Temporal regulation of the Dictyostelium glycogen phosphorylase 2 gene

Charles L. Rutherford, Ornella Selmin, Sandra Peters-Weigel

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

The product of the glycogen phosphorylase-2 gene in Dictyostelium functions to provide the glucose units that are used to construct the structural components of the terminal stage of development. In this report, we link a 1233 bp upstream gp2 fragment to a luciferase reporter gene in order to study the sequences that are involved in the temporal expression of the gene. Various deletions of the promoter-luciferase fusion were then transformed into Dictyostelium cells. All deletion constructs, from -1216 to -486 nucleotides from the translational start codon, showed the same temporal pattern of expression as the authentic gp2 gene, as well as similar luciferase activities. Removal of an additional 37 nucleotides resulted in nearly 100-fold decrease in activity, yet retained the normal temporal expression of luciferase. Analysis of DNA binding proteins with the gel shift assay revealed a stage-dependent pattern of proteins that bound to the gp2 promoter. A similar pattern of temporal expression of the binding proteins was observed with either the full-length probe or with oligonucleotide probes that contained sequences that were identified as putative regulatory sites. Likewise, the full-length and oligonucleotide probes demonstrated identical binding patterns during several steps of purification of the DNA binding proteins. SDS-PAGE and Southwestern blot analysis of a DNA-affinity purified fraction, identified a 23 kDa peptide as the binding protein.

Original languageEnglish (US)
Pages (from-to)111-125
Number of pages15
JournalBBA - Gene Structure and Expression
Volume1351
Issue number1-2
DOIs
StatePublished - Mar 20 1997
Externally publishedYes

Fingerprint

Glycogen Phosphorylase
Dictyostelium
Luciferases
Genes
Oligonucleotide Probes
DNA-Binding Proteins
Carrier Proteins
Southwestern Blotting
Nucleotides
Gene Order
Initiator Codon
Reporter Genes
Purification
Polyacrylamide Gel Electrophoresis
Assays
Fusion reactions
Gels
Gene Expression
Glucose
Peptides

Keywords

  • Development
  • Dictyostelium
  • Glycogen phosphorylase
  • Transcription factor

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology
  • Biophysics

Cite this

Temporal regulation of the Dictyostelium glycogen phosphorylase 2 gene. / Rutherford, Charles L.; Selmin, Ornella; Peters-Weigel, Sandra.

In: BBA - Gene Structure and Expression, Vol. 1351, No. 1-2, 20.03.1997, p. 111-125.

Research output: Contribution to journalArticle

Rutherford, Charles L. ; Selmin, Ornella ; Peters-Weigel, Sandra. / Temporal regulation of the Dictyostelium glycogen phosphorylase 2 gene. In: BBA - Gene Structure and Expression. 1997 ; Vol. 1351, No. 1-2. pp. 111-125.
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