The Herbicide-Resistant Species of the Cyanobacterial Dl Protein Obtained by Thorough and Random in vitro Mutagenesis

Random mutations were introduced into the DNA fragment of thepsbA2 gene of Synechocystis sp. PCC 6803, which encodes thecarboxyl-terminal 178 amino acid region of the Dl protein ofthe PSII reaction center, by in vitro random mutagenesis toobtain Dl species resistant to herbicides and to understandthe protein-herbicide interactions. The mutants were screenedon the criterion of resistance to either 1 µM DCMU or10 µM atrazine. In these mutants, amino acid substitutionswere distributed throughout the entire area of the targetedregion in the Dl protein. However, in every mutant, except forone case, the substitution was present in the region describedas the "herbicide-binding niche", i.e., between Phe211 and Leu275,although some amino acid substitutions which were not previouslydescribed were found at residues known to be involved with herbicideaffinity. Thus, the result of random mutagenesis basically supportsthe validity of the proposed structural model for the Dl protein,as well as of the herbicide-binding niche. Preliminary characterizationof the herbicide-resistant mutants obtained in this study hasalso been conducted. (Received December 8, 1997; )     

Immunological Cross-Reactivity among Corresponding Proteins of Photosystems I and II from Widely Divergent Photosynthetic Organisms

Immunological cross-reactivity among corresponding proteinsassociated with photosystems I and II in higher plants, greenalgae, red algae and cyanobacteria were examined with antiseraraised against the proteins from Synechococcus elongatus andspinach. (1) Generally, the cross-reactivity was very high betweenclosely related species but decreased with increasing phylogeneticdistances between organisms. Exceptionally, proteins from redalgae showed lower reactivities with the antisera against thecyanobacterial proteins than did corresponding proteins fromgreen algae and higher plants. (2) The extent of the cross-reactivitywas found to vary with the antisera used. Three antisera preparedagainst large chlorophyll-carrying proteins of photosystem Iand photosystem II reaction center complexes of Synechococcusreacted with the corresponding proteins of all the organismsexamined. By contrast, an antiserum raised against the extrinsic35 kDa protein of the cyanobacterium reacted with none of corresponding33 kDa proteins of other species. The antiserum against thespinach 33 kDa protein showed a wider range of cross-reactivity.Antisera raised against the Dl and D2 proteins from spinachwere highly reactive with corresponding proteins from otherphotosynthetic organisms, whereas an antiserum against a well-conservedsequence of the spinach D2 protein showed limited cross-reactivity.The results show that, although the extent of immunologicalcross-reactivity is determined mainly by the homology betweenproteins, caution is indicated in the application of immunologicalmethods to determinations of the distribution of various proteinsrelated to photosystems I and II in very different organisms. (Received December 8, 1989; Accepted March 12, 1990)     

Reality of P-680 chlorophyll protein – Identification of the site of primary photochemistry in oxygenic photosynthesis

Until recently nearly all available experimental evidence seemed to indicate that the largest subunit of about 50 kDa in the photosystem II core complex ( psb B gene product) is the site of primary photochemistry, and thus the name "P-680 apoprotcin" has been given to this subunit. The notion, however, has also been challenged on the basis of deduced amino acid sequence homology between the proteins in the photosystem II and those of the purple bacterial reaction center. The actual preparation of a functionally active photosystem II reaction center completely devoid of the psb B gene product, but consisting of D-1 and D-2 proteins and cytochrome b -559, has now been achieved.     

Purification and Characterization of Phospholipase D (PLD) from Rice (Oryza sativa L.) and Cloning of cDNA for PLD from Rice and Maize (Zea mays L.)

Phospholipase D (PLD) was purified to high homogeneity fromrice bran (Oryza sativa L.). Two peaks of PLD activity wereresolved by Mono Q anion-exchange chromatography. The molecularmass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDain gel filtration. Antibodies raised against the protein inone of the peaks precipitated the enzyme activities in bothpeaks. Enzymatic characteristics of PLD in the two peaks wereidentical except for a difference of 0.1 in the isoelectricpoints. Sequence analysis covering more than 10% of the aminoacids of the proteins and peptide mapping did not detect anydifference in the primary structure of the proteins. A cDNAfor PLD was isolated from rice and it encoded a protein of 812residues. The N-terminal sequences of purified PLDs matchedthe deduced amino acid sequence starting from residue 47. ANorthern blot showed this gene was expressed in leaves, roots,developing seeds and cultured cells, and a Southern blot detecteda single band of rice genomic DNA hybridizing to the cDNA. AcDNA for PLD was also isolated from maize. The similarity ofthe deduced amino acid sequences of PLD was 90% between riceand maize, 73% between the cereals and castor bean. ²Present address: Agribusiness Division, Japan Tobacco Inc.2-1,Toranomon, 2-chome, Minato-ku, Tokyo, 140 Japan     

