The differential expression of alternatively spliced transcripts and imprinting status of MEG9 gene in cows

Meg9/Mirg (maternally expressed gene 9/microRNA containing gene), a non‐coding RNA (ncRNA) comprising many alternatively splicing isoforms, has been identified as maternally expressed in mouse and sheep, but its imprinting status and splicing variants are still unknown in cattle. In this study, we found three splicing variants of the cattle MEG9 gene expressed in a tissue‐specific manner. A single nucleotide polymorphism site (SNP c.1354C>G) was identified in exon 3 of cattle MEG9 and used to distinguish between monoallelic and biallelic expression. Our results showed that MEG9 exhibited monoallelic expression in all examined cattle tissues by comparing sequencing results between genomic DNA and cDNA levels at the c.1354C>G SNP site, suggesting that MEG9 is imprinted in cattle.     

Paternal expression of a novel imprinted gene, Peg12/Frat3, in the mouse 7C region homologous to the Prader-Willi syndrome region.

Paternally expressed imprinted genes (Pegs) were systematically screened by comparing gene expression profiles of parthenogenetic and normal fertilized embryos using an oligonucleotide array. A novel imprinted gene, Peg12/Frat3, was identified along with 10 previously known Pegs. Peg12/Frat3 is expressed primarily in embryonic stages and might be a positive regulator of the Wnt signaling pathway. It locates next to the Zfp127 imprinted gene in the mouse 7C region, which has syntenic homology to the human Prader-Willi syndrome region on chromosome 15q11-q13, indicating that this imprinted region extends to the telomeric side in the mouse.     

Preliminary study on the molecular structure of 3'''' region of Duchenne muscular dystrophy (DMD) gene in Chinese

Number and order of HindⅢ exon-containing fragments (Hd) at 3' region of DMD gene were studied systematically using 16 partly-overlapping cDNA subprobes which were produced from dystrophin cDNA 9- 14 with each of 9 restriction endonudeases. There are 25 Hd fragments corresponding to cDNA 9 -14 in DMD gene. Since then, the exact length and the new order of Hd fragments are established. A new 2.1 kb fragment (Hd 55) is revealed, a 5.2 kb fragment (formely designated as Hd 59) is excluded and the existence of a controversial 3.2 kb fragment (Hd 64) is confirmed. Besides, three new exons were revealed by comparing the PvuⅡ and the XbaⅠ hybridization patterns with the Hindlll hybridization patterns for these cDNA subprobes. It is concluded that there are at least 66 Hd fragments, or 79 exons in DMD gene basing on the discovery of three additional exons. The corresponding relationship between the 66 Hd fragments and the SfiⅠ large scale physical map has been studied, and at least 17 Hd fragments or 19 exo     

Understanding the relationships between molecule structure and imprinting effect of two acetyl‐nitrogen heterocyclic compounds

The molecularly imprinted polymers (MIPs) for two structural analogs, 1,3,5‐triacetyl‐1,3,5‐triazacyclohexane (TRAT) and 1,3,5,7‐tetraacetyl‐1,3,5,7‐tetraazacyclooctane (TAT), have been synthesized respectively under the same conditions. The TAT‐MIP showed excellent imprinting effect, whereas the TRAT‐MIP did not. To understand the different imprinting effects of the MIPs prepared from these two templates, the geometric structures and energetic properties of complexes formed around TAT and TRAT were studied computationally. The results indicate that in liquid phase, for the complexes formed with TAT and its nearest neighbor molecules, the magnitude of the binding energy increases with the number of surrounding TAT, methacrylic acid, and acetonitrile (ACT), whereas for the cases of TRAT, the magnitude of the binding energy increases with the number of surrounding TRAT and trimethylolpropane trimethacrylate. The studied systems form stronger and thus more stable networks encapsulating TAT than with TRAT. ACT may also play an important role in the polymerization phase in stabilizing the shapes of the cavities that TATs reside in. We propose these as the major factors that affect the different imprinting effects of the two MIPs. Copyright © 2016 John Wiley & Sons, Ltd.     

The 5′-flanking region of the E1 α form of the murine p16 (MTS1) gene

We have PCR-amplified and sequenced the immediate (841 bp) 5′-flanking region of murine p16^INK4a (MTS1, CDKN2) tumor suppressor gene. Comparing to recently published 5'-flanking region of the human α form of p16^INK4a, homologies were found in several regions of murine p16^INK4a-α putative promoter sequence.     

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.     

Influence of variants in the NPY gene on obesity and metabolic syndrome features in Spanish children

Variants in the neuropeptide Y (NPY) gene have been associated with obesity and its traits. The objective of the present study was to evaluate the association of single nucleotide polymorphisms (SNPs) in the NPY gene with obesity, metabolic syndrome features, and inflammatory and cardiovascular disease (CVD) risk biomarkers in Spanish children. We recruited 292 obese children and 242 normal-body mass index (BMI) children. Height, weight, BMI, waist circumference, clinical and metabolic markers, adipokines, and inflammatory (PCR, IL-6, IL-8 and TNF-α) and CVD risk biomarkers (MPO, MMP-9, sE-selectin, sVCAM, sICAM, and PAI-1) were analyzed. Seven SNPs in the NPY gene were genotyped. The results of our study indicate that anthropometric measurements, clinical and metabolic markers, adipokines (leptin and resistin), and inflammatory and CVD risk biomarkers were generally elevated in the obese group. The exceptions to this finding included cholesterol, HDL-c, and adiponectin, which were lower in the obese group, and glucose, LDL-c, and MMP-9, which did not differ between the groups. Both rs16147 and rs16131 were associated with the risk of obesity, and the latter was also associated with insulin resistance, triacylglycerols, leptin, and HDL-c. Thus, we confirm the association of rs16147 with obesity, and we demonstrate for the first time the association of rs16131 with obesity and its possible impact on the early onset of metabolic syndrome features, mainly triacylglycerols, in children.     

A comparison of Bonus and Quota mating systems for utilising the sex-determining region Y gene in terminal sire beef cattle breeding

The use of the sex-determining region Y gene in terminal sire beef cattle breeding was investigated, assuming that fertile transgenic bulls carrying this gene on an autosome can be created. The benefit of these transgenic bulls arises from having an increased proportion of calves with a male phenotype. Two mating strategies utilising the transgenic bulls were devised and compared; the Quota scheme whereby a quota of normal bulls is used alongside the transgenic bulls in a breeding nucleus, and the Bonus scheme in which a phenotypic bonus is assigned to transgenic bulls indicating the added value of their offspring. Bonus and Quota breeding schemes were comparable, in terms of the value of offspring of the bulls, for the first 6 years of selection, after which time the Quota breeding scheme was superior. For time horizons less than 10 years, and large assumed phenotypic superiorities of male calves, breeding schemes with transgenic bulls were superior to traditional breeding schemes without transgenic bulls. If the time horizon was longer, or if the assumed superiority of male calves was small, then traditional breeding schemes were generally superior to those utilising transgenic bulls. Scenarios were observed, however, where transgenic bulls were always superior to normal bulls, in terms of their value as sires. Equations were derived to predict genetic gain and the equilibrium genetic lag between normal and transgenic bulls in Quota breeding schemes.     

Description and molecular analysis of SRY and AR genes in a patient with 46,XY pure gonadal dysgenesis (Swyer syndrome)

46,XY pure gonadal dysgenesis, first described in 1955 by Swyer, results from testicular tissue loss during the first 8 weeks of fetal life, a critical period for male differentiation. We describe a case of an 18 years old patient presented to us with a chief complain of primary amenorrhea. Chromosomal analysis revealed a 46,XY karyotype. A molecular investigation was undertaken in an attempt to determine mutations in SRY and AR genes through DNA sequencing. Mutations were shown to be absent. The molecular basis of Swyer syndrome is still unknown, although the presence of mutations in testicular organizing genes downstream of SRY is still to rule out. The patient, who is considered as female, was placed on estrogen replacement therapy, while bilateral prophylactic laparoscopic gonadectomy was programmed due to the high prevalence of gonadal tumors in this syndrome. No signs of malignance were detected in the gonadal tissue, which predicts that an intact SRY gene is usually, but not always, not related to the formation of malignancies like dysgeminomas or gonadoblastomas.     

大林姬鼠两亚种线粒体DNA 细胞色素b 基因和控制区的序列多样性

为了研究大林姬鼠两亚种(韩国的指名亚种及中国东北和内蒙古地区的东北亚种)线粒体DNA 的变异程度并确定朝鲜亚种的分类地位,我们分别将来自韩国和中国东北长白山地区的两亚种的线粒体DNA 的细胞色素b 基因和控制区进行了测序分析。我们将测序所得到细胞色素b 基因序列与来自基因库的大林姬鼠5 个亚种的相应的单倍型进行了分析,结果显示大林姬鼠可分为4 个类群[类群1:韩国大林姬鼠指名亚种;类群2:中国长白山和内蒙古地区的东北亚种、俄罗斯外加贝尔的majuculus 亚种;类群3:中国长春的东北亚种、俄罗斯Primorye(俄罗斯远东地区) rufulus 亚种、俄罗斯库页岛(俄罗斯远东地区)和日本北海道地区的giliacus 亚种;类群4:中国黑龙江海林地区的东北亚种]。线粒体的控制区序列分析显示韩国指名亚种也不同于中国东北地区的东北亚种。本研究的类群1,2 和3 与Serizawa et al. (2002)的研究的K、S 和R 的分支相对应。这表明韩国指名亚种(类群1 和分支K)的线粒体DNA 与其他类群不同。另外,我们还发现在细胞色素b 基因构建的系统树中,东北亚种可以与类群2 (分支S)及类群3 (分支R 的不同亚种聚合在一起。我们认为线粒体DNA 的母性遗传与两个相邻亚种的个体之间的种内杂交造成了基于细胞色素b 序列对东北亚种的聚类分析结果与基于形态学特征的分类结果的不一致。因此,我们提出对这些显示出核苷酸序列多样性的东北亚种不能只用细胞色素b的数据进行亚种分类,还应该结合形态学和核DNA 特征进行进一步分析。最后,我们还发现韩国的指名亚种的细胞色素b 序列在平均距离16. 93% 的基础上不同于来自基因库的A. speciosus。Jones and Johnson (1965)指出了韩国的大林姬鼠在形态上的区别,所以我们认为韩国的大林姬鼠指名亚种A. p. peninsulae 是一种具有形态和遗传特异性的地方亚种。     

The taxonomic status of some Atlanto‐Mediterranean species in the subgenus Holothuria (Echinodermata: Holothuroidea: Holothuriidae) based on molecular evidence

Molecular and morphological data were used to evaluate the taxonomic status of the species Holothuria tubulosa Gmelin, 1790 , Holothuria stellati Delle Chiaje, 1823 , Holothuria mammata Grube, 1840 , and Holothuria dakarensis Panning, 1939, belonging to the nominate subgenus Holothuria (Holothuria) (family Holothuriidae) from the Mediterranean Sea and Atlantic Ocean. A 16S rRNA marker distinguished three well‐supported clades with clear genetic differentiation amongst them. The morphometric characters, although they reflected the clades, showed great variability, and some specimens from different clades overlapped. The morphological data and the literature suggest that the clades correspond to H. dakarensis (from Cape Verde Islands), H. mammata (from the Atlanto‐Mediterranean area) and H. tubulosa (from the Mediterranean Sea). Holothuria stellati is considered here to be a junior subjective synonym of H. tubulosa. Great morphological intraspecific variation within H. tubulosa and H. mammata explains the confusion in the literature. Holothuria tubulosa includes specimens with distinctive ossicles, but others are similar to H. mammata. In these cases, the presence or absence of Cuvierian tubules proved a reliable indicator to the identity of these species; unfortunately this character is difficult to assess in preserved material. According to the results of discriminant analysis we propose a set of ossicle morphometric variables that permit the optimum assignation of individuals to the clades. Our results present a new perspective on the taxonomic status of species in Holothuria (Holothuria), and show how a molecular approach, combined with a morphological approach, can solve taxonomic problems.     

A novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers for the enrichment and determination of polychlorinated biphenyls in fish samples

The novel reductive graphene oxide‐based magnetic molecularly imprinted poly(ethylene‐co‐vinyl alcohol) polymers (rGO@m‐MIPs) were successfully synthesized as adsorbents for six kinds of polychlorinated biphenyls (PCBs) in fish samples. rGO@m‐MIPs was prepared by surface molecular imprinting technique. Besides, Fe₃O₄ nanoparticles (NPs) were employed as magnetic supporters, and rGO@Fe₃O₄ was in situ synthesis. Different from functional monomer and cross‐linker in traditional molecularly imprinted polymer, here, 3,4‐dichlorobenzidine was employed as dummy molecular and poly(ethylene‐co‐vinyl alcohol) was adopted as the imprinted polymers. After morphology and inner structure of the magnetic adsorbent were characterized, the adsorbent was employed for disperse solid phase extraction toward PCBs and exhibited great selectivity and high adsorption efficiency. This material was verified by determination of PCBs in fish samples combined with gas chromatography‐mass spectrometry (GC‐MS) method. According to the detection, the low detection limits (LODs) of PCBs were 0.0035–0.0070 µg l⁻¹ and spiked recoveries ranged between 79.90 and 94.23%. The prepared adsorbent can be renewable for at least 16 times and expected to be a new material for the enrichment and determination of PCBs from contaminated fish samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Overexpression of a novel imprinted gene,<Emphasis Type="Italic">PEG10</Emphasis>, in human hepatocellular carcinoma and in regenerating mouse livers

Many of the promising applications of the microarray technology are pertinent to identifying abnormalities in gene expression that contribute to malignant progression. We developed a bioinformatics tool to identify differentially expressed genes in human hepatocellular carcinoma (HCC). This involved the construction of a liver EST database (http://lestdb.nhri.org.tw) and in silico verification of differentially expressed genes with a human hepatoma microarray database. The stringency of the search was reinforced with a statistical analysis. A novel imprinted gene,Paternally Expressed 10 (PEG10) was identified as having an elevated level of expression in the majority of the HCC samples and was also induced to express during G₂/M phase of regenerating mouse liver. Ectopic expression ofPEG10 in 293T cells affects cell cycle progression. PEG10 is distributed in the cytosol and associates with the nuclear membrane. This is the first time that an imprinted gene has been found to reexpress in both human HCC and in the regenerating mouse liver. This result indicates that the induction of the paternally imprinted gene may play an important role during liver regeneration or carcinogenesis of the human hepatocyte. Understanding the molecular basis of the abnormal imprinting ofPEG10 will shed new light on the process that leads to liver disease.