Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.     

Accumulation of DNA damages in aging Paramecium tetraurelia

Summary Paramecium tetraurelia cells of ages 4, 15, and 27 days were labeled with [¹⁴C]-thymidine. In addition, cells were grown clonally for 27 days (108 generations) and labeled with [¹⁴C]-thymidine in the presence of 0.5 or 7.5 g/ml of mitomycin-C (MMC) or no MMC. These cells were gently deposited on a filter membrane, which impedes the passage of DNA strands. The cells were then lysed with detergents and the cellular components washed through the filters, leaving double-stranded DNA intact on the surface. Proteinase K was used to remove histone or DNA-bound proteins. The DNA was then eluted under alkaline conditions, which denatures double-stranded DNA and converts apurinic/apyrimidinic sites into single-strand breaks. The results obtained with the cells of ages 4, 15, and 27 days (16, 60, and 108 generations, respectively) indicate that as Paramecium tetraurelia ages during asexual reproduction, apurinic/apyrimidinic lesions, strand breaks or single-strand gaps accumulate. This accumulation may be the basic mechanism of aging in such cells. In the MMC-treated cells of 27 days (108 generations), the MMC reduced elution of DNA fragments more at the higher than at the lower pH's used; random MMC cross-links should occur more often in longer strands than in shorter strands. The reductions in elution preferentially at higher pH, at which longer single strands would be eluted, confirmed the pH-versuslength relationship for Paramecium DNA eluted under our conditions.     

A Comparison of Internal Eliminated Sequences in the Genes that Encode Two K+-Channel Isoforms in Paramecium tetraurelia

We examined both the somatic (macro-) and the germinal (micronuclear) DNAs that encode two K⁺-channel isoforms. PAK1 and PAK11 , in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5'-most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206-bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K⁺-channel isoforms.     

High-throughput enhancer trap by remobilization of transposon Minos in Ciona intestinalis

The enhancer trap approach utilizing transposons yields us information about gene functions and gene expression patterns. In the ascidian Ciona intestinalis, transposon-based transgenesis and insertional mutagenesis were achieved with a Tc1/mariner transposon Minos. We report development of a novel technique for enhancer trap in C. intestinalis. This technique uses remobilization of Minos in the Ciona genome. A Minos vector for enhancer trap was constructed and a tandem array insertion of the vector was introduced into the Ciona genome to create a mutator line. Minos was remobilized in Ciona chromosomes to create new insertions by providing transposases. These transposase-introduced animals were crossed with wild-type animals. Nearly 80% of F1 families showed novel GFP expression patterns. This high-throughput enhancer trap screen will be useful to create new marker transgenic lines showing reporter gene expression in specific tissues and to identify novel patterns of gene expression.     

DNA transposons: nature and applications in genomics

Repeated DNA makes up a large fraction of a typical mammalian genome, and some repetitive elements are able to move within the genome (transposons and retrotransposons). DNA transposons move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant role in the evolution of many genomes. As genetic tools, DNA transposons can be used to introduce a piece of foreign DNA into a genome. Indeed, they have been used for transgenesis and insertional mutagenesis in different organisms, since these elements are not generally dependent on host factors to mediate their mobility. Thus, DNA transposons are useful tools to analyze the regulatory genome, study embryonic development, identify genes and pathways implicated in disease or pathogenesis of pathogens, and even contribute to gene therapy. In this review, we will describe the nature of these elements and discuss recent advances in this field of research, as well as our evolving knowledge of the DNA transposons most widely used in these studies.     

Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP

Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo.     

Identification of mutations in Caenorhabditis elegans that cause resistance to high levels of dietary zinc and analysis using a genomewide map of single nucleotide polymorphisms scored by pyrosequencing

Zinc plays many critical roles in biological systems: zinc bound to proteins has structural and catalytic functions, and zinc is proposed to act as a signaling molecule. Because zinc deficiency and excess result in toxicity, animals have evolved sophisticated mechanisms for zinc metabolism and homeostasis. However, these mechanisms remain poorly defined. To identify genes involved in zinc metabolism, we conducted a forward genetic screen for chemically induced mutations that cause Caenorhabditis elegans to be resistant to high levels of dietary zinc. Nineteen mutations that confer significant resistance to supplemental dietary zinc were identified. To determine the map positions of these mutations, we developed a genomewide map of single nucleotide polymorphisms (SNPs) that can be scored by the high-throughput method of DNA pyrosequencing. This map was used to determine the approximate chromosomal position of each mutation, and the accuracy of this approach was verified by conducting three-factor mapping experiments with mutations that cause visible phenotypes. This is a generally applicable mapping approach that can be used to position a wide variety of C. elegans mutations. The mapping experiments demonstrate that the 19 mutations identify at least three genes that, when mutated, confer resistance to toxicity caused by supplemental dietary zinc. These genes are likely to be involved in zinc metabolism, and the analysis of these genes will provide insights into mechanisms of excess zinc toxicity.     

