Assessment of genomic imprinting of <Emphasis Type="Italic">PPP1R9A</Emphasis>, <Emphasis Type="Italic">NAP1L5</Emphasis> and <Emphasis Type="Italic">PEG3</Emphasis> in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kiduey, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it’s expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

Molecular characterization and association analysis of porcine adipose triglyceride lipase (<Emphasis Type="Italic">PNPLA2</Emphasis>) gene

The adipose triglyceride lipase (PNPLA2, also known as ATGL) is a novel triacylglycerol (TG) lipase which specifically removes the first fatty acid from the triglyceride molecule generating free fatty acid and diglyceride (DG) in mammalian cells. Here we describe the molecular characterization of the porcine ATGL gene. The full-length cDNA sequence contains a 1,461 bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90. The porcine ATGL protein shares high identity with other mammalian ATGL. The ATGL gene contains 9 coding exons, spans approximately 6 kb. The porcine ATGL mRNA was expressed predominantly in backfat, mildly in muscle, small intestine and heart, and almost absent in liver, spleen, lung, stomach, kidney and ovary. Statistical analysis showed the ATGL gene polymorphism (G/A³⁹²) was different between Chinese indigenous and introduced commercial western pig breeds, and was highly associated with almost all the fat deposition and carcass traits, including subcutaneous fat thickness, viscera adipose tissue, lean percentage, loin eye traits and even rib numbers.     

Molecular characterization,chromosomal localization and association analysis with back-fat thickness of porcine <Emphasis Type="Italic">LPIN2</Emphasis> and <Emphasis Type="Italic">LPIN3</Emphasis>

The products of mammalian LPIN2 and LPIN3 are phosphatidate phosphatase type 1 enzymes, which play an important role in the de novo biosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolamine. In this study, we obtained a 2,985-bp cDNA sequence of porcine LPIN2, which contains a 2,676-bp open reading frame flanked by an 11-bp 5′UTR and a 298-bp 3′UTR, and a 2,843-bp cDNA sequence of porcine LPIN3, which contains a 111-bp 5′UTR, a 2,580-bp open reading frame and a 152-bp 3′UTR. RT-PCR analysis showed that both LPIN2 and LPIN3 mRNA were ubiquitously expressed with a very high level in liver. By using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel, porcine LPIN2 and LPIN3 were assigned to 6q24-(1/2)q31 and 17(1/2)q21-q23, respectively. One T2193C single nucleotide polymorphism in LPIN2 was identified and was detected by Hin6I PCR-RFLP. Association analysis showed that different genotypes of LPIN2 were associated with back-fat thickness between the 6th and 7th ribs (P < 0.01).     

The porcine <Emphasis Type="Italic">ANG</Emphasis>, <Emphasis Type="Italic">RNASE1</Emphasis> and <Emphasis Type="Italic">RNASE6</Emphasis> genes: molecular cloning,polymorphism detection and the association with haematological parameters

Angiogenin (ANG) [also known as ribonuclease, RNase A family, 5 (RNASE5)], ribonuclease, RNase A family, 1 (pancreatic) (RNASE1) and ribonuclease, RNase A family, k6 (RNASE6) are three members of the RNase A superfamily. It has been suggested that these three genes play important roles in host defense. In this study, we obtained the whole open reading frame (ORF) of each gene and found the deduced proteins contain some similar structures harboring a catalytic triad and an invariant “CKXXNTF” signature motif. One single nucleotide polymorphism (SNP) was detected in each gene (g. 149G>T polymorphism in the porcine ANG gene, which resulted in an amino acid change from glycine to valine, g. 296A>G polymorphism in the porcine RNASE1 gene and g. 389C>T polymorphism in the porcine RNASE6 gene). Association analyses revealed the significant associations (P < 0.05) between the porcine ANG g. 149G>T polymorphism and mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean platelet volume (MPV) and platelet-large cell ratio (P-LCR) measured on 0-day-old pigs and MCV measured at 32 days after birth. The porcine RNASE6 g. 389C>T polymorphism was significantly associated (P < 0.05) with MCV, MCH and neutrophil percentage (NEI %) measured on 0-day-old pigs, respectively. Our current findings, if confirmed by other studies, might shed some light on the roles of the investigated genes in host defense.     

Porcine <Emphasis Type="Italic">CSRP3</Emphasis>: polymorphism and association analyses with meat quality traits and comparative analyses with <Emphasis Type="Italic">CSRP1</Emphasis> and <Emphasis Type="Italic">CSRP2</Emphasis>

CRP3 is the muscle-specific form of the cysteine and glycine-rich protein family and plays an important role in myofiber differentiation. Here we isolated and characterized its coding gene CSRP3 from porcine muscle. Phylogenic analyses demonstrated that CSRP3 diverged first and is distinguished from two other members, CSRP1 and CSRP2. CSRP3 mRNA was up-regulated during the development of porcine embryonic skeletal muscle, indicating its potential importance in muscle growth. Genetic variant analyses detected multiple variations in an approximately 400 bp region covering exon 4 and its downstream intron, and two haplotypes were identified by sequencing. One of synonymous substitutions C1924T was used for linkage and association analyses. It was revealed that the substitution of C1924T had significant associations with firmness (P < 0.01), Lab Loin pH, Off Flavor Score and Water Holding Capacity (P < 0.05), and a suggestive effect (P < 0.1) on Flavor Score and Average Glycolytic Potential in a Berkshire × Yorkshire F2 population. The association analyses results agreed with the gene’s localization to a QTL region for meat quality traits on porcine chromosome 2p14-17 demonstrated by both linkage mapping and RH mapping. These results provide fundamental evidence for CSRP3 as a functional candidate gene affecting pig meat quality.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

<Emphasis Type="Italic">Xanthium italicum</Emphasis>, <Emphasis Type="Italic">Xanthium strumarium</Emphasis> and <Emphasis Type="Italic">Arctium lappa</Emphasis> as new hosts for <Emphasis Type="Italic">Diaporthe helianthi</Emphasis>

Sunflower (Helianthus annuus) stem canker caused by Diaporthe helianthi is one of the most important sunflower diseases in Croatia. Until recently, sunflower was the only known host for D. helianthi. In our research carried out in the area of Eastern Croatia, isolates of Diaporthe/Phomospis were collected from Xanthium italicum, X. strumarium and Arctium lappa. Using morphological, cultural and molecular ITS rDNA data, isolates from these weeds were identified as D. helianthi. The following isolates were used in the pathogenicity test: one isolate originated from sunflower (Su5/04), three from X. italicum (Xa2, Xa3 and Xa5), two from X. strumarium (Xa9 and Xa12), one from Xanthium sp. (Xa13) and one from A. lappa (Ar3). According to the results, it was determined that isolate Xa5 (originated from X. italicum) was the most pathogenic to sunflower stems. The average length of the lesion was 11.3 cm. The lowest level of pathogenicity was found in Xa9 (isolated from X. strumarium). The length of the lesion was 0.1 cm.     

Association between <Emphasis Type="Italic">PNPLA3</Emphasis> rs738409 polymorphism and hepatocellular carcinoma risk: an updated meta-analysis

Hepatocellular carcinoma (HCC) is a common malignant tumor and the leading cause of cancer-related death worldwide. The protein encoded by patatin-like phospholipase domain-containing protein 3 (PNPLA3) plays important roles in liver fatty metabolism. Recent studies have indicated associations of PNPLA3 rs738409 with various liver diseases, including HCC. This meta-analysis was performed to investigate and update the association between rs738409 polymorphism and the risk of HCC, and to test the association between rs738409 and HCC specifically in patients within chronic hepatitis B and/or C infection, alcoholic liver disease, or other diseases. Studies were searched from the literature database up to March 31, 2016. The meta-analysis was conducted based on statement of preferred reporting items for systematic reviews and meta-analyses. Pooled odds ratios (OR) and 95 % confidence intervals (CI) were estimated the strength of associations between rs738409 polymorphism and HCC risk. Fifteen published studies, consisting of 2264 HCC patients (case) and 5802 without HCC individuals (control), were included in the present study. Meta-analysis revealed that rs738409 polymorphism contributed to HCC risk under the allelic effect model (C vs. G: OR 1.73; 95 % CI   1.53–1.96), the dominant effect model (CC vs. CG+GG: OR 1.61; 95 % CI 1.44–1.81), and the recessive effect model (CC+CG vs. GG: OR 2.66; 95 % CI 2.28–3.11). Furthermore, the effect of rs738409 G allele on liver oncogenesis was higher in alcoholic liver disease (OR 2.55), compared to chronic hepatitis C/B (OR 1.32) and other diseases (OR 2.27). The results suggested that rs738409 polymorphism was significantly associated with HCC risk and it could be used as one risk factor for HCC.     

Probiotic <Emphasis Type="Italic">Lactobacillus</Emphasis> strains: <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Purification and properties of aminoaldehyde dehydrogenase from <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5′-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS–PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a pI value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that Avena AMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K _m values for APAL, ABAL, and GBAL were 1.5×10^–6, 2.2×10^–6, and 1.3×10^–5 M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH. Electronic Publication     

Leaf-rust resistance in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.). 2. Genetic analysis and mapping of resistance genes <Emphasis Type="Italic">Pr3</Emphasis>, <Emphasis Type="Italic">Pr4</Emphasis>, and <Emphasis Type="Italic">Pr5</Emphasis>

Three dominant resistance genes, Pr3, Pr4, and Pr5, were identified by genetic analysis of resistance to leaf rust in rye (Puccinia recondita f. sp. secalis). Each of the three genes confers resistance to a broad scale of single-pustule isolates (SPIs), but differences could be observed for specific Pr gene/SPI combinations. Resistance conferred by the three genes was effective in both detached-leaf tests carried out on seedlings and in field tests of adult plants. Molecular marker analysis mapped Pr3 to the centromeric region of rye chromosome arm 1RS, whereas Pr4 and Pr5 were assigned to the centromeric region of 1RL. Chromosomal localization and reaction patterns to specific SPIs provide evidence that the three Pr genes represent distinct and novel leaf-rust resistance genes in rye. The contributions of these genes to resistance breeding in rye and wheat are discussed.The authors dedicate this paper to Prof. Dr. H.H. Geiger, University of Hohenheim, on the occasion of his 65th birthday.An erratum to this article can be found at     

<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

Regioselective glucosidation of <Emphasis Type="Italic">trans</Emphasis>-resveratrol in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing glucosyltransferase from <Emphasis Type="Italic">Phytolacca americana</Emphasis>

A glucosyltransferase (GT) of Phytolacca americana (PaGT3) was expressed in Escherichia coli and purified for the synthesis of two O-β-glucoside products of trans-resveratrol. The reaction was moderately regioselective with a ratio of 4′-O-β-glucoside: 3-O-β-glucoside at 10:3. We used not only the purified enzyme but also the E. coli cells containing the PaGT3 gene for the synthesis of glycoconjugates. E. coli cell cultures also have other advantages, such as a shorter incubation time compared with cultured plant cells, no need for the addition of exogenous glucosyl donor compounds such as UDP-glucose, and almost complete conversion of the aglycone to the glucoside products. Furthermore, a homology model of PaGT3 and mutagenesis studies suggested that His-20 would be a catalytically important residue.     

Strong Expression and Conserved Regulation of <Emphasis Type="Italic">ACT2</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Arabidopsis ACT2 represents an ancient class of vegetative plant actins and is strongly and constitutively expressed in almost all Arabidopsis sporophyte vegetative tissues. Using the beta glucuronidase report system, the studies showed that ACT2 5′ regulatory region was significantly more active than CaMV 35S promoter in Arabidopsis seedlings and gametophyte vegetative tissues of Physcomitrella patens. Its activity was also observed in rice and maize seedlings. Thus, the ACT2 5′ regulatory region could potentially serve as a strong regulator to express a transgene in divergent plant species. ACT2 5′ regulatory region contained 15 conserved sequence elements, an ancient intron in its 5′ un-translated region (5′ UTR), and a purine-rich stretch followed by a pyrimidine-rich stretch (PuPy). Mutagenesis and deletion analysis illustrated that some of the conserved sequence elements and the region containing PuPy sequences played regulatory roles in Arabidopsis. Interestingly, mutation of the conserved elements did not lead a dramatic change in the activity of ACT2 5′ regulatory region. The ancient intron in ACT2 5′ UTR was required for its strong expression in both Arabidopsis and P. patens, but did not fully function as a canonical intron. Thus, it was likely that some of the conserved sequence elements and gene structures had been preserved in ACT2 5′ regulatory region over the course of land plant evolution partly due to their functional importance. The studies provided additional evidences that identification of evolutionarily conserved features in non-coding region might be used as an efficient strategy to predict gene regulatory elements.