Non-viral gene transfer: liposomes and nanospheres promise to deliver the goods

Effective treatment of early endobronchial cancer with regional administration of liposome–p53 complexesZou, Y. et al. (1998)J. Natl. Cancer Inst. 90, 1130–1137Controlled gene delivery by DNA–gelatin nanospheresTruong-Le, V.L. et al. (1998)Hum. Gene Ther. 9, 1709–1717     

An SH2 domain stands between infectious mononucleosis and a fatal lymphoproliferative disease

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding geneCoffey, A.J. et al. (1998)Nat. Genet. 20, 129–135The X-linked lymphoproliferative-disease gene product SAP regulates signals induced through the co-receptor SLAMSayos, J. et al. (1998)Nature 395, 462–469     

Electronic zoo blotting identifies a modifier gene for spinal muscular atrophy

Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomicsScharf, J.M. et al. (1998)Nat. Genet. 20, 83–86     

Making an impression on X-linked mental retardation

Oligophrenin-1 encodes a rhoGAP protein involved in X-linked mental retardationBilluart, P. (1998)Nature 392, 923–926PAK3 mutation in nonsyndromic X-linked mental retardationAllen, K.M. et al. (1998)Nat. Genet. 20, 25–30Mutations in GDI1 are responsible for X-linked non-specific mental retardationD'Adamo, P. et al. (1998)Nat. Genet. 19, 134–139Non-specific X-linked semidominant mental retardation by mutations in a Rab GDP-dissociation inhibitorBienvenu, T. et al. (1998)Hum. Mol. Genet. 7, 1311–1315     

Borna disease virus takes the acid test

Mechanism of Borna disease virus entry into cellsGonzalez-Dunia, D. et al. (1998)J. Virol. 72, 783–788     

Increasing the options for inflammatory bowel disease treatment?

Curative treatment of an experimentally induced colitis by a CD44 variant V7-specific antibodyWittig, B. et al. (1998)J. Immunol. 161, 1069–1073     

Better mutation detection

Mutation detection and typing of polymorphic loci through double-strand conformation analysisArgüello, J.R. et al. (1998)Nat. Genet. 18, 192–194     

Crosstalk among cancer signalling pathways

Identification of c-MYC as a target of the APC pathwayHe, T-C. et al. (1998)Science 281, 1509–1512Smad3 mutant mice develop metastatic colorectal cancerZhu, Y. et al. (1998)Cell 94, 703–714     

Unusual imprinting defects with low recurrence risks

Sporadic imprinting defects in Prader–Willi syndrome and Angelman syndrome: implications for imprint-switch models, genetic counselling, and prenatal diagnosisBuiting, K. et al. (1998)Am. J. Hum. Genet. 63, 170–180     

Aspirin as a cancer-preventing drug

Aspirin suppresses the mutator phenotype associated with hereditary nonpolyposis colorectal cancer by genetic selectionRuschoff, J. et al. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 11301–11306     

Baldness as a mendelian genetic trait

Alopecia universalis associated with a mutation in the human hairless geneAhmad, W. et al. (1998)Science 279, 720–724     

Systematic synthesis of nonviral gene therapy vectors

Lipitoids—novel cationic lipids for cellular delivery of plasmid DNA in vitroHuang, C-Y. et al. (1998)Chem. Biol. 5, 345–354     

Uric acid might herald new hopes for multiple sclerosis patients

Uric acid: a natural scavenger of peroxynitrite, in experimental allergic encephalomyelitis and multiple sclerosisHooper, D.C. et al. (1998)Proc. Natl. Acad. Sci. U. S. A. 95, 675–680     

Distinct Regulation of Mlh1p Heterodimers in Meiosis and Mitosis in Saccharomyces cerevisiae

