Molecular cloning and characterization of a putative proline dehydrogenase gene in the Colorado potato beetle,Leptinotarsa decemlineata

Leptinotarsa decemlineata adults exhibit a season-dependent activity. In spring, post-diapause beetles often fly a long distance from overwintering sites to potato fields. In summer and autumn, the flight ability is sharply reduced. Proline is the main energy substrate ofL. decemlineata during flight and proline dehydrogenase （ProDH） catalyzes the first step in proline catabolism. Here we identified a putative LdProDHgene; it had three cDNA isoforms which shared the same 5＇UTR and coding region, but differed in the lengths of 3＇UTRs （515, 1 092 and 1 242 bp for isoforms-1, -2 and -3, respectively）. LdProDH encoded a 616 amino acid protein that showed high sequence similarity to ProDH-like proteins from other insect species. LdProDHwas expressed in the third and fourth instars larvae and adults, but not in pupae. Dietary ingestion of bacterially expressed LdProDH- dsRNA by adults significantly decreased its messenger RNA （mRNA） level, and caused an elevation of free proline content in the hemolymph. Further observation revealed that three canonical polyadenylation signals （AATAAA） were tandemly located in the 3＇UTR of isoform-3. The first, second and third polyadenylation sites gave rise to isoforms-1, -2 and -3, respectively. Analysis of the genomic DNA uncovered that the three isoforms resulted from alternative polyadenylation. The mRNA level of isoform-1, which expressed at low levels in pre-diapause adults, became abundant in post-diapause beetles. It is indicated that the LdProDH expression is fine-tuned through 3＇UTR to control proline catabolism for the season-dependent activity ofL. decemlineata adults.     

Oral immunogenicity of porcine reproductive and respiratory syndrome virus antigen expressed in transgenic banana

Porcine reproductive and respiratory syndrome virus (PRRSV) is a persistent threat of economically significant influence to the swine industry worldwide. Recombinant DNA technology coupled with tissue culture technology is a viable alternative for the inexpensive production of heterologous proteins in planta. Embryogenic cells of banana cv. ‘Pei chiao’ (AAA) have been transformed with the ORF5 gene of PRRSV envelope glycoprotein (GP5) using Agrobacterium‐mediated transformation and have been confirmed. Recombinant GP5 protein levels in the transgenic banana leaves were detected and ranged from 0.021%–0.037% of total soluble protein. Pigs were immunized with recombinant GP5 protein by orally feeding transgenic banana leaves for three consecutive doses at a 2‐week interval and challenged with PRRSV at 7 weeks postinitial immunization. A vaccination‐dependent gradational increase in the elicitation of serum and saliva anti‐PRRSV IgG and IgA was observed. Furthermore, significantly lower viraemia and tissue viral load were recorded when compared with the pigs fed with untransformed banana leaves. The results suggest that transgenic banana leaves expressing recombinant GP5 protein can be an effective strategy for oral delivery of recombinant subunit vaccines in pigs and can open new avenues for the production of vaccines against PRRSV.     

Identification of the interaction between bta‐miR‐370 and OLR1 gene in bovine adipocyte

It has been shown that the oxidized low density lipoprotein receptor 1 (OLR1) gene plays an important role in the degradation of oxidized low density lipoprotein. Previous studies found a SNP in the 3′‐untranslated region (3′‐UTR) of the OLR1 gene associated with milk production traits in different dairy cattle populations and with loin eye area and marbling depth in beef cattle. MicroRNAs can regulate gene expression by binding the 3′‐UTR of target genes to degrade or to repress the translation of target genes. Bioinformatics have shown that there is a binding site of bta‐miR‐370 in the 3′‐UTR of the OLR1 gene, and a previous luciferase reporter assay system showed that the A/C mutation occurring in the 3′‐UTR of this gene caused the binding sites of bta‐miR‐370 to disappear in HEK293 cells. To further validate whether OLR1 was the target gene of bta‐miR‐370, the over‐expression and interference expression of bta‐miR‐370 were determined by transfecting bta‐miR‐370 mimics and inhibitor supplementations into bovine adipocyte. The qRT‐PCR result showed that the relative expression of OLR1 gene significantly decreased in the mimics group compared to the control, whereas the expression level in inhibitor group was higher than its control group. The above results were further verified by a Western blot at the protein level. In addition, lipid formation analysis of bovine adipocytes was performed via oil red O staining, and we found that cytoplasm lipid droplets in the inhibitor group showed a tendency to increase compared to the control group, whereas in the mimics group, we observed an obvious decrease of cytoplasm lipid droplets compared to the control and inhibitor groups. Taken together, our data here suggest that bta‐miR‐370 has a negative regulation role for OLR1 both at the gene expression and protein levels and bovine adipocytes cytoplasm lipid droplets formation, which provides a reference for illustrating how the OLR1 gene affects milk production and beef quality traits in cattle.     

