The transcription factor Bright associates with Bruton's tyrosine kinase, the defective protein in immunodeficiency disease.

Binding of the transcription factor Bright to Ig heavy chain loci after B cell activation is associated with increased heavy chain transcription. We now report that Bright coprecipitates with Bruton's tyrosine kinase (Btk), the defective enzyme in X-linked immunodeficiency disease (xid). Furthermore, we observed Btk in the nucleus of activated murine B cells, and mobility shift assays suggest that it is a component of the Bright DNA-binding complex. While BRIGHT protein was synthesized in activated spleen cells from xid mice, it did not bind DNA or associate stably with Btk. These data suggest that deficiencies in BRIGHT DNA-binding activity may contribute to the defects in Ig production seen in xid mice.

Zinc finger and carboxyl regions of adenovirus E1A 13S CR3 are important for transactivation of the cytomegalovirus major immediate early promoter by adenovirus.

Reactivation of latent cytomegalovirus (CMV) is an important cause of disease in susceptible patients. We previously demonstrated that an adenovirus early gene product can transactivate the CMV major immediate early (IE) promoter in inflammatory cells. This effect was due to the conserved region 3 (CR3) of the adenovirus E1A 13S gene product. There are two domains in the CR3 region, a zinc finger (aa 147-177) and a carboxyl (aa 180-188) domain. Both are crucial for transactivation of downstream promoter elements of adenovirus in E1A 13S. We sought to determine if either or both of these specific domains is also necessary for transactivation of the CMV IE promoter by the adenovirus E1A 13S gene product. We cotransfected T-lymphocyte Jurkat cells and monocyte/macrophage-like THP-1 cells with plasmids expressing wild-type (WT) or CR3 mutant E1A 13S and a CMV IE chloramphenicol acetyltransferase (CAT) reporter construct. With extracts of cells coinfected with E1A WT set to 100%, mutation in the zinc finger domain, the carboxyl domain, or both domains decreased CMV IE CAT activity by >/= 96%. In contrast, a mutation in the region between the zinc finger and carboxyl domains reduced CMV IE CAT activity by only 24 to 26%. Mixing studies in Jurkat cells confirmed the importance of these domains. We also evaluated the active site of the CMV IE promoter involved in transactivation in THP-1 cells using CMV IE promoter deletions and single promoter element constructs. These studies showed that progressive deletion of the 19-bp CMV IE repeats containing cyclic AMP response element binding protein/activating transcription factor (CREB/ATF) sites resulted in progressive loss of activity. The importance of this element was confirmed using single promoter elements containing CMV IE 16-, 18-, 19-, and 21-bp repeats. Finally, using a 19-bp single promoter element construct and the CR3 mutants we demonstrated that mutations in the zinc finger (C171S) carboxyl region (S185N) or both regions (C171S/ S185N) resulted in significant (83, 94, and 85%) loss of activity. We conclude that the zinc finger and carboxyl domains of the CR3 region of E1A 13S are necessary for transactivation of the CMV promoter and that this occurs mainly through activation of the 19-bp CREB/ATF site of the promoter.

Rate measurements by the pulsed-accelerated-flow method.

A pulsed-accelerated-flow spectrophotometer with UV-visible capability is described that permits measurement of pseudo-first-order rate constants as large as 500 000 s(-)(1) (t(1/2) = 1.4 µs). Chemical rate processes are resolved from physical mixing rate processes by variation of flow velocities under conditions of turbulent flow. Two mixing processes are found in the mixing/observation tube. One mixing rate constant, valid for the full length of the tube, is directly proportional to the flow velocity. The other mixing behavior, proportional to the square of the flow velocity, is found only in the immediate vicinity of the 10 inlet reactant streams that collide with one another in the middle of the observation tube. Contributions from the latter mixing become more important for very fast reactions that take place close to the inlet jets. These mixing models and improved signal/noise permit the measurement of rate constants for very fast reactions. Applications of the PAF method to electron-transfer, proton-transfer, hydrolysis, and non-metal redox reactions are reported for pseudo-first-order and second-order reactions.

Pulsed-accelerated-flow spectrometer with position-resolved observation.

The title instrument (PAF-PRO) permits the progress of rapid reactions to be monitored at 128 positions along a 2 cm observation cell as reactants flow down the cell. A decelerated push is used to give velocity changes from 21 to 2 m s(-1) during a time interval of 384 ms. Variation of flow velocity allows reaction rate constants to be resolved from the physical mixing process. The flow system brings the reacting solutions together in a 10-jet radial mixer 0.32 cm before the mixture enters the observation tube (0.203 cm width, 1.945 cm length). A masked charge-coupled device (CCD) is the array detector used to obtain transmittance data as a function of position and velocity. The masked portion of the CCD serves as a dynamic memory buffer for fast data acquisition. Pseudo-first-order rate constants are measured from 200 to 12 000 s(-1). The instrument also is calibrated for second-order reactions (equal concentrations) with initial half-lives of 0.3-1.5 ms. Applications of the PAF-PRO system for the study of fast multistep reactions are presented.