Fourth Intron Research Articles

Molecular characterization of a new waxy allele with partial expression in spelt wheat

Starch composition which is dependent on the waxy protein, the enzyme responsible for amylose synthesis in the grain, is an important aspect of the wheat quality. In this report, we describe the characterization of a novel Wx-A1 allele (Wx-A1g formerly known as -Wx-A1a) in Spanish spelt wheat lines which is responsible for a remarkable decline in the concentration of Wx-A1 protein found in the endosperm. Comparison of the DNA sequences in the Wx-A1a and Wx-A1g alleles showed the presence of a 160-bp insertion within the fourth intron in the latter. This insertion had some characteristics of a transposable-like element. RT-PCR analysis showed the presence of normal and aberrant mRNA transcripts in the Wx-A1g lines, indicating that the aberrant transcripts are un-spliced and contained the longer fourth intron. This may be related to the low level of Wx-A1 protein in these lines. In addition, a simple and fast PCR assay was designed for differentiating among different Wx-A1 alleles (a, b, f and g). The mutation described here is not related to either of the Wx-A1 mutations identified previously in common and durum wheats and could help to extend the range of amylose content of wheats.

Analysis of the formation of flower shapes in wild species and cultivars of tree peony using the MADS-box subfamily gene

Tree peony ( Paeonia suffricotisa) cultivars have a unique character compared with wild species; the stamen petalody results in increased whorls of petals and generates different flower forms, which are one of the most important traits for cultivar classification. In order to investigate how petaloid stamens are formed, we obtained the coding sequence (666 bp) and genomic DNA sequence of the PsTM6 genes (belongs to B subfamily of MADS-box gene family) from 23 tree peony samples, Five introns and six exons consisted of the genomic DNA sequence. The analysis of cis-acting regulatory elements in the third and fourth intron indicated that they were highly conserved in all samples. Partial putative amino acids were analyzed and the results suggested that functional differentiation of PsTM6 paralogs apparently affected stamen petalody and flower shape formation due to due to amino acid substitution caused by differences in polarity and electronic charge. Sliding window analysis indicated that the different regions of PsTM6 were subjected to different selection forces, especially in the K domain. This is the first attempt to investigate genetic control of the stamen petalody based on the PsTM6 sequence. This will provide a basis for understanding the evolution of PsTM6 and its the function of in determining stamen morphology of tree peony.

HDLG Associated Protein 1 (DLGAP1) Supports Mpl-Driven Cell Proliferation and Differentiation Through Its Centrosomal Function,

Abstract 3402The Mpl proto-oncogene functions as a basic factor in megakaryocytic development and platelet production as well as contributing to hematopoietic stem cell (HSC) homeostasis and self-renewal. Several mutations in Mpl have been shown to be associated with the myeloproliferative neoplasms. We used retroviral insertional mutagenesis to screen for factors that might give a proliferative and/or survival advantage for cells dependent on Mpl signaling. We developed a retroviral construct expressing a dimerizable form of Mpl that can interrupt genes through insertion, and can activate adjacent genes with its long terminal repeats. We transduced the human leukemia cell line K562 with this construct and blocked the endogenous transforming BCR-ABL kinase using Imatinib. Cells dependent on Mpl signaling were selected by the addition of the dimerizing drug, AP20187. In the absence of Mpl signaling the cells underwent erythroid differentiation and died. Cells that acquired a proliferative advantage and were dependent on Mpl function were expanded. Cloning of retroviral integration sites (RIS) from the selected populations allowed the identification of recurrent RIS that co-localized in the genome. We have identified 668 RIS from 36 independent transductions with a presumed ∼ 1.9 × 106 independent insertions in the initial non-selected, cell population. Among these, 203 RIS represented independent insertion events. Three independent RIS were located in the fourth intron of the Discs large homolog-associated protein 1 (DLGAP1). This protein is a member of the Discs-large/Scribble/Lethal Giant Larvae pathway. DLGAP1, in cooperation with DLG1 and CDC42, has recently been shown to control centrosome positioning and cell polarity in astrocytes (Manneville J-B et al. 2010, JBC, v.191, no 3, pp. 585–598). We investigated the role of DLGAP1 in MPL signaling.Overexpression of full length DLGAP1 had minimal effect on plain K562 cells, but it significantly slowed down the proliferation of K562 cells switched to Mpl signaling. The identified insertion sites would predict the increased expression of an amino-end truncated isoform. When this truncated form was overexpressed in K562 cells and Mpl dependent K562 cells, the cells proliferated at an increased rate. Immunofluorescent studies revealed that the full length DLGAP1 colocalized with major centrosomal markers including gamma-tubulin, PCM1, and APC in K562, HEL, UT7/TPO and Mo7e cell lines. We did not observe colocalization with centrosomes when the truncated isoform of DLGAP1 was overexpressed. Immunoflourescent microscopy revealed that endogenous DLGAP1 in myeloid cell lines was localized in the centriolar satellites, a cellular structure essential for centrosome integrity and function. The immunoflourescent pattern using an anti DLGAP1 antibody showed cell cycle dependent assembly and disassembly. Protein sequence analyses of DLGAP1 indicated consensus sites for presumptive Jak2 phosphorylation as well as consensus sites for SRC tyrosine kinases. To test the possible influence of the two types of kinases on DLGAP1 we treated K562, HEL and UT7/TPO cell lines with an inhibitor of Jak2 (AG490) and independently with an inhibitor of SRC (SU6656). In each case the centrosomal staining of DLGAP1 was diminished with minimal effect on the cytoplasmic fraction of DLGAP1. Interestingly, treatment with SU6656 of the K562 cells switched to Mpl signaling and overexpressing full length GFP-DLGAP1 fusion resulted in a significant increase in the number of polyploid cells. The changes correlated with upregulation of the megakaryocytic marker CD41a and downregulation of the erythroid marker Glycophorin A as assessed by FACS analyses. As centrosomal aberrations are frequent markers of the myeloproliferative neoplasms, we intend to study the role of DLGAP1 in these bone marrow disorders. Disclosures:No relevant conflicts of interest to declare.

