Resolution of end-to-end distance distributions of flexible molecules using quenching-induced variations of the Forster distance for fluorescence energy transfer.

We describe a new method to recover the distribution of donor-to-acceptor (D-A) distances in flexible molecules using steady-state measurements of the efficiency of fluorescence energy transfer. The method depends upon changes in the Forster distance (Ro) induced by collisional quenching of the donor emission. The Ro-dependent transfer efficiencies are analyzed using nonlinear least squares to recover the mean D-A distance and the width of the distribution. The method was developed and tested using three synthetic D-A pairs, in which the chromophores were separated by alkyl chains of varying lengths. As an example application we also recovered the distribution of distances from the single tryptophan residue in troponin I (trp 158) to acceptor-labeled cysteine 133. The half-width of the distribution increases from 12 A in the native state to 53 A when unfolded by guanidine hydrochloride. For both TnI and the three model compounds the distance distributions recovered from the steady-state transfer efficiencies were in excellent agreement with the distributions recovered using the more sophisticated frequency-domain method (Lakowicz, J.R., M.L. Johnson, W. Wiczk, A. Bhat, and R.F. Steiner. 1987. Chem. Phys. Lett. 138:587-593). The method was found to be reliable and should be generally useful for studies of conformational distributions of macromolecules.     

Potassium channel "inactivation" induced by soft-glass patch pipettes

We have studied the potassium currents of rat pituitary pars intermedia cells kept in primary culture using whole-cell recording with patch pipettes. The potassium current recorded with hard-glass pipettes is mainly carried by voltage-dependent channels that show slow inactivation in the presence of 0.5 mM internal EGTA. Fast "inactivation" of the potassium current is seen with patch pipettes fabricated from soft glass (soda glass or potash lead glass), and is probably caused by block of the potassium channels by di- or multivalent cations released from the glass.     

Stereocontrolled routes to cis-hydroxyamino sugars, part VII: synthesis of daunosamine and ristosamine

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-- -erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-- -altropyranoside)-[3,4-d]-2-oxazoline. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-- -ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.     

A Quantitative Model for Nonrandom Generalized Transduction, Applied to the Phage P22–SALMONELLA TYPHIMURIUM System

A mathematical model for nonrandom generalized transduction is proposed and analyzed. The model takes into account the finite number of transducing particle classes for any given marker. The equations for estimation of the distance between markers from contransduction frequency data are derived and standard errors of the estimates are given. The obtained relationships depend significantly on the number of classes of transducing fragments. The model was applied to estimate the number of transducing fragment classes for a given marker in transduction with phage P22 of Salmonella typhimurium. It was found that the literature data on frequencies of contransduction in crosses with mutual substitution of selective and nonselective markers can be rationalized most accurately by assuming that the mean number of classes is equal to 2. An improved method for analysis of cotransduction data is proposed on the basis of our model and the results of calculation. The method relies on solving a set of algebraic equations for cotransduction frequencies of markers located within one phage length. The method allows a relatively precise determination of distances between markers, positions of transducing particle ends and deletion or insertion lengths. The approach is applied to the trp-cysB-pyrF and aroC-hisT-purF-dhuA regions of the Salmonella typhimurium chromosome.     

Genealogy of Principal Strains of the Yeast Genetic Stock Center

We have constructed a genealogy of strain S288C, from which many of the mutant and segregant strains currently used in studies on the genetics and molecular biology of Saccharomyces cerevisiae have been derived. We have determined that its six progenitor strains were EM93, EM126, NRRL YB-210 and the three baking strains Yeast Foam, FLD and LK. We have estimated that approximately 88% of the gene pool of S288C is contributed by strain EM93. The principal ancestral genotypes were those of segregant strains EM93-1C and EM93-3B, initially distributed by C. C. Lindegren to several laboratories. We have analyzed an isolate of a lyophilized culture of strain EM93 and determined its genotype as MATa/MATα SUC2/SUC2 GAL2/gal2 MAL/MAL mel/mel CUP1/cup1 FLO1/flo1. Strain EM93 is therefore the probable origin of genes SUC2, gal2, CUP1 and flo1 of S288C. We give details of the current availability of several of the progenitor strains and propose that this genealogy should be of assistance in elucidating the origins of several types of genetic and molecular heterogeneities in Saccharomyces.     

On the Divergence of Alleles in Nested Subsamples from Finite Populations

Within-population variation at the DNA level will rarely be studied by sequencing of loci of randomly chosen individuals. Instead, individuals will usually be chosen for sequencing based on some knowledge of their genotype. Data collected in this way require new sampling theory. Motivated by these observations, we have examined the sampling properties of a finite population model with two mutation processes and with no selection or recombination. One mutation process generates new alleles according to an infinite-alleles model, and the other generates polymorphisms at sites according to an infinite-sites model. A sample of n genes is considered. The stationary distribution of the number of segregating sites in a subsample from one of the allelic classes in the sample conditional on the allelic configuration of the sample is studied. A recursive scheme is developed to compute the moments of this distribution, and it is shown that the distribution is functionally independent of the number of additional alleles in the sample and their respective frequencies in the sample. For the case in which the sample contains only two alleles, the distribution of the number of segregating sites in a subsample containing both alleles conditional on the sample frequencies of the alleles is studied. The results are applied to the analysis of DNA sequences of two alleles found at the Adh locus of Drosophila melanogaster. No significant departure from the neutral model is detected.     

Immunohistochemical aspects of immunological cross-reaction and masking of epitopes for localization studies on pregnancy-associated plasma protein A

Summary The influence of antibody absorption procedures and proteolytic pre-treatment of formaldehyde-fixed placental tissue on the localization of pregnancy-associated plasma protein A by immunoperoxidase technique was examined.Apparently monospecific IgG fraction of the anti-plasma protein applied directly on fixed tissue resulted in staining of connective tissue and a thin apical rim of the syncytiotrophoblast. Further absorption of the antibody with foetal connective tissue abolished this staining reaction. Pre-treatment of the fixed placental tissue with trypsin prior to application of the antibody, which had been absorbed with connective tissue, resulted in staining within the cytoplasm of the syncytiotrophoblast exclusively. Identical staining was seen when this IgG preparation was used directly on frozen placental tissue.The results point to the importance of the specificity of the antibody preparations and of proteolytic unmasking of epitopes when fixed tissues are used for localization studies of pregnancy-associated plasma protein A by immunoperoxidase technique.