枯草杆菌启动子－信号肽序列的克隆及序列分析

利用含红霉素抗性基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢｏｍＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ的双脱氧链终止法测定了１０个克隆片段的ＤＮＡ顺序,结果表明,克隆的片段都含有启动子、核糖体结合优点及信号肽序列。克隆片段可以在大肠杆菌和枯草杆菌中恢复氨苄青霉素抗性的表型。β－内酰胺酶活力测定结果证明：大肠杆菌的酶活力主要积累在周质空间内而枯草杆菌的酶活力主要分泌到胞外。     

枯草杆菌启动子—信号肽序列的克隆及序列分析

利用含红霉素抗生基因和缺启动子－信号肽序列的氨苄青霉素抗性基因的双功能质粒ｐＧＰＢ１４为探针载体,克隆了枯草杆菌的启动子－信号肽序列并对克隆的片段进行序列分析。枯草杆菌染色体ＤＮＡ经Ｓａｕ３Ａ酶解后与ＢａｎＨＩ酶切的质粒ｐＧＰＢ１４连接,转化大肠杆菌Ｃ６００,筛选抗氨苄青霉素及抗红霉素的转化子,从双抗性转化子中提取重组质粒并经酶切分析,显示克隆的ＤＮＡ片段在０．２７－１．５ｋｂ之间。用Ｓａｎｇｅｒ     

大肠杆菌青霉素G酰化酶基因及其邻近区域的核苷酸全序列

我们由E.coli AS1.76克隆了青霉素G酰化酶的基因,并且测定了其全部核苷酸序列。青霉素G酰化酶结构基因是由下述功能片段组成的:(1)编码信号肽(26个氨基酸残基)的78个碱基对;(2)编码α-亚基(209个氨基酸残基)的627个碱基对;(3)编码间隔肽(54个氨基酸残基)的162个碱基对;(4)编码β亚基(557个氨基酸残基)的1671个碱基对。此外,我们还发现起始密码子(ATG)前有个核糖体结合位点和启动子序列以及在终止密码子(TAA)之后有个转录终止信号。与最近发表的青霉素G酰化酶基因的DNA序列比较,同源性达99.7%。     

低温菌启动子探针质粒的构建

为了在宿主菌Acinetobacter sp.DWC6中构建低温菌蛋白表达载体,以pBR322质粒为基础,去除质粒上β-内酰胺酶基因的启动子片段,取而代之为来源于质粒pJRD215的卡那霉素抗性基因片段,并在pBR322中插入Acinetobacter菌属特异性ori的DNA片段,构建了能在Acinetobacter sp.DWC6和E.coli中正常复制的启动子探针质粒pBAP1。通过在质粒pBAP1中的β-内酰胺酶基因上游随机导入Acinetobacter sp.DWC6基因组片段,通过检测宿主细胞的氨苄青霉素抗性和β-内酰胺酶活性,来筛选强启动子片段,并分析了启动子探针质粒载体的功能及启动子的强度。     

利用大肠杆菌质粒检测与分离痘苗病毒的启动子

本文发现,痘苗病毒DNA一些巳知的启动子序列和一些功能尚不清楚的DNA片段,可以在大肠杆菌中起始氯霉素乙酰基转移酶(Chloramphenicol Acetyltrsnsferase,简称CAT)基因的转录和表达,使转化细菌呈现氯霉素抗性表型,这一结果证明,痘苗病毒的启动子可以被大肠杆菌的RNA多聚酶所识别并有效工作。同时发现不同启动子具有不同的强度,利用大肠杆菌质粒分离和检测痘苗病毒的启动子序列,不仅可以研究痘苗病毒基因组的表达调节特点,而且也为组建痘苗病毒表达载体提供了一个快速、简便可靠的方法。     

乙型肝炎病毒adr和adw亚型表面抗原在非洲绿猴肾细胞中的表达

用pSV2-gPt作载体,将乙型肝炎病毒adr亚型的全序列基因和adw亚型的Bgl Ⅱ大片段DNA分别插入PSV 2-gpt BamHI及Bgl Ⅱ切口,获得4种重组质粒。它们都含有SV40DNA复制起始点、早期启动子、大肠杆菌黄嘌呤鸟嘌呤磷酸核糖转移酶(XGPRT)和氨苄青霉素抗性基因(Apr)。经酶切鉴定,选出正向重组质粒PSV 2-gbr1和pSV 2-gbw1,并分别转化Vero细胞,在选择培养基的压力下,两个质粒所含乙型肝炎表面抗原DNA均得到表达。     

