Three distinct messenger RNAs can encode the human immunosuppressant-binding protein FKBP12.

FKBP12 is an 11.8-kDa protein that binds the potent immunosuppressants FK506 and rapamycin. When bound to FK506, FKBP12 forms an inhibitory complex with calcineurin and interferes with signal transduction in activated T lymphocytes. In studying human FKBP12 cDNAs and the human FKBP12 gene, we found that three distinct transcripts can encode human FKBP12. The transcripts, which we designate FKBP 12A, 12B and 12C, contain identical open reading frames, but vary in abundance and are distinguished by unique 3' untranslated regions. The mature transcripts derive from either four or five exons and are generated by the differential use of one splice junction and three cleavage-polyadenylation sites within FKBP12. FKBP12A and 12B populations increase in abundance and/or stability when T-cell populations are mitogenically activated in vitro, implying that one result of T-cell stimulation is increased demand for the FKBP12 message. These transcripts are also present in a variety of human tissues, suggesting that FKBP12 and/or the mRNAs encoding it might affect physiological function(s) in a diverse array of cells.

Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 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 amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. 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 and a potential calmodulin-binding site, implying that posttranslational phosphorylation could generate multiple isoforms of the protein and that calmodulin and intracellular Ca2+ levels could affect FKBP52 function. FKBP52 transcripts are present in a variety of human tissues and could vary in abundance and/or stability.

Mutagenesis by (+)-anti-B[a]P-N2-Gua, the major adduct of activated benzo[a]pyrene, when studied in an Escherichia coli plasmid using site-directed methods.

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.

Construction of a human shuttle vector containing a single nitrogen mustard interstrand, DNA-DNA cross-link at a unique plasmid location.

DNA cross-linking reagents are frequently unusually cytotoxic, and many, including the nitrogen mustards, are potent chemotherapeutic agents, presumably because DNA cross-links effectively block DNA replication. Most of these reagents form both inter- and intrastrand DNA cross-links, but it is unknown which is more effective at blocking replication and why. To evaluate the role of interstrand cross-links, a human shuttle vector was constructed that contains a single, nitrogen mustard interstrand cross-link at a unique site. In previous work (J.O. Ojwang, D. A. Grueneberg, and E. L. Loechler, Cancer Res., 49: 6529-6537, 1989) a duplex oligonucleotide was synthesized that had an interstrand cross-link derived from a nitrogen mustard moiety bound at the N(7)- position of the guanines in the opposing strands of a 5'-GAC-3' 3'-CTG-5' sequence. Herein, a procedure is described to incorporate this oligonucleotide into an SV40-based human shuttle vector, which was designed for these experiments. The purified cross-linked vector was characterized and shown: (a) to have a chemical (i.e., a nitrogen mustard) modification at the anticipated genome location; (b) to have a modification that covalently joins the two duplex strands of the vector together; and (c) to contain a single interstrand cross-link per genome. The methodologies described to construct this vector are expected to be generally applicable and, thus, site-specific incorporation of an interstrand cross-link derived from any appropriate chemical should be possible. These procedures complement existing methodologies that permit the incorporation of monoadducts and intrastrand cross-links into vectors in a site-specific manner.

Construction of an Escherichia coli vector containing the major DNA adduct of activated benzo[a]pyrene at a defined site.

The mutagenic and carcinogenic substance benzo[a]pyrene reacts with DNA following activation to its corresponding 7,8-diol 9,10-epoxide (BPDE), and the major DNA adduct (BP-N2-Gua) is formed when the C(10)-position of BPDE reacts with the N2-position of guanine. It is unknown if this adduct is a premutagenic lesion in vivo. Herein, the construction and characterization of an M13mp19-based, E. coli vector that contains BP-N2-Gua located in the unique PstI restriction endonuclease recognition site at nucleotide position 6249 in the (-)-strand is described (designated, BP-N2-Gua-M13mp19). First, the oligonucleotide 5'-TGCA-3' was reacted with BPDE and a product (5'-T(BP-N2)GCA-3') was isolated by HPLC that, when enzymatically digested to deoxynucleosides, yielded an adduct that comigrated on HPLC with an authentic BP-N2-Gua deoxynucleoside standard. Second, the 5'-hydroxyl group of 5'-T-(BP-N2)GCA-3' was phosphorylated with ATP and T4 polynucleotide kinase, and the product (5'-pT(BP-N2)GCA-3') was purified by HPLC. This product is stable when heated at 80 degrees C at both neutral and alkaline pH. Third, M13mp19 was manipulated such that the sequence 5'-pTGCA-3' was selectively removed from the (-)-strand in its unique PstI recognition site, and 5'-pT(BP-N2)GCA-3' was ligated into this gap with T4 DNA ligase and ATP. The product of this reaction (BP-N2-Gua-M13mp19) was shown to be insensitive to cleavage by PstI, which suggests that a modification is located in the PstI recognition site. The most likely modification is the adduct BP-N2-Gua.

Mapping the binding site of aflatoxin B1 in DNA: systematic analysis of the reactivity of aflatoxin B1 with guanines in different DNA sequences.

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.