cDNA cloning and overexpression of acidic ribosomal phosphoprotein P1 gene (RPLP1) from the giant panda

RPLP1 is one of acidic ribosomal phosphoproteins encoded by RPLP1 gene, which plays an important role in the elongation step of protein synthesis. The cDNA of RPLP1 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology, which was also sequenced, analyzed preliminarily and expressed in E.coli. The cDNA fragment cloned is 449bp in size, containing an open reading frame of 344bp encoding 114 amino acids. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other five species studied, including Homo sapiens, Mus musculus, Rattus norvegicus, Bos Taurus and Sus scrofa. The homologies for nucleotide sequences of Giant Panda PPLP1 to that of these species are 92.4%, 89.8%, 89.0%, 91.3% and 87.5%, while the homologies for amino acid sequences are 96.5%, 94.7%, 95.6%, 96.5% and 88.6%. Topology prediction showed there are three Casein kinase II phosphorylation sites and two N-myristoylation sites in the RPLP1 protein of the Giant Panda (Ailuropoda melanoleuca). The RPLP1 gene was overexpressed in E. coli and the result indicated that RPLP1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 18kDa polypeptide, which was in accordance with the predicted protein and could also be used to purify the protein and study its function.     

Giant panda ribosomal protein S14: cDNA, genomic sequence cloning, sequence analysis, and overexpression

RPS14 is a component of the 40S ribosomal subunit encoded by the RPS14 gene and is required for its maturation. The cDNA and the genomic sequence of RPS14 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology and touchdown-PCR, respectively; they were both sequenced and analyzed. The length of the cloned cDNA fragment was 492 bp; it contained an open-reading frame of 456 bp, encoding 151 amino acids. The length of the genomic sequence is 3421 bp; it contains four exons and three introns. Alignment analysis indicates that the nucleotide sequence shares a high degree of homology with those of Homo sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, and Danio rerio (93.64, 83.37, 92.54, 91.89, 87.28, 84.21, and 84.87%, respectively). Comparison of the deduced amino acid sequences of the giant panda with those of these other species revealed that the RPS14 of giant panda is highly homologous with those of B. taurus, R. norvegicus and D. rerio (85.99, 99.34 and 99.34%, respectively), and is 100% identical with the others. This degree of conservation of RPS14 suggests evolutionary selection. Topology prediction shows that there are two N-glycosylation sites, three protein kinase C phosphorylation sites, two casein kinase II phosphorylation sites, four N-myristoylation sites, two amidation sites, and one ribosomal protein S11 signature in the RPS14 protein of the giant panda. The RPS14 gene can be readily expressed in Escherichia coli. When it was fused with the N-terminally His-tagged protein, it gave rise to accumulation of an expected 22-kDa polypeptide, in good agreement with the predicted molecular weight. The expression product obtained can be purified for studies of its function.     

Comparative Analysis and Molecular Characterization of a Gene BANF1 Encoded a DNA-Binding Protein During Mitosis from the Giant Panda and Black Bear

Barrier to autointegration factor 1 (BANF1) is a DNA-binding protein found in the nucleus and cytoplasm of eukaryotic cells that functions to establish nuclear architecture during mitosis. The cDNA and the genomic sequence of BANF1 were cloned from the Giant Panda (Ailuropoda melanoleuca) and Black Bear (Ursus thibetanus mupinensis) using RT-PCR technology and Touchdown-PCR, respectively. The cDNA of the BANF1 cloned from Giant Panda and Black Bear is 297 bp in size, containing an open reading frame of 270 bp encoding 89 amino acids. The length of the genomic sequence from Giant Panda is 521 bp, from Black Bear is 536 bp, which were found both to possess 2 exons. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to some mammalian species studied. Topology prediction showed there is one Protein kinase C phosphorylation site, one Casein kinase II phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Giant Panda, and there is one Protein kinase C phosphorylation site, one Tyrosine kinase phosphorylation site, one N-myristoylation site, and one Amidation site in the BANF1 protein of the Black Bear. The BANF1 gene can be readily expressed in E. coli. Results showed that the protein BANF1 fusion with the N-terminally His-tagged form gave rise to the accumulation of an expected 14 kD polypeptide that formed inclusion bodies. The expression products obtained could be used to purify the proteins and study their function further.     

