Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation.

An expression vector containing a cDNA complementary to 1.3 kb of the 5′ coding sequences of the fibronectin gene in the antisense orientation with respect to its promoter was introduced by electroporation into hybrids between melanoma cells and normal fibroblasts in which malignancy was suppressed. Immunofluorescence analysis of clones transfected with the antisense cDNA showed a dramatic decrease in the amount of fibronectin on the cell surface compared to that seen on the surface of the untransfected hybrid cells or of cells transfected with fibronectin cDNA in the sense orientation or with the expression vector alone. Four out of five clones transfected with the antisense cDNA were highly tumorigenic, whereas transfectants containing either the sense fibronectin construct or the expression vector alone remained non-tumorigenic. These results suggest that antisense RNA to fibronectin may be able to abrogate the suppression of malignancy imposed on the hybrid cells by the fibroblast parent.

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Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0030015 ◽

1989 ◽

Vol 3 (1) ◽

pp. 15-21 ◽

Cited By ~ 14

Author(s):

M. C. Hanks ◽

R. T. Talbot ◽

H. M. Sang

Keyword(s):

Escherichia Coli ◽

Cdna Sequence ◽

Expression Vector ◽

Total Cell ◽

Biologically Active ◽

Cell Protein ◽

Western Blots ◽

E Coli ◽

Ring Dove

ABSTRACT The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK233-2 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL). Expression of this manipulated cDNA sequence in E. coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots. The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active. R-chPRL was expressed at a level of approximately 1·5% of total cell protein.

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Myogenic differentiation induces taurine transporter in association with taurine-mediated cytoprotection in skeletal muscles

Biochemical Journal ◽

10.1042/bj20051303 ◽

2006 ◽

Vol 394 (3) ◽

pp. 699-706 ◽

Cited By ~ 20

Author(s):

Yoriko Uozumi ◽

Takashi Ito ◽

Yuki Hoshino ◽

Tomomi Mohri ◽

Makiko Maeda ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Atrophy ◽

Promoter Activity ◽

Expression Vector ◽

Myogenic Differentiation ◽

Consensus Sequence ◽

Biological Significance ◽

Muscle Differentiation ◽

C2c12 Cells ◽

Taurine Transporter

Skeletal muscle homoeostasis is maintained by a variety of cytoprotective mechanisms. Since ablation of the TauT (taurine transporter) gene results in susceptibility to exercise-induced muscle weakness in vivo, it has been suggested that TauT is essential for skeletal muscle function. However, the regulatory mechanisms of TauT expression remain to be elucidated. In the present study, we demonstrated that TauT was up-regulated during myogenesis in C2C12 cells. Treatment with bFGF (basic fibroblast growth factor), which inhibited muscle differentiation, abrogated myogenic induction of TauT. The promoter activities of TauT were up-regulated during muscle differentiation in C2C12 cells. Database analyses identified an MEF2 (myocyte enhancer binding factor 2) consensus sequence at −844 in the rat TauT gene. Truncation of the promoter region containing the MEF2 site significantly reduced the promoter activity, demonstrating the functional importance of the MEF2 site. Electrophoretic mobility-shift assays confirmed that MEF2 bound to the MEF2 consensus sequence and that DNA–protein complex levels were increased during differentiation. Promoter analyses using mutated promoter-reporter plasmids demonstrated that this site was functional. Importantly, transfection with a MyoD expression vector markedly enhanced TauT promoter activity in the (non-myogenic) 10T1/2 cells. Moreover, co-transfection with an MEF2 expression vector augmented MyoD-induced TauT promoter activity, suggesting that MEF2 is required for full activation of TauT expression. Finally, we examined the effects of taurine on myotube atrophy to clarify the biological significance of the up-regulation of TauT, and demonstrated that taurine attenuated muscle atrophy induced by dexamethasone. TauT expression is regulated under the control of the myogenic programme, and we propose that this is the mechanism for taurine-mediated resistance to muscle atrophy.

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The α2 cDNA sequence of human haptoglobin carries a bacterial promoter functional in vivo

Bioscience Reports ◽

10.1007/bf01116423 ◽

1986 ◽

Vol 6 (4) ◽

pp. 363-373 ◽

Cited By ~ 2

Author(s):

Ariane van der Straten ◽

Rosette Loriau ◽

Albert Herzog ◽

Alex Bollen

Keyword(s):

Computer Analysis ◽

Binding Sites ◽

Cdna Sequence ◽

Expression Vector ◽

Β Subunit ◽

Hybrid Protein ◽

Ribosome Binding ◽

Ribosome Binding Sites ◽

Efficient Expression

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete α2FSβ precursor protein or only for the β subunit have been placed under the control of the λPR promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the PR promoter, the complete α2FSβ constructions constitutively express a smaller polypeptide of ∼30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (α2PF and α2PS) located in the duplicated α2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hpα2 cDNA sequence, when fused upstream to the cDNA coding for α1-antitrypsin, constitutively promotes in vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against α1-antitrypsin or haptoglobin.

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Three-Dimensional Structure of Cloned T3 Connector Protein at 1.6nm Resolution

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100180161 ◽

1990 ◽

Vol 48 (1) ◽

pp. 282-283

Author(s):

José L. Carrascosa ◽

José M. Valpuesta ◽

Hisao Fujisawa

Keyword(s):

Dna Sequences ◽

Expression Vector ◽

Three Dimensional ◽

Deae Cellulose ◽

Dna Packaging ◽

Dimensional Structure ◽

Two Dimensional ◽

Freezing And Thawing ◽

Three Dimensional Structure ◽

Dimensional Reconstruction

The head to tail connector of bacteriophages plays a fundamental role in the assembly of viral heads and DNA packaging. In spite of the absence of sequence homology, the structure of connectors from different viruses (T4, Ø29, T3, P22, etc) share common morphological features, that are most clearly revealed in their three-dimensional structure. We have studied the three-dimensional reconstruction of the connector protein from phage T3 (gp 8) from tilted view of two dimensional crystals obtained from this protein after cloning and purification.DNA sequences including gene 8 from phage T3 were cloned, into Bam Hl-Eco Rl sites down stream of lambda promotor PL, in the expression vector pNT45 under the control of cI857. E R204 (pNT89) cells were incubated at 42°C for 2h, harvested and resuspended in 20 mM Tris HC1 (pH 7.4), 7mM 2 mercaptoethanol, ImM EDTA. The cells were lysed by freezing and thawing in the presence of lysozyme (lmg/ml) and ligthly sonicated. The low speed supernatant was precipitated by ammonium sulfate (60% saturated) and dissolved in the original buffer to be subjected to gel nitration through Sepharose 6B, followed by phosphocellulose colum (Pll) and DEAE cellulose colum (DE52). Purified gp8 appeared at 0.3M NaCl and formed crystals when its concentration increased above 1.5 mg/ml.

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