Mlh1p forms three heterodimers that are important for mismatch repair (Mlh1p/Pms1p), crossing over during meiosis (Mlh1p/Mlh3p), and channeling crossover events into a specific pathway (Mlh1p/Mlh2p). All four proteins contain highly conserved ATPase domains and Pms1p has endonuclease activity. Studies of the functional requirements for Mlh1p/Pms1p in Saccharomyces cerevisae revealed an asymmetric contribution of the ATPase domains to repairing mismatches. Here we investigate the functional requirements of the Mlh1p and Mlh3p ATPase domains in meiosis by constructing separation of function mutations in Mlh3p. These mutations are analogous to mutations of Mlh1p that have been shown to lead to loss of ATP binding and/or ATP hydrolysis. Our data suggest that ATP binding by Mlh3p is required for meiotic crossing over while ATP hydrolysis is dispensable. This has been seen previously for Mlh1p. However, when mutations that affect ATP hydrolysis by both Mlh3p and Mlh1p are combined within a single cell, meiotic crossover frequencies are reduced. These observations suggest that the function of the Mlh1p/Mlh3p heterodimer requires both subunits to bind ATP but only one to efficiently hydrolyze it. Additionally, two different amino acid substitutions to the same residue (G97) in Mlh3p affect the minor mismatch repair function of Mlh3p while only one of them compromises its ability to promote crossing over. These studies thus reveal different functional requirements among the heterodimers formed by Mlh1p.CROSSING over during meiosis not only generates variation but is also important for providing the necessary interactions between homologous chromosomes that ensure correct segregation at division I of meiosis. Recombination is initiated by the production of programmed double-strand breaks (DSBs), catalyzed by the covalently attached Spo11p (Bergerat et al. 1997; Keeney et al. 1997), aided by a number of proteins (reviewed in Keeney and Neale 2006). DSBs are made at a much higher frequency than crossovers, and designation of only a subset to yield crossovers is thought to occur during early stages of DSB repair (Borner et al. 2004). At least two distinct pathways contribute to the production of crossover events in Saccharomyces cerevisiae. The major pathway is dependent on Msh4p/Msh5p and the mismatch repair proteins Mlh1p and Mlh3p (Ross-MacDonald and Roeder 1994; Hollingsworth et al. 1995; Hunter and Borts 1997; Wang et al. 1999; Abdullah et al. 2004) and the second pathway is dependent on Mus81p/Mms4p endonuclease (de los Santos et al. 2001, 2003).Mitotic mismatch repair (MMR) is the process by which mutations that arise during DNA replication and recombination are recognized and removed (reviewed in Kolodner 1996; Harfe and Jinks-Robertson 2000). Msh2p forms a heterodimer with Msh6p (MutSα) to repair base–base mismatches and small insertions and/or deletions and with Msh3p (MutSβ) to repair large insertions and/or deletions (reviewed in Jiricny 2006). Mlh1p forms heterodimers with Pms1p, Mlh2p, and Mlh3p to coordinate the removal of these mismatches (Prolla et al. 1994; Wang et al. 1999). Mlh1p/Pms1p (MutLα) are involved in the repair of all types of mismatches in combination with MutSα and MutSβ, and in the absence of either protein a mutator phenotype is observed (Habraken et al. 1997, 1998). Mlh1p/Mlh2p (MutLβ) and Mlh1p/Mlh3p (MutLγ) are involved in the MutSβ pathway only, which repairs frameshift mutations caused by insertions or deletions. Consequently mlh3Δ mutants only exhibit a weak mutator phenotype, due to a lesser involvement in mismatch repair and a partial overlap in function with Pms1p (Flores-Rozas and Kolodner 1998; Harfe et al. 2000).Although the MutL homologs interact primarily through their C-terminal domains (Pang et al. 1997; Ban and Yang 1998), it is thought that the N-terminal domains must also interact for the complex to be fully functional (Ban and Yang 1998). Binding of ATP causes the proteins to undergo conformational changes, which are essential for the interaction between the N termini (Ban et al. 1999; Tran and Liskay 2000; Sacho et al. 2008). ATP hydrolysis and subsequent release of