Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat

More than 100 resistance genes against wheat rust pathogens have been described in wheat and its relatives. Although many of them have been extensively used in wheat resistance breeding, none of these resistance loci has yet been analyzed at the molecular level. By screening a set of near-isogenic lines carrying different leaf rust resistance genes with a wheat probe encoding a serine/threonine protein kinase, we detected a polymorphic DNA fragment in the line with the Lr10 resistance gene. This fragment mapped to the Lr10 disease resistance locus and encodes a receptor-like protein kinase which we called LRK10. LRK10 contains a new type of extracellular domain not found in known plant or animal receptor kinases. Several conserved amino acids in S-domain glycoproteins and receptor-like kinases were also found in LRK10, suggesting that LRK10 and S-domain proteins belong to the same superfamily of specific recognition molecules in plants. Lrk10 was expressed at low levels in young seedlings and belongs to a gene family. Analysis of wheat lines with and without the Lr10 gene demonstrated that Lrk10 and Lr10 belong to the same genetic locus. We conclude that gene isolation based on protein kinase homology can identify new receptor domains and provide candidates for disease resistance genes in the complex wheat genome.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

Maintenance of transposable element copy number in natural populations of Drosophila melanogaster and D. simulans

To investigate the main forces controlling the containment of transposable elements (TE) in natural populations, we analyzed the copia, mdg1, and 412 elements in various populations of Drosophila melanogaster and D. simulans. A lower proportion of insertion sites on the X chromosome in comparison with the autosomes suggests that selection against the detrimental effects of TE insertions is the major force containing TE copies in populations of Drosophila. This selection effect hypothesis is strengthened by the absence of the negative correlation between recombination rate and TE copy number along the chromosomes, which was expected under the alternative ectopic exchange model (selection against the deleterious rearrangements promoted by recombination between TE insertions). A cline in 412 copy number in relation to latitude was observed among the natural populations of D. simulans, with very high numbers existing in some local populations (around 60 copies in a sample from Canberra, Australia). An apparent absence of selection effects in this Canberra sample and a value of transposition rate equal to 1–2 × 10^-3 whatever the population and its copy number agree with the idea of recent but temporarily drastic TE movements in local populations. The high values of transposition rate in D. simulans clearly disfavor the hypothesis that the low amount of transposable elements in this species could result from a low transposition rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Two Intracellular Signaling Pathways for the Dopamine D3 Receptor: Opposite and Synergistic Interactions with Cyclic AMP

Abstract: As cerebral neurons express the dopamine D₁ receptor positively coupled with adenylyl cyclase, together with the D₃ receptor, we have investigated in a heterologous cell expression system the relationships of cyclic AMP with D₃ receptor signaling pathways. In NG108-15 cells transfected with the human D₃ receptor cDNA, dopamine, quinpirole, and other dopamine receptor agonists inhibited cyclic AMP accumulation induced by forskolin. Quinpirole also increased mitogenesis, assessed by measuring [³H]thymidine incorporation. This effect was blocked partially by genistein, a tyrosine kinase inhibitor. Forskolin enhanced by 50–75% the quinpirole-induced [³H]thymidine incorporation. This effect was maximal with 100 n M forskolin, occurred after 6–16 h, was reproduced by cyclic AMP-permeable analogues, and was blocked by a protein kinase A inhibitor. Forskolin increased D₃ receptor expression up to 135%, but only after 16 h and at concentrations of >1 µ M . Thus, in this cell line, the D₃ receptor uses two distinct signaling pathways: it efficiently inhibits adenylyl cyclase and induces mitogenesis, an effect possibly involving tyrosine phosphorylation. Activation of the cyclic AMP cascade potentiates the D₃ receptor-mediated mitogenic response, through phosphorylation by a cyclic AMP-dependent kinase of a yet unidentified component. Hence, transduction of the D₃ receptor can involve both opposite and synergistic interactions with cyclic AMP.     

Instantaneous force-velocity-length relationship in diaphragmatic sarcomere

Coirault, Catherine, Denis Chemla, Jean-Claude Pourny,Francine Lambert, and Yves Lecarpentier. Instantaneousforce-velocity-length relationship in diaphragmatic sarcomere.J. Appl. Physiol. 82(2): 404-412, 1997.The simultaneous analysis of muscle force, length, velocity, andtime has been shown to precisely characterize the mechanicalperformance of isolated striated muscle. We tested the hypothesis thatthe three-dimensional force-velocity-length relationship reflectsmechanical properties of sarcomeres. In hamster diaphragm strips,instantaneous sarcomere length (SL) and muscle length were simultaneously measured during afterloaded twitches. SL was measured by means of laser diffraction. Wealso studied the influence of initialSL, abrupt changes in total load, and2 × 10⁷ M dantrolene.Baseline resting SL at the apex of thelength-active tension curve was 2.2 ± 0.1 µm, whereasSL at peak shortening was 1.6 ± 0.1 µm in the preloaded twitch and 2.1 ± 0.1 µm in the "isometric" twitch. Over the whole load continuum and at anygiven level of isotonic load, there was a unique relationship between instantaneous sarcomere velocity and instantaneousSL. Part of this relationship was timeindependent and initial SL independent and was markedly downshifted after dantrolene. When five different muscle regions were considered, there were no significant variations ofSL and sarcomere kinetics along themuscle. These results indicate that the time- and initiallength-independent part of the instantaneous force-velocity-lengthrelationship previously described in muscle strips reflects intrinsicsarcomere mechanical properties.

