Abstract
By cloning the cDNA coding for the membrane associated actin-binding protein p24, we identified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. Using a cDNA probe corresponding to this motif, we isolated cDNA clones coding for a protein of the annexin family. On the basis of a long NH2-terminal sequence encompassing the Gly/Tyr/Pro/Gln motifs, the Dictyostelium annexin was identified as a homolog of vertebrate annexin VII (synexin). The mRNA coding for the Dictyostelium annexin VII has a size of 1.6 kilobases and is present during all developmental stages. Annexin VII is coded for by a single gene in Dictyostelium. A mutant deficient in annexin VII was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VII cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon. Using this approach, homologous recombination in the annexin VII gene led to an expression of the viral epitope under the control of the endogenous annexin VII promoter. Lack of annexin VII is not a lethal event for D. discoideum, and the cells are able to undergo development on agar plates.
Highlights
By cloning the cDNA coding for the membrane associated actin-binding protein p24, weidentified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum
Following the of genes in Dictyosteliumdiscoideum contains a repeated nomenclature suggested by Crumpton and Dedman (1990), sequencemoticfoding foramino acid stretches of the we will refer in this report to synexin as annexiVnII
Annexin V, named endonexin 11, lipocortin encoding part of one of these genes as a probe, we isolated a V, or 35K calelectrin and described as an inhibitor of blood cDNA coding for the annexin VII-homolog of D. discoideum. coagulation hasbeen crystallized inthepresence of Ca2+ In analogy to human annexin V for which a detailed three(Huber et al, 1990a, 1990b).The x-ray structuroef the protein dimensional structure has been obtained (Huetbaelr., 1990a), reveals a symmetrical arrangement of the four repeats, each the core region of Dictyostelium annexin VI1 contains three one composed of five short a-helices
Summary
From the Max-Planck-Institute for BiochemistryA, m Klopferspitz 18a, 8033 Martinsried, Federal Republic of Germany. By cloning the cDNA coding for the membrane associated actin-binding protein p24, weidentified a repetitive sequence motif consisting of the amino acids Gly, Tyr, Pro, Gln which is characteristic for a gene family in Dictyostelium discoideum. A mutant deficient in annexin VI1 was isolated using a vector which carried the amino-terminal third of the Dictyostelium annexin VI1 cDNA followed by a viral epitope specific for a monoclonal antibody and a stop codon Using this approach,homologousrecombination in the annexin VI1 gene led to an expression of the viral epitope under the control of the endogenous annexin VI1 promoter.Lack ofannexin VI1 is not a lethal event for D. discoideum, and the cellsare able to (Iwasacki et al, 1987) and the formation of membrane-cytoskeletal contacts.
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