Diversity of the glycine/tyrosine-rich keratin-associated protein 6 gene (<Emphasis Type="Italic">KAP6</Emphasis>) family in sheep

Keratin-associated proteins (KAPs) are structural components of wool and variation in them may affect wool characteristics. In this study, we used PCR-SSCP to analyse the ovine KAP6 family which encodes glycine and tyrosine-rich KAPs. Five unique PCR-SSCP patterns were detected in the 250 sheep investigated. Between two and five patterns were observed in individual sheep and none with only one pattern was detected. This suggests the amplicons were heterogeneous and derived from more than one locus. To analyse these heterogeneous PCR amplicons, a sequencing approach using SSCP to separate individual amplified sequences, was developed. Using this approach, five DNA sequences (A–E) representing five unique PCR-SSCP patterns were obtained. D was identical to a published ovine KAP6-1 sequence (GenBank accession no. M95719), whereas the others were novel, but the closest homology was with KAP6 sequences from human, sheep, goats and cattle. The five ovine KAP6 sequences could be assigned into three distinct groups. B and D were identical to each other, with the exception of a 57-bp deletion/insertion and a single nucleotide polymorphism (SNP) in the 3′-UTR region. These appear to be allelic variants of ovine KAP6-1. A and C could form another group, as they were similar to each other (with only one synonymous SNP), but different to the other sequences. This group appears to be related to a sheep KAP6 amino acid sequence, and represent allelic variation at another KAP6 locus (designated KAP6-2). The remaining sequence E did not show high sequence homology with either the KAP6-1 or KAP6-2 sequences, but exhibited homology with a bovine KAP6-3 sequence, with the exception of a deletion/insertion of 30 nucleotides. This suggests that E represents ovine KAP6-3. This sequence was detected in only 11% of the sheep investigated, suggesting either a KAP6-3 null allele, or failure to amplify allleles. These results suggest that ovine KAP6 is a complex gene family, that is not only comprised multiple loci, but that is also polymorphic.     

成年核移植山羊生长相关基因的表达分析

体细胞核移植过程有可能影响克隆动物生长相关基因尤其是印迹基因的表达水平。本研究运用同源引物 PCR 扩增、RACE 技术并结合同源克隆策略, 克隆了 7 个山羊生长相关基因包括 3 个印迹基因(H19、IGF2 和 IGF2R)和 4 个非印迹基因(IGF1、IGF1R、GHR 和 GHSR)的完全 CDS 或者部分 cDNA 序列, 经生物信息学技术确认后, 用荧光实时定量 PCR 对 8只成年克隆山羊中这些基因的表达水平进行分析, 结果表明 3 个印迹基因中 IGF2R 基因表达水平极显著高于对照组的自然繁殖山羊(P<0.01), 而 H19 和 IGF2 的表达则没有很大区别; 4 个非印迹基因中只有 IGF1R 的表达水平极显著高于对照组(P<0.01), IGF1、GHR 和 GHSR 的表达与对照组相似。表明即使在表型正常的成年克隆动物也存在一定的表观遗传异常。通过对获得完全 CDS 和 3′UTR 的 IGF2 基因经过生物信息学分析表明, 山羊 IGF2 基因包含一个 540 bp 的开放阅读框 (ORF)编码 179 个氨基酸。IGF2 基因 cDNA 序列和氨基酸序列以及其它基因部分序列比较分析表明, 山羊所有这些基因与绵羊的同源性要高于同牛的同源性。     

Characterization and SNP Identification of Part of the Goat Melanophilin Gene

The melanophilin (MLPH) gene has been characterized as the candidate gene for dilute coat color in some species, but little is known about it in the goat. In this study, part of the genomic DNA sequence (19,289 bp) containing the whole coding region of the MLPH gene from goat, as well as from sheep, was determined. We found 16 exons and 15 introns; the coding region was 1767 bp distributed in 15 exons (2–16). In sheep, the length of part of the genomic DNA sequence was 16,988 bp, with 16 exons and 15 introns, and the coding region was 1833 bp, distributed in 15 exons (2–16). Dozens of SNPs as well as some noticeable motifs in the goat MLPH gene were found during the process of sequencing and polymorphism screening. Based on the SSR Tool, three simple sequence repeat motifs were detected in the goat and sheep DNA sequences. Compared with cattle, we found insertions of 4 amino acids in goats and 26 amino acids in sheep.     

Polymorphisms of caprine <Emphasis Type="Italic">GDF</Emphasis>9 gene and their association with litter size in Jining Grey goats

The exons 1, 2 and flanking region of growth differentiation factor 9 (GDF9) gene in five randomly selected does of Jining Grey, Boer and Liaoning Cashmere goats were amplified and analyzed. Thirteen nucleotide differences were identified in GDF9 gene between sheep (AF078545) and goats. Four SNPs (G3288A in intron 1, G423A, A959C [Gln320Pro] and G1189A [Val397Ile] in exon 2) were detected in four goat breeds with different prolificacy, in which G3288A was a new SNP in goats. The results showed that loci 3288, 423 and 1189 in Boer goats, loci 3288 and 423 in Guizhou White goats, loci 423 and 1189 in Liaoning Cashmere goats were all in complete linkage disequilibrium (D′ = 1, r ² = 1), respectively. In moderate (Boer goat) and low prolificacy (Liaoning Cashmere goat) breeds, linkage analysis indicated that there were more fervent linkage disequilibrium among loci 3288, 423 and 1189 than high prolificacy (Jining Grey and Guizhou White goats) breeds. For the 959 locus, the genotype distribution showed obvious difference between high prolificacy breeds and moderate or low prolificacy breeds (P < 0.05 or P < 0.01). The Jining Grey goat does with genotype CC or AC had 0.81 (P < 0.01) or 0.63 (P < 0.01) kids more than those with genotype AA, respectively. The present study preliminarily showed an association between allele C at 959 locus of GDF9 gene and high litter size in Jining Grey goats. These results provide further evidence that the GDF9 gene may be significantly correlated with high prolificacy in goats.     

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.     

Analysis on DNA sequence of <Emphasis Type="Italic">TSHB</Emphasis> gene and its association with reproductive seasonality in goats

Thyroid stimulating hormone beta chain (TSHB) is mainly expressed in pituitary and its expression is closely related to photoperiodic control of seasonal reproduction in animals. In the present study, ten primer pairs have been used to clone the DNA sequence and to detect genetic mutations of goat TSHB gene. Two DNA fragments of goat TSHB gene were obtained, which were 2,614 and 1,031 bp in length, respectively. They comprised about 2.5 kb 5′ regulatory region, all of the three exons and two introns. Goat TSHB gene has a coding region of 417 bp, encoding 138 amino acids which was predicted to be a secretory protein with a signal peptide of 16 amino acids. The sequence of TSHB gene is highly conserved among mammals. In addition, five mutations (C53A, 3 bp Indel at the 287–289 locus, 34 bp Indel at the 584–617 locus, A1819C and E2_72TA) were found in goat TSHB gene and they were shown to be in strong linkage disequilibrium. Interestingly, the genotype distributions for both single locus and haplotype have shown to be significant different between seasonal and nonseasonal goat breeds. And haplotype H2 and diplotype H2/H4 may be related to year-round estrus. We preliminarily presumed that the five closely linked mutations of goat TSHB gene may be part of the causal sources for the diversities of reproductive seasonality in goats. Our study may provide a possible efficient genetic way to decrease seasonality in goats.     

Functional characterization of a new holin-like antibacterial protein coding gene <Emphasis Type="Italic">tmp1</Emphasis> from goat skin surface metagenome

We have identified a holin-like gene from a goat skin surface metagenome. The ORF designated tmp1 coding for 34 amino acids shared sequence similarity with putative holin-like toxin genes. To analyze the antibacterial activity of tmp1 encoded protein, this ORF was cloned and expressed in Escherichia coli BL21(DE3). The expressed gene product Tmp1 exhibited antibacterial activity against Gram-positive bacteria but not to Gram-negative bacteria. A single transmembrane domain (TMD) was identified within Tmp1 and deletion analysis of the N-terminal region and TMD indicated TMD to be responsible for antibacterial activity. The TMD-dependent antibacterial activity was validated using a synthetic peptide with the amino acid sequence of TMD. Besides antibacterial activity, Tmp1 also complemented the function of holin in a lysis-defective bacteriophage lambda. To broaden the spectrum of antibacterial activity, a mutant library of tmp1 was generated by random mutagenesis. Four mutants with amino acid substitutions at the N-terminus of Tmp1 exhibited increased antibacterial activity against Gram-positive and Gram-negative bacteria and were not hemolytic. An improved activity of these mutant proteins is attributed to their increased hydrophobicity.     

Molecular Characterization of the Full-Length Coding Sequence of the Caprine Laminin Receptor Gene (RPSA)

Scrapie is a prion disease in sheep and goats. Ribosomal protein SA (RPSA), also called 37 kDa laminin receptor precursor/67 kDa laminin receptor has been demonstrated to be a putative cell surface receptor for prion. To investigate the caprine RPSA, we cloned the full-length coding sequence of the gene of goat and submitted it to GenBank. The length of the open reading frame is 888 bp, encoding 295 amino acids. The putative amino acid sequence is highly similar to that of other mammals. The caprine amino acid sequence of RPSA is shown to be identical to the sequence of species susceptible to scrapie at positions 241, 272, and 291. The phylogenetic tree analysis revealed that the genetic distance between sheep and goat is the smallest. Moreover, RT-PCR results of 11 tissues indicated that RPSA mRNA is expressed in all selected caprine 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.