Molecular cloning and expression analysis of bovine programmed death‐1

Recent work has shown that PD‐1, an immune inhibitory receptor, is involved in mechanisms for down‐regulating immune responses during tumor progression or chronic viral infection. However, in the case of bovine diseases, there have been no reports on this molecule due to lack of information about bovine PD‐1. In this study, we performed identification and preliminary characterization of the bovine PD‐1 gene in two breeds of cattle. We cloned full cDNA sequences encoding for PD‐1 from both Holstein‐Friesian and Japanese Black breeds, and found that both of the genes encoded a 282‐amino acid protein, which had a signal sequence, transmembrane domain and an immunoreceptor tyrosine‐based inhibitory motif. This bovine PD‐1 showed 72.9% and 65.6% homology to human and mouse PD‐1, respectively, both of which have been well characterized and documented. Quantitative real‐time PCR analysis showed that bovine PD‐1 is expressed predominantly in T‐cells (such as CD4⁺ and CD8⁺ cells) and among PBMCs, and is strongly upregulated on T‐cell stimulation via ConA. A limited number of cattle were tested yet, as expected, the degree of PD‐1 mRNA expression in CD4⁺ and CD8⁺ T‐cells was greater in cattle with bovine leukemia virus‐induced lymphoma than in uninfected cattle. Further studies to characterize the functions of bovine PD‐1 are therefore warranted, in order to elucidate the mechanism of the immunosuppression associated with progression of several diseases and therapy in cattle.     

Molecular cloning and expression analysis of CD82 in pig

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.     

Molecular cloning, expression and evolutionary analysis of the avian tyrosine kinase JAK1

The Janus protein tyrosine kinases (JAK) constitute a protein family that plays a pivotal role in signalling of a large number of cytokine receptors. The cDNA of the chicken homologue of JAK1 was cloned and its nucleotide sequence determined. Chicken JAK1 protein comprises 1150 amino acids as deduced from its cDNA sequence with a calculated molecular mass of 133 kDa. The overall structure of JAK proteins exemplified by the JAK homology domains JH1–JH7 is also preserved in chicken JAK1. Additionally, phylogenetic analysis demonstrates that chicken JAK1 is more closely related to mammalian JAK1 than to those of fish, exhibiting 80%, 79% and 63% identity in amino acid sequence to human, mouse and zebrafish JAK1, respectively. JAK1 proteins were found to be most conserved in the kinase (JH1) and pseudokinase (JH2) domains. This data is supported by Southern hybridization studies of ZOO blots. Chicken JAK1 shows a ubiquitous expression pattern and is transcribed as a 5.5 kb mRNA in various tissues and cell types. JAK1 expression was particularly high in lymphoid cells.     

Molecular cloning and expression analysis of novel wheat cysteine protease

A cDNA clone encoding a novel papain-like cysteine protease was isolated from wheat germ (Triticum aestivum). This cDNA encoded a 371-residue protein, designated WCP2, composed of signal peptide followed by a propeptide and a mature protease containing active site residues that are highly conserved among the papain family. The mature WCP2 protein (26 kDa) was detected in the quiescent embryo and its level of expression in the germinating embryo was greatly increased.     

Molecular cloning and expression of phospholipase C epsilon 1 in zebrafish

Phospholipase C (PLC) is a key enzyme that generates inositol 1,4,5-triphosphate (IP₃) and diacylglycerol (DAG), two second messengers in signal transduction. Phospholipase Cε (PLCE1) is a unique member of the PLC family in that it carries both RasGEF and Ras-associating (RA) domains and may serve as an activator and an effector of small GTPases. Recently, mutations in PLCE1 have been associated with early-onset nephrotic syndrome. We have identified the zebrafish ortholog of PLCE1 and characterized its expression pattern in zebrafish embryos by in situ hybridization. Zebrafish plce1 gene encodes a protein of 2248 amino acids that shares 56% identity with mammalian PLCε. During zebrafish embryonic development, plce1 is expressed in a dynamic pattern in a variety of organs, such as brain, muscle, liver, and pronephric glomeruli. Our results suggest that zebrafish may be used as a model organism to address the function of PLCε during the development of these organs.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

Molecular cloning of preproacrosin and analysis of its expression pattern in spermatogenesis

Complementary DNA clones for the boar preproacrosin have been isolated from a randomly primed testis cDNA library in lambda gt10 and from an oligo(dT)-primed testis cDNA in lambda gt11. The nucleotide sequence of the 1418-bp cDNA insert includes a 46-bp 5'-untranslated region, an open reading frame of 1248 bp corresponding to 416 amino acids (45.59 kDa) and a 121-bp 3'-untranslated region. The deduced amino acid sequence includes the active-site residues histidine, asparagine and serine of the catalytic triad of the serine proteinase super-family and is colinear with that determined by amino acid sequencing of the boar acrosin light chain and of a small region of the NH2-terminal sequence of the heavy chain. The preproacrosin cDNA contains at the 3' end a 381-bp sequence which codes for an amino acid sequence not yet found in any other serine proteinase. This amino acid sequence is rich in proline (42 out of 127 amino acids) and is suggested to be involved in the recognition and binding of the spermatozoa to the zona pellucida of the ovum. The mRNA for preproacrosin is synthesized as an approximately 1.6-kb-long molecule only in the postmeiotic stages of boar and bull spermatogenesis.     

管氏肿腿蜂毒液精氨酸激酶基因的克隆与表达分析

为了解精氨酸激酶作为寄生蜂毒液蛋白的功能,本研究克隆了管氏肿腿蜂毒液精氨酸激酶基因的开放阅读框,分析其表达特征,并测定了该毒液蛋白在毒液中的酶活性。克隆获得的毒液精氨酸激酶基因的开放阅读框长1 068 bp,编码了355个氨基酸,其理论分子量为39.71 kDa,等电点为5.86,并具有精氨酸激酶典型的N端和C端结构域,以及精氨酸和ADP结合位点、ATP-胍基磷酸转移酶活性位点和高度保守的天冬氨酸和精氨酸残基。系统进化分析表明,膜翅目精氨酸激酶分为两个亚家族,管氏肿腿蜂精氨酸激酶属于亚家族1。荧光定量PCR分析结果显示,该毒液蛋白基因在卵、幼虫、蛹、雌成虫中的表达量逐渐上升,至羽化10 d时达最高。在不同组织中,该基因在头部和毒液器官中的表达量较高。通过酶活测定发现,管氏肿腿蜂毒液中精氨酸激酶的酶活性为5.18 U/g蛋白。该研究有助于今后揭示寄生蜂毒液精氨酸激酶的功能与作用机制。