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PubMed1995

Systematic screening of an arrayed cDNA library by PCR.

We have developed a PCR-based method for rapid and effective screening of arrayed cDNA libraries. This strategy directly addresses the limitations of conventional hybridization-based schemes and provides a more rapid, cost-effective, and sensitive method compatible with large-scale and routine cDNA clone recovery. To prepare arrayed libraries, 1-2 x 10(6) cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within "blocks" of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion beginning with the plate pools. Next, the row/column pools corresponding to each positive plate pool were screened. Finally, isolated clones from within each positive well were identified by hybridization. We have applied this approach to the screening of an arrayed human brain cDNA library resulting in the recovery of cDNAs corresponding to > 25 genes and expressed sequence tags.

Adult↗

PubMed1991

Complete cDNA and gene sequence of the developmentally regulated arylphorin of Calliphora vicina and its homology to insect hemolymph proteins and arthropod hemocyanins.

Two cDNA libraries were prepared from poly(A)+ RNA isolated from fat bodies of last instar larvae of the blowfly Calliphora vicina. The libraries were probed with a genomic clone containing the coding sequence for an arylphorin subunit. Two cDNA clones as well as the genomic clone were mapped and their nucleotide sequences were determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 759 amino acid residues. The deduced primary structure of Calliphora arylphorin and hemolymph proteins of other insect species and arthropod hemocyanine show nearly 30% identity. Highly conserved regions could be also identified.

Adipose Tissue↗

PubMed1992

Isolation and sequence of sheep immunoglobulin E heavy-chain complementary DNA.

A complementary DNA (cDNA) corresponding to sheep immunoglobulin E heavy (IgE H) chain messenger RNA (mRNA) was isolated and sequenced. A fragment of sheep IgE constant H (epsilon)-chain was initially synthesized using the polymerase chain reaction (PCR) and single-stranded cDNA prepared from the mRNA of parasite-stimulated sheep lymph nodes. This fragment was then used to probe a cDNA library, again prepared from a parasite-stimulated lymph node. A recombinant clone containing cDNA encoding a sheep IgE H-chain of 1802 base pairs was isolated and the H-chain cDNA-sequenced. The nucleotide sequence was found to code for a complete sheep IgE H-chain, consisting of both variable (V) and constant (C) regions. When the amino acid sequence derived from the nucleotide sequence was compared to other species epsilon-chains, interesting homologies and differences between corresponding domains were found, including conservation in cysteine and tryptophan residues and variable glycosylation sites.

Amino Acid Sequence↗

PubMed2002

Development of new transformation-competent artificial chromosome vectors and rice genomic libraries for efficient gene cloning.

The transformation-competent artificial chromosome vector (TAC) system has been shown to be very useful for efficient gene isolation in Arabidopsis thaliana (Proc. Natl. Acad. Sci. USA 96 (1998) 6535). To adapt the vector system for gene isolation in crops, two new TAC vectors and rice genomic libraries were developed. The new vectors pYLTAC17 and pYLTAC27 use the Bar gene and Hpt gene driven by the rice Act1 promoter as the plant selectable markers, respectively, and are suitable for transformation of rice and other grasses. Two representative genomic libraries (I and II) of an Indica rice variety Minghui63, a fertility restorer line for hybrid rice, were constructed with pYLTAC17 using different size classes of partially digested DNA fragments. Library I and library II consisted of 34,560 and 1.2 x 10(5) clones, with average insert sizes of approximately 77 and 39 kb, respectively. The genome coverage of the libraries I and II was estimated to be about 5 and 11 haploid genome equivalents, respectively. Clones of the library I were stored individually in ninety 384-well plates, and those of the library II were collected as bulked pools each containing 30-50 clones and stored in eight 384-well plates. A number of probes were used to hybridize high-density colony filters of the library I prepared by an improved replicating method and each detected 2-9 positive clones. A method for rapid screening of the library II by pooled colony hybridization was developed. A TAC clone having an 80 kb rice DNA insert was successfully transferred into rice genome via Agrobacterium-mediated transformation. The new vectors and the genomic libraries should be useful for gene cloning and genetic engineering in rice and other crops.