Expression, purification, and evaluation for anticancer activity of ribosomal protein L31 gene (RPL31) from the giant panda (Ailuropoda melanoleuca)

Ribosomal protein L31 gene is a component of the 60S large ribosomal subunit encoded by RPL31 gene, while ribosomal protein L31 (RPL31) is an important constituent of peptidyltransferase center. In our research, the cDNA and the genomic sequence of RPL31 were cloned successfully from the giant panda (Ailuropoda melanoleuca) using RT-PCR technology respectively, following sequencing and analyzing preliminarily. We constructed a recombinant expression vector contained RPL31 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product was purified to obtain recombinant protein of RPL31 from the giant panda. Recombinant protein of RPL31 obtained from the experiment acted on human laryngeal carcinoma Hep-2 and human hepatoma HepG-2 cells for study of its anti-cancer activity by MTT [3-(4, 5-dimehyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide] method. Then observe these cells growth depressive effect. The result indicated that the cDNA fragment of the RPL31 cloned from the giant panda is 419 bp in size, containing an open reading frame of 378 bp, and deduced protein was composed of 125 amino acids with an estimated molecular weight of 14.46-kDa and PI of 11.21. The length of the genomic sequence is 8,091 bp, which was found to possess four exons and three introns. The RPL31 gene can be readily expressed in E.coli, expecting 18-kDa polypeptide that formed inclusion bodies. Recombinant protein RPL31 from the giant panda consists of 157 amino acids with an estimated molecular weight of 17.86 kDa and PI of 10.77. The outcomes showed that the cell growth inhibition rate in a time- and dose-dependent on recombinant protein RPL31. And also indicated that the effect at low concentrations was better than high concentrations on Hep-2 cells, and the concentration of 0.33 μg/mL had the best rate of growth inhibition, 44 %. Consequently, our study aimed at revealing the recombinant protein RPL31 anti-cancer function from the giant panda, providing scientific basis and resources for the research and development of cancer protein drugs anti-cancer mechanism research. Further studies of the mechanism and the signal transduction pathways are in progress.     

A newly identifiedMinute locus,M(2)32D,encodes the ribosomal protein L9 inDrosophila melanogaster

A gene encoding a ubiquitously expressed mRNA inDrosophila melanogaster was isolated and identified as the gene for ribosomal protein L9 (rpL9) by its extensive sequence homology to the corresponding gene from rat. TherpL9 gene is localized in polytene region 32D where two independent P element insertions flanking the locus are available. Remobilization of either P element generated lines with a typicalMinute phenotype, e.g. thin and short bristles, prolonged development, and female semisterility in heterozygotes as well as homozygous lethality. All these characteristics can be rescued when a 3.9 kb restriction fragment containing therpL9 gene is reintroduced by P element-mediated germline transformation. This result confirms thatM(2)32D codes for ribosomal protein L9.     

Nucleotide sequence of cDNA encoding the mitochondrial precursor protein of the ATPase inhibitor from the giant panda (Ailuropoda melanoleuca)

Mitochondrial ATP synthase (F1Fo-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In the present study, using RT-PCR combined with in silico cloning, we isolated and sequenced the cDNA encoding the inhibitor protein of the giant panda (Ailuropoda melanoleuca). The deduced protein sequence showed that the protein is composed of 106 amino acids and the estimated molecular weight of the ATPIF(1) protein is 12.32 kDa with an isoelectric point (pI) of 10.17. Alignment analysis revealed that the deduced protein sequence shares 66%, 78.3%, 66%, 72.6%, 77.4%, and 78.3% homology with that of Mus musculus, Pan troglodytes, Rattus norvegicus, Bos taurus, Macaca mulatta, and Homo sapiens, respectively. Topology prediction showed that there are three protein kinase C phosphorylation sites, one amidation site, three N-myristoylation sites, one casein kinase II phosphorylation site, and one tyrosine kinase phosphorylation site in the ATPase inhibitor. In particular, amino acids in the region between 39 and 72, which is the minimum sequence showing ATPase inhibitory activity, were highly conserved in the protein.     

