东亚钳蝎中一个中性哺乳动物神经毒素全长cDNA的克隆和分析

构建了东亚钳蝎毒腺cDNA文库,根据东亚钳蝎中性哺乳动物神经毒素BmKM4的氨基酸序列设计并合成引物,用PCR方法从文库中筛选到BmKM4全长cDNA序列.它由5′UTR、可读框和3′UTR组成.与其他东亚钳蝎哺乳动物神经毒素cDNA的相应区域相比,BmKM4cDNA的5′UTR高度保守,而其3′UTR则变异较大.AUG的旁侧序列为AAAATGAA,与绝大多数蝎毒素基因一致.在BmKM4mRNApoly(A)尾上游17nt处,有一典型的腺苷化信号(AATAAA).可读框编码84个氨基酸的毒素前体,包括N端19个氨基酸残基组成的信号肽,中间64个氨基酸残基组成的成熟毒素,以及C末端的额外碱性氨基酸Arg.椐据一般规律,尾端Arg在毒素前体的成熟过程中会被切除。 Abstract:A full-length cDNA sequence encoding the precursor of a neutral mammalian neurotoxin,BmKM4,was first isolated from a cDNA library made from thc venom gland of Chinese scorpion Buthus martensii Karsch.ABmKM4-specific primer and a primer corresponding to the partial sequence of pSPORT1 vector were used as forward primer and reverse primer,respectively,to screen the cDNA library by PCR reaction.Sequence analysis of positive clones showed that the BmKM4 cDNA is composed of three parts:5'UTR,open reading frame and 3'UTR.Compared with the corresponding regions of other scorpion mammalian neurotoxin cDNAs,the 5'UTR of BmKM4 cDNA is highly conservative versus highly variable for 3'UTR.The lateral sequence of initiation codon (AUG) is AAATGAA which is in consistent with that of most scorpion toxin genes.On the 3'-end,a putative polyadenylation signal (AATAAA) was 1Tnt upstream of Poly (A) tail.The open reading frame encodes a precursor of 84 amino acid residues,including a signal peptide of 19 residues,a mature toxin(BmKM4) of 64 residues,and a basic residue (Arg) tailwhich would be removed in the processing step.     

一个新的东亚钳蝎毒素(BmKT_1)全长cDNA的克隆和分析

首先构建了东亚钳蝎毒腺组织 c DNA文库 ;根据已知的东亚钳蝎哺乳动物毒素氨基酸序列保守区设计引物 ,并用 PCR从 c DNA文库中扩增出一个 c DNA片段作为筛选 c DNA文库的探针 ;从 c DNA文库中筛选到二个编码同一个新的蝎毒素多肽的 c DNA,它们除 3′- UTR外 ,其余序列完全一致 .它们均含有 2 55bp长的开放阅读框 ,编码 85肽的前体毒素 ,包括 1 9个氨基酸残基的信号肽 ,66个残基的成熟毒素 (命名为 Bm KT1) ;Bm KT1氨基酸序列与已知的蝎毒素具有较大的同源性 ,与 Bm KM1,Lqq ,Lqhα IT和 Bm K M10 的同源性分别为 77%、67%、67%和 65% .Bm KT1的 C端不存在末端修饰步骤且具有一个与这些毒素不相同的特征结构 ,即在末端延伸了两个氨基酸残基 - P- S,推测 Bm KT1具有新的活性功能特征 .     

中国对虾蜕皮抑制激素全长cDNA的克隆及序列分析

对虾的蜕皮活动由蜕皮抑制激素和蜕皮激素调控,蜕皮抑制激素是甲壳动物CHH家族神经肽的成员之一,通过抑制Y器官蜕皮激素的合成而调节蜕皮,以中国对虾（Fennropenaeus chinensis）眼柄总RNA为材料,采用cDNA末端快速扩增（RACE）方法。首次得到蜕皮抑制激素的全长cDNA（GenBank登录号：AF469187）。该全长cDNA大小为697bp,是由320bp的3′RACE产物和468bp的5′RACE产物拼接而成,Blast搜索结果显示,该全长cDNA与甲壳动物的MIH基因序列具有较高的相似性,用Clustal X进行多序列比较结果表明,由该全长cDNA推导的氨基酸序列与对虾类的MIH的氨基酸序列同源性最高,其中与日本对虾,斑节对虾,刀额新对虾MIH的同源性分别为95.1%,83.1%,79.1%,根据以上数据,推断该697bp的全长cDNA为编码中国对虾MIH前体的cDNA。进一步序列分析表明,编码中国对虾MIH前体cDNA包括312bp的开放阅读框,81bp的3′UTR和302bp的5′UTR;编码103个氨基酸的MIH前体分子包括信号肽和成熟肽,信号肽由28个氨基酸组成,成熟肽由75个氨基酸组成,成熟肽中6个半胱氨酸非常保守。     