Base Sequence↗

PubMed2005

Identification of a mitochondrial ATP synthase small subunit gene (RMtATP6) expressed in response to salts and osmotic stresses in rice (Oryza sativa L.).

Large areas of northern China have alkaline soil due to the accumulation of sodium carbonates (NaHCO3, Na2CO3). To understand better how plants can tolerate alkaline soil, a cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. A cDNA clone isolated from this library was identified by a homology search as a mitochondrial ATP synthase 6 kDa subunit gene (RMtATP6; GenBank accession nos AB055076, BAB21526). In transformed yeast and tobacco protoplasts, the RMtATP6 protein was localized in mitochondria using the green fluorescent protein (GFP) marker. Analysis of RMtATP6 mRNA levels suggested that the expression of this gene was induced by stress from sodium carbonates and other sodium salts. Transgenic tobacco overexpressing the RMtATP6 gene had greater tolerance to salt stress at the seedling stage than untransformed tobacco. Among the other genes for F1F0-ATPase of rice, some were found to be up-regulated by some environmental stresses and some were not. These data suggest that the RMtATP6 protein acts as a subunit of ATP synthase, and is expressed in response to stress from several salts, with the other genes coding for the subunits of the same ATP-synthase.

Adaptation, Physiological↗

PubMed1987

A recombinant molecule from a disseminating strain of Neisseria gonorrhoeae that confers serum bactericidal resistance.

A cosmid gene library was prepared from Neisseria gonorrhoeae JC1, a serum-resistant clinical isolate from a patient with disseminated gonococcal infection. From this library a recombinant molecule, pWM3, was isolated which had the ability to transform F62, a serum-sensitive strain of N. gonorrhoeae, to serum resistance. This plasmid contained 2.2 kilobases of insert gonococcal DNA that coded for two peptides, one of 29 kilodaltons (kDa) and one of 17.5 kDa. Deletion of the region coding for the 29-kDa peptide resulted in the loss of the ability of the plasmid to transform F62 to serum resistance. N. gonorrhoeae F62 acquired the ability to bind blocking antibody when transformed with pWM3 or subclones that code for only the 29-kDa protein. Although similar in size, the cloned 29-kDa protein and protein III are antigenically distinct.

Antibodies, Bacterial↗

PubMed1986

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.

Animals↗

PubMed1997

Synthesis utilizing insoluble polymers: new reactions and small molecules.

It can be argued that solid-phase organic chemistry is the preferred method of preparing screening libraries for lead compound discovery and lead optimization within the pharmaceutical industry. Solid-phase peptide chemistry is the most reliable and general of the synthetic methods available today; however, the purities of products are inadequate for the needs of the current market and the screening departments of pharmaceutical companies. Recent synthetic advances in solid-phase organic chemistry have started to increase the range of chemistry that can be used to prepare 'drug-like' small molecules in library format.

Chemistry, Organic↗

PubMed1990

Mouse embryonic stem cells express the cardiac myosin heavy chain genes during development in vitro.

In the mouse embryo, early organogenesis is characterized by the formation of a functional cardiac muscle, such that 9-day embryos exhibit beating, although not fully developed hearts. In light of this observation, we found it intriguing that mouse embryoid bodies (EB), which can develop in vitro from totipotential embryonic stem cells, undergo spontaneous contractile activity. To determine if these cells are capable of recapitulating aspects of cardiogenesis, a cDNA library was prepared from beating EB and screened with a chicken skeletal myosin heavy chain cDNA. We found that the predominant myosin transcripts in the library encode the alpha- and beta-cardiac isoforms. In addition, an embryonic skeletal myosin cDNA was isolated. The myosin heavy chain transcripts in both EB and 9-day embryonic hearts were found to be the same. Transcript-specific primers were prepared, and polymerase chain reaction analyses on single EB were carried out. The data show that a single EB is capable of expressing both the alpha- and beta-isoforms as well as very low amounts of the embryonic skeletal transcript. These data indicate that EB transcribe the appropriate tissue- and developmental stage-specific myosin heavy chain genes and therefore serve as a model system for studying early cardiogenic processes at the molecular level.

Animals↗

PubMed2002

Serial analysis of gene expression in renal carcinoma cells reveals VHL-dependent sensitivity to TNFalpha cytotoxicity.

