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At least 685 records · Page 38Linked to original sources
PubMed1991

Isolation of a complementary DNA encoding the bean PR4 chitinase: an acidic enzyme with an amino-terminus cysteine-rich domain.

The amino acid sequences of peptides generated by trypsin and chymotrypsin digestions of the acidic PR4 chitinase from bean were determined. Oligonucleotide primers derived from this sequence were used to synthesize a PR4 chitinase-specific probe by PCR-amplification. This probe allowed the isolation of cDNA clones encoding PR4 chitinase that have been sequenced. This acidic and extracellular chitinase shows some homology to the basic isoform from the same plant, and differs from other known acidic chitinases by the presence of an amino-terminal cysteine-rich domain. Southern blot analysis of bean genomic DNA revealed that PR4 chitinase is encoded by a single gene.

Amino Acid Sequence↗
PubMed1990

Molecular cloning, sequencing and expression of an alpha 2-adrenergic receptor complementary DNA from rat brain.

We have isolated a cDNA clone from rat brain using a human platelet alpha 2-adrenergic receptor genomic clone as a probe. Comparison of the deduced amino acid sequence (450 residues) corresponding to the rat brain cDNA with that of the human platelet and human kidney alpha 2-adrenergic receptors showed 84% and 44% sequence similarity, respectively. The major sequence difference between the rat brain and human platelet proteins, was a stretch of 48 amino acids within the third cytosolic loop in which the similarity was only 42%. Analysis of the 48 amino acid-region indicated that the two receptors significantly differ in terms of their primary amino acid sequence and the predicted secondary and tertiary structural features. There was no sequence similarity between the human platelet and rat brain clone over the 177 bases of 3'-noncoding sequence and a less than 50% similarity over a stretch of 210 nucleotides in the 5'-untranslated region. Southern-blot analysis with a human platelet alpha 2-adrenergic receptor probe revealed the existence of a single 5.2 kb restriction fragment (KpnI/SacI) in both human and rat genomic DNA; the rat brain alpha 2-receptor probe, however, hybridized to a single 1.9 kb band in rat DNA. Northern-blot analysis of rat brain poly(A+) RNA with the rat brain cDNA probe under stringent hybridization conditions revealed a single 4.5 kb mRNA; none was detected by the human platelet receptor probe. The rat brain 4.5 kb mRNA was not detected in any (other than brain) tested rat tissues utilizing either rat brain or human platelet DNA probes. The rat brain cDNA was expressed in a mammalian cell line (COS-2A) and found to bind the alpha 2-adrenergic antagonist [3H]yohimbine; based on the binding-affinity for prazosin, the presently cloned receptor was pharmacologically closer to the alpha 2A subclass. We conclude that the rat brain cDNA encodes a new alpha 2-adrenergic receptor subtype that may be brain-specific.

Amino Acid Sequence↗
PubMed1983

Androgen regulation of prostatic binding protein messenger RNA studied by using cloned complementary DNA.

The construction of a double-stranded cDNA library using rat prostatic poly(A)RNA and pBR322/kappa 1776 system and the isolation of three prostatic binding protein (PBP) cDNA clones are described. These cDNA clones were characterized and identified by in situ hybridization, mRNA selection-translation and immuno-precipitation as coding for the three subunit components, C1, C2, and C3, of PBP. These clones were used in hybridization experiments with prostatic poly(A)RNA to determine the effect of testosterone on the levels of PBP-mRNA. The results showed that synthesis of these mRNAs varied in response to either androgen withdrawal or replacement. Accumulation of PBP-mRNAs coding for C2 and C3 components occurred 1 hr after androgen administration to castrated rat, whereas the mRNA coding for the C1 component did not appear until 4 hr after androgen replacement. Quantitation of PBP-mRNA sequences in nuclear and polysomal poly(A)RNAs showed that they did not vary coordinately in response to androgen withdrawal. These results indicate differential regulation of PBP genes and suggest possible multiple levels of androgen control of PBP synthesis.

