摘要:

ABSTRACTThe interaction of the estradiol receptor with cloned DNA fragments from the prolactin gene was investigated using a competitive binding assay. A DNA fragment from the 5′-flanking region of the rat prolactin gene was able to bind the estradiol receptor selectively. DNA fragments representing most of the remaining 10 kb of the prolactin gene showed little or no selective binding to the estradiol receptor. The fragment from the 5′-flanking region which selectively binds the receptor is located between 1.2 and 2.0 kb upstream from the transcription initiation site rather than in a more proximal position as has been observed for the interaction of several other steroid hormone receptors with specific genes. Nucleotide sequence analysis demonstrated that this region of DNA contains two large segments of alternating purine-pyrimidine sequence. One of the alternating purine-pyrimidine regions is very large, containing more than 160 nucleotides of almost perfect poly(dG-dT). Further studies will be required to determine if the receptor interacts specifically with these interesting sequences. However, the ability of the estradiol receptor to bind to an upstream 5′-flanking region of the prolactin gene may be part of the mechanism which allows the receptor to stimulate the transcription of this gene select

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.1

年代:1985

数据来源: MAL

摘要:

ABSTRACTMessenger RNAs (mRNAs) were prepared from MCF-7 breast cancer cells grown in the presence of estradiol. Complementary DNAs (cDNAs) were inserted into pBR322 plasmid and a library of 4400 recombinant bacterial clones was prepared. The clones were screened byin situdifferential hybridization with cDNAs prepared from RNAs of MCF-7 cells grown either in the presence or absence of estradiol. Several estrogen-induced or estrogen-repressed clones were identified. One of them corresponded to a relatively frequent mRNA (0.8% of recombinant plasmids) of 650 nucleotides. The concentration of this mRNA was increased by estradiol (half maximal induction ~0.05 nM) but not by progesterone, dexamethasone, or dihydrotestosterone. Tamoxifen inhibited the effect of estradiol but was devoid of any agonistic activity when administered separately. This messenger was present in biopsies of breast cancer, but not in endometrium or liver.The cloned cDNA was sequenced. An open reading frame was found corresponding to a protein of less than 100 amino acids. A search of data banks showed no identity or marked similarity to previously published DNA or protein sequences, particularly to those of growth factors evoked by some characteristics of the coded polypeptide.The cloned cDNA probe was used to screen a library of Charon 4A phage containing human genomic fragments. Screening of 300,000 phages yielded two different recombinants hybridizing to the cDNA. Southern blot experiments using DNA from recombinant phage, MCF-7 cells, and placenta showed the presence of a unique gene exhibiting a similar restriction pattern in DNAs from malignant and nonmalignant tissues.

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.11

年代:1985

数据来源: MAL

摘要:

ABSTRACTA variety of recombinant DNA molecules were constructed in which an avian retroviral long terminal repeat (LTR) was ligated to the bovine growth hormone (bGH) gene. The retroviral LTR was derived from a plasmid clone of a Schmidt Ruppin B strain of Rous sarcoma virus while the bGH gene was subcloned from a λ bacteriophage genomic library. Using a transient eukaryotic expression assay system, recombinant plasmid constructs were screened for their ability to direct expression and secretion of bGH. One such plasmid DNA construct, termed pBGH-4, was found to be active in the production of bGH. Stable mouse fibroblast cell lines were generated containing pBGH-4 DNA integrated into the mouse cell genome. Many of these mouse cell lines express and secrete bGH. One line, L-Pdλ-BGH4-13, was found to secrete bGH at a rate of 75 μg per 5 × 106cells per 24 hr. Bovine growth hormone derived from this cell line is biologically act

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.23

年代:1985

数据来源: MAL

摘要:

ABSTRACTThe construction of a plasmid, pl66.9, for the controlled synthesis of the A α-chain of human fibrinogen inEscherichia coliis described. The plasmid combines thetacpromoter, constructed for controlled high-level peptide expression, with the promoter and signal peptide codons from anE. coliplasmid β-laclamase gene and a cDNA of the A α-chain of human fibrinogen. Thetacpromoter is repressed inlaclQstrains ofE. coliand induced by isopropylthio-β-D-galactoside (IPTG). Protein blot analysis of lysates of cells carrying pl66.9 demonstrates the IPTG-dependent synthesis of polypeptides which cross react with antisera to the A α-chain of human fibrinogen. The largest and predominant species corresponds to an apparent molecular weight of 63,000. When the cell growth media or cell lysates are treated with thrombin, the enzyme which normally releases fibrinopeptide A (FPA) from the A α-chain, FPA-like peptides are detectable by radioimmunoassay with antiserum prepared against human FPA. Thrombin-treated cell growth media prepared 4 hr after IPTG induction contained 340 ng/ml of FPA-like material; using a mass ratio of 40 for FPA to A α, this indicates that the A α-peptide concentration in the culture media is

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.33

年代:1985

数据来源: MAL

摘要:

ABSTRACTA new method for preparing small quantities of λ DNA from phage lysates has been developed. The protocol is based on the concentration and purification of bacteriophage particles from crude lysates using small DEAE-cellulose columns. This Chromatographie step gives an absolute separation of the λ DNA from the cellular nucleic acids and a 20-fold enrichment relative to the major soluble proteins in crude lysates, while effecting a 10-fold concentration of the phage. Final deproteinization and concentration of the λ DNA is achieved by conventional precipitation steps. The λ DNA produced by this method is shown to be nondegraded, biologically active, and an excellent substrate for restriction enzymes. A detailed protocol is provided for starting with individual plaques and using the method to obtain purified DNA from large numbers of λ cl

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.39

年代:1985

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.52

年代:1985

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.55

年代:1985

数据来源: MAL

ISSN:1044-5498    

DOI:10.1089/dna.1985.4.68

年代:1985

数据来源: MAL