Multiple sclerosis: in situ evidence for antibody- and complement-mediated demyelination

Blue light responses in higher plants can be mediated not only by specific blue light receptors, but also by the red/far-red photoreversible phytochrome system. The question of interdependence between these photoreceptors has been debated over many years. The availability of Arabidopsis mutants for the blue light receptor CRY1 and for the two major phytochromes phyA and phyB allows a reinvestigation of this question. The analysis of photocontrol of seed germination, inhibition of hypocotyl growth and anthocyanin accumulation clearly demonstrates that (i) phyA shows a strong control in blue light responses especially at low fluence rates; (ii) phyB mediated induction reactions can be reversed by subsequent blue light irradiations; and (iii) CRY1 mediates blue light controlled inhibition of hypocotyl growth only at fluence rates higher than 5 mumol m-2s-1 and independently of phytochrome A and B.     

HYPOSENSITIVE TO LIGHT,an Alpha/Beta Fold Protein,Acts Downstream of ELONGATED HYPOCOTYL 5 to Regulate Seedling De-Etiolation

Ambient light has profound effects on early seedling de-etiolation through red and far-red light-absorbing phytochromes and blue and UV-A light-absorbing cryptochromes.Subsequent integration of various light signal transduction pathways leads to changes in gene expression and morphogenic responses.Here,we report the isolation of a new Arabidopsis light-signaling component,HYPOSENSITIVE TO LIGHT or HTL.Both htl-1 and htl-2 alleles displayed a long hypocotyl phenotype under red,far-red,and blue light,whereas overexpression of HTL caused a short hypocotyl phenotype under similar light conditions.The mutants also showed other photomorphogenic defects such as elongated petioles,retarded cotyledon and leaf expansion,reduced accumulation of chlorophyll and anthocyanin pigments,and attenuated expression of light-responsive CHLOROPHYLL A/B BINDING PROTEIN 3 and CHALCONE SYNTHASE genes.HTL belongs to an alpha/beta fold protein family and is localized strongly in the nucleus and weakly in the cytosol.The expression of HTL was strongly induced by light of various wavelengths and this light induction was impaired in elongated hypocotyl 5.HY5directly bound to both a C/G-box and a G-box in the HTL promoter but with a greater affinity toward the C/G-box.HTL.therefore,represents a new signaling step downstream of HY5 in phy-and cry-mediated de-etiolation responses.     

Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene

The plant hormone auxin controls many aspects of development and acts in part by inducing expression of various genes. Arabidopsis thaliana semidominant shy2 (short hypocotyl) mutations cause leaf formation in dark-grown plants, suggesting that SHY2 has an important role in regulating development. Here we show that the SHY2 gene encodes IAA3, a previously known member of the Aux/IAA family of auxin-induced genes. Dominant shy2 mutations cause amino acid changes in domain II, conserved among all members of this family. We isolated loss-of-function shy2 alleles including a putative null mutation. Gain-of-function and loss-of-function shy2 mutations affect auxin-dependent root growth, lateral root formation, and timing of gravitropism, indicating that SHY2/IAA3 regulates multiple auxin responses in roots. The phenotypes suggest that SHY2/IAA3 may activate some auxin responses and repress others. Models invoking tissue-specificity, feedback regulation, or control of auxin transport may explain these results.     

Control of cell elongation and stress responses by steroid hormones and carbon catabolic repression in plants

Molecular analysis of Arabidopsis mutants displaying hypocotyl elongation defects in both the dark and light revealed recently that steroids play an essential role as hormones in plants. Deficiencies in brassinosteroid biosynthesis and signalling permit photomorphogenic development and light-regulated gene expression in the dark, and result in severe dwarfism, male sterility and de-repression of stress-induced genes in the light. A cytochrome P450 steroid hydroxylase (CYP90) controls a rate limiting step in brassinosteroid biosynthesis and appears to function as a signalling factor in stress responses. Another key step in steroid biosynthesis is controlled by the Arabidopsis SNF1 kinases that phosphorylate the 3-hydroxy-3methylglutaryl-CoA reductase. The activity of SNF1 kinases is regulated by PRL1, an evolutionarily conserved alpha-importin-binding nuclear WD-protein. The prl1 mutation results in cell elongation defects, de-repression of numerous stress-induced genes, and augments the sensitivity of plants to glucose, cold stress and several hormones, including cytokinin, ethylene, auxin, and abscisic acid.     

