Functional study of genes essential for autogamy and nuclear reorganization in Paramecium.

Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.

Efficacy of antioxidants in the yeast Saccharomyces cerevisiae correlates with their effects on protein thiols.

We have found previously that only a limited number of antioxidants are able to protect yeast cells against endogenous and exogenous oxidative stress. In search of factors determining this selectivity of antioxidant action we compared the ability of a set of antioxidants to: (i) protect a thiol-dependent enzyme alcohol dehydrogenase (ADH) against inactivation by superoxide, peroxynitrite and hydrogen peroxide; (ii) prevent H(2)O(2)-induced activation of Yap1 p; and (iii) decrease extracellular redox potential of the medium. The results obtained provide demonstration with respect to yeast that the ability to lower redox potential and to maintain critical thiol groups in the reduced state is an important facet of the action of antioxidants.

Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia.

The duplication of entire genomes has long been recognized as having great potential for evolutionary novelties, but the mechanisms underlying their resolution through gene loss are poorly understood. Here we show that in the unicellular eukaryote Paramecium tetraurelia, a ciliate, most of the nearly 40,000 genes arose through at least three successive whole-genome duplications. Phylogenetic analysis indicates that the most recent duplication coincides with an explosion of speciation events that gave rise to the P. aurelia complex of 15 sibling species. We observed that gene loss occurs over a long timescale, not as an initial massive event. Genes from the same metabolic pathway or protein complex have common patterns of gene loss, and highly expressed genes are over-retained after all duplications. The conclusion of this analysis is that many genes are maintained after whole-genome duplication not because of functional innovation but because of gene dosage constraints.

Analysis of CFTR, SPINK1, PRSS1 and AAT mutations in children with acute or chronic pancreatitis.

OBJECTIVES: Defects of PRSS1, SPINK1, CFTR and AAT are considered causative or predisposing to pancreatitis. The aim of this study was to evaluate the impact of these defects into molecular pathology of chronic pancreatitis (CP) and acute recurrent pancreatitis (ARP). METHODS: Ninety-two children with CP or ARP, 55 family members and 50 controls were investigated. The subjects were screened for PRSS1 mutations: R122H, R122C, A16V, N29I; SPINK1 N34S variant; panel of 14 CFTR defects: INNOLiPA CFTR12, CFTRdele2,3 and IVS8-T variant or panel of 3 CFTR defects-F508del, CFTRdele2,3 and IVS8-T; AAT mutations: E264V, E342K. RESULTS: We identified 1 mutated allele in at least 1 of 4 genes in 31 of 92 patients and 12 of 50 controls (P = 0.157). Mutations in SPINK1 and PRSS1 were most frequent. PRSS1 mutations were identified mainly in CP patients (9.6% of CP vs 2.5% of ARP alleles, P = 0.094), whereas N34S SPINK1 mutation was present with comparable frequency in CP and ARP patients (7.7% vs 10.0%, P = 0.768). The frequency of mutations in CFTR alleles was similar to controls (4.9% vs 5%, P = 0.587). Overall frequency of AAT mutations was lower than in the controls. Family studies showed that defects in the examined genes did not always segregate with disease. CONCLUSIONS: PRSS1 defects seem to be causative for pancreatitis, whereas defects in SPINK1 are suggested to be associated with the disease. No association between CFTR mutations and pancreatitis was observed. The importance of AAT variants remains speculative.

(AC)n microsatellite polymorphism and 14-nucleotide deletion in exon 42 ankyrin-1 gene in several families with hereditary spherocytosis in a population of South-Western Poland.

