噬菌体随机肽库筛选抗志贺样毒素Ⅱ结合亚单位Stx2B的单抗的识别表位

目的利用噬菌体随机肽库技术筛选志贺样毒素Ⅱ结合亚单位Stx2B的单抗的识别表位。方法以抗志贺样毒素Ⅱ结合亚单位Stx2B的单克隆抗体筛选噬菌体随机12肽库,挑取阳性克隆测定DNA序列,推导其氨基酸序列并进行同源性分析。通过ELISA鉴定获得的噬菌体短肽与单抗之间的结合特性。结果从噬菌体随机12肽库中筛选出20株可与抗志贺样毒素Ⅱ结合亚单位Stx2B的单抗特异结合的噬菌体克隆,其中多数克隆呈现核心序列WTSRW（Q）,该序列与志贺样毒素Ⅱ结合亚单位Stx2B的一级序列具有一定的同源性。结论WTSRW（Q）序列是志贺样毒素Ⅱ结合亚单位Stx2B单抗的识别表位。     

Isolation of genes expressed in specific tissues of Arabidopsis thaliana by differential screening of a genomic library

The small genome size of Arabidopsis thaliana allows the isolation of genes expressed in specific tissues and under controlled conditions by the differential screening of a genomic library, as has been shown previously for yeast and Drosophila. cDNA probes, based on poly(A)+ mRNA isolated from different Arabidopsis organs, were used in colony hybridizations with 1145 randomly chosen genomic clones, representing 27,000 kb of Arabidopsis DNA. Twenty percent of the clones containing low-copy-number sequences hybridized with one or more of the cDNA probes that were synthesized from mRNA isolated from leaves, stems, seed pods, inflorescences, callus tissue, and light-grown and dark-grown plants. Comparison of the colony hybridizations led to the identification of a large variety of clones which contain differentially expressed genes. The pattern of expression was confirmed by Northern analysis. The advantage of the described method is that it yields directly genomic sequences that contain specifically expressed or induced genes. In particular, it circumvents the construction and differential screening of cDNA libraries for every tissue or environmental parameter to be analyzed.     

The expression of Ia antigenic determinants on macrophages required for the in vitro antibody response.

A subpopulation of antigen-presenting macrophages required for an in vitro antibody response to burro erythrocytes was deleted by pretreating the splenic macrophages with anti-Ia serum and complement (C). The in vitro response of the macrophage depleted T-B cell population could not be restored by the addition of macrophages resistant to anti-Ia antibodies and C (Ia-). The response of Ia- macrophages and the macrophage-depleted T-B cells was only reconstituted by the addition of Ia+ macrophages. Macrophages pretreated with anti-Ia antibodies restricted to react with determinants of one I subregion could not support the in vitro antibody response when added to cultures whose macrophages were pretreated with anti-Ia serum and C specific for the I-J subregion. These results confirmed that Ia determinants of the I-A, the I-E, and the I-C subregions were all expressed on the I-J+ macrophage required for an in vitro antibody response.     

Specific glycosidase activity isolated from a random phage display antibody library

Carbohydrates serve as key receptor sites in various cellular events such as viral attachment, tumor formation, and tissue inflammation. A potential route to control these events is to manipulate targeted carbohydrate structures in vivo using specifically designed glycohydrolases. Here we show that a stereospecific catalytic activity designed toward a particular sugar and linkage can be readily isolated from a phage display antibody library derived from a nonimmunized host. The activity was isolated using a transition-state analogue mimicking an alpha-glucosidasic linkage as antigen and showed a 20-fold specificity for that sugar and linkage. The DNA sequence, however, contains a large deletion in the antibody gene, which also changes the downstream reading frame, resulting in a translated sequence containing only 57 amino acids that has a predominantly hydrophobic amino terminal and a strongly hydrophilic carboxy terminal. The isolated catalytic activity has a strong pH dependence, attributable to one or more of the numerous potentially charged groups in the carboxyl terminal. While the protein readily forms more stable multimers, the 7.3-kD monomer represents by far the smallest glycosidase enzyme reported to date and can provide substantial new information toward understanding and modifying glycosidase activity.     

Retro-recombination screening of a mouse embryonic stem cell genomic library

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for ‘rolling out’ plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage–plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed ‘retro-recombination screening’ (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a λTK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage λ into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.     

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library

AIMS: Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. METHODS AND RESULTS: We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. CONCLUSIONS: Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.     

Variable nuclear markers for Melanoplus oregonensis identified from the screening of a genomic library

We report the isolation of seven single‐copy nuclear loci from the montane grasshopper Melanoplus oregonensis. With an average length of approximately 930 bp and between five and 67 variable sites, these loci are useful for population genetic analyses within M. oregonensis. Amplification of nuclear loci in additional species of western Melanoplus grasshoppers suggests that they will also be useful for a variety of population genetic, phylogeographic and phylogenetic studies.     

Biologic, antigenic, and full-length genomic characterization of a bovine-like coronavirus isolated from a giraffe

Coronaviruses (CoVs) possess large RNA genomes and exist as quasispecies, which increases the possibility of adaptive mutations and interspecies transmission. Recently, CoVs were recognized as important pathogens in captive wild ruminants. This is the first report of the isolation and detailed genetic, biologic, and antigenic characterization of a bovine-like CoV from a giraffe (Giraffa camelopardalis) in a wild-animal park in the United States. CoV particles were detected by immune electron microscopy in fecal samples from three giraffes with mild-to-severe diarrhea. From one of the three giraffe samples, a CoV (GiCoV-OH3) was isolated and successfully adapted to serial passage in human rectal tumor 18 cell cultures. Hemagglutination assays, receptor-destroying enzyme activity, hemagglutination inhibition, and fluorescence focus neutralization tests revealed close biological and antigenic relationships between the GiCoV-OH3 isolate and selected respiratory and enteric bovine CoV (BCoV) strains. When orally inoculated into a BCoV-seronegative gnotobiotic calf, GiCoV-OH3 caused severe diarrhea and virus shedding within 2 to 3 days. Sequence comparisons and phylogenetic analyses were performed to assess its genetic relatedness to other CoVs. Molecular characterization confirmed that the new isolate belongs to group 2a of the mammalian CoVs and revealed closer genetic relatedness between GiCoV-OH3 and the enteric BCoVs BCoV-ENT and BCoV-DB2, whereas BCoV-Mebus was more distantly related. Detailed sequence analysis of the GiCoV-OH3 spike gene demonstrated the presence of a deletion in the variable region of the S1 subunit (from amino acid 543 to amino acid 547), which is a region associated with pathogenicity and tissue tropism for other CoVs. The point mutations identified in the structural proteins (by comparing GiCoV-OH3, BCoV-ENT, BCoV-DB2, and BCoV-Mebus) were most conserved among GiCoV-OH3, BCoV-ENT, and BCoV-DB2, whereas most of the point mutations in the nonstructural proteins were unique to GiCoV-OH3. Our results confirm the existence of a bovine-like CoV transmissible to cattle from wild ruminants, namely, giraffes, but with certain genetic properties different from those of BCoVs.     

Isolation of a large number of novel mammalian genes by a differential cDNA library screening strategy.

As part of the ongoing human and mouse genome projects, the aim of this study was to isolate novel, previously uncharacterized, genes from mouse testis. Two approaches were compared for their effectiveness in isolating novel genes: random, vs differential, complementary DNA (cDNA) cloning methods. In the differential approach, only the cDNA clones containing rare sequences (as determined by preliminary clone hybridization) are further analyzed; in the random approach, cDNA clones are isolated at random from the cDNA library. More than two hundred cDNA clones altogether were analyzed, using a PCR-mediated amplification and sequencing strategy. A comparison of these sequences to nucleic acid and protein sequence databases, revealed that 84% of the isolated rare cDNA clones represented new, previously uncharacterized mouse genes. In contrast, less than 63% of the cDNA clones isolated at random from cDNA libraries, contained novel genes. Thus, the probability of isolating new, previously uncharacterized, mammalian genes from cDNA libraries can be markedly improved by focusing efforts on clones containing rare sequences.     

Polymorphic microsatellite markers isolated from partially enriched genomic library of Chitala chitala

A total eight polymorphic microsatellite loci were obtained from genomic library of Indian feather back, Chitala chitala (order Osteoglossiformes, family Notopteridae) and the 46 samples were analysed to determine genetic variation. The mean number of alleles per locus ranged from 4.50 to 5.25, and expected heterozygosity ranged from 0.124 to 0.852. Deviation from Hardy–Weinberg equilibrium expectations (P < 0.002) was observed at loci Cch2, Cch9 (Bhaghirathi) and Cch9 (Brahmaputra). The identified microsatellite loci were found promising for population genetics studies of C. chitala and related species Notopterus notopterus (family Notopteridae).     

General method for cloning amplified DNA by differential screening with genomic probes.

