The structure and conservation of Pam. (A) Amino acid sequence of human Pam predicted from overlapping cDNA clones. The sequence includes the following motifs: LZ1 and LZ2, potential leucine zippers; RHD-1 and RHD-2, RCC1 homology domains; PR1 and PR2, Pam repeats; CDSM, cell division sequence motif; Myc-binding Region, insert in the original phage λgt11-PAM0; HHD, histone-binding protein homology domain; NLS, putative nuclear localization signal; RZF, potential ring zinc finger domain; and Zn, two putative C2H2-type zinc finger motifs. (B) Topography of human Pam. All abbreviations are as in A, except for SR, a serine-rich region occupying residues 2643–3057 (20% serine).

Pam RNA in human tissues. Northern blots prepared with RNAs from multiple human tissues were obtained from CLONTECH (MTN I and II). The filters were analyzed with radioactive probe prepared from PAM cDNA following the manufacturer’s instructions.

Myc interacts with Pam in vivo. (A) Binding of Myc to GST-Pam0. GST and GST-Pam0 proteins were incubated with a nuclear extract of CB33-Myc cells, recovered on glutathione agarose beads, and analyzed by Western blotting by using antibody directed against Myc (9E10). Lane 1, binding with GST-Pam0 protein. Lane 2, binding with GST protein. Lane 3, total nuclear extract of CB33-Myc cells. (B) Binding of Pam to GST-Myc1. Purified GST or GST-Myc1 protein was incubated with a nuclear extract of Hela cells, recovered on glutathione agarose beads, and analyzed by Western blotting with an antiserum against Pam. Lane 1, binding with GST-Myc1 protein. Lane 2, binding with GST protein. Lane 3, the supernatant after adsorption of the nuclear extract with GST protein. Lane 4, the supernatant after adsorption of the nuclear extract with GST-Myc1 protein. (C) Coprecipitation of Pam and Myc from nuclear extracts of Hela cells. Extracts were subjected to immunoprecipitation with various antisera. The precipitates were analyzed by Western blotting with antiserum against Myc (9E10). Lane 1, total nuclear extract. Lanes 2–5, immunoprecipitates. Lanes 6–9, supernatants after immunoprecipitation. Lanes 2 and 6, immunoprecipitation with anti-Pam. Lanes 3 and 7, polyclonal anti-Myc from UBI. Lanes 4 and 8, polyclonal anti-Myc prepared by investigators (cv3). Lanes 5 and 9, nonspecific rabbit IgG.

Mapping the PAM gene to the chromosome 13q22 region between markers AC224 and COLAC1. BTF3 gene and a pseudogene for RNA helicase A (DDX9P) were identified by random sequencing of PAC224a14 (22).

Pam binds specifically to Myc but not NMyc. Lyso-PAM0 and Lyso-PAM0′ represent two lysogens isolated from the positive phage λgt11- PAM0 by using the host bacterial strain Y1089r-. Equal amounts of extracts from Lyso-PAM0, Lyso-PAM0′ or host bacteria Y1089r- were fractionated by electrophoresis through a PAGE gel. The proteins then were transferred to a nitrocellulose filter, and the filter was incubated with various probes.

Sequence alignments of the RCC1 homology domains (RHD-1 and RHD-2) and two additional direct repeats (PR1 and PR2). (A) Alignment of RHD-1 of Pam with RCC1 proteins. RCC_XENLA and RCC_HUMAN are the RCC1 proteins from Xenopus and human (P25183 and P18754). RCC_XLRP3 is the X-linked retinitis pigmentosa 3 protein (O92834), which belongs to the RCC1 family. p619RLD-1 is the first RCC1-like domain in p619 protein (U50078). The underlining indicates the signature sequences of RCC1 family (PS00625 and PS00626). The arrow indicates the amino acid insertion of WKLEQCMVC. (B) Alignment of RHD-2 of Pam with RCC1 proteins. The arrow indicates the amino acid insertion of FLRI. All details are as in A. (C) Alignment of the two direct repeats of human Pam (PR1 and PR2).

Subcellular localization of Pam. Immunofluorescence microscopy was performed on normal human aortic endothelial cells with antibody directed against Pam. (A) and (D) Immunofluorescence with Pam-specific antiserum. (B) and (E) Staining with 4′,6-diamidino-2-phenylindole (DAPI). (C) and (F) Phase contrast microscopy. The bars represent 40 μm in C and 20 μm in F.

Localization of the Pam-binding site within Myc. (A) Binding of ³²P-labeled truncations of GST-Myc to LacZ-Pam0. Binding was assayed by using the Far-Western assay. (B) Summary of results. The binding data for some of the truncation mutants listed here are not shown in A. The numbers indicate the amino acid residues in human Myc.