Abstract
Transcription of the three yolk protein genes of Drosophila melanogaster is under strict developmental regulation. Understanding the mechanism of this regulation requires examination of the DNA sequences and protein factors necessary for normal transcriptional control. We have identified a sequence-specific DNA-binding protein, yolk protein factor 1 (YPF1), that has high affinity for a 31-bp sequence in the yolk protein 1 gene. This sequence is within the translated region at a site beginning 148 bp downstream of the transcription initiation site. DNA deletion and substitution analysis demonstrated that this sequence is necessary and sufficient for DNA/YPF1 interaction in vitro and is necessary for normal steady state levels of yolk protein 1 gene RNA in vivo. YPF1 binding activity was detected in extracts from late stage egg chambers and early stage embryos but not from tissues that express yolk protein genes.
Highlights
The threeyolk protein genes’ of Drosophila melanogaster form a small multigene family and are coordinately regulated in a sex, developmental stage, and tissuespecific manner
1,2, and 3; YPF1, yolk protein factor I; PMSF, phenylmethanesulfonyl fluoride; EGTA, [ethylenebis(oxyethylenenitri1o)ltetraaceticacid bp, base pair
In this paper we describe the identification of YPF1, the localization of its binding site, and the demonstration that thisbinding site is required for normal steady state levels of ypl RNA
Summary
Homogenized in 2:l volume (buffer/material) in the homogenization Assay-The gel binding assay has been used to examine a buffer plus 400 mM KCl, 100 pg/ml PMSF, 1 pg/ml leupeptin and 1 pg/ml pepstatin A. We have modified the assay conditions to allow detection of specific protein/DNA interactions in crude extracts of Drosophila cells and tissues. DNA concentrations are given in terms of moles of polynucleotide conclude that a sequence-specific DNA-binding protein is present in the extract This protein, YPFl, interacts with a 340-bp fragment which spans the yptlranscription start site. (Fig. 4A).This DNA construct has all the sequence elements necessary for the normal tissue and developmental expression of ypl and yp2 It contains bacteriophage M13 fragments in the transcribed portions of each gene to distinguish the introduced copy of the gene from the endogenous copy[8].This DNA was clonedinto a P-element vector and transformed into the germlineof Drosophila.Three independent lines of transformed flies were recovered. Deletion of the binding sequence has no effect on the expression of the endogenous
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