1. Volume 87, Issue 7, Pages 1225-1235 (December 1996)
Dacapo, a Cyclin-Dependent Kinase Inhibitor, Stops Cell Proliferation during Drosophila Development 
Mary Ellen Lane, Karsten Sauer, Kenneth Wallace, Yuh Nung Jan, Christian F Lehner, Harald Vaessin 
Cell 
Volume 87, Issue 7, Pages (December 1996)
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2. Figure 1 Characterization of dap in Wild Type and Mutants
(A) The structure of the dap cDNA is shown schematically. The cDNA sequence (accession number U68477) was found to start 27 bp downstream of the P element insertion present in dap1. The open reading frame is represented by a black box. In addition, the binding sites of the primer A, B, C, and P that were used for the characterization of w− chromosomes derived from dap1 (see [C]) are indicated.
(B) An alignment of the amino acid sequence derived from the dap cDNA (DAP) with other members of the Kip family of cdk inhibitors. The C. elegans sequence (CE) was identified by the genome sequencing project (accession number Z50796). In addition, the sequence of human p21Cip1 (HS CIP1, accession number U03106) and human p27Kip1 (HS KIP1, accession number U10906) were included. The alignment was achieved with the program CLUSTAL of the PCGENE software package (Intelligenetics). Stars indicate identity in all sequences. Amino acids identical with the DAP sequence are shaded. The black line above the first line denotes a cluster of conserved amino acids that was deleted in the mutant protein (DAPΔLFG) used for biochemical control experiments (see Figure 2).
(C) PCR experiments with the primer A, B, C, and P (see [A]) were used for the characterization of dap1 derivatives. The primer pair AB amplifies from genomic DNA of control flies (w) a product (lane 1) with the same size as also obtained from a fully viable derivative (lane 2) indicating that this derivative (prec) has resulted from a precise excision event. The absence of the P element from this derivative is also evidenced by the lack of a product after amplification with the BP primer combination (lane 3), while this primer combination amplifies the expected product from the original P element insertion line dap1 (lane 5). The size of this P element in dap1, however, precludes a successful amplification with the AB primer combination (lane 4). The absence of a product after amplification from the derivative dap4 with the primer combinations AB (lane 6) and
BP (lane 7) and the size of the product obtained with the primer combination AC (lane 10) demonstrate that this almost completely lethal derivative has resulted from an imprecise excision event. A HaeIII digest of ΦX174 DNA is run in the marker lane (M) of the left gel; the size of the fragments (kb) on the right gel is indicated by arrowheads on the right side.
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3. Figure 2 DAP Binds and Inhibits Cyclin E/cdk2 Complexes
(A) Wild-type dap protein (dap) and a mutant (dapΔLFG) lacking the first cluster of conserved amino acids (see Figure 1B) were expressed in E. coli with a hexa-histidine tag at the C-terminus and purified (see Experimental Procedures). The purification of wild-type (lanes 1–3) and mutant DAP (lanes 4–6) was analyzed by SDS–PAGE and Coomassie blue staining. Total bacterial extracts before (lanes 1 and 6) and after induction (lanes 2 and 5) of DAP expression, as well as the purified proteins (lanes 3 and 4) were loaded on the gel. The position of wild-type and mutant DAP is indicated by arrowheads on the left and right side, respectively.
(B) The cdk inhibitor activity of DAP towards cdk2 (cdk2), cyclin E (cycE), cdk1 (cdk1), cyclin A (cycA), and cyclin B complexes (cycB) was assayed. Buffer (b) or purified mutant (m) or wild-type (w) DAP was added to embryo extracts. DAP protein was added to a concentration corresponding to a 1:1 (1), 10:1 (10), or a 100:1 (100) molar ratio of DAP to cdk2 (estimated to be 1 nM in the embryo extracts) in the experiment shown in the upper panels (cdk2) and to a 100:1 molar ratio in the experiments shown in the lower panels (cycE, cdk1, cycA, cycB). Histone H1 kinase activity of the cdk complexes was assayed after immunoprecipitation with antibodies against
cdk2, cyclin E, cdk1, cyclin A, or cyclin B. The specificity of the immunoprecipitations was controlled by competition with the peptide (peptide: +) used for immunization in the case of anti-cdk2, or by control immunoprecipitations using preimmune sera (not shown). The relative histone H1 kinase activites are indicated below the panels showing the relevant regions after SDS–PAGE and analysis by a PhosphoImager.
(C) Immunoprecipitates obtained from 6 to 8 hr embryo extracts with two different, affinity-purified rabbit anti-DAP antibodies (lanes 1 and 2), with anti-cdk2 (lanes 3 and 4) in the presence (+) or absence (−) of competing peptide, anti-cdk1 (lanes 5 and 6) in the presence (+) or absence (−) of competing peptide, or with an affinity-purified rabbit antibody against an irrelevant protein (THR) used for control (lane 7) were analyzed by immunoblotting for the presence of DAP (panel dap), cdk2 (panel cdk2), cyclin E (panel cycE), cdk1 (panel cdk1), cyclin A (panel cycA), and cyclin B (panel cycB). The positions of these proteins are indicated by arrowheads on the left or right side. Additional bands visible on the immunoblots represent cross-reactions with immunoglobulin used for the immunoprecipitations.
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4. Figure 3
dap Transcript and Protein Distribution during Drosophila Development
The distribution of dap transcripts was analyzed by whole mount in situ hybridization with embryos (A–I) and third-instar eye imaginal discs (J). The embryos shown are at stage 2 during the syncytial cycles (A), at stage 5 during cellularization (B), at stage 6 during early gastrulation (C), at stage 10 (D and E), at early stage 11 before the onset of the terminal division (mitosis 16) in the epidermis as revealed by double labeling with a DNA stain (not shown) (F), at late stage 11 after completion of mitosis 16 in the epidermis (G), at stage 13 during dorsal closure (H), and at stage 15 after dorsal closure (I). (D) and (E) show a central focal plane (with central nervous system and anterior and posterior midgut) and a tangential focal plane (with epidermis), respectively, of the same embryo. The eye imaginal disc (J) is oriented anterior down and posterior up; the position of the morphogenetic furrow is indicated by the arrowhead on the right side. The distribution of DAP protein was analyzed immunocytochemically with affinity-purified anti-DAP antibodies (K–O). The embryos shown in (K)–(O) are at the same developmental stage or slightly older as the embryos shown in (B), (E)–(G), and (I). The inset in (O) shows a high magnification view illustrating the predominantly nuclear signal obtained after anti-DAP immunolabeling. All embryos are oriented anterior to the left and dorsal up. Bars in (I) and (J) correspond to 50 μm.
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5. Figure 4
dap Is Required to Stop the Epidermal Cell Proliferation during Embryogenesis
Control (A and C) and dap4/dap4 embryos (B and D–F) are shown after BrdU pulse-labeling and immunofluorescent anti-BrdU labeling (A and B), or after anti-tubulin labeling (C and D). While BrdU is incorporated in proliferating cells of the nervous system in the control embryo, it is not incorporated at stage 12 in the postmitotic epidermal cells (A). In contrast, BrdU is incorporated in the epidermis of dap4/dap4 embryos after mitosis 16 at stage 12 (B). While no mitotic spindles are observed in the postmitotic epidermis of control embryos at stage 13 (C), mitotic figures are abundant in the epidermis of dap4/dap4 embryos (D). The high magnification views of a double labeling of tubulin (E) and DNA (F) do not reveal DNA bridges during anaphase of the extra division in the dap4/dap4 epidermis. Bars in (A), (C), and (F), correspond to 50, 5, and 1 μm, respectively.
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6. Figure 5
Premature Overexpression of dap Is Sufficient to Stop the Epidermal Cell Proliferation in a G1 Phase
Premature dap overexpression in epidermal stripes was achieved by crossing together the prd-GAL4 transgene (directing the expression of GAL4 under the control of the prd regulatory region) and the GAL4-dependent UAS-dap transgene (A, B, and E–H). Simultaneous expression of dap and cyclin E was achieved by crossing together the prd-GAL4 transgene and the GAL4-dependent transgenes UAS-dap and UAS-CycE (C and D). Embryos were BrdU pulse labeled followed by immunofluorescent anti-BrdU labeling (A–D). Epidermal regions from (A) and (C) are shown at high magnification in (B) and (D), respectively. A merged image showing double labeling with anti-BrdU (red) and anti-DAP (green) is shown in (E), and the boundary between ventral (ve) and dorsal (de) epidermis is indicated by the broken line. At the stage shown in (E), ventral epidermal cells (including the DAP-expressing cells) complete S phase 15 and incorporate BrdU into the late replicating heterochromatin resulting in a characteristic, speckled staining. At the same stage, dorsal epidermal cells except the DAP-expressing cells have already progressed into S phase 16 and incorporate BrdU throughout the cell nuclei. A merged image showing double labeling with anti-tubulin (red) and anti-DAP (green) is shown in (F). At the stage shown in (F), the cells in the ventral epidermis are progressing through mitosis 15, while dorsal epidermal cells are already at the stage of S phase 16. Immunofluorescent anti-cyclin A labeling (G) before mitosis 16 is almost undetectable in the DAP-expressing epidermal stripes (right side), while it is readily detectable in the interstripes (left side). Conversely, immunofluorescent anti-cyclin E labeling before mitosis 16 (H) is stronger in the DAP expressing cells (right side) than in the interstripes (left side). Bars in (A), (B), and (G) correspond to 50, 10, and 5 μm, respectively.
Cell  , DOI: ( /S (00) )
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