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.     

Molecular and DNA methylation analysis of <Emphasis Type="Italic">Peg10</Emphasis> and <Emphasis Type="Italic">Xist</Emphasis> gene in sheep

Peg10 is a maternally imprinted gene located in the imprinted domain of human chromosome 7q21 and mouse proximal chromosome 6. It is predominantly expressed in, and participates in the formation of, the placenta. Moreover, Peg10 is overexpressed in hepatocellular carcinoma, and is involved in hepatocarcinogenesis. The large noncoding RNA Xist has been shown to direct the female mammalian X chromatosome dosage compensation pathway. In the present study, we obtained partial cDNA sequences of sheep Peg10 and Xist. mRNA expression analysis in nine organs showed that they were universally expressed in two-day old lambs. The mRNA expression profile of Peg10 showed similar tissue specificity to pig, but was different compared with human and mouse. We concluded that the Peg10 mRNA expression profile was species specific. However, there was little difference in Xist expression between nine tissues of female lambs. Using bisulfite sequencing, we revealed that the first exon of Xist was either completely methylated or completely unmethylated, indicating that the newly obtained fragment of Xist was also differentially methylated in sheep as the DMR of Peg10. We did not find tissue specific DNA methylation of Xist, consistent with the Xist mRNA expression profile.     

Identification of the ovine KAP11-1 gene (<Emphasis Type="Italic">KRTAP11</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) and genetic variation in its coding sequence

Keratin-associated proteins (KAPs) are a structural component of the wool fibre and form the matrix between the keratin intermediate filaments (KIFs). The gene encoding high sulphur-protein KAP11-1 has been identified in human, cattle and mouse, but not yet in sheep, despite the economic importance of wool. In this study, PCR using primers based on the cattle KAP11-1 gene sequence produced an amplicon of the expected size with sheep DNA. Upon using PCR–Single Stranded Conformational Polymorphism (PCR–SSCP) analysis in 260 sheep, six different PCR–SSCP patterns were detected. Either one or a combination of two banding patterns was observed for each sheep, suggesting they were either homozygous or heterozygous for this gene. Sequencing of the amplicons confirmed the occurrence of six DNA sequences. All of these were unique, and the greatest homology was with KRTAP11-1 sequences from cattle, human and mouse, suggesting that they were derived from the ovine KAP11-1 gene and were allelic variants. The ovine KAP11-1 gene had an open reading frame of 477 nucleotides encoding 159 amino acids. The putative protein was rich in serine, cysteine, and threonine which account for 18.2–18.9, 12.6 and 12.0 mol%, respectively. Of these, approximately 20 of the serine and threonine residues might be phosphorylated. Five nucleotide substitutions were identified, and one was non-synonymous and would result in an amino acid change at a potential phosphorylation site. The genetic variation found in KRTAP11-1 may influence its expression, protein structure, and/or post-translational modifications, and consequently affect wool fibre structure and wool traits.     

Molecular characterization of the sheep <Emphasis Type="Italic">CIB1</Emphasis> gene

The calcium and integrin binding protein 1(CIB1), is an EF-hand-containing protein that binds many effector proteins including the platelet αIIbβ3 integrin and potentially regulates their functions. Here we report the cloning and characterization of the sheep CIB1 gene. The CIB1 cDNA is 885-bp in size, containing a 45-bp of 5′ untranslated region (UTR), a 264-bp long 3′-UTR and a 576-bp open reading frame that encodes 191 amino acids. The sheep CIB1 cDNA shows 98.3, 92.0, 91.8, 91.3, 90.5 and 90.1% of similarity, at the nucleotide level, to its equivalents in cattle, pigs, rhesus monkey, humans, rats and mice, respectively at the deduced protein level, the corresponding values are more than 94%. The sheep CIB1 gene consisted of seven exons. Quantitative PCR (Q-PCR) showed that CIB1 was widely expressed in different tissues with the highest level in the testis, suggesting that it may play a role in ram fertility. We cloned the sheep CIB2, CIB3 and CIB4 genes and detected their expression patterns in different tissues.     

Molecular Cloning,Expression and Chromosomal Localization of Mouse <Emphasis Type="Italic">MM-1</Emphasis>

The protooncogene product Myc associates with many proteins. The isolation of the mouse MM-1; c-Myc binding protein (Myc-Modulator 1) cDNA is described. The cDNA contains a 462 bp open reading frame that encodes a polypeptide of 154 amino acid residues. The deduced amino acid sequence indicates that mouse MM-1 has a 99% identity with the sequence of human MM-1. The expression of mouse MM-1 mRNA was detected in the fetal liver, but its level was 3-fold higher than that in the normal adult liver, and was slightly increased after a partial hepatectomy. It is expressed widely in a variety of adult mouse tissues. Thus, MM-1 may play a role in liver development and growth. A bioinformatics analysis indicates that mouse MM-1 gene consists of 6 exons. Furthermore, the chromosomal location of the mouse MM-1 gene was on the F2-F3 band of chromosome 15, as determined by fluorescence in situ hybridization. The nucleotide sequence data reported in this paper appear in DDBJ, EMBL, and the GenBank nucloetide sequence databases with the following accession number, AF108357.     

