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PubMedDate not supplied

A straightforward, one-pot protocol for the preparation of libraries of 2-oxazolines.

A practical protocol for the parallel synthesis of 2-oxazolines using polymer-supported reagents is described. Polymer-supported Mukaiyama reagent is used to couple a carboxylic acid with an amino alcohol, giving a beta-hydroxyamide, which is then cyclized in situ using either polymer-supported sulfonyl chloride resin or polymer-bound 2-fluoropyridinium triflate. Both 2,4-disubstituted and 2,4,5-trisubstituted 2-oxazolines are obtained in high yields and excellent purities after a simple resin filtration and solvent evaporation routine.

Adjuvants, Immunologic↗
PubMed1999

A multiplasmid approach to preparing large libraries of polyketides.

A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs). The eryA PKS genes encoding the three DEBS subunits were individually cloned into three compatible Streptomyces vectors carrying mutually selectable antibiotic resistance markers. A strain of Streptomyces lividans transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes. The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations. Combinations of these vector sets were introduced into S. lividans, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs. This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.

Anti-Bacterial Agents↗
PubMed2001

Combined use of 16S ribosomal DNA and 16S rRNA to study the bacterial community of polychlorinated biphenyl-polluted soil.

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study.

Bacteria↗
PubMed1987

Cloning and expression of murine lymphotoxin cDNA.

The murine lymphotoxin (LT) gene has been cloned and used to identify cDNA clones in a library prepared from activated murine T cell mRNA. A recombinant murine genomic library was screened with a human lymphotoxin cDNA probe, resulting in the isolation of the entire LT gene. The murine LT gene structure is similar to the human gene, containing three intervening sequences. An activated murine T cell cDNA library was prepared with poly(A)+ RNA isolated 7 hr after concanavalin A stimulation of an L3T4+ interleukin 2-dependent murine T cell clone. Two colonies of the cDNA library that contained inserts that hybridized with the murine LT gene probe were sequenced and were used to construct expression plasmids. The amino acid sequence deduced from the cDNA indicates that murine LT is highly homologous to human LT (74%) and is related to murine tumor necrosis factor (35% homology). The cDNA was transcribed and was translated in vitro, and was expressed in COS-1 cells. This has resulted in the production of LT biological activity.

Amino Acid Sequence↗
PubMed1996

Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans.

A gene lineage (SAR406) related to Chlorobium and Fibrobacter species was found in 16S rRNA gene clone libraries prepared from samples from two oceans. The clone libraries were constructed from total picoplankton genomic DNA to assess bacterial diversity in the lower surface layer. The samples were collected by filtration from a depth of 80 m at a site in the western Sargasso Sea and from a depth of 120 m at a site in the Pacific Ocean, approximately 70 km from the Oregon coast. The PCR and primers which amplified nearly full-length 16S rRNA genes were used to prepare the clone libraries. Among the diverse gene clones in these libraries were two related clones (SAR406 and OCS307) which could not be assigned to any of the major bacterial phyla. Phylogenetic analyses demonstrated that these genes were distant relatives of the genus Fibrobacter and the green sulfur bacterial phylum, which includes the genus Chlorobium. The inclusion of SAR406 in phylogenetic trees inferred by several methods resulted in support from bootstrap replicates for the conclusion that Fibrobacter and Chlorobium species and SAR406 are a monophyletic group. An oligonucleotide probe that selectively hybridized to clone SAR406 was used to examine the distribution of this gene lineage in vertical profiles from the Atlantic and Pacific Oceans and in monthly time series at 0 and 200 m in the Atlantic Ocean. During stratified periods, the genes were most abundant slightly below the deep chlorophyll layer. Seasonal changes in the surface abundance of SAR406 rDNA were highly correlated with chlorophyll a levels (r = 0.75).

Bacteria↗
PubMed1986

Trehalose and maltose metabolism in yeast transformed by a MAL4 regulatory gene cloned from a constitutive donor strain.

