Identification of bacterial clones that encode cow's caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant DNA plasmids carrying cDNA fragments of bovine caseins in the cDNA library from rRNA of bovine mammary glands. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated filter paper. Antigens covalently bound to the CNBr-activated paper or bound to the nitrocellulose filters were detected by reaction with antiserum to caseins, followed by 125I-labeled protein A from Staphylococcus aureus and autoradiography. Four clones were positive among 5400 bacterial clones of the cDNA library--al, b2, b5, h7. Molecular weights of chimeric proteins pre-beta-lactamase:casein synthesized in Escherichia coli were determined by immunoblotting. Colony hybridization and DNA sequence analysis showed that clone b5 contained cDNA fragment of bovine kappa-caseins and clone h7 cDNA fragment of beta-casein. The last clone was designated pKcas beta-7.     

Identification of bacterial clones encoding bovine caseins by direct immunological screening of the cDNA library

A sensitive immunoassay was used to identify recombinant plasmids carrying cDNA fragments of bovine caseins in the cDNA library from bovine mammary gland mRNA. Colonies grown on nitrocellulose filters were lysed in situ and proteins from the lysates were blotted onto CNBr-activated cellulose filter paper. Antigens covalently bound to CNBr-activated paper or bound to nitrocellulose filters were detected by reaction with antiserum to caseins, followed by ¹²⁵I-labelled Staphylococcus aureus protein A and autoradiography. Six clones were found positive among 5400 of the cDNA library: 3-A1, 3-B2, 3-B5, 3-H7, 2-A5 and 2-C9. The molecular weights of chimeric pre-β-lactamase: casein proteins synthesized in Escherichia coli were estimated by immunoblotting. Colony hybridization and nucleotide sequence analysis showed that clone 3-B5 contained a cDNA fragment of bovine χ-casein, clone 3-H7 contained a cDNA fragment of β-casein, while clones 2-A5 and 2-C9 carried cDNA fragments of α_si-casein.     

Direct and general selection for lysogens of Escherichia coli by phage lambda recombinant clones.

We report a simple in vivo technique for introducing an antibiotic resistance marker into phage lambda. This technique could be used for direct selection of lysogens harboring recombinant phages from the Kohara lambda bank (a collection of ordered lambda clones carrying Escherichia coli DNA segments). The two-step method uses homologous recombination and lambda DNA packaging to replace the nonessential lambda DNA lying between the lysis genes and the right cohesive (cos) end with the neomycin phosphotransferase (npt) gene from Tn903. This occurs during lytic growth of the phage on a plasmid-containing host strain. Neomycin-resistant (npt+) recombinant phages are then selected from the lysates containing the progeny phage by transduction of a polA1 lambda lysogenic host strain to neomycin resistance. We have tested this method with two different Kohara lambda phage clones; in both cases, neomycin resistance cotransduced with the auxotrophic marker carried by the lambda clone, indicating complete genetic linkage. Linkage was verified by restriction mapping of purified DNA from a recombinant phage clone. We also demonstrate that insertion of the npt+ recombinant phages into the lambda prophage can be readily distinguished from insertion into bacterial chromosomal sequences.     

Screening recombinant clones containing sequences homologous to Escherichia coli genes using single-stranded bacteriophage vector

Detection and isolation of Escherichia coli clones carrying vectors with foreign DNA sequences partially homologous to specific E. coli genes is difficult because denatured DNA in the host genome can hybridize with the probe. In this paper we present a procedure which simplifies this task by using bacteriophage M13 as the cloning vector. The procedure takes advantage of the secretory properties of the phage, as well as the property of nitrocellulose membrane to bind protein and single-stranded DNA but not double-stranded DNA. This procedure is shown to be effective in identifying E. coli clones containing sequences of Chlamydomonas reinhardtii chloroplast DNA that are homologous to the rpoC gene of E. coli. We suggest that this procedure can be used generally for rapid isolation of DNA sequences that are homologous to E. coli genes.     

Characterization of a pollen-specific gene family from Brassica napus which is activated during early microspore development

In this paper we describe the isolation and characterization of a genomic clone (Bp4) from Brassica napus which contains three members of a pollen-specific multigene family. This family is composed of 10 to 15 closely related genes which are expressed in early stages of microspore development. The complete nucleotide sequence of the clone Bp4 and of three homologous cDNA clones is reported. One of the genes (Bp4B) contained in the genomic clone is believed to be non-functional because of sequence rearrangements in its 5 region and intron splicing sites. The remaining genes (Bp4A and Bp4C), as well as the cDNA clones, appear to code for small proteins of unique structure. Three different types of proteins can be predicted as a result of the deletion of carboxy or amino terminal portions of a conserved core protein. These proteins all share a common alternation of hydrophobic and hydrophilic domains. A fragment of the genomic clone containing the gene Bp4A, as well as the non-functional gene Bp4B, was introduced into tobacco plants via Agrobacterium-mediated transformation. The functional gene Bp4A is expressed in transgenic tobacco plants and shows spatial and temporal regulation consistent with the expression patterns seen in Brassica napus.     

Cloning of genes encoding redox proteins of known amino acid sequence from a library of the Desulfovibrio vulgaris (Hildenborough) genome

A library of 900 recombinant phages has been constructed for the genome of Desulfovibrio vulgaris Hildenborough (1.7 x 10(6) bp) by cloning size-fractionated Sau3A fragments (15-20 kb) into the replacement vector lambda-2001. When a hydrogenase gene probe, a 4.7-kb SalI-EcoRI fragment of known nucleotide sequence, was used to screen the plaque lifted library, 23 positive clones were found, which together span 31 kb of D. vulgaris DNA. To facilitate the cloning of genes with oligodeoxynucleotides as probes, DNA was purified for all clones in the library and spotted on a 16 x 16-cm grid of nitrocellulose. This grid was incubated sequentially to identify lambda clones containing the gene for redox proteins of known amino acid sequence: cytochrome c3 (one 18-mer----four clones), flavodoxin (one 17-mer and one 26-mer----one clone) and rubredoxin (one 44-mer----21 clones). The four cyc-positive clones are also recognized by the rubredoxin oligodeoxynucleotide probe. Restriction mapping defines a 35-kb region of the D. vulgaris chromosome in which the rub and cyc loci are separated by 17.5 kb. The nucleotide sequence of the rubredoxin gene was determined and the deduced amino acid sequence found to agree with that determined in Bruschi [Biochim. Biophys. Acta 434 (1976) 4-17] with the exception of Thr-21 which is found to be encoded by GAC, an Asp codon. A plausible ribosome-binding site precedes the N-terminal initiator methionine residue. Rubredoxin does not have an N-terminal signal sequence which is in agreement with the cytoplasmic location of this redox protein.     

Construction of cloned libraries from RNA of human fetal tissues

We have constructed libraries of recombinant DNA plasmids containing sequences complementary to polyadenylated RNA from a variety of human midtrimester fetal tissues. The bacterial colonies containing these plasmids have been grown and replicated on nitrocellulose filters in a manner that facilitates permanent storage, rapid screening, and transportability to other laboratories. We screened a portion of the library for the presence of repetitive sequences and found that approximately 20% of the clones contain repetitive sequences. We have also shown that some clones contain nonrepetitive sequences. Pools of recombinant cDNA-containing plasmids devoid of repetitive sequences have been constructed to permit the chromosomal localization of a variety of actively transcribed sequences. The construction of such large, tissue-specific clone banks should facilitate the direct isolation, mapping, and characterization of normal and abnormal human genes.     

Fine specificity and T-cell receptor β-chain gene rearrangements of five H-2Db-specific cytotoxic T-cell clones

A panel of cytotoxic T lymphocyte clones that recognize H-2^b target cells has been established. Six different clones were distinguished according to the following criteria. First, the fine specificity of the clones was determined by testing proliferation and cytotoxicity on target cells of recombinant mice. Clone 221 recognized H-2K^b, and five other clones recognized H-2D^b. Clone 433 distinguished itself from the other five D^b-specific clones by cross-reacting with an antigen on H-2^k cells. Second, the presence of an idiotypic determinant as defined by the 3179 clone-specific monoclonal antibodies was investigated in cytotoxicity inhibition experiments. One of the D^b-specific clones, 653, was inhibited by these antibodies and was therefore clearly different from the other D^b-specific clones. The third criterion involved the rearrangement pattern of the DNA coding for the chain of the T-cell receptor. Southern blot analysis showed that each clone had a unique pattern. Interestingly, clone 653 , which expresses the same idiotypic determinant as clone 3F9, had deleted the C 1 gene cluster, whereas this gene is functionally expressed in clone 3179.Abbreviations used in this paper C constant gene segment - Con A concanavalin A - CTLs cytotoxic T lymphocytes - D diversity gene segment - ³H-dThd tritiated thymidine - J joining gene segment - kb kilobase pairs - LPS lipopolysaccharide - MHC major histocompatibility complex - MLC mixed lymphocyte culture - SDS sodium dodecyl sulfate - V ariable gene segment     

