinogen A, pepsinogen C and anti-H. pylori antibodies. The histological features and the ureA and cagA genes were also considered. RESULTS: The frequency of organ-specific autoantibodies was higher in patients with H. pylori infection than in uninfected patients (chi2-test p < .0001). Specifically gastric autoantibodies were significantly higher: seven of the 56 H. pylori-positive children were PCA-positive and one was IFA-positive (chi2-test p = .0004). The presence of autoantibodies was not associated with any clinical or biohumoral signs of disease. CONCLUSIONS: Our study detected a relationship between H. pylori infection in childhood and the presence of organ-specific autoantibodies unassociated with any clinical or biohumoral signs of disease. Helicobacter pylori infection in childhood could trigger the onset of clinical autoimmune gastritis, and/or other clinical autoimmune diseases.     

Parallel Double Helices of DNA. Conformational Analysis of Regular Helices with the Second Order Symmetry Axis

Abstract

We have performed a conformational analysis of DNA double helices with parallel directed backbone strands connected with the second order symmetry axis being at the same time the helix axis. The calculations were made for homopolymers poly(dA) · poly(dA), poly(dC) · poly(dC), poly(dG) poly(dG), and poly(dT) · poly(dT). All possible variants of hydrogen bonding of base pairs of the same name were studied for each polymer. The maps of backbone chain geometrical existence were constructed. Conformational and helical parameters corresponding to local minima of conformational energy of “parallel” DNA helices, calculated at atom-atom approximation, were determined. The dependence of conformational energy on the base pair and on the hydrogen bond type was analysed. Two major conformational advantageous for “parallel” DNA's do not depend much on the hydrogen-bonded base pair type were indicated. One of them coincided with the conformational region typical for “antiparallel” DNA in particular for the B-form DNA Conformational energy of “parallel” DNA depends on the base pair type and for the most part is similar to the conformational energy of “antiparallel” B-DNA.     

Grouping and Inhibition of Oogenesis in Two Strains of Acanthoscelides obtectus Say (Coleoptera Bruchidae) of Different Geographical Origin-Specificity of This Inhibitory Effect

Summary

Reproduction of A. obtectus females originating from Rubona (Rwanda) was inhibited after grouping (2 females, or 1 male—1 female). Only a few females produced mature oocytes; vitellogenin was synthesized, released into the haemolymph, but not incorporated into the oocytes. When females produced mature oocytes, their ovarian production was lower than in isolated virgin females. After pairing of a male and a female, 38% of the females mated, but mating under these conditions did not stimulate oogenesis.

Oogenesis of females originating from Tours (France) was only slightly inhibited by the presence of males or females of the same origin. “France” males and females inhibited the reproduction of “Rwanda” females under grouping conditions. However, “France” females showed only a slight sensitivity to grouping with “Rwanda” bruchids. In the presence of Phaseolus vulgaris seeds, grouping had no effect and all females, whatever their origin, produced mature oocytes. Inhibition weis relatively specific as cohabitation with bruchids of other genera (Callosobruchus maculatus-Zabrotes subfasciatus) had no effect. Cohabitation with two species of the same genus (Acanthoscelides obvelatus—A. argilaceus) inhibited oogenesis of A. obtectus “Rwanda” strain. The significance of this regulation and its importance in the maintenance of populations in nature are also analyzed.     

Premelting and the hydrogen-exchange open state in synthetic RNA duplexes

Here we attempt to relate equilibrium temperature-dependent spectral changes in two synthetic RNA homopolymer duplexes—poly(rA) · poly(rU) and poly(rI) · poly(rC)—to the conformational opening detected in stopped-flow hydrogen–deuterium exchange experiments on these molecules. We are concerned with changes in several spectral properties that occur well below onset of the thermally induced helix-coil “melting” transition in these systems. These are known as “premelting” transitions, and can be detected in uv CD spectra as well as in vibrational bands of the bases in the ir. Both CD and ir spectra exhibit isoelliptic or isosbestic points consistent with a well-defined two-state premelting process. Application of a least-squares analysis to two-state models for premelting using data from different bands in the CD and ir shows that the enthalpies are substantially greater than that of the hydrogen-exchange opening. Thus the hydrogen-exchange open state represents only one premelting reaction among several that lead to equilibrium changes in helix geometry or base vibrational modes. The latter include processes that occur on a rapid time scale, including potential base-pair openings not productive for the exchange reaction. It appears that the former, and not the hydrogen-exchange opening, dominates the premelting alterations monitored by ir and CD spectroscopy.     

Deletion mutants of simian virus 40 generated by enzymatic excision of DNA segments from the viral genome

Deleted genomes of simian virus 40 have been constructed by enzymatic excision of specific segments of DNA from the genome of wild-type SV40². For this purpose, a restriction endonuclease from Hemophilus influenzae (endo R · HindIII) was used. This enzyme cleaves SV40 DNA into six fragments, which have cohesive termini. Partial digest products were separated by electrophoresis in agarose gel and subsequently cloned by plaque formation in the presence of complementing temperature-sensitive mutants of SV40. Individual deletion mutants generated in this way were mapped by analysis of DNA fragments produced by endo R · Hind digestion of their deleted genomes, and by heteroduplex mapping. Two types of deletions were found: (1) “excisional” deletions, in which the limits of the deleted segment corresponded to HindIII cleavage sites, and (2) “extended” deletions, in which the deleted segment extended beyond HindIII cleavage sites. Excisionally deleted genomes presumably arose by cyclization of a linear fragment via cohesive termini generated by endo R · HindIII whereas genomes with extended deletions probably were generated by intramolecular recombination near the ends of linear fragments. Of the nine mutants analyzed, two had deletions in the “early” region of the SV40 genome, six had deletions in the “late” region, and one had a deletion that spanned both regions.