Plant proteome analysis

Proteome analysis is becoming a powerful tool in the functional characterization of plants. Due to the availability of vast nucleotide sequence information and based on the progress achieved in sensitive and rapid protein identification by mass spectrometry, proteome approaches open up new perspectives to analyze the complex functions of model plants and crop species at different levels. In this review, an overview is given on proteome studies performed to analyze whole plants or specific tissues with particular emphasis on important physiological processes such as germination. The chapter on subcellular proteome analysis of plants focuses on the progress achieved for plastids and mitochondria but also mentions the difficulties associated with membrane-bound proteins of these organelles. Separate chapters are dedicated to the challenging analysis of woody plants and to the use of proteome approaches to investigate the interaction of plants with pathogens or with symbiotic organisms. Limitations of current techniques and recent conceptual and technological perspectives for plant proteomics are briefly discussed in the final chapter.     

Altered myogenesis in Six1-deficient mice

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.     

Chromatin boundaries in budding yeast: the nuclear pore connection

Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.     

Immunocytochemical localisation of GABA in endocrine cells of the rat entero-pancreatic system

The occurence of GABA-containing cells in the rat entero-pancreatic system was investigated by using anti-GABA-glutaraldehyde antibodies at the light and electron microscope level. In the pancreas, the B cells showed intense immunoreactivity, contrary to non-B and exocrine cells. Moreover, post-embedding immunogold staining was localised mostly in mitochondria, close to rough endoplasmic reticulum and in the nucleus. The insulin granules appeared non-significantly stained, which suggests the lack of cosecretion of GABA together with insulin. In the duodenum, GABA immunoreactivity was detected in certain endocrine cell types, suggesting a possible interaction with this amino acid. The well established GABAergic innervation in the enteric system was also confirmed by immunolabelling.     

Polymorphism ofTcrb andTcrg genes in Biozzi mice: Segregation analysis of a newTcrg haplotype with antibody responsiveness

Tcrb andTcrg gene polymorphism was investigated in high (H) and low (L) responder Biozzi mice from selection I, II, and GS by Southern blot analysis with appropriateV andC probes. No polymorphism of theTcrb haplotype was detected between H and L mice in all selections which were all found to be of the BALB/c type. The H-I and H-II g genotype was of BALB/c and DBA/2 type, respectively. In contrast, a newTcrg haplotype shared by L-I and L-II mice was identified and characterized by C1, 2, 3, C4, V1, 2, 3, V5, and V6 restriction fragment length polymorphisms (RFLPs).Tcrg genotypes were not fixed in the GS selection and two additional new haplotypes were identified in two L-GS mice. An attempt was made to correlate the L-Ig genotype with the low responder status by analyzingg haplotypes among highest and lowest responder (H-1 x L-I)F₂ hybrids immunized with sheep red blood cells (SRBC). No correlation was found in this segregation study, whereas a highly significant one was established with theH-2 haplotype, a locus already known to participate in the genetic control of H-I/L-I difference. The lack of correlation between SRBC response and theTcrg genotype was consistent with the heterogenousg haplotypes found in mice of the GS selection. Together, the present results suggest that H and L mice have the sameTcrab potential repertoire and that T-cell receptor (Tcr) genes cannot be considered as immune response genes in this model. Our results also indicate that the F₂ segregation analysis, given a polymorphic gene, is suitable for an investigation of its immune response functions.     

Huntingtin proteolysis releases non‐polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N‐ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full‐length HTT remain elusive. Moreover, the contribution of non‐polyQ C‐terminal fragments is unknown. Using time‐ and site‐specific control of full‐length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N‐ter fragments, the C‐ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C‐ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis‐induced alteration of this function may be relevant to disease.