Molecular cloning of the mouse CCK gene: expression in different brain regions and during cortical development.

In this paper we describe experiments that address specific issues concerning the regulation of the mouse cholecystokinin gene in brain and intestine. The mouse cholecystokinin gene was cloned and sequenced. Extensive homology among the mouse, man and rat genes was noted particularly in the three exons and the regions upstream of the RNA start site. RNAse protection assays for each of the three exons were used to demonstrate that CCK is expressed in only a subset of tissues and that the same cap site and splice choices are used in brain, intestine as well as in cerebellum, cortex, midbrain, hypothalamus and hippocampus. CCK RNA was also noted to be detectable in kidney. Thus the same gene using the same promoter is expressed in subsets of cells that differ in their biochemical, morphologic and functional characteristics. The level of expression of CCK was also monitored during mouse cortical development and the appearance of CCK RNA was compared to glutamate decarboxylase (GAD), enkephalin and somatostatin. It was noted that each of these cortical markers was first expressed at different times during cortical development. The appearance of CCK RNA during intestinal development was also measured and found to precede appearance in cortex by several days.     

Molecular cloning and embryonic expression of the Xenopus Arnt gene

Versican, a proteoglycan recently implicated in hair follicle induction, has been shown to influence axon outgrowth in vitro and in vivo. We used immunohistochemistry to study the relationship between versican expression and innervation, during rat vibrissa follicle development and the adult hair cycle. During development, nerve fibres were commonly associated with areas of weak versican expression, and the path of axons appeared to be delineated by sharp boundaries of versican expression. Versican expression changed in the lower follicle dermis during the adult hair follicle cycle but remained strong around the follicle neck reflecting the constant innervation. Our observations show a correlation between versican expression and peripheral innervation indicating that versican may have a dual role in hair follicle biology.     

皮层神经发生的分子调控机制

在高等动物神经发育过程中,背侧端脑神经上皮细胞在多种调控基因及环境调控因子的作用下经历了错综复杂的发育事件,逐步形成具有精细的分层结构和高级功能的神经皮层。这些发育中的调控具有严格的时间和区域特性。本文主要就皮层形成中神经干细胞增殖、分化方向及皮层神经元亚型的特化等方面的调控机制予以论述。     

香鱼hepcidin基因的克隆、序列分析及组织表达特征

Hepcidin是一类富含半胱氨酸的抗菌肽,在鱼类非特异性免疫中起重要作用,具有调节铁代谢的功能。该研究克隆了香鱼hepcidin基因cDNA序列,全长763个核苷酸,包含一个完整的开放阅读框,推测编码一个由85个氨基酸组成的相对分子质量为9.7k的多肽,N端24个氨基酸是信号肽序列。香鱼hepcidin的成熟肽序列由25个氨基酸组成,含有8个半胱氨酸,可形成4个分子内二硫键结构。系统进化树分析表明,hepcidin的物种进化关系与目前接受的物种分类关系基本一致,香鱼hepcidin位于鱼类hepcidin簇中,与大西洋鲑hepcidin的亲缘关系最近,达60%。香鱼hepcidin在肝脏中表达量最大,在脾、肾、心脏和肌肉中也有表达。实时荧光定量PCR(qRT-PCR)结果表明,鳗利斯顿氏菌(Listonella anguillarum)感染以后香鱼肝组织中hepcidin基因mRNA表达量显著增加,在12h增加了8.26倍。hepcidin基因可能在香鱼抗外界病原物感染过程中起重要作用。     

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.     

Molecular cloning,expression pattern and chromosomal mapping of pig CD69

CD69 is a type II membrane protein belonging to C-type lectin family receptor, and expressed on activated leukocytes. Pig CD69 was cloned by RT-PCR using degenerate primers. Pig CD69 cDNA contains a 600 bp open reading frame with its predicted polypeptide sequence of 200 amino acids. Pig CD69 has 75%, 67%, and 57% sequence identity with cow, human, and mouse CD69, respectively. A splicing isoform, which lacks exon 2 encoding the transmembrane domain, was detected. Pig CD69 gene is located on Chromosome (Chr) 5q25 where the NKG2D gene was mapped. In RT-PCR analysis, pig CD69 mRNA was detected in activated PBL, NK cells, macrophages, monocytes, and granulocytes, but not in resting cells. The inducers for CD69 gene expression were PMA, PHA, LPS, G7 mAb, PNK-E mAb, PM16-6 mAb and the K562 cell line. Moreover, CD69 mRNA is expressed in bone marrow, spleen, thymus and lymph nodes but not in muscle, mammary gland, or the pig kidney cell line (LLC-PK(1)). These results indicate that pig Chr 5q25 contains the NK gene complex and CD69 can be used as an activation marker in pig cells of innate as well as acquired immune systems.     

Molecular cloning and mRNA expression pattern of Sox10 in Paramisgurnus dabryanus

A number of genetic studies have established that Sox10 involved in a wide range of developmental processes including sex differentiation and neurogenesis in vertebrates. A Sox10 homologue was cloned from brain of Paramisgurnus dabryanus by using homologous cloning and RACE method, designated as PdSox10. The full-length cDNA of PdSox10 contains a 312 bp 5′ UTR, a 1,476 bp open reading frame (ORF) encoding 492 amino acids and a 262 bp 3′ UTR (Accession no.: JQ217143). The overall topology of the phylogenetic tree shows that the PdSox10 fits within the Sox10 clade. During embryogenesis, PdSox10 gene seemed to be de novo synthesized in the embryos from gastrulae stage. From the somitogenesis stage and thereafter, distinct expression of PdSox10 was observed in the medial neural tube, extending from the hindbrain through the posterior trunk. In adult, PdSox10 mRNA was detected primarily in the gonads, as well as in brain and heart by RT-PCR. In situ hybridization on gonadal sections further demonstrated that PdSox10 is expressed especially in premature germ cells, in early perinucleolus stage oocytes and cortical–alveolar stage oocytes in ovaries and in spermatogonia and spermatocytes in testes. These preliminary findings suggested that PdSox10 is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including neurogenesis and sex differentiation in vertebrates.     

Molecular cloning and mRNA expression pattern of Sox4 in Paramisgurnus dabryanus

Sox4 belonged to the SoxC subfamily of the Sox family, which play important roles in the development of the vertebrate gonad and nervous system. A Sox4 homologue was cloned from brain of Paramisgurnus dabryanus by using homologous cloning and rapid amplification of cDNA ends (RACE), designated as PdSox4. The full-length cDNA was 2163bp, containing the 759bp 5'-untranslated region, 267bp 3'-untranslated region and encoding a putative protein of 378 amino acids with a characteristic high mobility group box (HMG-box) DNA-binding domain of 79 amino acids with the specific motif (RPMNAFMVW). Alignment and phylogenetic analyses indicated that PdSox4 shares highly identical sequence with Sox4 homologues from different species. The signal peptide analysis predicted that PdSox4 is a non-secretory protein. The hydropathy profile of PdSox4 protein revealed that this protein is hydrophilic in nature. The expression profiles of PdSox4 in different developmental stages and various adult tissues of sexs were analyzed by quantitative real-time RT-PCR (qRT-PCR) and In situ hybridization (ISH). The results showed that PdSox4 was ubiquitously expressed during embryogenesis and various adult tissues, especially in central nervous system. Tissue distribution analyses revealed that PdSox4 was expression in developing germ cells. Taken together, these preliminary findings suggested that PdSox4 is highly conserved during vertebrate evolution and involved in a wide range of developmental processes including embryogenesis, neurogenesis and gonad development.