Validation and further characterization of a major quantitative trait locus associated with host response to experimental infection with porcine reproductive and respiratory syndrome virus

Infectious diseases are costly to the swine industry; porcine reproductive and respiratory syndrome (PRRS) is the most devastating. In earlier work, a quantitative trait locus associated with resistance/susceptibility to PRRS virus was identified on Sus scrofa chromosome 4 using approximately 560 experimentally infected animals from a commercial cross. The favorable genotype was associated with decreased virus load and increased weight gain (WG). The objective here was to validate and further characterize the association of the chromosome 4 region with PRRS resistance using data from two unrelated commercial crossbred populations. The validation populations consisted of two trials each of approximately 200 pigs sourced from different breeding companies that were infected with PRRS virus and followed for 42 days post‐infection. Across all five trials, heritability estimates were 0.39 and 0.34 for viral load (VL; area under the curve of log‐transformed viremia from 0 to 21 days post‐infection) and WG to 42 days post‐infection respectively. Effect estimates of SNP WUR10000125 in the chromosome 4 region were in the same directions and of similar magnitudes in the two new trials as had been observed in the first three trials. Across all five trials, the 1‐Mb region on chromosome 4 explained 15 percent of genetic variance for VL and 11 percent for WG. The effect of the favorable minor allele at SNP WUR10000125 was dominant. Ordered genotypes for SNP WUR10000125 showed that the effect was present irrespective of whether the favorable allele was paternally or maternally inherited. These results demonstrate that selection for host response to PRRS virus infection could reduce the economic impact of PRRS.     

A Polymorphic 3’UTR Element in ATP1B1 Regulates Alternative Polyadenylation and Is Associated with Blood Pressure

Although variants in many genes have previously been shown to be associated with blood pressure (BP) levels, the molecular mechanism underlying these associations are mostly unknown. We identified a multi-allelic T-rich sequence (TRS) in the 3’UTR of ATP1B1 that varies in length and sequence composition (T_22-27 and T₁₂GT ₃GT₆). The 3’UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRS is downstream of the proximal polyadenylation site (A2). Therefore, we hypothesized that alleles of this TRS might influence ATP1B1 expression by regulating alternative polyadenylation. In vitro, the T₁₂GT ₃GT₆ allele increases polyadenylation at the A2 polyadenylation site as compared to the T₂₃ allele. Consistent with our hypothesis, the relative abundance of the A2-polyadenylated ATP1B1 mRNA was higher in human kidneys with at least one copy of the T₁₂GT ₃GT₆ allele than in those lacking this allele. The T₁₂GT ₃GT₆ allele is also associated with higher systolic BP (beta = 3.3 mmHg, p = 0.014) and diastolic BP (beta = 2.4 mmHg, p = 0.003) in a European-American population. Therefore, we have identified a novel multi-allelic TRS in the 3’UTR of ATP1B1 that is associated with higher BP and may mediate its effect by regulating the polyadenylation of the ATP1B1 mRNA.     

CRISPRpas: programmable regulation of alternative polyadenylation by dCas9

Most human protein-coding genes produce alternative polyadenylation (APA) isoforms that differ in 3′ UTR size or, when coupled with splicing, have variable coding sequences. APA is an important layer of gene expression program critical for defining cell identity. Here, by using a catalytically dead Cas9 and coupling its target site with polyadenylation site (PAS), we develop a method, named CRISPRpas, to alter APA isoform abundance. CRISPRpas functions by enhancing proximal PAS usage, whose efficiency is influenced by several factors, including targeting strand of DNA, distance between PAS and target sequence and strength of the PAS. For intronic polyadenylation (IPA), splicing features, such as strengths of 5′ splice site and 3′ splice site, also affect CRISPRpas efficiency. We show modulation of APA of multiple endogenous genes, including IPA of PCF11, a master regulator of APA and gene expression. In sum, CRISPRpas offers a programmable tool for APA regulation that impacts gene expression.     