ADP is required to allow the protein complex to return to its initial state, completing the cycle so that the subunits are ready to bind ATP again if required. Using mutants of MLH1 and PMS1 that are presumed to be defective for ATP binding and/or ATP hydrolysis, it has been shown that both of these functions are essential for fully effective mismatch repair (Tran and Liskay 2000). However, the ATP binding and ATP hydrolysis mutants of PMS1 exhibited lower mitotic mutation rates than the corresponding MLH1 ATPase mutants, suggesting that there is functional asymmetry within the Mlh1p/Pms1p heterodimer (Tran and Liskay 2000; Hall et al. 2002). Another example of the asymmetry in the contributions of these subunits to function can be seen in assays that measure recombination between diverged sequences (homeologous recombination). The Mlh1p ATPase activity has been shown to be more important for the suppression of homeologous recombination than Pms1p ATPase activity (Welz-Voegele et al. 2002). This functional asymmetry is supported by in vitro biochemical analysis that demonstrated Pms1p has a lower ATP binding affinity than Mlh1p (Hall et al. 2002).As mentioned above, Mlh1p/Mlh3p function in the Msh4p/Msh5p pathway for meiotic recombination (Hunter and Borts 1997; Santucci-Darmanin et al. 2000). The Msh4p/Msh5p complex is thought to act in the stabilization of Holliday junction intermediates to allow their resolution in a crossover configuration (Snowden et al. 2004). The Mlh1p/Mlh3p complex has been suggested to act in the resolution of these structures, either directly or indirectly. Human Pms2 and its yeast homolog, Pms1p, have been shown to possess a latent endonuclease activity, conferred by a motif that is conserved among some of the MutL homologs, including Mlh3p (Kadyrov et al. 2006, 2007). Mutations in the DHQA(X)₂E(X)₄E motif in yeast MLH3 cause defects in both mismatch repair and meiotic recombination equivalent to mlh3Δ, suggesting that Mlh3p may also possess an endonuclease activity that is important for the generation of crossovers (Nishant et al. 2008).ATP binding by Mlh1p has been shown to be important for both of its meiotic functions (crossing over and repair of heteroduplex DNA) (Pang et al. 1997; Tran and Liskay 2000; Hoffmann et al. 2003). In contrast, the ATP hydrolysis mutant mlh1-E31A/mlh1-E31A appears to have no effect on meiotic recombination (Tran and Liskay 2000; Hoffmann et al. 2003). This may partly be explained by in vitro studies demonstrating that this mutant exhibits a low level of ATPase activity (Hall et al. 2002).The meiotic functions of MLH1 can be functionally separated as shown by mutating the same residue, G98, to different amino acids (Hoffmann et al. 2003). The residue G98 is situated in the ATPase motif in the GFRGEAL box (GYRGDAL in Mlh3p), which forms the lid of the ATP binding pocket. Mutations in this motif are predicted to affect ATP binding and/or heterodimerization with Pms1p (Ban and Yang 1998; Ban et al. 1999). Mutating the residue G98 in the ATP binding lid to alanine resulted in defective repair of heteroduplex DNA while crossing over was unaffected, but when the same residue was mutated to valine both mismatch repair and crossover functions were defective (Hoffmann et al. 2003). The mlh1-G98V mutant disrupts the interaction of Mlh1p with Pms1p, while mlh1-G98A does not (Pang et al. 1997). This may contribute to the difference observed in the effect on crossing over as Mlh1p is thought to interact with Pms1p and Mlh3p through the same residues (Wang et al. 1999; Kondo et al. 2001). Consequently if the interaction with Pms1p is affected then it is likely that the interaction with Mlh3p is also disrupted.We constructed mlh3 mutants corresponding to the ATP binding and ATP hydrolysis mutants of mlh1 to explore the role of Mlh3p in meiotic recombination. We also constructed mlh3-G97A and mlh3-G97V mutants, equivalent to the mlh1-G98A/V pair that has been shown to differentially affect the mitotic and meiotic functions of Mlh1p. All mutants were assayed for mitotic mismatch repair, meiotic heteroduplex repair, crossing over, and chromosome segregation.     

Toxic triplets provide clues to neurodegeneration

Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophyMerry, D.E. et al. (1998)Hum. Mol. Genet. 7, 693–701Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell cultureCooper, J.K. et al. (1998)Hum. Mol. Genet. 7, 783–790Aggregation of N-terminal huntingtin is dependent on the length of its glutamine repeatsLi, S.H. and Li, X.J. (1998)Hum. Mol. Genet. 7, 777–782     

Using RNA Interference to Identify Specific Modifiers of a Temperature-Sensitive,Embryonic-Lethal Mutation in the Caenorhabditis elegans Ubiquitin-Like Nedd8 Protein Modification Pathway E1-Activating Gene rfl-1

The essential Caenorhabditis elegans gene rfl-1 encodes one subunit of a heterodimeric E1-activating enzyme in the Nedd8 ubiquitin-like protein conjugation pathway. This pathway modifies the Cullin scaffolds of E3 ubiquitin ligases with a single Nedd8 moiety to promote ligase function. To identify genes that influence neddylation, we used a synthetic screen to identify genes that, when depleted with RNAi, enhance or suppress the embryonic lethality caused by or198ts, a temperature-sensitive (ts) mutation in rfl-1. We identified reproducible suppressor and enhancer genes and employed a systematic specificity analysis for each modifier using four unrelated ts embryonic lethal mutants. Results of this analysis highlight the importance of specificity controls in identifying genetic interactions relevant to a particular biological process because 8/14 enhancers and 7/21 suppressors modified lethality in other mutants. Depletion of the strongest specific suppressors rescued the early embryonic cell division defects in rfl-1(or198ts) mutants. RNAi knockdown of some specific suppressors partially restored Cullin neddylation in rfl-1(or198ts) mutants, consistent with their gene products normally opposing neddylation, and GFP fusions to several suppressors were detected in the cytoplasm or the nucleus, similar in pattern to Nedd8 conjugation pathway components in early embryonic cells. In contrast, depletion of the two strongest specific enhancers did not affect the early embryonic cell division defects observed in rfl-1(or198ts) mutants, suggesting that they may act at later times in other essential processes. Many of the specific modifiers are conserved in other organisms, and most are nonessential. Thus, when controlled properly for specificity, modifier screens using conditionally lethal C. elegans mutants can identify roles for nonessential but conserved genes in essential processes.UBIQUITIN-mediated proteolysis regulates many biological processes (Nandi et al. 2006). In the early Caenorhabditis elegans embryo, these include oocyte maturation, cell cycle progression, cell polarization, and cell fate patterning, all of which require the timely destruction of maternally expressed proteins (Bowerman and Kurz 2006; Greenstein and Lee 2006). One C. elegans protein targeted for proteolysis early in embryogenesis is MEI-1, the AAA-ATPase subunit of the microtubule-severing complex called katanin (Mains et al. 1990; Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002; Pintard et al. 2003a; Xu et al. 2003). Katanin is a heterodimer of two subunits called p60 and p80 in vertebrates and MEI-1 and MEI-2 in C. elegans. Katanin in C. elegans is required for proper assembly and function of the small, barrel-shaped meiotic spindles (Albertson and Thomson 1993; McNally et al. 2006) and must be degraded after meiotic divisions to permit assembly of the much larger first mitotic spindle in the one-cell zygote. In mutants that fail to degrade katanin after the completion of meiosis, the first mitotic spindle is fragmented and mis-oriented, cytokinesis is defective, and the embryos die without hatching (Dow and Mains 1998; Srayko et al. 2000; Kurz et al. 2002).The katanin subunit MEI-1 is targeted for poly-ubiquitylation and proteolytic destruction by a Cullin-based E3 ligase (Kurz et al. 2002). This complex includes the Cullin scaffolding protein CUL-3 and a substrate-specific adaptor called MEL-26 that binds to CUL-3 through a BTB domain and to MEI-1 through a MATH domain (Pintard et al. 2003b). Cullin 3-based E3 ligases in mammals also utilize substrate-specific adaptor proteins that, like MEL-26, have both a Cullin-binding BTB/POZ domain and another protein–protein interaction domain that binds to the substrate (Geyer et al. 2003; Cullinan et al. 2004; Angers et al. 2006). While MEI-1/Katanin downregulation by the CUL-3/MEL-26 E3 ligase is essential at most growth temperatures, a mel-26 null mutation is viable at the low growth temperature of 15° (Lu and Mains 2007). This bypass of mel-26 at 15° depends at least in part on the anaphase-promoting complex and its targeting of MEI-1 for proteolytic degradation (Lu and Mains 2007). Phosphorylation by the kinase MBK-2 primes MEI-1 for proteolysis (Quintin et al. 2003; Stitzel et al. 2007) and also promotes the downregulation of MEI-1 by the anaphase-promoting complex (Lu and Mains 2007).CUL-3 is the only C. elegans Cullin thus far identified that requires modification by the ubiquitin-like protein Nedd8 (Bowerman and Kurz 2006). In contrast, C. elegans CUL-2 is required for progression through meiosis and for the localized degradation of cell fate determinants in one-cell-stage embryos (Liu et al. 2004; Sonneville and Gonczy 2004), but neddylation-defective mutants do not exhibit these early defects (Bowerman and Kurz 2006). Cullin neddylation is mediated by the Nedd8 protein conjugation pathway, which begins with a heterodimeric E1-activating enzyme consisting of ULA-1 and RFL-1 (Uba3p in budding yeast) and also includes the E2-conjugating enzyme UBC-12 (Jones and Candido 2000; Srayko et al. 2000; Kurz et al. 2002) and the E3 ligase DCN-1 (Kurz et al. 2005).The downregulation of MEI-1/katanin by the CUL-3/MEL-26 E3 ligase requires a balance of both CUL-3 neddylation, which is mediated by the Nedd8 conjugation pathway, and deneddylation, which is mediated by the conserved COP-9 Signalosome (Pintard et al. 2003a). Other Cullin-based E3 ubiquitin ligases also require a balance of neddylation and deneddylation (Lyapina et al. 2001; Schwechheimer et al. 2001; Bornstein et al. 2006; Hetfeld et al. 2008). Deneddylation may modulate activation of the E3 ligase and thereby prevent the premature degradation of substrate adaptor proteins that also can become poly-ubiquitylated and degraded as a result of E3 ligase function.To identify additional factors that influence neddylation, and the downregulation of MEI-1/katanin after the completion of meiosis in C. elegans, we report here our use of RNA interference (RNAi) to reduce gene functions in a temperature-sensitive (ts) neddylation-defective mutant, rfl-1(or198ts). The discovery of RNAi and its systemic properties in C. elegans have made it possible to systematically target C. elegans genes for depletion by feeding worms bacterial strains that express double-strand RNAs corresponding to C. elegans gene sequences (Fire et al. 1998; Timmons et al. 2001; Feinberg and Hunter 2003; Baugh et al. 2005; Lehner et al. 2006; van Haaften et al. 2006). Furthermore, chemical mutagenesis screens have identified temperature-sensitive mutations in many essential C. elegans genes, which can be used for synthetic screens by choosing intermediate-growth temperatures that sensitize the genetic background and also optimize visual scoring of embryonic viability. Recently, genomewide RNAi screens have been used to identify C. elegans genes that, when reduced in function, restore viability to temperature-sensitive, embryonic-lethal mutants (Labbe et al. 2006; O''Rourke et al. 2007). Because a loss of suppressor function restores mutant viability, the suppressors may negatively regulate either the wild-type gene product or the process that requires the wild-type gene product.Here we report our identification of C. elegans genes that, when reduced in function by feeding RNAi, reproducibly suppressed or enhanced rfl-1(or198ts) embryonic lethality. Most suppressors were specific for rfl-1(or198ts), while specific enhancement was less common. Many of the rfl-1-specific suppressors and enhancers are conserved but appear nonessential. GFP fusions to several specific suppressors exhibit localization patterns that resemble those known for neddylation pathway components, and depletion of some of these partially restored CUL-3 neddylation in rfl-1(or198ts) mutants. In addition to identifying possible roles for conserved genes in cullin neddylation, we report the first quantitative analysis of specificity for both the enhancement and the suppression of a conditionally lethal mutant in C. elegans. Our results highlight the importance of testing genetic modifiers of conditionally lethal mutants for locus specificity.     

Suppression of Mitochondrial DNA Instability of Autosomal Dominant Forms of Progressive External Ophthalmoplegia-Associated ANT1 Mutations in Podospora anserina

Maintenance and expression of mitochondrial DNA (mtDNA) are essential for the cell and the organism. In humans, several mutations in the adenine nucleotide translocase gene ANT1 are associated with multiple mtDNA deletions and autosomal dominant forms of progressive external ophthalmoplegia (adPEO). The mechanisms underlying the mtDNA instability are still obscure. A current hypothesis proposes that these pathogenic mutations primarily uncouple the mitochondrial inner membrane, which secondarily causes mtDNA instability. Here we show that the three adPEO-associated mutations equivalent to A114P, L98P, and V289M introduced into the Podospora anserina ANT1 ortholog dominantly cause severe growth defects, decreased reactive oxygen species production (ROS), decreased mitochondrial inner membrane potential (Δψ), and accumulation of large-scale mtDNA deletions leading to premature death. Interestingly, we show that, at least for the adPEO-type M106P and A121P mutant alleles, the associated mtDNA instability cannot be attributed only to a reduced membrane potential or to an increased ROS level since it can be suppressed without restoration of the Δψ or modification of the ROS production. Suppression of mtDNA instability due to the M106P and A121P mutations was obtained by an allele of the rmp1 gene involved in nucleo-mitochondrial cross- talk and also by an allele of the AS1 gene encoding a cytosolic ribosomal protein. In contrast, the mtDNA instability caused by the S296M mutation was not suppressed by these alleles.THE maintenance and expression of mitochondrial DNA (mtDNA) depend on many nuclear-encoded gene products. Recent studies have shown that defects in this maintenance can have devastating consequences for the cell and the organism. In humans, these defects are an important cause of neurological diseases including autosomal dominant (or recessive) progressive external ophthalmoplegia (adPEO) (Chinnery 2003; Copeland 2008). These disorders are characterized by multiple large-scale deletions of mtDNA. Three different genes that can cause PEO with multiple mtDNA deletions have been identified: the mtDNA polymerase (POLG), the heart/muscle isoform of the adenine nucleotide translocator (ANT1), and the mitochondrial DNA helicase, Twinkle.The adenine nucleotide translocator (ANT), also known as the ADP/ATP mitochondrial translocator, is the most abundant protein in the inner mitochondrial membrane (Riccio et al. 1975; Nury et al. 2006; Klingenberg 2008). It exports ATP produced by mitochondrial oxidative phosphorylation toward the cytosol to meet the energy requirements of the cell; in exchange, it transports ADP into the mitochondrial matrix to fuel the conversion of ADP to ATP by the F₁F_O-ATP synthase. In humans, four isoforms of the ANT protein exist, and they are differently expressed in a tissue-specific manner (Stepien et al. 1992; Palmieri 2004; Dolce et al. 2005). The human ANT1 isoform is predominantly expressed in skeletal and cardiac muscle, and specific ANT1 mutations are associated with adPEO characterized by mtDNA instability (Kaukonen et al. 1999, 2000; Napoli et al. 2001; Komaki et al. 2002; Siciliano et al. 2003). In mice, Ant1 knockout induces mitochondrial myopathy (Graham et al. 1997), increased H₂O₂ production, and mtDNA damage and inhibits oxidative phosphorylation (Esposito et al. 1999). Some of these mutations were introduced in the AAC2 gene of Saccharomyces cerevisiae that encodes the major ADP/ATP mitochondrial translocator isoform in this organism. Numerous and sometimes contradictory effects have been reported depending in particular on the yeast laboratory strains examined (Kaukonen et al. 2000; Chen 2002, 2004; Fontanesi et al. 2004; Palmieri et al. 2005; Wang et al. 2008b).In an attempt to better understand how these mutations affect mitochondrial DNA stability and their functional consequences on mitochondrial metabolism, we decided to introduce them in the unique ADP/ATP translocator gene of Podospora anserina, PaAnt. Like S. cerevisiae, the filamentous fungus P. anserina is an excellent system for genetic and molecular analyses. In contrast to S. cerevisiae, it is a strict multicellular aerobe that can display heteroplasmic states in which intact and rearranged mitochondrial genomes coexist. In this organism, life span is a reflection of mtDNA stability, and death is always associated with large mtDNA rearrangements. “Natural death” or aging is accompanied by large-scale reorganizations of the mtDNA whereas a nuclear-controlled premature death syndrome is accompanied by the accumulation of site-specific mtDNA deletions (Belcour et al. 1999; Silar et al. 2001 for reviews). P. anserina therefore occupies an interesting position among model systems for studying the cellular consequences of mutations in the ADP/ATP translocase gene.We show here that the mutations M106P, A121P, and S296M, equivalent to the L98P, A114P (familial), and V289M (sporadic) human mutations, severely impair the vegetative and sexual development of the fungus and are responsible for decreased ROS production and for decreased inner membrane potential (Δψ). The severity of the phenotypes differs according to the mutation. The three mutations show mtDNA instability, which leads to premature death. All these mutated traits are dominant. Interestingly, the mtDNA instability associated with the M106P and A121P mutations depends on the rmp1 gene. This gene exists under two naturally occurring alleles, rmp1-1 and rmp1-2, which control mtDNA integrity in some genetic contexts (Belcour et al. 1991; Contamine et al. 1996, 2004). When associated with the rmp1-1 allele, the M106P and A121P mutations lead to rapid mtDNA instability whereas, in the presence of the rmp1-2 allele, mtDNA instability is suppressed, and life span is considerably increased. Surprisingly, suppression is not accompanied by a restoration of the Δψ or a modification in the ROS level, demonstrating that these parameters are not sufficient to explain the M106P and A121P mtDNA instability. Mitochondrial DNA instability due to the M106P and A121P mutations is also suppressed by a mutation in the AS1 gene encoding a ribosomal protein. The suppressor effects are not observed for the S296M mutation.     

In Planta Mutagenesis Determines the Functional Regions of the Wheat Puroindoline Proteins

In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs'' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F₂:F₃ populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.NATURAL selection has captured a relatively small subset of potentially useful protein sequences. Unraveling the critical features of proteins via understanding the process of their evolution is a powerful approach for proteins present in many diverse species (Bashford et al. 1987; Hampsey et al. 1988). However, this approach is not feasible for the wheat puroindolines (PINs) that are present only in hexaploid wheat and related species (Massa and Morris 2006). The PINs are unique in structure in having a tryptophan-rich domain and are members of the protease inhibitor/seed storage/lipid transfer protein family (PF00234) (Finn et al. 2008).The tryptophan-rich domain has been hypothesized to control PIN function (Giroux and Morris 1997), but there is no unbiased direct evidence for this since previous studies have focused on the tryptophan box alone (Evrard et al. 2008). A nonbiased approach would consist of random mutagenesis followed by functional analysis (Bowie et al. 1990). This approach has been used extensively for proteins that can be expressed in vitro using either random (Tarun et al. 1998; Guo et al. 2004; Smith and Raines 2006; Georgelis et al. 2007) or site-directed mutations (Miyahara et al. 2008; Osmani et al. 2008). However, functional analysis of many plant proteins in vitro may not be comparable to in planta analysis. In the case of puroindolines, there is no in vitro assay that properly mimics the synergistic binding of PINA and PINB to starch granules or is as easy to measure as grain hardness. Therefore, creation and analysis of a large number of new alleles in wheat in planta is an ideal approach to dissect PIN function.The absence of high-throughput transformation and/or functional screening methods in most crop plants is the largest obstacle in the way of in planta protein functional analysis. However, high-throughput in vitro random or targeted mutagenesis followed by functional analysis has been demonstrated in Arabidopsis thaliana (Dunning et al. 2007) and Nicotiana benthamiana (Boter et al. 2007). Traditional in planta mutagenesis followed by analysis of loss-of-function mutations has been used to clone unknown genes (Xiong et al. 2001) or to define function for candidate genes (Haralampidis et al. 2001; Qi et al. 2006). A high-throughput in planta functional approach for PINA and PINB seems attractive for three reasons. First, the EMS mutation rate in wheat is higher than in any other plant (Slade et al. 2005; Feiz et al. 2009a). Second, PINs control the vast majority of variation in grain hardness (Campbell et al. 1999). Finally, a small-scale preliminary study indicated the feasibility of this approach (Feiz et al. 2009a).PINA and PINB are cysteine-rich proteins unique in having a tryptophan-rich domain (Blochet et al. 1993) and together compose the wheat Hardness (Ha) locus (Giroux and Morris 1998; Wanjugi et al. 2007a). Ha is located on chromosome 5DS and is the major determinant of wheat endosperm texture (Mattern et al. 1973; Law et al. 1978; Campbell et al. 1999). Soft texture (Ha) results when both Pin genes are wild type (Pina-D1a, Pinb-D1a) while hard texture (ha) results from mutations in either Pin (Giroux and Morris 1997, 1998). Transgenic studies in rice (Krishnamurthy and Giroux 2001), wheat (Beecher et al. 2002; Martin et al. 2006), and corn (Zhang et al. 2009) have demonstrated that Pin mutations are causative to hard grain texture. PINA and PINB are not functionally interchangeable and control grain hardness via cooperative binding to starch granules (Hogg et al. 2004; Swan et al. 2006; Wanjugi et al. 2007a; Feiz et al. 2009b). PIN binding to starch granules is mediated by polar lipids (Greenblatt et al. 1995) and PIN abundance is correlated with seed polar lipid content (Feiz et al. 2009b). Variation in PIN function affects grain hardness along with nearly all end product quality traits (Hogg et al. 2005; Martin et al. 2007, 2008; Wanjugi et al. 2007b; Feiz et al. 2008). Determining PINs'' function-determining regions could lead to greater knowledge of their mode of action and to wheat quality improvements. Current PIN functional analyses have been limited to in vitro tests of binding to each other (Ziemann et al. 2008) or to yeast membranes (Evrard et al. 2008).Here, we report the creation and functional analysis in planta of new alleles of PINA and PINB. This is the first successful in planta functional analysis of a crop plant protein.