A bHLH transcription factor, DvIVS, is involved in regulation of anthocyanin synthesis in dahlia (Dahlia variabilis)

Dahlias (Dahlia variabilis) exhibit a wide range of flower colours because of accumulation of anthocyanin and other flavonoids in their ray florets. Two lateral mutants were used that spontaneously occurred in ‘Michael J’ (MJW) which has yellow ray florets with orange variegation. MJOr, a bud mutant producing completely orange ray florets, accumulates anthocyanins, flavones, and butein, and MJY, another mutant producing completely yellow ray florets, accumulates flavones and butein. Reverse transcription–PCR analysis showed that expression of chalcone synthase 1 (DvCHS1), flavanone 3-hydroxylase (DvF3H), dihydroflavonol 4-reductase (DvDFR), anthocyanidin synthase (DvANS), and DvIVS encoding a basic helix–loop–helix transcription factor were suppressed, whereas that of chalcone isomerase (DvCHI) and DvCHS2, another CHS with 69% nucleotide identity with DvCHS1, was not suppressed in the yellow ray florets of MJY. A 5.4 kb CACTA superfamily transposable element, transposable element of Dahlia variabilis 1 (Tdv1), was found in the fourth intron of the DvIVS gene of MJW and MJY, and footprints of Tdv1 were detected in the variegated flowers of MJW. It is shown that only one type of DvIVS gene was expressed in MJOr, whereas these plants are likely to have three types of the DvIVS gene. On the basis of these results, the mechanism regulating the formation of orange and yellow ray florets in dahlia is discussed.

Dissection of Splicing Regulation at an Endogenous Locus by Zinc-Finger Nuclease-Mediated Gene Editing

Sequences governing RNA splicing are difficult to study in situ due to the great difficulty of traditional targeted mutagenesis. Zinc-finger nuclease (ZFN) technology allows for the rapid and efficient introduction of site-specific mutations into mammalian chromosomes. Using a ZFN pair along with a donor plasmid to manipulate the outcomes of DNA repair, we introduced several discrete, targeted mutations into the fourth intron of the endogenous BAX gene in Chinese hamster ovary cells. Putative lariat branch points, the polypyrimidine tract, and the splice acceptor site were targeted. We recovered numerous otherwise isogenic clones carrying the intended mutations and analyzed the effect of each on BAX pre-mRNA splicing. Mutation of one of three possible branch points, the polypyrimidine tract, and the splice acceptor site all caused exclusion of exon five from BAX mRNA. Interestingly, these exon-skipping mutations allowed usage of cryptic splice acceptor sites within intron four. These data demonstrate that ZFN-mediated gene editing is a highly effective tool for dissection of pre-mRNA splicing regulatory sequences in their endogenous context.

