RAPD-based screening of genomic libraries for positional cloning

RAPD markers are frequently used for positional cloning. However, RAPD markers often contain repeated sequences which prevent genomic library screening by hybridisation. We have developed a simple RAPD analysis of genomic libraries based on the identification of cosmid pools and clones amplifying the RAPD marker of interest. Our method does not require the cloning or characterisation of the RAPD marker as it relies on the analysis of cosmid pools or clones using a simple RAPD protocol. We applied this strategy using four RAPD markers composed of single copy or repeated sequences linked to avirulence genes of the rice blast fungus Magnaporthe grisea . Cosmids containing these RAPD markers were easily and rapidly identified allowing the construction of physical contigs at these loci.     

Cytogenetic and phenotypic effects of a chromosomal rearrangement involving the Z-chromosome and micro-chromosome in the chicken

Measurements demonstrated that the Z-chromosome was truly metacentric. Forty-six percent of one arm of a female's Z-chromosome had been translocated to a microchromosome (Z-micro) by irradiation of semen. The bread was 23 crossover units distal to the late feathering (K) locus. The barring (B) locus on the non-broken arm assorted almost independently of the Z-micro segment. Semen from eight sons of this Z-micro female was used to inseminate 98 dwarf (dw) broiler-type females. From karyotypes of 147 male and 149 female progeny, we identified 69 males heterozygous and 79 females hemizygous for Z-micro. Body weight of 43 males heterozygous for Z-micro was significantly greater than that of 45 normal Z paternal half-brothers at all ages from 2 to 24 weeks. In contrast, body weight of 57 Z-micro females compared with their 56 normal Z paternal half-sisters was depressed significantly at 2, 4, and 6 weeks but not significantly at 8, 12, 16, and 24 weeks of age. Age at first egg was retarded 8 days and egg production over a 153 day test period was reduced 19.6%, primarily due to a reduction of egg laying sequence from 2.7 to 2.1 days in the Z-micro females.     

Generation of chromosome fragment specific bovine DNA sequences by microdissection and DOP-PCR

Trends in causative organisms and sources of infection were studied in a series of 288 episodes of bacteremia in neutropenic cancer patients observed in a single institution from 1986 to 1993. The incidence of bacteremia increased significantly from 20 episodes per 1000 admissions in 1986 to 50 episodes per 1000 admissions in 1993 (p = 0.00001). Over the study period, a continuous increment in gram-positive bacteremia, which reached 81% of episodes in 1993 (p = 0.000001), was observed. Conversely, the incidence of gram-negative bacteremia remained stable. Coagulase-negative staphylococci and viridans group streptococci were the most commonly isolated pathogens. Bacteremia caused by coagulase-negative staphylococci increased from 3 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.0001), and viridans group streptococci bacteremia increased from 0 episodes per 1000 admissions to 19 episodes per 1000 admissions (p = 0.000001). The upward trend in gram-positive bacteremia appeared to be related to a significant increase in both intravascular catheters (p = 0.003) and oral mucositis (p = 0.003) as sources of infection. Specific strategies to prevent chemotherapy-induced mucositis and catheter-related bacteremia merit further investigations.     

Generation of cDNA expression libraries enriched for in-frame sequences

Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.     

Generation and utilization of synthetic combinatorial libraries

The use of combinatorial chemistry is fundamentally changing the pace and scope of basic research and drug discovery. Since the introduction of synthetic peptide libraries several years ago, combinatorial chemistry has proven to be a powerful tool for the generation of immense molecular diversities of peptides, peptidomimetics and new organic compounds. This article briefly reviews methods for the generation and application of combinatorial libraries, with particular emphasis on soluble synthetic combinatorial libraries. The utility of these molecular diversities for basic research and drug discovery has been demonstrated through the identification of numerous highly active compounds such as antigenic peptides, receptor ligands, antimicrobial compounds and enzyme inhibitors.     

