Development of a new approach for screening combinatorial libraries using MALDI-TOF-MS and HPLC-ESI-MS/MS

Mass spectrometric techniques play a prominent role in the rapidly expanding field of high-throughput screening (HTS). In this paper, the authors present a novel qualitative approach for the screening of a small library of compounds using MALDI-TOF-MS and HPLC-ESI-MS/MS. Chymotrypsin (CT), a serine protease, was selected as the target protein. A well-known inhibitor of CT is chymostatin (CS), a naturally occurring peptide aldehyde, which is reported to be a mixture of 3 components-A, B, and C-differing only in one of the amino acids. The authors report that native CS mixture consists of 3 additional ring hydroxylated components and that each compound exists in 2 epimeric forms. In case of protein-binding compounds, only 1 of the epimers was found to be active. A unique feature of this study is the generation of a combinatorial library of CS derivatives applying a one-pot strategy followed by identification and structural elucidation of the library components. Analytical investigation of the library resulted in the identification of 22 compounds. After incubation with CT and centrifugal ultrafiltration, 10 compounds were detected as protein-binding ligands. Finally, the complementary potentials of MALDI-TOF-MS and HPLC-MS/MS in the screening of complex ligand mixtures have been discussed.     

Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of microsatellite markers in peach [Prunus persica (L.) Batsch]

Microsatellites have emerged as an important system of molecular markers. We evaluated the potential of microsatellites for use in genetic studies of peach [Prunus persica (L.) Batsch]. Microsatellite loci in peach were identified by screening a pUC8 genomic library, a λZAPII leaf cDNA library, as well as through database searches. Primer sequences for the microsatellite loci were tested from the related Rosaceae species apple (Malus×domestica) and sour cherry (Prunus cerasus L.). The genomic library was screened for CT, CA and AGG repeats, while the cDNA library was screened for (CT)_n- and (CA)_n-containing clones. Estimates of microsatellite frequencies were determined from the genomic library screening, and indicate that CT repeats occur every 100 kb, CA repeats every 420 kb, and AGG repeats every 700 kb in the peach genome. Microsatellite- containing clones were sequenced, and specific PCR primers were designed to amplify the microsatellite- containing regions from genomic DNA. The level of microsatellite polymorphism was evaluated among 28 scion peach cultivars which displayed one to four alleles per primer pair. Five microsatellites were found to segregate in intraspecific peach-mapping crosses. In addition, these microsatellite markers were tested for their utility in cross-species amplification for use in comparative mapping both within the Rosaceae, and with the un- related species Arabidopsis thaliana L. Received: 18 June 1999 / Accepted: 6 December 1999     

A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein

Phage display is a widely used technology for the isolation of peptides and proteins with specific binding properties from large libraries of these molecules. A drawback of the common phagemid/helper phage systems is the high infective background of phages that do not display the protein of interest, but are propagated due to non-specific binding to selection targets. This and the enhanced growth rates of bacteria harboring aberrant phagemids not expressing recombinant proteins leads to a serious decrease in selection efficiency. Here we describe a VCSM13-derived helper phage that circumvents this problem, because it lacks the genetic information for the infectivity domains of phage coat protein pIII. Rescue of a library with this novel CT helper phage yields phages that are only infectious when they contain a phagemid-encoded pIII-fusion protein, since phages without a displayed protein carry truncated pIII only and are lost upon re-infection. Importantly, the CT helper phage can be produced in quantities similar to the VCSM13 helper phage. The superiority of CT over VCSM13 during selection was demonstrated by a higher percentage of positive clones isolated from an antibody library after two selection rounds on a complex cellular target. We conclude that the CT helper phage considerably improves the efficiency of selections using phagemid-based protein libraries.     

Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin

Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8-32), whereas SUN B8155 did not displace the specific binding of [(125)I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.     

Phage display screening in low dielectric media

Here we report the first application of phage display screening in low dielectric media. Two series of phage clones with affinity for alpha-chymotrypsin (CT) were selected from a Ph.D.(TM)-C7C library, using either a buffer or acetonitrile in buffer (50%, v/v). The affinity of lysates, individual clones or selected cyclic peptides for the enzyme was studied by examining their influence on CT activity. Peptides displayed on phage selected in buffer provided significant protection from enzyme autolysis resulting in marked increase in CT activity (>100%). Phage selected in ACN provided some, albeit weak, protection from the detrimental influence on CT from ACN. In conclusion, the results demonstrate the potential for the application of phage display screening protocols to targets in media of low dielectricity.     

Humoral detection of leukaemia-associated antigens in presentation acute myeloid leukaemia

The serological analysis of recombinant cDNA expression libraries (SEREX) technique was used to immunoscreen a testes cDNA expression library with sera from newly diagnosed acute myeloid leukaemia (AML) patients. We used a testis cDNA library to aid our identification of cancer-testis (CT) antigens. We identified 44 antigens which we further immunoscreened with sera from AML, chronic myeloid leukaemia (CML), and normal donors. Eight antigens were solely recognised by patient sera including the recently described CT antigen, PASD1, and the cancer-related SSX2 interacting protein, SSX2IP. RT-PCR analysis indicated that we had identified three antigens which were expressed in patient bone marrow (BM) and peripheral blood (PB) but not in normal donor samples (PASD1, SSX2IP, and GRINL1A). Real-time PCR (RQ-PCR) confirmed the restricted expression of PASD1 in normal donor organs. Antigen presentation assays using monocyte-derived dendritic cells (mo-DCs) showed that PASD1 could stimulate autologous T-cell responses, suggesting that PASD1 could be a promising target for future immunotherapy clinical trials.     

Isolation and Characterization of Microsatellites in Snap Bean

The objectives of this study were to isolate and characterize microsatellites from a heat tolerant variety of snap bean (Phaseolus vulgaris L.) in order to generate polymorphic genetic markers linked to quantitative trait loci for heat tolerance. A genomic library contained 400-800 bp inserts was constructed and screened for the presence of (GA/CT)n and (CA/GT)n repeats. The proportion of positive clones yielded estimated of 3.72×10 4 such dinucleotide repeats per genome, roughly comparable to the abundance reported in other eukaryotic genomes. Twenty-six positive clones were sequenced. In contrast to mammalian genomes, the (GA/CT)n motif was much more abundant than the (CA/GT)n motif in these clones. The (GA/CT)n repeats also showed longer average repeat length (mean n=10.4 versus 6.5), suggesting that they are better candidates for yielding polymorphic genetic markers in the snap bean genome.     

Microsatellite markers for Bromus tectorum (cheatgrass)

Bromus tectorum (cheatgrass) is a flourishing invasive weed in the western United States. The objective of our study is to characterize its genetic diversity. We made a B. tectorum genomic library in lambda phage and screened approximately 4000 clones for poly CA and poly CT dinucleotide repeats. Of 38 sequences with dinucleotide repeats isolated from the library, we designed primer sets for 18 loci. A preliminary screen of 40 individuals from four populations indicated that seven loci were polymorphic. These loci will be valuable for elucidation of cheatgrass genetic diversity and population structure.     

Monoclonal antipeptide antibodies as tools to dissect closely related gene products. A model using peptides encoded by the calcitonin gene

We have explored the possibility of using mAb as tools for distinguishing between closely related gene products. We utilized calcitonin (CT) gene products as a model, because this 32-amino-acid-amidated hormone is biosynthesized by post-translational processing of a larger precursor. By using CT as a hapten, we had previously identified a mAb (CT07) with restricted specificity to mature CT, and had shown that another mAb (CT08) directed to a different epitope bound to both CT and the CT precursor. In this study, we used synthetic peptides analogous to various regions of biosynthetic intermediates of CT as haptens, and generated a library of mAb which define distinct epitopes. First, we identified two separate epitopes located in either the 1-11 or the 12-21 region of the C-terminal flanking peptide of CT (katacalcin, KC), and which were recognized by mAb KC01 and KC04, respectively. Second, we identified a conformational epitope in the C-terminal region of the putative glycine-extended form of CT (CT-Gly). This epitope was recognized by mAb CT19 and was shared by mature CT but not by CT precursors. Third, we identified an epitope restricted to CT-Gly and recognized by mAb CT20. For dissecting between related products of the CT gene, we designed different monoclonal immunoradiometric assays (m-IRMA) based on CT08 as the radiolabeled indicator antibody. A first m-IRMA based on CT07 as the capture antibody specifically recognized mature CT and did not cross-react with CT precursors. Conversely, another m-IRMA with KC01 as the capture antibody was specific for CT precursors and did not cross-react with either mature CT or CT-Gly. A third assay based on CT20 as the capture mAb was specific for CT-Gly and was not affected by the presence of either CT precursors or mature CT. We also used these antibodies to demonstrate that neoplastic C cells incompletely released processed CT precursors in serum, in addition to mature CT. This study demonstrates that mAb can be used as tools to selectively recognize closely related gene products. These findings might be applied to the study of other molecules biosynthesized by enzymatic modifications of a larger precursor.     

Cloning, nucleotide sequence and molecular evolution of a rabbit processed metallothionein MT-2 pseudogene

A rabbit metallothionein-2 pseudogene (MT-2 psi) has been isolated from a partial rabbit genomic library. Its unusual sequence shows evidence of complex rearrangements involving recombination and deletion events. There are no intervening sequences, 3' poly A tract or 5' regulatory DNA sequences. The pseudogene is flanked by two sets of direct repeats (CT)3 GT(CT)4 and CTGG(G)CTC. They are most probably the sites of insertion of MT-2 psi in the rabbit genome. In addition, a number of repetitive DNA sequences are observed flanking the MT-2 psi gene. These are features of a processed retrogene.     

Characterization of 13 polymorphic microsatellite loci for an evergreen tree,Myrica rubra

We developed 13 microsatellite markers from a genomic library enriched for dinucleotide (CT) repeats in a dioecious evergreen tree, Myrica rubra. We screened loci from 32 adult trees. The number of alleles ranged from two to 14, and the expected heterozygosity ranged from 0.324 to 0.884. The total paternity exclusionary power where the mother was known equalled 0.99965. These loci are of clear value in studying mating system and parentage analysis of this species.     

Isolation of microsatellite loci in green abalone (Haliotis fulgens) and cross‐species amplification in two other North American red (Haliotis rufescens) and pink (Haliotis corrugata) abalones

Microsatellite loci were isolated in Haliotis fulgens using a (CT)_n enriched‐genomic library. From 33 sequenced clones, 21 microsatellites regions were identified, 15 with the expected (CT)_n. Eight microsatellite loci were amplified, six of which were polymorphic with a range of three to 20 alleles, and five cross‐amplified in two other species (Haliotis rufescens and Haliotis corrugata). These microsatellites will be useful as population genetic markers in the three species.     

Characterization of 15 polymorphic microsatellite loci in an endangered tropical tree Hopea bilitonensis (Dipterocarpaceae) in Peninsular Malaysia

This study reports the isolation and characterization of 15 polymorphic microsatellite loci in an endangered tropical tree Hopea bilitonensis (Dipterocarpaceae). The primers were designed from a genomic library enriched for dinucleotide (CT) repeats and screened on 24 adult trees from a natural population. The number of alleles ranged from two to eight, and the observed heterozygosity ranged from 0.042 to 1.000. These loci will allow mating system and population analyses to be carried out in this species.     

Isolation and characterization of microsatellite markers in Bonelli's eagle (Hieraaetus fasciatus)

To obtain polymorphic molecular markers for population genetics and conservation studies in Bonelli's eagle populations, we screened a partial genomic library enriched for microsatellites with di‐ and tri‐nucleotide motifs [(GT) (CT) (AAC) and (GCC)]. A total of 15 polymorphic markers were obtained. The number of alleles ranged from two to eight. These markers will be very useful for paternity tests and population structure studies as well as for evaluating the outcome of conservation programs.     

Assessing species diversity of Coral Triangle artisanal fisheries: A DNA barcode reference library for the shore fishes retailed at Ambon harbor (Indonesia)

The Coral Triangle (CT), a region spanning across Indonesia and Philippines, is home to about 4,350 marine fish species and is among the world's most emblematic regions in terms of conservation. Threatened by overfishing and oceans warming, the CT fisheries have faced drastic declines over the last decades. Usually monitored through a biomass‐based approach, fisheries trends have rarely been characterized at the species level due to the high number of taxa involved and the difficulty to accurately and routinely identify individuals to the species level. Biomass, however, is a poor proxy of species richness, and automated methods of species identification are required to move beyond biomass‐based approaches. Recent meta‐analyses have demonstrated that species richness peaks at intermediary levels of biomass. Consequently, preserving biomass is not equal to preserving biodiversity. We present the results of a survey to estimate the shore fish diversity retailed at the harbor of Ambon Island, an island located at the center of the CT that display exceptionally high biomass despite high levels of threat, while building a DNA barcode reference library of CT shore fishes targeted by artisanal fisheries. We sampled 1,187 specimens and successfully barcoded 696 of the 760 selected specimens that represent 202 species. Our results show that DNA barcodes were effective in capturing species boundaries for 96% of the species examined, which opens new perspectives for the routine monitoring of the CT fisheries.     

Simple‐sequence repeat (SSR) markers of Japanese plum (Prunus salicina Lindl.) are highly polymorphic and transferable to peach and almond

Thirty‐five polymorphic microsatellites were developed using a CT/AG enriched genomic library of Japanese plum cv. Santa Rosa. Twenty‐seven of them detected a single locus and eight two or more loci. A high level of variability was observed in a set of eight cultivars for the 27 single‐locus microsatellites: 5.7 average number of alleles per locus; 73% mean observed heterozygosity and 74% discrimination power. Most SSRs were transferable to peach (85%) and almond (78%).