Inactive Allele Research Articles

Essential Role for endogenous siRNAs during meiosis in mouse oocytes.

The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Both small RNA species silence gene expression post-transcriptionally in association with the ARGONAUTE (AGO) family of proteins. In mammals, there are four AGO proteins (AGO1-4), of which only AGO2 possesses endonucleolytic activity. siRNAs trigger endonucleolytic cleavage of target mRNAs, mediated by AGO2, whereas miRNAs cause translational repression and mRNA decay through association with any of the four AGO proteins. Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because mouse oocytes express both miRNAs and endo-siRNAs, this phenotype could be due to the absence of either class of small RNA, or both. However, we and others demonstrated that miRNA function is suppressed in mouse oocytes, which suggested that endo-siRNAs, not miRNAs, are essential for female meiosis. To determine if this was the case we generated mice that express a catalytically inactive knock-in allele of Ago2 (Ago2ADH) exclusively in oocytes and thereby disrupted the function of siRNAs. Oogenesis and hormonal response are normal in Ago2ADH oocytes, but meiotic maturation is impaired, with severe defects in spindle formation and chromosome alignment that lead to meiotic catastrophe. The transcriptome of these oocytes is widely perturbed and shows a highly significant correlation with the transcriptome of Dicer null and Ago2 null oocytes. Expression of the mouse transcript (MT), the most abundant transposable element in mouse oocytes, is increased. This study reveals that endo-siRNAs are essential during meiosis I in mouse females, demonstrating a role for endo-siRNAs in mammals.

95 DOSAGE COMPENSATION OF X CHROMOSOME INACTIVATION CENTER (XIC)-LINKED GENES IS ALREADY ACHIEVED IN PORCINE BLASTOCYST

X-chromosome inactivation (XCI) is an epigenetically essential process for balancing dosage of X-linked genes between male and female eutherian. Importance of this complex and species-specific event has been highlighted recently in developmental and stem cell biology. However, the process has been confirmed only in restricted species, even though the species-specific studies are needed for comprehensive understanding of XCI in specific species. XCI is regulated by the various genes, many of which are coded on the X chromosome inactivation centre (XIC). Among the XIC-linked genes, especially non-coding RNA (ncRNA) like XIST, which is master gene for XCI, are known to regulate XIC. But the centre is not identified in various species. In this study, we identified XIC in pig and analysed the dosage differences of XIC-linked gene in porcine embryos. At first, the centre was searched in pig. The genomic length of the porcine XIC was similar to human XIC and the order and coding strand of the counterparts in pig XIC were same as the human XIC-linked genes. However, sequence comparison between human XIC-linked gene and its porcine counterpart showed that ncRNA around XIST were less conserved rather than protein-coding genes. This would be caused by rapid evolution of genomic region harboring ncRNA. The expression of XIC-linked genes was compared between male and female porcine embryonic fibroblast (PEF) to confirm that dosage compensation is completed in PEF. Most of the genes were not expressed sex-specifically, but two genes, XIST and an uncharacterized gene, LOC102165544, were expressed female preferentially in PEF. Interestingly, LOC102165544, which had low sequence homology with human JPX, was expressed about 2-fold higher in female PEF. This means that XIST and LOC102165544 are XCI-escaping genes. Among the XIC-linked genes, CHIC1, XIST, LOC102165544, and RLIM were stably expressed in embryonic stage, and XIST and LOC102165544 were up-regulated after morula formation. As XIST accumulation is a requisite for XCI initiation, expression levels of the 4 genes between male and female blastocysts were compared. Interestingly, expression levels of CHIC1 and RLIM were not different in male and female blastocysts. This means their dosage would be already compensated in porcine blastocyst. Additionally, to confirm loci of the 2 genes CHIC1 and RLIM harbor one of the inactive alleles in female blastocyst, the DNA methylation pattern was examined. One of the CHIC1 alleles was inactive but RLIM CpG site was hypo-methylated in female blastocyst. This would indicate that one of the RLIM alleles is transcriptionally inactivated by chromatin modification rather than by DNA methylation of the allele. Regulatory regions of XIST and LOC102165544 were demethylated in blastocyst and this showed XCI was not finished in porcine blastocyst. Conclusively, our results demonstrate the XCI already occurs in porcine blastocyst at least one gene but it is not completed.This work was supported by Next BioGreen21 program (PJ009493), Rural Development Administration, Republic of Korea.

