Hypomethylation trends in the intergenic region of the imprinted IGF2 and H19 genes in cloned cattle

Bos taurus is a good model for embryo biotechnologies such as nuclear transfer. However, animals produced from these technologies often suffer from large calf syndrome, suggesting fetal growth dysregulation. The imprinted fetal mitogen IGF2 is clustered with H19 and the two genes are co-regulated in humans and mice. Although the allelic expression pattern of IGF2/H19 has been elucidated in agricultural species such as sheep and cattle, the underlying mechanism of their imprinting regulation has not been characterized. Using bisulfite sequencing the methylation status of 44 CpG sites in a CpG rich intergenic region of IGF2/H19 in the liver, brain, lung, kidney and placenta of control calves (produced by conventional breeding). One fragment containing 16 CpG sites was differentially methylated region (DMR), and thus may be important in regulating IGF2/H19 allelic expression.The DMR in tissues from cloned term calves that either died immediately after birth or were sacrificed due to complications shortly thereafter were examined. There were significant variations in the methylation of this DMR in some of the cloned animals compared to the controls. Most of the observed variations tended toward hypomethylation. The hypomethylation of this DMR in the liver and placenta of clones correlates with the previous observation of abnormal, biallelic expression of the H19 allele in those clones [Zhang, S., Kubota, C., Yang, L., Zhang, Y., Page, R., O’Neill, M., Yang, X., Tian, X.C., 2004. Genomic imprinting of H19 in naturally reproduced and cloned cattle. Biol. Reprod.] but not with allelic expression of IGF2 (as determined in this study). These data suggest that this DMR is involved in H19 allelic expression, but that other mechanisms probably regulate the expression of IGF2/H19. Contrary to global hypermethylation observed in cloned embryos, putative imprinting control regions can display hypomethylation trends in specific organs of cloned calves.     

Expression of KCNQ1OT1, CDKN1C,H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 imprinting control regions is conserved between human and bovine

Background

Beckwith-Wiedemann syndrome (BWS) is a loss-of-imprinting pediatric overgrowth syndrome. The primary features of BWS include macrosomia, macroglossia, and abdominal wall defects. Secondary features that are frequently observed in BWS patients are hypoglycemia, nevus flammeus, polyhydramnios, visceromegaly, hemihyperplasia, cardiac malformations, and difficulty breathing. BWS is speculated to occur primarily as the result of the misregulation of imprinted genes associated with two clusters on chromosome 11p15.5, namely the KvDMR1 and H19/IGF2. A similar overgrowth phenotype is observed in bovine and ovine as a result of embryo culture. In ruminants this syndrome is known as large offspring syndrome (LOS). The phenotypes associated with LOS are increased birth weight, visceromegaly, skeletal defects, hypoglycemia, polyhydramnios, and breathing difficulties. Even though phenotypic similarities exist between the two syndromes, whether the two syndromes are epigenetically similar is unknown. In this study we use control Bos taurus indicus X Bos taurus taurus F1 hybrid bovine concepti to characterize baseline imprinted gene expression and DNA methylation status of imprinted domains known to be misregulated in BWS. This work is intended to be the first step in a series of experiments aimed at determining if LOS will serve as an appropriate animal model to study BWS.

Results

The use of F1 B. t. indicus x B. t. taurus tissues provided us with a tool to unequivocally determine imprinted status of the regions of interest in our study. We found that imprinting is conserved between the bovine and human in imprinted genes known to be associated with BWS. KCNQ1OT1 and PLAGL1 were paternally-expressed while CDKN1C and H19 were maternally-expressed in B. t. indicus x B. t. taurus F1 concepti. We also show that in bovids, differential methylation exists at the KvDMR1 and H19/IGF2 ICRs.

Conclusions

Based on these findings we conclude that the imprinted gene expression of KCNQ1OT1, CDKN1C, H19, and PLAGL1 and the methylation patterns at the KvDMR1 and H19/IGF2 ICRs are conserved between human and bovine. Future work will determine if LOS is associated with misregulation at these imprinted loci, similarly to what has been observed for BWS.     

