Molecular cloning and characterization of a C-type lectin in roughskin sculpin (Trachidermus fasciatus)

C-type lectins, as the members of pattern-recognition receptors (PRRs), play significant roles in innate immunity responses through binding to the pathogen-associated molecular patterns (PAMPs) presented on surfaces of microorganisms. In our study, a C-type lectin gene (TfCTL1) was cloned from the roughskin sculpin using expression sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length of TfCTL1 was 696 bp, consisting of a 95 bp 5′ untranslated region (UTR), a 498 bp open reading frame (ORF) encoding a 165 amino acid protein, and a 103 bp 3′ UTR with a polyadenylation signal sequence AATAAA and a poly(A) tail. The deduced amino acid sequence of TfCTL1 contained a signal peptide and a single carbohydrate recognition domain (CRD) which had four conserved disulfide-bonded cysteine residues (Cys⁶¹-Cys¹⁵⁸, Cys¹³⁴-Cys¹⁵⁰) and a Ca²⁺/carbohydrate-binding site (QPD motif). Results from the qRT-PCR indicated that TfCTL1 mRNA was predominately expressed in the liver. The temporal expression of TfCTL1 was obviously up-regulated in the skin, blood, spleen and heart in time dependent manners by lipopolysaccharide (LPS) challenge, whereas in the liver, TfCTL1 was initially down-regulated from 2 h to 48 h followed by an abrupt up-regulation at 72 h. Recombinant TfCTL1 CRD purified from Escherichia coli BL21 was able to agglutinate some Gram-positive bacteria, Gram-negative bacteria and a yeast in a Ca²⁺-dependent manner. Further analysis showed that TfCTL1 can bind to several kinds of microorganisms selectively in a Ca²⁺-independent manner. These results suggested that TfCTL1 might be involved in the innate response as a PRR.     

pHG276: A multiple cloning site pBR322 copy number vector expressing a functional lacα peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter P_R has been used to direct the synthesis of lacα in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI⁸⁵⁷ gene for temperature regulation of lacα expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lacα and consequently, a Lac⁻ phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.     

Pheromone 4 gene of Euplotes octocarinatus

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5′ and 3′ splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3′ end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macro-nuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes. © 1992 Wiley-Liss, Inc.     

A complete sequence of the pGA1611 binary vector

We report the nucleotide sequence of the binary vector pGA1611, which is used for the transformation of foreign DNA into rice. This vector is 13,476 bp long. The 5577- bp T- DNA region consists of a 1987- bp ubiquitine promoter region, 45 bp for the multiple cloning site, a 253- bpnos terminator region, and the 2045- bpCaMV35S- hph- T7 chimaeric gene. The vector backbone (7004 bp) carriesoriT,traJ,trfA,tetA,tetR, andoriV. An 892- bp RB region and the 489- bp LB region are also present The T- DNA possesses 15 unique sites, six of which are at the multiple cloning site. This information will be valuable for cloning foreign DNA and modifying the vector.     

pHG276: a multiple cloning site pBR322 copy number vector expressing a functional lac alpha peptide from the bacteriophage lambda PR promoter

The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.     

扩展青霉PF898碱性脂肪酶基因组DNA的克隆及序列分析

扩展青霉 (Penicilliumexpansum)PF898可产生一种具有重要工业生产价值的碱性脂肪酶(PEL) .在通过 3′RACE和 5′RACE获得PEL完整的cDNA序列的基础上 ,通过PCR方法首次克隆了该脂肪酶的完整的基因组DNA序列 (GenBank登录号为AF330 6 35 ) .该脂肪酶DNA全长 14 0 4bp ,包括PEL编码区、3′非翻译区和部分 5′非翻译区基因的序列 .编码区DNA由 1135个碱基组成 ,含有 5个内含子 ,大小分别为 5 8bp、4 7bp、5 0bp、5 6bp和 6 9bp .在已报道的丝状真菌脂肪酶中 ,PEL基因的内含子数量最多 ,而其大小与其它丝状真菌脂肪酶基因的内含子一样 ,均为只有几十个碱基的小内含子 .PCR扩增获得的PLEDNA序列还包括由 195个碱基组成的 3′端非编码区序列 ,74个碱基的部分 5′端非编码区序列 .PELDNA全长序列中的 - 2 4至 - 2 7nt为TATAbox ,终止码TGA下游15 6nt出现AATAAA序列 ,TGA下游 182位出现poly(A)尾 ,为典型的真核基因结构 .同源性序列分析表明 ,PEL与其它真菌来源脂肪酶的基因组DNA序列同源性约为 39%～ 4 9% ,PEL内含子之间或PEL内含子与其它丝状真菌脂肪酶基因的内含子之间的序列同源性约 4 2 %～ 5 7% .     