Diagnosis of MECP2 duplication syndrome with molecular genetic techniques

To investigate whether the four boys with delayed motor development and intellectual disability suffer from MECP 2 duplication syndrome. Blood specimens and clinical data of four patients and mothers of patient 2 and patient 4 were collected. Genomic DNA was extracted from peripheral blood using DNA extraction kit. At first multiplex ligation-dependent probe amplification (MLPA) was employed in 4 patients, two distinct kits SALSA P036 and P070 for sub-telomere screening, and SALSA P245 for the 22 common microdeletion and microduplication syndromes. Then array-CGH analysis was carried out. Two mothers of patients were tested by array- comparative genomic hybridization (CGH) and X chromosome inactivation analysis. All the 4 patients presented with severe hypotonia, delayed motor development, intellectual disability and absent or limited language. Three patients manifested recurrent pneumonia in infancy except patient 2. Four patients had duplication on chromosome Xq28 with MLPA kit SALSA P245. Array-CGH identified the size of each duplication on Xq28. The precise size of each duplication was different in the four patients: patient 1, 14.931 Mb, patient 2, 0.393 Mb, patient 3, 0.482 Mb and patient 4, 0.299 Mb. To compare Xq28 duplications with UCSC database (http://genome.ucsc.edu/) revealed that each duplication harbors the MECP 2 and HCFC 1 gene. Mothers of patient 2 and patient 4 also carried microduplication on Xq28. X chromosome inactivation analysis demonstrated completely skewed inactivation (0: 100) and it is the inactive allele that passed on to the patients. For patients that present with delayed motor development, intellectual disability, hypotonia, absent or limited language and recurrent infection, combination of MLPA and array- CGH is effective and specific diagnostic methods of MECP 2 duplication syndrome.

The catalytic activity of the mitogen-activated protein kinase extracellular signal-regulated kinase 3 is required to sustain CD4+ CD8+ thymocyte survival.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family whose function is largely unknown. Given the central role of MAPKs in T cell development, we hypothesized that ERK3 may regulate thymocyte development. Here we have shown that ERK3 deficiency leads to a 50% reduction in CD4(+) CD8(+) (DP) thymocyte number. Analysis of hematopoietic chimeras revealed that the reduction in DP thymocytes is intrinsic to hematopoietic cells. We found that early thymic progenitors seed the Erk3(-/-) thymus and can properly differentiate and proliferate to generate DP thymocytes. However, ERK3 deficiency results in a decrease in the DP thymocyte half-life, associated with a higher level of apoptosis. As a consequence, ERK3-deficient DP thymocytes are impaired in their ability to make successful secondary T cell receptor alpha (TCRα) gene rearrangement. Introduction of an already rearranged TCR transgene restores thymic cell number. We further show that knock-in of a catalytically inactive allele of Erk3 fails to rescue the loss of DP thymocytes. Our results uncover a unique role for ERK3, dependent on its kinase activity, during T cell development and show that this atypical MAPK is essential to sustain DP survival during RAG-mediated rearrangements.

Genome wide association study of cholesterol and poly- and monounsaturated fatty acids, protein, and mineral content of beef from crossbred cattle

The objectives were to determine the variation explained by the BovineSNP50v2 BeadChip for cholesterol (CH), polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), protein, and minerals in beef cattle, and to identify chromosomal regions that harbor major allelic variants underlying the variation of these traits. Crossbred steers and heifers (n=236) segregating at the inactive myostatin allele on BTA2 were harvested and steaks were sampled from the M. semitendinosus and the M. longissimus thoracis et lumborum for nutrient analysis. A Bayes C algorithm was employed in genome-wide association analysis. The resulting posterior heritability (SD) estimates ranged from 0.43 (0.10) to 0.71 (0.08) for lipid traits and 0.05 (0.08) to 0.75 (0.06) for mineral traits. Across cuts, correlations between genomic estimated breeding values (GEBV) were similar for CH, MUFA and PUFA. The top 0.5% 1-Mb windows for all traits explained up to 9.93% of the SNP variance. Slight differences did exist between cuts and between different measurement scales of fatty acids.

The histone demethylase activity of Rph1 is not essential for its role in the transcriptional response to nutrient signaling.