Complex probes for high-throughput parallel genetic mapping of genomic mouse BAC clones

We investigated the effect of centrally administered pituitary adenylate cyclase activating polypeptide (PACAP) on feeding in rats, and the involvement of hypothalamic neuropeptide gene expression using in situ hybridization. Intracerebroventricular injection of PACAP (1000 pmol/rat) significantly decreased food intake in a dose-dependent manner. In PACAP-treated rats, neuropeptide Y (NPY) mRNA levels in the arcuate nucleus and galanin mRNA levels in the paraventricular nucleus increased, and corticotropin-releasing hormone (CRH) mRNA levels in the paraventricular nucleus decreased. In rats fasted for 72 h, NPY mRNA levels increased, and CRH mRNA levels decreased, but galanin mRNA levels were unchanged. These results indicate that the anorectic function of PACAP is not mediated by NPY or CRH, and that PACAP increases galanin synthesis.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.     

Enhancement of ELISAs for screening peptides in epitope phage display libraries

The complete analysis of epitope phage display libraries requires sensitive assays capable of detecting peptides expressed on phage that have a wide range of affinities for antibody. We have compared two ELISAs, a 'direct' assay where the phage is captured by an anti-phage antibody and the peptide detected by the antibody used for screening, and a 'reverse' assay where the antibody used for screening is first coated on the well and the binding of phage detected by the anti-phage antibody. We demonstrate, by comparing two fUSE5 derived phage bearing five peptides reacting with the anti-cryptococcal polysaccharide antibody 2H1, that the reverse ELISA is the more sensitive assay. Further, there is a limit in affinity, here around 1 microM, above which phage clones are negative by the direct ELISA. We describe an enhancement of the direct assay by mixing 2H1 with 3-fold excess of anti-heavy or anti-light chain antibody. The resulting polymerization of 2H1 induces an increase in antibody avidity that is responsible for the enhancement. The enhanced direct ELISA allowed rapid and sensitive detection of positive clones and is easily inhibited by free peptide, while the reverse ELISA is not. The enhanced ELISA has also been used successfully for immunological screening of intermediate libraries, allowing detection of rare positive clones that would otherwise be lost. The combination of the three ELISAs, reverse, direct, and enhanced direct, should provide a way to rank phage clones into three classes: very low, low, and high affinity, providing information previously obtained only by the synthesis and testing of many peptides.     

Generation of type-specific probes for the detection of single-copy human papillomavirus by a novel in situ hybridization method

The integration of human papillomavirus (HPV) DNA is associated with the pathogenesis of HPV-associated malignancies. The ability, however, of standard in situ hybridization (ISH) to detect low-copy integrated HPV DNA is limited. We describe the generation of HPV type-specific biotin-labeled DNA probes and a novel ISH method that uses the catalyzed reporter deposition (CARD) system for the detection of single-copy target HPV DNA in formalin-fixed, paraffin-embedded tissue. Consensus primers flanking the noncoding region of HPVs were used to generate biotin-labeled HPV-6b, -11, -16 and -18 probes by polymerase chain reaction (PCR). The probes were used for ISH with the novel technique of CARD to increase the sensitivity of the assay. Tissue blocks were prepared from CaSki (500-600 copies of HPV-16), SiHa (1-2 copies of HPV-16), and HeLa (10-50 copies of HPV-18) cell lines, as well as from an HPV-negative cell line, C33A, and then tested to demonstrate the sensitivity and specificity of the probes. Surgical specimens were used to show the clinical applicability of this technique. We successfully detected HPV-16 DNA in CaSki and SiHa cells but not in HeLa or C33A cells. HPV-18 DNA was detected in HeLa cells but not in CaSki, SiHa, or C33A cells. Sensitivity was increased when ISH was performed using probes with more biotin incorporation or when more cycles of signal amplification were employed, but significant nonspecific background was observed after more than two cycles of signal amplification. The probes generated in this study detected specific types of HPV in surgical specimens with much higher sensitivity than did conventional ISH. We concluded that our new method was highly sensitive and could be applied to formalin-fixed, paraffin-embedded clinical material for the detection of HPV.     