Animals↗
PubMed1999

Metal ions in non-complementary DNA base pairs: an ab initio study of Cu(I), Ag(I), and Au(I) complexes with the cytosine-adenine base pair.

Ab initio calculations have been carried out to characterize the structure and energetics of a silver(I) complex with the cytosine-adenine DNA base pair and an aqua ligand in the coordination sphere of Ag. In addition, we have also studied analogous complexes with Cu(I) and Au(I), and structures in which adenine has been replaced by purine in order to investigate the structural role of the adenine amino group. The calculations revealed that all metal-modified structures are dominated by the metal-base interactions, while the water-metal ion interaction and many-body interligand repulsion are less important contributions. Nevertheless, the structural role of the water molecule in the complex is quite apparent and in agreement with an earlier crystallographic study. The metal-modified base pairs exhibit large conformational flexibility toward out-of-plane motions (propeller twist and buckle), comparable or, in some cases, even larger than that observed in the base pairs without metal ions. All structures have been optimized within the Hartree-Fock approximation, while interaction energies were evaluated with the inclusion of electron correlation.

Adenine↗
PubMed2005

Shear stress regulates gene expression in vascular endothelial cells in response to tumor necrosis factor-alpha: a study of the transcription profile with complementary DNA microarray.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Cell Adhesion↗
PubMed1983

Molecular cloning of complementary DNA: preparation of a plasmid vector with low transformation background.

A simple method that allows the rapid preparation of oligo dG-tailed plasmid vectors is presented. The procedure involves purification of the tailed molecules by hybridization to oligo dC-cellulose followed by a stepwise thermal elution. The resulting plasmid is virtually devoid of transformation activity in the absence of oligo dC-tailed DNA fragments. It allows construction of cDNA libraries with as low as 1% of colonies harboring wild-type plasmids.

Cellulose↗
PubMed1989

Rapid amplification of complementary DNA from small amounts of unfractionated RNA.

We have combined, in a rapid and straightforward manner, the synthesis and subsequent amplification of individual cDNA sequences from microgram quantities of unfractionated total RNA. Taq1 polymerase, a thermostable DNA polymerase, and Moloney murine leukemia virus (MMLV) reverse transcriptase share similar buffer conditions; these reactions can be performed sequentially, in a single tube, without the need for purification or changes of buffer after the synthesis of cDNA. In this way, nonspecific losses of material are minimized and the required number of cells is reduced. Cell numbers, particularly from human tissues, can be limiting; the requirement for only small amounts of unfractionated RNA makes possible the isolation and characterization of cDNAs from biological materials available in limited quantities. As a demonstration system, we report the rapid synthesis and amplification of cDNA sequences corresponding to the first exon of human immunoglobulin E (IgE). MMLV reverse transcriptase is used with specific (i.e., IgE) or generic (i.e., oligo-[dT(12-18)]) oligomers to prime first strand cDNA synthesis from unfractionated RNA isolated from a human myeloma line, U-266. The necessary primers, deoxynucleotides and Taq1 polymerase, required for second strand cDNA synthesis and the subsequent logarithmic amplification process, are then added to the reaction mixture. This technique provides a useful means of characterizing expressed and processed gene transcripts.

DNA↗
PubMed1987

Complementary DNA for the mouse homolog of the human amyloid beta protein precursor.

The human amyloid beta protein is a major component of brain amyloid found in patients with Alzheimer's disease. As an initial step to understand the biological function of its precursor protein, we have isolated cDNA for the mouse homolog of the human beta protein precursor. Comparison of the predicted amino acid sequence with that of human revealed a quite high degree of homology (96.8%), and the calculated evolutionary rate of the mRNA at amino acid substitution site was relatively low (0.1 x 10(-9)/site/year). The mRNA was abundant in brain and kidney, and also detected in other tissues at low level. These results indicated that this protein is highly conserved through mammalian evolution and may be involved in a basic biological process(es).

Amino Acid Sequence↗