Mosquito ribosomal RNA genes: Characterization of gene structure and evidence for changes in copy number during development

A family of nine recombinant bacteriophages containing rRNA genes from cultured cells of the mosquito, Aedes albopictus, has been characterized by restriction mapping, Southern-blotting and S1-nuclease analyses. The 18S rRNA coding region measured 1800 bp and contained a conserved Eco RI site near the 3′-end. The 28S rRNA coding region was divided into α and β sequences, comprising 1750 and 2000 bp, respectively, which were separated by a 350 bp sequence that is removed from the rRNA precursor during processing. The entire rDNA repeat unit had a minimum length of 15.6 kb, including a nontranscribed spacer region that contained a series of PvuI repeats upstream of the 18S rRNA coding sequence. During development of the mosquito, Aedes aegypti, the rRNA gene copy number per haploid genome increased from about 400 in larvae to about 1200 in adults.     

牛性别决定新基因Fgf9的克隆及生物信息学分析

Fgf9基因是脊椎动物性别决定中重要的信号因子,它在睾丸发育过程中参与Sertoli细胞的增殖和睾丸索的形成。基于表达序列标签（expressed sequence tags, ESTs）克隆原理,采用序列拼接和 RTPCR方法获得了荷斯坦奶牛Fgf9基因的cDNA序列,并对其组织表达特征进行分析。利用生物信息学方法对Fgf9基因序列和蛋白结构进行分析。结果显示：Fgf9基因定位于牛12号染色体上,cDNA全长为697bp,开放阅读框为627bp,编码208个氨基酸,分子量23.38245kDa,等电点7.0600。RTPCR证实该开放阅读框正确,在牛的各组织中均有表达,且与牛其他cDNA无同源性,获得GenBank登陆号为：EU693028。功能结构分析显示Fgf9蛋白具有典型的FGF家族保守结构域,包括受体相互作用位点和肝素结合位点。信号肽预测显示牛Fgf9蛋白可能不存在信号肽序列。
学     

西伯利亚蓼rd22基因的克隆与序列分析

以3% NaHCO₃溶液胁迫处理48 h的西伯利亚蓼为试材,利用RACE技术,从其茎部组织克隆了脱水应答蛋白RD22的全长cDNA序列。测序后的结果分析表明,该cDNA序列全长为1 302 bp,5′非翻译区为59 bp,3′非翻译区为25 bp,开放读码框为1 218 bp,编码405个氨基酸。在氨基酸序列的C端含有一个比较保守的BURP结构域,N端含有5个重复序列THV-VGKGGV-V。信号肽检测证明该蛋白为分泌性蛋白,前21个氨基酸区域为信号肽结构。其推演的氨基酸序列与葡萄的同源性最高,达到60%。该基因已在GenBank上注册,基因序列登录号为DQ836050。     

A newEscherichia coli: Bacteroides shuttle vector,pRRI207, based on theBacteroides ruminicola plasmid replicon pRRI2

A 3.4kb cryptic plasmid, pRRI2, was isolated fromBacteroides ruminicola 223/M2/7 and used as the basis for a newBacteroides/Escherichia shuttle vector. Constructs were made incorporating pRRI2, aBacteroides erythromycin/clindamycin resistance marker and theE. coli pUC8-derived vector pHG165. One of these, pRRI207 (11 kb), was capable of introduction into strains belonging to four different species ofBacteroides (B. uniformis, B. distasonis, B. thetaiotaomicron, orB. ruminicola) either by conjugal transfer fromE. coli or by electrotransformation. pRRI207 carries several unique restriction sites derived from the pUC8 multiple cloning site. Only one of sixB. ruminicola strains tested was used successfully as a recipient for pRRI207, indicating that further modifications to transfer procedures or marker genes may be needed for wider application in this species.     

西伯利亚蓼铜伴侣蛋白基因在盐胁迫条件下的表达分析

铜伴侣蛋白是细胞质中负责传递铜离子的一种小分子转运蛋白,在逆境胁迫过程中铜伴侣蛋白的ATX1家族具有消除活性氧的作用．本研究应用RACE技术从西伯利亚蓼中克隆了具有完整编码区的铜伴侣蛋白全长cDNA序列,命名为PsATX-1．PsATX-1基因全长516 bp,其中开放读码框为228 bp,编码75个氨基酸,5′非编码区为83 bp,3′非编码区为204 bp．GenBank中登录号为EU620702．经比对发现,该基因所编码蛋白拥有重金属结合位点MXCXXC,缺少多数高等植物铜伴侣蛋白(CCH)所特有的C末端结构域（CTD）．实时荧光定量PCR分析显示,PsATX-1基因在西伯利亚蓼的地下茎、茎、叶中皆有表达,其中叶中表达量最高;PsATX-1基因受3% NaHCO3胁迫诱导,在不同部位表达模式有差异．     