Antibody response against a Leishmania donovani amastigote-stage-specific protein in patients with visceral leishmaniasis

Shoots of higher plants grow upward in response to gravity. To elucidate the molecular mechanism of this response, we have isolated shoot gravitropism (sgr) mutants in Arabidopsis thaliana. In this report, we describe three novel mutants, sgr4-1, sgr5-1 and sgr6-1 whose inflorescence stems showed abnormal gravitropic responses as previously reported for sgr1, sgr2 and sgr3. These new sgr mutations were recessive and occurred at three independent genetic loci. The sgr4-1 mutant showed severe defect in gravitropism of both inflorescence stem and hypocotyl but were normal in root gravitropism as were sgr1 and sgr2. The sgr5-1 and sgr6-1 mutants showed reduced gravitropism only in inflorescence stems but normal in both hypocotyls and roots as sgr3. These results support the hypothesis that some mechanisms of gravitropism are genetically different in these three organs in A. thaliana. In addition, these mutants showed normal phototropic responses, suggesting that SGR4, SGR5 and SGR6 genes are specifically involved in gravity perception and/or gravity signal transduction for the shoot gravitropic response.     

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.     

Receptor editing occurs frequently during normal B cell development

Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that lambda light chain genes usually rearrange after kappa genes. This allowed us to analyze kappa loci in IgMlambda+ cells to determine how frequently in-frame kappa genes fail to suppress lambda gene rearrangements. To do this, we analyzed recombined VkappaJkappa genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the kappa locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged kappa loci, as nearly half (47%) of the RS-inactivated VkappaJkappa joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.     

High frequency of interactions between lung cancer susceptibility genes in the mouse: mapping of Sluc5 to Sluc14

Although several genes that cause monogenic familial cancer syndromes have been identified, susceptibility to sporadic cancer remains unresolved. Animal experiments have demonstrated multigenic control of tumor susceptibility. Recently, we described four mouse lung cancer susceptibility (Sluc) loci, the main effects of which are masked by their mutual interactions. Because such interactions can considerably affect the strategies for identification of cancer susceptibility genes in humans, it is necessary to establish whether they are common or rare. Here, we report the mapping of 10 additional Sluc loci and show that 13 of the 14 Sluc loci are involved in one or more interactions, demonstrating that interactions of tumor susceptibility genes are frequent and that they probably form complex networks.     

Localization to chromosome 11 of a gene encoding a human minor histocompatibility antigen

The graft-versus-host disease (GVHD) seen in human leukocyte antigen (HLA)-matched sibling bone marrow transplants is by definition due to the "minor" histocompatibility antigens (mHAs) encoded outside the HLA region of human chromosome 6. Few of these antigens have been characterized in humans, and in general the locations of the encoding loci are unknown. Genetic experiments performed in mice have identified many mHAs, but only a few genes have been identified. Using T lymphocyte clones reactive with specific mHAs, combined with genetic linkage analysis, we identified two distinct loci in a single patient, each locus encoding an antigen presented to a T cell clone by HLA-B7. The technique used in this study should allow a rough enumeration of the number of mHAs in humans that are capable of eliciting T cell responses in vivo. Whether these T cell responses correlate with clinical GVHD is not yet clear.     

Molecular characterization of a novel amplicon at 1q21-q22 frequently observed in human sarcomas

Suppressors of position-effect variegation (Su(var)s) in Drosophila melanogaster are usually studied in the presence of chromosomal rearrangements, which exhibit variegated expression of euchromatic genes moved near to, or heterochromatic genes moved away from, centromeric heterochromatin. However, the effects of Su(var) mutations on heterochromatic gene expression in the absence of a variegating re-arrangement have not yet been defined. Here we present a number of results which suggest that Su(var) gene products can interact to affect the expression of the light gene in its normal heterochromatic location. We initially observed that eye pigment was reduced in several Su(var) double mutants; the phenotype resembled that of light mutations and was more severe when only one copy of the light gene was present. This reduced pigmentation could be alleviated by a duplication for the light gene or by a reduction in the amount of cellular heterochromatin. In addition, the viability of most Su(var) double mutant combinations tested was greatly reduced in a genetic background of reduced light gene dosage, when extra heterochromatin is present. We conclude that Su(var) gene products can affect expression of the heterochromatic light gene in the absence of any chromosomal rearrangements. However, it is noteworthy that mutations in any single Su(var) gene have little effect on light expression; we observe instead that different pairings of Su(var) mutations are required to show an effect on light expression. Interestingly, we have obtained evidence that at least two of the second chromosome Su(var) mutations are gain-of-function lesions, which also suggests that there may be different modes of interaction among these genes. It may therefore be possible to use this more sensitive assay of Su(var) effects on heterochromatic genes to infer functional relationships among the products of the 50 or more known Su(var) loci.     