3′非翻译区靶向人工微小RNA对PRRSV在猪肺巨噬细胞中复制的抑制作用（英文）北大核心CSCD

【目的】研究重组腺病毒(rAd)传送的3′非翻译区(UTR)靶向amiR3UTR对猪繁殖与呼吸综合征病毒(PRRSV)在猪肺巨噬细胞(PAM)中复制的抑制作用。【方法】用表达amiR3UTR或对照amiRcon的腺病毒载体转染AAV-293细胞,获得rAd-amiR3UTR-GFP和rAd-amiRcon-GFP,用定量RT-PCR检测amiR3UTR在rAd转导细胞中的表达,用定量RT-PCR、Western blotting和病毒滴定检测amiR3UTR对PRRSV复制的抑制作用。【结果】原代PAM及其细胞系3D4/163均能被rAd-amiR3UTR-GFP转导,但前者转导效率很低;rAd-amiR3UTR-GFP转导细胞能有效表达amiR3UTR,且表达具有剂量和时间依赖性;rAd表达的amiR3UTR能显著抑制不同毒株PRRSV在PAM细胞中的复制,且抑制作用具有剂量依赖性。【结论】amiR3UTR能抑制不同毒株PRRSV在PAM中的复制,其rAd有望作为抗PRRSV新策略进行深入研究。     

The gene structure and expression of human ABHD1: overlapping polyadenylation signal sequence with Sec12

Background

Overlapping sense/antisense genes orientated in a tail-to-tail manner, often involving only the 3'UTRs, form the majority of gene pairs in mammalian genomes and can lead to the formation of double-stranded RNA that triggers the destruction of homologous mRNAs. Overlapping polyadenylation signal sequences have not been described previously.

Results

An instance of gene overlap has been found involving a shared single functional polyadenylation site. The genes involved are the human alpha/beta hydrolase domain containing gene 1 (ABHD1) and Sec12 genes. The nine exon human ABHD1 gene is located on chromosome 2p23.3 and encodes a 405-residue protein containing a catalytic triad analogous to that present in serine proteases. The Sec12 protein promotes efficient guanine nucleotide exchange on the Sar1 GTPase in the ER. Their sequences overlap for 42 bp in the 3'UTR in an antisense manner. Analysis by 3' RACE identified a single functional polyadenylation site, ATTAAA, within the 3'UTR of ABHD1 and a single polyadenylation signal, AATAAA, within the 3'UTR of Sec12. These polyadenylation signals overlap, sharing three bp. They are also conserved in mouse and rat. ABHD1 was expressed in all tissues and cells examined, but levels of ABHD1 varied greatly, being high in skeletal muscle and testis and low in spleen and fibroblasts.

Conclusions

Mammalian ABHD1 and Sec12 genes contain a conserved 42 bp overlap in their 3'UTR, and share a conserved TTTATTAAA/TTTAATAAA sequence that serves as a polyadenylation signal for both genes. No inverse correlation between the respective levels of ABHD1 and Sec12 RNA was found to indicate that any RNA interference occurred.     

TPH2 5′‐ and 3′‐regulatory polymorphisms are differentially associated with HPA axis function and self‐injurious behavior in rhesus monkeys

Tryptophan hydroxylase‐2 (TPH2) synthesizes neuronal serotonin and is linked to numerous behavioral traits. We have previously characterized the functionality of polymorphisms (especially 2051A>C) in 3’‐untranslated region (3’‐UTR) of rhesus monkey TPH2 (rhTPH2). This study further assessed the functionality of additional polymorphisms (–1605T>C, –1491Tn, –1485(AT)n, –1454A>G, –1325In>Del and –363T>G) in rhTPH2 5’‐flanking region (5’‐FR), and evaluated the effects of rhTPH2 5’ and 3’ genotypes on central serotonin turnover, hypothalamic–pituitary–adrenal (HPA) axis function and self‐injurious behavior (SIB) in 32 unrelated adult male monkeys of Indian origin. Haplotypes of the rhTPH2 5’‐FR polymorphisms exert a significant, cell‐dependent effect on reporter gene expression, primarily conferred by –1485(AT)n. The –1485(AT)n and 2051A>C polymorphisms interact to influence cerebrospinal fluid (CSF) 5‐HIAA and plasma adrenocorticotropic hormone (ACTH) in the afternoon. While –1485(AT)n exerts significant main effects on the afternoon cortisol level and nocturnal HPA negative feedback, 2051A>C has significant main effects on the morning cortisol level and cortisol response to ACTH challenge, as well as marginally significant main effects on the daytime HPA negative feedback and self‐biting rate. In addition, the genotype/allele frequency of the 5’‐FR –1325Ins>Del differed significantly between the self‐wounders and non‐wounders, whereas 3’‐UTR 2128S>L polymorphism differed significantly in genotype/allele frequency between the high‐ and low‐frequency biters. This study shows the functionality of rhTPH2 5’‐FR polymorphisms, and provides evidence for the differential association of rhTPH2 5’‐FR and 3’‐UTR polymorphisms with HPA axis function and SIB. Our findings shed light on the role of TPH2 gene variance in physiology and behavioral traits, and also contribute to the understanding of the pathophysiology and genetics of SIB