Corticosteroid binding globulin, testosterone-estradiol binding globulin, and androgen binding protein belong to protein families distinct from steroid receptors

The cDNA nucleotide sequences and the deduced amino acid sequences of human corticosteroid binding globulin (hCBG), human testosterone-estradiol binding globulin (hTeBG), and rat androgen binding protein (rABP) were determined. Studies of the steroid binding sites suggest they are toward the carboxy-terminus in hTeBG and rABP and more central in hCBG. hCBG has remarkable sequence homology with members of a superfamily whose functions have diverged; these include thyroxine-binding protein, serine protease inhibitors, egg white proteins, and angiotensinogen. hTeBG and rABP have a 68% amino acid sequence identity. Hybridization studies suggest that hTeBG is probably even more closely related, if not identical, to hABP. The carboxy-terminal sequences of hTeBG and rABP are also similar to that of protein S, a vitamin-K-dependent clotting factor. There were no nucleotide or amino acid sequence homologies between hCBG, hTeBG, or rABP and other steroid binding proteins such as steroid receptors, albumin, alpha-fetoprotein, and vitamin D binding protein. We conclude that the "extracellular steroid binding proteins" and steroid receptors do not appear to have descended from a common ancestor.     

N-Terminal Amino Acid Sequence and Processing Site of Sweet Potato Cytochrome c Oxidase Subunit II

The N-terminal amino acid sequence of sweet potato cytochromec oxidase subunit II polypeptide was determined. Comparisonsbetween the sequence and amino acid sequences deduced from thenucleotide sequences of other higher plant subunit II genesindicate a post-translational clevage of N-terminal extensionpart. ¹Present address: Institute of Low Temperature Science, HokkaidoUniversity, Sapporo, 060 Japan. (Received June 13, 1989; Accepted September 8, 1989)     

烟实夜蛾触角普通气味结合蛋白Ⅱ cDNA的克隆、序列分析及在大肠杆菌中的表达

利用RT PCR技术扩增了编码烟实夜蛾Helicoverpa assulta雌、雄虫触角普通气味 结合蛋白Ⅱ的Cdna片段,将其克隆至Pgem-T Easy载体,获得了普通气味结合蛋白Ⅱ基因成熟蛋白阅读框序列。将该基因重组到表达型质粒Pet-30a(+)中,并转化入原核细胞中表达。序列 测定结果表明,烟实夜蛾触角普通气味结合蛋白基因的成熟蛋白阅读框全长489 bp,编码162个 氨基酸残基,预测分子量和等电点分别为18.2 kD和5.35。推导的氨基酸序列与已报道的10种昆虫普通气味结合蛋白Ⅱ高度同源（73%～98%）,并具有气味结合蛋白的典型特征。SDS-PAGE和Western印迹分析表明,经IPTG诱导,普通气味结合蛋白Ⅱ基因能在大肠杆菌BL21(DE3)中表达,电泳检测到一条约23 kD大小的外源蛋白,与预测的融合蛋白分子量大小相应。     

