刀鲚POMC基因的cDNA克隆及其应激应答

为了研究刀鲚(Coilia nasus)阿黑皮素原(proopiomelanocortin,POMC)基因的应激应答及调控,克隆了刀鲚POMC基因的c DNA全长。刀鲚POMC基因的c DNA全长2030 bp,编码区699 bp,编码232个氨基酸。二级结构预测显示,刀鲚POMC包括信号肽(signal peptide)、N末端区(N-terminal region,NPP)、促肾上腺皮质激素结构域(adrenocorticotropic hormone,ACTH)、β-脂解素(β-LPH)、α-促黑素(α-MSH)、促肾上腺皮质激素样垂体中叶多肽(CLIP)、γ-脂解素(γ-LPH)、β-促黑素(β-MSH)和β-内咖肽(β-EP)结构域。运用荧光定量的方法检测了该基因在刀鲚不同组织中的表达分布。结果显示,POMC在健康刀鲚的脑高表达,鳃、肾、精巢相对高表达,肝、脾、肠、头肾、肌肉和卵巢中微量表达。运输胁迫后,POMC基因的表达量显著下降(P0.05);10‰ NaCl组4 h和6 h POMC基因的表达量显著上调(P0.05),6 h的表达量恢复至应激前。POMC基因在脂肪代谢及应激调控中发挥重要的功能,本研究结果可为该基因在刀鲚后续育种及应激调控工作提供重要基础。     

团头鲂促肾上腺皮质激素释放激素受体基因序列特征及表达分析

促肾上腺皮质激素释放激素受体(corticotropin-releasing hormone receptor,CRHR)是鱼类下丘脑–垂体–头肾调控轴上的重要应激调节因子。本研究通过c DNA末端快速扩增(RACE)技术成功克隆出团头鲂(Megalobrama amblycephala)CRHR1 m RNA全长序列,应用生物信息学方法对其序列特征进行解析;同时采用荧光定量PCR技术分析了团头鲂CRHR1的组织分布图谱及外源性皮质醇注射模拟应激处理下团头鲂CRHR1 m RNA的表达变化。研究结果表明,CRHR1 m RNA序列开放阅读框(open reading frame,ORF)为1290 bp,编码429个氨基酸。团头鲂CRHR1氨基酸序列与鲤科鱼类的CRHR1氨基酸序列同源性最高;该受体具有7次横跨膜结构和氨基端激素受体结构域。CRHR1在垂体中表达量最高,其次为下丘脑,在心脏、肝、脾等组织中表达丰度较低。经外源性皮质醇注射后,实验组血糖和血清皮质醇显著高于对照组,在处理2 h后达到峰值;实验组血清ACTH水平与对照组差异总体不显著,但呈现先升高后下降。应激处理后,CRHR1转录水平在4种组织中的表达变化存在差异;垂体中CRHR1在早期出现明显的表达抑制,在下丘脑中则呈现先缓慢升高后缓慢下降的趋势,而心脏和头肾中CRHR1在早期则表现出表达迅速上调而后缓慢下降的趋势。本研究进一步丰富了CRHR在鱼类研究方面的基础资料,为鱼类应激调控提供了理论基础。     

Neuropeptides regulating the activity of goldfish corticotropes and melanotropes

The goldfish (Carassius auratus) has proven an advantageous model for investigations of the neuroendocrine regulation of pituitary hormone secretion in teleost fishes. Investigations examining the secretion of adrenocorticotropin (ACTH) and melanocyte-stimulating hormone (MSH) from pituitary cellsin vitro have been used to identify neuropeptides influencing goldfish corticotrope and melanotrope activity. Ovine CRF, urotensin I (UI), arginine vasotocin (AVT), isotocin and angiotensins I and II stimulate the release of ACTH from corticotropesin vitro. Thyrotropin-releasing hormone (TRH), oCRF, UI and neuropeptide Y stimulate the release of MSH from melanotropesin vitro. Immunocytochemical studies have revealed the presence of separate CRF- and UI-immunoreactive perikarya in the hypothalamus suggesting the existence of two structurally similar, yet distinct, hypothalamic CRF-UI-like peptides. Interactions of AVT and CRF in the regulation of ACTH secretion is suggested from studies demonstrating the co-localization of AVT- and CRF-immunoreactivities in perikarya of the preoptic-hypophyseal system. These investigations demonstrate that the secretory activity of goldfish corticotropes and melanotropes is influenced by a diversity of neuropeptides of hypothalamic origin.     

