One-Lane Chemical Sequencing of 3′-Fluorescent-Labeled DNA

Ferraboli, Sergio; Negri, Rodolfo; Mauro, Ernesto Di; Barlati, Sergio

doi:10.1006/abio.1993.1539

Cited by 17 publications

(17 citation statements)

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“…Nucleotide sequence of PCR products from HEL cells was determined with the aid of the automatic ABI 377 sequencer (Applied Biosystems, Foster City, CA, USA). 30 In both cases 100% homology was observed with the human kidney MCR gene and with the human lung or kidney ENaC gene (data not shown). These provide unequivocal demonstration for the presence of the MCR and of the ENaC (a member of the ASSC superfamily) in the HEL cell line.…”

Section: Data Inmentioning

confidence: 95%

“…The mixture was heated to 95°C for 5 min at the end of the reaction. [28][29][30] Polymerase chain reaction (PCR) was performed with the aid of the primers 5′-AGGCTACCACAGTCTCCCTG-3′ (sense) and 5′-GCAGTGTAAAATCTCCAGTC-3′ (antisense) for the MCR, synthesized by Genosys (UK). The ASSC was similarly analyzed with the aid of the primers 5′-CTGCCTTTATGGAT-GATGGT-3′ (sense) and 5′-GTTCAGCTCGAAGAAGA-3′ (antisense).…”

Section: Analysis By Pcrmentioning

confidence: 99%

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Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

Mirshahi

Golestaneh

et al. 2000

Leukemia

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We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the ␣ subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5′-AGGCTAC-CACAGTCTCCCTG-3′ and 5′-GCAGTGTAAAATCTCCAGTC-3′ (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5′-CTGCCTTTATG GATGATGGT-3′ (sense) and 5′-GTTCAGCTCGAAGAAGA-3′ (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells. Leukemia (2000) 14, 1097-1104.

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Section: Data Inmentioning

confidence: 95%

Section: Analysis By Pcrmentioning

confidence: 99%

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

Mirshahi

Golestaneh

et al. 2000

Leukemia

View full text Add to dashboard Cite

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“…Total RNA was dissolved in 30 ml RNase free water and quantified by ultraviolet (UV) absorption. The ratio OD 260 nm /OD 280 nm was 1.6 -2.0 in all cases, and the RNA yield varied from 20 g. RNA samples were kept frozen at Ϫ80°C until use (Chomczinsky and Sacchi, 1987;Mirshahi et al, 1998;Ferraboli et al, 1993).…”

Section: Pcr Analysismentioning

confidence: 99%

Epithelial sodium channel and the mineralocorticoid receptor in cultured rat M�ller glial cells

Golestaneh

Kozak

Klein³

et al. 2001

Glia

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“…The nucleotide sequence of the PCR products from keratocytes was determined with the aid of four different chain terminators in a single electrophoretic run using the automatic ABI 377 sequencer [24]. The sequence of reverse and forward strands was aligned, the undetermined N bases were corrected and finally compared with all of the nucleotide sequences for the ENaC and the MCR found in different databases using the PC/Gene software.…”

Section: )-Gttcagctcgaagaaga-3) (Antisense) Data Inmentioning

confidence: 99%

“…The 3)-end-labeled DNA was separated from the free nucleotide triphosphates by gel filtration using C-100 spin columns (Clontech) and vacuum dried. The purified 3)-end-labeled fragments were thereafter degraded by 10% (v/v) aqueous piperidine (30 min at 95°C), followed by 80% (v/v) formamide (10 min at 110°C) and finally quantitated in an ABI 377 automatic sequencer [24].…”

Section: Sequence Analysismentioning

confidence: 99%

Mineralocorticoid Hormone Signaling Regulates the ‘Epithelial Sodium Channel’ in Fibroblasts from Human Cornea

Mirshahi

Golestaneh

et al. 2000

Ophthalmic Res

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We investigated the regulation of sodium absorption by steroid hormones in embryologically diverse cells from the human eye. A cell extract from human corneal fibroblasts was positive for both the epithelial sodium channel (ENaC) and the mineralocorticoid receptor (MCR) as 82- to 85-kD and 102-kD bands, respectively, by the Western blot technique. In fluorescent, confocal and electron microscopy, the MCR was revealed as a nucleocytoplasmic protein, whereas the ENaC was almost exclusively membrane bound; both appeared aligned along actin filaments of corneal keratocytes, and both were widely colocalized in various cell types of human cornea in situ. Following reverse transcription and amplification of total RNA isolated from corneal fibroblasts, the ENaC and MCR genes in the PCR product were evident as predicted bands of 520 and 843 bp, respectively, whose sequence exhibited 100% identity with those from known human sources. The multiplication of corneal fibroblasts was influenced by both the MCR-specific antagonist RU 26752 and the natural hormone aldosterone, and these steroids also stimulated protein phosphorylation. In quantitative PCR, both the basal and aldosterone-induced levels of ENaC were diminished by the MCR-specific antagonist ZK 91587. Consequently, the ocular sodium channel appears to be regulated by steroid signalling in cells of diverse embryological origins, contrary to the existing notions where (a) this process would be limited exclusively to the epithelial cells and (b) ocular sodium transport would be regulated via the Na⁺-K⁺-ATPase in the basolateral membrane.

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One-Lane Chemical Sequencing of 3′-Fluorescent-Labeled DNA

Cited by 17 publications

References 0 publications

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines

Epithelial sodium channel and the mineralocorticoid receptor in cultured rat M�ller glial cells

Mineralocorticoid Hormone Signaling Regulates the ‘Epithelial Sodium Channel’ in Fibroblasts from Human Cornea

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