Cloning by functional complementation,and inactivation,of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^? mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Cloning by functional complementation, and inactivation, of theSchizosaccharomyces pombe homologue of theSaccharomyces cerevisiae geneABC1

TheSaccharomyces cerevisiae geneABC1 is required for the correct functioning of thebc ₁ complex of the mitochondrial respiratory chain. By functional complementation of aS. cerevisiae abc1 ^– mutant, we have cloned aSchizosaccharomyces pombe cDNA, whose predicted product is 50% identical to the Abc1 protein. Significant homology is also observed with bacterial, nematode, and even human amino acid sequences of unknown function, suggesting that the Abc1 protein is conserved through evolution. The cloned cDNA corresponds to a singleS. pombe geneabc1Sp, located on chromosome II, expression of which is not regulated by the carbon source. Inactivation of theabc1Sp gene by homologous gene replacement causes a respiratory deficiency which is efficiently rescued by the expression of theS. cerevisiae ABC1 gene. The inactivated strain shows a drastic decrease in thebc ₁ complex activity, a decrease in cytochromeaa3 and a slow growth phenotype. To our knowledge, this is the first example of the inactivation of a respiratory gene inS. pombe. Our results highlight the fact thatS. pombe growth is highly dependent upon respiration, and thatS. pombe could represent a valuable model for studying nucleo-mitochondrial interactions in higher eukaryotes.     

Isolation of a novel rmn1 gene genetically linked to spnab2 with respect to mRNA export in fission yeast

In fission yeast, Schizosaccharomyces pombe, the spnab2 gene encodes an ortholog of the budding yeast nuclear abundant poly(A)⁺ RNA-binding protein 2 (Nab2) that is an essential protein required for both mRNA biogenesis and nuclear export of mRNA to the cytoplasm. We have previously isolated three mutants (SLnab1–3) that showed synthetic lethality under the repressed condition of spnab2 expression. In this study, we isolated a novel rmn1 gene as a multicopy suppressor that complemented the defects in growth and mRNA export of SLnab1 mutant cells. The rmn1 gene contained three introns and encoded a 589 amino-acid protein with the RNA recognition motif (RRM) in the central region. The Δrmn1 null mutant was viable but showed a s light mRNA export defect. However, its over-expression caused a deleterious effect on growth accompanied by intense accumulation of poly(A)⁺ RNA in the nucleus. The combination of Δrmn1 with Δspnab2 or Δspmex67 also inhibited growth. In addition, Rmn1p was associated with Rae1p in vivo. These results suggest that rmn1 is a novel gene that is functionally linked to spnab2.     

Genome-wide association analyses reveal significant loci and strong candidate genes for growth and fatness traits in two pig populations

Background

Recently, genome-wide association studies (GWAS) have been reported on various pig traits. We performed a GWAS to analyze 22 traits related to growth and fatness on two pig populations: a White Duroc × Erhualian F₂ intercross population and a Chinese Sutai half-sib population.

Results

We identified 14 and 39 loci that displayed significant associations with growth and fatness traits at the genome-wide level and chromosome-wide level, respectively. The strongest association was between a 750 kb region on SSC7 (SSC for Sus scrofa) and backfat thickness at the first rib. This region had pleiotropic effects on both fatness and growth traits in F₂ animals and contained a promising candidate gene HMGA1 (high mobility group AT-hook 1). Unexpectedly, population genetic analysis revealed that the allele at this locus that reduces fatness and increases growth is derived from Chinese indigenous pigs and segregates in multiple Chinese breeds. The second strongest association was between the region around 82.85 Mb on SSC4 and average backfat thickness. PLAG1 (pleiomorphic adenoma gene 1), a gene under strong selection in European domestic pigs, is proximal to the top SNP and stands out as a strong candidate gene. On SSC2, a locus that significantly affects fatness traits mapped to the region around the IGF2 (insulin-like growth factor 2) gene but its non-imprinting inheritance excluded IGF2 as a candidate gene. A significant locus was also detected within a recombination cold spot that spans more than 30 Mb on SSCX, which hampered the identification of plausible candidate genes. Notably, no genome-wide significant locus was shared by the two experimental populations; different loci were observed that had both constant and time-specific effects on growth traits at different stages, which illustrates the complex genetic architecture of these traits.

Conclusions

We confirm several previously reported QTL and provide a list of novel loci for porcine growth and fatness traits in two experimental populations with Chinese Taihu and Western pigs as common founders. We showed that distinct loci exist for these traits in the two populations and identified HMGA1 and PLAG1 as strong candidate genes on SSC7 and SSC4, respectively.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0089-5) contains supplementary material, which is available to authorized users.     

Soluble fumarate reductase isoenzymes from Saccharomyces cerevisiae are required for anaerobic growth

In Saccharomyces cerevisiae, the cytosolic and promitochondrial isoenzymes of fumarate reductase are encoded by the FRDS and OSM1 genes, respectively. The product of the OSM1 gene is reported to be required for growth in hypertonic medium. Simultaneous disruption of the FRDS and OSM1 genes resulted in the inability of the yeasts to grow anaerobically on glucose as a carbon source, and disruption of the OSM1 gene caused poor growth under anaerobic conditions. However, the disruption of both the FRDS and/or OSM1 genes had no effect on aerobic growth or growth under hypertonic conditions. These results suggest that the fumarate reductase isoenzymes in Saccharomyces cerevisiae are essential for anaerobic growth but not for growth under hypertonic conditions.     

Regulation of nitrogen starvation responses by the alarmone (p)ppGpp in rice

Nitrogen is an essential macronutrient for all living organisms and is critical for crop productivity and quality.In higher plants, inorganic nitrogen is absorbed through roots and then assimilated into amino acids by the highly conserved glutamine synthetase/glutamine:2-oxoglutarate aminotransferase(GS/GOGAT) cycle.How nitrogen metabolism and nitrogen starvation responses of plants are regulated remains largely unknown. Previous studies revealed that mutations in the rice ABNORMAL CYTOKININ RES...     

Novel 12-bp deletion in the coding region of the bovineNPM1 gene affects growth traits

The nucleophosmin 1 gene (NPM1) encodes a multifunctional nucleolar phosphoprotein that plays a crucial role in the control of various aspects of cell growth and homeostasis. In this study, the coding region of theNPM1 gene was screened in 1035 individuals of 4 Chinese cattle breeds by DNA sequencing and polyacrylamide gel electrophoresis. A novel 12-bp deletion mutation was identified in the coding region of theNPM1 gene. The PCR products of primerNPM1-P2 exhibited 3 genotypes and 2 alleles: 178 bp (denoted asW) and 166 bp (denoted asD). GenotypeDD and alleleD were predominant in the studied populations. Association analysis with growth traits in the Nanyang breed (N = 265) showed that the animals with genotypeDD had significantly greater birth weight, body weight, body length, and heart girth than those with genotypeWD (P < 0.01 orP < 0.05) at birth and after 6 months and 12 months, but not at 18 and 24 months of age. Results of this study suggest that theNPM1 gene is a candidate gene for growth traits in cattle.     

Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E^ΔP5abc). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E^ΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.     

A root-specific wall-associated kinase gene, HvWAK1, regulates root growth and is highly divergent in barley and other cereals

Wall-associated receptor-like kinases (WAKs) are important candidates for directly linking the extracellular matrix with intracellular compartments and are involved in developmental processes and stress response. WAK gene family has been identified in plants such as Arabidopsis and rice. Here, we present a detailed analysis of the WAK1 gene from barley cv. Golden Promise, mapped to chromosome 5H. Three BAC clones corresponding to the WAK fragment were sequenced and the full-length WAK1 gene was characterized. The gene has three exons and two short introns with a coding region of 2,178 bp encoding a protein of 725 amino acids. A regulatory region was analyzed in ?1,000 bp sequence upstream to start codon. Using conserved domains database and SMART, various conserved domains such as GUB WAK Bind, epidermal growth factor CA, and protein kinase C as well as other regions like signal peptides, active sites, and transmembrane domains were identified. The gene organization of HvWAK1 was compared with wheat (TaWAK1) and Arabidopsis (AtWAK1), suggesting that the WAK1 gene organization has remained highly conserved. Nonetheless, WAK1 was found to be highly divergent when compared with sequences available from barley cv. Haruna Nijo (50 %), rice (46 %), wheat (21 %), Arabidopsis (25 %), and maize (19 %). This divergence may have facilitated a better adaptation to surrounding environments due to its role in communication between the extracellular matrix, cell, and outer environment. Semiquantitative RT-PCR-based expression analysis indicates HvWAK1 expression is specific to roots. Significant differences in root growth between GP wild type and GP-Ds mutant seedlings were observed under control and salt stress conditions.     

Abc3-Mediated Efflux of an Endogenous Digoxin-like Steroidal Glycoside by Magnaporthe oryzae Is Necessary for Host Invasion during Blast Disease

Magnaporthe oryzae, which causes the devastating rice-blast disease, invades its host plants via a specialized infection structure called the appressorium. Previously, we showed that the ATP-Binding Cassette 3 transporter is necessary for appressorial function (host penetration) in M. oryzae. However, thus far, the molecular basis underlying impaired appressorial function in the abc3Δ remains elusive. We hypothesized that the abc3Δ appressoria accumulate excessive amounts of specific efflux substrate(s) of the Abc3 transporter in M. oryzae. We devised an innovative yeast-based strategy and identified Abc3 Transporter efflux Substrate (ATS) to be a digoxin-like endogenous steroidal glycoside that accumulates to inhibitory levels in M. oryzae abc3Δ appressoria. Exogenous ATS altered cell wall biogenesis and viability in wild-type Schizosaccharomyces pombe, but not in S. pombe expressing M. oryzae Abc3. We show that ATS associates with the Translation Elongation factor Tef2 in M. oryzae, and propose that ATS regulates ion homeostasis during pathogenesis. Excessive ATS accumulation, either intracellularly due to impaired efflux in the abc3Δ or when added exogenously to the wild type, renders M. oryzae nonpathogenic. Furthermore, we demonstrate that the host penetration defects in the abc3Δ are due to aberrant F-actin dynamics as a result of altered Tef2 function and/or ion homeostasis defects caused by excess accumulation of ATS therein. Rather surprisingly, excessive exogenous ATS or digoxin elicited the hypersensitive response in rice, even in the absence of the blast fungus. Lastly, reduced disease symptoms in the inoculated host plants in the presence of excessive digoxin suggest a potential use for such related steroidal glycosides in controlling rice-blast disease.     

Genetic control of Rous sarcoma regression in chickens: Linkage with the major histocompatibility complex

Studies with two closely related inbred chicken strains show that regression of Rous sarcoma virus-induced tumors is a dominantly inherited trait, controlled by a gene within, or closely linked to, the major histocompatibility complex (B region). Strain G-B2 birds are capable of regressing Rous tumors, while strain G-B1 birds are uniformly susceptible to progressive Rous tumor development. Evidence for crossing over between the genes controlling serologically determined MHC antigens on erythrocytes and genes controlling Rous sarcoma growth was obtained. The MHC-linked gene which confers the ability to regress Rous sarcomas is designatedR-Rs-1. The allelic gene which allows for progressive tumor growth in homozygous birds is designatedr-Rs-1.     

A single nucleotide polymorphism at the Vrn-D1 promoter region in common wheat is associated with vernalization response

Facultative wheat varieties adapt to a particular environment. But the molecular basis for the facultative growth habit is not clear relative to winter and spring growth habit. Two sets of wheat varieties were chosen for this study. Set 1 comprised ten spring accessions and Set 2 comprised ten facultative accessions. All accessions had been tested by the previously described allele-specific markers and shown having the same allelic composition of vrn-A1 vrn-B1 Vrn-D1 and vrn-B3. Here we examined whether differences in growth habit might be associated with as yet unidentified sequence variation at Vrn-D1 locus. A region including the intron 1 deletion, the entire reading frame from a cDNA template and a part of promoter region of the dominant Vrn-D1 gene in each of the accessions was sequenced, and a single nucleotide polymorphism was found between facultative accessions and spring accessions in the CArG-box at the promoter region. The novel allele in facultative accessions was designated as Vrn-D1b. The investigation of an F₂ population segregating for Vrn-D1b and Vrn-D1a (previously, Vrn-D1) in the greenhouse under long days without vernalization showed that the plants with Vrn-D1b homozygous allele headed 32?days later and had about three more leaves than the plants with Vrn-D1a homozygous allele. As Vrn-D1b has the same deletion in intron 1 as Vrn-D1a, and, in addition, a single nucleotide mutation at promoter region, and is associated with facultative growth habit, we suggest that the promoter mutation may modify the basal activity level of an allele of VRN1 that is already active (due to the loss of segments in intron 1). Our finding further supports that both the promoter and intron 1 regulatory affect vernalization response and work independently.