Possible Origin of Two Variants of a Carnation 1-Aminocyclopropane-1-carboxylate Synthase Gene, DcACS1a and DcACS1b, as Suggested by Intron Structure in Homologous Genes in Dianthus Species

The intron structures of two variants of 1-aminocyclopropane-1-carboxylate synthase (ACS) genes (DcACS1a and DcACS1b) in carnation (Dianthus caryophyllus) and genes homologous to them (ACS1 homologous genes) in other 10 Dianthus species (16 strains in total) were studied by comparing the sizes of the PCR amplificates and nucleotide sequence of the introns. All 16 sequenced homologous ACS1 genes, including DcACS1 genes themselves, had five exons and four introns. The exons had similar nucleotide sequences and consequently similar deduced amino-acid sequences. The sizes of three introns (intron-1, -2, -3) were variable among the homologous genes, whereas that of the fourth intron (intron-4) was almost identical. The variation in introns was probably caused by the insertion (and deletion) of nucleotide fragments of given lengths. Interestingly, the 3'-UTR of DcACS1a was different from that of DcACS1b, and the latter was similar to other 14 ACS1 homologous genes. Moreover, the length of Thr repeat in the C-terminal region was long in DcACS1a protein but short in DcACS1b protein, and the latter resembled ACS1 homologous proteins in other Dianthus species. The present findings suggest that (1) the variation in intron structure between two variants of carnation DcACS1 is reminiscent of the variation that occurred universally in Dianthus species, (2) DcACS1a is probably a gene intrinsic to carnation, and (3) DcACS1b was acquired from another, as yet unknown, Dianthus species, in the course of breeding modern carnation cultivars.

Molecular genetic analysis of autosomal dominant late-onset cataract in a Chinese Family

Congenital cataract is a highly heterogeneous disorder at both the genetic and the clinical-phenotypic levels. A unique cataract was observed in a 4-generation Chinese family, which was characterized by autosomal dominant inheritance and late-onset. Mutations in the 13 known genes (CRYAA, CRYAB, CRYBB1, CRYBB2, CRYGC, CRYBA1/A3, CRYGD, Connexin50, Connexin46, intrinsic membrane protein LIM2, cytoskeletal protein BFSP2, the major intrinsic protein-MIP and the heat shock factor HSF4) have previously been demonstrated to be the frequent reason for isolated congenital cataracts, but the exact molecular basis and underlying mechanisms of congenital cataract still remain unclear. This study was designed to find whether these 13 genes developed any mutation in the family members and to identify the disease-causing gene. Polymerase chain reaction (PCR) and direct DNA sequence analysis were carried out to detect the 13 genes. The results showed that no mutation causing amino acid alternations was found in these potential candidate genes among all patients in the family, and only several single-nucleotide polymorphisms (SNPs) were identified. A transitional mutation in the fourth intron of CRYBB2 and some silent mutations in the first exon of BFSP2 and CRYGD were found in the cataract family, but further study showed that these mutations could also be found in normal controls. It was concluded that some unidentified genes may underlie the occurrence of late-onset cataract in this family. A genome-wide screening will be carried out in the next study.

Mastl Kinase Gene Disruption Causes Embryonic Lethality.

AbstractAbstract 1567Microtubule-associated serine/threonine like kinase (MASTL kinase) was originally identified by genetic linkage as the probable mutant gene responsible for the platelet phenotype in a family with autosomal-dominant thrombocytopenia. MASTL kinase is the mammalian ortholog of Drosophila Greatwall kinase, which is required for cell cycle regulation. Affected members of the thrombocytopenic family carried a mutant MASTL kinase allele with a single nucleotide change that converted a glutamic acid codon to one specifying aspartic acid at amino acid 167 of the protein; no unaffected members of the family carried the mutant allele. Affected family members had a 60% reduction in the numbers of circulating platelets and megakaryocytes that were less mature and less polyploid than normal megakaryocytes. We have sought to determine how MASTL kinase affects platelet production and circulation by generating a strain of mice deficient in MASTL kinase. For this, we obtained embryonic stem (ES) cells containing a gene trap mutation within the murine Mastl gene from the Mutant Mouse Regional Resource Center of the National Center for Research Resources (MMRRC, University of California, San Diego). The mutation was generated by an insertional disruption, and the resulting mutant allele carries a beta–galactosidase/neomycin fusion expression cassette within the fourth intron of the Mastl kinase gene. Chimeric animals were bred and their offspring were genotyped by Southern blotting, confirming germ-line transmission. F1 heterozygote offspring were produced and appear developmentally normal and healthy, with platelet counts indistinguishable from those of their wild-type littermates. We have yet to observe a single weaned animal that is homozygous for the mutant allele in the F1 mice, indicating that MASTL kinase deficiency results in embryonic lethality. We observed decidual swellings at 13.5 days post coitus (dpc), which contained no traces of either embryos or yolk sac membranes. While this has made it impossible to genotype these malformed embryos, it does indicate that the embryos are viable to 3.5–5.5 dpc and are able to implant within the uterine horn. This unexpected phenotype demonstrates a significant and previously unknown role for Mastl kinase in embryonic development. We hypothesize that Mastl kinase may be involved in regulating cell differentiation during embryogenesis. In terms of role of MASTL kinase in familial thrombocytopenia, the mutant animals have allowed us to conclude that the thrombocytopenic syndrome is very unlikely to be the result of haploinsufficiency of MASTL kinase because heterozygous animals have normal platelet counts. Because of the dominant nature of the syndrome, this suggests that the mechanism involves either gain-of-function of the mutant allele or a dominant-negative inhibition of MASTL function specific to the megakaryocyte, two possibilities we are examining. Disclosures:No relevant conflicts of interest to declare.

Novel SNPs in the ATP1B2 gene and their associations with milk yield, milk composition and heat-resistance traits in Chinese Holstein cows

Genetic association analysis was applied to examine the effect of the Na(+)/K(+)-ATPase beta 2 subunit (ATP1B2) gene on rectal temperature, milk traits, K(+) levels and Na(+)/K(+)-ATPase (NKA) activity in the red blood cells of 1001 Chinese Holstein cows under normal and heat-stress conditions. We detected two novel single nucleotide polymorphisms, G2258A and C2833T, in the second and fourth introns, respectively, of ATP1B2. G2258A significantly affected milk fat content (P < 0.05) and 305-day milk yield (P < 0.01), but not milk protein content. C2833T significantly affected milk protein content (P < 0.01) and 305-day milk yield (P < 0.05), but not milk fat content. Calculated gene substitution effects suggested that A to G substitution in G2258A, and T to C substitution in C2833T, positively affected milk fat content, 305-day milk yield and somatic cell score, but negatively affected milk protein content. We also detected significant variation in milk fat content, milk protein content, 305-day milk yield and somatic cell scores (P < 0.05 or P < 0.01) among the nine ATP1B2 haplotypes. Under heat-stress, the C2833T polymorphism was significantly related to rectal temperature (P < 0.01), red blood cell K(+) levels, NKA activity and milk yield (P < 0.05). Cows with the TT genotype showed the desirable characteristics of low rectal temperature and red blood cell K(+), low decline rate in milk yield and red blood cell NKA activity. This study suggests that the ATP1B2 single nucleotide polymorphism C2833T is a genetic marker of heat-resistance traits in Chinese Holstein cows.

Regulation of Adipocyte Differentiation by Activation of Serotonin (5-HT) Receptors 5-HT2AR and 5-HT2CR and Involvement of MicroRNA-448-Mediated Repression of KLF5

Retrovirus insertion-mediated random mutagenesis was applied in 3T3-L1 preadipocyte cells to better understand the molecular basis of obesity (the expansion of individual adipocytes). We found that tryptophan hydroxylase-1, a rate-limiting enzyme for the synthesis of serotonin (5-HT), is expressed in adipocytes and is required for their differentiation. A 5-HT type 2A receptor (5-HT(2A)R) antagonist, ketanserin, and a 5-HT(2c)R antagonist, SB-242084, inhibited adipocyte differentiation. Because 5-HT(2c)R mRNA levels are up-regulated during adipocyte differentiation and micro-RNA (miR)-448 is located in the fourth intron of Htr2c, we also studied the role of miR-448 in 3T3-L1 cells. Through a bioinformatics approach, Krüppel-like factor 5 (KLF5) was identified as a potential target of miR-448. Using a luciferase reporter assay, we confirmed that miR-448 targets the Klf5 3'-intranslated region. Overexpression of miR-448 reduced the expression of Klf5 and adipocyte differentiation, which was confirmed by the reduced expression of adipogenic genes and triglyceride accumulation. To examine the loss of miR-448 function, we constructed a decoy gene that had tandem complementary sequences for miR-448 in the 3'-untranslated region of a luciferase gene under the control of a cytomegalovirus promoter. When the miR-448 decoy gene was introduced into 3T3-L1 preadipocytes, KLF5 was up-regulated and triglyceride concentration was increased. In this study, we identified the regulation of adipocyte differentiation by 5-HT, 5-HT(2A)R, and 5-HT(2C)R. miR-448-mediated repression of KLF5 was identified as a negative regulator for adipocyte differentiation.

