SEARCH · PubMed Health

Results for “Library preparation”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 541 records · Page 30Linked to original sources

PubMed1995

Functional analysis of aryl hydrocarbon receptor nuclear translocator interactions with aryl hydrocarbon receptor in the yeast two-hybrid system.

The aryl hydrocarbon receptor (AHR) mediates dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced transcriptional activation of a battery of genes by interaction with a cofactor, called aryl hydrocarbon receptor nuclear translocator (ARNT) protein. Both AHR and ARNT belong to a family of proteins that includes the Drosophila circadian-rhythm protein and "single-minded" protein. These proteins share a domain called the PAS domain. In addition to the PAS domain, both AHR and ARNT contain basic helix-loop-helix (bHLH) and glutamine (Q)-rich domains. The roles of these domains in the receptor-mediated transcriptional activation are not understood completely. By using the yeast two-hybrid system with the N-terminal half of AHR as a probe, which contains the bHLH and PAS regions, to screen cDNA libraries prepared from human lymphocytes and C57BL mouse liver for clones encoding proteins capable of binding to these regions, we isolated a partial ARNT cDNA clone. These results demonstrated that the N-terminal half of AHR is capable of interacting with ARNT in yeast (probably through the bHLH motif). A fusion protein containing the GAL4 DNA binding domain (DB) linked to the full-length AHR was not capable of activating expression of a reporter gene containing the GAL4 DNA binding site, suggesting that ligand-free AHR alone has no transactivating properties in yeast. However, the C-terminal portion (amino acid residues 580-797) of the AHR, including the Q-rich domain, could confer transactivation of the reporter gene expression in the same system, suggesting that the N-terminal portion of the AHR contains transcription repression properties. In contrast, GAL4(DB)-ARNT fusion protein was able to activate expression of the same reporter gene. Deletion analysis of ARNT revealed that the C-terminal 75 amino acids, including the Q-rich domain, exhibited full transactivation function in yeast and mammalian cells. These results revealed different structural organizations for the transactivation properties between AHR and ARNT, although both contained transactivation domains at the C-termini.

Animals↗

PubMed1990

Purification of the growth-associated protein GAP-43 by reversed phase chromatography: amino acid sequence analysis and cDNA identification.

GAP-43 is a neuronal phosphoprotein. Increased synthesis and axonal transport of GAP-43 has been associated with axon growth, and altered phosphorylation of GAP-43 has been associated with changes in synaptic efficacy. Here we report a rapid and effective procedure employing reverse-phase HPLC for the purification of GAP-43 from rat brain. To characterize the protein purified by this procedure, we generated proteolytic fragments and determined their amino acid sequences. These directly determined sequences, corresponding to 56% of the GAP-43 amino acids, confirm recently reported sequences deduced from the nucleotide sequences of cDNAs. Using oligonucleotide probes constructed according to these amino acid sequences, we identified GAP-43 cDNAs in a library prepared from neonatal rat superior cervical ganglion cells. One of these cDNAs was 1.1 kB in size; it hybridized specifically with a 1.5 kB RNA from brain, but not from liver, and contained the entire coding sequence for GAP-43. This cDNA differed from recently reported cDNAs in its 3' untranslated region.

Amino Acid Sequence↗

PubMed1987

Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues.

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.

Animals↗

PubMed1988

Molecular consequences of awdb3, a cell-autonomous lethal mutation of Drosophila induced by hybrid dysgenesis.

The abnormal wing disc locus, which is at salivary gland chromosome position 100C-D of the Drosophila melanogaster genome, has been identified by a recessive lethal mutation, awdb3, induced by hybrid dysgenesis. When homozygous, this mutation causes abnormal development of the brain, the ovaries, and the larger imaginal discs as described in the preceding paper (C.R. Dearolf, E. Hersperger, and A. Shearn, 1988, Dev. Biol. 129, 159-168). The DNA corresponding to this locus was isolated from a genomic library prepared from awdb3 heterozygotes by screening with a P-element probe. The awdb3 allele resulted from the insertion of a P-element fragment into a gene that encodes an 0.8-kb poly(A)+ RNA. In mutant larvae, that 0.8-kb transcript is replaced by two chimeric transcripts that are 0.7 and 1.3 kb in length, both of which contain P-element and awd sequences. The wild-type awd+ gene transcript is most abundant during the second and third larval instars but is found at a lower level during every developmental stage as well as in continuous cell lines. Thus the awd+ gene transcript can be detected in normal larvae at a developmental stage long before defects are expressed in mutant larvae. Moreover, some tissues, for example the salivary gland of nonmutant, third-instar larvae, contain high levels of this transcript, even though these tissues appear to develop normally in mutant larvae.

Animals↗

PubMed1988

Regulation of expression of arylphorin and female-specific protein mRNAs in the tobacco hornworm, Manduca sexta.

Two non-cross-hybridizing cDNA clones were isolated from a lambda gt11 cDNA library prepared from Day 2 fifth instar female fat body of Manduca sexta and shown by hybrid selection to code respectively for the two storage proteins arylphorin and female-specific protein (FSP). Analysis of the developmental expression of arylphorin showed its presence during the feeding phases of the penultimate (fourth) and final (fifth) larval instars and its absence during the molt. Abdominal ligation of larvae followed by infusion of Grace's medium showed that this amino acid-rich medium was able to maintain arylphorin expression in fourth instar larvae, but not continued high expression in fifth instar larvae. This nutrient medium however was sufficient to allow initiation of expression in newly ecdysed fifth larval abdomens. Infusion of 5 micrograms 20-hydroxyecdysone (20HE) caused a significant reduction of arylphorin RNA in ligated fourth larval abdomens, whereas 50 micrograms was required in Day 2 fifth larval abdomens to suppress this RNA. Thus, both the lack of incoming nutrients and the rising titer of ecdysteroid contribute to the loss of arylphorin mRNA at the molts and at wandering. By contrast, FSP mRNA was first detected in females on Day 2 of the fifth instar, but not in males until wandering, and then was present throughout the prepupal period. In females allatectomy caused the precocious appearance of FSP mRNA which was prevented by application of 10 micrograms methoprene, a juvenile hormone analog. Expression of FSP mRNA in males however appeared to be independent of hormonal milieu.

Adipose Tissue↗

PubMed1992

A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.

To obtain gene sequences controlling the early steps of amphibian neurogenesis, we have performed differential screening of a subtractive cDNA library prepared by a novel PCR-based method from a single presumptive neural plate of a Xenopus laevis late-gastrula embryo. As a result we have isolated a fragment of a novel homeobox gene (named XANF-1, for Xenopus anterior neural folds). This gene is expressed predominantly in the anterior part of the developing nervous system. Such preferential localization of XANF-1 mRNA is established from its initially homogenous distribution in ectoderm of early gastrula. This change in the expression pattern is conditioned by a differential influence of various mesoderm regions on ectoderm: anterior mesoderm activates XANF-1 expression in the overlying ectoderm, whereas posterior axial and ventral mesoderm areas inhibit it. The data obtained demonstrate for the first time that selection of genes for specific expression in the CNS of the early vertebrate embryo is affected not only by chordamesoderm (a neural inductor) but also by ventral mesoderm.

Amino Acid Sequence↗

PubMed1989

Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors.

With a cDNA library prepared from skin of Phyllomedusa sauvagei, the sequence of the precursor of dermorphin was elucidated recently. The sequence suggested the existence of another peptide, distantly related to dermorphin. Two variants of this peptide have now been synthesized containing either L- or D-methionine as the second amino acid. The peptide containing the D-methionine exhibited high-affinity and selectivity for delta opioid receptors in the mouse vas deferens and in rat brain homogenates. Moreover, using the synthetic peptide as marker, we could isolate small quantities of the corresponding natural peptide containing D-methionine as the second amino acid from skin extracts of Phyllomedusa sauvagei. The name deltorphin is proposed for this new peptide and its sequence is Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2.

Animals↗

PubMed1989

Coordinate induction of fibronectin, fibronectin receptor, tropomyosin, and actin genes in serum-stimulated fibroblasts.

From a collection of more than 80 nonoverlapping clones, isolated by differential screening of a lambda cDNA library prepared from serum-stimulated cells in the presence of cycloheximide, we have identified four clones that encoded for components of the cytoskeleton and extracellular matrix. DNA sequencing of clones B2, V58, TT1, and P38 demonstrated that they corresponded to beta-actin, alpha-tropomyosin, fibronectin, and the beta-subunit of fibronectin receptor. All four mRNA levels showed a detectable increase 30 min after stimulation and remained at high levels for at least 8 h. The half-lives of these mRNAs were found to be very long in contrast to those of other growth factor-inducible genes. An increase in transcription was observed for the four genes. Actin and fibronectin showed nearly maximal increase at 15 min, while fibronectin receptor and tropomyosin reached their maximum transcription at 1 h. These results demonstrated that four interacting components of the cytoskeleton and extracellular matrix are rapidly induced in stimulated quiescent cells, possibly reflecting part of the coordinate changes in gene expression that occur during embryogenesis and wound healing.

Actins↗

PubMed1988

Eimeria acervulina: DNA cloning and characterization of recombinant sporozoite and merozoite antigens.

Genes encoding antigens of Eimeria acervulina were cloned from cDNA expression libraries prepared from the sporozoite and merozoite stages in order to examine humoral and cellular immune responses to this protozoan parasite. Two clones expressing surface antigens were characterized by DNA hybridization studies to identify homologous genomic DNA fragments. The proteins they encode were identified by 125I-labeling, immunoblotting, immunofluorescence, and T-cell activation experiments. One, designated cSZ-1, encodes a 130-kDa beta-galactosidase fusion protein which represents a portion of a p240/p160 immunodominant sporozoite surface antigen. Immunofluorescence studies using anti-cSZ-1 sera and live or 1% paraformaldehyde-fixed E. acervulina sporozoites have confirmed this surface locale. Purified cSZ-1 fusion protein, which is not recognized by sera from E. acervulina-infected chickens, induced the activation of immune T lymphocytes in vitro. Another cDNA clone, designated cMZ-8, gives rise to a 150-kDa fusion protein and encodes a portion of a p250 immunodominant merozoite surface antigen. This was established by immunoblotting of 125I-labeled merozoite proteins with anti-cMZ-8 sera and immunofluorescence staining of live and 1% paraformaldehyde-fixed E. acervulina merozoites. Purified cMZ-8 is recognized by sera from E. acervulina-infected chickens and induces a significant activation of immune T lymphocytes in vitro.

Animals↗

PubMed1986

Isolation of partial cDNAs for rat liver and muscle glycogen phosphorylase isozymes.

cDNA clones for the rat liver and muscle glycogen phosphorylase isozymes have been isolated using isozyme-selective antibodies and libraries prepared in the expression vector, lambda gt11. A 1.2 kb cDNA coding for the carboxy-terminal domain of rat liver phosphorylase was found to have 82% homology with the amino acid sequence of rabbit muscle phosphorylase. Limited sequencing of rat muscle phosphorylase cDNA indicated a 95% homology with the rabbit muscle enzyme. The rat liver clone has eight additional amino acid residues at the COOH-terminus compared to the rat muscle clone. Furthermore, 17 of 26 (65%) residues between amino acids 815-840 differ between liver and muscle isozymes. The similarity in enzymatic properties and conservation of structure except at the COOH-terminus suggest that the liver and muscle phosphorylase isozymes do not exist in order to have significant differences in the regulation of glycogen breakdown in the two tissues. Rather, the phosphorylase isozymes probably evolved for tissue-specific transcriptional regulation of the genes in liver and muscle.

Animals↗

PubMed1986

Cloning of rat brain protein kinase C complementary DNA.

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.

Amino Acid Sequence↗

PubMed1987

Neuronal nicotinic acetylcholine receptor beta-subunit is coded for by the cDNA clone alpha 4.

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for alpha-bungarotoxin, which have been purified from rat and chicken brains by immuno-affinity chromatography, consist of two types of subunits, alpha and beta. The beta-subunits form the ACh binding sites. Putative nicotinic AChR subunit cDNAs alpha 3 and alpha 4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR alpha-subunit. Here we determine the amino-terminal amino acid sequence of the rat brain AChR beta-subunit by protein microsequencing to be the same as amino acid residues 27-43 of the protein which could be coded by alpha 4. Further, we present evidence consistent with a subunit stoichiometry of alpha 3 beta 2 for this neuronal nicotinic AChR.

Amino Acid Sequence↗

PubMed1987

Isolation of several cDNAs encoding yeast peroxisomal enzymes.

Several candidate clones carrying partial cDNAs for yeast peroxisomal enzymes, such as catalase, carnitine acetyltransferase, isocitrate lyase, malate synthase and acyl-CoA oxidase, were efficiently isolated at a single plating from a phage lambda gt11 recombinant cDNA library prepared with poly(A)-rich RNA from an n-alkane-grown yeast, Candida tropicalis, with a mixture of antibodies against the respective purified enzymes. Among them, one candidate clone carrying partial cDNA for catalase was subcloned and subjected to nucleotide sequence analysis. We succeeded in determining that the amino acid sequence deduced from the nucleotide analysis included the sequences derived from the two peptide fragments obtained from the purified enzyme.