Defects in ankyrin-1 have been implicated in approximately half of all patients with hereditary spherocytosis. However, not all polymorphisms in this gene lead to the changes in expressed protein or to the changes of the level of its expression. In this study, we report on several cases of the (AC)n microsatellite polymorphism in 3' untranslated region of ANK1 gene found in nine families (19 patients) with hereditary spherocytosis (HS) and also in ten healthy individuals from the same territory. We also found that 14-nucleotide deletion in this region of ANK1 which was shifted by five nucleotides in relation to another 14-nucleotide deletion listed in Single Nucleotide Polymorphism National Center for Biotechnology Information (SNP NCBI) database. This deletion seems to be present only in individuals with 11/14 and 13/14 AC repeats what would be an interesting correlation between these two features. However, comparison of the data obtained for HS patients and healthy individuals indicates that both polymorphisms are not connected to the pathology of hereditary spherocytosis.

Antioxidants protect the yeast Saccharomyces cerevisiae against hypertonic stress.

Yeast (Saccharomyces cerevisiae) mutants lacking CuZnSOD have been reported to be hypersensitive to hypertonic media and to show increased oxidative damage. This study demonstrates that hypertonic medium (containing 0.8 M NaCl) increases the generation of superoxide and other reactive species in yeast cells. Other sequelae of exposure to hypertonic medium include oxidation of cellular low-molecular weight thiols and decrease in total antioxidant capacity of cellular extracts. deltasod1 mutant is more sensitive than a wild-type strain to colony growth inhibition on a hypertonic medium. Anaerobic conditions, ascorbate, glutathione, cysteine and dithiothreitol are able to ameliorate this growth inhibition but a range of other antioxidants does not protect. The protective ability of the antioxidants does not correlate with the rate of their reactions with superoxide but seems to be conditioned by low redox potential for one-electron oxidation of free radicals of the antioxidants. It suggests that repair of low-redox potential targets rather than prevention of their damage by superoxide is important in the antioxidant protection against oxidative stress induced by hypertonic conditions.

High coding density on the largest Paramecium tetraurelia somatic chromosome.

Paramecium, like other ciliates, remodels its entire germline genome at each sexual generation to produce a somatic genome stripped of transposons and other multicopy elements. The germline chromosomes are fragmented by a DNA elimination process that targets heterochromatin to give a reproducible set of some 200 linear molecules 50 kb to 1 Mb in size. These chromosomes are maintained at a ploidy of 800n in the somatic macronucleus and assure all gene expression. We isolated and sequenced the largest megabase somatic chromosome in order to explore its organization and gene content. The AT-rich (72%) chromosome is compact, with very small introns (average size 25 nt), short intergenic regions (median size 202 nt), and a coding density of at least 74%, higher than that reported for budding yeast (70%) or any other free-living eukaryote. Similarity to known proteins could be detected for 57% of the 460 potential protein coding genes. Thirty-two of the proteins are shared with vertebrates but absent from yeast, consistent with the morphogenetic complexity of Paramecium, a long-standing model for differentiated functions shared with metazoans but often absent from simpler eukaryotes. Extrapolation to the whole genome suggests that Paramecium has at least 30,000 genes.

Random sequencing of Paramecium somatic DNA.

We report a random survey of 1 to 2% of the somatic genome of the free-living ciliate Paramecium tetraurelia by single-run sequencing of the ends of plasmid inserts. As in all ciliates, the germ line genome of Paramecium (100 to 200 Mb) is reproducibly rearranged at each sexual cycle to produce a somatic genome of expressed or potentially expressed genes, stripped of repeated sequences, transposons, and AT-rich unique sequence elements limited to the germ line. We found the somatic genome to be compact (>68% coding, estimated from the sequence of several complete library inserts) and to feature uniformly small introns (18 to 35 nucleotides). This facilitated gene discovery: 722 open reading frames (ORFs) were identified by similarity with known proteins, and 119 novel ORFs were tentatively identified by internal comparison of the data set. We determined the phylogenetic position of Paramecium with respect to eukaryotes whose genomes have been sequenced by the distance matrix neighbor-joining method by using random combined protein data from the project. The unrooted tree obtained is very robust and in excellent agreement with accepted topology, providing strong support for the quality and consistency of the data set. Our study demonstrates that a random survey of the somatic genome of Paramecium is a good strategy for gene discovery in this organism.