Primary structure and thermostability of bigeye tuna myoglobin in relation to those of other scombridae fish

ABSTRACT:   In the present study, the cDNA encoding myoglobin (Mb) of bigeye tuna Thunnus obesus was cloned and its amino acid sequence deduced in order to investigate the relationship between the primary structure and thermostability of scombridae fish Mb. An open reading frame of bigeye tuna Mb cDNA contained 444 nucleotides encoding 147 amino acids. The primary structure of bigeye tuna Mb was highly conserved when compared with those of bluefin tuna and yellowfin tuna Mb, the sequence identity being 95.2–100.0%. It also showed relatively high identity (82.3–89.1%) with the counterparts of scombridae fish. Myoglobin was then isolated from the dark muscle of four scombridae fish including bigeye tuna. Differential scanning calorimetry and circular dichroism measurements on these Mb revealed that the thermostability of bigeye tuna Mb was lowest and that of skipjack Katsuwonus pelamis Mb highest among the scombridae fish Mb examined. The α-helical contents of scombridae fish Mb at 10°C were in the range of 39.8–44.8%, clearly lower than that of horse Mb (55.3%), suggesting instability of fish Mb. The melting temperatures of these Mb fell in the range of 75.7–79.9°C, lower than that of horse Mb (84.2°C). These results strongly suggest the instability of fish Mb.     

Molecular cloning of a cDNA encoding vitellogenesis-inhibiting hormone in the whiteleg shrimp Litopenaeus vannamei and preparation of its recombinant peptide using an E. coli expression system

The cDNA encoding a precursor of Liv-SGP-G, the most abundant crustacean hyperglycemic hormone-family peptide in the sinus gland of the whiteleg shrimp Litopenaeus vannamei, was cloned by RT-PCR coupled with 5′- and 3′-RACE. The determined cDNA sequence consisted of 621 nucleotides comprising a 69-bp 5′-untranslated region, a 357-bp open reading frame, and a 195-bp 3′-untranslated region. The deduced sequence of 72 amino acid residues corresponding to mature Liv-SGP-G was completely identical to that of natural Liv-SGP-G, determined in our previous study. The recombinant Liv-SGP-G was thereafter heterologously expressed in Escherichia coli, and its vitellogenesis-inhibiting activity in ex vivo cultured ovarian fragments was examined. The recombinant peptides inhibited vitellogenin gene expression with almost the same efficacy as that of natural Liv-SGP-G purified from sinus glands.     

Tumors of the hypophysis as the cause of both Cushing's syndrome and diabetes insipidus in dogs]

Invasive tumors of the pituitary gland associated with Cushing's disease and diabetes insipidus are described in ten dogs. All patients showed typical clinical symptoms including polyuria/polydipsia and acanthosis nigricans. All tumors led to compression und partial destruction of the posterior lobe of the pituitary gland and the infundibular stalk; two of them infiltrated the hypothalamic region. All tumors produced ACTH and caused hyperadrenocorticism. In six cases, additional expression of beta-lipoprotein and MSH were found: in pars intermedia adenomas many cells stained strongly for MSH and/or beta-lipoprotein, whereas in tumors of the pars distalis only occasional cells stained positive. The purpose of the present study was to describe the neuropathological findings and the immunohistochemistry of hormone excretion in pituitary tumors in dogs resulting in Cushing's disease associated with D.i., to review the literature and to discuss the pathogenesis.     

Distinct expression of GnRH genes in the red seabream brain

This paper reports the molecular cloning of a cDNA encoding the precursor of seabream gonadotropin-releasing hormone (prepro-sbGnRH) and the localization of salmon GnRH (sGnRH) and seabream GnRH (sbGnRH) expressing neurons in the brain of the red seabream (Pagrus major). The cloned prepro-sbGnRH cDNA has a 285 bps open reading frame encoding a 23 amino acid signal peptide, a 10 amino acid sbGnRH, the cleavage site (Gly-Lys-Arg), and a 59 amino acid GnRH-associated peptide. The expression of sGnRH and sbGnRH peptides, and prepro-sGnRH and prepro-sbGnRH mRNA were studied using immunocytochemistry and non-radioactive in situ hybridization, respectively. We found cell bodies that reacted positively with both the sGnRH cRNA probe and anti-sGnRH serum, but not with the sbGnRH cRNA probe or anti-sbGnRH serum in the ganglion of the terminal nerve. Cell bodies that reacted positively with the sbGnRH cRNA probe, anti-sbGnRH serum, and anti-sGnRH serum, but negatively with the sGnRH cRNA probe were found in the preoptic area (POA). Immunocytochemistry showed that a distinct bundle of axons arises in the POA which projected to the pituitary gland. These results suggest that sbGnRH is the most relevant hypophysiotropic form of GnRH.     

Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression

ABSTRACT:   Antimicrobial peptides serve as an important component of the innate immune system of all species by functioning to provide a rapid first line defense against infection. Arthropod antimicrobial peptides have been well described in insects, whereas only a few molecules have been identified in crustaceans. Five variants (types 1–5) of Marsupenaeus japonicus crustin-like peptide cDNA that were obtained from a hemocyte cDNA library and polymerase chain reaction (PCR) amplification are reported here. Marsupenaeus japonicus crustin-like peptide type 1, the predominant type, has a cDNA consisting of 679 nucleotides and an open reading frame consisting of 573 base pairs coding for 191 amino acid residues. Other types contain varying glycine-rich repeats at the N-terminal amino acid sequences. The deduced amino acid sequences of these variants are highly similar to those of Litopenaeus setiferus (80% identity), Litopenaeus vannamei (80% identity) and Carcinus maenas crustins (44% identity). Expression of Marsupenaeus japonicus crustin-like peptide mRNA was detected in hemocytes, but not in the heart, hepatopancreas, gill, fore-gut, mid-gut, muscle, subcuticular epithelium or ovary. The expression level of crustin-like peptide mRNA increased significantly 1, 3 and 7 days post-peptidoglycan feeding as determined by quantitative real-time PCR. These results suggest that crustin-like peptide could have an important role in shrimp defense mechanisms.     

草鱼胰岛素样生长因子_基因克隆及序列分析

采用逆转录－聚合酶链式反应（ＲＴ－ＰＣＲ）方法,从草鱼肝脏的总ＲＮＡ中扩增出胰岛素样生长因子－Ｉ（ＩＧＦ－Ｉ）基因序列,定向克隆至质粒pUC18,测宇了该基因序列,推导期编码的蛋白序列,克隆的cDNA序列编码包括Ｂ,Ｃ,Ａ,Ｄ和Ｅ五个区域的１７个氨基酸,与鲤ＩＧＦ－Ｉ成熟肽比较,核酸序列和氨基酸序列的同源性分别为９３．８％和９７．１％,E区域分析结果表明,所克隆的草鱼ＩＧＦ－Ｉ序列属于ＩＧＦ－ＩＥa-2亚型。     

Immunohistochemical investigation of the pituitary of the sturgeon (Acipenser baeri,Chondrostei)

An immunohistochemical study of the sturgeon (Acipenser baeri) pituitary was undertaken using antisera directed against hormones from various classes of vertebrates, including the only pituitary hormone available from sturgeon, gonadotrophin. A positive reaction was obtained after application of antisera towards the following hormones 1–24 synthetic ACTH (1-24 ACTH), melanophore stimulating hormone (MSH), ovine prolactin (oPRL), ovine growth hormone (oGH), salmon growth hormone (sGH), carp gonadotrophin (cGTH) and its beta subunit (cGTH), sturgeon gonadotrophin (aciGTH), carp thyrotrophin (cTSH) and subunit of the human thyrotrophin (hTSH). The results demonstrate that, in general, the sturgeon pituitary resembles that of teleosts as regards the distribution of the different cell types: ACTH and PRL cells in the rostral pars distalis, GTH, TSH and GH cells in the proximal pars distalis and MSH and PAS-cells in pars intermedia. In addition to the topographical organization of the sturgeon pituitary, this study provides data on the immunological relationships between sturgeon pituitary hormones and those of other vertebrates.     

半滑舌鳎促滤泡激素基因全长cDNA的克隆与组织表达分析

从半滑舌鳎Cynoglossus semilaevis Günther脑垂体中提取总RNA,利用RT-PCR和RACE技术首次克隆得到半滑舌鳎促滤泡激素（FSH）基因全长cDNA序列。半滑舌鳎FSHcDNA全长为541bp,开放阅读框为393bp,编码130个氨基酸（GenBank序列登录号：JQ277933）。发现一个N-糖基化位点：24～27NTT。与其他脊椎动物的FSH成熟肽氨基酸序列同源性比较表明,半滑舌鳎FSH与鲽形目和鲈形目鱼类FSH同源性为42％～49％,与鲤形目和高等脊椎动物FSH同源性为27％～319/5。用MEGA4．0软件构建了NJ树,获得的进化树表明,半滑舌鳎FSH与其他鱼类FSH聚类,亲缘关系较近。实时荧光定量组织表达分析表明,FSHmRNA除在垂体中大量表达外,其他组织也有表达,尤以脑、性腺表达量较高。半滑舌鳎FSHmRNA在垂体以外组织中的广泛表达,暗示FSH可能具有广泛的生理功能。     