Amino Acid Sequence↗

PubMed1989

The constituents of storage granules in the dermal glands of Xenopus laevis. Structure of a basic polypeptide deduced from cloned cDNA.

Mature dermal glands of Xenopus laevis contain storage granules with a characteristic ellipsoid shape. A few major proteins are present in these granules, including two with the same amino-terminal sequence and apparent molecular masses of 26 and 28 kDa. Using antibodies against these proteins, positive clones were isolated from a cDNA expression library prepared from skin of X. laevis. One cDNA encodes a preprotein with a typical signal sequence and a mature part of 187 amino acids. The protein shows internal homology at both the amino and carboxyl end. The latter part has a very high content of basic amino acids.

Amino Acid Sequence↗

PubMed1989

The hexose carrier from Chlorella. cDNA cloning of a eucaryotic H+-cotransporter.

The cDNA coding for the inducible H+/hexose cotransporter of Chlorella kessleri has been cloned and sequenced. It was isolated by differential screening of a cDNA library prepared from glucose-induced cells. The increase in expression of the gene correlates quantitatively with the increase in uptake activity due to induction; it is not expressed in a hexose transport mutant. An open reading frame allows for a membrane protein of 533 amino acids with a relative molecular mass of 57 kDa. This protein is highly homologous to the human and rat glucose transporters catalyzing facilitated diffusion and to the bacterial H+/pentose cotransporters. It is not related to the H+/lactose cotransporter of E. colli and to the mammalian Na+/glucose cotransporter.

Base Sequence↗

PubMed1989

Human embryonic myosin heavy chain cDNA. Interspecies sequence conservation of the myosin rod, chromosomal locus and isoform specific transcription of the gene.

A 3.6 kilobase cDNA clone coding for the human embryonic myosin heavy chain has been isolated and characterized from an expression library prepared from human fetal skeletal muscle. The derived amino acid sequence for the entire rod part of myosin shows 97% sequence homology between human and rat and a striking interspecies sequence conservation among the charged amino acid residues. The single copy gene is localized to human chromosome 17 and its expression in fetal skeletal muscle is developmentally regulated. The sequence information permits the design of isoform-specific probes for studies on the structure of the gene and its role in normal and defective human myogenesis.

Amino Acid Sequence↗

PubMed1990

Dermal glands of Xenopus laevis contain a polypeptide with a highly repetitive amino acid sequence.

Mature dermal glands of Xenopus laevis contain storage granules with a characteristic ellipsoid shape. These granules contain, as a minor component, a heat-stable, acidic polypeptide with an apparent molecular mass of 75 kDa. Using antibodies against this protein, positive clones were isolated from a cDNA expression library prepared from skin of X. laevis. One of the cloned cDNAs encodes a pre-protein with a typical signal sequence and a mature part of 396 amino acids. The protein contains 33 copies of the sequence Gly-Gly/Glu-(Ala-Pro)2-4-Ala-Glu. Using the single-letter code for the four predominant amino acids, we have termed this polypeptide the APEG protein. Near its carboxy-terminus, one segment has been found with an amino acid sequence similar to that of spasmolytic polypeptide from porcine pancreas and to the human protein pS2.

Amino Acid Sequence↗

PubMed1990

Molecular cloning and sequence determination of four different cDNA species coding for alpha-subunits of G proteins from Xenopus laevis oocytes.

A cDNA library prepared from Xenopus laevis oocytes in lambda gt10 was screened with a mixture of three oligonucleotide probes designed to detect sequences found in different mammalian genes coding for alpha-subunits of G-proteins. In addition to a clone coding for a G alpha o-type subunit previously reported [(1989) FEBS Lett. 244, 188-192] four additional clones have been found coding for different G alpha protein subunits. By comparison with mammalian alpha-subunits, these oocyte cDNAs correspond to two closely related G alpha s-1a, to a G alpha i-1 and to a G alpha i-3 species. The derived amino acid sequences showed that both G alpha s species contain 379 residues, corresponding to the short species without the serine residue and with a calculated Mr of 42720. The G alpha i-1 gene encodes a 354 amino acid protein with an Mr of 39,000 and the G alpha i-3 encodes an incomplete open reading frame of 345 residues, lacking the first 9 amino acid residues at the NH2 terminus. All these G alpha-subunits showed high identity with their respective mammalian counterparts (75-80%), indicating a great degree of conservation through the evolution and the important cellular regulatory function that they play.

Amino Acid Sequence↗