Distribution of olfactory receptor genes in the human genome.

We demonstrate that members of the olfactory receptor (OR) gene family are distributed on all but a few human chromosomes. Through FISH analysis, we show that OR sequences reside at more than 25 locations in the human genome. Their distribution is biased for terminal bands. Flow-sorted chromosomes were used to isolate 87 OR sequences derived from 16 chromosomes. Their sequence-relationships are indicative of the inter- and intrachromosomal duplications responsible for OR family expansion. The human genome has accumulated a striking number of dysfunctional copies: 72% of the sequences are pseudogenes. ORF-containing sequences predominate on chromosomes 7, 16 and 17.

Calmodulin regulation of the ATP-dependent calcium uptake by inverted vesicles prepared from rabbit synaptosomal plasma membranes.

Calmodulin has been shown to activate the ATP-dependent Ca2+ uptake in inside-out vesicles which have been prepared from rabbit synaptosomal plasma membranes by the methodology of Gill et al. (Gill, D.L., Grollman, E.F. and Kohn, L.D. (1981) J. Biol. Chem. 256, 184-192). Following extensive washings of these membranes with EGTA/EDTA solutions, the Ca2+ uptake activity demonstrated an affinity for calmodulin of 30 nM and an affinity for Ca2+ of 2 microM. The activity was completely inhibited by the anticalmodulin compound R24571 (Ki congruent to 8 microM). The molecular weight of the ATPase molecule, revealed by a combination of the [125I]calmodulin overlay technique and [32P]phosphoenzyme electrophoresis, was 145 000. The overlay technique also revealed that the mechanism of activation is via a direct binding of calmodulin to the pump molecule.

Cyclic adenosine 3',5'-monophosphate-dependent regulation of purified bovine aortic calcium/calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase was extracted from bovine aortic muscularis by a low ionic strength buffer containing 50% glycerol. It was purified 130-fold with a 10% yield by anion-exchange chromatography followed by affinity chromatography on calmodulin-Sepharose. The enzyme was 95% calcium/calmodulin-dependent and exhibited a specific activity of 2-6 mumol/min per mg. It phosphorylated the myosin regulatory light chain exclusively. The apparent Kd for calmodulin was 6.3 nM. Upon phosphorylation of the enzyme by the catalytic subunit of cyclic AMP-dependent protein kinase, its affinity for calmodulin decreased 4-fold, without alteration of the V. When examined by SDS-polyacrylamide gel electrophoresis, the purified enzyme was made up of two major peptides (Mr 142 000 and 131 000, respectively), with a minor 80 000 dalton peptide. All these peptides were 32P-labeled after incubation with [gamma-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. Also, after non-denaturing polyacrylamide gel electrophoresis, they all exhibited myosin light chain kinase activity, suggesting that the 131 000 and 80 000 dalton species are proteolytic products of the native enzyme of Mr 142 000. Vascular smooth muscle myosin light chain kinase is therefore soluble, calcium/calmodulin dependent and phosphorylatable by cyclic AMP-dependent protein kinase with concomitant decrease in its affinity for calmodulin. These features account for the beta-adrenergic relaxation of vascular smooth muscle.

Isolation of cardiac membrane proteolipids by high pressure liquid chromatography. A comparison of reticular and sarcolemmal proteolipids, phospholamban and calciductin.

Membrane-bound phosphorylatable proteolipids were reported to play a role in the regulation of transmembrane Ca2+ fluxes by catecholamines. A generally applicable purification procedure is described by which such proteolipids as the cardiac sarcoplasmic reticulum phospholamban is purified by solvent extraction followed by high pressure liquid chromatography on microparticulate silica. Phospholamban is thereby purified with a yield of 3.37 mg from 100 mg of sarcoplasmic reticulum proteins, significantly higher than that obtained by any of the previously reported procedures. It appeared homogeneous upon dodecyl sulfate-polyacrylamide gel electrophoresis where it is stained by Coomassie blue and detected by autoradiography. The same procedure is applicable to cardiac sarcolemmal calciductin. Both proteolipids exhibit the same Mr 11 000 and pI 3.7 upon two-dimensional gel electrophoresis. Their amino acid compositions are very similar if not identical. This raises the intriguing possibility that phospholamban and calciductin are identical though they obviously belong to different membranes.

Radioelectrophoresis: a specific microassay for parvalbumins. Application to muscle biopsies from man and other vertebrates.