Primary structure of the reaction center from Rhodopseudomonas sphaeroides

The reaction center is a pigment-protein complex that mediates the initial photochemical steps of photosynthesis. The amino-terminal sequences of the L, M, and H subunits and the nucleotide and derived amino acid sequences of the L and M structural genes from Rhodopseudomonas sphaeroides have previously been determined. We report here the sequence of the H subunit, completing the primary structure determination of the reaction center from R. sphaeroides. The nucleotide sequence of the gene encoding the H subunit was determined by the dideoxy method after subcloning fragments into single-stranded M13 phage vectors. This information was used to derive the amino acid sequence of the corresponding polypeptide. The termini of the primary structure of the H subunit were established by means of the amino and carboxy terminal sequences of the polypeptide. The data showed that the H subunit is composed of 260 residues, corresponding to a molecular weight of 28,003. A molecular weight of 100,858 for the reaction center was calculated from the primary structures of the subunits and the cofactors. Examination of the genes encoding the reaction center shows that the codon usage is strongly biased towards codons ending in G and C. Hydropathy analysis of the H subunit sequence reveals one stretch of hydrophobic residues near the amino terminus; the L and M subunits contain five such stretches. From a comparison of the sequences of homologous proteins found in bacterial reaction centers and photosystem II of plants, an evolutionary tree was constructed. The analysis of evolutionary relationships showed that the L and M subunits of reaction centers and the D1 and D2 proteins of photosystem II are descended from a common ancestor, and that the rate of change in these proteins was much higher in the first billion years after the divergence of the reaction center and photosystem II than in the subsequent billion years represented by the divergence of the species containing these proteins.     

Import of Modified Dl Protein and its Assembly into Photosystem II by Isolated Chloroplasts

A portion of the rbcS gene that encoded the transit peptideand 20 amino acid residues of the N-tenninal region of the smallsubunit of ribulosebisphosphate carb-oxylase/oxygenase was fusedto the 5' end of the psbA gene which encodes the Dl proteinof PSII reaction center. The chimeric gene was expressed invitro as a 42-kDa protein, which was imported into chloroplastsisolated from pea leaves. The imported protein was processedsuch that the transit peptide was lost in the stroma, the resultantprotein was translocated into thylakoid membranes, and the C-ter-minalpeptide was then removed to yield a mature protein with an N-terminalextension originated from the small sub-unit. The mature proteinappeared to be assembled into the PSII core complex, resemblingthe native Dl protein in terms of protein structure and topologywithin the membrane. Our observations indicate that the structureof the precursor to the Dl protein includes information forthe proper assembly of the protein into the PSII core complex. (Received September 10, 1996; Revision received December 14, 1996. )     

吸血诱导的埃及伊蚊海口株后期胰蛋白酶基因测序及与美国株同源性比较

应用逆转录-聚合酶链式反应（RT-PCR）技术从吸血后24 h埃及伊蚊海口株总RNA中扩增出了后期胰蛋白酶编码区cDNA序列。采用自动DNA分析仪进行序列分析,并与已知埃及伊蚊美国株后期胰蛋白酶基因及推导的氨基酸序列进行了同源性比较。结果表明：埃及伊蚊海口株后期胰蛋白酶基因序列与美国株同源性达98%,有11个碱基发生变异;氨基酸同源性达99%,仅有3个氨基酸发生变异,但与催化位点密切相关的氨基酸及N末端氨基酸序列完全一致。以上结果显示,埃及伊蚊胰蛋白酶不同地理株间存在微小的差异。     

Molecular cloning and characterization of N-syndecan, a novel transmembrane heparan sulfate proteoglycan

A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.     

Modeling the quinone-B binding site of the photosystem-II reaction center using notions of complementarity and contact-surface between atoms

Functional identity and significant similarities in cofactors and sequence exist between the L and M reaction center proteins of the photosynthetic bacteria and the D1 and D2 photosystem-II reaction center proteins of cyanobacteria, algae, and plants. A model of the quinone (Q_B) binding site of the D1 protein is presented based upon the resolved structure of the Q_B binding pocket of the L subunit, and introducing novel quantitative notions of complementarity and contact surface between atoms. This model, built -without using traditional methods of molecular mechanics and restricted to residues in direct contact with Q_B, accounts for the experimentally derived functional state of mutants of the Dl protein in the region of Q_B. It predicts the binding of both the classical and phenol-type PSII herbicides and rationalizes the relative levels of tolerance of mutant phenotypes. © 1995 Wiley-Liss, Inc.     

