[1] High-efficiency cloning of full-length cDNA; Construction and screening of cDNA expression libraries for mammalian cells

Okayama, Hiroto; Kawaichi, Marisa Emiko; Brownstein, Michael J.; Lee, F; Yokota, Takashi; Arai, Ken

doi:10.1016/0076-6879(87)54067-8

Cited by 307 publications

(58 citation statements)

References 15 publications

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“…Total RNAs were extracted from adult rat cortex, cerebellum and hippocampus using guanidinium thiocyanate as in [14]. 10-/~g samples were electrophoresed through denaturing 2.2 M formaldehyde/1 °7, agarose gels and transferred to GeneScreen (NEN).…”

Section: Northern Blot Analysismentioning

confidence: 99%

Cloning and expression of a novel rat GABA_A receptor

Lolait

O’Carroll

Kusano³

et al. 1989

FEBS Letters

105

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Two full-length cDNA clones encoding ~t-and fl-subunits of a GABAA receptor have been isolated from a rat cerebral cortex eDNA library. The mature ~t-subunit protein consists of 428 amino acids with a calculated Mr of 48680. This protein is highly homologous (~ 99% amino acid identity) with the bovine brain ~q-subunit receptor [(1988) Nature 335, 76-79]. The mature rat fl-subunit receptor is a 448 amino acid polypeptide and shares ~ 80% amino acid identity with the previously characterized bovine GABAA receptor fl-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA ~t-and fl-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.

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Section: Northern Blot Analysismentioning

confidence: 99%

Cloning and expression of a novel rat GABA_A receptor

Lolait

O’Carroll

Kusano³

et al. 1989

FEBS Letters

105

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“…Sensory epithelia were carefully dissected from surrounding tissues and frozen in liquid nitrogen. Total RNA was isolated by isopycnic centrifugation through a cesium trifluoroacetate gradient (9). After enrichment for poly(A)ϩ RNA (PolyATtract, Promega), 5 g of poly(A)ϩ RNA was used as starting material for the construction of a unidirectional cDNA library (HybriZAP Two-Hybrid cDNA Cloning Kit, Stratagene).…”

Section: Methodsmentioning

confidence: 99%

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders

Heller

Sheane

Javed

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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To identify genes expressed in the vertebrate inner ear, we have established an assay that allows rapid analysis of the differential expression pattern of mRNAs derived from an auditory epithelium-specific cDNA library. We performed subtractive hybridization to create an enriched probe, which then was used to screen the cDNA library. After digoxigenin-labeled antisense cRNAs had been transcribed from hybridization-positive clones, we conducted in situ hybridization on slides bearing cryosections of late embryonic chicken heads, bodies, and cochleae. One hundred and twenty of the 196 clones analyzed encode 12 proteins whose mRNAs are specifically or highly expressed in the chicken's inner ear; the remainder encode proteins that occur more widely. We identified proteins that have been described previously as expressed in the inner ear, such as ␤-tectorin, calbindin, and type II collagen. A second group of proteins abundant in the inner ear includes five additional types of collagens. A third group, including Coch-5B2 and an ear-specific connexin, comprises proteins whose human equivalents are candidates to account for hearing disorders. This group also includes proteins expressed in two unique cell types of the inner ear, homogene cells and cells of the tegmentum vasculosum.Developmental analysis of the inner ear is hindered by the paucity of molecular markers for specific cell types. At present, most cells in the cochlea and vestibular organs cannot be unambiguously identified before they display their characteristic mature forms. The cloning of cDNAs that encode proteins specific to hair cells, supporting cells, and other cells of the inner ear is, in turn, retarded by the limited numbers of these cells in an animal. This disadvantage potentially can be circumvented through enrichment of specific cDNAs by subtractive hybridization (1-3) and library normalization (4). That only a few inner ear-specific cDNAs have been obtained from libraries constructed by this approach (2, 3), however, suggests that the efficient cloning of cDNAs specific to sensoryepithelial cell types requires libraries constructed from more abundant sources than whole cochleae. To address this problem, we constructed a cDNA library from sensory epithelia of the chicken's cochlea and developed an enriched probe with which to identify cDNAs encoding proteins expressed by specific cell types of the inner ear.The identification of proteins specific to the internal ear is also potentially useful in the study of heritable human deafness, which afflicts about one individual in a thousand. Several proteins that have been identified as markers for specific aural cell types recently have been advanced as candidate genes for human hearing disorders (for reviews see refs. 5-8). Some of the genes identified by the strategy described in this paper are likely to have human equivalents that cause hearing disorders when mutated. MATERIALS AND METHODSLibrary Construction and Screening. Basilar papillae from 250 late-embryonic (E, embryonic day) (8% E14...

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“…Total RNA was extracted from cultured cells by the guanidium/ caesium trifluoroacetate method (Okayama et al, 1987) with a RNA extraction kit (Pharmacia). RNA samples (10,lg) were fractionated by 1.2 %-agarose-gel electrophoresis and transferred to nylon filters.…”

Section: Northern Blotfingmentioning

confidence: 99%

Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress

Petronini

Angelis

Borghetti

et al. 1993

Biochemical Journal

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Induced expression of the HSP70 gene in 3T3 and SV-3T3 cells was monitored by measurements of the synthesis of HSP70 and of the cellular contents of both HSP70 and its mRNA. The presence of betaine (N-trimethylglycine) at concentrations of 2.5-25 mM decreased the induction of HSP70 gene expression caused by incubation of 3T3 and SV-3T3 cells in hypertonic (0.5 osM) medium. This effect was accompanied by an enhancement of SV-3T3 cell adaptation, assayed by colony formation, to the hyperosmotic conditions. In contrast, the presence of betaine did not affect HSP70 gene expression induced in these

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[1] High-efficiency cloning of full-length cDNA; Construction and screening of cDNA expression libraries for mammalian cells

Cited by 307 publications

References 15 publications

Cloning and expression of a novel rat GABA_A receptor

Cloning and expression of a novel rat GABA_A receptor

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders

Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress

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[1] High-efficiency cloning of full-length cDNA; Construction and screening of cDNA expression libraries for mammalian cells

Cited by 307 publications

References 15 publications

Cloning and expression of a novel rat GABAA receptor

Cloning and expression of a novel rat GABAA receptor

Molecular markers for cell types of the inner ear and candidate genes for hearing disorders

Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress

Contact Info

Product

Resources

About

Cloning and expression of a novel rat GABA_A receptor

Cloning and expression of a novel rat GABA_A receptor