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PubMed1990

Association of fibronectin-like antigens in chromatin preparations from rat hepatoma cells.

High molecular weight hepatoma-associated nonhistone chromosomal proteins (NHPs) in transplantable rat hepatoma cells were reported previously from this laboratory. A cDNA library prepared from Morris hepatoma 7777 cells was screened with the polyclonal antibodies against hepatoma NHPs and a positive cDNA clone (lambda P2A1) was isolated. DNA sequence analysis revealed that the cDNA clone was identical to that of rat fibronectin (FN). The polyclonal antibodies against hepatoma NHPs were shown to bind specifically to both rat plasma FN and the fusion proteins encoded by lambda P2A1. A monoclonal antibody specific to rat plasma FN also recognized high molecular weight antigens of hepatoma NHPs in a pattern similar to that demonstrated with the polyclonal antibodies. These results suggest the existence of FN or FN-like antigens in the chromatin preparations from rat hepatoma cells. The antigenic proteins are localized in the nuclei of neoplastic foci of liver undergoing hepatocarcinogenesis.

Animals↗

PubMed1986

Construction, analysis, and application to 46,XY gonadal dysgenesis of a recombinant phage DNA library from flow-sorted human Y chromosomes.

The analysis of a recombinant human Y-enriched Hind III total digest phage library prepared from the DNA of flow sorted human Y chromosomes is described. Out of 43 phage inserts from the library thus far mapped, 25 revealed hybridization with Y chromosomal DNA. These inserts may be divided into five groups according to their degree of Y specific hybridization: inserts that hybridize with one single copy or slightly repeated Y-specific DNA sequence, Y-specific repeated sequences of various restriction fragment lengths, Y-chromosomal DNA sequence(s) shared by a sequence on the X and/or on autosomes, Y-specific DNA sequences in addition to multiple X and/or autosomal sequences, or Y-specific repeated DNA in addition to multiple X and/or autosomal sequences. Application of probes from this library for diagnostic purposes is shown in two 46,XY patients with gonadal dysgenesis and small deletions of the Y short arm.

Cloning, Molecular↗

PubMed2000

Gene expression in mouse cerebellum during its development.

Genes expressed during the cerebellar development of the mouse were identified in 3'-directed cDNA libraries prepared from the postnatal day 4, day 12, and week 6 cerebellar tissues. Among about 5500 clones selected randomly from each library, there were approximately 3500 distinct species. A total of 7728 species were identified in the three libraries, 1346 of which were known genes in the GenBank, 3041 EST-matching genes, and 3341 new genes. Relative expression levels at the three postnatal stages were quantitated by adapter-tagged competitive PCR for 130 known genes that appeared six times or more in one of the libraries. Genes for ribosomal proteins and some cytoskeletal and nuclear proteins were abundantly expressed at the early stage, coincidently with extensive proliferation of granule cells as the major cerebellar component. Genes related to brain functions, including those for mitochondrial activities and some ion channel systems, were more active at a later stage when the majority of granule cells were engaged in axon extension and synapse formation or the cerebellum had reached maturity. Compared to these stage-specifically expressed genes, genes for transcriptional regulation, signal transduction, protein modification, and basic cellular functions, in general, were not abundantly expressed at any stage of development.

Animals↗

PubMed1987

Estrogen regulation of alpha 1(I)-procollagen messenger ribonucleic acid in the rat uterus.

A cDNA library, prepared from mRNA isolated from the uteri of 3-day estradiol-stimulated immature rats, was constructed in pBR322. From this library an estrogen-regulated clone, pERU3, was isolated. This clone contained sequences complementary to uterine mRNA that migrated during gel electrophoresis as a double band of about 5.0 and 5.8 kilobases. Little of this mRNA was seen in several other tissues examined. An increase in the amount of this RNA in uterus was seen 2 h after estradiol treatment, with maximum hybridization occurring, in different experiments, between 18 and 36 h, followed by a decline. Hybridization of the cDNA insert of the pERU3 plasmid with known probes indicated that it coded for alpha 1(I)-procollagen. This conclusion was supported by in vitro translation experiments in which the hybrid-selected mRNA complementary to pERU3 DNA was shown to code for a collagenase-sensitive protein with a size corresponding to that of alpha 1(I)-procollagen. This system, therefore, provides an additional tool for the study of the estrogen regulation of gene expression in the uterus.

Animals↗

PubMed1999

Solid-phase and combinatorial synthesis in beta-lactam chemistry.

Combinatorial chemistry has became a core technology for the rapid development of novel lead compounds in the pharmaceutical industry and for the optimization of therapeutic efficacy. The effort to prepare libraries of compounds by combinatorial chemistry has led to an unprecedented growth in solid phase organic synthesis (SPOS), particularly for the preparation of non-oligomeric small molecules. In this context, the clinically valuable b-lactam compounds are very attractive targets for research using these new techniques. In recent years, b-lactam compounds have been recognized not only as unique antibacterial agents but also as potent enzyme inhibitors, drug delivery agents, and versatile synthetic intermediates. This review gives a comprehensive up-date for the application of solid-phase and combinatorial synthesis to b-lactam compounds.

Combinatorial Chemistry Techniques↗

PubMed1988

Gene expression in the embryonic nervous system of Xenopus laevis.

Development of the nervous system in the amphibian embryo is initiated during gastrulation by an inductive interaction between chordamesoderm and dorsal ectoderm. The induced ectoderm forms the neural plate while uninduced ectoderm generates epidermis. We screened for genes activated during gastrulation and expressed specifically in the nervous system of Xenopus laevis in the expectation that clones representing such genes will constitute useful markers for the study of early neurogenesis. Probes were prepared from adult brain RNA by subtraction with RNA from ovary and from different combinations of adult kidney, muscle, and skin; cDNA libraries prepared from early to late neurula embryo RNA were screened with these probes. Six clones were chosen for further study. Three of these clones are not represented in the maternal RNA population but are activated at the late gastrula stage; the other three increase from a maternal base. Expression of five of the genes is restricted to the neural plate during embryogenesis, and all six are restricted to the central nervous system in premetamorphic tadpoles and adults. One of the clones encodes an apparently neurospecific isoform of beta-tubulin; the identity of the other clones is unknown. Expression of all six genes is suppressed in axis-deficient embryos that lack dorsal structures including the brain.

Animals↗

PubMed1990

Improved genetic selection for screening bacteriophage libraries by homologous recombination in vivo.

Three major difficulties have hindered the general application of in vivo recombination techniques to library screening: (i) the original selection could not be applied to libraries prepared in phage vectors lacking amber mutations, (ii) nonirradiated packaging extracts gave high backgrounds even when amber mutated vectors were used, and (iii) most red- vectors lacked rap, a recently discovered phage gene promoting phage-plasmid recombination. Here we describe a selection scheme for phage bearing suppressor tRNA plasmids, which relies upon an Escherichia coli host bearing an amber mutation in the dnaB gene. The selection is tight enough to allow library screening by recombination, is applicable to almost every phage vector in common use, and overcomes the background associated with nonirradiated packaging extracts. We also describe an ancillary plasmid that supplies the rap gene function in trans, permitting the recombination level to be raised fruitfully in phage libraries lacking endogenous rap. Finally, we demonstrate simple procedures for preparing and detecting phages that have lost integrated suppressor tRNA plasmids by homologous recombination.

Base Sequence↗

PubMed1998

A new deleted allele in the human cytochrome P450 2A6 (CYP2A6) gene found in individuals showing poor metabolic capacity to coumarin and (+)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502).

The S-oxidation of (4)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one hydrochloride (SM-12502) and the 7-hydroxylation of coumarin are primarily catalyzed by cytochrome P450 2A6 (CYP2A6). The activities of SM-12502 S-oxidase and coumarin 7-hydroxylase were investigated with liver microsomes from 20 human individuals. Liver microsomes from individual H16 showed the lowest activities of both enzymes. The expression of CYP2A6 protein was not detectable in liver microsomes from individuals H4, H5, H7, H8, H12 and H16. CYP2A6 mRNA was hardly detectable in the liver of the individual H16. A new SacI-restriction fragment length polymorphism showing the lack of a 2.6 kb fragment was found in two of forty genomic DNA preparations from individuals H16 and No. 594, using CYP2A6 cDNA as a probe. This deletional 2.6 kb fragment was isolated from a genomic library prepared from one individuals showing normal coumarin 7-hydroxylase activity and was sequenced. This fragment contained a CYP2A6 gene region from 319 bp upstream of a putative exon 6 to a SacI site in exon 9, indicating that this region was deleted in the two individuals in this study. We also demonstrated by polymerase chain reaction analysis that the exon 8 of CYP2A6 gene was deleted in individuals H16 and No. 594. These results indicate that the reduced activity of SM-12502 S-oxidase and no activity of coumarin 7-hydroxylase are caused by the lack of CYP2A6 mRNA and CYP2A6 protein caused by the CYP2A6 gene deletion.

Aryl Hydrocarbon Hydroxylases↗

PubMed2000

SH2-directed ligands of the Lck tyrosine kinase.

Two separate libraries, prepared via parallel synthesis, were employed to identify low-molecular-weight SH2-targeted ligands for the Lck tyrosine protein kinase. These libraries were constructed to furnish non-amino acid analogues of the (1) Glu-Glu and (2) Ile residues of the Lck SH2 domain peptide ligand Ac-pTyr-Glu-Glu-Ile-amide. The lead compound acquired in this study exhibits a dissociation constant for the Lck SH2 domain that is comparable to that displayed by Ac-pTyr-Glu-Glu-Ile-amide. These results demonstrate that the standard amino acid residues Glu-Glu-Ile can be completely replaced with non-amino acid moieties without loss of SH2 affinity.

Amides↗

PubMed2001

An unusual seed-specific 3-ketoacyl-ACP synthase associated with the biosynthesis of petroselinic acid in coriander.

Petroselinic acid (18:1 delta6) is the major component of the seed oil of Umbelliferae species such as coriander (Coriandrum sativum) as well as Araliaceae and Garryaceae species. This unusual fatty acid is synthesized in plastids by the delta4 desaturation of palmitoyl-acyl carrier protein (16:0-ACP) and subsequent elongation of delta4-hexadecenoyl (16:1 delta4)-ACP. To characterize the enzymatic nature of the elongation reaction, an in vitro assay was developed with 16:1 delta4-ACP and 16:0-ACP as substrates. Extracts from developing coriander seeds elongated 16:1 delta4-ACP in a competitive assay at rates ten-fold greater than that with 16:0-ACP. In contrast, extracts from castor seeds, which do not synthesize petroselinic acid, displayed a strong preference for the elongation of 16:0-ACP rather than 16:1 A4-ACP. In addition, the elongation of 16:1 A4-ACP and 16:0-ACP by coriander seed extracts was strongly inhibited by cerulenin at concentrations as low as 10 microM. This finding suggested that the elongation of 16:1 A4-ACP and 16:0-ACP in coriander seed is catalyzed by a 3-ketoacyl-ACP synthase (KAS) 1-type enzyme(s), rather than a KAS II-type activity that is typically associated with 16:0-ACP elongation. Consistent with this, a cDNA for a diverged form of KAS I was isolated from a cDNA library prepared from developing coriander seed. Using a variety of heterologous probing techniques, no KAS II-type cDNAs could be identified in this library. Multiple alignment of KAS amino acid sequences indicated that, although the polypeptide corresponding to the coriander cDNA is more closely related to KAS I. its active site motif deviates from those found in both KAS I and KAS II enzymes. Also suggestive of a possible role in petroselinic acid synthesis, antibodies raised to the recombinant protein recognize an abundant 45 kDa polypeptide in coriander endosperm that is not detected in coriander leaves. These antibodies also recognize a major band of similar size in developing seeds of English ivy (Hedera helix), an Araliaceae species that also accumulates petroselinic acid in a seed-specific manner.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1983

Cloning and expression in Escherichia coli of full-length complementary DNA coding for human alpha 1-antitrypsin.

A cDNA library prepared from human liver was screened for alpha 1-antitrypsin, a major constituent of plasma which functions as inhibitor of proteolytic enzymes. The library was screened using a 12-base-long synthetic oligodeoxyribonucleotide corresponding to a known DNA fragment of human alpha 1-antitrypsin and by hybrid-selection of alpha 1-antitrypsin mRNA. A plasmid, pULB1523, was identified carrying a cDNA insert of about 1400 bp coding for human alpha 1-antitrypsin. Restriction mapping and DNA sequence analysis indicated that the 1400 bp code for the signal peptide and for the complete mature alpha 1-antitrypsin molecule. In addition, a solid-phase enzyme-linked immunoassay showed that pULB1523 expresses human alpha 1-antitrypsin in bacteria. Fusion of the alpha 1-antitrypsin sequence to the leader sequence of the beta-lactamase gene (plasmid pKT287) resulted also in the expression of the protein in bacteria.

Base Sequence↗

PubMedDate not supplied

Preparation of trifluoromethylpyridine libraries.

S-Alkylation followed by heterocyclization of trifluoromethyl-3-cyano-2(1H)-pyridinethiones was used for preparation of libraries of S-alkyl trifluoromethylpyridines and thieno[2,3-b]pyridines. The S-alkylation (in water--DMF mixtures) was successful for all 18 alkylating agents employed (yields typically > 50%). S-Alkyl derivatives were further converted to corresponding thieno[2,3-b]pyridines via heterocyclization in base conditions (yields > 65%). Structures of new compounds were elucidated by a combination of IR and 1H NMR spectroscopy and elemental analysis and were confirmed by means of single-crystal X-ray diffraction analysis.

Dihydropyridines↗

PubMed2026

A contextual activity score (CAS) for inferring ADAR-associated transcriptional activity across RNA-seq, single-cell, and spatial transcriptomics.

BACKGROUND AND OBJECTIVE: Adenosine-to-inosine RNA editing, catalyzed by Adenosine Deaminases Acting on RNA (ADARs), is a widespread modification involved in neural function, immune regulation, and cancer. The Alu Editing Index (AEI) is the standard metric to estimate ADAR activity but requires raw sequencing reads and is poorly suited for single-cell and spatial transcriptomic data. This study aimed to develop an alternative framework for inferring ADAR-associated transcriptional activity from gene expression data across diverse transcriptomic technologies. METHODS: We developed the Contextual Activity Score (CAS), a framework based on transcriptional signatures from ADAR perturbation experiments. Context-specific signatures were generated for human neurons, mouse neurons, and cancer models to infer ADAR1 and ADAR2 activity. CAS was computed from normalized gene expression matrices using regulon-based enrichment analysis. Performance was evaluated by comparing with the Alu Editing Index across bulk RNA sequencing datasets, simulated sequencing depths, and library preparation protocols. RESULTS: CAS showed strong concordance with the Alu Editing Index across multiple datasets, while remaining robust to reduced sequencing depth and different library protocols. Unlike the Alu Editing Index, CAS can be applied to single-cell and spatial transcriptomic data and enables the independent assessment of ADAR2 activity. In cancer and neuronal contexts, CAS captured biologically meaningful variations in ADAR-associated transcriptional activity at sample, cell-type, and spatial levels. CONCLUSION: CAS provides a scalable approach applicable across multiple RNA-seq protocols for estimating ADAR-associated transcriptional activity using gene expression data. This method, implemented in an open-source R package for broad adoption, expands the ability to study ADAR-associated transcriptional activity across transcriptomic modalities where direct editing quantification is challenging, such as single-cell and spatial transcriptomics.

Adenosine Deaminase↗

PubMed1995

Structure and organization of the bovine oxytocin receptor gene.

A DNA probe specific for the V and VI transmembrane domains of the bovine oxytocin receptor was initially prepared by reverse transcription PCR, and its structure and specificity confirmed by DNA sequencing. This probe was then used to screen a bovine genomic DNA library in bacteriophage lambda, and three positive clones were purified, subjected to restriction analysis and relevant fragments sequenced. Parallel to this, a cDNA library prepared using bovine endometrial RNA at the time of ovulation was screened by PCR employing the same primers as above. The longest cDNA clone was also fully sequenced. This clone still lacked, however, a substantial stretch of 5'sequence. The full transcript structure, and hence also the exon-intron organization, was then obtained by RT-PCR using primer oligonucleotides deduced from the cloned genomic sequence. All nucleotide sequence information was derived from a combination of two independent genomic clones, a cDNA clone and several independent RT-PCR reactions programmed by myometrial RNA, all in both strand orientations. The structural organization of the bovine oxytocin gene essentially conforms to that described for the human gene. Unlike the human gene, however, the 5'non-coding region of the primary transcript is interrupted by only a single intron, with a further intron in the coding region separating the sequences encoding the transmembrane domains VI and VII. The difference between this structure and that for the human gene suggests the existence of a differential splicing of 5' non-coding sequences.

Amino Acid Sequence↗

PubMed2005

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation.

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.

Chryseobacterium↗

PubMed2000

The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice.

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

Amino Acid Sequence↗

PubMed2001

Combinatorial and Evolution-Based Methods in the Creation of Enantioselective Catalysts.

Combinatorial methods in the development of enantioselective homogeneous catalysts constitute a new branch of catalysis research. The goal is to prepare libraries of potential asymmetric catalysts, rather than choosing the traditional one-catalyst-at-a-time approach. Several conceptional advancements have been reported in the parallel preparation of chiral ligands. Currently the most meaningful systems constitute modularly constructed ligands on solid supports, which allow high degrees of structural diversity and thus the maximum probability of finding enantioselective catalysts or even new types of ligands for asymmetric catalysis. Search strategies have been developed which amongst other things, lead to catalysts not likely to have been discovered by traditional methods. Genuine application of such strategies involve thousands of catalysts and require high-throughput screening systems capable of assaying enantioselectivity. The first high-throughput ee-screening systems were in fact developed for use in the directed evolution of enantioselective enzymes, a process based on "evolution in the test tube" in which the appropriate methods of random mutagenesis, gene expression, and ee assays are combined. Since no screening system is likely to be universal, different approaches are necessary. Thus far these include assays based on UV/Vis, fluorescence, circular dichroism, mass spectrometry, and even modified gas chromatography as well as special forms of capillary electrophoresis. One of the most efficient systems involves the concept of the mass-spectrometric detection of deuterium-labeled pseudo-enantiomers and pseudo-prochiral compounds with which about 1000 exact ee determinations can be achieved per day, although the assay is restricted to kinetic resolution and/or reactions of prochiral compounds bearing enantiotopic groups. Super-high-throughput screening for enantioselectivity is possible in many cases by making use of chirally modified capillary array electrophoresis in a parallel step. Accordingly, 7000 to 30 000 ee determinations can be carried out per day. These and other analytical developments are expected to stimulate further research in the combinatorial search for asymmetric homogeneous catalysts and in the directed evolution of enantioselective enzymes for use in organic chemistry.

Journal Article↗

PubMed1997

Retrofitting YACs for direct DNA transfer into plant cells.

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.

Chromosomes, Artificial, Yeast↗