Quick-Blot: Selective mRNA or DNA Immobilization from Whole Cells

Bresser, Joel; Doering, Jeffrey L.; Gillespie, David

doi:10.1089/dna.1983.2.243

Cited by 91 publications

(27 citation statements)

References 22 publications

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“…Colonies appeared after 2 to 3 weeks in selective medium, and all of the colonies for each construct were pooled to establish a stable cell line. The average number of copies of the CAT constructs integrated into the genome of each cell line was estimated by the dot blot method of Bresser et al (5), by using 106 cells. The blots were probed with a 32P-labelled fragment containing the CAT-encoding gene.…”

Section: Methodsmentioning

confidence: 99%

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

Ouyang¹,

Bommakanti²,

Miskimins³

1993

Mol. Cell. Biol.

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A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-0-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-0-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.Transferrin receptors (TR) mediate uptake of iron into cells by binding and endocytosis of transferrin. Expression of TR is coupled to cell proliferation, and blocking of receptor function prevents cells from entering the S phase. This is most likely the result of an increased need for iron during the DNA-synthetic phase of the cell cycle. Rapidly proliferating cells have elevated levels of TR compared with quiescent, noncycling cells (13,22). When quiescent cells are activated by growth factors or mitogens, the level of cell surface TR increases. Increased expression of TR in mitogen-activated cells involves increased transcription of the TR-encoding gene (21). This is a delayed response that occurs several hours after mitogen stimulation and reaches a maximum just prior to entry into the S phase.Previously we have characterized the promoter region of the TR-encoding gene and analyzed the DNA-protein interactions within this region. We found two elements that are protected in DNase I footprinting experiments (3,26,28). These protein recognition sites are indicated by the boxes labelled A and B in Fig. 1A. Element A is GC rich and contains a consensus binding sequence for transcription factor Spl. However, we have demonstrated that multiple factors can interact within this region (26). Element B contains a sequence that is similar to the sequences recognized by both the Apl and cyclic AMP (cAMP) response element-binding protein (CREB) families of transcription factors. Microinjection of oligonucleotides that span both elements A and B was able to block serum induction of DNA synthesis (27). However, oligonucleotides for either site alone had no effect. These results suggest that the factors that recognize these sequences in the TR promoter have cooperative effects that are critical for the signalling pathways involved in transition to the S phase.

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Section: Methodsmentioning

confidence: 99%

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

Ouyang¹,

Bommakanti²,

Miskimins³

1993

Mol. Cell. Biol.

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“…To assess the expression of the alAT gene in small cell samples, the Quick-blot method was used as described by Bresser et al (68), according to the manufacturer's recommendations (Schleicher & Schuell). Filters were hybridized as above.…”

Section: Northern Analysismentioning

confidence: 99%

Expression of the alpha-1-antitrypsin gene in mononuclear phagocytes of normal and alpha-1-antitrypsin-deficient individuals.

Mornex¹,

Chytil-Weir²,

Martinet³

et al. 1986

J. Clin. Invest.

166

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To evaluate the contribution of mononuclear phagocytes, and particularly alveolar macrophages, to alpha-l-antitrypsin (alAT) production in normal and alAT-deficient individuals, Northern analysis with a human alAT complementary DNA was used to demonstrate that alAT messenger RNA (mRNA) can be detected in liver, blood monocytes, and alveolar macrophages.Quantification of alAT mRNA expression demonstrated that: (a) type PiMM monocytes and alveolar macrophages expressed, respectively, 200-fold and 70-fold less alAT mRNA per cell than the liver, (b) the level of expression of the alAT gene was increased during the in vitro maturation of blood monocytes; and (c) blood monocyte and alveolar macrophage levels of expression of the alAT gene were the same in PiMM and PiZZ individuals. However, the amount of newly synthesized alAT secreted by ZZ alveolar macrophages was 10 times lower than that secreted by MM alveolar macrophages. Thus, mononuclear phagocytes of PiZZ individuals express a secretory defect in alAT in a fashion similar to hepatocytes. Not only do mononuclear phagocytes provide a readily accessible cell to evaluate the regulation of alAT gene expression, but these cells may contribute to the levels of alAT present in the lower respiratory tract in the nornul and ZZ states.

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“…The supernatant was centrifuged at 50,000 X g to obtain a high speed pellet. Both pellets were treated according to Bresser et al (1983)andspottedonnitrocellulose paper. The washing procedures were similar to Bresser et al (1983), except that the air dried filter was baked for 2 h at 8O'C.…”

Section: Preparation Of Nitrocellulose Membranes With Test Dnamentioning

confidence: 99%

“…In combination with simple devices for the collection of clinical specimens (Richter et al, 1983) and protocols for rapid sample preparation (e.g. Bresser et al, 1983) nucleic acid hybridization should become acceptable for routine laboratory use and for applications in diagnostic laboratories where very high sensitivity is frequently not important.…”

Section: Introductionmentioning

confidence: 99%

Sandwich nucleic acid hybridization: A method with a universally usable labeled probe for various specific tests

Wolf¹,

Leser²,

Haus³

et al. 1986

Journal of Virological Methods

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Quick-Blot: Selective mRNA or DNA Immobilization from Whole Cells

Cited by 91 publications

References 22 publications

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

Expression of the alpha-1-antitrypsin gene in mononuclear phagocytes of normal and alpha-1-antitrypsin-deficient individuals.

Sandwich nucleic acid hybridization: A method with a universally usable labeled probe for various specific tests

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