Melting behavior of a covalently closed, single-stranded, circular DNA

We synthesized the 26-residue deoxynucleotide sequence d(TTCCT5GGAATTCCT5GGAA) which folds intramolecularly to form a dumbbell-shaped, double-hairpin structure with a gap between the 3' and the 5' ends. We used T4 polynucleotide kinase to phosphorylate the 5' end followed by T4 DNA ligase to close the 3' and 5' ends. Melting of the dumbbell structure formed by this ligated sequence produces a covalently closed, single-stranded, circular final state. We employed calorimetric and spectroscopic techniques to characterize thermodynamically the melting behavior of the ligated molecule and compared it with the corresponding melting behavior of its unligated precursor. This comparison allowed us to characterize uniquely the influence of single-stranded ring closure on intramolecular duplex melting. The data reveal that ring closure produces a thermally more stable structure which exhibits significantly altered melting thermodynamics. We rationalize these thermodynamic differences in terms of differential solvation and differential counterion association between the ligated and unligated molecules. We also note the importance of such constrained dumbbell structures as models for hairpins, cruciforms, and locally melted domains within naturally occurring DNA polymers.     

Genetics of leaf blight resistance in wheat

Summary Studies on the genetics of leaf blight caused byAlternaria triticina using generation mean analysis revealed that additive components played a major role, but that dominance components also contributed significantly in controlling the variability for leaf blight resistance in wheat crosses. Furthermore, the additive x additive type of epistasis was predominant in the first three crosses, whereas in the fourth cross additive x dominance (j) and dominance x dominance (1) components of epistasis were most significant. Because of this it may be desirable to follow a simple recurrent selection scheme for higher tolerance, to isolate resistant plants from the segregating populations derived from crosses of parents of diverse origin following the pedigree method of breeding. CPAN-1887 was very tolerant to leaf blight in the present study and should be utilized in hybridization programs to develop leaf-blight-resistant varieties.     

Ras oncogene mediated induction of a 92 kDa metalloproteinase; strong correlation with the malignant phenotype

We have previously demonstrated that activated ras oncogenes can induce the metastatic phenotype and type IV collagenolytic activity in NIH/3T3 cells (Thorgeirsson et al. Mol. Cell. Biol. 5:259-262, 1985). The present study demonstrates ras-mediated induction of a 92 kDa metalloproteinase, which degrades gelatin and type IV collagen. Association of the 92 kDa proteinase expression with the malignant phenotype was also observed in human tumor cell lines. Our data indicate that the 92 kDa gelatin-collagen IV degrading metalloproteinase is an important participant in the proteolytic process involving tumor cell invasion and metastasis.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K

Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.     

Characterization of Bacteria by Particle Beam Mass Spectrometry

A technique is described for detecting and characterizing bacteria on a single-particle basis by mass spectrometry. The method involves generation of a particle beam of single whole cells which are rapidly volatilized and ionized in vacuum in the ion source of a quadrupole mass spectrometer. The particle beam can be generated, with minimal sample handling, from a naturally occurring aerosol or from a solution of bacteria that can be dispersed as an aerosol. The mass spectrum is generated by successively measuring the average intensities of different mass peaks. The average intensity is obtained by measuring the ion intensity distribution at the particular mass (m/e) for ion pulses from more than 1,000 bacteria particles. Bacillus cereus, Bacillus subtilis, and Pseudomonas putida samples were analyzed to test the capability of the instrument for differentiating among species of bacteria. Significant ion-intensity information was produced over the m/e range of 50 to 300, an improvement over previous pyrolysis-mass spectrometry results. The complex mass spectra contained a few unique peaks which could be used for the differentiation of the bacteria. A statistical analysis of the variations in peak intensities among the three bacteria provided a quantitative measure of the reproducibility of the instrument and its ability to differentiate among bacteria. The technique could lead to a new rapid method for the analysis of microorganisms and could be used for the detection of airborne pathogens on a continuous, real-time basis.     

Regulation of toxin biosynthesis by plasmids in Vibrio cholerae

Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 degrees C, were isolated. One ts plasmid was unstable at 42 degrees C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.     

Transplacental micronucleus inducing ability of arecoline, a betel nut alkaloid, in mice

Arecoline, a major betel nut alkaloid, was tested for its effectiveness in inducing micronuclei in fetal mouse blood after transplacental exposure late in the gestation period. Positive results were obtained and a linear dose-response relationship was expressed when pregnant mice were treated with arecoline at dose levels of 20, 40 and 80 mg/kg and micronucleated polychromatic erythrocytes from fetal blood were subsequently analysed.     

Senescence of attached leaves: Regulation by developing pods

Analysis of total nitrogen, chlorophyll content, ribulose-1,5-bisphosphate carboxylase/oxygenase activity and net photosynthesis rate was carried out on the leaves that support the developing pods in pigeon pea [ Cajanus cajan (L.) Millsp. cv. Prabhat] at several stages during pod filling. A continuous loss in all the above-mentioned parameters was observed during the course of pod development. When no pods were allowed to develop by continuous flower removal treatment, there was a considerable delay in loss of all these metabolic parameters. Excision of pods after their mid-development resulted not only in no further loss, but also in a significant recovery both of total nitrogen and of other investigated characteristics.