A newly identifiedMinute locus,M(2)32D, encodes the ribosomal protein L9 inDrosophila melanogaster

A gene encoding a ubiquitously expressed mRNA inDrosophila melanogaster was isolated and identified as the gene for ribosomal protein L9 (rpL9) by its extensive sequence homology to the corresponding gene from rat. TherpL9 gene is localized in polytene region 32D where two independent P element insertions flanking the locus are available. Remobilization of either P element generated lines with a typicalMinute phenotype, e.g. thin and short bristles, prolonged development, and female semisterility in heterozygotes as well as homozygous lethality. All these characteristics can be rescued when a 3.9 kb restriction fragment containing therpL9 gene is reintroduced by P element-mediated germline transformation. This result confirms thatM(2)32D codes for ribosomal protein L9.     

大熊猫和亚洲黑熊TNNC1 基因的克隆、表达与序列分析

慢肌肌钙蛋白C (Troponin C type 1,TNNC1)具有高度保守性,调控骨骼肌慢肌和心肌的收缩,影响肌蛋白的生成,从而可能导致动物肌肉的生长、进化和功能的差异。本研究以大熊猫和亚洲黑熊骨骼肌为材料,提取总RNA 和基因组DNA,运用RT-PCR 和Touch-down PCR 分别扩增出TNNC1 基因的cDNA 序列和结构基因序列,并且构建了含有TNNC1 cDNA 的重组表达载体,转化进入E. coli BL21 进行超表达研究。结果表明大熊猫TNNC1 基因的cDNA 片段长602 bp,包含一个编码161 个氨基酸的开放阅读框,其结构基因全长2 831 bp,包含6 个外显子和5 个内含子。亚洲黑熊TNNC1 基因的cDNA 片段长486 bp,亦包含一个编码161 个氨基酸的开放阅读框,其结构基因全长2 758 bp,同样包含6 个外显子和5 个内含子。该两个物种的TNNC1 基因与已报道的13种动物的TNNC1 基因具有很高的相似性。拓扑预测表明,大熊猫和亚洲黑熊TNNC1 蛋白有1 个蛋白激酶C 磷酸化位点,5 个酪蛋白激酶Ⅱ磷酸化位点,1 个N-豆蔻酰化位点,3 个EF 手性钙结合域及1 个N - 糖基化位点。将TNNC1 基因在大肠杆菌中表达发现TNNC1 蛋白与氮端多聚组氨酸标签蛋白(His6) 融合成大小为23. 5kD 左右的多肽,这与预期结果相一致。本研究结果为进一步深入探讨大熊猫和亚洲黑熊TNNC1 基因及蛋白的结构、功能和进化关系提供资料。     

内蒙古白绒山羊TSC2基因克隆与表达模式分析

旨在克隆内蒙古白绒山羊TSC2基因cDNA并分析其特性及基本表达模式.利用RT-PCR分段克隆TSC2基因cDNA片段并测序,将得到的cDNA各片段核苷酸序列拼接后获得绒山羊TSC2基因编码区全长序列(HQ684023)并进行生物信息学分析.半定量RT-PCR方法检测TSC2基因在不同组织中的表达特异性.结果表明内蒙古白绒山羊TSC2基因cDNA编码区核苷酸序列为5184 bp,包含了编码1727个氨基酸残基的全长ORF.核苷酸序列与牛、猪、马、大熊猫、犬、恒河猴、人、小鼠及大鼠的同源性分别为97％、90％、89％、88％、87％、87％、87％、86％和86％.NCBI CDD程序预测该基因编码的蛋白质有一个Tuberin结构域和一个Rap-GAP结构域;Psite程序分析有5个N糖基化位点、2个cAMP和cGMP依赖蛋白激酶磷酸化位点、16个蛋白激酶C磷酸化位点、25个酪蛋白激酶磷酸化位点.PSORT程序预测其定位于胞内体膜.TSC2基因在内蒙古白绒山羊的睾丸、脑、肝脏、肺、乳腺、脾和肾脏等组织中都有表达,mRNA丰度在睾丸中较高,乳腺中较低.     

The primary structure of rat ribosomal protein L9

The amino acid (aa) sequence of rat ribosomal (r) protein L9 was deduced from the nucleotide (nt) sequence in a recombinant cDNA and confirmed from the N-terminal aa sequence of the protein. L9 contains 192 aa and has an Mr of 21879. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 20-23 copies of the L9 gene. The mRNA for the protein is about 800 nt in length. Rat L9 is related to Saccharomyces cerevisiae YL11, Methanococcus vannielii L6, Escherichia coli L6 and other members of the prokaryotic L6 family. The protein contains a possible internal duplication of 11 aa.