黄鳝PLA2基因的克隆及表达分析

利用同源克隆技术和RACE技术克隆黄鳝(Monopterus albus)磷脂酶A2(PLA2)基因的cDNA全长;并采用荧光定量RT-PCR法检测PLA2基因在黄鳝各组织中的表达量情况。结果显示:黄鳝PLA2基因全长cDNA大小为2 606 bp(Gen Bank登录号:KX852397),其中开放阅读框2 205 bp,编码736个氨基酸,预测分子大小为83.623 kD,理论等电点5.84;5'和3'非编码区长度分别为208 bp和193 bp。该蛋白序列没有信号肽和跨膜结构。氨基酸序列比对和系统树分析显示,黄鳝PLA2基因的结构十分保守,黄鳝与大黄鱼和金头鲷的PLA2基因亲缘关系最近,与鼠类和蟾蜍的亲缘关系较远。RT-PCR分析表明,PLA2基因在组织中的表达具有组织特异性,在消化器官前肠组织中的表达最高,在性腺及肌肉组织中表达很少。     

Cloning of a second variant of somatolactin-encoding cDNA from the gilthead seabream Sparus aurata

cDNA clones which include coding sequences of the gilthead seabream (Sparus aurata) somatolactin (SL) have been isolated from a cDNA library prepared from seabream pituitary gland poly (A)⁺ RNA. Flounder – SL cDNA was used as a hybridization probe. The complete nucleotide (nt) sequence of the seabream somatolactin has been determined. The clone encodes a polypeptide of 231 amino acid (aa) residues including 24 amino acid residues of signal peptide. Northern blot hybridization detected one band of approximately 1.8 kb mRNA. By comparing the sequences of this SL cDNA to the one recently published, it is suggested that two variants of the SL exist in seabream. By comparing the sequences of the aa of SL to the deduced aa sequences, it is possible that even a third variant of SL exists in this species.     

Structural and thermodynamic characterization of tropomyosin from fast skeletal muscle of bluefin tuna

ABSTRACT:   Tuna tropomyosin is a mixture of nearly equimolar amounts of two isoforms (designated α and β). cDNA encoding the α form was cloned from bluefin tuna Thunnus thynnus fast skeletal muscle. The full-length cDNA contained 1220 bp, comprising an open reading frame of 855 bp encoding 284 amino acid residues, flanked by 5'-untranslational regions (156 bp) and 3'-untranslational regions (209 bp). The deduced amino acid sequence showed considerably high homology in a range of 93.7–98.6% to those of other vertebrate α-type tropomyosins. In phylogenetic analysis, bluefin tuna tropomyosin showed the closest relationship with the white croaker counterpart. The predicted mass was 32 919 Da, and isoelectric point was 4.50, assuming acetylation of the N-terminus. By differential scanning calorimetry, bluefin tuna tropomyosin gave two major endothermic peaks at 29.3 and 41.5°C, probably caused by the presence of two isoforms. Circular dichroism spectra supported such a unique denaturation profile.     

蓝太阳鱼生长激素全长cDNA的克隆与序列分析

The full length cDNA encoding growth hormone of a freshwater fish, Lepomis cyanellus, (LcGH) was cloned from pituitary RNA with RT-PCR, 3‘ and 5‘ RACE (rapid amplification of cDNA ends). The LcGH cDNA (Genbank No. AY530822), about 989nt (nucleotide) long, consisted of a open reading frame with 615nt long, 5‘and 3‘untranslated regions with 93nt and 224nt long respectively, and a 57nt poly (A) tail. The DNA sequence analysis showed that there are typical Kozak sequence and polyadenylation signal. The pregrowth hormone peptide of 204aa deduced from LcGH cDNA included a putative signal peptide (17aa) locating in its Nterminal. There exist a Asn-Cys-Thr glycosylation site at amino acid 201, and 4 cysteine residues (No. 69, 177, 194, 202) that are essential to construct two S-S bonds in this pregrowth hormone peptide. Homological comparision among LcGH and other species growth hormones showed that There is high homology (more than 85%) between growth hormone of Lepomis cyanellus and that of most perciformes fish, but low homology (less than 70%) in comparison with other species such as Siluriformes and Cypriniformes fish.     

Molecular characterization of southern bluefin tuna myoglobin (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

The primary structure of southern bluefin tuna Thunnus maccoyii Mb has been elucidated by molecular cloning techniques. The cDNA of this tuna encoding Mb contained 776 nucleotides, with an open reading frame of 444 nucleotides encoding 147 amino acids. The nucleotide sequence of the coding region was identical to those of other bluefin tunas (T. thynnus and T. orientalis), thus giving the same amino acid sequences. Based on the deduced amino acid sequence, bioinformatic analysis was performed including phylogenic tree, hydropathy plot and homology modeling. In order to investigate the autoxidation profiles, the isolation of Mb was performed from the dark muscle. The water soluble fraction was subjected to ammonium sulfate fractionation (60–90 % saturation) followed by preparative gel electrophoresis. Autoxidation profiles of Mb were delineated at pH 5.6, 6.5 and 7.4 at temperature 37 °C. The autoxidation rate of tuna Mb was slightly higher than that of horse Mb at all pH examined. These results revealed that tuna myoglobin was unstable than that of horse Mb mainly at acidic pH.