CRTF is a novel transcription factor that regulates multiple stages of Dictyostelium development
Xiuqian Mu*,
Seth A. Spanos
,
Joseph Shiloach and
Alan Kimmel
Laboratory of Cellular and Developmental Biology, NIDDK (MMDS; Bldg 50/3351), National Institutes of Health, Bethesda, MD 20892-8028, USA
* Present address: Department of Biochemistry and Molecular Biology, 1515 Holcombe, M. D. Anderson Cancer Center, University of Texas, Houston, TX 77030, USA
Present address: School of Medicine, 50 North Medical Drive, University of Utah, Salt Lake City, UT 84132, USA
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Fig. 1. Purification scheme for CRTF. Final pools of 0.4 M and 0.5 M NaCl eluates from the CAR1 DNA affinity column represent an activity enrichment of 10,000-fold (see Materials and Methods).
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Fig. 2. Analysis of purified CRTF. (A) Purified CRTF was separated by SDS-polyacrylamide gel electrophoresis and detected by silver staining. (B) HPLC analysis of tryptic peptides for the p35 and p40 forms of CRTF. (C) Deduced peptide sequences of P1, P2 and P3. The P3 fraction is a mixture of peptides P3a and P3b.
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Fig. 3. Amino acid sequence of full-length CRTF. Deduced amino acid sequence (with numbers indicated) from the full-length CRTF cDNA. --- represents the position of the single intron. The poly asparagine (N)- and poly glutamine (Q)-rich runs present in the full-length but not the truncated form of CRTF are shown in red and blue, respectively. The DNA-binding domain (DBD) is boxed. The P1, P2, P3a and P3b peptides are underlined. The H/C residues that may form an atypical zinc-finger motif are in bold. The asterisks denote positions for C-terminal deletions to amino acids 870, 865 and 859 (see Fig. 4). A putative nuclear localization sequence at the C terminus is in green. ^ indicates cloning position of BLAST gene within the DBD for the gene disruption construct.
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Fig. 4. Deletion mapping of the DNA-binding domain of CRTF. A series of truncated CRTF forms were expressed in E. coli and equal amounts of protein assayed for ability to complex (bracket) with the CAR1 element by electrophoretic mobility shift.
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Fig. 5. CRTF expression in WT and crtf-null strains. (A) CRTF mRNA expression at various hours of development. (B) CRTF mRNA expression in wild-type (WT) and crtf-null strains. (C) CRTF DNA-binding activity (arrow) from nuclear extracts of wild-type (WT) and crtf-null strains treated with 20 nM cAMP at 6 minutes intervals for 5 hours. Control (-) had no added extract (see Fig. 7).
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Fig. 6. Abnormal development of crtf nulls. (A) Wild-type and crtf-null strains were developed (time in hours) on agar at 0.7x105 cells/cm2. (B) Wild-type and crtf-null strains were developed (time in hours) on agar at 4.5x105 cells/cm2. (C) Wild-type and crtf-null strains were developed to fruiting bodies as in Fig. 6B and spores analyzed by phase microscopy.
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Fig. 7. Expression of CRTF-HA protein rescues crtf-null development. (A) crtf-null strain and crtf-null strain expressing the CRTF-HA tagged protein (crtf-null::act15/CRTF-HA) were developed on agar for 24 hours. (B) crtf-null and crtf-null::act15/CRTF-HA strains were developed to fruiting bodies as in Fig. 7A and spores analyzed by phase microscopy. (C) Wild-type, crtf-null and crtf-null::act15/CRTF-HA cells were differentiated in shaking culture in the presence of exogenous pulses of cAMP. Nuclear extracts were prepared from cells differentiated for 0 and 5 hours, and analyzed by immunoblot using anti-HA sera. Migration positions of molecular weight markers are indicated. Arrow indicates presence of the 40 kDa-Ha form of CRTF. A nonspecific band of  60 kDa is detected in all cells.
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Fig. 8. Aggregation stage gene expression defects in crtf-nulls. WT and crtf-null strains were differentiated in shaking culture in the presence or absence of exogenous pulses of cAMP. RNA expression was examined after various hours of differentiation.
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Fig. 9. Cell-specific gene expression patterns in crtf-nulls. (A) Expression of prestalk (ecmA) and prespore (cotB, psA) genes in wild-type and crtf-null strains differentiated in shaking culture in the presence of exogenous pulses of cAMP (P) followed by treatment without (-) or with (+) 500 µM cAMP. (B) spiA mRNA expression in wild-type and crtf-null strains at various stages of development (hours). (C) Spatial expression of prestalk ecmA/lacZ and prespore psA/lacZ in crtf-nulls; red arrows indicate prestalk regions of slugs.
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Fig. 10. CAR1-independent defects of crtf-nulls. (A) Wild-type and crtf-null strains differentiated in shaking culture for 5 hours in the presence of exogenous 30 nM pulses of cAMP for 5 hours followed by treatment with 500 µM cAMP for an additional 5 hours. Cells were then plated on filters and examined at 10 and 24 hours. (B) Spores obtained from cells developed to fruiting bodies in Fig. 8A were analyzed. (C,D) Constitutive expression of CAR1 in crtf-nulls will rescue aggregation but not late development or sporulation of crtf-nulls. crtf-nulls carrying act15/CAR1 were developed (C) on filters for times shown and spores analyzed (D).
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Fig. 11. Cell autonomous defects of crtf-nulls. (A) Co-development of crtf-nulls with WT fails to rescue development (top). The percentage of phase bright spores (as determined for various wild-type/crtf-null developmental mixes) directly reflects the percentage wild-type input (bottom); <1% of spores are crtf-null, regardless of input ratio. (B) 8-Bromo cAMP treatment (right) of crtf-nulls fails to rescue sporulation.
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© The Company of Biologists Ltd 2001