A two-dimensional radioelectrophoretic method is described, by which parvalbumins from minute biopsy samples (approx. 50 mg) can be detected and quantitated by their 45Ca2+-binding properties. In the first dimension, parvalbumins are purified by sieving through a gradient polyacrylamide gel and collected at the bottom of the electrophoresis tubes. The second dimension is a disc electrophoresis in the presence of 45Ca2+. Parvalbumins can thus be identified and quantitated by three criteria: low molecular weight, acidic character and calcium-binding properties, since they are never exposed to denaturing conditions. Validity of the technique was demonstrated on carp myogen, and on extracts from rabbit psoas and heart muscles. Application of this method to the shrew fast beating myocardium shows that it does contain parvalbumin, in agreement with the proposed role of soluble relaxing factor (Pechère et al. (1977) FEBS Lett. 75, 111--1141. When applied to human muscle biopsies, radioelectrophoresis points to an uneven distribution of parvalbumin among different skeletal muscles. For the human limb muscles tested in this study, the parvalbumin content is similar to that of rabbit psoas muscle.

Immunocytochemical and biochemical evidence for the presence of calmodulin in bull sperm flagellum. Isolation and characterization of sperm calmodulin.

Upon fluorescent staining with a goat antibody anti-ram testis calmodulin, washed bull sperm appears to contain calmodulin in the acrosome, in the post acrosomal region, in the neck region probably associated with the implantation plates and thin laminated fibers, and in a sheath around the upper part of the flagellum. Heads and midpieces + tails were separated by elutriation of sonicated sperm. Immunofluorescent labeling of fragments confirms the presence of calmodulin in implantation plates, where sonication disrupted heads from midpieces, and in a sheath around the midpiece and the upper part of the principal piece. These results were confirmed by electrophoretic and radioenzymatic assays of calmodulin in the fragments, using calmodulin-deficient Ca2+/calmodulin-dependent myosin light chain kinase. Small but significant amounts (approx. 3 micrograms per 10 (10) sperm) are found in midpieces + tails vs. approx. 280 micrograms in the same number of heads. These results are in agreement with a recent report from Jones et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2772-2776. Sperm calmodulin was purified from a whole sperm 1 M KCl extract and found to exhibit the same characteristics as other mammalian calmodulins isolated so far in terms of ultraviolet absorption spectrum and amino acid composition, including one residue of epsilon-N-trimethyllysine. Its behavior upon SDS-polyacrylamide gel electrophoresis was dependent on the presence or absence of Ca2+. The high performance liquid chromatography tryptic peptide maps were similar, if not identical, to mammalian calmodulin maps (Autric et al. (1980) Biochim. Biophys. Acta 631, 139-147). Sperm calmodulin is therefore probably identical to the somatic cell protein.

Regulation of calcium accumulation and efflux from platelet vesicles. Possible role for cyclic-AMP-dependent phosphorylation and calmodulin.

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a (Ca2+ + Mg2+)-ATPase activity of about 10 nmol (min . mg)-1 and an ATP-dependent Ca2+ uptake of about 10 nmol (min . mg)-1. When incubated in the presence of Mg[gamma-32P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [32P]phosphate-labeled Ca2+ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca2+ or Ca2+ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of 125I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol (Mr = 94 000, 87 000, 60 000 and 43 000). Some minor calmodulin-binding proteins were enriched in the membrane fractions (Mr = 69 000, 57 000, 39 000 and 37 000). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca2+ uptake is essentially unaltered, while the Ca2+ capacity is diminished as a consequence of a doubling in the rate of Ca2+ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca2+ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 microM calmodulin result in increased levels of vesicle phosphorylation.

The first green lineage cdc25 dual-specificity phosphatase.

The Cdc25 protein phosphatase is a key enzyme involved in the regulation of the G(2)/M transition in metazoans and yeast. However, no Cdc25 ortholog has so far been identified in plants, although functional studies have shown that an activating dephosphorylation of the CDK-cyclin complex regulates the G(2)/M transition. In this paper, the first green lineage Cdc25 ortholog is described in the unicellular alga Ostreococcus tauri. It encodes a protein which is able to rescue the yeast S. pombe cdc25-22 conditional mutant. Furthermore, microinjection of GST-tagged O. tauri Cdc25 specifically activates prophase-arrested starfish oocytes. In vitro histone H1 kinase assays and anti-phosphotyrosine Western Blotting confirmed the in vivo activating dephosphorylation of starfish CDK1-cyclinB by recombinant O. tauri Cdc25. We propose that there has been coevolution of the regulatory proteins involved in the control of M-phase entry in the metazoan, yeast and green lineages.

MEFV mutations in Behçet's disease.