Rph1 and Gis1 are two related yeast zinc finger proteins that function as downstream effectors in the Ras/PKA, TOR and Sch9 nutrient signaling pathways. Both proteins also contain JmjC histone demethylase domains, but only Rph1 is known to be an active enzyme, demethylating lysine 36 of histone H3. We have studied to what extent the demethylase activity of Rph1 contributes to its role in nutrient signaling by performing gene expression microarray experiments on a yeast strain containing a catalytically inactive allele of RPH1. We find that the enzymatic activity of Rph1 is not essential for its role in growth phase dependent gene regulation. However, the ability of Rph1 to both activate and repress transcription is partially impaired in the active site mutant, indicating that the demethylase activity may enhance its function in vivo. Consistent with this, we find that the Rph1 mutation and a deletion of the histone H3 methylase Set2 affect the same target genes in opposite directions. Genes that are differentially expressed in the Rph1 mutant are also enriched for binding of Rpd3, a downstream effector in silencing, to their promoters. The expression of some subtelomeric genes and genes involved in sporulation and meiosis are also affected by the mutation, suggesting a role for Rph1-dependent demethylation in regulating these genes. A small set of genes are more strongly affected by the active site mutation, indicating a more pronounced role for the demethylase activity in their regulation by Rph1.

TIR1-like auxin-receptors are involved in the regulation of plum fruit development.

Ethylene has long been considered the key regulator of ripening in climacteric fruit. Recent evidence showed that auxin also plays an important role during fruit ripening, but the nature of the interaction between the two hormones has remained unclear. To understand the differences in ethylene- and auxin-related behaviours that might reveal how the two hormones interact, we compared two plum (Prunus salicina L.) cultivars with widely varying fruit development and ripening ontogeny. The early-ripening cultivar, Early Golden (EG), exhibited high endogenous auxin levels and auxin hypersensitivity during fruit development, while the late-ripening cultivar, V98041 (V9), displayed reduced auxin content and sensitivity. We show that exogenous auxin is capable of dramatically accelerating fruit development and ripening in plum, indicating that this hormone is actively involved in the ripening process. Further, we demonstrate that the variations in auxin sensitivity between plum cultivars could be partially due to PslAFB5, which encodes a TIR1-like auxin receptor. Two different PslAFB5 alleles were identified, one (Pslafb5) inactive due to substitution of the conserved F-box amino acid residue Pro61 to Ser. The early-ripening cultivar, EG, exhibited homozygosity for the inactive allele; however, the late cultivar, V9, displayed a PslAFB5/afb5 heterozygous genotype. Our results highlight the impact of auxin in stimulating fruit development, especially the ripening process and the potential for differential auxin sensitivity to alter important fruit developmental processes.

Aldehyde dehydrogenase 2—a potential genetic risk factor for lung function among southern Chinese: evidence from the Guangzhou Biobank Cohort Study

PurposeIn Asia, moderate alcohol users have better lung function. Never users have more inactive aldehyde dehydrogenase 2 (ALDH2) alleles (A) potentially generating confounding because inactive alleles may increase acetaldehyde exposure and reduce lung function. MethodsWe examined the association of ALDH2 genotypes with percentage predicted lung function (forced expiratory volume in 1 second; forced vital capacity) for age, sex, and height among 5641 older Chinese using multivariable linear regression. ResultsALDH2 genotypes were associated with alcohol use and height but not other attributes. Inactive alleles were inversely associated with lung function (percentage predicted forced expiratory volume in 1 second −1.52%, 95% confidence interval [CI], −2.52% to −0.51% for one inactive allele and −2.05%, 95% CI, −3.85% to −0.26% for two inactive alleles compared with two active alleles; and for percentage predicted forced vital capacity −1.25%, 95% CI −2.15% to −0.35% and −1.65%, 95% CI, −3.25% to −0.04%). The association of moderate use with lung function was attenuated after adjusting for ALDH2, in addition to other potential confounders. ConclusionsPrevious findings in Chinese may be confounded by ALDH2. High frequency of inactive ALDH2 alleles in East Asia may exacerbate the effect of environmental acetaldehyde exposure on lung function and potentially on chronic obstructive pulmonary disease.

Maternal inheritance of an inactive type III deiodinase gene allele affects mouse pancreatic β-cells and disrupts glucose homeostasis.

