Search results for "recombinant"

Characterization and DNA-binding properties of GRF, a novel monomeric binding orphan receptor related to GCNF and betaFTZ-F1

1999

0014-2956 (Print) Comparative Study Journal Article Research Support, Non-U.S. Gov't; A PCR approach has been used to isolate, from Bombyx mori, a cDNA encoding a novel orphan receptor (GRF) that is most closely related to Bombyx betaFTZ-F1 and to the vertebrate germ cell nuclear factor. The major GRF mRNA is detected in most tissues as an 8-kb transcript whose amount follows the circulating ecdysteroid concentration with a delay. The expression pattern of GRF is similar to that of the Bombyx homologue of the Drosophila early-late gene DHR3, and precedes that of betaFTZ-F1 in all stages and tissues examined. The GRF protein is thus likely to be required in many tissues, but in a temporally …

ADR1 and SNF1 Mediate Different Mechanisms in Transcriptional Regulation of Yeast POT1 Gene

1994

We studied the consequences of adr1 and snf1 mutations on POT1 gene expression in different growth conditions. The results obtained reveal that ADR1 and SNF1 genes affect POT1 transcription in different ways: ADR1 has a minor role in derepression in low concentration of glucose but is essential for activation in stationary phase whereas SNF1 is essential for derepression and activation, although it does not seem to be directly involved in the molecular mechanism of activation in stationary phase.

Analysis of expression of the gene encoding for the nuclear autoantigen La/SS-B using reporter gene constructs.

1998

In earlier studies mRNA isoforms encoding for the nuclear autoantigen La were identified. In an alternative La mRNA form the exon 1 was replaced with the exon 1'. Moreover, exon 1' La mRNAs were found to start at different 5'-regions. In dependence on the 5'-start the exon 1' La mRNAs encoded for up to three open reading frames upstream of the La frame, which starts in the exon 2. The exon 1' was located in the intron about 70 nts downstream of the exon 1. The exon 1' La mRNA was proposed to be the result of a promoter switch in combination with an alternative splicing mechanism. The commonly used technique to study the expression of a eucaryotic gene is to fuse a reportergene immediately d…

Interleukin-4 induces secretion of CSF for granulocytes and CSF for macrophages by peripheral blood monocytes.

1989

Abstract T cells are known to interact cooperatively with monocytes to produce Colony-Stimulating Factors (CSF), although T cell-mediated signals leading to CSF secretion by monocytes are not completely understood. We have made use of Northern blot hybridization and specific bioassays to study the effects of the T cell product interleukin-4 (IL-4) on monocyte CSF expression. The results suggest a previously unrecognized role of IL-4 as a CSF inducer since exposure of monocytes to IL-4 resulted in accumulation of transcripts for granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF). Consequently, IL-4-activated monocytes released factors in their culture supernatants biologically and antigenica…

Activation and methotrexate-mediated suppression of the TNF alpha promoter in T cells and macrophages.

1998

Role of hepatocyte nuclear factor 3γ in the expression of human CYP2C genes

2004

Hepatocyte nuclear factor 3 gamma (HNF-3 gamma) is an important transcription factor for the maintenance of specific liver functions. However, its relevance in the expression of human cytochrome P450 (CYP) genes has not yet been explored. Several HNF3 putative binding sites can be identified in human CYP2C 5'-flanking regions. Gene reporter experiments with proximal promoters revealed that HNF-3 gamma transactivated CYP2C8, CYP2C9, and CYP2C19 (25-, 4-, and 4-fold, respectively), but it did not transactivate CYP2C18. However, overexpression of HNF-3 gamma in hepatoma cells by means of a recombinant adenovirus induced CYP2C9, CYP2C18, and CYP2C19 mRNA (4.5-, 20-, and 50-fold, respectively) b…

Transfection of Primary Hepatocytes with Liver-Enriched Transcription Factors Using Adenoviral Vectors

2014

Primary cultured hepatocytes are probably the best model to study endogenous metabolic pathways, toxicity, or drug metabolism. Many of these studies require expression of ectopic genes. It would be desirable to use a method of transfection that allows dose-response studies, high efficiency of transfection, and the possibility to express several genes at the same time. Adenoviral vectors fulfill these requirements, becoming a valuable tool for primary hepatocyte transfection. Moreover, they are easy to generate and do not require a high level of biocontainment. In the present chapter, we describe the generation, cloning, amplification, and purification of an adenoviral vector capable of infe…

Immunological Methods for Analysis of Recombinant Proteins

1998

We want to introduce researchers to techniques that help to solve some problems in the work of the molecular biologist. After transformation of recombinant DNA in E. coli cells many clones are usually produced, and the same situation appears if recombinant DNA expression libraries are available. Furthermore, if appropriate monoclonal or polyclonal antibodies are available, the method of immunoscreening of colonies for direct immunological detection of translational products of cloned genes can be used. Selected clones could be chosen for further studies such as determination of their primary structure by DNA sequencing and for characterization of an appropriate expressed protein.

A facile chemoenzymatic approach: one-step syntheses of monoterpenoid indole alkaloids.

2010

Facile chemoenzymatic syntheses of cytotoxic monoterpenoid indole alkaloids with novel skeletons and multiple chiral centers are described. Synthesis of these alkaloids was achieved by a simple one-step reaction using strictosidine and 12-aza-strictosidine as the key intermediates. Strictosidines were prepared by coupling of secologanin with tryptamine and 7-aza-tryptamine, respectively, using the immobilized recombinant Rauvolfia strictosidine synthase. A detailed stereochemical analysis is presented herein. The results provide an opportunity for a chemoenzymatic approach that leads to an increased diversification of complex alkaloids with improved structures and activities.

Improved Expression of His6-Tagged Strictosidine Synthase cDNA for Chemo-Enzymatic Alkaloid Diversification

2010

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of stricto…