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.     

Identifying loci required for follicular patterning using directed mosaics

We have developed a 'directed mosaic' system in Drosophila by using the GAL4 system to control the expression of the yeast recombinase, FLP, in a spatial and temporal fashion. By directing FLP expression, we show that it is possible to efficiently and specifically target loss-of-function studies for vital loci to the developmental pathway of interest. A simple F1 adult phenotypic screen demonstrated that most adult tissues can be analyzed with this approach. Using GAL4 lines expressed during oogenesis, we have refined the system to examine the roles of vital loci in the development of the follicular epithelium. We have identified essential genes involved in egg chamber organization, cell migration and cell shape. Further, we have used this technique to gain insights into the role of the Drosophila EGF receptor pathway in establishing the egg axes. Finally, using different UAS-FLP, GAL4 and existing FRT lines, we have built stocks that permit the analysis of approximately 95% of the genome in follicular mosaics.     

Relationship between the genomic organization and the overlapping embryonic expression patterns of the zebrafish dlx genes

To understand the relationship between the expression and the genomic organization of the zebrafish dlx genes, we have determined the genomic structure of the dlx2 and dlx4 loci. This led to the identification of the zebrafish dlx1 and dlx6 genes, which are closely linked to dlx2 and dlx4, respectively. Therefore, the inverted convergent configuration of Dlx genes is conserved among vertebrates. Analysis of the expression patterns of dlx1 and dlx6 showed striking similarities to those of dlx2 and dlx4, respectively, the genes to which they are linked. Furthermore, the expression patterns of dlx3 and dlx7, which likely constitute a third pair of convergently transcribed genes, are indistinguishable. Thus, the overlapping expression patterns of linked Dlx genes during embryonic development suggest that they share cis-acting sequences that control their spatiotemporal expression. The evolutionary conservation of the genomic organization and combinatorial expression of Dlx genes in distantly related vertebrates suggest tight control mechanisms that are essential for their function during development.     

Social relations and extent and severity of coronary artery disease. The Stockholm Female Coronary Risk Study

Peak bone mass is a major determinant of risk of osteoporotic fracture. Family and twin studies have found a strong genetic component to the determination of bone mineral density (BMD). However, BMD is a complex trait whose expression is confounded by environmental influences and polygenic inheritance. The number, locations, and effects of the individual genes contributing to natural variation in this trait are all unknown. Experimental animal models provide a means to circumvent complicating environmental factors, and the development of dense genetic maps based on molecular markers now provides opportunities to resolve quantitative genetic variation into individual regions of the genome influencing a given trait (quantitative trait loci, QTL). To begin to identify the heritable determinants of BMD, we have examined genetically distinct laboratory mouse strains raised under strict environmental control. Mouse whole-body bone mineral content by dual-energy X-ray absorptiometry (DXA) correlated strongly with skeletal calcium content by ashing, and peak whole-body BMD by DXA in female mice occurred at approximately 80-90 days of age. We therefore determined mean body weight and peak whole body BMD values in 12-week-old female mice from a panel of 24 recombinant inbred (RI) BXD strains, derived from a cross between C57BL/6 and DBA/2 progenitors. The distribution of body weight and BMD values among the strains clearly indicated the presence of strong genetic influences on both of these traits, with an estimated narrow sense heritability of 60% and 35%, respectively. The patterns of differences in body weight and peak whole body BMD in the BXD strains were then integrated with a large database of genetic markers previously defined in the RI BXD strains to generate chromosome map sites for QTL. After correction for redundancy among the significant correlations, QTL analysis of the BXD RI strain series provisionally identified 10 chromosomal sites linked to peak bone mass development in the female. Several of the identified sites map near genes encoding hormones, structural proteins, and cell surface receptors that are intricately involved in skeletal homeostasis. Four QTL for body weight were also identified. One of these loci was also strongly linked to inherited variation in BMD. This finding suggests that body weight and peak BMD may be influenced by linked genes or perhaps by common genes with pleiotropic effects. Our phenotyping in the RI BXD strains has allowed us to map a number of specific genetic loci strongly related to the acquisition of peak BMD. Confirmation of these findings will likely result in the understanding of which genes control skeletal health.     

NPH4, a conditional modulator of auxin-dependent differential growth responses in Arabidopsis

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein.