Fine mapping of porcine chromosome 6 QTL and LEPR effects on body composition in multiple generations of an Iberian by Landrace intercross

The leptin receptor gene (LEPR) is a candidate for traits related to growth and body composition, and is located on SSC6 in a region where fatness and meat composition quantitative trait loci (QTL) have previously been detected in several F2 experimental designs. The aims of this work were: (i) to fine map these QTL on a larger sample of animals and generations (F3 and backcross) of an Iberian x Landrace intercross and (ii) to examine the effects of LEPR alleles on body composition traits. Eleven single nucleotide polymorphisms (SNPs) were detected by sequencing LEPR coding regions in Iberian and Landrace pig samples. Three missense polymorphisms were genotyped by pyrosequencing in 33 F0, 70 F1, 418 F2, 86 F3 and 128 individuals coming from the backcross of four F2 males with 24 Landrace females. Thirteen microsatellites and one SNP were also genotyped. Traits analysed were: backfat thickness at different locations (BF(T)), intramuscular fat percentage (IMF(P)), eye muscle area (EM(A)), loin depth (LO(D)), weight of shoulder (SH(W)), weight of ribs (RIB(W)) and weight of belly bacon (BB(W)). Different statistical models were applied in order to evaluate the number and effects of QTL on chromosome 6 and the possible causality of the LEPR gene variants with respect to the QTL. The results support the presence of two QTL on SSC6. One, at position 60-100 cM, affects BF(T) and RIB(W). The other and more significant maps in a narrow region (130-132 cM) and affects BF(T), IMF(P), EM(A), LO(D), SH(W), RIB(W) and BB(W). Results also support the association between LEPR alleles and BF(T) traits. The possible functional implications of the analysed polymorphisms are considered.     

Malic enzyme 1 genotype is associated with backfat thickness and meat quality traits in pigs

Malic enzyme 1 (ME1) is a part of the tricarboxylate shuttle that provides NADPH and acetyl-CoA required in fatty acid biosynthesis. The pig ME1 locus maps on the proximal end of chromosome 1, where a quantitative trait loci (QTL) affecting fat deposition has been previously described. We amplified fragments of 1457 and 1459 bp that corresponded to the complete coding region and the 3'-untranslated region (UTR), respectively, of the pig ME1 gene. The sequences of these two fragments in pigs from three breeds (Landrace, Large White and Piétrain) contained five single nucleotide polymorphisms (SNP) in the 3'-UTR: C1706T, G1762T, A1807C, C1857A and T1880A. Three haplotypes were found in two generations of a selected Landrace population: H1 (C1706 G1762 A1807 C1857 A1880), H2 (C1706 G1762 A1807 C1857 T1880) and H3 (T1706 T1762 C1807 A1857 T1880). Using Bayesian association analyses, significant associations (highest posterior density at 95%) between ME1 genotype and backfat (BF) thickness at 171 days and muscular pH were found in a Landrace population.     

A novel LIM and SH3 protein (lasp-2) highly expressing in chicken brain

From eluates of F-actin affinity chromatography of chicken brain, we identified a novel actin-binding protein (lasp-2) whose gene was predicted in silico. We cloned cDNA of chicken lasp-2 and analyzed its structure, expression, activity, and localization with lasp-1 (LIM and SH3 protein 1), a previously identified actin-binding protein closely related to lasp-2. Chicken lasp-2 showed high homology to mammalian putative lasp-2. Both chicken lasp-1 and chicken lasp-2 have N-terminal LIM domains, C-terminal SH3 domains, and internal nebulin repeats. However, lasp-2 is greatly different from lasp-1 in the sequence between the second nebulin repeat and a SH3 domain, and the region is conserved in chicken, mouse, and human. As expected from its structural similarity to lasp-1, lasp-2 possessed actin-binding activity and localized with actin filament in filopodia of neuroblastoma. In contrast to lasp-1, which is widely distributed in non-muscle tissues, lasp-2 was highly expressed in brain.     

Association of inhibitory tyrosine protein kinase p50csk with protein tyrosine phosphatase PEP in T cells and other hemopoietic cells.

p50csk is a tyrosine protein kinase (TPK) that represses the activity of Src family TPKs. We previously showed that Csk is a potent negative regulator of antigen receptor signaling in T lymphocytes and that its Src homology (SH) 3 and SH2 domains are required to inhibit these signals. To test the idea that the Csk SH3 and SH2 domains mediate interactions with other cellular proteins, we attempted to identify Csk-associated polypeptides using the yeast two-hybrid system. The results of our experiments demonstrated that Csk physically associates with PEP, a protein tyrosine phosphatase (PTP) expressed in hemopoietic cells. Further analyses revealed that this interaction was mediated by the Csk SH3 domain and by a proline-rich region (PPPLPERTP) in the non-catalytic C-terminal portion of PEP. The association between Csk and PEP was documented in transiently transfected Cos-1 cells and in a variety of cells of hemopoietic lineages, including T cells. Additional analyses demonstrated that the association between Csk and PEP is highly specific. Together, these data indicated that PEP may be an effector and/or a regulator of p50csk in T cells and other hemopoietic cells. Moreover, they allowed the identification of PEP as the first known ligand for the Csk SH3 domain.     

Fine mapping of porcine SSC14 QTL and SCD gene effects on fatty acid composition and melting point of fat in a Duroc purebred population

The stearoyl-CoA desaturase (delta-9-desaturase; SCD) gene is a candidate gene for fatty acid composition. It is located on pig SSC14 in a region where quantitative trait loci (QTL) for fatty acid composition were previously detected in a Duroc purebred population. The objective of the present study was to fine map the QTL, to identify polymorphisms of the pig SCD gene and to examine the effects of SCD polymorphisms on fatty acid composition and melting point of fat in the population. The pigs were examined for fatty acid composition and melting point of inner and outer subcutaneous fat and inter- and intramuscular fat; the number of pigs examined was 479-521. Two SNPs (g.-353C>T and g.-233T>C) were identified in the promoter region of the SCD gene and were completely linked in the pigs from the base generation. In all pigs, 19 microsatellite markers and SCD haplotypes were then genotyped. Different statistical models were applied to evaluate the effects of QTL and the possible causality of the SCD gene variants with respect to the QTL. The results show that all significant QTL for C14:0, C18:0, C18:1 and melting point of fat were detected in the same region, located near the SCD gene. The results also show a significant association between SCD haplotypes and fatty acid composition and fat melting point in this population. These results indicate that the haplotype of the SCD gene has a strong effect on fatty acid composition and melting point of fat.     

The v-Src SH3 domain binds phosphatidylinositol 3''-kinase.

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.     

Highly conserved eight amino acid sequence in SH2 is important for recognition of phosphotyrosine site.

Src homology region 2(SH2) has been demonstrated to recognize phosphotyrosine site. To clarify the precise mechanism of the recognition, we developed in vitro binding assay system using EGF receptor and SH2/SH3 region of phospholipase C(PLC) gamma 1. Phosphorylated EGF receptor bound to immobilized SH2/SH3 of PLC gamma 1 in Sepharose beads, while nonphosphorylated EGF receptor did not bind. In SH2 domain of PLC gamma 1, there are several highly conserved amino acid sequences that are common in a variety of SH2-containing proteins. Especially the eight amino acid sequence, G(S/T)FLVR(E/D)S is highly conserved in these proteins. We synthesized several peptides related to these sequences and examined the effect of peptides on the binding of EGF receptor to SH2 of PLC gamma 1. P1, GSFLVRES was the most effective inhibitor to suppress the binding. P2, GSFLVAES in which one amino acid, arginine of P1 is substituted by alanine is still effective. But a peptide, P3, SFLVRE in which two amino acids are deleted from P1 did not inhibit markedly. Moreover, P1 peptide immobilized in Sepharose beads also bound phosphorylated EGF receptor. These data suggest that highly conserved amino acid sequence GSFLVRES is the minimum essential unit to recognize tyrosine phosphorylated site.     

SH3GL1在紫杉醇耐药乳腺癌中的表达及临床意义

膜结合蛋白SH3GL1参与调控某些肿瘤细胞生物学行为,而其对紫杉醇耐药乳腺癌细胞的恶性生物学行为影响尚未见报道.为阐明 SH3GL1对紫杉醇耐药敏感性的影响以及潜在的分子机制,本研究首先采用免疫组化法证实SH3GL1在紫杉醇耐药乳腺癌组织高表达(P<0.001).Western印迹法证实,SH3GL1在紫杉醇耐药细胞株MCF-7/PTX高表达(P<0.01);随后,采用MTT分别检测（0,50,100 nmol/L）紫杉醇处理后MCF-7细胞株增殖情况,同时Western印迹法检测SH3GL1表达,发现100 nmol/L紫杉醇能够抑制MCF-7细胞增殖(P<0.05);同时抑制SH3GL1表达(P<0.01); 敲减SH3GL1表达后,紫杉醇耐药细胞株MCF-7/PTX和MCF-7增殖速率降低(P<0.05),耐药基因MDR1表达降低(P<0.05),p-AKT和p-gp水平下降(P<0.05).上述结果表明,降低SH3GL1表达可以减弱紫杉醇耐药性,增加乳腺癌对紫杉醇的敏感性,这为临床上紫杉醇耐药乳腺癌患者的治疗提供了新的靶点.     

Molecular cloning, expression profile, polymorphism and the genetic effects of the dopamine D1 receptor gene on duck reproductive traits

The dopamine D1 receptor (DRD1), a member of the dopamine receptor (DR) gene family, participates in the regulation of reproductive behaviors in birds. In this study, a 1,390 bp fragment covering the complete coding region (CDS) of duck DRD1 gene was obtained. The cDNA (GenBank: JQ346726) contains a 1,353 bp CDS and a 37 bp 3'- UTR including a TGA termination codon (nucleotides 1,354-1,356 bp). The duck DRD1 shares about 76-96 % nucleic acid identity and 82-98 % amino acid identity with their counterparts in other species. A phylogenetic tree based on amino acid sequences displays that duck DRD1 protein is closely related with those of chicken and zebra finch. The quantitative real-time PCR analysis indicates that the DRD1 mRNA is widely expressed in all examined tissues. Five single nucleotide polymorphisms (SNPs) (c.189A > T, c.507C > T, c.681C > T, c.765A > T, c.1044A > G) in the CDS of duck DRD1 gene were indentified, c.681C > T and c.765A > T were genotyped and analyzed in a two generations duck population by using of PCR-RFLP. Association analysis demonstrated that the c.681C > T genotypes were significantly associated with body weight at sexual maturity (when laying their first egg) (P < 0.01), egg production within 360 days (P < 0.05) and 420 days (P < 0.01); the c.765A > T genotypes were significantly associated with egg shape index and egg shell strength (P < 0.05). Those results suggest that the DRD1 gene may be a potential genetic marker to improve some reproductive traits in ducks.     

Evidence of association between methylenetetrahydrofolate reductase gene and susceptibility to breast cancer: a candidate-gene association study in a South-eastern European population

The methylenetetrahydrofolate reductase (MTHFR) gene has been proposed as a candidate gene for breast cancer (BC). However, the specific role of MTHFR polymorphisms and haplotypes has not been fully clarified and replicated. We examined the association of two common MTHFR polymorphisms (C677T and A1298C) and their haplotypes in a candidate-gene association study, involving 300 female patients with BC and 283 healthy women. The single locus analysis for the two polymorphisms revealed an association only for the C677T polymorphism [odds ratio (95% confidence interval), OR=2.05 (1.21-3.48)], but adjustment for age diminished this association [OR=1.76 (0.92-3.42)]. The menopausal status showed no significant effect in the association between the MTHFR polymorphisms and BC. The analysis of haplotypes showed an association for the C677-A1298 haplotypes (p=0.04). The available evidence from our study may support a contributory role of MTHFR polymorphisms in BC development. Future larger studies may help in elucidating the genetics of BC further.     

Effect of luteinizing hormone/choriogonadotropin receptor (LHCGR) gene on chicken reproductive traits

Luteinizing hormone/choriogonadotropin receptor (LHCGR) gene, potentially related to reproductive traits in chickens, was genotyped by using the Pooled DNA Sequencing, PCR–SSCP and Directing Sequencing techniques. 306 Erlang Mountain chickens form one line (SD03, a line that has been selected for egg quality from a local chicken breed in Sichuan province, China) were genotyped in this study. The associations between LHCGR polymorphisms and six reproductive traits [body weight at first egg (BWAFE), weight of first egg, age at first egg (AFE), number of eggs at 300 days of age (EN), body weight at 300 days of age and egg weight at 300 days of age (EWTA)] were estimated using the one-way analysis of variance method. Results showed that SNP +G4058A and SNP +T4099G of the LHCGR gene were significantly associated with BWFE and AFE. Birds with the AG genotype for the +G4058A SNP exhibited shorter AFE (P < 0.05) and greater EN than those of the GG and AA genotypes, suggesting a balancing selection (overdominance); the effect of allele C in SNP +C3021T and allele C in SNP +T4490C on EN and AFE is additive and may reflect the influence of positive selection. These alleles have promise as genetic markers for future marker-assisted selection.     

Multivariate Analysis of Dopaminergic Gene Variants as Risk Factors of Heroin Dependence

Background

Heroin dependence is a debilitating psychiatric disorder with complex inheritance. Since the dopaminergic system has a key role in rewarding mechanism of the brain, which is directly or indirectly targeted by most drugs of abuse, we focus on the effects and interactions among dopaminergic gene variants.

Objective

To study the potential association between allelic variants of dopamine D2 receptor (DRD2), ANKK1 (ankyrin repeat and kinase domain containing 1), dopamine D4 receptor (DRD4), catechol-O-methyl transferase (COMT) and dopamine transporter (SLC6A3) genes and heroin dependence in Hungarian patients.

Methods

303 heroin dependent subjects and 555 healthy controls were genotyped for 7 single nucleotide polymorphisms (SNPs) rs4680 of the COMT gene; rs1079597 and rs1800498 of the DRD2 gene; rs1800497 of the ANKK1 gene; rs1800955, rs936462 and rs747302 of the DRD4 gene. Four variable number of tandem repeats (VNTRs) were also genotyped: 120 bp duplication and 48 bp VNTR in exon 3 of DRD4 and 40 bp VNTR and intron 8 VNTR of SLC6A3. We also perform a multivariate analysis of associations using Bayesian networks in Bayesian multilevel analysis (BN-BMLA).

Findings and conclusions

In single marker analysis the TaqIA (rs1800497) and TaqIB (rs1079597) variants were associated with heroin dependence. Moreover, –521 C/T SNP (rs1800955) of the DRD4 gene showed nominal association with a possible protective effect of the C allele. After applying the Bonferroni correction TaqIB was still significant suggesting that the minor (A) allele of the TaqIB SNP is a risk component in the genetic background of heroin dependence. The findings of the additional multiple marker analysis are consistent with the results of the single marker analysis, but this method was able to reveal an indirect effect of a promoter polymorphism (rs936462) of the DRD4 gene and this effect is mediated through the –521 C/T (rs1800955) polymorphism in the promoter.     

A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail

A quantitative trait loci (QTL) study was undertaken to identify genome regions involved in the control of fearfulness in Japanese quail (Coturnix japonica). An F2 cross was made between two quail lines divergently selected over 29 generations on duration of tonic immobility (DTI), a catatonic-like state of reduced responsiveness to a stressful stimulation. A total of 1065 animals were measured for the logarithm of DTI (LOGTI), the number of inductions (NI) necessary to induce the immobility reaction, open-field behaviour including locomotor activity (MOVE), latency before first movement (LAT), number of jumps (JUMP), dejections (DEJ) and shouts (SHOUT), corticosterone level after a contention stress (LOGCORT) and body weight at 2 weeks of age (BW2). A total of 310 animals were included in a genome scan using selective genotyping with 248 AFLP markers. A total of 21 suggestive or genome-wide significant QTL were observed. Two highly significant QTL were identified on linkage group 1 (GL1), one for LOGTI and one for NI. In the vicinity of the QTL for LOGTI, a nearly significant QTL for SHOUT and a suggestive QTL for LAT were also identified. On GL3, genome-wide significant QTL were observed for JUMP and DEJ as well as suggestive QTL for LOGTI, MOVE, SHOUT and LAT. A significant QTL for BW2 was observed on GL2 and a nearly significant one on GL1. These results may be useful in the understanding of fearfulness in quail and related species provided that fearfulness has the same genetic basis.     

Structural insights into the specific binding of huntingtin proline-rich region with the SH3 and WW domains

The interactions of huntingtin (Htt) with the SH3 domain- or WW domain-containing proteins have been implicated in the pathogenesis of Huntington's disease (HD). We report the specific interactions of Htt proline-rich region (PRR) with the SH3GL3-SH3 domain and HYPA-WW1-2 domain pair by NMR. The results show that Htt PRR binds with the SH3 domain through nearly its entire chain, and that the binding region on the domain includes the canonical PxxP-binding site and the specificity pocket. The C terminus of PRR orients to the specificity pocket, whereas the N terminus orients to the PxxP-binding site. Htt PRR can also specifically bind to WW1-2; the N-terminal portion preferentially binds to WW1, while the C-terminal portion binds to WW2. This study provides structural insights into the specific interactions between Htt PRR and its binding partners as well as the alteration of these interactions that involve PRR, which may have implications for the understanding of HD.     