Provisional assignment of the gene for uridine monophosphatase-2 (Umph-2) to mouse chromosome 11

The segregation of the mouse gene for uridine monophosphatase-2 (Umph-2) was examined in 14 independent mouse-Syrian hamster hybrids and 10 hybrid subclones. Umph-2 cosegregated with the mouse galactokinase (Glk) gene in 23 of the 24 hybrids and showed at least four discordances with all other mouse marker isozymes examined. The observed synteny of Umph-2 and Glk, which has also been observed in humans, indicates that the mouse Umph-2 gene is on chromosome 11.     

Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.     

Association study of the oestrogen signalling pathway genes in relation to age at natural menopause

Genetic factors play a significant role in influencing the variation of age at natural menopause (AANM). Estrogen receptor β (ESR2), is an important factor in the mechanism of action of estrogen, while the aromatase gene (CYP19) and the 17-alphahydroxylase gene (CYP17) are involved in the biosynthesis of estrogen. We tested whether polymorphisms of ESR2, CYP19 and CYP17 genes are associated with AANM in Caucasian females. A total of 52 SNPs (17 for ESR2, 28 for CYP19, and 7 for CYP17) were successfully genotyped for 229 Caucasian women having experienced natural menopause. Comprehensive statistical analyses focusing on the association of these genes with AANM were conducted. The effects of age, height and age at menarche on AANM were adjusted when conducting association analyses. We found that six SNPs (2, 6–7, 9, 13 and 16) within ESR2 were not significantly associated with AANM after Bonferroni correction. However, two blocks of ESR2 were associated with AANM. For CYP19, two SNPs (24 and 27) were nominally associated with AANM. No significant association was observed between CYP17 and AANM. Our results suggest that genetic variation in the ESR2 and CYP19 genes may influence the variation in AANM in Caucasian women.     

Haploinsufficient and predominant expression of multiple endocrine neoplasia type 1 (MEN1)-related genes, MLL, p27 and p18 in endocrine organs

Multiple endocrine neoplasia type 1 (MEN1) is a rare autosomal dominantly inherited syndrome characterized by parathyroid, gastro-entero-pancreatic and anterior pituitary tumors. Although the tissue selectivity of tumors in specific endocrine organs is the very essence of MEN1, the mechanisms underlying the tissue-selectivity of tumors remain unknown. The product of the Men1 gene, menin, and mixed lineage leukemia (MLL) have been found to cooperatively regulate p27^Kip1/CDKN1B (p27) and p18^Ink4C/CDKN2C (p18) genes. However, there are no reports on the tissue distribution of these MEN1-related genes. We investigated the expression of these genes in the endocrine and non-endocrine organs of wild-type, Men1 knockout and MLL knockout mice. Men1 mRNA was expressed at a similar level in endocrine and non-endocrine organs. However, MLL, p27 and p18 mRNAs were predominantly expressed in the endocrine organs. Notably, p27 and MLL mRNAs were expressed in the pituitary gland at levels approximately 12- and 17-fold higher than those in the liver. The heterozygotes of Men1 knockout mice the levels of MLL, p27 and p18 mRNAs did not differ from those in the wild-type mice. In contrast, heterozygotes of MLL knockout mice showed significant reductions in p27 mRNA as well as protein levels in the pituitary and p27 and p18 in the pancreatic islets, but not in the liver. This study demonstrated for the first time the predominant expression MEN1-related genes, particularly MLL and p27, in the endocrine organs, and a tissue-specific haploinsuffiency of MLL, but not menin, may lead to a decrease in levels of p27 and p18 mRNAs in endocrine organs. These findings may provide basic information for understanding the mechanisms of tissue selectivity of the tumorigenesis in patients with MEN1.     

Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5-8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 degrees C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.     

日龄及性别对橘小实蝇实验种群飞行能力的影响

利用飞行磨系统对实验种群橘小实蝇的1、6、11、16、21、26、31日龄的7个雌雄个体分别进行22h连续吊飞试验,测定其累计飞行距离、累计飞行时间、平均飞行速度。橘小实蝇原始种群采自湖南长沙,经室内连续饲养约30代,成为试验种群。试验环境条件为连续光照条件,温度25℃,RH 60%—80%,光照强度为990 lx。整个吊飞过程不补充食物和水份。共得到179组吊飞数据,其中雌虫86组,雄虫93组,每个日龄处理不少于9头。结果显示随着日龄的增加,橘小实蝇雌蝇和雄蝇的飞行能力均逐渐增加,到达16日龄后雌蝇飞行能力迅速下降,表现为单峰型;雄蝇的飞行能力在16—31日龄一般维持较高水平,但在26日龄有一定波动。16日龄雌蝇和雄蝇的累计飞行距离分别是(2485.6±2287.2)m和(2152.3±1773.3)m。其中1头21日龄雄性个体的累计飞行距离最大,完成了8795.80 m的飞行。雌蝇累计飞行距离最远的发生在16日龄,为8116.6 m。日龄对橘小实蝇的累计飞行距离和平均飞行速度的影响都达到了显著水平(前者,F=2.88,P0.05;后者,F=4.98,P0.01)。雌蝇和雄蝇的平均飞行速度的差异达到了显著水平(F=4.10,P0.05)。日龄和性别的互作对平均飞行速度的影响也达到了显著水平(F=2.74,P0.05)。尽管实验种群不能完全代表野生种群,研究结果对研究橘小实蝇扩散规律及综合防治,特别是改进橘小实蝇雄性不育技术仍有重要的参考价值。     

Heterologous expression of the mutated melon ethylene receptor gene Cm-ERS1/H70A produces stable sterility in transgenic lettuce (Lactuca sativa)

The mutated melon ethylene receptor gene Cm-ERS1/H70A was introduced into tobacco and induced stable sterility in transgenic lines. This gene contains a missense mutation that converts the His(70) residue to Ala in the melon ethylene receptor gene Cm-ERS1. To test the applicability of this inducible sterility system to other plants, lettuce (Lactuca sativa) was transformed with the gene using Agrobacterium, and putative transformants containing Cm-ERS1/H70A were obtained. Thirteen randomly selected putative transformants were grown in a growth room under constant conditions, and seven of the lines showed sterility or significantly reduced fertility. DNA gel blot analysis confirmed the integration of the Cm-ERS1/H70A gene into the genomes of the putative transformants, and RT-PCR and protein gel blot analysis confirmed the expression of Cm-ERS1/H70A mRNA and protein in all of the transformants. Five transformants showing sterility or reduced fertility when grown in a growth room under constant conditions were randomly selected to be grown in an open-air greenhouse under various environmental conditions. All five showed stable sterility under the various conditions. These results suggest that Cm-ERS1/H70A can induce sterility in heterologous transgenic plants.     

Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution

The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event. Correspondence to: M. Pauly     

Stoneworts (Characeae) and associated macrophyte species as indicators of water quality and human activities in the Pays-de-la-Loire region, France

Two ecologically and morphologically distinct forms of Furcellaria lumbricalis and loose form of Coccotylus truncatus were experimentally tested to obtain information on the growth rates and influence of the habitat depth on their eco-physiological activity. Incubations were carried out in the area inhabited by a loose F. lumbricalis–C. truncatus community in Kassari Bay of the West-Estonian Archipelago Sea, the NE Baltic Sea. During the incubation period (20.04–21.10.2002) loose forms of F. lumbricalis and C. truncatus showed similar dynamics of both growth rate of biomass and primary production while attached form of F.␣lumbricalis had, as a rule, significantly lower growth rate and primary production values. The highest eco-physiological activity was recorded from the shallowest incubation depth (4 m) for all three algal forms. All three tested algal forms had similar pattern of growth during the incubation period – highest growth rates were detected in spring and early summer while during the rest of the incubation period algal biomass was in a steady state when production balanced degradation processes.     