Familial Mediterranean fever (FMF) and Behçet's disease (BD), both inflammatory diseases, are highly prevalent in the Middle Eastern and Mediterranean populations. FMF is a Mendelian autosomic recessive disease linked to MEFV, a gene of unknown function. BD in contrast is a polyfactorial disease associated with the major histocompatibility complex. Because FMF and BD have epidemiological similarities, we asked whether the FMF gene was implicated in BD. We screened for the common MEFV mutations a cohort of 114 chromosomes from definite BD patients [meeting the criteria of the International study group] and probable cases [meeting at least two of these criteria]. We screened in parallel an ethnically matched cohort of FMF and control chromosomes. The M694V, V726A and E148Q mutations tended to be more frequent in definite BD (2.6%, 2.6%, and 5.2%, respectively) than in controls (0%, 0%, and 2.2%). The P706 polymorphism was found in 10.5% of the probable BD chromosomes, but in only 1.6% of the controls (p=0.01). Because some MEFV mutations were more frequent in BD than in controls, we suggest that they may act as additional susceptibility factors in BD.

Four novel dystrophin point mutations: detection by protein truncation test and transcript analysis in lymphocytes from Duchenne muscular dystrophy patients.

About 30% of cases of Duchenne muscular dystrophy (DMD) result from point mutations randomly distributed in the immense dystrophin gene. As already observed for the gross rearrangements, most of the DMD point mutations identified so far give rise to truncated proteins. Here, we report results of a comprehensive search for point mutations within the dystrophin gene based on illegitimate transcript analysis by using the RT-PCR technique in combination with a method capable of selectively detecting translation-termination mutations, called the protein truncation test (PTT). The RT-PCR-PTT procedure was successful in detecting mutations in 4 out of the 6 DMD patients who were investigated. These mutations, Q2972X in exon 59, 3474insC in exon 24, delT393-G394+5 in exon/intron 3, and 2436delAG in exon 18, had not been previously described. Moreover, several alternatively spliced forms of ectopic dystrophin mRNA were characterized in normal controls or in DMD patients. Most of these differentially spliced messages consisting of exon skipping or intronic sequence insertion are reported here for the first time.

Familial Mediterranean fever in the 'Chuetas' of Mallorca: a question of Jewish origin or genetic heterogeneity.

Familial Mediterranean fever (FMF) is a hereditary disease commonly found among Jews, Armenians, Turks and Arabs. Recently, FMF was found in the 'Chuetas', a unique community on the island of Mallorca (Spain). To address the question of their possible Jewish origin, we analysed markers known to be linked to the gene responsible for FMF in Jews (MEFV) in this population. We found that 1/3 of the 16p13.3 chromosomes of the 'Chuetas' FMF patients bore the major ancestral haplotypes (S,S2) and their corresponding M694V and E148Q mutations, displayed by Jews from North Africa. Furthermore, we also detected a novel mutation (L110P) in this community. Yet 2/3 of these patients bore S negative haplotypes and lack the mutations commonly known to cause FMF. These results confirm that at least some of the 'Chuetas' share a common origin with Jews. However, they also provide evidence for the possibility of genetic heterogeneity in this disorder.

Phenotype-genotype correlation in Jewish patients suffering from familial Mediterranean fever (FMF).

Familial Mediterranean Fever is one of the most frequent recessive disease in non-Ashkenazi Jews. The gene responsible for the disease (MEFV) has very recently been identified. The M694V ('MED') mutation was found in about 80% of the FMF Jewish (Iraqi and North African) chromosomes. To see if the presence of this mutation could be correlated with particular traits of the disease, we examined a number of clinical features in a panel of 109 Jewish FMF patients with 0, 1 or 2 MED mutations. We showed that homozygosity for this mutation was significantly associated with a more severe form of the disease. In homozygous patients, the disease started earlier (mean age 6.4 +/- 5 vs 13.6 +/- 8.9) and both arthritis and pleuritis were twice as frequent as in patients with one or no M694V mutation. Moreover, 3/3 patients with amyloidosis displayed two MED mutations. No association was found with fever, peritonitis, response to colchicine and erysipeloid eruption. The present result strongly suggests the potential prognostic value of the presence of this mutation.

Evidence for aggregation of endothelin 1 in water.

In this report it is shown by CD spectroscopy that endothelin 1, when dissolved in water, is able to present intermolecular interactions leading to formation of aggregates. Surface tension and conductivity measurements suggest that the aggregation occurs through formation of micelles with a CMC of about 2.2 x 10(-5) M.

The protein inhibitor of adenosine 3':5'-monophosphate-dependent protein kinases. Isolation and characterization of three isoinhibitors.

The protein inhibitor of adenosine 3':5 monophosphate-dependent protein-kinases has been purified from rabbit skeletal muscle by affinity chromatography on Sepharose-bound catalytic subunit of the kinase (Demaille et al. (1977) Biochemistry 16, 3080-3086). The inhibitory material can be separated into three species A, B and C either by polyacrylamide gel electrophoresis or by anion-exchange on DEAE-cellulose. However, the isoinhibitors display the same molecular weight and isoelectric point, and the same absence of acid-stable covalently bound phosphate. Their amino acid compositions are strikingly similar and their NH2-terminus are blocked. Also, their COOH-terminus appear to be very close to one another when studied by carboxypeptidase Y digestion. Their major difference lies in their inhibitory properties which are significantly weaker in inhibitor C (Ki = 0.87 nM) than in A and B (Ki = 0.18 and 0.23 nM, respectively). This is the first report on the existence of various forms of the protein-kinase inhibitor that exhibit different inhibitory properties and may play a role in the regulation of the protein-kinase system.