小鼠精胺氧化酶基因启动子结构和功能研究

目的:克隆和鉴定小鼠精胺氧化酶(mSMO)启动子DNA序列.方法:用Trizol试剂法从小鼠成纤维细胞中提取总RNA,应用5'-RACE法获得mSMO的转录起始位点;巢式PCR方法克隆含有mSMO启动子的DNA序列,并由此构建5'端系列截短的mSMO启动子荧光素酶报告质粒;将报告质粒瞬时转染Cos7细胞后,用荧光素酶分析法测定启动子活性.结果:确定mSMO基因至少有6个转录起始位点(+1,+4,+27,+31,+51,和+63),且均定位于外显子1中.克隆获得mSMO基因转录起始位点上游大约2.1kb的DNA片断,以此片段为基础构建了11个5'端系列截短的启动子报告质粒.报告基因分析证实,该上游DNA片段具有启动子活性,截短至-615和-176bp时,获得2个启动子活性峰值,截短至-373bp时启动子活性最低.结论:mSMO基因含有多个转录起始位点,其上游-176～+124bp为mSMO核心启动子区,-615～-176bp区为重要的转录调控区.     

麦迪霉素产生菌中具强启动活性DNA片段的结构与功能分析

用启动子探针质粒ｐＩＪ４８６从麦迪霉素产生菌（Ｓ．ｍｙｃａｒｏｆａｃｉｅｎｓ）中克隆到１个具启动功能的ＨｉｎｄＩＩＩ－ＨｉｎｄＩＩＩ２．０ｋｂ片段,含该片段的重组质粒ｐ４Ｈ２转化子在ＭＭ基本培养基上对卡那霉素（Ｋｍ）抗性可达５００μｇ／ｍｌ以上。亚克隆缺失分析结果表明,该片段不同部分的缺失对启动活性有不同程度的影响,说明它具有较复杂的转录调控机制。ＤＮＡ序列分析结果显示,该片段含有１９８４个核苷酸,其Ｇ＋Ｃ％为４７．７％,不存在典型的链霉菌的可读框;进一步的分析发现,其６５０ｂｐ、１１５０ｂｐ和１５００ｂｐ区域分别与麦迪霉素酮基还原酶基因等链霉菌基因的启动子序列具有同源性;在５２０－５７０ｂｐ区域与大肠杆菌ｔＲＮＡ基因上游激活序列（ＵＡＳ）有较好的同源性,提示链霉菌中可能存在可增强基因转录的ＤＮＡ元件。     

绿豆种子8S球蛋白基因启动子的克隆及分析

根据绿豆种子8S球蛋白α'亚基基因的末端序列设计3个特异反向引物.以绿豆基因组DNA为模板,采用基因组步移法,获得了8S球蛋白α'亚基基因起始密码子上游784 bp的DNA片段,通过序列测定和生物信息学分析,发现该序列含有启动子核心区以及大量的种子特异表达相关的顺式作用元件,表明此序列为种子特异启动子序列.通过PCR方法在启动子3'端加上翻译增强序列TMV-omega序列,构建了植物双元表达载体pBI-8SG α'-omega-gus,并成功将其转化进入农杆菌.     

木霉几丁质酶基因克隆及植物转化载体构建

利用PCR技术从绿色木霉LTR-2(Trichoderma virede)基因组DNA中扩增到一段序列,测序结果表明,该编码基因片段大小为 1 508 bp,其中包括一个 1 459 bp的开放阅读框,起始密码子位于 45 bp,终止密码子位于 1 501 bp,共编码氨基酸 424 个.在Genbank中进行序列比对,发现该序列同已发表的Trichoderma viride 42 ku几丁质酶氨基酸序列具有99%的同源性.将该片段同pCAMBIA1300中的35S启动子和35S-polyA终止子连接后,插入载体pCAMBIA1302多克隆位点中,构建成植物转化载体,最后将构建好的载体pCHI1302-42通过转化导入根癌农杆菌LBA4404中,为进一步构建转基因植物奠定了基础.     

Chlorophyll isolation,structure and function: major landmarks of the early history of research in the Russian Empire and the Soviet Union

This paper covers major events of the early history of chlorophyll research in the Russian Empire and the Soviet Union from 1771 until 1952, when the modern period of studies on photosynthesis began in full swing. Short biographical sketches of key scientists, reviews of their major research contributions and some selected photographs are included. This revised version was published online in August 2006 with corrections to the Cover Date.     

