Characterization of a radish introgression carrying the Ogura fertility restorer gene Rfo in rapeseed,using the Arabidopsis genome sequence and radish genetic mapping

The radish Rfo gene restores male fertility in radish or rapeseed plants carrying Ogura cytoplasmic male-sterility. This system was first discovered in radish and was transferred to rapeseed for the production of F1 hybrid seeds. We aimed to identify the region of the Arabidopsis genome syntenic to the Rfo locus and to characterize the radish introgression in restored rapeseed. We used two methods: amplified consensus genetic markers (ACGMs) in restored rapeseed plants and construction of a precise genetic map around the Rfo gene in a segregating radish population. The use of ACGMs made it possible to detect radish orthologs of Arabidopsis genes in the restored rapeseed genome. We identified radish genes, linked to Rfo in rapeseed and whose orthologs in Arabidopsis are carried by chromosomes 1, 4 and 5. This indicates several breaks in colinearity between radish and Arabidopsis genomes in this region. We determined the positions of markers relative to each other and to the Rfo gene, using the progeny of a rapeseed plant with unstable meiotic transmission of the radish introgression. This enabled us to produce a schematic diagram of the radish introgression in rapeseed. Markers which could be mapped both on radish and restored rapeseed indicate that at least 50 cM of the radish genome is integrated in restored rapeseed. Using markers closely linked to the Rfo gene in rapeseed and radish, we identified a contig spanning six bacterial artificial chromosome (BAC) clones on Arabidopsis chromosome 1, which is likely to carry the orthologous Rfo gene.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by H. C. BeckerS. Giancola and S. Marhadour contributed equally to this work     

Wild type and <Emphasis Type="Italic">H43Y</Emphasis> variant of human <Emphasis Type="Italic">TRIM5α</Emphasis> show similar anti-human immunodeficiency virus type 1 activity both in vivo and in vitro

Polymorphisms in human genes have been shown to affect the rate of disease progression to acquired immune deficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Recently, tripartite motif 5α (TRIM5α) was identified as a factor that confers resistance to HIV-1 infection in Old World monkey cells. Subsequently, Sawyer et al. (Curr Biol 16:95–100, 2006) reported a single nucleotide polymorphism (H43Y) in the human TRIM5α gene and TRIM5α protein with 43Y was found to lose its ability to restrict HIV-1. In the present study, we reevaluated effects of this allele on in vitro anti-HIV-1 activity as well as on HIV-1 disease progression in European and Asian cohorts of HIV-1-infected individuals. Our epidemiological and molecular biological findings clearly indicate H43Y has a very minor effect on anti-HIV-1 activity of TRIM5α, suggesting that this allele is immaterial, at least in HIV-1-infected Europeans and Asians.     

Sporadic Infantile Epileptic Encephalopathy Caused by Mutations in PCDH19 Resembles Dravet Syndrome but Mainly Affects Females

Dravet syndrome (DS) is a genetically determined epileptic encephalopathy mainly caused by de novo mutations in the SCN1A gene. Since 2003, we have performed molecular analyses in a large series of patients with DS, 27% of whom were negative for mutations or rearrangements in SCN1A. In order to identify new genes responsible for the disorder in the SCN1A-negative patients, 41 probands were screened for micro-rearrangements with Illumina high-density SNP microarrays. A hemizygous deletion on chromosome Xq22.1, encompassing the PCDH19 gene, was found in one male patient. To confirm that PCDH19 is responsible for a Dravet-like syndrome, we sequenced its coding region in 73 additional SCN1A-negative patients. Nine different point mutations (four missense and five truncating mutations) were identified in 11 unrelated female patients. In addition, we demonstrated that the fibroblasts of our male patient were mosaic for the PCDH19 deletion. Patients with PCDH19 and SCN1A mutations had very similar clinical features including the association of early febrile and afebrile seizures, seizures occurring in clusters, developmental and language delays, behavioural disturbances, and cognitive regression. There were, however, slight but constant differences in the evolution of the patients, including fewer polymorphic seizures (in particular rare myoclonic jerks and atypical absences) in those with PCDH19 mutations. These results suggest that PCDH19 plays a major role in epileptic encephalopathies, with a clinical spectrum overlapping that of DS. This disorder mainly affects females. The identification of an affected mosaic male strongly supports the hypothesis that cellular interference is the pathogenic mechanism.     