Alternative splicing of the maize Ac transposase transcript in transgenic sugar beet (Beta vulgaris L.)

The maize Activator/Dissociation (Ac/Ds) transposable element system was introduced into sugar beet. The autonomous Ac and non-autonomous Ds element excise from the T-DNA vector and integrate at novel positions in the sugar beet genome. Ac and Ds excisions generate footprints in the donor T-DNA that support the hairpin model for transposon excision. Two complete integration events into genomic sugar beet DNA were obtained by IPCR. Integration of Ac leads to an eight bp duplication, while integration of Ds in a homologue of a sugar beet flowering locus gene did not induce a duplication. The molecular structure of the target site indicates Ds integration into a double strand break. Analyses of transposase transcription using RT–PCR revealed low amounts of alternatively spliced mRNAs. The fourth intron of the transposase was found to be partially misspliced. Four different splice products were identified. In addition, the second and third exon were found to harbour two and three novel introns, respectively. These utilize each the same splice donor but several alternative splice acceptor sites. Using the SplicePredictor online tool, one of the two introns within exon two is predicted to be efficiently spliced in maize. Most interestingly, splicing of this intron together with the four major introns of Ac would generate a transposase that lacks the DNA binding domain and two of its three nuclear localization signals, but still harbours the dimerization domain.

Distinct enhancers at the Pax3 locus can function redundantly to regulate neural tube and neural crest expressions

Pax3 is a transcription factor expressed in somitic mesoderm, dorsal neural tube and pre-migratory neural crest during embryonic development. We have previously identified cis-acting enhancer elements within the proximal upstream genomic region of Pax3 that are sufficient to direct functional expression of Pax3 in neural crest. These elements direct expression of a reporter gene to pre-migratory neural crest in transgenic mice, and transgenic expression of a Pax3 cDNA using these elements is sufficient to rescue neural crest development in mice otherwise lacking endogenous Pax3. We show here that deletion of these enhancer sequences by homologous recombination is insufficient to abrogate neural crest expression of Pax3 and results in viable mice. We identify a distinct enhancer in the fourth intron that is also capable of mediating neural crest expression in transgenic mice and zebrafish. Our analysis suggests the existence of functionally redundant neural crest enhancer modules for Pax3.

Identification of Genes Cooperating with Mpl Signaling Using Retroviral Insertional Mutagenesis.

Abstract 4558The thrombopoietin receptor (Mpl) regulates the proliferation of hematopoietic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. Mutations in the Mpl gene have been shown to be associated with myeloproliferative neoplasms (MPN). We attempted to identify genes cooperating with Mpl signaling that could provide cell survival and proliferative advantage using MSCV based retroviral insertional mutagenesis coupled with an inducible Mpl signaling system. The retroviral Mpl vectors used in our screening system were based on the well-described retroviral vector MGIFM. That vector carried a bacterial shuttle plasmid coding for the Neo gene and a bacterial origin of replication in its 3′ untranslated region, and also coding sequences of a drug dependent, dimerizable, fusion protein containing the cytoplasmic domain of Mpl. This vector can interrupt gene structure through insertion, and its intact long terminal repeats can activate adjacent genes. The shuttle plasmid allows nonbiased recovery of proviral genomic integration sites (RIS). The retroviral Mpl vectors were used to transduce the human leukemia cell line K562. Selection for Mpl signaling dependent cells required blocking of the endogenous transforming Bcr-Abl kinase with imatinib and supplying the dimerizer drug, AP20187. Similar selection approach was applied in another leukemia cell line, a cytokine dependent UT7 cells, after transduction depriving them of exogenous cytokines and supplying the AP20187. In multiple independent transductions only a few percent of the transduced cells survived in the presence of AP20187, as assessed by the GFP expression, which was co-expressed with the Mpl fusion gene through an internal ribosome entry site. The surviving cells in long term culture were dependent on Mpl signaling and were sensitive to a Jak2 inhibitor, AG490. A wide variation in the mean fluorescent intensity of the surviving cells (from 37.5 to 238.8) in independent transductions indicated that a high level of Mpl expression was not the critical factor of selection. Transduction conditions were designed so that each experiment started with 30,000 independent insertion events from which selection of an Mpl-dependent cell population could be derived. Thirty six independent transductions have been performed with a presumed ∼ 1.89 × 106 independent insertions in the initial non-selected, cell population. After selection, 668 retroviral insertion sites (RIS) have been recovered that represent 203 independent insertion events. Within each independent transduction, a small number of RISs were identified suggesting selection of insertion events that result in a proliferative advantage. This was not observed in an independent experiment in K562 cells, in which the cells were not switched to Mpl dependent growth. Insertion events that occurred in close proximity are considered to indicate a potential cooperating mutation that results in a proliferative and/or survival advantage to the cell. Insertional events in several instances fell in regions of known frequent chromosomal aberrations in MPN. The distribution of insertions appears to be non-random. Many sites are adjacent to genes that have been identified as regulators of apoptosis, proliferation and myeloid development (data not shown). We are validating this conclusion by performing a parallel screening of RIS in multiple (40) populations of independent transductions of the K562 and UT7 cells transduced with retrovirus/Mpl vector without selection on AP20187. This screening will provide sufficient data to statistically compare the outcome of the two sets of data, AP20187- selected versus non selected cell populations. To date, we have a number of candidate genes. The Discs large homolog-associated protein 1 (DLGAP1) gene is implicated secondary to three independent insertion events at 18p11.3. All insertions are in the fourth intron. This protein interacts with a known tumor suppressor and is being investigated as a possible novel hematopoietic regulator. Disclosures:No relevant conflicts of interest to declare.

Molecular Cloning and Linkage Mapping of Cryptochrome Multigene Family in Soybean

The cryptochromes are a family of blue light photoreceptors that play important roles in the controls of plant development. Seven full‐length cryptochrome cDNAs (GmCRY1a, GmCRY1b, GmCRY1c, GmCRY1d, GmCRY2a, GmCRY2b, and GmCRY2c) were isolated by cDNA library screening and reverse transcriptase–polymerase chain reaction from ‘Williams’ soybean [Glycine max (L.) Merr.], indicating that soybean cryptochrome genes comprise a multigene family. They had homologies ranging from 60 to 89% with CRY1 and CRY2 genes of Arabidopsis and pea (Pisum sativum L.). Two types of transcripts were isolated in GmCRY1b, GmCRY1c, GmCRY1d, and GmCRY2a. One type was derived from four exons, whereas the other type was derived from five exons. Occurrence of the former transcript could be explained by retention of the fourth intron, suggesting existence of alternative splicing. Gene sequences were compared between a soybean line, Tokei 780, and an accession of soybean wild relative, Glycine soja, Hidaka 4. Based on a 10‐bp indel, an amplicon length polymorphism (ALP) marker was designed for mapping of GmCRY2b. For mapping of the other cryptochrome genes, derived cleaved amplified polymorphic sequence (dCAPS) markers were constructed. The cryptochrome genes were individually assigned to different molecular linkage groups (MLG) (GmCRY1a: MLG C1; GmCRY1b: C2; GmCRY1c: B2; GmCRY1d: F; GmCRT2a: O; GmCRY2b: D1b; GmCRY2c: I). The distribution of cryptochrome genes that was deduced from the soybean genome database was consistent with mapping results.

Molecular identification ofTrichodermaspecies associated withPleurotus ostreatusand natural substrates of the oyster mushroom

Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1alpha gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.