两个东亚钳蝎抗哺乳动物神经毒素的cDNA序列

从我国山东东亚马氏钳蝎尾腺中分离纯化ｍＲＮＡ,经逆转录构建了ＢｍＫ蝎毒ｃＤＮＡ文库。利用ＰＣＲ扩增,筛选到两个抗哺乳动物毒素的ｃＤＮＡ基因,并测定了序列。这两个ｃＤＮＡ阅读框均为２５２ｂｐ组成,可翻译８４肽的毒素前体,包括Ｎ端１９个氨基酸组成的信号肽,６４个氨基酸残芭的成熟毒蛋白,以及Ｃ端一个额外的碱性氨基酸Ａｒｇ。其中由一ＣＤＮＡ所推导的氨基酸序列（ＢｍＫＭ１）一已知的天然毒素ＢｍＫ１蛋白序列完     

东亚钳蝎K+通道阻断肽cDNA的克隆与表达

目的：克隆东亚钳蝎毒素基因,以进一步研究其生物学和药理学功能。方法：利用已知蝎神经毒素基因序列,设计引物,用RT-PCR方法克隆从蝎毒腺组织蝎毒素cDNA。结果：成功地克隆了一个新的东亚钳蝎毒素基因,该基因开放阅读框架编码59个氨基酸残基,其中前22个为信号肽,成熟肽为37个氨基酸残基,经PCR扩增除去信号肽序列,克隆到pTreHisA质粒中,在E．coli中表达了分子质量为7ku左右融合蛋白,表达产物占菌体总蛋白的21％左右。结论：其结构中含有三对二硫链,6个Cys残基组成蝎K^ 通道毒素共同特征序列-CXXXC-、-GXC-、-CXC-,推断其为K^ 通道阻断肽,命名为KChTX1。已被Gene-bank收录,收录号为AY129234。     

东亚钳蝎蝎毒素BmKBT基因组序列的克隆及其分析

东亚钳蝎 (ButhusmartensiiKarsch ,BmK)蝎毒素BmKBT(又名BmKabT)是一个在初级结构上相似于β类哺乳动物毒素和功能接近于α类哺乳动物毒素的Na+ 通道毒素 .基于从毒腺cDNA文库中筛选得到的全长BmKBT前体核苷酸序列设计引物 ,以蝎基因组总DNA为模板进行聚合酶链式反应 (PCR) ,将PCR产物克隆至T载体、测序 .序列分析表明 :在BmKBT信号肽编码区的 3′端的- 4位Gly密码子的第 1位与第 2位碱基中有 1个长 2 2 5nt的内含子 ,插入位点距离该基因的起始密码子 4 6nt ,AT含量为 78 7% ,其内含子可能的剪接分枝位点距离 3′剪接受体位点 4 7nt.内含子的大小及其基因组织结构分析表明 :BmKBT具有与α类哺乳动物毒素类似的基因组织结构 ,进一步说明BmKBT是一个介于α类和β类Na+ 通道毒素之间的中间型蝎毒素 ,可以作为研究蝎毒素分子进化的合适材料     

大口黑鲈脂代谢相关基因NPY、UCP2、LPL、HL克隆与分子进化分析

采用RT-PCR和RACE方法克隆了大口黑鲈NPY基因cDNA全序列及UCP2、LPL、HL基因cDNA核心片段。序列分析结果表明,大口黑鲈NPY基因cDNA全序列长664 bp,其中5′端非翻译区(5′-UTR)长53 bp,3′端非翻译区(3′-UTR)长311 bp,开放阅读框(ORF)长300 bp,编码99个氨基酸,即前体NPY。大口黑鲈前体NPY包括三个部分,28个氨基酸组成的信号肽、36氨基酸组成的成熟NPY以及32个氨基酸组成的由Gly-Lys-Arg指示的NPY C端肽（CPON）。大口黑鲈UCP2、LPL、HL基因cDNA核心片段长度分别为737 bp、509 bp和666 bp,各自编码245个氨基酸、169个氨基酸和222个氨基酸。将4个基因的氨基酸序列分别与其他物种的氨基酸序列进行同源性比较,并通过MEGA3构建系统树,对这4个脂代谢相关基因的分子进化特征进行了探讨。     

