BiochemistryExpression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes Communicated by Etienne-Emile Baulieu, August 17, 1992 ABSTRACT Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent Mr 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amlifed a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the done to deduce the hFKBP52 sequence (calculated Mr 51,810) and to express hFKBP52 in Escherichia coi. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 Is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs MATERIALS AND METHODSPreparation of an FK506 Affinity Matrix and Isolation of FKBPs. An amino derivative of FK506 was prepared (10) and coupled to Affi-Gel 10 (Bio-Rad) in methanol overnight; unreacted groups were blocked with 50 mM ethanolamine. FKBPs from calf thymus cytosol were prepared by using the matrix as described (11), dialyzed at 40C against 10 mM Tris-HCl (pH 7.0), lyophilized, reconstituted in SDS sample buffer, and resolved in a 12.5% acrylamide gel. Proteins were stained (Fig. 1A) or electroblotted.Protein Sequence Determination of bFKBP52. The Mr 55,000 band, later called bFKBP52, was stained on and excised from Immobilon-P (Millipore) for automated aminoterminal sequencing (26). Peptides were generated with endoproteinase Lys-C (Wako Chemicals USA, Richmond, VA) and separated by microbore C18 HPLC and eluted at 200A.l/min from 5% B at 0 min to 33% B at 65 min, 60%6 B at 90 min, and 100% B at 105 min (solvent A, 0.09%o trifluoroacetic acid in water; solvent B, 0.06% trifluoroacetic acid in acetonitrile). Effluent fractions corresponding to absorption peaks at 214 nm were collected, stored immediately at -200C, and applied later to a Polybrene-precycled glass-fiber filter for sequencing.Isolation of a Human cDNA Encoding FKBP52. Using BLAST (27) to search GenPept (translated GenBank Release 64.3 with daily updates, searched on July 11, 1991) with the deduced hFKBP12 sequence (7, 8), we identified two related murine polypeptides (encoded by GenBank sequences X17068 and X17069) that shared significant sequence identity with our bFKBP52 sequence (Fig. 2). Two DNA oligomers...

The suspected major mutagenic adduct of benzo[a]pyrene, (+)-anti-B[a]P-N2-Gua, is built into the unique PstI recognition site of the Escherichia coli plasmid, pUC19, in order to study its mutagenic potential. The adduct can either be at G437, which is replicated during leading strand DNA synthesis, or at G438, which is replicated during lagging strand DNA synthesis. The DNA strand complementary to the strand containing the (+)-anti-B[a]P-N2-Gua adduct is saturated with UV lesions to minimize its potential to generate progeny. Although all in-frame mutations could have been detected, a G437----T transversion mutation is virtually exclusively obtained at a frequency of approximately 0.04% per adduct following transformation into Uvr+ E. coli when SOS is not induced, and approximately 0.18% when SOS is induced. The mutation frequency of the adduct in a Uvr- background is estimated to be approximately 0.2% when SOS is not induced, and approximately 0.9% when SOS is induced. The absence of G438----T mutations is rationalized. G----T mutations from (+)-anti-B[a]P-N2-Gua are compared to the mutational specificity of the ultimate mutagenic form of activated benzo[a]pyrene.

The mutagenic and carcinogenic chemical aflatoxin B1 (AFB1) reacts almost exclusively at the N(7)-position of guanine following activation to its reactive form, the 8,9-epoxide (AFB1 oxide). In general N(7)-guanine adducts yield DNA strand breaks when heated in base, a property that serves as the basis for the Maxam-Gilbert DNA sequencing reaction specific for guanine. Using DNA sequencing methods, other workers have shown that AFB1 oxide gives strand breaks at positions of guanines; however, the guanine bands varied in intensity. This phenomenon has been used to infer that AFB1 oxide prefers to react with guanines in some sequence contexts more than in others and has been referred to as "sequence specificity of binding". Herein, data on the reaction of AFB1 oxide with several synthetic DNA polymers with different sequences are presented, and (following hydrolysis) adduct levels are determined by high-pressure liquid chromatography. These results reveal that for AFB1 oxide (1) the N(7)-guanine adduct is the major adduct found in all of the DNA polymers, (2) adduct levels vary in different sequences, and, thus, sequence specificity is also observed by this more direct method, and (3) the intensity of bands in DNA sequencing gels is likely to reflect adduct levels formed at the N(7)-position of guanine. Knowing this, a reinvestigation of the reactivity of guanines in different DNA sequences using DNA sequencing methods was undertaken. The reactivities of 190 guanines were determined quantitatively and considered in a pentanucleotide context, 5'-WXGYZ-3', where the central, underlined G represents the reactive guanine and W, X, Y, and Z can be any of the nucleotide bases. Methods are developed to determine that the X (5'-side) base and the Y (3'-side) base are most influential in determining guanine reactivity. The influence of the bases in the 5'-position (X) is 5'-G (1.0) greater than C (0.8) greater than A (0.3) greater than T (0.2), while the influence of the bases in the 3'-position (Y) is 3'-G (1.0) greater than T (0.8) greater than C (0.4) greater than A (0.3). These rules in conjunction with molecular modeling studies (to be published elsewhere) were used to assess the binding sites that might be utilized by AFB1 oxide in its reaction with DNA.

Gene amplification is known to be critical for upregulating gene expression in a few cases, but the extent to which amplification is utilized in the development of diverse organisms remains unknown. By quantifying genomic DNA hybridization to microarrays to assay gene copy number, we identified two additional developmental amplicons in the follicle cells of the Drosophila ovary. Both amplicons contain genes which, following their amplification, are expressed in the follicle cells, and the expression of three of these genes becomes restricted to specialized follicle cells late in differentiation. Genetic analysis establishes that at least one of these genes, yellow-g, is critical for follicle cell function, because mutations in yellow-g disrupt eggshell integrity. Thus, during follicle cell differentiation the entire genome is overreplicated as the cells become polyploid, and subsequently specific genomic intervals are overreplicated to facilitate gene expression.