Overall Informativity,OI, in DNA Polymorphisms Revealed by inter-AluPCR: Detection of Genomic Rearrangements

We studied two systems of multilocus markers revealed by PCR using primers directing amplification betweenAlurepeats in a tail-to-tail orientation. Genomic polymorphisms were detected as the presence or absence of the electrophoretic bands representing DNA fragments of a given length. A total of 104 such fragments segregating as Mendelian markers in a panel of eight CEPH families were analyzed by two-point linkage analysis. Fifty-one of these fragments were localized with respect to CEPH markers; they represented 33 loci, 7 of which were multiallelic. Locus-specific oligonucleotides were developed and used as hybridization probes to identify the mapped loci within a complex pattern of inter-AluPCR products. A great proportion of inter-AluPCR polymorphisms represented length variants within amplified DNA segments, while others were presumably due to mutations within the priming sites. To describe the expected number of informative loci per typing experiment we introduced a parameter called overall informativity (OI), which provides a single measure of the multiplex ratio and the informativity of markers contributing to a multilocus system (OIof a single locus is equivalent to its heterozygosity and cannot exceed 0.5 for a biallelic codominant marker). HighOIvalues (5.8 and 11.5) of the two presented systems of inter-AluPCR markers of random chromosomal distribution render them suitable for mapping genomic rearrangements such as genomic deletions in tumoral tissues. This was illustrated by the detection of loss of heterozygosity in the 9q22–qter region in sporadic colon cancer.     

JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region.

The high-affinity receptor for granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a unique alpha chain and a beta c subunit that is shared with the receptors for interleukin-3 (IL-3) and IL-5. Two regions of the beta c chain have been defined; these include a membrane-proximal region of the cytoplasmic domain that is required for mitogenesis and a membrane-distal region that is required for activation of Ras, Raf-1, mitogen-activated protein kinase, and S6 kinase. Recent studies have implicated the cytoplasmic protein tyrosine kinase JAK2 in signalling through a number of the cytokine receptors, including the IL-3 and erythropoietin receptors. In the studies described here, we demonstrate that GM-CSF stimulation of cells induces the tyrosine phosphorylation of JAK2 and activates its in vitro kinase activity. Mutational analysis of the beta c chain demonstrates that only the membrane-proximal 62 amino acids of the cytosolic domain are required for JAK2 activation. Thus, JAK2 activation is correlated with induction of mitogenesis but does not, alone, activate the Ras pathway. Carboxyl truncations of the alpha chain, which inactivate the receptor for mitogenesis, are unable to mediate GM-CSF-induced JAK2 activation. Using baculovirus-expressed proteins, we further demonstrate that JAK2 physically associates with the beta c chain but not with the alpha chain. Together, the results further support the hypothesis that the JAK family of kinase are critical to coupling cytokine binding to tyrosine phosphorylation and ultimately mitogenesis.     

Evolution of secondary structure in the family of 7SL-like RNAs

Primate and rodent genomes are populated with hundreds of thousands copies of Alu and B1 elements dispersed by retroposition, i.e., by genomic reintegration of their reverse transcribed RNAs. These, as well as primate BC200 and rodent 4.5S RNAs, are ancestrally related to the terminal portions of 7SL RNA sequence. The secondary structure of 7SL RNA (an integral component of the signal recognition particle) is conserved from prokaryotes to distant eukaryotic species. Yet only in primates and rodents did this molecule give rise to retroposing Alu and B1 RNAs and to apparently functional BC200 and 4.5S RNAs. To understand this transition and the underlying molecular events, we examined, by comparative analysis, the evolution of RNA structure in this family of molecules derived from 7SL RNA.RNA sequences of different simian (mostly human) and prosimian Alu subfamilies as well as rodent B1 repeats were derived from their genomic consensus sequences taken from the literature and our unpublished results (prosimian and New World Monkey). RNA secondary structures were determined by enzymatic studies (new data on 4.5S RNA are presented) and/or energy minimization analyses followed by phylogenetic comparison. Although, with the exception of 4.5S RNA, all 7SL-derived RNA species maintain the cruciform structure of their progenitor, the details of 7SL RNA folding domains are modified to a different extent in various RNA groups. Novel motifs found in retropositionally active RNAs are conserved among Alu and B1 subfamilies in different genomes. In RNAs that do not proliferate by retroposition these motifs are modified further. This indicates structural adaptation of 7SL-like RNA molecules to novel functions, presumably mediated by specific interactions with proteins; these functions were either useful for the host or served the selfish propagation of RNA templates within the host genome.Abbreviations FAM fossil Alu element - FLAM free left Alu monomer - FRAM free right Alu monomer - L-Alu left Alu subunit - R-Alu right Alu subunit Correspondence to: D. LabudaDedicated to Dr. Robert Cedergren on the occasion of his 25th anniversary at the University of Montreal     

Use of random amplified polymorphic DNA (RAPD) markers in the discrimination and verification of genotypes in Eucalyptus

We carried out four separate studies using random amplified polymorphic DNA (RAPD) markers to analyse samples of Eucalyptus supplied by several different organisations. The objective was to examine the reproducibility of the RAPD technique and its ability to discriminate between individual genotypes for verification of clonal identities. We found that RAPD profiles that are unique to a genotype can be generated reliably and simply and that even closely related genotypes can be distinguished. In addition, in each of the four studies, we detected cases where the plant material studied had been mis-sampled or mis-labelled (i.e. the RAPD profiles were not consistent with the identification numbers): (1) ramets of a Eucalyptus grandis clone were found to be derived from 2 different clones; (2) ramets labelled as 2 different Eucalyptus hybrid clones were found to be the same clone, owing to a mis-planted clonal hedge; (3) samples supplied as a single progeny of a controlled E. nitens cross were derived from two crosses involving different pairs of parents; (4) mis-labelling was detected for ramets of 4 of a set of 10 clones of E. grandis and E. camaldulensis. For three of the four studies, the detection of genotype mis-identifications was unexpected, suggesting that labelling or sampling errors during the handling of plant material are a frequent occurrence, with potentially serious economic consequences.     

