High-Efficiency Cloning of Full-Length cDNA; Construction and Screening of cDNA Expression Libraries for Mammalian Cells

Okayama, Hiroshi; Kawaichi, Marisa Emiko; Brownstein, Michael J.; Lee, F.; Yokota, Toshihiko; Arai, Kohei

doi:10.1016/b978-0-12-765560-4.50018-6

Cited by 59 publications

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“…One of the amplified sequences was the starting point for isolating the cDNA of jShaw1. Total RNA was isolated from P. penicillatus medusae collected at the Bamfield Marine Sciences Center using the RNAeasy Midiprep kit (Qiagen, Mississauga, ON, Canada) or by density gradient centrifugation using cesium trifluoroacetate (Okayama et al, 1987). The majority of the bell, which is rich in mesoglea but has few cells, was dissected away, approximately 2mm above the marginal ring.…”

Section: Materials and Methods Jshaw1 Cloningmentioning

confidence: 99%

jShaw1, a low-threshold, fast-activating Kv3 from the hydrozoan jellyfish Polyorchis penicillatus

Sand

Atherton

Spencer

et al. 2011

Journal of Experimental Biology

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Section: Materials and Methods Jshaw1 Cloningmentioning

confidence: 99%

jShaw1, a low-threshold, fast-activating Kv3 from the hydrozoan jellyfish Polyorchis penicillatus

Sand

Atherton

Spencer

et al. 2011

Journal of Experimental Biology

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“…Total RNA was extracted from myometrial tissues by the cesium-trifluoroacetate gradient method (Okayama et al 1987) and poly(A) + RNA was purified using oligo(dT) columns. Fifteen micrograms total RNA (G blotting) or 10 µg poly(A) + RNA ( 2 -AR blotting) were denatured by formaldehyde, fractionated by 1% agarose gel electrophoresis and transferred to GeneScreen-Plus membranes (Dupont-New England Nuclear) by overnight capillary blotting.…”

Section: Rna Preparation and Northern Blottingmentioning

confidence: 99%

In vivo stimulation of the beta(2)-adrenergic pathway increases expression of the Gi proteins and the alpha(2)A-adrenergic receptor genes in the pregnant rat myometrium

Lecrivain

Mhaouty-Kodja²,

Cohen‐Tannoudji³

et al. 1998

Journal of Endocrinology

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Cross-regulations between G s and G i mediated pathways controlling the adenylyl cyclase activity have been clearly demonstrated in vitro. To elucidate whether activation of the -adrenergic pathway in the pregnant myometrium might affect G i proteins and 2 -adrenergic receptors (ARs), we treated late pregnant rats from day 18 to day 21 with twice-daily administration of isoproterenol (8 mg/ kg). This treatment increased myometrial cAMP levels and led after 76 h to a significant and maximal rise in the immunoreactive amount of myometrial G i 2 and G i 3 proteins (1·4-and 1·7-fold respectively) associated with a parallel increase of the steady-state levels of both G i 2 and G i 3 mRNA (1·6-and 1·9-fold respectively). Propranolol antagonized this response indicating the implication of the -adrenergic pathway. Nuclear run-on assays demonstrated that isoproterenol enhanced respectively by 1·3-and 1·2-fold the transcription rate of the G i 2 and G i 3 genes. Quantification of myometrial 2 -ARs by [3 H]rauwolscine binding revealed that the total number of receptors was also increased at 76 h by 1·7-fold when compared with controls, with no change in the affinity of the 2 -ARs for the ligand. This effect was antagonized by propranolol. Quantification of both 2A -and 2B -subtypes by Northern blotting analysis demonstrated that this elevation was due to a selective increase of the 2A -subtype mRNAs. The present results indicate that in vivo stimulation of the -adrenergic pathway by isoproterenol increases both G i 2 /G i 3 and 2A -AR expression in the pregnant rat myometrium. The possible contribution of such a mechanism in pregnancy-related changes of both entities is discussed.