Genetic identification and characterization of three genes that prevent accumulation of oxidative DNA damage in Drosophila adult tissues

Reactive oxygen species generated in the process of energy production represent a major cause of oxidative DNA damage. Production of the oxidized guanine base, 8-oxo-guanine (8-oxoG), results in mismatched pairing with adenine and subsequently leads to G:C to T:A transversions after DNA replication. Our previous study demonstrated that Drosophila CG1795 encodes an ortholog of Ogg1, which is essential for the elimination of 8-oxoG. Moreover, the Drosophila ribosomal protein S3 (RpS3) possesses N-glycosylase activity that eliminates 8-oxoG in vitro. In this study, we show that RpS3 heterozygotes hyper-accumulate 8-oxoG in midgut cell nuclei after oxidant feeding, suggesting thatRpS3 is required for the elimination of 8-oxoG in Drosophila adults. We further showed that several muscle-aging phenotypes were significantly accelerated in RpS3 heterozygotes. Ogg1 is localized in the nucleus, while RpS3 is in the cytoplasm, closely associated with endoplasmic reticulum networks. Results of genetic analyses also suggest that these two proteins operate similarly but independently in the elimination of oxidized guanine bases from genomic DNA. Next, we obtained genetic evidence suggesting that CG42813 functions as the Drosophila ortholog of mammalian Mth1 in the elimination of oxidized dGTP (8-oxo-dGTP) from the nucleotide pool. Depletion of this gene significantly increased the number of DNA damage foci in the nuclei of Drosophila midgut cells. Furthermore, several aging-related phenotypes such as age-dependent loss of adult locomotor activities and accumulation of polyubiquitylated proteins in adult muscles were also significantly accelerated in CG42813-depleted flies. Lastly, we investigated the phenotype of adults depleted of CG9272, which encodes a protein with homology to mammalian Nth1 that is essential for the elimination of oxidized thymine. Excessive accumulation of oxidized bases was observed in the epithelial cell nuclei after oxidant feeding. In conclusion, three genes that prevent accumulation of oxidative DNA damage were identified in Drosophila.     

Expression of a 26S proteasome ATPase subunit, MS73, in muscles that undergo developmentally programmed cell death, and its control by ecdysteroid hormones in the insect Manduca sexta

MS73, an ATPase regulatory subunit of the 26S proteasome in the moth Manduca sexta, is shown to be expressed at a high level only in muscles that are undergoing developmentally programmed cell death, or which are destined to do so. The amount of MS73 is increased by more than two-fold just before death in each of three different muscles that die at different times, under different developmental controls. An ecdysteroid (moulting hormone) agonist, RH-5849, that prevents the occurrence of programmed cell death in two of these muscles also prevents the normally occurring rise in level of MS73 in these muscles. This evidence is consistent with a role for MS73 in programmed cell death.     

Compelling evidence that a single nucleotide substitution in TYRP1 is responsible for coat-colour polymorphism in a free-living population of Soay sheep

Identifying the genes that underlie phenotypic variation in natural populations is a central objective of evolutionary genetics. Here, we report the identification of the gene and causal mutation underlying coat colour variation in a free-living population of Soay sheep (Ovis aries). We targeted tyrosinase-related protein 1 (TYRP1), a positional candidate gene based on previous work that mapped the Coat colour locus to an approximately 15cM window on chromosome 2. We identified a non-synonymous substitution in exon IV that was perfectly associated with coat colour. This polymorphism is predicted to cause the loss of a cysteine residue that is highly evolutionarily conserved and likely to be of functional significance. We eliminated the possibility that this association is due to the presence of strong linkage disequilibrium with an unknown regulatory mutation by demonstrating that there is no difference in relative TYRP1 expression between colour morphs. Analysis of this putative causal mutation in a complex pedigree of more than 500 sheep revealed almost perfect co-segregation with coat colour (chi2-test, p<0.0001, LOD=110.20), and very tight linkage between Coat colour and TYRP1 (LOD=29.50).     

Identification of two novel missense WFS1 mutations, H696Y and R703H,in patients with non-syndromic low-frequency sensorineural hearing loss

Non-syndromic low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of hearing loss in which frequencies ≤2000 Hz predominantly are affected. To date, different mutations in two genes, DIAPH1 and WFSI, have been found to be associated with LFSNHL.Here, we report a five-generation Chinese family with postlingual and progressive LFSNHL. We mapped the disease locus to a 2.5 Mb region on chromosome 4p16 between markers SNP_A-2167174 and D4S431, overlapping with the DFNA6/14/38 locus. Sequencing of candidate gene revealed a heterozygous c.2086C＞T substitution in exon 8 of WFS1, leading to p. H696Y substitution at the C-terminus of Wolframin (WFS1).In addition, we performed mutational screening of WFS1 in 37 sporadic patients, 7-50 years of age, with LFSNHL. We detected a heterozygous c.2108G＞A substitution in exon 8 of WFS1, leading to p. R703H substitution in a patient. The H696 and R703 in WFS1 are highly conserved across species, including human, orangutan, rat, mouse, and frog (Xenopus). Sequence analysis demonstrated the absence of c.2086C＞T or c.2108G＞A substitutions in the WFS1 genes among 200 unrelated control subjects of Chinese background, supporting the hypothesis that they represent causative mutations, and not rare polymorphisms. Our data provide additional molecular and clinical information for establishing a better genotype-phenotype correlation for LFSNHL.     

Identification of two novel missense WFS1 mutations,H696Y and R703H,in patients with non-syndromic low-frequency sensorineural hearing loss

Non-syndromic low-frequency sensorineural hearing loss(LFSNHL) is an unusual type of hearing loss in which frequencies≤2000 Hz predominantly are affected.To date,different mutations in two genes,DIAPH1 and WFS1,have been found to be associated with LFSNHL. Here,we report a five-generation Chinese family with postlingual and progressive LFSNHL.We mapped the disease locus to a 2.5 Mb region on chromosome 4p16 between markers SNP_A-2167174 and D4S431,overlapping with the DFNA6/14/38 locus.Sequencing of cand...     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.