Does Chi Give or Take?

In lytic cycle crosses with Red-Gam-lambda phage, particles were examined that had undergone an Int-mediated exchange. It was assumed that this exchange dimerized the circular lambda, making it packageable. Among these Int-mediated recombinants, particles were identified that had, in addition, enjoyed a close double exchange mediated by the RecBC pathway. Such close double exchanges indicate localized negative interference and are analogous to eukaryotic conversions that have retained parental configuration of flanking markers. These events are stimulated by Chi, a recombinator specific to the RecBC pathway. When Chi is present in only one parent in the cross, the complementary double exchange recombinants are Chi stimulated to the same degree. This behavior of Chi contrasts with that of characterized eukaryotic recombinators.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

A general theory of chromosome pairing based on the palindromic DNA model of Sobell with modifications and amplifications

A model for chromosome pairing is presented. It is based on the presence of segments of symmetric sequences of bases (palindromes) in the DNA at specific places in the chromosome. Palindromic DNA has been characterized by Wilson &; Thomas (1974), who state that these sequences are a regular feature of eukaryotic DNA. Sobell (1972) has suggested that they may be involved in synapsis and genetic recombination. Sobell's model is modified and amplified in an attempt to develop a general theory of chromosome pairing that explains congressional pairing, synapsis, non-homologous pairing, the initiation of crossing over, and interference.     

Saccharomyces cerevisiae Mer3 is a DNA helicase involved in meiotic crossing over

Crossing over is regulated to occur at least once per each pair of homologous chromosomes during meiotic prophase to ensure proper segregation of chromosomes at the first meiotic division. In a mer3 deletion mutant of Saccharomyces cerevisiae, crossing over is decreased, and the distribution of the crossovers that occur is random. The predicted Mer3 protein contains seven motifs characteristic of the DExH box type of DNA/RNA helicases. The mer3G166D and the mer3K167A mutation, amino acid substitutions of conserved residues in a putative nucleotide-binding domain of the helicase motifs caused a defect in the transition of meiosis-specific double-strand breaks to later intermediates, decreased crossing over, and reduced crossover interference. The purified Mer3 protein was found to have DNA helicase activity. This helicase activity was reduced by the mer3GD mutation to <1% of the wild-type activity, even though binding of the mutant protein to single- and double-strand DNA was unaffected. The mer3KA mutation eliminated the ATPase activity of the wild-type protein. These results demonstrate that Mer3 is a DNA helicase that functions in meiotic crossing over.     

Energetics of cyclodextrin-induced dissociation of insulin

The energetics of dissociation of bovine insulin in aqueous solution have been investigated by sensitive dilution microcalorimetry. Cyclodextrins increase dissociation of insulin oligomers in a manner consistent with their interaction with protein side chains. For example, assuming monomer-dimer equilibrium, in the absence of cyclo-dextrins the calorimetric dilution data (25 °C, pH 2.5) indicate a dimer dissociation constant (K_diss) of about 12 µM and an endothermic dissociation enthalpy (H_diss) of +41 kJ mol^–1. Addition of methyl--cyclodextrin (up to 200 mm) makes dissociation significantly more endothermic (H_diss = 79 kJ mol^–1) and reduces the apparent dimer dissociation constant by more than two orders of magnitude (K_diss 1.7 mm). Qualitatively similar results are observed with -cyclodextrin and other -cyclodextrin derivatives. Cyclodextrin-induced insulin dissociation is also observed at pH 7.4.     

Oligosaccharide mapping of proteoglycan-bound and xyloside-initiated dermatan sulfate from fibroblasts

The copolymeric structure of dermatan sulfate chains synthesized by skin fibroblasts has been examined. Chains initiated onto exogeneousp-nitrophenyl--D-xylopyranoside or attached to protein in a large proteoglycan, PG-L, and two small proteoglycans, PG-S1 and PG-S2, have been compared by using high resolution electrophoresis and gel chromatography of oligosaccharides generated by specific enzymatic or chemical degradations. The results confirm that chains attached to PG-L are glucuronate-rich, whereas novel findings indicate that chains attached to either of the two PG-S variants yield closely similar oligosaccharide maps, have approximately equal glucuronate and iduronate content and contain over 90% 4-sulfated disaccharide repeating units. Dermatan sulfate chains built onto xyloside at concentrations of 50 µm and below have a copolymeric structure similar to that of chains from the two PG-S variants. These findings indicate that the polymer-modifying machinery can generate chains with extended iduronate-containing repeats also when the xylose primer is not linked to core protein.     

Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5′-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3′-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blmp15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (>90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.     

mus309 mutation, defective in DNA double-strand break repair, affects intergenic but not intragenic meiotic recombination in Drosophila melanogaster

The effect was investigated of the hypomorphic DNA double-strand break repair, notably synthesis-dependent strand annealing, deficient mutation mus309 on the third chromosome of Drosophila melanogaster on intergenic and intragenic meiotic recombination in the X chromosome. The results showed that the mutation significantly increases the frequency of intergenic crossing over in two of three gene intervals of the X chromosome studied. Interestingly the increase was most prevalent in the tip of the X chromosome where crossovers normally are least frequent per physical map unit length. In particular crossing over interference was also affected, indicating that the effect of the mus309 mutation involves preconditions of crossing over but not the event of crossing over itself. On the other hand, the results also show that most probably the mutation does not have any effect on intragenic recombination, i.e. gene conversion. These results are fully consistent with the present molecular models of meiotic crossing over initiated by double-strand breaks of DNA followed by formation of a single-end-invasion intermediate, or D-loop, which is subsequently processed to generate either crossover or non-crossover products involving formation of a double Holliday junction. In particular the results suggest that the mus309 gene is involved in resolution of the D-loop, thereby affecting the choice between double-strand-break repair (DSBR) and synthesis-dependent strand annealing (SDSA) pathways of meiotic recombination.     

The monoclonal antibody,anti-asialoglycophorin from human erythrocytes,specific forβ-d-Gal-1-3-α-d-GalNAc-chains (Thomsen-Friedenreich receptors)

The monoclonal antibody 22.19 of IgM class obtained after immunization of BALB/c mice with asialoglycophorin of human erythrocyte membranes is described. The specificity of this antibody for -d-Gal-1-3--d-GalNAc- disaccharide chains (Thomsen-Friedenreich receptors) was established by studying its reactivity against various erythrocytes, glycoproteins and oligosaccharides and by comparison with two lectins, peanut agglutinin andVicia graminea lectin, which recognize these disaccharide chains.Abbreviations PNA peanut agglutinin - VgL Vicia graminea lectin - TF Thomsen-Friedenreich - HSA human serum albumin - MoAb monoclonal antibody     

Gene Conversion at the Gray Locus of SORDARIA FIMICOLA: Fit of the Experimental Data to a Hybrid DNA Model of Recombination

A hybrid DNA (hDNA) model of recombination has been algebraically formulated, which allows the prediction of frequencies of postmeiotic segregation and conversion of a given allele and their probability of being associated with a crossing over. The model considered is essentially the "Aviemore model." In contrast to some other interpretations of recombination, it states that gene conversion can only result from the repair of heteroduplex hDNA, with postmeiotic segregation resulting from unrepaired heteroduplexes. The model also postulates that crossing over always occurs distally to the initiation site of the hDNA. Eleven types of conversion and postmeiotic segregation with or without associated crossover were considered. Their theoretical frequencies are given by 11 linear equations with ten variables, four describing heteroduplex repair, four giving the probability of hDNA formation and its topological properties and two giving the probability that crossing over occurs at the left or right of the converting allele. Using the experimental data of Kitani and coworkers on conversion at the six best studied gray alleles of Sordaria fimicola, we found that the model considered fit the data at a P level above or very close (allele h4) to the 5% level of sampling error provided that the hDNA is partly asymmetric. The best fitting solutions are such that the hDNA has an equal probability of being formed on either chromatid or, alternatively, that both DNA strands have the same probability of acting as the invading strand during hDNA formation. The two mismatches corresponding to a given allele are repaired with different efficiencies. Optimal solutions are found if one allows for repair to be more efficient on the asymmetric hDNA than on the symmetric one. In the case of allele g1, our data imply that the direction of repair is nonrandom with respect to the strand on which it occurs.     