Pulsed ultrafiltration mass spectrometry: a new method for screening combinatorial libraries

In response to the need for rapid screening of combinatorial libraries to identify new lead compounds during drug discovery, we have developed an on-line combination of ultrafiltration and electrospray mass spectrometry, called pulsed ultrafiltration mass spectrometry, which facilitates the identification of solution-phase ligands in library mixtures that bind to solution-phase receptors. After ligands contained in a library mixture were bound to a macromolecular receptor, e.g., human serum albumin or calf intestine adenosine deaminase, the ligand-receptor complexes were purified by ultrafiltration and then dissociated using methanol to elute the ligands into the electrospray mass spectrometer for detection. Ligands with dissociation constants in the micromolar to nanomolar range were successfully bound, released, and detected using this method, including warfarin, salicylate, furosemide, and thyroxine binding to human serum albumin, and erythro-9-(2-hydroxy-3-nonyl)adenine binding to calf intestine adenosine deaminase. Repetitive bind- and-release experiments demonstrated that the receptor could be reused. Thus, pulsed ultrafiltration mass spectrometry was shown to provide a simple and powerful new method for the screening of combinatorial libraries in support of new drug discovery.     

The tetramethylammonium chloride method for screening of cDNA libraries using highly degenerate oligonucleotides obtained by backtranslation of amino-acid sequences

We describe a method for screening of cDNA libraries with highly degenerate oligonucleotides using tetramethylammonium chloride (TMAC). This method is a convenient alternative to using probes generated by the polymerase chain reaction (PCR), especially when these cannot easily be made. Nylon filters were prehybridized in buffered sodium chloride and hybridized with labelled oligonucleotide in buffer containing 3 M TMAC. In TMAC the melting temperature of the oligonucleotide is independent of the G + C content, thus only depending on the length. This was confirmed by the cloning of 13 specific cDNAs with G + C contents between 27% and 61% using 15- to 20-mer oligonucleotides with a degeneracy up to 512. The method was further improved for highly degenerate oligonucleotides by testing hybridization of four 18-mer oligonucleotides, each containing one deoxyinosine (I) instead of A, G, C or T, respectively. Oligonucleotides containing I pairing with A, G or T may have slightly lower melting temperatures than those pairing with C. At practical circumstances all oligonucleotides hybridize about equally well at hybridization temperatures 10 degrees C below the irreversible melting temperature. This was further confirmed by the cloning of four cDNAs with oligonucleotides containing deoxyinosines at 3 or 4 ambiguous positions.     

Affinity-based screening of combinatorial libraries using automated, serial-column chromatography

We have developed an automated serial chromatographic technique for screening a library of compounds based upon their relative affinity for a target molecule. A "target" column containing the immobilized target molecule is set in tandem with a reversed-phase column. A combinatorial peptide library is injected onto the target column. The target-bound peptides are eluted from the first column and transferred automatically to the reversed-phase column. The target-specific peptide peaks from the reversed-phase column are identified and sequenced. Using a monoclonal antibody (3E-7) against beta-endorphin as a target, we selected a single peptide with sequence YGGFL from approximately 5800 peptides present in a combinatorial library. We demonstrated the applicability of the technology towards selection of peptides with predetermined affinity for bacterial lipopolysaccharide (LPS, endotoxin). We expect that this technology will have broad applications for high throughout screening of chemical libraries or natural product extracts.     

Triple-color FISH analysis of 12p amplification in testicular germ-cell tumors using 12p band-specific painting probes

Forty-nine surgical specimens and nine germ cell tumor lines were analyzed by triple-color FISH using microdissected probes for the cytogenetic bands of chromosome arm 12p (12p11.2, p12, and p13). FISH analysis demonstrated amplification of material from all three bands in all tumors. This amplification was in the form of increased copy number of 12p or i(12p) and/or 12p amplified regions (AMP12p). The number of copies of 12p was variable (4-11 copies) from case to case but tended to remain relatively constant in all clones of the same tumor, even when the amplification took the form of an amplified region composed of 12p material. In tumors with multiple clones, i(12p) and AMP12p were never found in the same cell. No correlation was found between 12p copy number and tumor type. We describe, for the first time, a relative overrepresentation of 12p13 or 12p12-p13 regions in six tumors (two surgical samples and four cell lines), either as "partial 12p" (five cases) or within a 12p amplified region (one case). The ubiquitous amplification of all three 12p bands in germ-cell tumors supports the hypothesis that 12p harbors more than one gene important for oncogenesis of adult male germ-cell tumors, and that these genes may be located in different areas of 12p.     