Cloning and ontogenetic expression of the uncoupling protein 1 gene <Emphasis Type="Italic">UCP1</Emphasis> in sheep

The uncoupling protein 1 (UCP1) is an indicator of brown adipocytes and is involved in the control of body temperature and regulation of energy balance. It abundantly expresses in newborns and has important functions in adults. However, little information was known on UCP1 gene expression in young and adolescent sheep. In this study, we cloned and identified the full-length DNA and cDNA sequences of the ovine UCP1 gene, which were 6659 bp and 1621 bp, respectively, and predicted the location of the gene on chromosome 17. Forty-eight animals with an equal number of males and females each for both Guangling Large Tail sheep (GLT) and Small Tail sheep Han (STH) sheep were used to study the ontogenetic expression of UCP1 mRNA in eight adipose tissues by quantitative real-time polymerase chain reaction (PCR). The results showed that the mRNA was expressed in all tissues studied and at all stages from 2 to 12 months of age. Nevertheless, the mRNA in perirenal fat was expressed significantly higher than that in other tissues and lower in superficial fat than in deep deposits. The highest expression was observed in animals at 2 months of age and then decreased gradually with age. Global expression in GLT was significantly higher than that in STH. Interactions between tissue and breed and age also influenced the mRNA expression significantly. In addition, the mRNA expression was associated with the single nucleotide polymorphism (SNP) haplotypes detected in the cDNA of the gene.     

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.     

Class I major histocompatibility complex cDNA clones from sheep thymus: alternative splicing could make a long cytoplasmic tail

To investigate the class I major histocompatibility complex (MHC) genes expressed in the young sheep thymus, a cDNA library was screened with a human HLA-B7 cDNA probe under conditions of relaxed stringency. Thirteen clones were isolated and found by partial sequences to fall into five classes, requiring the expression of at least three loci. One sequence was found six times, almost half of the total, and may thus represent the major message expressed in the young sheep thymus. One of the clones was found to have failed to excise the intron between cytoplasmic exons 7 and 8, leading to the predicted synthesis of a cytoplasmic domain 23 amino acids longer than the other sheep sequences, and 15 amino acids longer than any cytoplasmic domain previously described. The sequences of all the clones were found to be most similar to bovine, and least similar to mouse class I MHC sequences.The nucleotide sequence data reported in this paper have been sunmitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M 34672-6.     

Imprinting regulation of the murine Meg1/Grb10 and human GRB10 genes; roles of brain-specific promoters and mouse-specific CTCF-binding sites

The imprinted mouse gene Meg1/Grb10 is expres sed from maternal alleles in almost all tissues and organs, except in the brain, where it is expressed biallelically, and the paternal allele is expressed preferentially in adulthood. In contrast, the human GRB10 gene shows equal biallelic expression in almost all tissues and organs, while it is almost always expressed paternally in the fetal brain. To elucidate the molecular mechanisms of the complex imprinting patterns among the different tissues and organs of humans and mice, we analyzed in detail both the genomic structures and tissue-specific expression profiles of these species. Experiments using 5′-RACE and RT–PCR demonstrated the existence in both humans and mice of novel brain- specific promoters, in which only the paternal allele was active. The promoters were located in the primary differentially methylated regions. Interest ingly, CTCF-binding sites were found only in the mouse promoter region where CTCF showed DNA methylation-sensitive binding activity. Thus, the insulator function of CTCF might cause reciprocal maternal expression of the Meg1/Grb10 gene from another upstream promoter in the mouse, whereas the human upstream promoter is active in both parental alleles due to the lack of the corresponding insulator sequence in this region.     

A pore-forming protein,perforin, from a non-mammalian organism,Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.     

Genomic imprinting of the insulin-like growth factor 2 gene in sheep

A number of genes in the human and mouse genomes are subject to genomic imprinting, with selective inactivation of one allele of a gene in a parent-of-origin specific manner. One of the first imprinted genes identified was the Insulin-like Growth Factor 2 gene (IGF2), which promotes growth of the fetus and is expressed from only the paternal allele in most tissues in both the mouse and human. The aim of this study was to establish the imprinting status of IGF2 in sheep (Ovis aries). Sheep provide an interesting model to study imprinting, owing to differences in their placental development and the fact that they have been subject to strong artificial selection for various production traits. We report the identification of a length polymorphism in the transcribed 3′-untranslated region of the ovine IGF2 gene. This polymorphism was used to map IGF2 to sheep Chromosome (Chr) 21 and demonstrate that IGF2 is indeed imprinted in sheep, being expressed from the paternal allele. We also report that the developmental switch from imprinted IGF2 expression in the fetal liver to biallelic IGF2 expression in the adult liver, which occurs in the human but not mouse, also occurs in sheep. Differences in male- and female-specific recombination values reported around the IGF2 locus in the human were also observed around the ovine IGF2 locus. The techniques developed in this study will enable the imprinting status of IGF2 to be assessed in a variety of tissues and stages of development in normal sheep. Received: 3 October 1998 / Accepted 29 January 1999     

<Emphasis Type="Italic">Cdkn1c</Emphasis> (<Emphasis Type="Italic">p57</Emphasis><Superscript><Emphasis Type="Italic">Kip2</Emphasis></Superscript>) is the major regulator of embryonic growth within its imprinted domain on mouse distal chromosome 7

Background  

Cdkn1c encodes an embryonic cyclin-dependant kinase inhibitor that acts to negatively regulate cell proliferation and, in some tissues, to actively direct differentiation. This gene, which is an imprinted gene expressed only from the maternal allele, lies within a complex region on mouse distal chromosome 7, called the IC2 domain, which contains several other imprinted genes. Studies on mouse embryos suggest a key role for genomic imprinting in regulating embryonic growth and this has led to the proposal that imprinting evolved as a consequence of the mismatched contribution of parental resources in mammals.     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22