马氏珠母贝galectin-4基因cDNA的克隆及表达分析

半乳糖凝集素(Galectins)属于一种多功能凝集素家族,在机体抵抗微生物的感染中起重要作用。本研究利用cDNA末端快速扩增(rapid-amplification of cDNA ends,RACE)技术克隆获得马氏珠母贝galectin-4(PmGal-4)基因cDNA的全长,并对其序列进行分析。PmGal-4基因cDNA全长1 071 bp,5′非翻译区(5′UTR)长75 bp,3′非翻译区(3′UTR)长72 bp;其开放阅读框(open reading frame,ORF)长度为924 bp,编码307个氨基酸组成的前体肽,理论分子量约为34.6 kD,理论等电点为8.80。多序列比对结果显示各物种间galectin-4具有高保守性。序列分析结果显示PmGal-4的氨基酸序列具有典型富含半胱氨酸(cysteine-rich domain,CRD)的结构域。实时荧光定量PCR分析表明,PmGal-4基因在马氏珠母贝所检测的组织中呈组成型表达特征,但在闭壳肌和外套膜中表达量最高。上述结果表明PmGal-4基因可能参与了马氏珠母贝多种生理功能,特别是在免疫防御反应中具有重要的作用。     

鸡含锰超氧化物歧化酶cDNA克隆及序列分析

 为弄清鸡含锰超氧化物歧化酶 (manganese containingsuperoxidedismutase ,MnSOD)的cDNA序列 ,以开展动物锰营养学的深入研究 ,根据已知鸡MnSOD的N端氨基酸序列设计简并引物 ,应用 3′RACE(rapidamplificationofcDNAends)技术 ,扩增克隆了鸡心肌MnSOD 990bp的 3′cDNA片段 .再根据 3′RACE片段测序结果设计引物进行 5′RACE ,结果获取了一个与 3′RACE片段相互重叠的鸡心肌MnSOD 52 1bp的 5′RACE片段 ,并对其进行了克隆测序 .最后根据 3′RACE片段和 5′RACE片段序列信息进行拼接 ,从而获取鸡MnSODcDNA的全序列信息 .研究结果表明 :鸡MnSODcDNA全长为 110 8个核苷酸 ,其中 5′非翻译区 2 5个核苷酸 ,编码区 675个核苷酸 ,3′非翻译区 4 0 8个核苷酸 ,编码一个长 2 2 4个氨基酸残基的蛋白质前体 .其中信号肽长 2 6个氨基酸残基 ,成熟肽长 198个氨基酸残基 ,分子量为 2 2kD .与人、大鼠、线虫、果蝇等真核生物MnSOD氨基酸序列的同源性分别为82 4 %、84 .7%、62 .4 %、59.3% .     

不同地域卵黄萤荧光素酶基因的克隆与序列分析

为了比较不同地域萤火虫荧光素酶基因的进化关系,通过GenBank中已知的荧光素酶基因保守区段设计引物,利用5′ RACE（rapid amplification of cDNA ends）和3′ RACE技术克隆了来自云南省文山州和西双版纳州的同种卵黄萤荧光素酶基因cDNA和全基因序列.来自不同地域的2种卵黄萤荧光素酶在基因序列上存在3个不同碱基位点,但是它们编码的荧光素酶只存在1个不同的氨基酸.卵黄萤荧光素酶基因全长（从起始密码子到终止密码子）1998 bp,包含7个外显子,6个内含子,其cDNA序列共1976 bp,包含102 bp 5′ UTR (untranslated region)、1635 bp的荧光素酶基因开放阅读框和239 bp的3′ UTR序列.卵黄萤荧光素酶基因的开放阅读框编码1个544个氨基酸的蛋白质,比同属的其它几种荧光素酶少4个氨基酸. 来自2个不同地域的卵黄萤荧光素酶在进化上是比较保守的,它们与北美萤火虫Photinus pyralis荧光素酶在碱基序列上分别有629%和63%相似性.     