Construction and characterization of the transformation-competent artificial chromosome (TAC) libraries of Leymus multicaulis

Transformation-competent artificial chromosome system is able to clone and transfer genes efficiently in plants.In order to clone genes highly tolerant to barley yellow dwarf virus(BYDV),Aphids,drought and salt from Leymus multicaulis,the two TAC genomic libraries I and II were constructed in vector pYLTAC17 and pYLTAC747H/sacB,which contain about 165000 and 236000 recombinant clones sepa-rately.The genome coverage of the two libraries was totally estimated to be about 3―5 haploid genome equivalents,as size selection of genomic DNA fragments was approximately from 9 to 300 kb.Clones of the genomic libraries were collected as bulked pools each containing 500 clones or so,stored in twelve 96-deep-well plates and then were gridding in triplicate onto a high-density colony hybridization filter with a 3×3 pattern using a GeneTAC?G3 arraying robot after being transferred manually into three 384-well plates.Meanwhile 2501 and 2890 clones of Library in pYLTAC17 and in pYLTAC747H/sacB were stored individually in fourteen 384-well plates and then were automatically gridding in duplicate onto a high-density colony hybridization filter with a 6×6 pattern after a replication of plates.Nineteen positive clones were detected by using the probe glutahione reductase gene of L.multicaulis.TAC libraries constructed here can be used to isolate genomic clones containing target genes,and to carry out genome walking for positional cloning.Once the target TAC clones were isolated,they could be immediately transferred into plant genomes with the Agrobacterium system.     

Analysis of sequences from human brain cDNA gene bank which are functionally active in nervous tissue and tumor cells

Construction of a human cortex cDNA bank is described as well as the isolation from this bank of pBH71 and pBH3 clones with preferential expression in nervous and in tumor cells. The clones can be included into the third class of cDNA according to Sutcliff's classification. The mRNA corresponding to this cDNA class is considered to play the key role in determination of specificity of nervous tissue. Expression of the pBH71 sequence was revealed in human cortex and in tissues of different genesis (from neuroblastoma to uterus myoma), a 2 kb mRNA which corresponds to one and the same cDNA chain having been found in all tissues under analysis. The nucleotide sequence of cDNA insertion into the pBH71 clone of 447 n.p. was determined, and particular features of cDNA nucleotide composition and possible schemes of its translation were analysed. Weak homology was found between the 3'-end of cDNA insertion of the pBH71 clone and the 3'-end region of human proopiomelanocortine. The cDNA of the pBH3 clone hybridizes with the 0.8 kb mRNA revealed in human cortex and neuroendocrine tumors of different nature. No homology was revealed between the cDNA sequence of the pBH3 clone and any genes deciphered.     

Two-dimensional electrophoresis/phage panning (2D-PP): a novel technology for direct antibody selection on 2-D blots

We describe a novel method, two-dimensional electrophoresis/phage panning (2D-PP), for the generation of antibodies against proteins in crude biochemical samples, such as cellular membrane fractions. These sources have traditionally presented problems as to the development of antibodies by conventional techniques. 2D-PP involves two-dimensional resolution of proteins, blotting of the proteins onto a nitrocellulose membrane, and screening of a phage antibody library and isolation of corresponding antibodies. By 2D-PP with detergent-insoluble "lipid rafts" as a target protein complex, we obtained specific phage pools against eight antigen spots (from a total of 39 spots). These antibodies were functional in Western blotting, enzyme-linked immunosorbent assaying (ELISA), and immunoscreening of a cDNA expression library. Propagation of anti-nitrocellulose phages was the major problem in 2D-PP, but was overcome by the use of the soluble anti-nitrocellulose antibody fragment. 2D-PP constitutes a key tool for functional analysis of proteins in complex fractions.     

A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, lambda pGY97

It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.     

Conditions that allow for effective transfer of membrane proteins onto nitrocellulose membrane in Western blots

A major hurdle in characterizing bacterial membrane proteins by Western blotting is the ineffectiveness of transferring these proteins from sodium dodecyl sulfate -- polyacrylamide gel electrophoresis (SDS-PAGE) gel onto nitrocellulose membrane, using standard Western blot buffers and electrophoretic conditions. In this study, we compared a number of modified Western blotting buffers and arrived at a composition designated as the SDS-PAGE-Urea Lysis buffer. The use of this buffer and specific conditions allowed the reproducible transfer of highly hydrophobic bacterial membrane proteins with 2-12 transmembrane-spanning segments as well as soluble proteins onto nitrocellulose membranes. This method should be broadly applicable for immunochemical studies of other membrane proteins.     

Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans.

A genomic clone bank of Paracoccus denitrificans DNA has been constructed in the expression vector set pEX1, pEX2, and pEX3. Screening of this clone bank with antibodies raised against P. denitrificans methanol dehydrogenase resulted in the isolation of a clone, pNH3, that synthesized methanol dehydrogenase cross-reactive proteins. The nucleotide sequence of the P. denitrificans DNA fragment inserted in this clone has been determined and shown to contain the full methanol dehydrogenase structural gene. DNA cross-hybridization was found with DNA fragments which have been reported to contain the methanol dehydrogenase structural genes from Methylobacterium sp. strain AM1 and Methylobacterium organophilum.     

Construction of a Tc1-like transposon Sleeping Beauty-based gene transfer plasmid vector for generation of stable transgenic mammalian cell clones

We have constructed a single plasmid-, Tc1-like transposon-based gene transfer vector, termed the Prince Charming vector (pPC). The pPC vector was constructed by ligating the CMV-driven "Sleeping Beauty" transposase gene downstream to the Tc1-like transposon inverted repeat (IR) elements and by inserting the RSV promoter (to drive expression of the gene-of-interest) along with a multiple cloning site (MCS), a polyadenylation signal, and the SV40 promoter-driven neomycin gene, at a site flanked by the transposon IR elements. To assess the utility of the pPC vector, we cloned a red fluorescent protein (RFP) gene into the pPC vector at the MCS and transfected human TE85 osteosarcoma cells with the pPC-RFP expression vector using Effectene. Stable transgenic cell clones expressing RFP were selected with G418 sulfate and individual clones were isolated. After 4 weeks of clonal isolation and expansion, 99% of cells in each randomly selected clone expressed RFP strongly. Aliquots of each clone were then maintained in either the presence or the absence of G418 sulfate and were passaged weekly. Even after 6 months in culture in the absence of G418 sulfate, approximately 90% of the cells in each clone still maintained a strong expression level of RFP, indicating that these transgenic cell clones were stable and that the clonal stability of these clones did not require a constant selection pressure. In conclusion, we have developed a single plasmid-, Tc1-like transposon-based gene transfer vector that can be used to generate stable transgenic mammalian cell clones.     

Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry.

A total of 896 samples of retail fresh meats and poultry was assayed for Escherichia coli serogroup O157:H7 by a hydrophobic grid membrane filter-immunoblot procedure developed specifically to isolate the organism from foods. The procedure involves several steps, including selective enrichment, filtration of enrichment culture through hydrophobic grid membrane filters, incubation of each filter on nitrocellulose paper on selective agar, preparation of an immunoblot (by using antiserum to E. coli O157:H7 culture filtrate) of each nitrocellulose paper, selection from the filters of colonies which corresponded to immunopositive sites on blots, screening of isolates by a Biken test for precipitin lines from metabolites and antiserum to E. coli O157:H7 culture filtrate, and confirmation of isolates as Vero cell cytotoxic E. coli O157:H7 by biochemical, serological, and Vero cell cytotoxicity tests. E. coli O157:H7 was isolated from 6 (3.7%) of 164 beef, 4 (1.5%) of 264 pork, 4 (1.5%) of 263 poultry, and 4 (2.0%) of 205 lamb samples. One of 14 pork samples and 5 of 17 beef samples contaminated with the organism were from Calgary, Alberta, Canada, grocery stores, whereas all other contaminated samples were from Madison, Wis., retail outlets. This is the first report of the isolation of E. coli O157:H7 from food other than ground beef, and results indicate that the organism is not a rare contaminant of fresh meats and poultry.     

