用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。     

Isolation of cDNA clones and complete amino acid sequence of human erythrocyte glycophorin C

Two cDNA clones for glycophorin C, a transmembrane glycoprotein of the human erythrocyte which carries the blood group Gerbich antigens, have been isolated from a human reticulocyte cDNA library. The clones were identified with a mixture of 32 oligonucleotide probes (14-mer) which have been synthetized according to the amino acid sequence Asp-Pro-Gly-Met-Ala present in the N-terminal tryptic peptide of the molecule. The primary structure of glycophorin C deduced from the nucleotide sequence of the 460 base-pair insert of the pGCW5 clone indicates that the complete protein is a single polypeptide chain of 128 amino acids clearly organized in three distinct domains. The N-terminal part (residues 1-57, approximately) which is N- and O-glycosylated is connected to a hydrophilic C-terminal domain (residues 82-128, approximately) containing 4 tyrosine residues by a hydrophobic stretch of nonpolar amino acids (residues 58-81, approximately) probably interacting with the membrane lipids and permitting the whole molecule to span the lipid bilayer. Northern blot analysis using a 265-base-pair restriction fragment obtained by DdeI digestion of the inserted DNA shows that the glycophorin C mRNA from human erythroblasts is approximately 1.4 kilobases long and is present in the human fetal liver and the human K562 and HEL cell lines which exhibit erythroid features. The glycophorin C mRNA, however, is absent from adult liver and lymphocytes, indicating that this protein represents a new erythrocyte-specific probe which might be useful to study erythroid differentiation.     

Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins

An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution.     

Molecular cloning of a cDNA for human delta-aminolevulinate dehydratase

A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues.     

Deoxynucleotide sequence of an insect cDNA codes for an unreported member of the Chironomus thummi globin family

Synthetic oligonucleotides served as probes to isolate insect globin clones from a Chironomus thummi cDNA bank. The cDNA insert of one clone (pC-S9) was completely sequenced by the dideoxy termination procedure. Beginning at the 5' end of the coding region, the 584 base pair sequence encodes most of an N-terminal hydrophobic signal sequence and the complete sequence for a mature secreted globin, and contains a polyadenylation recognition site 3' to an appropriate stop codon. The inferred amino acid sequence is that of an unreported variant of hemoglobin VIIB. Based on the number of differences between Hb VIIB chains, the pC-S9 gene has been evolutionarily independent longer than the other (two) members of the globin VIIB subfamily.     

cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain

A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

Isolation of a full-length cDNA encoding mouse aromatase P450

A full-length cDNA clone for aromatase P450 has been isolated from a pregnant mouse ovarian cDNA library. The insert of this clone (2394 bp) contains a 1509-bp open reading frame encoding 503 amino acid residues together with a 46-bp 5'-untranslated stretch and an 839-bp 3'-untranslated region to which a poly(A) tract is attached. Northern blot analysis of ovarian RNA from pregnant mice reveals a major mRNA band of 2.5 kb with a minor band of 2.1 kb. Comparison of mouse aromatase P450 with that of rat, human, and chicken shows 91, 81, and 69% identity in the nucleotide sequence and 92, 79, and 69% identity in the deduced amino acid sequence, respectively. The membrane-spanning domain of mouse aromatase P450 is estimated to be an extremely hydrophobic segment located within the N-terminal region of the molecule. Furthermore, a highly conserved heme-binding domain is noticed.     

Characterization of Novel Secreted and Membrane Proteins Isolated by the Signal Sequence Trap Method

We recently described a method, called the signal sequence trap (SST) method, to clone cDNAs of secreted proteins and/or type I transmembrane proteins containing N-terminal signal sequences by using an epitope-tagging expression plasmid vector. In this paper we describe the summary of a large-scale screening of approximately 5900 clones of an SST cDNA library constructed from mouse bone marrow stromal cell line ST-2 cells. Of 26 positive clones obtained and sequenced, 11 clones appeared to contain authentic signal sequences. Five of the clones corresponded to the 5′ ends of the cDNA of known genes containing N-terminal signal sequences. The full-length cDNA clones of the 6 other unknown clones were isolated and sequenced. One clone, termed SDF3, encoded a mouse counterpart of human pigment epithelium-derived factor. Another clone, termed SDR1, had considerable homology with basigin, a member of the immunoglobulin superfamily. A third clone, termed SDF5, had partial homology with aDrosophilatissue polarity genefrizzled(fz) and its rat homologues,fz-1andfz-2.The other three clones had no significant homology with sequences in the databases. These results indicate that the SST method is effective and useful for the isolation of secreted and membrane proteins without knowledge of their functions.     

Isolation and characterization of recombinant DNA clones of avian retroviruses: size heterogeneity and instability of the direct repeat

Unintegrated proviral DNA of Schmidt-Ruppin B Rous sarcoma virus was cloned in the bacteriophage lambda vector Charon 21A. A total of 12 independent recombinant lambda SRBtd clones which were derived from the transformation-defective component in the viral preparation were analyzed with restriction endonucleases and molecular hybridization techniques. Three classes of clones were observed. Type I clones contained a 5.0-megadalton insert of viral DNA, type II clones contained phage with two size classes of inserts (5.0 and 5.2 megadaltons), and one type III clone contained only a 5.2-megadalton insert. The smaller insert present in type II clones appeared to be derived by deletion of one copy of a directly repeated sequence which was present in the larger insert. Mapping data indicated that the deletion includes all or part of the terminal repeat found in linear double-stranded proviral DNA. Similar results were obtained from lambda RAV2 recombinant clones derived from Rous-associated virus type 2. Analysis of DNA from type II and type III clones of lambda SRBtd and lambda RAV2 revealed limited heterogeneity in the size of the direct repeat.     

Screening and characterization of a cellulase gene from the gut microflora of abalone using metagenomic library

A metagenomic fosmid library was constructed using genomic DNA isolated from abalone intestine. Screening of a library of 3,840 clones revealed a 36 kb insert of a cellulase positive clone (pAMHElO). A shotgun clone library was constructed using the positive clone (pAMHElO) and further screening of 3,840 shotgun clones with an approximately 5 kb insert size using a Congo red overlay revealed only one cellulase positive clone (pAMHL9). The pAMHL9 consisted of a 5,293-bp DNA sequence and three open reading frames (ORFs). Among the three ORFs, cellulase activity was only shown in the recombinant protein (CelAMll) coded by ORF3, which showed 100% identity with outer membrane protein A from Vibrio alginolyticus 12G01, but no significant sequence homology to known cellulases. The expressed protein (CelAMll) has a molecular weight of approximately 37 kDa and the highest CMC hydrolysis activity was observed at pH 7.0 and 37°C. The carboxymethyl cellulase activity was determined by zymogram active staining and different degraded product profiles for CelAMll were obtained when cellotetraose and cellopentaose were used as the substrates, while no substrate hydrolysis was observed on oligosaccharides such as cellobiose and cellotriose.     

Nucleotide sequences of two pea cDNA clones encoding the small subunit of ribulose 1,5-bisphosphate carboxylase and the major chlorophyll a/b-binding thylakoid polypeptide

Two major chloroplast proteins are encoded by nuclear genes and synthesized on free cytoplasmic ribosomes: the small subunit of ribulose 1,5-bisphosphate carboxylase and the apoprotein components of the chlorophyll a/b light harvesting complex. We have recently reported the isolation of two cDNA clones from pea which encode both the small subunit of ribulose 1,5-bisphosphate carboxylase (pSS15) and the polypeptide 15 (pAB96), the major chlorophyll a/b binding protein (Broglie, R., Bellemare, G., Bartlett, S., Chua, N.-H., and Cashmore, A. R. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 7304-7308). To further characterize these clones, we determined their nucleotide sequence. Clone pSS15 contains a 691-base pair cDNA insert which encodes the entire 123 amino acids of the mature small subunit protein. In addition, this clone also encodes 33 amino acids of the NH2-terminal transit peptide extension and 148 nucleotides of the 3' noncoding region preceding the poly(A)tail. A second cDNA clone (pAB96) contains an 833-nucleotide insert which encodes most of polypeptide 15. The DNA sequence of this cloned cDNA was used to deduce the previously undetermined amino acid sequence of this integral thylakoid membrane protein. The nucleotide sequence of the cDNA clone, pSS15, should provide information concerning the role of the transit sequence in the transport of cytoplasmically synthesized chloroplast proteins. Similarly, the deduced amino acid sequence of polypeptide 15 will provide information for predicting its orientation in thylakoid membranes as well as its role in binding chlorophyll.