We have used serial analysis of gene expression (SAGE) to investigate the influence of the von Hippel-Lindau (VHL) gene on global gene expression profiles. SAGE libraries were prepared from renal cell carcinoma (RCC) lines that either lack (parental) or express wild-type VHL (wtVHL). Comparison of these libraries revealed some differentially expressed genes (Glut-1, for example) that were known to be influenced by VHL, but the majority of genes had not previously been reported to be affected by the cell's VHL status. The identification of several genes involved in TNFalpha-mediated events prompted us to compare the sensitivity of cells with different VHL status in TNFalpha cytotoxicity assays. Strikingly, VHL-deficient cells were much more resistant to the toxic influence of TNFalpha. We propose that VHL-dependent sensitization of RCC cells to TNFalpha-mediated killing may contribute to VHL's growth suppressive function.

Blotting, Northern↗

PubMed1996

Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae.

We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non-typable Haemophilus influenzae (NTHI), mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to 70-75% of NTHI strains. NTHI strains that lack HMW1/HMW2-like proteins remain capable of efficient attachment to cultured human epithelial cells, suggesting the existence of additional adhesion molecules. We reasoned that characterization of high-molecular-weight immunogenic proteins from an HMW1/HMW2-deficient strain might identify additional adhesion proteins. A genomic library was prepared in lambda EMBL3 with chromosomal DNA from non-typable Haemophilus strain 11, a strain that lacks HMW1/HMW2-like proteins. The library was screened immunologically with convalescent serum from a child naturally infected with strain 11, and phage clones expressing high-molecular-weight recombinant proteins were identified by Western blot analysis. One clone was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non-adherent Escherichia coli strain DH5 alpha with plasmids containing the genetic locus encoding this protein gave rise to E. coli transformants that adhered avidly to Chang conjunctival cells. Subcloning and mutagenesis studies localized the DNA conferring the adherence phenotype to a 4.8 kbp fragment, and nucleotide sequence analysis further localized the gene encoding the adhesion protein to a 3.3 kbp open reading frame predicted to encode a protein of 114 kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2-deficient non-typable H. influenzae strains. In contrast, the hia gene was not present in any of 23 non-typable H. influenzae strains which expressed HMW1/HMW2-like proteins. Identification of this second family of high-molecular-weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HMW1/HMW2-like proteins and Hia-like proteins which would be protective against disease caused by most or all non-typable H. influenzae.

Adhesins, Bacterial↗

PubMed1988

Nucleotide sequences of chimpanzee MHC class I alleles: evidence for trans-species mode of evolution.

To obtain an insight into the evolutionary origin of the major histocompatibility complex (MHC) class I polymorphism, a cDNA library was prepared from a heterozygous chimpanzee cell line expressing MHC class I molecules crossreacting with allele-specific HLA-A11 antibodies. The library was screened with human class I locus-specific DNA probes, and clones encoding both alleles at the A and B loci have been identified and sequenced. In addition, the sequences of two HLA-A11 subtypes differing by a single nucleotide substitution have been obtained. The comparison of chimpanzee and human sequences revealed a close similarity (up to 98.5%). The chimpanzee A locus alleles showed greatest similarity to the human HLA-A11/A3 family of alleles, one of them being very close to HLA-A11. Similarly, segments of the ChLA-B alleles displayed greatest similarity to certain HLA-B alleles. The calculated evolutionary branch point for the A11-like alleles is 7 x 10(6) to 9 x 10(6) years, whereas the other A locus alleles diverged between 12 x 10(6) and 17 x 10(6) years ago. Since the human and chimpanzee lineages separated 5 x 10(6) to 7 x 10(6) years ago, our data support the notion that during evolution, MHC alleles are transmitted from one species to the next.

Alleles↗

PubMed1996

Human antibody responses to HIV type 1 glycoprotein 41 cloned in phage display libraries suggest three major epitopes are recognized and give evidence for conserved antibody motifs in antigen binding.