Ectogenesis and the case against the right to the death of the foetus

Ectogenesis, or the use of an artificial womb to allow a foetus to develop, will likely become a reality within a few decades, and could significantly affect the abortion debate. We first examine the implications for Judith Jarvis Thomson’s violinist analogy, which argues for a woman’s right to withdraw life support from the foetus and so terminate her pregnancy, even if the foetus is granted full moral status. We show that on Thomson’s reasoning, there is no right to the death of the foetus, and abortion is not permissible if ectogenesis is available, provided it is safe and inexpensive. This raises the question of whether there are persuasive reasons for the right to the death of the foetus that could be exercised in the context of ectogenesis. Eric Mathison and Jeremy Davis have examined several arguments for this right, doubting that it exists, while Joona Räsänen has recently criticized their reasoning. We respond to Räsänen’s analysis, concluding that his arguments are unsuccessful, and that there is no right to the death of the foetus in these circumstances.     

On the diversity of the Cladocera in the tropics

The mythical concept of an impoverished tropical cladoceran fauna is refuted. On a planetary scale, around half of the cladoceran species presently known occur exclusively in the tropics-subtropics, often with considerable restriction to particular geographical subzones. On a regional (political) scale, the situation is often unclear because of the continued fragmentary nature of studies, and because political units are not a good basis for biogeographical comparisons. At the finest level of resolution (lake-perlake comparisons), there appears to be an upper limit of c. 50 cladoceran species per individual lake. No significant difference between lakes in the temperate zone and in the tropics could be established here. Daphnia is largely absent from the tropics, but is replaced by more Sidids, Moinids, and Bosminids, such that the average cladoceran community in the limnetic zone of a tropical lake is not characterized by less species but rather by lower population densities. This, in turn, is considered a consequence of higher prevalent predation levels in the tropics.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Assessing the Physiological State of the Fucoids from the Barents Sea Littoral by the End of the Polar Night

Methods of amperometry and potentiometric titration were used to follow dark respiration (DR) and apparent photosynthesis (AP) in the fucoids Ascophyllum nodosum (L.) Le Jol, Fucus vesiculosus L., and F. serratus L. from the Barents Sea littoral by the end of the 40-day-long polar night. The macroalgae were shown to manifest species-specific low rates of photosynthesis and respiration. However, in spite of their low photosynthetic status due to the effects of subzero temperature and prolonged low or zero illumination, the macroalgae have been able to restore DR and AP to the initial level already by the day 9; the ability to restore AP depended on the level of illumination. The study of the changes in the carbonate–bicarbonate system in the light and darkness demonstrated that the macroalgae grown in darkness, in contrast to those grown in twilight, could absorb bicarbonate in darkness; however, they lost this capacity after two-day-long illumination at an irradiance of 7 mol/(m² s). Bicarbonate uptake in darkness and the capacity to restore the systems of photosynthesis and respiration in fucoid cells are discussed in the context of algal energy metabolism under the polar night conditions.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Feeding the vertebrate retina from the Cambrian to the Tertiary

The retina of the vertebrate eye is metabolically active and requires nutritive support. During the last 540 million years it has evolved into forms as complicated and nutritionally demanding as those found in avian or primate eyes. Diffusion from the choroid is generally able to supply the metabolic needs of thin retinae. However, when the thickness exceeds the limits of diffusion, structures are needed to supplement the vascular supply from the choroid. These supplemental nutritive devices include the choroidal gland, the falciform process and preretinal vascular plexus of fish, the conus papillaris of lizards, the pecten oculi of birds, the intraretinal vessels of mammals and a few novel systems that remain difficult to classify. These vascular systems are among the most variable features of the vertebrate eye. Here, we review classical and recent findings regarding such retinal nutrition systems, propose a three category classification for them based on histologic origins and speculate on the evolutionary forces which drove their development.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Separation of the pineal vesicle from the wall of the third ventricle during the post-hatching development of the chicken

Summary According to light- and electron-microscopic observations the pineal organ of the 3-day-old chicken consists of a prominent end vesicle and a tapering parenchymal stalk. During this stage the pineal lumen is in open communication with the third ventricle. However, in the 40-day-old chicken, which still possesses a well-developed end vesicle, the proximal portion of the pineal stalk displays regressive changes leading to local fragmentation. At this stage the pineal stalk is reduced, and the pineal lumen is missing. In 1-year-old chickens the parenchyma of the proximal portion of the stalk is further diminished, and in 3-year-old domestic fowl is completely displaced by bundles of collagenous fibers, only some nerve fibers being present. This post-hatching pineal development may reflect the sequence of changes leading from pineal sense organs to pineal glands.This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan