Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: Member of an ancient channel family

Gregory M. Preston, Peter C Agre

Research output: Contribution to journalArticle

Abstract

CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5′ untranslated nucleotide sequence, an 807-bp open reading frame, and ≈ 2 kilobases of 3′ untranslated sequence containing a polyadenylylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DN A sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

Original languageEnglish (US)
Pages (from-to)11110-11114
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume88
Issue number24
StatePublished - 1991

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Aquaporin 1
Erythrocyte Membrane
Membrane Proteins
Complementary DNA
Ion Channels
Polymerase Chain Reaction
Amino Acid Sequence
Bone Marrow
Crystallins
DNA Primers
Protein Sequence Analysis
Protein C
Gene Library
Glycosylation
Immunoblotting
Base Pairing
Open Reading Frames
Organism Cloning
Erythrocytes
Databases

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: Member of an ancient channel family",
abstract = "CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5′ untranslated nucleotide sequence, an 807-bp open reading frame, and ≈ 2 kilobases of 3′ untranslated sequence containing a polyadenylylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DN A sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.",
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T1 - Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons

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AB - CHIP28 is a 28-kDa integral membrane protein with similarities to membrane channels and is found in erythrocytes and renal tubules. A cDNA for CHIP28 was isolated from human fetal liver cDNA template by a three-step polymerase chain reaction (PCR) cloning strategy, starting with degenerate oligonucleotide primers corresponding to the N-terminal amino acid sequence determined from purified CHIP28 protein. Using the third-step PCR product as a probe, we isolated a recombinant from a human bone marrow cDNA library. The combined sequence of the PCR products and bone marrow cDNA contains 38 base pairs of 5′ untranslated nucleotide sequence, an 807-bp open reading frame, and ≈ 2 kilobases of 3′ untranslated sequence containing a polyadenylylation signal. This corresponds to the 3.1-kilobase transcript identified by RNA blot-hybridization analysis. Authenticity of the deduced amino acid sequence of the CHIP28 protein C terminus was confirmed by expression and immunoblotting. Analysis of the deduced amino acid sequence suggests that CHIP28 protein contains six bilayer-spanning domains, two exofacial potential N-glycosylation sites, and intracellular N and C termini. Search of the DN A sequence data base revealed a strong homology with the major intrinsic protein of bovine lens, which is the prototype of an ancient but recently recognized family of membrane channels. These proteins are believed to form channels permeable to water and possibly other small molecules. CHIP28 shares homology with all known members of this channel family, and it is speculated that CHIP28 has a similar function.

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