Noncanonical meiosis in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> as a model for studying the molecular bases of the homologous chromosome synapsis,crossing over,and segregation

The nematode C. elegans is a classic study object of developmental biology and genetics, which is particularly suitable for studying the molecular bases of meiosis. Developing meiocytes are located in the threadlike gonads of C. elegans in linear gradient order of the stages of meiosis, which facilitates studying the order of intracellular events during meiosis. C. elegans has polycentric chromosomes. This causes a special order of events during meiosis, and as a consequence, meiosis in C. elegance differs from canonical meiosis of most eukaryotes. In the meiotic prophase I, all chromosomes carry single protein “pairing centers.” They are responsible for joining homologous chromosomes in pairs. This initiates the formation of synaptonemal complexes (SCs). Programmed double-stranded DNA breaks appear after initiation of the SC assembly, and they give rise to meiotic recombination. The initiation of meiotic recombination after the chromosome pairing distinguishes the C. elegans meiotic pattern from those in the absolute majority of eukaryotes studied. C. elegans has strict crossing over interference, which allows for the formation of one chiasma per bivalent. In the late prophase I, the polycentric centromeres are remodeled, one of the chromosome ends acquires a cuplike kinetochore, and during two meiotic divisions, chromosomes behave as monocentric. The study of meiosis in C. elegans allows for separate investigation of synapsis and recombination of homologous chromosomes and provides material for studying the evolution of meiosis.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Monitoring the (photo)genotoxicity of photosensitizer drugs: direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (93.6 kJ/m²) chlorpromazine (0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04–0.20 mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.     

FAB CID-MS/MS characterization of tetrasaccharide tri- and tetrasulfate derived from the antigenic determinant recognized by the anti-chondroitin sulfate monoclonal antibody MO-225

The fast atom bombardment (FAB) collision induced dissociation (CID)-mass spectrometry/mass spectrometry (MS/MS) technique was successfully applied to characterize and identify the structures of the immunoreactive trisulfated and tetrasulfated tetrasaccharides that were obtained from the chondroitin sulfate in a shark fin using a treatment with chondroitinase ABC.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MS/MS mass spectrometry/mass spectrometry - UA2S-GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA-GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA-GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose     

Unequal Crossing Over as the Pathway of Adaptive Homologous Recombination between the Direct DNA Repeats in Tandem Duplications of Escherichia coli

Homologous recombination between direct DNA repeats within the extended tandem duplications in E. coli results from unequal sister-chromosome exchanges. This conclusion follows from the observations on the segregation of completely or partly homozygous diploid segregants by heterozygous duplications. The formation of diploid segregants with preserved heterozygosity for the unselected markers could also result from symmetrical intrachromosomal recombination. Analysis of the segregant genotypes, however, confirmed their formation via unequal crossing over. The data obtained indicated that in tandem duplications segregation of diploid recombinants of different types was preceded by the formation of triplications as the products of unequal sister-chromosome exchanges. In heterozygous duplications, unequal crossing over is manifested as a highly frequent adaptive exchange, providing the survival of the most part of the duplication-carrying cells on selective medium. It is suggested that adaptive mutagenesis can be the consequence of unequal sister crossing over.     

Relation between the transforming activity of a marker and its proximity to the end of the DNA particle

Summary Tranforming pneumococcal DNA is inactivated by treatment with restriction enzymes. For mutations belonging to the same locus (amiA locus), the extent of inactivation depends strongly upon the mutations and the enzymes. Two EcoRI and one BamHI restriction sites have been located within the amiA locus. After treatment of donor DNA with either one of these enzymes, the lowest transforming activity is observed for mutations that map near restriction sites. This effect of proximity to the nearest end of the DNA fragment extends over a distance of 1,400 nucleotides. The curve of transforming activity versus DNA size obtained with endonuclease-generated DNA fragments is very similar to that obtained previously with mechanically sheared DNA. Both curves show a striking slope change for donor DNA size around 2,700 base pairs, i.e. twice the length found for the extent of the end effect. We suggest that for donor DNA fragments larger than 2,700 base pairs the transforming activity depends mainly upon the size of donor whereas for donor DNA fragments shorter than 2,700 base pairs both a size-dependent phenomenon and the end effect contribute to reduce drastically the transforming activity.     

Predictivity in taxonomy and the probability of a tree

The contribution ofJ. S. L. Gilmour to numerical taxonomy is reviewed. His important concept of natural classification, as being general-purpose classifications with high predictivity, led to the development of ideas of information content, unit characters and equal character-weighting. The concept of predicitivity is extended to taxonomic trees (phenograms or cladograms). Under certain assumption of random sampling of characters it is shown that the probability of recovering the correct tree topology or tree-form may be small if characters are few. There may be very many topologies or tree-forms, every one of which has individually a low probability. It is, however, possible to estimate the aggregate probability of trees which have more than some specified resemblance to the correct tree. The practical prospects of estimating the distribution of tree probabilities are discussed.Dedicated to the memory of JohnS. L. Gilmour.