Polycystic Kidney Disease in the Medaka (Oryzias latipes) pc Mutant Caused by a Mutation in the Gli-Similar3 (glis3) Gene

Polycystic kidney disease (PKD) is a common hereditary disease in humans. Recent studies have shown an increasing number of ciliary genes that are involved in the pathogenesis of PKD. In this study, the Gli-similar3 (glis3) gene was identified as the causal gene of the medaka pc mutant, a model of PKD. In the pc mutant, a transposon was found to be inserted into the fourth intron of the pc/glis3 gene, causing aberrant splicing of the pc/glis3 mRNA and thus a putatively truncated protein with a defective zinc finger domain. pc/glis3 mRNA is expressed in the epithelial cells of the renal tubules and ducts of the pronephros and mesonephros, and also in the pancreas. Antisense oligonucleotide-mediated knockdown of pc/glis3 resulted in cyst formation in the pronephric tubules of medaka fry. Although three other glis family members, glis1a, glis1b and glis2, were found in the medaka genome, none were expressed in the embryonic or larval kidney. In the pc mutant, the urine flow rate in the pronephros was significantly reduced, which was considered to be a direct cause of renal cyst formation. The cilia on the surface of the renal tubular epithelium were significantly shorter in the pc mutant than in wild-type, suggesting that shortened cilia resulted in a decrease in driving force and, in turn, a reduction in urine flow rate. Most importantly, EGFP-tagged pc/glis3 protein localized in primary cilia as well as in the nucleus when expressed in mouse renal epithelial cells, indicating a strong connection between pc/glis3 and ciliary function. Unlike human patients with GLIS3 mutations, the medaka pc mutant shows none of the symptoms of a pancreatic phenotype, such as impaired insulin expression and/or diabetes, suggesting that the pc mutant may be suitable for use as a kidney-specific model for human GLIS3 patients.

Ccm1p/Ygr150cp, a pentatricopeptide repeat protein, is essential to remove the fourth intron of both COB and COX1 pre-mRNAs in Saccharomyces cerevisiae

YGR150C gene product (Ygr150cp) is one of the three mitochondrially located Saccharomyces cerevisiae proteins with pentatricopeptide repeat (PPR) motifs. Ygr150cp is essential for mitochondrial functionality but its molecular targets are still unknown. This study was undertaken to define the role of Ygr150cp in mitochondria biogenesis. Repression of Ygr150cp expression in complemented mutants prevented their use of glycerol or lactate, but allowed limited growth on ethanol-containing medium. RNA hybridization studies showed that Deltaygr150c meiotic segregants produced COB and COX1 transcripts but failed to process them into the mature forms. Detailed RT-PCR assays revealed that Deltaygr150c specifically failed to remove the fourth intron of both COB and COX1 pre-mRNAs while all other group I introns were excised. Expression of Ygr150cp mutants without any of the PPR motifs did not complement the growth phenotype. Accordingly, we designate YGR150C as CCM1 (COB and COX1 mRNA maturation). This report provides the first evidence of PPR protein involvement in the specific removal of group I introns in mitochondria of S. cerevisiae.

Association of the tyrosine hydroxylase (TH) gene polymorphism and activity trait measured by questionnaire and behavioral tests in German shepherd dogs

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of dopamine and norepinephrine, which can be converted to norepinephrine and epinephrine. Dopamine plays a role in activity in mice, humans, and in dogs. The polymorphism in the TH gene in dogs was recently published, yet the association between behavior and genotype had not been studied. In this study, 131 German shepherd dogs were genotyped for the length polymorphism of a 36 bp part in the fourth intron of the TH gene. The animals were kept as pets by Hungarian families, and owners were asked to fill out a questionnaire about their dogs’ activity-impulsivity and attention skills (Dog ADHD-RS questionnaire). Eighty-one of the 131 dogs and owners participated in direct behavioral observations in 3 different situations: (1) ‘‘friendly approach’’: a person who was unfamiliar to the dog approached the animal in a friendly way and petted the dog; (2) ‘‘attention’’: a piece of food or a ball was held in front of the dog so that the dog could almost reach the object; and (3) ‘‘lying down’’: owners asked their dogs to lie down on their sides and stay in this position. Allele-wise comparisons were performed both for behaviors evaluated by owners and for those based on direct observation. Two different alleles were identified: allele 1 (14.9% of the 262 alleles analyzed) contained 1 copy of the 36 bp sequence, whereas 2 of them were included in allele 2 (85.1%). The prevalence of the different genotypes in the study population fit well with the Hardy-Weinberg equilibrium. According to the questionnaire scores given by the owners, the 2 alleles differed in activity-impulsivity phenotype at the behavioral level (Mann-Whitney U test, U 5 3106, z 5 22.848, P 5 0.004) but not in attention deficit (U 5 4245, z 5 20.238, P 5 0.812). The shorter allele was associated with higher activity-impulsivity scores. In the ‘‘friendly approach’’ test from direct observations, the shorter allele was associated with a higher probability to initiate contact with the approaching person instead of waiting passively for the stranger to initiate contact (c5 6.990, df5 1, P5 0.008). During the ‘‘attention’’ test, activity of the 2 alleles was found to be the same (U 5 1665, z 5 -0.804, P 5 0.421), but the number of gaze alternations between object-owner-other directions differed (U 5 1403.5, z 5 -1.999, P 5 0.046). The shorter allele was associated with a higher number of gaze alternations. In the ‘‘lying-down’’ test, a significant difference was found between the 2 alleles; the shorter allele corresponded to a higher lying-down latency (U 5 1180, z 5 22.155, P 5 0.031). In summary, the results based on the opinion of the owner and the direct observational situations were in concordance. The shorter allele of the tyrosine hydroxylase gene was associated with more active behavior in Hungarian German shepherd dogs.