Sequencing and genotypic analysis of the triosephosphate isomerase (TPI1) locus in a large sample of long-lived Germans

Background

With crucial roles on the differentiation of anterior pituitary and the regulation of the prolactin (PRL), growth hormone (GH) and thyroid-stimulating hormone-β (TSH-β) genes, the chicken PIT1 gene is regarded as a key candidate gene for production traits. In this study, five reported polymorphisms (MR1-MR5) of the PIT1 gene were genotyped in a full sib F₂ resource population to evaluate their effects on growth, carcass and fatty traits in chickens.

Results

Marker-trait association analyses showed that, MR1 was significantly associated with shank diameters (SD) at 84 days (P < 0.05), hatch weight (HW) and shank length (SL) at 84 days (P < 0.01), MR2 was significantly associated with BW at 28, 42 days and average daily gain (ADG) at 0–4 weeks (P < 0.05), and MR3 was significantly associated with ADG at 4–8 weeks (P < 0.05). MR4 was associated with SL at 63, 77, 84 days and BW at 84 days (P < 0.05), as well as SD at 77 days (P < 0.01). Significant association was also found of MR5 with BW at 21, 35 days and SD at 63 days (P < 0.05), BW at 28 days and ADG at 0–4 weeks (P < 0.01). Both T allele of MR4 and C allele of MR5 were advantageous for chicken growth. The PIT1 haplotypes were significantly associated with HW (P = 0.0252), BW at 28 days (P = 0.0390) and SD at 56 days (P = 0.0400). No significant association of single SNP and haplotypes with chicken carcass and fatty traits was found (P > 0.05).

Conclusion

Our study found that polymorphisms of PIT1 gene and their haplotypes were associated with chicken growth traits and not with carcass and fatty traits.     

Dopamine receptors D1 and D2 are related to observed maternal behavior

The dopamine pathway and especially the dopamine receptors 1 and 2 (DRD1 and DRD2) are implicated in the regulation of mothering in rats. Evidence for this in humans is lacking. Here, we show that genetic variation in both DRD1 and DRD2 genes in a sample of 187 Caucasian mothers predicts variation in distinct maternal behaviors during a 30-min mother-infant interaction at 6 months postpartum. Two DRD1 single-nucleotide polymorphisms (SNPs rs265981 and rs686) significantly associated with maternal orienting away from the infant (P = 0.002 and P = 0.003, respectively), as did DRD1 haplotypes (P = 0.03). Two DRD2 SNPs (rs1799732 and rs6277) significantly associated with maternal infant-directed vocalizing (P = 0.001 and P = 0.04, respectively), as did DRD2 haplotypes (P = 0.01). We present evidence for heterosis in DRD1 where heterozygote mothers orient away from their infants significantly less than either homozygote group. Our findings provide important evidence that genetic variation in receptors critical for mothering in non-human species also affect human maternal behaviors. The findings also highlight the importance of exploring multiple dimensions of the complex human mothering phenotype.     

INCENP基因功能性单倍型可调控启动子活性并影响公牛精液品质

为研究内着丝粒蛋白(Inner centromere protein, INCENP)基因启动子区单核苷酸多态性(SNPs)与精液品质的相关性,本文利用聚合酶链反应-限制性片段长度多态性(PCR-RFLP)方法检测了250头中国荷斯坦公牛INCENP基因的基因型。在INCENP基因启动子区鉴定出两个SNPs (g.-556 G>T,rs 136823901和g.-692 C>T,rs 211010999),发现了3种单倍型(CG、TT、TG)。分析两个SNP位点的基因型频率和等位基因频率,各SNP及单倍型组合与中国荷斯坦公牛精液品质的相关性,结果表明SNP位点g.-556 G>T GT基因型个体的鲜精活力显著高于GG基因型个体(P<0.05),单倍型组合H1H1(CCGG)、H1H3(CTGT)、H2H3(TTGT)和H3H3(TTTT)个体的鲜精活力和冻精解冻后活力均显著高于H1H2个体(P<0.05)。为进一步研究g.-556 G>T和g.-692 C>T影响精液品质的可能机理,本文将3种单倍型质粒分别转染小鼠睾丸间质细胞(MLTC-1),结果显示含TG单倍型的载体荧光素酶活性最高。由此推测,g.-556 G>T和g.-692 C>T为启动子区功能性突变位点,可通过调节启动子活性来调控INCENP基因表达,进而影响精液品质。