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Section: Methodsmentioning

confidence: 99%

“…Total cellular RNA was prepared from XhoC cells as described by Okayama et al (37), and poly(A) ϩ RNA was reverse transcribed by using an oligo(dT) primer with an XhoI site. The RNA strand of the mRNA-cDNA hybrid was replaced with the corresponding DNA strand by using Escherichia coli RNase H, E. coli DNA polymerase I, and E. coli DNA ligase (37), and the cDNA was ligated to EcoRI linkers after both termini were blunt ended. After cleavage with EcoRI and XhoI, the cDNA (100 ng) was ligated to 100 ng of EcoRI/SalI-digested yeast expression plasmid pGAD424 (Clontech) by incubation at 16°C for 48 h. The ligated cDNA was extracted with phenol-chloroform, ultrafiltered by using micron 10 (Millipore), and electrotransformed into E. coli DH10B with an E. coli Pulser (Bio-Rad) to generate the pGAD-XhoC cDNA library (Fig.…”

Section: Methodsmentioning

confidence: 99%

Cloning and Characterization of a GC-Box Binding Protein, G10BP-1, Responsible for Repression of the Rat Fibronectin Gene

Oda

Shirasuna

Suzuki

et al. 1998

Molecular and Cellular Biology

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Fibronectin (FN) is an extracellular matrix protein that connects the extracellular matrix to intracellular cortical actin filaments through binding to its cell surface receptor, ␣5␤1, a member of the integrin superfamily. The expression level of FN is reduced in most tumor cells, facilitating their anchorage-independent growth by still unclarified mechanisms. The cDNA clone encoding G-rich sequence binding protein G10BP-1, which is responsible for repression of the rat FN gene, was isolated by using a yeast one-hybrid screen with the G10 stretch inserted upstream of the HIS3 and lacZ gene minimal promoters. G10BP-1 comprises 385 amino acids and contains two basic regions and a putative zipper structure. It has the same specificity of binding to three G-rich sequences in the FN promoter and the same size as the G10BP previously identified in adenovirus E1A-and E1B-transformed rat cells. Expression of G10BP-1 is cell cycle regulated; the level was almost undetectable in quiescent rat 3Y1 cells but increased steeply after growth stimulation by serum, reaching a maximum in late G 1 . Expression of FN mRNA is inversely correlated with G10BP-1 expression, and the level decreased steeply during G 1 -to-S progression. This down regulation was strictly dependent on the downstream GC box (GCd), and base substitutions within GCd abolished the sensitivity of the promoter to G10BP-1. In contrast, the level of Sp1, which competes with G10BP for binding to the G-rich sequences, was constant throughout the cell cycle, suggesting that the concentration of G10BP-1 relative to that of Sp1 determines the expression level of the FN gene. Preparation of glutathione S-transferase pulldowns of native proteins from the cell extracts containing exogenously or endogenously expressed G10BP-1, followed by Western blot analysis, showed that G10BP-1 forms homodimers through its basic-zipper structure.Fibronectin (FN) is a large glycoprotein of the extracellular matrix that binds to its cell surface receptor, ␣5␤1, a member of the integrin superfamily, as a dimer (2,15,39). FN consists of multiple rodlike domains, and each domain binds to a component of the extracellular matrix such as collagen and heparin and to its receptor (16). The cytoplasmic domain of the receptor binds to bundles of actin filaments known as stress fibers indirectly through binding to the attachment proteins (14). The binding of FN to its receptor at so-called focal contact sites therefore connects the cytoskeleton with the extracellular matrix (3, 17, 21, 39), governing cell shape, adhesion, and movement. FN is also involved in the regulation of cell proliferation and differentiation through organization of the extracellular matrix and cytoskeleton (7, 15).The expression level of FN is closely linked to the growth potential of cells. The level increases when cells cease growing, and senescent cells inevitably express FN at high levels (11,28,33). On the contrary, FN expression is greatly inhibited upon neoplastic transformation (10,20,35), and most tumor cells express ...

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High-Efficiency Cloning of Full-Length cDNA; Construction and Screening of cDNA Expression Libraries for Mammalian Cells

Cited by 59 publications

References 0 publications

jShaw1, a low-threshold, fast-activating Kv3 from the hydrozoan jellyfish Polyorchis penicillatus

jShaw1, a low-threshold, fast-activating Kv3 from the hydrozoan jellyfish Polyorchis penicillatus

In vivo stimulation of the beta(2)-adrenergic pathway increases expression of the Gi proteins and the alpha(2)A-adrenergic receptor genes in the pregnant rat myometrium

Cloning and Characterization of a GC-Box Binding Protein, G10BP-1, Responsible for Repression of the Rat Fibronectin Gene

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