Methanogenic diversity and activity in municipal solid waste landfill leachates

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.     

Gastric Electrical Stimulation Decreases Gastric Distension-Induced Central Nociception Response through Direct Action on Primary Afferents

Background & Aims

Gastric electrical stimulation (GES) is an effective therapy to treat patients with chronic dyspepsia refractory to medical management. However, its mechanisms of action remain poorly understood.

Methods

Gastric pain was induced by performing gastric distension (GD) in anesthetized rats. Pain response was monitored by measuring the pseudo-affective reflex (e.g., blood pressure variation), while neuronal activation was determined using c-fos immunochemistry in the central nervous system. Involvement of primary afferents was assessed by measuring phosphorylation of ERK1/2 in dorsal root ganglia.

Results

GES decreased blood pressure variation induced by GD, and prevented GD-induced neuronal activation in the dorsal horn of the spinal cord (T9–T10), the nucleus of the solitary tract and in CRF neurons of the hypothalamic paraventricular nucleus. This effect remained unaltered within the spinal cord when sectioning the medulla at the T5 level. Furthermore, GES prevented GD-induced phosphorylation of ERK1/2 in dorsal root ganglia.

Conclusions

GES decreases GD-induced pain and/or discomfort likely through a direct modulation of gastric spinal afferents reducing central processing of visceral nociception.     

RAD51 haploinsufficiency causes congenital mirror movements in humans

Congenital mirror movements (CMM) are characterized by involuntary movements of one side of the body that mirror intentional movements on the opposite side. CMM reflect dysfunctions and structural abnormalities of the motor network and are mainly inherited in an autosomal-dominant fashion. Recently, heterozygous mutations in DCC, the gene encoding the receptor for netrin 1 and involved in the guidance of developing axons toward the midline, have been identified but CMM are genetically heterogeneous. By combining genome-wide linkage analysis and exome sequencing, we identified heterozygous mutations introducing premature termination codons in RAD51 in two families with CMM. RAD51 mRNA was significantly downregulated in individuals with CMM resulting from the degradation of the mutated mRNA by nonsense-mediated decay. RAD51 was specifically present in the developing mouse cortex and, more particularly, in a subpopulation of corticospinal axons at the pyramidal decussation. The identification of mutations in RAD51, known for its key role in the repair of DNA double-strand breaks through homologous recombination, in individuals with CMM reveals a totally unexpected role of RAD51 in neurodevelopment. These findings open a new field of investigation for researchers attempting to unravel the molecular pathways underlying bimanual motor control in humans.     

Expansion of functionally anergic CD21-/low marginal zone-like B cell clones in hepatitis C virus infection-related autoimmunity

Homeostasis of peripheral B cell subsets is disturbed during chronic hepatitis C virus (HCV) infection, leading to the occurrence of autoimmunity and B cell lymphoproliferation. However, mechanisms by which HCV causes lymphoproliferation remain controversial. We report in this article on the elevated number of clonal CD21(-/low)IgM(+)CD27(+) marginal zone (MZ)-like B cells, which correlates with autoimmunity and lymphoproliferation in HCV patients. We found an increase in autoreactive BCRs using V(H)1-69 and V(H)4-34 genes in CD21(-/low) MZ B cells. CD21(-/low) MZ B cells showed impaired calcium-mediated signaling, did not upregulate activation markers, and did not proliferate in response to BCR triggering. CD21(-/low) MZ B cells also were prone to dying faster than their CD21(+) counterparts, suggesting that these B cells were anergic. CD21(-/low) MZ B cells, in contrast, remained responsive to TLR9 stimulation. Gene array analyses revealed the critical role of Early growth response 2 and Cbl-b in the induction of anergy. Therefore, HCV patients who display high frequencies of unresponsive CD21(-/low) MZ B cells are more susceptible to developing autoimmunity and/or lymphoproliferation. These cells remain in peripheral blood controlled by functional anergy instead of being eliminated, and chronic antigenic stimulation through TLR stimulation may create a favorable environment for breaking tolerance and activating these cells.