To obtain insight into the function of dPcf11 in Drosophila, immunofluorescence microscopy was used to analyze the distribution on polytene chromosomes of a protein that had 37% amino acid sequence identity with the CTD interacting domain of yPcf11 (Meinhart, 2004). Antibodies were raised against an N-terminal polypeptide encompassing amino acids 1–238. In accordance with one form of dPcf11 predicted from cDNA sequence information, Western blotting detected a single polypeptide of ~60 kDa in whole-cell lysates from Drosophila salivary glands and tissue culture cells (Zhang, 2006).

Immunofluorescence analysis revealed that dPcf11 was concentrated at a small number of loci on polytene chromosomes. For chromosomes from either nonheat-shocked larvae or heat-shocked larvae, all locations staining intensely for dPcf11 also stained intensely for Pol II. The chromosomes from heat-shocked larvae provide the strongest indication that dPcf11 is involved in transcription. Heat shock causes the induction of a small collection of genes whose locations on the chromosomes are known. dPcf11 is concentrated at every one of these locations. In addition to the naturally occurring genes, dPcf11 was also detected on a heat shock-inducible transgene located at 87E (Wu, 2003). Interestingly, dPcf11 was not detected at the myriad of locations on the chromosome from nonheat-shocked larvae that modestly stain with antibody against Pol II. Many of these locations are likely to contain Pol II molecules that have paused as a result of the negative elongation factor Negative elongation factor E (NELF) (Wu, 2003; Wu, 2005; Zhang, 2006 and references therein).

Locus 87A on some polytene chromosomes from heat-shocked larvae exhibited very intriguing patterns of dPcf11 and Pol II staining. 87A contains two copies of the hsp70 gene that are divergently transcribed. When this region was stretched during preparation of the chromosomes, the staining pattern for each protein appeared as two bands. Antibody against Pol II stained two regions toward the center of the locus, whereas antibody against dPcf11 stained two regions toward the periphery. These patterns of staining suggest that dPcf11 is concentrated toward the 3′ ends of each copy of hsp70 (Zhang, 2006).


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Pcf11: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology

date revised: 10/12/2006

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