Towards structural models of molecular recognition in olfactory receptors.

The G protein coupled receptors (GPCR) are an important class of proteins that act as signal transducers through the cytoplasmic membrane. Understanding the structure and activation mechanism of these proteins is crucial for understanding many different aspects of cellular signalling. The olfactory receptors correspond to the largest family of GPCRs. Very little is known about how the structures of the receptors govern the specificity of interaction which enables identification of particular odorant molecules. In this paper, we review recent developments in two areas of molecular modelling: methods for modelling the configuration of trans-membrane helices and methods for automatic docking of ligands into receptor structures. We then show how a subset of these methods can be combined to construct a model of a rat odorant receptor interacting with lyral for which experimental data are available. This modelling can help us make progress towards elucidating the specificity of interactions between receptors and odorant molecules.

Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction?

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

Calcium and magnesium binding by parvalbumin. A proton magnetic resonance spectral study.

Proton magnetic resonance spectroscopy has been used to monitor the kinetics and nature of the conformational transitions induced by binding of calcium and magnesium to carp parvalbumin. Rate constants have been determined for the exchange between the cation dependent conformational states of the protein in solution. These data enable a description of the kinetics and mechanism controlling the calcium flux in vivo during contraction.

Expression and subcellular localization of dystrophin in skeletal, cardiac and smooth muscles during the human development.

Dystrophin, the product of the DMD gene, is present in all muscle types in normal individuals. Its function has yet to be elucidated, but its absence or the presence of a truncated version of the protein is responsible for the appearance of Duchenne and Becker muscular dystrophies. Using monoclonal antibodies raised against distinct regions of the dystrophin protein, we have examined its expression and subcellular distribution during the human development in skeletal and smooth muscles. We show that both dystrophin expression and its association to the plasma membrane take place earlier in cardiac and smooth muscles (8 weeks of gestation) than in skeletal muscle. In skeletal muscle, dystrophin is first detected in the cytoplasm, and progressively localizes to the plasma membrane from 10 weeks onwards. Since we have obtained marked differences in staining when using antibodies against either a central region of the protein or the C-terminal part, we suggest that different fetal and adult dystrophin isoforms are expressed, probably differing in their C-terminal domain. These findings are discussed in the context of the pathology of Duchenne muscular dystrophy.

Becker muscular dystrophy: demonstration of the carrier status of a female by immunoblotting and immunostaining.

Becker muscular dystrophy (BMD) often results from in-frame mutations of the dystrophin gene, leading to the production of an altered-sized protein. We examined the expression of dystrophin in a BMD patient and in his asymptomatic mother by Western blot and immunofluorescence. The combination of these techniques allowed us to demonstrate the presence of two different dystrophins, normal-sized or reduced-sized in the muscular fibers of the asymptomatic carrier. This result emphasizes the value of dystrophin analysis for carrier detection and genetic counselling of families with Becker muscular dystrophy.

Multiplex fluorescent RT-PCR to quantify leukemic fusion transcripts.

The detection of chimeric transcripts derived from aberrant chromosomal fusion events provides an exceptionally valuable toolfor the diagnosis of leukemia. We have developed a simple, inexpensive, reproducible, and automated method to quantify RT-PCR products. Our approach utilizesfluorescent PCRfor the co-ampification of the specific fusion transcript with an internal control (HPRT). We have also combined the advantages of real-time quantitative PCR, namely continuous fluorescent detection of PCR products with the low cost of an endpoint assay by examining in a novel manner the amount offluorescent PCR product generated during the exponential phase of amplification. This has been achieved by using the automated loading and quantification capacity of a laser-induced fluorescence capillary electrophoresis system, the ABI PRIsMS 310A, so that we can effectively monitor amplification during the exponential phase cheaply, reproducibly, and in a sensitive manner. We have carefully verified our new technique using five leukemia cell lines, each expressing a differentfusion transcript. Specificity and reproducibility (cy within 10%) have been examined and demonstrate the excellent precision of our technology. The high sensitivity levels of at least 10(-4) to 10(-6) obtainedfor the serial dilutions of the five cell lines validate the choice of our fluorescent PCR as a comparable method to other more complicated and expensive methods. Our results have allowed us to quantify PCR products and the amount of chimeric mRNA originating from the translocation breakpoint. We demonstrate that our novelfluorescent method is useful to detect and quantify residual leukemic cells in patients undergoing therapy.