Dio3 is the most distal gene of the imprinted Dlk1-Dio3 gene locus and is expressed according to parental origin. Dio3 encodes the type 3 deiodinase (D3), a thioredoxin-fold like containing selenoenzyme that inactivates thyroid hormone and dampens thyroid hormone signaling. Here we used heterozygous animals with disruption of the Dio3 gene to study the allelic expression pattern of Dio3 in pancreatic β-cells and the metabolic phenotype resulting from its inactivation. Adult heterozygous mice with disruption of the Dio3 gene with maternal inheritance of the inactive Dio3 allele exhibited a total loss of D3 activity in isolated pancreatic islets, approximately 30% reduction in total pancreatic islet area, a marked decrease in insulin2 mRNA and in vivo glucose intolerance. In contrast, inheritance of the inactive Dio3 allele from the father did not affect D3 activity in isolated pancreatic islets and did not result in a pancreatic phenotype. Furthermore, exposure of pancreatic explants, D3-expressing MIN6-C3 cells or isolated pancreatic islets to 100 nM T3 for 24 hours reduced insulin2 mRNA by approximately 50% and the peak of glucose-induced insulin secretion. An unbiased analysis of T3-treated pancreatic islets revealed the down-regulation of 21 gene sets (false discovery rate q value < 25%) involved in nucleolar function and transcription of rRNA, ribonucleotide binding, mRNA translation, and membrane organization. We conclude that the Dio3 gene is preferentially expressed from the maternal allele in pancreatic islets and that the inactivation of this allele is sufficient to disrupt glucose homeostasis by reducing the pancreatic islet area, insulin2 gene expression, and glucose-stimulated insulin secretion.

The frequency of alleles in the GSTT1 and GSTM1 genes involved in phase II of xenobiotic transformation in long-lived people of subcarpathia

Polymorphism of genes of Phase II of xenobiotic biotransformation was studied in 166 long-lived people and in 169 control subjects who live in the Ivano-Frankivsk region. The frequency of the functionally inactive allele of the GSTT1 gene among the long-lived people was 24.70% and in the control group it was 20.12%. The frequency of the functionally inactive allele of GSTM1 in the long-lived people was 46.99 and in the control group it was 54.44%. The area of residence of the subjects was divided into zones with regard to environmental factors: environmentally comfortable, under moderate environmental load, and environmentally uncomfortable. Analysis of the combinations of alleles of glutathione-S-transferase genes revealed an elevated frequency of the GSTM1+/GSTT1+ allele combination in the long-lived people of the uncomfortable zone in comparison to the control group of the same zone: 54.55 and 35.09%, respectively (χ2 = 4.29; OR = 2.22 (1.04–4.75)). The GSTM1−/GSTT1+ combination was significantly more frequent in the control group than in the long-lived people: 21.82 and 43.86%, respectively (χ2 = 6.15; OR = 0.36 (0.16–0.82)). Significant differences were detected between the frequencies of combinations GSTM1+/GSTT1+, GSTM1+/GSTT1−, GSTM1−/GSTT1+, and GSTM1−/GSTT1− in long-lived people of the environmentally comfortable and uncomfortable zones: χ2 = 6.44, OR = 0.36 (0.16–0.80); χ2 = 4.86, OR = 4.89 (1.28–18.72); χ2 = 5.89, OR = 2.82 (1.20–6.58); χ2 = 3.90; OR = 0.19 (0.16–6.58), respectively.

Random Monoallelic Gene Expression Increases upon Embryonic Stem Cell Differentiation

Random autosomal monoallelic gene expression refers to the transcription of a gene from one of two homologous alleles. We assessed the dynamics of monoallelic expression during development through an allele-specific RNA-sequencing screen in clonal populations of hybrid mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We identified 67 and 376 inheritable autosomal random monoallelically expressed genes in ESCs and NPCs, respectively, a 5.6-fold increase upon differentiation. Although DNA methylation and nuclear positioning did not distinguish the active and inactive alleles, specific histone modifications were differentially enriched between the two alleles. Interestingly, expression levels of 8% of the monoallelically expressed genes remained similar between monoallelic and biallelic clones. These results support a model in which random monoallelic expression occurs stochastically during differentiation and, for some genes, is compensated for by the cell to maintain the required transcriptional output of these genes.

CYP2D6 Gene Polymorphisms and Predicted Phenotypes in Eight Indigenous Groups from Northwestern Mexico

Polymorphisms in CYP2D6 impact the interindividual and interethnic variability of drug efficiency; therefore, we determined the CYP2D6 allele distribution in eight Amerindian groups from northwestern Mexico and compared them with the frequencies in Mexican Mestizos. A total of 508 Amerindians were studied. Genotyping of CYP2D6*5 and multiplication alleles was performed by long-range PCR, while CYP2D6*2, *3, *4, *6, *10, *17, *29, *35, *41 and copy number were evaluated by real-time PCR. The most frequent alleles were CYP2D6*2 (0.05-0.28), CYP2D6*4 (0.003-0.21) and multiplications (0.043-0.107). CYP2D6*5, *6, * 10 and *41 were not observed in the majority of Amerindians, and CYP2D6*3, *17, *35 and *29 were not detected. The poor metabolizer genotype ( *4/*5) was lower (0.2%) in Amerindians than in Mestizos (5%); conversely, the ultrarapid metabolizer genotype was higher (12.6%) in indigenous groups than in Mestizos (7%). Our data show a lower frequency of CYP2D6 inactive alleles and a higher frequency of duplication/multiplication of CYP2D6 active alleles in indigenous populations that in Mestizos. Original submitted 14 August 2013; Revision submitted 7 October 2013.

Inactive alleles of cytochrome P450 2C19 may be positively selected in human evolution

BackgroundCytochrome P450 CYP2C19 metabolizes a wide range of pharmacologically active substances and a relatively small number of naturally occurring environmental toxins. Poor activity alleles of CYP2C19 are very frequent worldwide, particularly in Asia, raising the possibility that reduced metabolism could be advantageous in some circumstances. The evolutionary selective forces acting on this gene have not previously been investigated.We analyzed CYP2C19 genetic markers from 127 Gambians and on 120 chromosomes from Yoruba, Europeans and Asians (Japanese + Han Chinese) in the Hapmap database. Haplotype breakdown was explored using bifurcation plots and relative extended haplotype homozygosity (REHH). Allele frequency differentiation across populations was estimated using the fixation index (FST) and haplotype diversity with coalescent models.ResultsBifurcation plots suggested conservation of alleles conferring slow metabolism (CYP2C19*2 and *3). REHH was high around CYP2C19*2 in Yoruba (REHH 8.3, at 133.3 kb from the core) and to a lesser extent in Europeans (3.5, at 37.7 kb) and Asians (2.8, at −29.7 kb). FST at the CYP2C19 locus was low overall (0.098). CYP2C19*3 was an FST outlier in Asians (0.293), CYP2C19 haplotype diversity < = 0.037, p <0.001.ConclusionsWe found some evidence that the slow metabolizing allele CYP2C19*2 is subject to positive selective forces worldwide. Similar evidence was also found for CYP2C19*3 which is frequent only in Asia. FST is low at the CYP2C19 locus, suggesting balancing selection overall. The biological factors responsible for these selective pressures are currently unknown. One possible explanation is that early humans were exposed to a ubiquitous novel toxin activated by CYP2C19. The genetic adaptation took place within the last 10,000 years which coincides with the development of systematic agricultural practices.

TPMT genetic variants are associated with increased rejection with azathioprine use in heart transplantation

Azathioprine (AZA) is an important immunosuppressant drug used in heart transplantation (HTX). Consensus guidelines recommend that patients with thiopurine S-methyltransferase (TPMT) genetic variants be started on lower AZA dose because of higher active metabolite levels and risk of adverse events. However, in-vitro lymphocyte proliferation assays performed in participants with inactive TPMT alleles have suggested that AZA use may result in decreased immunosuppressant efficacy as compared with wild-type (WT) individuals. The objective of this study was therefore to determine the effect of TPMT genetic variation on AZA efficacy or prevention of rejection in HTX recipients treated with AZA. We genotyped 93 HTX recipients treated with AZA and measured erythrocyte TPMT enzyme activity. Acute rejection was monitored by routine endomyocardial biopsies. There were 83 WT and 10 heterozygote (HZ) HTX recipients. TPMT activity level was lower in HZ compared with WT (13.1±2.8 vs. 21±4.5 U/ml red blood cell, P<0.001). Despite similar AZA dose, HZ developed severe rejection earlier (P<0.001), and the total rejection score was higher (P=0.02) than WT. AZA was discontinued more frequently in HZ (P=0.01) because of rejection. The incidence of leukopenia was similar between the groups (40 vs. 43%, P=1.0). HTX recipients with TPMT genetic variant alleles who are treated with AZA develop acute rejection earlier, more frequently, and of greater severity. These patients, despite having lower TPMT enzymatic activity, should be monitored carefully for possible increased risk of acute rejection.

At least nine independent natural mutations of the DFR-A gene are responsible for appearance of yellow onions (Allium cepa L.) from red progenitors

Inactivation of the gene (DFR-A) coding for dihydroflavonol 4-reductase (DFR) involved in the anthocyanin biosynthesis pathway results in a yellow bulb color in onion (Allium cepa L.). Three inactive alleles have previously been identified in onion, and in this study we identified three active and six inactive novel DFR-A alleles from extensive analyses of diverse onion germplasms. Of the germplasms analyzed, we identified a yellow mutant containing a 171-bp deletion in the promoter region, which we designated DFR-APD. Critically reduced transcription of this mutant allele and perfect co-segregation with color phenotypes in segregating populations were observed. A second yellow mutant (DFR-A5′DEL) which we identified contained a 518-bp deletion covering exons 1 and 2, which played important roles in DFR function. Both 2- and 4-bp insertions in the coding region leading to the creation of pre-mature stop codons were also identified and designated DFR-AGT and DFR-A2AT, respectively. A 1-bp substitution mutation (DFR-AK48N) which changed a positively charged lysine residue into a neutral asparagine was identified. This lysine residue, a NADPH binding site, was strictly conserved in other species. In addition, insertion of a leucine residue around substrate binding sites and catalytic triad was identified in several yellow accessions and designated DFR-ATTA. Phylogenetic analysis of DFR-A alleles showed that all inactive alleles were independently derived from four different active alleles. In addition, the close relatedness and diversity of DFR-A mutants implied that all these mutations might have occurred after domestication of onions and had probably been maintained by artificial selection.

CASPASE-12 is not a protective factor for rheumatoid arthritis in African-Americans (P4106)

Abstract CASPASE-12 (CASP12) modulates inflammation due to its ability to down-regulate inflammatory cytokines, particularly interleukin-1. Most humans possess the inactive pseudogene allele (CASP12p1) but a functional, non-truncated CASP12 allele [single nucleotide polymorphism #rs497116; A-&amp;gt;G; ter124R] is found in 20% of persons of African descent. CASP12 is a risk factor for sepsis in persons of African descent. Rheumatoid arthritis (RA) is a serious and complex autoimmune disease that involves significant joint inflammation and destruction, and interleukin-1 contributes to the pathogenesis of RA. To determine if CASP12 may be protective against RA due to its anti-inflammatory functions, we genotyped 742 patients with rheumatoid arthritis and 342 controls from the Consortium for Longitudinal Evaluation of African-Americans with Early Rheumatoid Arthritis and the Veterans Affairs Rheumatoid Arthritis registries. Of RA patients, 17 (2.29%) were homozygous for CASP12, 146 (19.7%) were heterozygous, and 579 (78%) were homozygous for CASP12p1. These numbers did not vary significantly from controls. No significant differences were observed for CASP12 genotype on age of disease onset, seropositivity for rheumatoid factor or anti-CCP, baseline joint erosion, joint space narrowing scores, overall disease scores, C-reactive protein levels or erythrocyte sedimentation rates. Thus, CASP12 does not protect against RA in African-American patients, nor mitigates disease parameters.

The Rice Resistance Protein Pair RGA4/RGA5 Recognizes the Magnaporthe oryzae Effectors AVR-Pia and AVR1-CO39 by Direct Binding

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.

A regulator of complex and hybrid N‐glycan synthesis expressed mainly in testis

Complex and hybrid N‐glycan synthesis is initiated by N‐acetylglucosaminyltransferase I (GlcNAcT‐I, MGAT1). MGAT1 is inhibited by membrane‐bound GlcNAcT‐I Inhibitory Protein, GnT1IP‐L. The C‐terminal 39 amino acids of GnT1IP‐L are required to inhibit MGAT1, but not for co‐immunoprecipitation with MGAT1. To determine where GnT1IP‐L is active in the secretory pathway, a series of chimeric proteins were generated. The stem and luminal domain (St+L) of GnT1IP‐L was attached to the cytoplasmic and transmembrane domain (Cy+TM) of proteins known to reside in a specific compartment of the secretory pathway. GnT1IP‐L (St+L) targeted to the endoplasmic reticulum (ER) did not inhibit MGAT1 activity. GnT1IP‐L targeted to the medial Golgi, to the trans Golgi or to the Trans Golgi Network, also did not inhibit MGAT1. However, targeting of GnT1IP‐L to medial Golgi via the (Cy+TM) domain of MGAT1, inhibited MGAT1 activity, and the synthesis of complex N‐glycans. This suggests that GnT1IP‐L and MGAT1 may reside in the kin recognition complex. We are investigating whether GnT1IP‐L inhibits other activities of the kin recognition complex. The GnT1IP gene is highly expressed in spermatocytes but not spermatids, and is regulated during spermatogenesis. We have generated inactive and conditional GnT1IP alleles to investigate the consequences of deleting GnT1IP in spermatogonia and during embryonic development.

Pharmacogenetics of Risperidone: A Systematic Review of the Clinical Effects of CYP2D6 Polymorphisms

To summarize and evaluate the pharmacogenetic literature pertaining to the effects of CYP2D6 polymorphism on clinical outcomes of risperidone therapy. A systematic literature search was performed using the search terms risperidone, pharmacogenetics, cytochrome P-450 enzyme system, cytochrome P-450 CYP2D6, and polymorphism (genetic) in MEDLINE (1946-October 2012), EMBASE (1980-October 2012), PubMed (1947-October 2012), International Pharmaceutical Abstracts (1970-October 2012), and Google Scholar. Identified articles were included if they measured the association between CYP2D6 genetic polymorphisms and clinical outcomes in at least 2 patients taking risperidone. The data elements extracted from these articles consisted of study design, number of subjects, indication for risperidone therapy, CYP2D6 phenotype status, mean daily dose of risperidone, and effects on clinical outcomes. The identified citations consisted of 10 prospective nonrandomized, uncontrolled cohort studies, 1 retrospective cohort study, 1 prospective case-control study, and 1 retrospective case series. Studies were of variable quality and none provided high-quality evidence; they included heterogeneous patient populations with varying clinical diagnoses and drug therapy regimens. Most studies reported nonsignificant trends but were limited by power to detect statistical significance and short trial duration. However, increased risk of adverse effects (including QT interval prolongation) was observed in patients with inactive alleles. While there were trends toward increased adverse effects in poor metabolizers, most outcomes were not significant. As such, routine genotyping should not be used for screening. Future usefulness cannot be ruled out, as many studies had significant limitations that preclude determination of clinical relevance. Adequately powered clinical and epidemiologic studies are warranted to clarify the role of CYP2D6 genotyping in practice.

Development of Functional Markers for Detection of Inactive DFR-A Alleles Responsible for Failure of Anthocyanin Production in Onions (Allium cepa L.)

Inactivation of the gene coding for dihydroflavonol 4-reductase (DFR) is responsible for the color difference between red and yellow onions (Allium cepa L.). Two inactive DFR-A alleles, DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX>, were identified in our previous study. A functional marker was developed on the basis of the premature stop codon that inactivated the DFR-<TEX>$A^{PS}$</TEX> allele. A derived cleaved amplified polymorphic sequences (dCAPS) primer was designed to detect the single nucleotide polymorphism, an A/T transition, which produced the premature stop codon. Digested PCR products clearly distinguished the homozygous and heterozygous red <TEX>$F_2$</TEX> individuals. Meanwhile, to develop a molecular marker for detection of the DFR-<TEX>$A^{DEL}$</TEX> allele in which entire DFR-A gene was deleted, genome walking was performed and approximately 3 kb 5' and 3' flanking sequences of the DFR-<TEX>$A^R$</TEX> coding region were obtained. PCR amplification using multiple primers binding to the extended flanking regions showed that more of the extended region of the DFR-A gene was deleted in the DFR-<TEX>$A^{DEL}$</TEX> allele. A dominant simple PCR marker was developed to identify the DFR-<TEX>$A^{DEL}$</TEX> allele using the dissimilar 3' flanking sequences of the DFR-A gene and homologous DFR-B pseudogene. Distribution of the DFR-<TEX>$A^{PS}$</TEX> and DFR-<TEX>$A^{DEL}$</TEX> alleles in yellow onion cultivars bred in Korea and Japan was surveyed using molecular makers developed in this study. Results showed predominant existence of the DFR-<TEX>$A^{PS}$</TEX> allele in yellow onion cultivars.