Mutant Syrian hamster cell lines resistant to N-(phosphonacetyl)-L-aspartate, a potent and specific inhibitor of aspartate transcarbamylase, have amplified the gene coding for the multifunctional protein (CAD) that includes this activity. The average amount of DNA amplified is approximately 500 kilobases per gene copy, about 20 times the length of the CAD gene itself. A differential screening method which uses genomic DNAs as probes was developed to isolate recombinant phage containing fragments of amplified DNA. One probe was prepared by reassociating fragments of total genomic DNA from 165-28, a mutant cell line with 190 times the wild-type complement of CAD genes, until all of the sequences repeated about 200 times were annealed and then isolating the double-stranded DNA with hydroxyapatite.This DNA was highly enriched in sequences from the entire amplified region, whereas the same sequences were very rare in DNA prepared similarly from wild-type cells. After both DNAs were labeled by nick translation, highly repeated sequences were removed by hybridization to immobilized total genomic DNA from wild-type cells. A library of cloned DNA fragments from mutant 165-28 was screened with both probes, and nine independent fragments containing about 165 kilobases of amplified DNA, including the CAD gene, have been isolated so far. These cloned DNAs can be used to study the structure of the amplified region, to evaluate the nature of the amplification event, and to investigate gene expression from the amplified DNA. For example, one amplified fragment included a gene coding for a 3.8-kilobase, cytoplasmic, polyadenylated RNA which was overproduced greatly in cells resistant to N-(phosphonacetyl)-L-aspartate. The method for cloning amplified DNA is general and can be used to evaluate the possible involvement of gene amplification in phenomena such as drug resistance, transformation, or differentiation. DNA fragments corresponding to any region amplified about 10-fold or more can be cloned, even if no function for the region is known. The method for removing highly repetitive sequences from genomic DNA probes should also be of general use.     

scFv antibodies against infectious bursal disease virus isolated from a combinatorial antibody library by flow cytometry

Infectious bursal disease is an economically important disease that affects chickens worldwide. Here, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry against VP2 protein through a bacteria display technology, resulting in the enrichment of scFv. Three scFv clones with different fluorescence intensity were obtained by random colony pick up. The isolated scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis. The potential use of the selected IBDV-specific scFv antibodies was demonstrated by the successful application of the isolated antibodies in western blotting assay and ELISA.     

Identification of three wheat globulin genes by screening a Triticum aestivum BAC genomic library with cDNA from a diabetes-associated globulin

Background

Plant systemic signaling characterized by the long distance transport of molecules across plant organs involves the xylem and phloem conduits. Root-microbe interactions generate systemic signals that are transported to aerial organs via the xylem sap. We analyzed the xylem sap proteome of soybean seedlings in response to pathogenic and symbiotic interactions to identify systemic signaling proteins and other differentially expressed proteins.

Results

We observed the increase of a serine protease and peroxidase in the xylem sap in response to Phytophthora sojae elicitor treatment. The high molecular weight fraction of soybean xylem sap was found to promote the growth of Neurospora crassa in vitro at lower concentrations and inhibit growth at higher concentrations. Sap from soybean plants treated with a P. sojae elicitor had a significantly higher inhibitory effect than sap from control soybean plants. When soybean seedlings were inoculated with the symbiont Bradyrhizobium japonicum, the abundance of a xyloglucan transendoglycosyl transferase protein increased in the xylem sap. However, RNAi-mediated silencing of the corresponding gene did not significantly affect nodulation in soybean hairy root composite plants.

Conclusion

Our study identified a number of sap proteins from soybean that are differentially induced in response to B. japonicum and P. sojae elicitor treatments and a majority of them were secreted proteins.     

Detection and isolation of RNA-binding proteins by RNA-ligand screening of a cDNA expression library.

A screening assay for the detection of RNA-binding proteins was developed. It allows the rapid isolation of cDNA clones coding for proteins with sequence-specific binding affinity to a target RNA. For developing the screening protocol, constituents of the human U1 snRNP were utilized as model system. The RNA partner consisted of the U1-RNA stem-loop II and the corresponding protein consisted of the 102 amino acid N-terminal recognition motif of the U1A protein, which was fused to beta-galactosidase and expressed by the recombinant lambda phage LU1A. Following binding of the fusion protein to nitrocellulose membranes, hybridization with a 32P-labeled U1-RNA ligand was carried out to detect specific RNA-protein interaction. Parameters influencing the specificity and the detection limit of binding were systematically investigated with the aid of the model system. Processing the nitrocellulose membranes in the presence of transition metals greatly increased the signal:background ratio. A simple screening protocol involving a single-buffer system was developed. Specific RNA-protein interaction could be detected in the presence of a large excess of recombinant phages from a cDNA library. Only moderate binding affinities (Kd = 10(-8) M) were required. The suitability of the RNA-ligand screening protocol was demonstrated by the identification of new viroid-RNA binding proteins from tomato.     

Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

Construction of a representative genomic library from a hexaploid

The large genome size and the great amount of DNA repeats make it rather difficult to obtain a representative hexaploid wheat genomic library. The protocol is given with modifications to phage isolation and to purification of vector and plant DNAs by electrogenomic libraries of the soft wheat variety carrying a translocated rye chromosome arm 1RS were constructed in arecD E. coli strain to reduce recombinational loss.