Amino- and carboxy-terminal deletion mutants of Gs alpha are localized to the particulate fraction of transfected COS cells

To elucidate the structural basis for membrane attachment of the alpha subunit of the stimulatory G protein (Gs alpha), mutant Gs alpha cDNAs with deletions of amino acid residues in the amino and/or carboxy termini were transiently expressed in COS-7 cells. The particulate and soluble fractions prepared from these cells were analyzed by immunoblot using peptide specific antibodies to monitor distribution of the expressed proteins. Transfection of mutant forms of Gs alpha with either 26 amino terminal residues deleted (delta 3-28) or with 59 amino terminal residues deleted (delta 1-59) resulted in immunoreactive proteins which localized primarily to the particulate fraction. Similarly, mutants with 10 (delta 385-394), 32 (delta 353-384), or 42 (delta 353-394) amino acid residues deleted from the carboxy terminus also localized to the particulate fraction, as did a mutant form of Gs alpha lacking amino acid residues at both the amino and carboxy termini (delta 3-28)/(delta 353-384). Mutant and wild type forms of Gs alpha demonstrated a similar degree of tightness in their binding to membranes as demonstrated by treatment with 2.5 M NaCl or 6 M urea, but some mutant forms were relatively resistant compared with wild type Gs alpha to solubilization by 15 mM NaOH or 1% sodium cholate. We conclude that: (a) deletion of significant portions of the amino and/or carboxyl terminus of Gs alpha is still compatible with protein expression; (b) deletion of these regions is insufficient to cause cytosolic localization of the expressed protein. The basis of Gs alpha membrane targeting remains to be elucidated.     

A new carboxylesterase from Brevibacterium linens IFO 12171 responsible for the conversion of 1,4-butanediol diacrylate to 4-hydroxybutyl acrylate: purification, characterization, gene cloning, and gene expression in Escherichia coli.

A carboxylesterase that is responsible for conversion of 1,4-butanediol diacrylate (BDA) to 4-hydroxybutyl acrylate (4HBA) was found in Brevibacterium lines IFO 12171, and purified to homogeneity. The purified enzyme was active toward a variety of diesters of ethylene glycol, 1,4-butanediol, and 1,6-hexanediol. The K(m) and kcat of the enzyme for BDA were 3.04 mM and 203,000 s-1, respectively. The reaction with the purified enzyme gave 98 mM 4HBA from 100 mM BDA for 60 min. The enzyme gene was cloned from the chromosomal DNA of the bacterium. The open reading frame encoding the enzyme was 1176 bp long, corresponding to a protein of 393 amino acid residues (molecular mass = 42,569 Da). The deduced amino acid sequence contained the tetra peptide motif sequence, STTK, and the serine residue was confirmed to be the catalytic center of BDA esterase by site-directed mutagenesis for several amino acid residues. The gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product (a fusion protein with 6 amino acid residues from beta-galactosidase) showed the same catalytic properties as the enzyme from the parent strain.     

B型烟粉虱和温室白粉虱热激蛋白90基因（hsp90）的全长cDNA克隆与系统发育分析

B型烟粉虱Bemisia tabaci (Gennadius) biotype B和温室白粉虱Trialeurodes vaporariorum均为全球普遍发生的重要害虫。本研究以其他昆虫热激蛋白90基因（hsp90）保守区域设计兼并引物扩增两种粉虱hsp90中间片段, 然后利用RACE技术获得全长cDNA。温室白粉虱hsp90全长cDNA的开放性阅读框2 166 bp, 编码722个氨基酸; 烟粉虱hsp90全长cDNA的开放性阅读框2 160 bp, 编码720个氨基酸。两种粉虱HSP90的完整氨基酸序列相似性高达92.94％, 并均具有定义HSP90家族签名序列的5个氨基酸保守区域和末尾基序“MEEVD”。通过real-time PCR技术, 探测到两个基因在mRNA水平上皆能高温诱导表达。采用昆虫纲所有完整HSP90氨基酸序列进行Kimura双参数遗传距离分析并构建NJ进化树, 结果显示hsp90在昆虫纲低级阶元水平和高级阶元水平系统进化上能得到一个较理想结果。本研究结果为B型烟粉虱和温室白粉虱抗逆适应性研究提供基础, 并进一步验证保守的功能基因hsp90可以作为研究生物系统发育的手段之一     

Cinnamyl Alcohol Dehydrogenase from Aralia cordata: Cloning of the cDNA and Expression of the Gene in Lignified Tissues

A full-length cDNA clone encoding a subunit of cinnamyl alcoholdehydrogenase (CAD) was isolated from a perennial dicot, Araliacordata. The identity of the clone was demonstrated by two criteria:(i) the amino acid sequences of peptides derived from the purifiedCAD protein of A. cordata were highly homologous to regionsof the amino acid sequence deduced from the nucleotide sequenceof the cDNA; and (ii) a fusion protein expressed from     

Sequence Analysis of Cloned cDNA and Proteolytic Fragments for Nitrate Reductase from Spinacia oleracea L.

Proteolytic fragments were obtained by limited proteolysis of120 kDa nitrate reductase from Spinacia oleracea L. using trypsinand Staphylococcus aureus V8 protease. Determination of NH₂-terminalsequences in 9 to 14 Edman degradation steps allowed the exactlocalization of the fragments within the amino-acid sequenceof spinach nitrate reductase was deduced from the nucleotidesequence of cDNA clone pSPNR117 which was initially identifiedby hybridization to squash nitrate reductase cDNA clone [Crawford,¹N. M., Campbell, W. H. and Davis, R. W. (1986) Proc. Natl. Acad.Sci. USA 83: 8073] and anti spinach nitrate reductase polyclonalantibodies. This clone has a 2324 base insert, and the aminoacid sequence deduced from its open reading frame, which contains640 residues. The predicted sizes 42.5 and 30 kDa were in reasonableagreement with previous determination of the apparent molecularsizes of the FAD-cyt-chrome b557-binding, and FAD-binding fragments,respectively. Arginine residue was the cleavage site for trypsin and glutamicacid was for S. aureus V8 protease. The amino acid residueswithin the linker regions which connect the functional domains,could be cleaved with trypsin or S. aureus V8 protease may bewell conserved in the amino acid sequences deduced from thenitrate reductase cDNA sequences. A sequence identity of 61.2-80.1 % was found in the amino acidsequences deduced from the cDNA sequences as obtained by spinachand other higher plant nitrate reductases. However, the aminoacid sequences surrounding the proteolytic cleavage sites ofnitrate reductase had poor homology. (Received March 30, 1991; Accepted July 24, 1991)     

Isolation of the D1 Protein and a 28-kDa Fragment of the D2 Protein from Spinach Photosystem II Membranes

The 32-kDa D1 protein, which contained no lysine in spinachchloroplasts, as deduced from its DNA code, was isolated byhigh-performance gel permeation chromatography in the presenceof 0.1% SDS and 4 M urea. Three proteins of the photosystemII reaction center complex have a molecular mass of 30–35kDa, and two, the D2 protein and the peripheral 33-kDa protein,were severed into peptide fragments by Achromobacter lysyl endopeptidase(EC 3.4.14.50 [EC] ) before the chromatography. The isolated D1 proteindid not contain chlorophylls and pheophytins but had an absorptionmaximum at 265 nm probably due to bound plastoquinone. A peptidefragment of 28 kDa from the D2 protein was also isolated fromspinach photosystem II membranes and the wheat photosystem IIreaction center. Antibodies raised against the 28-kDa peptidefrom wheat bound to the 34-kDa D2 protein, which suggested thatthis peptide was the largest sequence of Aspl4-Lys265. The fragmentof wheat D2 protein showed absorption maxima at 413 and 682nm attributable to bound pheophytin that probably had been convertedfrom chlorophyll a during the isolation process. (Received June 29, 1987; Accepted October 21, 1987)     

An unusual Group 2 LEA gene family in citrus responsive to low temperature

Six cDNAs representing unique cold-induced sequences have been cloned from the hardy citrus relative Poncirus trifoliata. Among these, pBCORc115 and pBCORc119 were found to belong to the same gene family. Sequencing data indicated that pBCORc115 and pBCORc119 each contained an open reading frame, coding for a 19.8 kDa protein (COR19) and a smaller 11.4 kDa protein (COR11) respectively. Inspection of the deduced amino acid sequences revealed three large repeats in COR19, but only one was present in the COR11. Two elements: a Q-clustered tract and a K-rich motif were identified in each repeat. The K-rich motifs were similar to those of cotton D-11 and Group 2 LEA proteins. A Serine-cluster, a common feature in many Group 2 LEA-like proteins, was also found in these proteins, but it was in an unusual position at the carboxy-terminus. A bipartite motif of basic residues, similar to known nuclear targeting sequences, was also present in COR19 and COR11, suggesting that members of this protein family may have a nuclear targeting function. The expression of COR19 mRNA in response to cold acclimation, drought, flooding, and salinization was examined. COR19 expression in leaf tissue was induced in response to cold acclimation, but repressed during drought and flooding stress.