Characterization of oligonucleotide probes for the identification of Acinetobacter spp., A. baumannii and Acinetobacter genomic species 3

The 16S-23S intergenic spacer regions of four Acinetobacter genomic species belonging to the A. calcoaceticus-A. baumannii (Acb) complex, i.e. genomic species 1 (A. calcoaceticus), genomic species 2 (A. baumannii), genomic species 3 and Tjernberg and Ursing (TU) genomic species 13, have been cloned and sequenced. Sequence analysis led to the discovery of a single copy of IIe and Ala tRNA genes within each spacer. Sequence comparison allowed the identification of a 192-base-pair long highly conserved sequence between the 3' end of the 16S rRNA and the 5' end of the tRNA(Ala) genes. Moreover, two short regions, which were specific to, respectively, genomic species 2 and 3, could be identified. Oligonucleotides corresponding to these sequences were constructed and tested for the ability to hybridize with chromosomal DNA extracted from Acinetobacter belonging to different genomic species and with chromosomal DNA of other bacterial genera. One of these oligonucleotides was demonstrated to be useful as a sensitive and specific probe for A. baumannii. A less sensitive probe for Acinetobacter genomic species 3 was also developed.     

Evaluation of oligonucleotide probes for simple tandem repeats (STR) to produce informative DNA fingerprints of the chicken

Early intervention programs are designed to enhance the developmental competence of participants and to prevent or minimize developmental delays. Children targeted for early intervention may either include environmentally or biologically vulnerable children, or those with established developmental deficits. There is growing consensus based on the best available evidence that early interventions can exert moderate positive effects. However, this literature is limited by substantial methodological weaknesses in most studies. Therefore further randomized clinical trials are needed to ascertain which programs best meet the needs of children with or at risk for developmental disability.     

Molecular screening for P-element insertions in a large genomic region of Drosophila melanogaster using polymerase chain reaction mediated by the vectorette

As an alternative to existing methods for the detection of new insertions during a transposon mutagenesis, we adapted the method of vectorette ligation to genomic restriction fragments followed by PCR to obtain genomic sequences flanking the transposon. By combining flies containing a defined genomic transposon with an excess of flies containing unrelated insertion sites, we demonstrate the specificity and sensitivity of the procedure in the detection of integration events. This method was applied in a transposon-tagging screen for BJ1, the Drosophila homolog of the vertebrate gene Regulator of Chromosome Condensation (RCCI). Genetic mobilization of a single genomic P element was used to generate preferentially new local insertions from which integrations into a genomic region surrounding the BJ1 gene were screened. Flies harboring new insertions were phenotypically selected on the basis of the zeste1-dependent transvection of white. We detected a single transposition to a 13-kb region close to the BJ1 gene among 6650 progeny that were analyzed. Southern analysis of the homozygous line confirmed the integration 3 kb downstream of BJ1.     

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken

Gene technology using polymerase chain reaction (PCR) has markedly advanced in recent year and has been introduced in clinical laboratories. In this paper, the genotypes of genomic DNAs of subjects with cisAB blood group were analysed using three methods, polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), and the PCR-direct sequencing method, and directly determined using the polymerase chain reaction (PCR) amplification of specific alleles (PASA)-method. The differences among the methods were as follows, PCR-RFLP and PCR-direct sequencing method require 2-step procedures, and are complicated for clinical laboratories. The PASA method is based on the fact that PCR amplification occurs only when the 3' endbase of the primer is matched to sites of the nucleotide substitution of ABO allelic cDNA. Three of five regions of allelic DNAs were co-amplified in a single PCR (multiplex-PCR) in this study. ABO and cisAB blood group genotypes were directly determined, based on the molecular size of allele-specific amplification products. The PASA method requires only about 4 hours from starting PCR to results, making it rapid, simple and useful for detecting the genotype of ABO and cisAB blood groups in comparison with PCR-RFLP and the direct sequencing methods and will allow this procedure to be very versatile and widely used throughout the research and clinical diagnostic communities. The analyses of the nucleotide sequence at nucleotides No. 261, 526, 703, 796 and 803 in 3 major subjects in the cisAB blood group (cisA2B3, cisA1B3 and cisA2B) revealed chimeric structures of the A allele and B allele on the same gene.