Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Transgenic expression of CD95 ligand on thyroid follicular cells confers immune privilege upon thyroid allografts

Constitutive Fas ligand (FasL) expression by specialized cells in the body participates in the immune privilege status of tissues containing these cells. This property has been used to prevent rejection of allogeneic grafts. Nevertheless, the mechanism responsible for such protection has not been fully elucidated. Unfortunately, grafting of FasL transgenic (TG) tissues has been unsuccessful. We have generated TG mice expressing FasL (soluble + membrane bound) on thyroid follicular cells (TFC), and used them to show that ectopic FasL expression prevents thyroid allograft rejection. FasL expression on TFC led to markedly decreased anti-allogeneic, cytotoxic, and helper T lymphocyte activities. The alloantibody response in TG thyroid recipients was either completely inhibited or switched toward a T2-Ab response. Surprisingly, the beneficial effect of FasL on TG thyroid grafts was abolished by host CD4(+) T cell depletion. Host CD8(+) T cell depletion improved nontransgenic (NTG), but not TG graft survival. Altogether, our results suggest that FasL-induced tolerance is concomitant with a move away from a T1 type response, and a CD4 T cell-mediated regulation of the allocytotoxic T cell response. These results were dependent upon the level of FasL expression on TFC, in that low expression of FasL led to a less marked effect compared with the effect observed with high expression of FasL. These results provide some insight into the role of FasL in regulating destructive alloimmune responses in the case of whole organ grafting, and they have important implications for the development of FasL-based immunotherapy in organ transplantation.     

Genotypic variation and chromosomal location of QTLs for somatic embryogenesis revealed by epidermal layers culture of recombinant inbred lines in the sunflower (Helianthus annuus L.)

The present study was conducted to identify the genetic factors controlling somatic embryogenesis in the sunflower. Two traits, the number of embryogenic explants per 40 explants plated (EE/40 E) and the number of embryos per 40 explants (E/40 E), were scored in 74 recombinant inbred lines (RILs) from a cross between ’PAC-2’ and ’RHA-266’. The experiment was designed as a randomized complete block with 76 genotypes (74 recombinant inbred lines and two parents) and three replications. Each replication consisted of three Erlenmeyer flasks with 40 epidermal layers (explants). Analyses of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritabilities for the somatic embryogenesis traits studied, EE/40 E and E/40 E, were high (0.64 and 0.77 respectively) and the genetic gain, in percentage of the best parent for 10% of selected RILs, was significant. Four QTLs for EE/40 E (tee) and seven for E/40 E (ete) were detected using composite interval mapping and AFLP mapping. The QTLs for EE/40 E explained 48% of the phenotypic variation while the QTLs for E/40 E explained about 89% of the variation. Received:14 December 1999 / Accepted:18 May 2000     

A new Saccharomyces cerevisiae strain with a mutant Smt3-deconjugating Ulp1 protein is affected in DNA replication and requires Srs2 and homologous recombination for its viability

The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37 degrees C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.     

Regional hydrodynamic gene delivery to the rat liver with physiological volumes of DNA solution

BACKGROUND: The major barrier to the clinical application of hydrodynamic gene delivery to the liver is the large volume of fluid required using standard protocols. Regional hydrodynamic gene delivery via branches of the portal vein has not previously been reported, and we have evaluated this approach in a rat model. METHODS: The pGL3 plasmid with the luciferase reporter gene was used at 50 micro g/ml in isotonic solutions, and was administered with a syringe pump for precise control of the hydrodynamic conditions evaluated. Gene expression was individually measured in six anatomically distinct liver lobes. The effect of systemic chloroquine to promote endocytic escape and a (Lys)(16)-containing peptide to condense the DNA into approximately 100-nm nanoparticles was also evaluated. RESULTS: Hydrodynamic conditions for excellent gene delivery were obtained by using 3-ml volumes ( approximately 12 ml/kg) of isotonic DNA solution delivered at 24 ml/min to the right lateral lobe ( approximately 20% of the liver mass). Under these conditions, >95% of gene delivery usually occurred in the targeted right lateral lobe. Outflow obstruction was essential for gene delivery, both at optimal and at very low levels of hydrodynamic gene delivery. The use of systemic chloroquine to promote endocytic escape did not augment hydrodynamic gene delivery, while condensation of DNA in non-ionic isotonic solutions (5% dextrose) to nanoparticles of approximately 100 nm completely abolished gene delivery. CONCLUSIONS: Regional hydrodynamic gene delivery via a branch of the portal vein offers a physiological model of liver gene therapy, for experimental and clinical application.     

Expression and functional role of peroxisome proliferator-activated receptor-gamma in ovarian folliculogenesis in the sheep

Peroxisome proliferator-activated receptor (PPARgamma) is a nuclear receptor that is activated by fatty acids and derivatives and the antidiabetic glitazones, which plays a role in the control of lipid and glucose homeostasis. In the present work, we tested the hypothesis that PPARgamma plays a role in reproductive tissues by studying its expression and function in the hypothalamo-pituitary-ovary axis in the sheep. PPARgamma 1 and PPARgamma 2 proteins and mRNAs were detected in whole ovine pituitary and ovary but not in hypothalamic extracts. In situ hybridization on ovarian section localized PPARgamma mRNA in the granulosa layer of follicles. Interestingly, PPARgamma expression was higher in small antral (1-3 mm diameter) than in preovulatory follicles (>5 mm diameter) (P < 0.001) and was not correlated with healthy status. To assess the biological activity of ovarian PPARgamma, ovine granulosa cells were transfected with a reporter construct driven by PPARgamma-responsive elements. Addition of rosiglitazone, a PPARgamma ligand, stimulated reporter gene expression, showing that endogenous PPARgamma is functional in ovine granulosa cells in vitro. Moreover, rosiglitazone inhibited granulosa cell proliferation (P < 0.05) and increased the secretion of progesterone in vitro (P < 0.05). This stimulation effect was stronger in granulosa cells from small than from large follicles. In contrast, rosiglitazone had no effect on LH, FSH, prolactin and growth hormone secretion by ovine pituitary cells in vitro. Overall, these data suggest that PPARgamma ligands might stimulate follicular differentiation in vivo likely through a direct action on granulosa cells rather than by modulating pituitary hormone secretion.     

Genome-wide nuclear morphology screen identifies novel genes involved in nuclear architecture and gene-silencing in Saccharomyces cerevisiae

Organisation of the cell nucleus is crucial for the regulation of gene expression but little is known about how nuclei are structured. To address this issue, we designed a genomic screen to identify factors involved in nuclear architecture in Saccharomyces cerevisiae. This screen is based on microscopic monitoring of nuclear pore complexes and nucleolar proteins fused with the green fluorescent protein in a collection of approximately 400 individual deletion mutants. Among the 12 genes identified by this screen, most affect both the nuclear envelope and the nucleolar morphology. Corresponding gene products are localised preferentially to the nucleus or close to the nuclear periphery. Interestingly, these nuclear morphology alterations were associated with chromatin-silencing defects. These genes provide a molecular context to explore the functional link between nuclear architecture and gene silencing.     

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.