Notch signalling in the paraxial mesoderm is most sensitive to reduced Pofut1levels during early mouse development

BackgroundThe evolutionarily conserved Notch signalling pathway regulates multiple developmental processes in a wide variety of organisms. One critical posttranslational modification of Notch for its function in vivo is the addition of O-linked fucose residues by protein O-fucosyltransferase 1 (POFUT1). In addition, POFUT1 acts as a chaperone and is required for Notch trafficking. Mouse embryos lacking POFUT1 function die with a phenotype indicative of global inactivation of Notch signalling. O-linked fucose residues on Notch can serve as substrates for further sugar modification by Fringe (FNG) proteins. Notch modification by Fringe differently affects the ability of ligands to activate Notch receptors in a context-dependent manner indicating a complex modulation of Notch activity by differential glycosylation. Whether the context-dependent effects of Notch receptor glycosylation by FNG reflect different requirements of distinct developmental processes for O-fucosylation by POFUT1 is unclear.ResultsWe have identified and characterized a spontaneous mutation in the mouse Pofut1 gene, referred to as "compact axial skeleton" (cax). Cax carries an insertion of an intracisternal A particle retrotransposon into the fourth intron of the Pofut1 gene and represents a hypomorphic Pofut1 allele that reduces transcription and leads to reduced Notch signalling. Cax mutant embryos have somites of variable size, showed partly abnormal Lfng expression and, consistently defective anterior-posterior somite patterning and axial skeleton development but had virtually no defects in several other Notch-regulated early developmental processes outside the paraxial mesoderm that we analyzed.ConclusionNotch-dependent processes apparently differ with respect to their requirement for levels of POFUT1. Normal Lfng expression and anterior-posterior somite patterning is highly sensitive to reduced POFUT1 levels in early mammalian embryos, whereas other early Notch-dependent processes such as establishment of left-right asymmetry or neurogenesis are not. Thus, it appears that in the presomitic mesoderm (PSM) Notch signalling is particularly sensitive to POFUT1 levels. Reduced POFUT1 levels might affect Notch trafficking or overall O-fucosylation. Alternatively, reduced O-fucosylation might preferentially affect sites that are substrates for LFNG and thus important for somite formation and patterning.

A Mucin-type O-Glycosyltransferase Modulates Cell Adhesion during Drosophila Development

Cell-cell and cell-matrix adhesion are crucial during many stages of eukaryotic development. Here, we provide the first example that mucin-type O-linked glycosylation is involved in a developmentally regulated cell adhesion event in Drosophila melanogaster. Mutations in one member of the evolutionarily conserved family of enzymes that initiates O-linked glycosylation alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member (pgant35A) did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of larval wing imaginal discs, which was restored with wild type pgant3 expression, suggesting that reduced glycosylation of basal proteins is responsible for disruption of adhesion in the adult wing blade. Glycosylation reactions demonstrated that PGANT3 glycosylates certain extracellular matrix (ECM) proteins. Immunoprecipitation experiments revealed that PGANT3 glycosylates tiggrin, an ECM protein known to bind integrin. We propose that this glycosyltransferase is uniquely responsible for glycosylating tiggrin in the wing disc, thus modulating proper cell adhesion through integrin-ECM interactions. This study provides the first evidence for the role of O-glycosylation in a developmentally regulated, integrin-mediated, cell adhesion event and reveals a novel player in wing blade formation during Drosophila development.