     

Ataxia with Vitamin E Deficiency: Refinement of Genetic Localization and Analysis of Linkage Disequilibrium by Using New Markers in 14 Families

Ataxia with vitamin E deficiency (AVED) is an autosomal recessive disease characterized clinically by neurological symptoms with often striking resemblance to those of Friedreich ataxia. This disorder has been reported previously as familial isolated vitamin E deficiency. We have mapped recently the AVED locus to a 5-cM confidence interval on chromosome 8q by homozygosity mapping in six Mediterranean families. We have now analyzed six new and two previously described families and demonstrate genetic homogeneity despite important clinical variability and wide geographic origins. Analysis of nine new tightly linked microsatellite markers, including four characterized in this study, revealed a predominant but not unique mutation in northern African populations, where this condition is more frequent. Haplotype analysis but also classical recombinations allowed us to refine the AVED position to a 1-cM interval. A YAC contig over this interval was constructed from marker STSs and YAC fingerprint data, in order to facilitate the search of the AVED gene.     

Chromosome 17 abnormalities and lack of TP53 mutations in paediatric central nervous system tumours

Central nervous system (CNS) tumours are the most common solid tumours in children. Cytogenetic and molecular genetic studies of these neoplasms have previously shown abnormalities of chromosome 17, implicating genes on this autosome in tumorigenesis. To identify mutations in the TP53 tumour suppressor gene (17p13.1), we have sequenced the five highly conserved regions of this gene in 29 mixed paediatric CNS tumors. No mutations were detected by this analysis. In order to identify other candidate disease loci on chromosome 17, we have carried out a detailed deletion mapping analysis using 16 polymorphic DNA markers on 19 of the above tumours and an additional four cases. Abnormalities of chromosome 17 occurred in nine cases (39%), six of which were primitive neuroectodermal tumour (PNET)-medulloblastomas. These findings suggest that it is unlikely that the TP53 gene is directly involved in the development of common paediatric brain tumours. This is in contrast to findings from adult brain and other tumour types. Moreover, the frequency of chromosome 17 aberrations, especially in PNET-medulloblastomas, suggests that other genes on this chromosome contribute to tumourigenesis.     

Carotenoid content in growing cells of Haematococcus pluvialis during a sunlight cycle

Under certain culture conditions, cells of the chlorophyte Haematococcus pluvialis accumulate significant amounts of astaxanthin. This study describes biomass and carotenoid production during a sunlight cycle in a continuous culture of growing cells of H. pluvialis and shows that these two parameters are under the control of irradiance. The hourly carotenoid production increases with light intensity and, in our culture conditions, carotenoid accumulation occurs in a few hours and without any morphological change in the algae. These carotenoids seem to be efficient in protecting algal cells against photoinhibition damage if their content is greater than 1% dry biomass. Below this concentration, that is to say in the early hours of high light intensity, dry biomass decreases due to cell lysis. The results demonstrate that secondary carotenoid accumulation in H. pluvialis may occur in the active growth phase and is stimulated from the first hours of sunlight illumination.     

Opa-typing: a high-resolution tool for studying the epidemiology of gonorrhoea

A single gonococcus possesses a family of 11 distinct and highly variable opa genes. The extensive variation and rapid evolution of the opa gene repertoire has been exploited to provide a high-resolution typing method for studies of the short-term transmission of gonorrhoea. The 11 opa genes are amplified with a single pair of primers by the polymerase chain reaction, digested with frequently-cutting restriction enzymes, and the fragments are fractionated on polyacrylamide to provide an opa-type. The method appeared to be highly discriminatory as the opa-types of gonococci, isolated world-wide over the last 30 years, were all different. Opa-typing discriminated between isolates of the same auxotype/serovar class. Similarly, there were 41 opa-types among 43 consecutive isolates from a sexually transmitted disease (STD) clinic. The two pairs of isolates from this clinic that gave the same opa-types were identical by other criteria and may have been from unsuspected sexual contacts. With one minor exception, identical opa-types were obtained from gonococci recovered from known sexual contacts. These results suggest that variation in the family of 11 opa genes evolves so rapidly that the opa-types of gonococci are distinguishable, unless the isolates are from sexual contacts or a short chain of disease transmission. The identification of gonococci with identical opa-types is therefore believed to be a good indicator that the individuals from which they were recovered were sexual partners, or part of a short chain of disease transmission.