A large panel of human Fab fragments against the gp41 subunit of the HIV-1 envelope glycoprotein was isolated by panning six phage-displayed antibody libraries against recombinant gp41. The libraries were prepared from HIV-1-seropositive donors. Twenty-three Fabs recognizing conformation-dependent determinants on gp41 were isolated. Further selection of libraries against (1) gp41 ligated with Fabs from the initial selection and against (2) a recombinant gp41-containing gp140 protein yielded five additional Fabs. Competition of members of the Fab panel with one another and with previously described antibodies revealed a series of overlapping specificities that were conveniently grouped into three major epitope clusters. The majority of Fabs recognized epitopes involving residues 649-668 (previously known as the cluster II region), numbered using the Los Alamos LAI sequence. A second set of Fabs reacted with an epitope involving residues 584-609 (known as the cluster I region). Another set of Fabs appeared to recognize a third conformational epitope that has been termed the cluster III region. This third Fab epitope group demonstrated some overlap with both clusters I and II in binding assays. None of the Fabs neutralized HIV-1 laboratory strains at biologically significant concentrations. This tends to support the opinion that a vaccine based on the gp41 molecule has the drawback that neutralizing epitopes of gp41 are rare and/or unfavorably presented to the immune system. Analysis of heavy chain sequences revealed common CDR3 motif sequences in several antibodies, which appears to be an interesting consequence of a persistent immune response to conserved antigen structures.

Amino Acid Sequence↗

PubMed2006

Using DNA microarrays to identify library-independent markers for bacterial source tracking.

Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers.

Animals↗

PubMed2000

[Generation of monoclonal antibody Fab fragments to parathyroid hormone related protein by phage display technology].

OBJECTIVE: To develop monoclonal antibody Fab fragments using display technique. METHODS: The mouse IgG Fab genes of heavy and light chains were amplified from spleen cells of a parathyroid hormone-related protein (PTHrP) immunized mouse. The combinatorial phage antibody library was prepared by inserting both heavy and light chain Fab genes into phagemid vector pComb3 and followed by infection of helper phage. The library was selected by purified recombinant PTHrP. RESULTS: The combinatorial phage antibody library was constructed successfully and the specific mouse Fabs to PThrP were selected and expressed in E. coli. CONCLUSIONS: The selected specific mouse Fabs can recognize PTHrP with high specificity.

Animals↗

PubMedDate not supplied

ZINC--a free database of commercially available compounds for virtual screening.

A critical barrier to entry into structure-based virtual screening is the lack of a suitable, easy to access database of purchasable compounds. We have therefore prepared a library of 727,842 molecules, each with 3D structure, using catalogs of compounds from vendors (the size of this library continues to grow). The molecules have been assigned biologically relevant protonation states and are annotated with properties such as molecular weight, calculated LogP, and number of rotatable bonds. Each molecule in the library contains vendor and purchasing information and is ready for docking using a number of popular docking programs. Within certain limits, the molecules are prepared in multiple protonation states and multiple tautomeric forms. In one format, multiple conformations are available for the molecules. This database is available for free download (http://zinc.docking.org) in several common file formats including SMILES, mol2, 3D SDF, and DOCK flexibase format. A Web-based query tool incorporating a molecular drawing interface enables the database to be searched and browsed and subsets to be created. Users can process their own molecules by uploading them to a server. Our hope is that this database will bring virtual screening libraries to a wide community of structural biologists and medicinal chemists.

Databases, Factual↗

PubMed2000

Discovery of selective metal-binding peptoids using 19F encoded combinatorial libraries.

A method for encoding solid-phase split/mix combinatorial libraries using the chemical shift of synthetic fluoroarenes ('F-codes') has been developed. They have wide chemical shift dispersion and are detectable at the sub-micromol level. 19F NMR is used for decoding. Nine fluoroarenes bearing linkers for attachment to solid-phase synthesis supports through a photocleavable group were prepared. A library of 90 N-alkylglycines bearing substituted succinamides was prepared on solid phase from nine amines, in which the amine is encoded by the fluorinated tag, and 10 anhydrides. Metal binding studies followed by decoding identified unique, specific binders of copper(II) and iron(III) with microM K(D)s.

Amides↗

PubMed1996

A genetic strategy for differential screening of meiotic germ-cell cDNA libraries.

The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis.

Animals↗