Characterisation and expression of the paired box protein 7 (Pax7) gene in polymorphic Arctic charr (Salvelinus alpinus)

Arctic charr (Salvelinus alpinus L.) from Lake Thingvallavatn, Iceland occur as four distinct morphs: large benthivorous (LB), dwarf benthivorous (DB), piscivorous (PI) and planktonivorous (PL). The morphs differ with respect to body size, head morphology, growth rate, and life history. The aim of this study was to investigate the paired box protein 7 (Pax7) gene as a candidate for such polymorphisms due to its importance in cranio-facial, skeletal muscle, and central nervous system development. No variation in coding and intronic sequences was found between morphs. We identified 10 alternate Pax7 isoforms with insertions/deletions: a four-residue (GNRT) deletion, a GEASS insertion truncated by the first serine residue (GEAS), and a thirteen-residue insertion (GQYA/TGPEYVYCGT). The latter insertion with a threonine (T) contains a putative casein kinase II (CK-2) phosphorylation site. Pax7 spatial expression patterns were identical in embryos of DB-, LB-, and PL-morphs, and were similar to those described for zebrafish Pax7c, but a difference in temporal expression for segmentation was observed between DB and LB morphs. At the end of segmentation, novel expression was observed in the mandibular region as two bilateral domains. The potential role of multiple alternative splicing of the Pax7 gene for the generation of different Arctic charr morphs is briefly discussed.     

Genetic mapping and protein product diversity of the self-incompatibility locus in wild tomato (Lycopersicon peruvianum)

Phenotypic diversity of self-incompatibility (S) alleles within nine natural populations ofLycopersicon peruvianum was investigated. Only 7 incompatible responses were observed of a total of 276 unique combinations tested, on the basis of controlled pollinations, indicating the large number of alleles that exist within these populations. Molecular weight polymorphism for specific major stylar proteins observed on SDS-PAGE was also evident in two of the populations examined. Five proteins were shown to map to theS locus and to be associated with differentS alleles through controlled pollinations and segregation of the proteins. Two of theseS related proteins had been described previously in terms of spatial and temporal expression consistent with their involvement in self-incompatibility (Mauet al., Planta 169, 184–191, 1986). A mapping population derived from a fully compatible cross was used to establish linkage of theS locus to two DNA markers,CD15 andTG184, that lie on chromosome 1. The order of the markers and estimates of map distances are given.     

Molecular cloning,expression analysis and chromosome localization of the <Emphasis Type="Italic">Tpt1</Emphasis> gene coding for the pig translationally controlled tumor protein (TCTP)

This work describes the cloning and structural analysis of a Tpt1 cDNA coding for the porcine translationally controlled tumor protein (TCTP) molecule and its expression in porcine cells and tissues. Pig Tpt1 cDNA is 842-pb long that displays typical features of translationally controlled mRNAs, including a 5′-UTR containing a 5′-terminal oligopyrimidine tract (5′-TOP), and a 3′-UTR with a high CG-content and one AU rich element (ARE). Both 5′-UTR and 3′-UTR are highly conserved when they are compared with those of other mammals. The pig Tpt1 cDNA contains a 516-b open reading frame that encodes a predicted TCTP protein composed of 172 amino acids that exhibits extensive conservation compared with TCTP sequences from other species and a common structural feature with all the other TCTP proteins analyzed in mammals. Expression analysis demonstrated that Tpt1 mRNA is ubiquitously expressed in normal porcine tissues and cells, showing a higher expression in spleen, lymph nodes and lung, and a lower one in skin and heart. The pig Tpt1 gene localizes on the porcine chromosome 11, region p11.