Models of the serine protease domain of the human antithrombotic plasma factor activated protein C and its zymogen.

Three-dimensional structural analysis of physiologically important serine proteases is useful in identifying functional features relevant to the expression of their activities and specificities. The human serine protease anticoagulant protein C is currently the object of many genetic site-directed mutagenesis studies. Analyzing relationships between its structure and function and between naturally occurring mutations and their corresponding clinical phenotypes would be greatly assisted by a 3-dimensional structure of the enzyme. To this end, molecular models of the protease domain of protein C have been produced using computational techniques based on known crystal structures of homologous enzymes and on protein C functional information. The resultant models corresponding to different stages along the processing pathway of protein C were analyzed for structural and electrostatic differences arising during the process of protein C maturation and activation. The most satisfactory models included a calcium ion bound to residues homologous to those that ligate calcium in the trypsin structure. Inspection of the surface features of the models allowed identification of residues putatively involved in specific functional interactions. In particular, analysis of the electrostatic potential surface of the model delineated a positively charged region likely to represent a novel substrate recognition exosite. To assist with future mutational studies, binding of an octapeptide representing a protein C cleavage site of its substrate factor Va to the enzyme's active site region was modeled and analyzed.     

Prevalence of Colonization Factor Antigens (CFAs) and Adherence to HeLa Cells in Enterotoxigenic Escherichia coli Isolated from Feces of Children in São Paulo

Fifty-eight enterotoxigenic Escherichia coli (ETEC) strains, isolated from children with and without diarrhea in Sao Paulo, were examined for the presence of colonization factor antigens (CFAs) and their ability to adhere to HeLa cells. Antisera to CFA/I, the coli surface (CS) antigens CS1CS3, CS2CS3, and CS2 of CFA/II, CFA/III, and CS5CS6 and CS6 of CFA/IV were used. CFAs were identified in 43% of the ETEC strains: 40% of the strains with CFAs harbored CFA/I, 24% carried CFA/II (CS1CS3), 24% carried CFA/IV (CS6), and 12% carried CFA/IV (CS5CS6). CFAs occurred mainly among ETEC strains producing only heat-stable (ST-I) enterotoxin and in strains also producing heat-labile toxin (LT-I). No ETEC strains tested expressed CFA/III. A marked change in serotypes of ST-I-producing strains was found in Sao Paulo between 1979 and 1990. Adherence to HeLa cells was detected in 14% of the ETEC strains. All of them had a diffuse adherence pattern and produced only ST-I, and 88% carried CS6 antigen.     

Effects of day of estrous cycle, time of day, luteolysis, and embryo on uterine contractility in mares

One-minute continuous ultrasonic scans of longitudinal sections of the uterine body were videotaped, and contractility scores (1 to 5, minimal to maximal contractility) were assigned without knowledge of mare identity, day of the estrous cycle or pregnancy status. Contractility was assessed, and plasma progesterone concentrations were determined for each of 3 daily examinations (at 0800, 1600 and 2400 hours) from Day 9 to Day 19 (Day 0 = day of ovulation). For both the nonbred (n=11) and pregnant (n=11) mares, there was no effect of hour of scan on the extent of uterine contractility. When data for the nonbred mares were normalized to the onset of luteolysis (defined for each mare as the first >/=25% decrease in plasma progesterone concentrations between successive samples), there was an abrupt increase (P<0.05) in contractility 24 hours prior to the onset of luteolysis. Contractility was also assessed daily in 20 nonbred and 27 pregnant mares from Day 0 to Day 17. For the nonbred mares, a biphasic profile in contractility occurred during the estrous cycle as indicated by the following significant changes: a decrease between Days 0 and 2, an increase between Days 2 and 4, a plateau between Days 4 and 7, a decrease between Days 7 and 11, an increase between Days 11 and 13, and a decrease between Days 14 and 16. For pregnant mares, contractility increased (P<0.05) prior to the late-diestrous increase for nonbred mares. In addition, a significant reduction in contractility was detected on Day 5 in these mares compared with that in the nonbred mares. Contractility in the uterine body in 7 mares was assessed every 5 minutes after departure of the embryonic vesicle from the uterine body. Levels of contractility in the uterine body were lower (P<0.05) 55 minutes after the vesicle had exited the body than 相似文献   

Solid state NMR study of [epsilon-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.     

Solid-state nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme

Tyrosyl ring motions in alpha-lytic protease were investigated by solid-state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (< or =10(3) s(-1)) and one at the fast (> or =10(7) s(-1)) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (< or =10(3) s(-1) for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert-butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 10(4) and 10(5) s(-1). The latter rate rose further to 10(6) s(-1) in the hydrated hydrophilic solvents and to > or =10(7) s(-1) in aqueous crystals. The deuterium spectrum of native alpha-lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr-123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions ("wobbling"), estimated by their effect on spin-lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, alpha-lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant.