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PubMed1991

Mouse protein kinase C-delta, the major isoform expressed in mouse hemopoietic cells: sequence of the cDNA, expression patterns, and characterization of the protein.

A complementary DNA (cDNA) of 2559 bp which encode all 674 amino acids of mouse protein kinase C-delta (PKC-delta) has been isolated from a cDNA library prepared from ABPL-2, a mouse myeloid tumor. The library was screened with a partial PKC-delta cDNA clone that had been created by polymerase chain reaction (PCR) amplification of ABPL-2 RNA using primers that are conserved among all rat PKC isozymes. This approach proved to be a distinct improvement over screening with synthetic oligonucleotides. Similar sets of cDNAs prepared from other hemopoietic cell lines were screened with this PKC-delta cDNA and with probes for the other PKC isoforms. These experiments revealed that the major isoform of PKC expressed in hemopoietic cells is PKC-delta. PKC-delta protein was purified from ABPL-3, a mouse myeloid tumor which expressed principally the delta isoform of PKC. The protein eluted from a hydroxylapatite column in the same position as PKC-beta and -epsilon would elute, if present. The kinase activity of purified PKC-delta showed strict dependence on the presence of phospholipids, but showed no activation by Ca2+.

Amino Acid Sequence↗

PubMed2006

Compositional properties of human cDNA libraries: practical implications.

The strikingly wide and bimodal gene distribution exhibited by the human genome has prompted us to study the correlations between EST-counts (expression levels) and base composition of genes, especially since existing data are contradictory. Here we investigate how cDNA library preparation affects the GC distributions of ESTs and/or genes found in the library, and address consequences for expression studies. We observe that strongly anomalous GC distributions often indicate experimental biases or deficits during their preparation. We propose the use of compositional distributions of raw ESTs from a cDNA library, and/or of the genes they represent, as a simple and effective tool for quality control.

Base Composition↗

PubMed1994

Cloning of the sole (Solea senegalensis) growth hormone-encoding cDNA.

We report here the complete nucleotide (nt) sequence of a cDNA clone encoding Solea senegalensis growth hormone (sGH) isolated from an expression library prepared from sole pituitary gland poly(A)+RNA. The library was screened using a flounder GH cDNA. The cDNA sequence containing an insert of 769 nt was found to encode a polypeptide of 203 amino acids (aa), including a signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 17 and 119-nt long, respectively. Northern blot hybridization detected a 0.9-kb RNA species. The sGH cDNA sequence shows homologies of 80.9, 76.9, 73.8 and 64.2% with the GH of tuna, gilthead seabream, flounder and rainbow trout.

Amino Acid Sequence↗

PubMed2000

Identification of chiral selectors from a 200-member parallel combinatorial library.

Selection of chiral selectors for the resolution of racemic N-(1-naphthyl)leucine ester 1 was studied with a 200-member parallel library prepared on polymeric synthesis resin. Through this study, the library screening procedure developed previously is improved and other pertinent issues concerning the screening process are also addressed. The equilibration time required for screening is reduced from 24 h to approximately 3 h. Excellent correlation of the outcome between the resin batch equilibration experiment and chromatographic separation is further demonstrated. It is also demonstrated that selectors with separation factors as low as 1.4 could be identified by this batch screening process. In addition, a great deal of information regarding enantioselective interactions was obtained in this parallel library study. Such information should prove useful in improving future library designs.

Chromatography, High Pressure Liquid↗

PubMed1994

cDNA approaches to isolation of the mouse mutant weaver gene.

The mouse autosomal recessive mutant gene weaver (wv) results in abnormalities in cerebellum, substantia nigra and testis. Although a substracted cDNA library prepared by removing P31 (wv/wv) sequences from a P1 (wv/+) library should contain mainly nonrepetitive neonatal sequences, unfortunately, repetitive sequences still appear during screening. Two clones, one repetitive, the other not, are used to illustrate the problems encountered in attempting to isolate the weaver gene from a substrated cDNA library.

Amino Acid Sequence↗

PubMed1991

Cloning and sequencing of the gilthead seabream (Sparus aurata) growth hormone-encoding cDNA.

The cDNA clones encoding gilthead seabream (gsb) (Sparus aurata) growth hormone (GH) have been isolated from a cDNA library prepared from seabream pituitary gland poly(A)+ RNA. The cDNA library was screened using red seabream and rainbow trout GH cDNAs. The complete nucleotide (nt) sequence of gsbGH has been determined. The cDNA sequence codes for a polypeptide of 204 amino acids (aa), including a putative signal peptide of 17 aa. The 5'- and 3'-untranslated regions of the message are 55 and 236 nt long, respectively. The predicted aa sequence of gsbGH revealed 97% homology with red seabream GH, 95% with tuna GH, 85% with yellowtail GH, and 65% with rainbow trout GH.

Amino Acid Sequence↗

PubMed1997

Spectrometrically monitored selection experiments: quantitative laser desorption mass spectrometry of small chemical libraries.

BACKGROUND: Selection experiments involving chemical libraries are routinely used in the pharmaceutical industry for finding and optimizing lead compounds. In principle, almost any process involving a binding event or a reaction could be probed systematically with chemical libraries prepared by combinatorial synthesis. Traditionally, however, the vast majority of library members cannot be monitored during the selection, making a systematic correlation of structure and activity difficult. To interpret selection experiments on the level of all library components, monitoring technologies are required that give a unique and quantitative spectroscopic signal for every compound in a mixture. RESULTS: Quantitative matrix-assisted laser desorption mass spectrometry of libraries of porphyrins and peptide-DNA hybrids consisting of 2-35 compounds is described. Porphyrin libraries were subjected to in vitro selections for liposome incorporation and binding to a protein pocket. It was shown that mesohydroxyphenyl substituted porphyrins, known high activity photosensitizers of tumors, are preferentially incorporated in liposome membranes. A mixture of peptide-DNA hybrids was assayed for the nuclease stability of its components. CONCLUSIONS: Small libraries of non-isobaric compounds can be exhaustively or near-exhaustively monitored by mass spectrometry. Monitored selection experiments can yield detailed structure-activity maps in a single experiment, speeding up drug discovery and the probing of biochemically relevant recognition events. It is proposed that monitored assays for target binding, membrane partitioning, and biostability could be run in parallel, to select drug candidates combining several favorable properties in 'multidimensional' selection experiments.

DNA↗

PubMed2006

Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.

Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.

Bacteria↗

PubMed1999

Cloning and sequencing of the gilthead sea bream estrogen receptor cDNA.

We report here the complete nucleotide sequence of a cDNA clone containing the full-coding sequence of the Sparus aurata estrogen receptor (ER) isolated from an expression library prepared from gilthead sea bream liver poly A+ RNA. The library was screened using a single strand rainbow trout ER cDNA probe, corresponding to the C-D domain. The cDNA sequence containing an insert of 2369 nucleotides was found to encode a protein of 579 amino acids. The 5'- and 3'-untranslated regions of the message are 186 and 392 nucleotides long, respectively. The gilthead sea bream ER shows the higher homology with the ER of another perciform, Chrysophrys major (93%), moderate to high homology with Oreocromis aureus (78%) medaka (77%) and rainbow trout (70.7%) ERs and lower homology with japanese eel (45%), amphibian (47%), avian (48.5%) and mammalian (47-47.5%) ERs. The sequence homologies and phylogenetic analysis of the various ERs suggest that gilthead sea bream ER should be considered as a ER alpha-like.

Amino Acid Sequence↗

PubMed1991

Study of transcription in measles virus-infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells.

From af primary plasmid cDNA library prepared from measles virus-infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08-0.01% were able to produce a hybridization signal above background. Unspecific cross-hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.

Animals↗

PubMed1986

Identification and characterization of neuron-specific and developmentally regulated gene transcripts in the chick embryo spinal cord.

Clones corresponding to neuron-specific and developmentally regulated messenger RNA species in the chick have been isolated from a complementary DNA library prepared using polyadenylated RNA from 7-day embryonic spinal cord. The library was initially screened by differential complementary DNA hybridization procedures for clones identifying polyadenylated RNAs present in embryonic spinal cord but absent from or at low abundance in liver tissue. A high proportion of selected recombinant plasmids were found to identify different RNA species which, although present in 14-day embryonic spinal cord, could not be detected in a corresponding region of the developing chick CNS that is devoid of neuronal cell bodies, the optic nerve. The neuron-specific assignment of these mRNAs within the developing neuroectoderm was confirmed using bulk-isolated neuronal and glial-enriched cell fractions from 7-day embryonic spinal cord. In addition, several distinctive patterns of developmentally regulated expression of neuron-specific messenger RNA species have been observed in the chick spinal cord. The studies lay a foundation for detailed examination of the regional and temporal distribution and control of neuronal gene expression in the chick spinal cord during embryogenesis.

Animals↗

PubMed1995

Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression.

Cultured human melanoma cells lose proliferative capacity and terminally differentiate after treatment with the combination of recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ). Subtraction hybridization of cDNA libraries prepared from actively proliferating human H0-1 melanoma cells from cDNA libraries produced from H0-1 cells treated with IFN-beta + MEZ identifies a novel melanoma differentiation-associated (mda) cDNA, mda-7, that displays elevated expression in differentiation inducer-treated H0-1 cells. mda-7 encodes a novel protein of 206 amino acids with a predicted size of 23.8 kDa. The level of mda-7 mRNA is elevated in actively proliferating normal human melanocytes versus primary and metastatic human melanomas. In the Matrigel-assisted melanoma progression model, mda-7 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. Treatment of human melanomas with IFN-beta + MEZ, and to a lesser extent with MEZ, results in growth suppression and induced or enhanced mda-7 expression. Immunoprecipitation analyses using peptide-derived rabbit polyclonal antibodies detect increases in mda-7 protein, and a higher molecular weight protein of approximately 90 to 100 kDa, in MEZ and IFN-beta + MEZ treated H0-1 cells. mda-7 is a highly conserved gene with an homologous sequence in the genome of yeast. Transfection of mda-7 expression constructs into H0-1 and C8161 human melanoma cells reduces growth and inhibits colony formation. These results confirm that mda-7 has antiproliferative properties in human melanoma cells and in this context may contribute to terminal cell differentiation. The mda-7 gene may also function as a negative regulator of melanoma progression.

Amino Acid Sequence↗

PubMed1994

Are vitellin and vitellogenin coded by one gene in the marine shrimp Penaeus semisulcatus?

A cDNA clone encoding a female-specific ovarian protein (presumably vitellin, Vt) has been isolated from a cDNA library prepared from poly (A)+ RNA extracted from vitellogenic ovaries of the shrimp Penaeus semisulcatus. The cDNA library was constructed and screened using a major cDNA band which was observed following analysis of total cDNA products by gel electrophoresis. This band, as well as the cDNA insert purified from the library, was estimated to have 1.1 kb. Both hybridized to mRNA prepared from ovaries or hepatopancreas (HEP) of vitellogenic females and showed a faint signal with ovaries from non-vitellogenic females, but did not hybridize to HEP from non-vitellogenic females or to HEP from males or testes. The size of the transcripts from the ovary and HEP was estimated to be 1.1 kb, similar to that of the cDNA insert, suggesting that a full length cDNA had been synthesized. Furthermore, the identical sizes of the transcripts from ovary and HEP and the ability of the ovarian cDNA to detect a transcript in HEP mRNA suggest that Vt from the ovary and vitellogenin (Vg) from HEP are the gene products of one gene. Alternatively, the homology between Vt and Vg is very high.

Animals↗

PubMed1995

Evidence for the overexpression of the VH4-34 (VH4.21) Ig gene segment in the normal adult human peripheral blood B cell repertoire.

The VH4-34 (VH4.21) gene has been repeatedly found to encode monoclonal anti-i/l cold agglutinins and is occasionally used by other autoantibodies with anti-DNA and rheumatoid factor specificity. To understand the basis for the frequent expression of the VH4-34 gene in autoimmunity, we prepared linker-based, amplified C mu and C gamma V gene (cDNA) libraries from the peripheral blood of two healthy adults (PBL-3 and PBL-4). The frequency of VH4 family gene-containing clones was determined with VH family-specific oligonucleotide probes, and by random sequencing we examined the frequency of VH4-34 gene expression among the VH4+ C mu clones. Collectively, these studies suggested that VH4-34 gene expression accounted for 7.8 and 10.3% of all IgM clones in the cDNA libraries of PBL-3 and PBL-4, respectively. In VH4 family-specific libraries prepared from genomic rearranged Ig DNA, the VH4-34 gene was also overrepresented relative to other VH4 family genes. In both the mu-cDNA as well as the rearranged Ig DNA libraries, the VH4-34-expressing clones were 95 to 100% homologous to the germline sequence and contained different CDR3 sequences. By contrast, the VH4-34-expressing C gamma clones showed somatic diversification from germline; in addition, the C gamma clones often had the same CDR3 sequences, suggesting that these clones were derived from either activated B cells or expanded, clonally related B cells. Multiparameter flow cytometric analysis of peripheral blood lymphocytes indicated that 3.2 to 6.2% of mu+CD20+ cells express the VH4-34-related Id 9G4. These current findings are relevant to the interpretation of restricted Ig gene usage reported in monoclonal cold agglutinin disease and other autoimmune disorders.

Adult↗

PubMed1994

Cloning and sequencing of the Atlantic salmon (Salmo salar) cytochrome c oxidase subunit III gene (coxIII) and analysis of coxIII expression during parr-smolt transformation.

Smoltification is the process whereby salmon alter their metabolism in preparation for movement from freshwater to seawater. Differential screening of a cDNA library prepared from post-smolt salmon liver mRNA led to the selection of a smoltification-induced sequence. Analysis of this cDNA revealed that it partially encoded subunit III of the enzyme cytochrome c oxidase. The complete coxIII sequence was amplified from salmon genomic DNA using consensus oligonucleotides based on ATPase 6 and tRNA(GLY) sequences from Pacific salmonid species. Cytochrome c oxidase subunit III liver mRNA levels were found to be significantly increased in salmon smolts. Northern blot analysis revealed a coxIII transcript of approximately 750 bp in all salmon tissues tested except blood. The DNA sequence of coxIII employs the mammalian mitochondrial genetic code and is strongly conserved when compared with that of other species.

Amino Acid Sequence↗

PubMed2003

Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses.

To identify cold-, drought-, high-salinity-, and/or abscisic acid (ABA)-inducible genes in rice (Oryza sativa), we prepared a rice cDNA microarray including about 1700 independent cDNAs derived from cDNA libraries prepared from drought-, cold-, and high-salinity-treated rice plants. We confirmed stress-inducible expression of the candidate genes selected by microarray analysis using RNA gel-blot analysis and finally identified a total of 73 genes as stress inducible including 58 novel unreported genes in rice. Among them, 36, 62, 57, and 43 genes were induced by cold, drought, high salinity, and ABA, respectively. We observed a strong association in the expression of stress-responsive genes and found 15 genes that responded to all four treatments. Venn diagram analysis revealed greater cross talk between signaling pathways for drought, ABA, and high-salinity stresses than between signaling pathways for cold and ABA stresses or cold and high-salinity stresses in rice. The rice genome database search enabled us not only to identify possible known cis-acting elements in the promoter regions of several stress-inducible genes but also to expect the existence of novel cis-acting elements involved in stress-responsive gene expression in rice stress-inducible promoters. Comparative analysis of Arabidopsis and rice showed that among the 73 stress-inducible rice genes, 51 already have been reported in Arabidopsis with similar function or gene name. Transcriptome analysis revealed novel stress-inducible genes, suggesting some differences between Arabidopsis and rice in their response to stress.

Abscisic Acid↗

PubMed1990

Molecular cloning and expression of the murine interleukin-5 receptor.

Murine interleukin-5 (IL-5) is known to play an essential role in Ig production of B cells and proliferation and differentiation of eosinophils. Here, we have isolated cDNA clones encoding a murine IL-5 receptor by expression screening of a library prepared from a murine IL-5 dependent early B cell line. A cDNA library was expressed in COS7 cells and screened by panning with the use of anti-IL-5 receptor monoclonal antibodies. The deduced amino acid sequence analysis demonstrates that the receptor is a glycoprotein of 415 amino acids (Mr 45,284), including an N-terminal hydrophobic region (17 amino acids), a glycosylated extracellular domain (322 amino acids), a single transmembrane segment (22 amino acids) and a cytoplasmic tail (54 amino acids). COS7 cells transfected with the cDNA expressed a 60 kd protein that bound IL-5 with a single class of affinity (KD = 2-10 nM). FDC-P1 cells transfected with the cDNA for murine IL-5 receptor showed the expression of IL-5 binding sites with both low (KD = 6 nM) and high affinity (KD = 30 pM) and acquired responsiveness to IL-5 for proliferation, although parental FDC-P1 cells did not show any detectable IL-5 binding. In addition, several cDNA clones encoding soluble forms of the IL-5 receptor were isolated. Northern blot analysis showed that two species of mRNAs (5.0 kb and 5.8 kb) were detected in cell lines that display binding sites for murine IL-5. Homology search for the amino acid sequence of the IL-5 receptor reveals that the IL-5 receptor contains a common motif of a cytokine receptor family that is recently identified.

Amino Acid Sequence↗

PubMed1964

[MEDICAL LIBRARIES OF THE SOVIET UNION].

Medical libraries are a part of the Soviet library system. The total number of medical libraries in the country is more than 4,000, with a collection of over 42,000,000 volumes that are used by over a million readers. The State Central Medical Library occupies a special place among these libraries. It carries out the functions of a methodological, bibliographic, and coordinating center. It has a collection of over 1,000,000 units of books and periodicals. All the bibliographic work of medical libraries as well as any other work is provided to help medical institutions. Libraries prepare special bibliographies of medical literature for publication according to a plan. The libraries also conduct reference and information work. The methodological work helps to solve the most important problems that arise in libraries with reference to the specific character of their work and tasks. The chief means of rendering methodological guidance are to analyze the work of various libraries, to hold conferences, to exchange visits with libraries, to give both field and correspondence consultations, and to organize qualification courses for librarians and bibliographers.

Bibliographies as Topic↗