Peptide mass fingerprinting by matrix-assisted laser desorption ionization mass spectrometry of proteins detected by immunostaining on nitrocellulose

We have developed an approach that allows peptide mass mapping by matrix-assisted laser desorption ionization-mass spectrometry of proteins visualized on a nitrocellulose membrane by immunochemical detection. Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electroblotted onto a nitrocellulose membrane and after blocking with a nonprotein-containing polymer such as polyvinylpyrrolidone 40 (PVP-40) or Tween 20, the proteins are stained with fount India ink. After incubation with primary and, if required, secondary peroxidase-coupled antibodies, immunochemically reactive proteins can be visualized using conventional enhanced chemiluminescence detection and assigned to the India ink-stained membrane by simple superposition. The proteins of interest are excised, submitted to "on-membrane" cleavage and the peptides are analyzed by mass spectrometry. Protein-based blocking reagents normally used in standard immunodetection protocols, such as skimmed milk, can be employed. We have obtained high-quality mass spectra of bovine serum albumin (BSA) detected on an immunoblot with an estimated amount of 100 fmol applied onto the gel, indicating the sensitivity of the present method. In addition, the approach is demonstrated with two other commercially available proteins, a serum protein, the successful identification of a tyrosine phosphorylated protein from total rat liver homogenate and serine phosphorylated proteins from an EcR 293 nuclear extract separated by two-dimensional (2-D) SDS-PAGE.     

自噬相关基因LC3-I的基因表达及单克隆抗体的制备

目的：通过克隆LC3．I基因,体外原核表达LC3-I蛋白后制备抗LC3单克隆抗体,作为自噬研究中的标记分子检测自噬的发生和发展过程。方法：RT．PCR方法从RAW264．7细胞基因组中克隆LC3基因,亚克隆至pQE80L原核表达载体后转化E．cobDH5a进行诱导表达,SDS—PAGE电泳及Westemblot鉴定表达蛋白。蛋白纯化后免疫BALB／c小鼠。采用淋巴细胞杂交瘤技术,制备分泌抗LC3．I杂交瘤细胞株,体内诱生腹水制备mAb,间接ELISA法测定其效价,辛酸一硫酸铵沉淀法及亲和层析法纯化mAb。结果：成功克隆了LC3一I基因,并对其在E．coilDH5a进行诱导表达,SDS-PAGE分析表明在相对分子量Mr为20×10^3有特异条带,Westernblot验证表达产物具有一定的生物学活性。建立了3株稳定分泌特异性抗LC3-ImAb的杂交瘤细胞株,诱导产生的腹水获得的抗体效价在10^5-10^7之间,结论：在E．coli中对LC3-I进行表达,并制备特异性较强抗LC3-I蛋白的单克隆抗体。为自噬研究提供了良好的标记分子,可对自噬形成和发展进行有效的检测。     

Simultaneous measurements of rates of protein de novo synthesis and its steady-state levels by immunoblotting

Immunoblotting was used for simultaneous measurements of rates of de novo synthesis and steady-state levels of a model protein-cytochrome P450r. This is the major phenobarbital-inducible P450 form in cultured chick-embryo hepatocytes (L. Oron, and S. Bar-Nun, 1984) Biochem. Biophys. Res. Commun. 125, 1096-1102). Cultured hepatocytes were metabolically labeled with [35S]methionine, and cellular proteins were resolved by gel electrophoresis and then quantitatively electroblotted onto nitrocellulose. The blot was directly exposed to autoradiography and the rates of de novo synthesis were estimated from the 35S label of P450r. The same blot was then incubated with specific anti-P450r antibodies, followed by 125I-labeled secondary antibodies, and the blot was reexposed to autoradiography through white paper. The paper masked the 35S-labeled proteins and the steady-state levels were estimated from the 125I-immunodecorated P450r. By this simple method, "true" inducers of cytochrome P450r, such as phenobarbital, can be defined, whereas "false" inducers, which primarily affect the degradation of P450r, can be detected.