Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans

Xiangyi Lu, Robert E. Grossi, Srilata Bagchi, Charles S. Rubin

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by non-covalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pi values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (̃6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5′-flanking region has no TATA box and contains preferred and minor transcription start sites.

Original languageEnglish (US)
Pages (from-to)3293-3303
Number of pages11
JournalJournal of Biological Chemistry
Volume265
Issue number6
StatePublished - Feb 25 1990

Fingerprint

Cloning
Caenorhabditis elegans
Cyclic AMP-Dependent Protein Kinases
Organism Cloning
Genes
Hatches
Gene Expression
Amino Acids
TATA Box
5' Flanking Region
Transcription Initiation Site
Detergents
Introns
Neurons
Urea
Epitopes
Exons
Carrier Proteins
Complementary DNA
Tissue

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. / Lu, Xiangyi; Grossi, Robert E.; Bagchi, Srilata; Rubin, Charles S.

In: Journal of Biological Chemistry, Vol. 265, No. 6, 25.02.1990, p. 3293-3303.

Research output: Contribution to journalArticle

Lu, Xiangyi ; Grossi, Robert E. ; Bagchi, Srilata ; Rubin, Charles S. / Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 6. pp. 3293-3303.
@article{357b3d1379b94fefa6a9fa26d4c33582,
title = "Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans",
abstract = "The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60{\%} of the C. elegans cAMP-binding protein is tightly associated with particulate structures by non-covalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pi values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69{\%} identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (̃6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5′-flanking region has no TATA box and contains preferred and minor transcription start sites.",
author = "Xiangyi Lu and Grossi, {Robert E.} and Srilata Bagchi and Rubin, {Charles S.}",
year = "1990",
month = "2",
day = "25",
language = "English (US)",
volume = "265",
pages = "3293--3303",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "6",

}

TY - JOUR

T1 - Cloning, structure, and expression of the gene for a novel regulatory subunit of cAMP-dependent protein kinase in Caenorhabditis elegans

AU - Lu, Xiangyi

AU - Grossi, Robert E.

AU - Bagchi, Srilata

AU - Rubin, Charles S.

PY - 1990/2/25

Y1 - 1990/2/25

N2 - The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by non-covalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pi values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (̃6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5′-flanking region has no TATA box and contains preferred and minor transcription start sites.

AB - The nematode Caenorhabditis elegans (C. elegans) expresses the regulatory subunit (R) of cAMP-dependent protein kinase at a level similar to the levels determined for R subunits in mammalian tissues. Approximately 60% of the C. elegans cAMP-binding protein is tightly associated with particulate structures by non-covalent interactions. Ionic detergents or 7 M urea solubilize particulate R. Solubilized and cytosolic R subunits have apparent Mr values of 52,000 and pi values of 5.5. cDNA and genomic DNA encoding a unique C. elegans R subunit were cloned and sequenced. The derived amino acid sequence contains 375 residues; carboxyl-terminal residues 145-375 are 69% identical with mammalian RI. However, residues 44-145 are markedly divergent from the corresponding regions of all other R sequences. This region might provide sufficient structural diversity to adapt a single R subunit for multiple functional roles in C. elegans. Antibodies directed against two epitopes in the deduced amino acid sequence of C. elegans R avidly bound nematode cytosolic and particulate R subunits on Western blots and precipitated dissociated R subunits and R2C2 complexes from solution. Immunofluorescence analysis revealed that the tip of the head, which contains chemosensory and mechanosensory neurons, and the pharyngeal nerve ring were enriched in R. The R subunit concentration is low during early embryogenesis in C. elegans. A sharp increase (̃6-fold) in R content begins several hours before the nematodes hatch and peaks during the first larval stage. Developmental regulation of R expression occurs at translational and/or post-translational levels. The 8-kilobase pair C. elegans R gene is divided into 8 exons by introns ranging from 46 to 4300 base pairs. The 5′-flanking region has no TATA box and contains preferred and minor transcription start sites.

UR - http://www.scopus.com/inward/record.url?scp=0025262242&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0025262242&partnerID=8YFLogxK

M3 - Article

C2 - 2303451

AN - SCOPUS:0025262242

VL - 265

SP - 3293

EP - 3303

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 6

ER -