We previously isolated the AML1 gene, which is rearranged by the t(8;21) translocation in acute myeloid leukemia. The AML1 gene is highly homologous to the Drosophila segmentation gene runt and the mouse transcription factor PEBP2 alpha subunit gene. This region of homology, called the Runt domain, is responsible for DNA-binding and protein--protein interaction. In this study, we isolated and characterized various forms of AML1 cDNAs which reflect a complex pattern of mRNA species. Analysis of these cDNAs has led to the identification of two distinct AML1 proteins, designated AML1b (453 amino acids) and AML1c (480 amino acids), which differ markedly from the previously reported AML1a (250 amino acids) with regard to their C-terminal regions, although all three contain the Runt domain. The large C-terminal region common to AML1b and AML1c is suggested to be a transcriptional activation domain. AML1c differs from AML1b by only 32 amino acids in the N-terminal. Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA. Northern blot analysis demonstrated the presence of six major transcripts, encoding AML1b or AML1c, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites. A minor transcript encoding AML1a which results from alternative splicing of a separate exon can be detected only by reverse transcription-polymerase chain reaction amplification. The distinct proteins encoded by the AML1 gene may have different functions, which could contribute to regulating cell growth and/or differentiation through transcriptional regulation of a specific subset of target genes.

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We recently reported the first pump−probe measurements on the hydrated electron with sufficient time resolution (∼35 fs) to directly observe the initial processes in the solvation dynamics of this key prototype for condensed-phase dynamics [Silva, C.; Walhout, P. K.; Yokoyama, K.; Barbara, P. F. Phys. Rev. Lett. 1998, 80, 1086]. An unprecedented relaxation process for the hydrated electron was observed that occurs on the 35−80 fs time scale and exhibits a solvent isotope effect (τ(D2O)/τ(H2O) ∼ 1.4). The new process was assigned to inertial/librational motion of the water surrounding the excess electron. The present paper reports a more extensive study of the ∼35 fs resolved dynamics of the hydrated electron in H2O and D2O at more probe wavelengths and as a function of pump-pulse intensity. The results are in agreement with the preliminary report and support the importance of librational water motion in the relaxation dynamics of the hydrated electron. New high excitation pulse intensity measurements reveal evidence of a high-intensity, two-photon channel involving ejection of the hydrated electron from its initial site to a different site in the solvent.

This study describes the mechanism of homodimer formation of the 90-kDa heat-shock protein (HSP90). In eukaryotic cells, there are two HSP90 isoforms, a and p, encoded by two separate genes.HSP90a exists predominantly as a homodimer, HSP90p mainly as a monomer. Analysis by native PAGE revealed that bacterially expressed HSP90a fused to glutathione S-transferase (GST) existed as a highmolecular-mass oligomer, and was converted to a homodimer following removal of the fusion enzyme by thrombin cleavage. A deletion mutant, HSP90aD44-603, formed a monomer and an N-terminal truncated mutant, HSP90aS33 -732, existed as a dimer, indicating that the dimer-forming ability resides somewhere in the C-terminal 200 amino acids. Limited proteolysis of the C-terminal 200 amino acids of HSP90a with chymotrypsin produced the C-terminal 16-kDa fragment (Met62WAla629-Asp732) and its adjacent more N-terminal 13-kDa fragment (Va1542-Tyr627/Met628). Size-exclusion HPLC and twodimensional PAGE analyses demonstrated that these two chymotryptic fragments bound each other. The C-terminal 198 amino acids as well as the full-length form of HSP90p revealed a lower dimer-forming activity than HSP90u. Expression of the chimeric proteins at the C-terminal 198 amino acids of the a and p isoforrns further indicated that the 16 amino acid substitutions locating between amino acids 561 and 685 account for the impeded dimerization of HSP90p. A leucine zipper motif (Met402-Leu423) was unlikely to be involved in the dimer formation. Taken together, these results indicate that the dimeric structure. of HSP90a is mediated by the C-terminal 191 amino acids and consists of duplicate interactions of the C-terminal region (Met62WAla629-Asp732) of one subunit and the adjacent more N-terminal region (Va1542-Try627/Met628) of the other subunit.Keywords: heat-shock protein 90; isoform; dimerization; leucine-zipper motif; amino acid substitution The 90-kDa heat shock protein (HSP90) is a highly conserved protein that is abundant in cytoplasm of most prokaryotic and eukaryotic cells even under unstressed conditions. In eukaryotic cells, there are two HSP90 genes and, hence, two HSP90 Ahhreviarions. GST, glutathione S-transferase ; HSP90n and HSP90p, the Q and isoforms of the 90-kDa heat-shock protein; GST-HSP90, GST fusion protein of HSP90; HSP90aD48-458 and HSP90aD44-603, mutants of HSP90 with residues 48-458 or 44-603 deleted; HSP90uS33-732, residues 533-732 of HSP90a; LZ, leucine zipper motif; HSP~OQDLZ, mutant of HSP90a with LZ deleted: TosLysCH,CI, N-tosyl-L-lysine chloromethane; TosPheCH2C1, N"-tosyl-1.-phenylalanine chloromethane.Enzymes. Trypsin (EC 3.4.21.4); thrombin (EC 3.4.21.5); chymotrypsin (EC 3.4.21 .1); factor Xa (EC 3.4.21.6); glutathione S-transferase (EC 2.5.1.18).teins [lo, 111. Several lines of evidence indicate a chaperon-like activity of HSP9O that modulates the activity of its associated proteins [9,. For instance, the high-affinity binding for steroid to the glucocorticoid receptor is maintained by the association of HSP90 with the recep...

Direct observation of the rotational fine structure levels of a vibrationally excited negative ion dipole-bound state (DBS) is reported. Autodetachment resonances of H2CCC− were observed for the 2A1−2B1 transition in one quanta of ν6, ν4, and ν2 and two quanta of ν6 vibrational modes of the DBS. Rotational assignments for both the electronic ground state and the DBS were performed. Strong type (a) Coriolis coupling between ν6 and ν9 in both the electronic ground and excited states was observed, and coupling constants were determined. QCISD ab initio calculations were performed for the ground state, the negative ion, and the neutral state of H2CCC. The calculations on the neutral agree well with measured vibrational frequencies of the dipole-bound state. The autodetachment resonances contain information about the detachment dynamics via the observed linewidths, showing, e.g., that motions along the dipole moment axis significantly enhance autodetachment, indicating that the DBS is unstable with respect to neutral core motion which modulates the magnitude value of the dipole moment.