Ypsilorchis and Ypsilorchidinae,a new genus and a new subtribe of Orchidaceae

A new orchid genus, Ypsilorchis Z. J. Liu, S. C. Chen & L. J. Chen, is established based on Ypsilor-chis fissipetala (Finet) Z. J. Liu, S. C. Chen & L. J. Chen (basionym: Liparis fissipetala Finet). The new genus differs from Liparis and its allies by having two granular-waxy pollinia each with a somewhat elastic caudicle, deeply bilobed petals and strongly crisped-margined leaves with an apical awn to 1 mm long. These features are an indication of its distant relation to the members of the subtribe Liparidinae, and thus a new subtribe, Ypsilor-chidinae Z. J. Liu, S. C. Chen & L. J. Chen, is proposed.     

兰科一新属和新亚族——丫瓣兰属和丫瓣兰亚族

基于丫瓣兰Ypsilorchis fissipetala (Finet)Z.J.Liu,S.C.Chen &L.J.Chen(基名为裂瓣羊耳蒜Liparis fissipetala Finet)建立了兰科新属--丫瓣兰属Ypsilorchis Z.J.Liu,S.C.Chen&.J.Chen.新属与羊耳蒜属Liparis的区别点为:新属有两个粉蜡质花粉团:每个花粉团具1个多少有弹性的花粉团柄;花瓣二深裂;叶具强烈波状的边缘,其先端有一个长达1 mm的芒尖.这些特征表明了其与羊耳蒜亚族Liparidinae有明显的差别,为此,建立了一个新的亚族--丫瓣兰亚族Ypsilorchidinae Z.J.Liu,S.C.Chen &L.J.Chert.     

Protein intake regulation in the weanling rat: Effects of additions of lysine,arginine and ammonia on the selection of gluten and energy

The effects of adding lysine, arginine and ammonia to gluten on the self-selection of protein and energy by the weanling rat simultaneously offered a choice of two diets differing only in gluten concentration (15 and 55%) were tested. Previous studies have shown that while lysine (6 g/100 g) additions to gluten decreased the amount of gluten selected by the rat from 40 to 20 g per 100 g of food eaten, selection was not related to the nutritional quality of the gluten. When graded levels of arginine (1.8, 3.6 or 7.2 g/100 g) were added to the gluten with or without lysine (0 or 6 g/100 g) the dietary protein selection was unaffected. The addition of ammonia (1.4 g/100 g as NH₄Cl) to gluten had initially the same effect as lysine (6 g/100 g) but with time protein intake returned to control levels. This effect of ammonia was unaltered by arginine additions. It is concluded that the mechanisms which lead to decreases in gluten selection caused by lysine or ammonia are not similar, and that the effects of lysine on gluten selection are not caused by an increased arginine requirement for urea cycle activity.     

Comparison of the effects of neuropeptide Y and adrenergic transmitters on LH release and food intake in male rats

In view of the recent demonstrations that Neuropeptide Y (NPY) and adrenergic transmitters coexist in neurons of the rat brain, we have compared the effects of intraventricular (Ivt) injections of NPY and catecholamines on LH release and food intake in intact male rats. Of the three catecholamines, dopamine (DA), norepinephrine (NE) and epinephrine (E), only E (5.3 micrograms or 15.9 micrograms/rat) significantly stimulated LH release, although NE and E (5.3 micrograms/rat) were equally effective in eliciting food intake in satiated rats. Ivt administration of 10 micrograms NPY significantly stimulated LH release, whereas either lower (0.5 or 2 micrograms/rat) or higher (25 micrograms/rat) doses were ineffective. In contrast, NPY at doses of 0.5 - 10 micrograms/rat increased cumulative food intake in a dose-related fashion. These findings present preliminary evidence of the physiological correlates of the neuronal coexistence of adrenergic transmitters and NPY in the brain and raise the possibility that NPY may normally act either independently, in concert with or via adrenergic systems to evoke LH release and feeding responses in the rat.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

巴戟天根的结构及其与蒽醌类化合物积累的关系

应用石蜡切片法、荧光显微镜和紫外分光光度法,对不同年生巴戟天根组织结构的变化进行了观察、对蒽醌类化合物在根中的分布场所及其积累动态进行了研究。结果表明:巴戟天根的结构类似一般多年生草本植物,薄壁细胞是巴戟天根中蒽醌类化合物的分布储存场所,蒽醌类化合物含量随着根生长年限的增加而增加。由以上研究总结出巴戟天以四年或四年以上采收为好,并以根皮厚、木心细者为上品。