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      Claudin-1 and -2: Novel Integral Membrane Proteins Localizing at Tight Junctions with No Sequence Similarity to Occludin

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          Abstract

          Occludin is the only known integral membrane protein localizing at tight junctions (TJ), but recent targeted disruption analysis of the occludin gene indicated the existence of as yet unidentified integral membrane proteins in TJ. We therefore re-examined the isolated junction fraction from chicken liver, from which occludin was first identified. Among numerous components of this fraction, only a broad silver-stained band ∼22 kD was detected with the occludin band through 4 M guanidine-HCl extraction as well as sonication followed by stepwise sucrose density gradient centrifugation. Two distinct peptide sequences were obtained from the lower and upper halves of the broad band, and similarity searches of databases allowed us to isolate two full-length cDNAs encoding related mouse 22-kD proteins consisting of 211 and 230 amino acids, respectively. Hydrophilicity analysis suggested that both bore four transmembrane domains, although they did not show any sequence similarity to occludin. Immunofluorescence and immunoelectron microscopy revealed that both proteins tagged with FLAG or GFP were targeted to and incorporated into the TJ strand itself. We designated them as “claudin-1” and “claudin-2”, respectively. Although the precise structure/function relationship of the claudins to TJ still remains elusive, these findings indicated that multiple integral membrane proteins with four putative transmembrane domains, occludin and claudins, constitute TJ strands.

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          Most cited references 50

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          Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

           U K Laemmli (1970)
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            Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.

            A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
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              Efficient selection for high-expression transfectants with a novel eukaryotic vector.

              We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.
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                Author and article information

                Affiliations
                [* ]Department of Cell Biology, []Department of Neurosurgery, and [§ ]Department of Anatomy, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan
                Journal
                J Cell Biol
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                29 June 1998
                : 141
                : 7
                : 1539-1550
                2132999
                9647647
                Categories
                Articles

                Cell biology

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