东亚钳蝎毒素BmKT四个同源基因的克隆及基因组织分析

BmKT是从本室构建的cDNA文库中筛选到的1个α钠通道毒素,根据其全长cDNA序列设计引物,采用PCR法以蝎总基因组DNA为模板,获得4个BmKT的同源基因,分别命名为BmKT′和BmKTa、BmKTb、BmKTb′.序列分析表明：BmKT′和BmKTa基因含有大小分别为509 bp和506 bp的内含子,位于信号肽编码区内,插入信号肽-4位残基Gly密码子的第一个碱基G之后;而BmKTb和BmKTb′ 的内含子大小均为418 bp．这4个BmKT的同源基因内含子符合GT/AG拼接规律,其中BmKT′和BmKTa的内含子A+T含量分别为61.7%和61.9%,低于目前已报导的大多数蝎毒素基因A+T含量,大大低于它们第一外显子A+T含量（71.7%）,略高于第二外显子A+T含量（55.5%）;而BmKTb,BmKTb′的内含子A+T含量分别为75.8%和76.1%,与目前已报导的大多数蝎毒素基因A+T含量相似．BmKT′基因的外显子与BmKT基因cDNA所对应氨基酸序列仅在信号肽中-7位有一个氨基残基的差异（BmKT: Leu→BmKTa: Val）;而BmKTa基因外显子所推断的氨基酸序列与BmKT前体比较,则在成熟肽的+54位发生了突变（BmKT: Lys→BmKTa: Asn）,是与BmKT同源的一个新基因．BmKTb基因和BmKTb′基因所编码的前体肽与BmKT基因对应的前体肽同源性约为65%,显然BmKTb和BmKTb′是不同于BmKT的2个新基因（GenBank登录号: BmKT′, AY786186; BmKTa, AY676142; BmKTb, AY676140; BmKTb′, AY676141）     

Ginkgo 11S seed storage protein family mRNA: unusual Asn-Asn linkage as post-translational cleavage site

By reducing the amount of ginkgo water-soluble polysaccharides, which occupy about 35% of the wet seed mass and interfere with the extraction of RNA, cDNA-quality mRNA was obtained from developing seeds of Ginkgo biloba. Based on the NH₂-terminal 17-amino acid sequence and an internal 12-amino acid sequence derived from the basic subunit of ginnacin, 11S-seed storage protein family of ginkgo, two degenerate oligonucleotide primers were synthesized and used for polymerase chain reaction (PCR). The resulting PCR product was used for screening the above endosperm cDNA library, and a plaque carrying the 1614 bp cDNA insert, which contained the entire coding region for a precursor of ginnacin was isolated. This is the first reported cloning of cDNA from ginkgo seeds. The deduced primary sequence is composed of a signal peptide segment (25 amino acid residues) and an acidic subunit (248 residues) followed by a basic subunit (187 residues). It was also found that the post-translational cleavage site in the ginnacin precursor is the Asn-Asn rather than the Asn-Gly bond found in a variety of the major subunit precursors in 11S seed protein family known to date. We showed that a purified soybean extract and an extract of ginkgo seeds can specifically hydrolyze-Asn²⁴⁸-Asn²⁴⁹- but not -Asn²⁴⁹-Val²⁵⁰-, in the heptapeptide Gly-Asn²⁴⁸-Asn-Val-Glu-Glu-Leu that corresponds to the ginnacin cleavage region.Abbreviations SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - CBB Coomassie Brilliant Blue - HPLC high-performance liquid chromatography - bp base pair(s) - PCR polymerase chain reaction     

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS1

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO₂ and HCO₃^?, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full‐length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5′ untranslated region (UTR) of 177 bp, a 3′ UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274‐amino‐acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the β‐CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real‐time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4‐fold higher and 10‐fold higher, respectively.     

Complete amino acid sequence of Bombyx egg-specific protein deduced from cDNA clone

Complementary (c)DNA coding for an insect yolk protein, the egg-specific protein of the silkworm Bombyx mori was cloned and the nucleotide sequence determined. The sequence covers the entire coding region of 1,677 base pairs with 5′ and 3′ noncoding regions (21 and 115 base pairs, respectively). The deduced amino acid sequence of the egg-specific protein consists of 559 amino acid residues. The NH₂-terminal 18 amino acid sequence is enriched in hydrophobic amino acids and assumed to be a signal peptide. A sequence, Asn-X-Thr, a potential N-linked glycosylation site, is found at positions 191 to 193. A serine-rich domain is localized in the region from 63 to 90, in which phosphorylation takes place. Cys His motif in 405 to 415 is analogous to a proposed metal binding sequence. Lys¹³²-Asn¹³³ and Arg²²⁸-Asp²²⁹ are probably the sites cleaved by the egg-specific protein protease that appears during embryogenesis. The derived amino acid sequence has no appreciable homology to other sequenced proteins.