A 6.8 kb fragment of DNA containing the regulatory sequence MAL4p has been cloned from a genomic library prepared from Saccharomyces cerevisiae strain 1403-7A which ferments maltose constitutively. The library was prepared by ligation of 5-20 kb Sau3AI restriction fragments of total yeast DNA into the BamH1 restriction site of shuttle vector YEp13. A restriction map of the cloned fragment indicates that it encompasses a 2.6 kb segment which closely resembles the regulatory MAL6 gene previously identified (Needleman et al. 1984). The hybrid plasmid, p(MAL4p)4, could transform maltose-nonfermenting strains which contain cryptic alpha-glucosidase and maltose permease genes (malp MALg), but could not transform strains containing a functional regulatory sequence and a defective maltase-permease region (MAlp malg). A correlated absence of maltase and permease DNA from the cloned fragment was indicated by the restriction map. Although the cloned DNA fragment was derived from a constitutive strain, maltose fermentation and alpha-glucosidase formation by yeast transformed with p(MAL4p)4 was largely inducible by maltose and sensitive to catabolite repression. Moreover, the active trehalose accumulation pattern (TAC(+) phenotype) linked to the complete MAL4 locus in strain 1403-7A and other constitutive MAL strains (Oliveira et al. 1981b) was not found in p(MAL4p)4 transformants. It may be concluded that constitutivity of maltose fermentation and the associated active trehalose accumulation are not merely consequences of a cis-dominant mutation causing constitutive formation of the MALp regulatory product. Moreover, constitutivity may not be caused solely by a mutation within the structural region of the MALp gene.

Cloning, Molecular↗
PubMed1998

Kinetic bias in estimates of coastal picoplankton community structure obtained by measurements of small-subunit rRNA gene PCR amplicon length heterogeneity

Marine bacterioplankton diversity was examined by quantifying natural length variation in the 5' domain of small-subunit (SSU) rRNA genes (rDNA) amplified by PCR from a DNA sample from the Oregon coast. This new technique, length heterogeneity analysis by PCR (LH-PCR), determines the relative proportions of amplicons originating from different organisms by measuring the fluorescence emission of a labeled primer used in the amplification reaction. Relationships between the sizes of amplicons and gene phylogeny were predicted by an analysis of 366 SSU rDNA sequences from cultivated marine bacteria and from bacterial genes cloned directly from environmental samples. LH-PCR was used to compare the distribution of bacterioplankton SSU rDNAs from a coastal water sample with that of an SSU rDNA clone library prepared from the same sample and also to examine the distribution of genes in the PCR products from which the clone library was prepared. The analysis revealed that the relative frequencies of genes amplified from natural communities are highly reproducible for replicate sets of PCRs but that a bias possibly caused by the reannealing kinetics of product molecules can skew gene frequencies when PCR product concentrations exceed threshold values.

Journal Article↗
PubMed1987

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.

Actins↗
PubMed1988

Studies on locus expansion, library representation, and chromosome walking using an efficient method to screen cosmid libraries.

We have developed an efficient screening method to search for clones in cosmid libraries prepared from human genomic DNA. Genomic, cDNA, and cosmid probes have been used to isolate homologous cosmids from human chromosomes 7, 10, 16, 17 and X as part of a search for polymorphic nucleotide sequences. This method has been successfully applied to chromosome walking experiments at the interstitial retinol-binding protein locus on chromosome 10, and may be a useful tool for investigating representation of cloned sequences in cosmid libraries. Our library was prepared in the vector c2RB (Bates and Swift, 1983), but the method is applicable to any cosmid cloning system in which the inserted DNA can be separated from the vector by restriction enzyme digestion. A cosmid library containing five human genome equivalents can be rapidly screened using three to four Southern hybridization filters. This results in substantial labor saving, particularly when screening genomes of high complexity with many different probes. Another advantage of the system is that it allows for the long-term storage of the cosmids so that they can be screened whenever necessary. As a consequence, cosmid screening can be made a routine laboratory procedure.

Blotting, Southern↗
PubMed1998

Molecular characterization of equine prostaglandin G/H synthase-2 and regulation of its messenger ribonucleic acid in preovulatory follicles.

To increase our understanding of the molecular control of PG synthesis in equine preovulatory follicles, the specific objectives of this study were to clone and determine the primary structure of equine prostaglandin G/H synthase-2 (PGHS-2) and to characterize the regulation of PGHS-2 messenger RNA (mRNA) in follicles before ovulation. A complementary DNA (cDNA) library prepared from follicular mRNA and a genomic library were screened with a mouse PGHS-2 cDNA probe to isolate the equine PGHS-2 cDNA and gene, respectively. The expression library yielded three nearly full-length clones that differed only in their 5'-ends; clones 3, 5, and 6 were 2946, 3138, and 3398 bp in length, respectively. The longest clone was shown to start 9 bp downstream of the transcription initiation site, as determined by primer extension analysis, and to contain 120 bp of 5'-untranslated region (UTR), 1812 bp of open reading frame, and 1466 bp of 3'-UTR. The open reading frame encodes a 604-amino acid protein that is more than 80% identical to PGHS-2 homologs in other species. Numerous repeats (n = 11) of the Shaw-Kamen's sequence (ATTTA) are present in the 3'-UTR, a motif typically indicative of mRNAs with a short half-life. The complete equine PGHS-2 gene was isolated and sequenced from a approximately 17-kilobase clone obtained from the genomic library. The equine PGHS-2 gene structure (10 exons and 9 introns; total length of 6991 bp) is similar to its human homolog except for lacking sequence elements in introns 4, 8, and 9 and in the 3'-UTR region of exon 10. To characterize the regulation of PGHS-2 mRNA in equine follicles before ovulation, preovulatory follicles were isolated during estrus, 0, 12, 24, 30, 33, 36, and 39 h (n = 4-5 follicles/time point) after an ovulatory dose of hCG. Results from Northern blots showed significant changes in steady state levels of PGHS-2 mRNA in preovulatory follicles after hCG treatment (P < 0.05). The transcript remained undetectable between 0-24 h post-hCG, first appeared (approximately 4 kilobases) only at 30 h, and reached maximal levels 33 h post-hCG. PGHS-2 mRNA was selectively induced in granulosa cells and not in theca interna. Thus, this study provides for the first time the primary structure of the equine PGHS-2 gene, transcript, and protein. It also demonstrates that the induction of PGHS-2 gene expression in equine granulosa cells is a long molecular process (30 h post-hCG), thereby providing a model to study the molecular basis for the late transcriptional activation of PGHS-2 in species with a long ovulatory process.

Amino Acid Sequence↗
PubMed2005

Polymer-assisted solution-phase chemical library synthesis.

Polymer-assisted solution-phase (PASP) synthesis has become a prevalent method for the parallel synthesis of chemical libraries. PASP methodology enables the chemist to prepare libraries of compounds more quickly and efficiently than previously. PASP technology encompasses a number of purification techniques for intermediate and final product purification. This methodology continues to progress, providing the practitioner with a broad range of ingenious purification methods, enabling multi-step syntheses of complex compounds. This review covers significant new advances in the use of tagged-reagents/substrates, resin capture-release techniques and PASP multi-step synthesis, some of which incorporate microwave heating and automation.

Anthracenes↗
PubMed2026

Systematic performance evaluation and application validation of an end-to-end NGS workstation.

Next-generation sequencing (NGS) library preparation is a core component of precision genomics, but it is commonly constrained by inefficiency, variability, and low throughput of manual protocols. To address these limitations, we developed and systematically evaluated a fully automated NGS workstations and further validated its performance across representative application scenarios. The automated system reduced total processing time from 8 to 10 to 4&#x2013;6&#xa0;h. At the same time, it maintained similar performance in pre-library metric, including DNA yield and fragment size, as well as post-capture sequencing metrics (Q30&#x2009;>&#x2009;90%, mapping rates&#x2009;>&#x2009;95%, on-target rates 85&#x2013;90%). The duplication rate was reduced to 5&#x2013;8%, compared with 10&#x2013;15% for manual methods, indicating increased library complexity. Bioinformatic evaluation of inter-species read mapping showed minimal cross-contamination, with a maximum contamination ratio of 0.0003%, indicating effective sample isolation in the automated workflow. High concordance in variant detection was observed between automated and manual workflows. Overall, this automated workstation provides a standardized and reproducible workflow that supports scalable precision genomics applications.

High-Throughput Nucleotide Sequencing↗
PubMed2025

Targeted Next-Generation Sequencing in Rare Diseases.

Targeted next-generation sequencing (NGS) in rare disease focuses on genetic analysis of specific regions in genome that are linked to a rare disease. In addition to library preparation, sequencing, and data analysis, targeted NGS includes an additional step of target enrichment of selected genes and regions. It allows for more sensitive and profound sequencing, as it is a fast and cost-effective approach with less data burden and is therefore often a method of choice for identifying rare variants in known genes, especially in diagnostics of rare diseases. Several in silico tools address the pathogenicity predictions of rare variants of unknown significance (VUS) and can therefore facilitate clinical interpretation.

Rare Diseases↗
PubMed1998

A new cytochrome P450 (CYP30) family identified in the clam, Mercenaria mercenaria.

A full-length clone with sequence similarity to genes in the cytochrome P450 superfamily was isolated from a cDNA library prepared from female Mercenaria mercenaria gonadal tissue. This clone was isolated while screening an expression library with an antibody prepared against a peptide sequence within the ligand-binding region of the murine Ah receptor. Comparison of the predicted amino acid sequence of this clone to those of other cytochrome P450 genes indicated that the closest overall sequence similarity (38%) was to proteins predicted from genes in the CYP3 family. Northern blots indicated the presence of a transcript of the appropriate size (3.0 kb) with homology to the clam cytochrome P450. In vitro translation of the cDNA clone produced a 50.7-kDa protein as determined by SDS-polyacrylamide gel electrophoresis. The in vitro translated protein was not recognized on Western blots by two polyclonal antibodies specific for members of the CYP3 family. Since the degree of similarity to existing cytochrome P450 families was below the 40% level required for membership, and the expressed protein was not recognized by CYP3-specific antibodies, this clam cytochrome P450 cDNA has been placed in a new family, cytochrome P450 30 (CYP30).

Amino Acid Sequence↗
PubMed1993

A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF.

Resistance to complement-mediated lysis in Trypanosoma cruzi is due to the expression of complement-regulatory factors by the virulent developmental forms of this protozoan parasite. An 87- to 93-kDa molecule, which we have termed T-DAF (trypomastigote decay-accelerating factor), is present on the surface of the parasite and inhibits complement activation in a manner functionally similar to the mammalian complement regulatory component, decay-accelerating factor. In this report, we characterized monospecific polyclonal and monoclonal antibodies which were obtained from mice and rabbits immunized with fast protein liquid chromatography-purified T-DAF. These polyclonal antibodies were shown to inhibit T-DAF activity and were capable of inducing lysis of the parasites. Both the polyclonal and monoclonal antibodies were used to screen a cDNA expression library prepared from T. cruzi trypomastigote mRNA. From this library, we obtained a partial lambda gt11 cDNA clone which showed genetic and functional similarity to the human C3 convertase inhibitor DAF (A. Nicholson-Weller, J. Burge, D. T. Fearon, P. F. Weller, and K. F. Austen, J. Immunol. 129:184-189, 1982).

Amino Acid Sequence↗
PubMed1987

Human chromosome-specific DNA libraries: use of an oligodeoxynucleotide probe to detect non-recombinants.

The frequencies of non-recombinants are directly determined in 21 human DNA libraries prepared from flow-purified chromosomes. A radiolabeled 18-base oligodeoxynucleotide, whose sequence spans the cloning site, stably hybridizes to non-recombinants. When the cloning site is interrupted by an insert, less stable hybridization occurs. Non-recombinant frequencies range from 1-34% with the frequency of false positives being less than 1%. Libraries are being prepared for each human chromosome as part of the National Laboratory Gene Library Project and the specifications of seven new libraries are presented.

Base Sequence↗