Establishment of order in the flow of genetic information in cells.

The activities related to the flow of genetic information encoded in DNA in a cell are very orderly. This order, in a living cell, is achieved through specific, but noncovalent, interactions of varieties of structurally dynamic macromolecules under constantly changing physiological conditions. Hence, it is expected that there should be some force that can stabilize the multicomponent reaction processes and establish (or maintain) order in genetic regulatory functions under far-from-equilibrium conditions. The genetic regulatory functions in a cell, however, are believed to be energetically coupled. Expression of genes in a cell is often modulated under changing environmental conditions, raising the possibility of a state controlled nature of the genetic regulatory functions. Adenosine triphosphate (ATP) is the major free-energy contributor for these energy-consuming cellular activities. Enzymatic transfer of high-energy phosphate group from ATP to other reactive components is considered to be the chief mode of energy-transduction in a cell for various biosynthetic processes, as well as other activities related to the flow of information. In an effort to find a solution of the paradox, we assessed the contribution of physiological state of a cell in the process of maintaining order in genetic regulatory functions. As an approach, we systematically perturbed the normal energy flow of a cellular system (bovine aortic endothelial [BAE] cell) by a protein kinase inhibitor (staurosporine), and then followed the expression patterns of several constitutively-expressed protein-encoding genes to measure the effects. Staurosporine, as a function of its concentration, disintegrated the membrane structure of these cells, and eventually caused their death. These secondary consequences of staurosporine treatment offered two additional grossly altered physiological states of the cell to study. Under all of these dramatically altered energy states of the system, an extreme degree of functional coherence prevailed at every level of genetic regulatory function. Integrity at the level of gene transcription remained unaffected. Degradation rate of specific mRNA remained unaltered. Translational activities involving varieties of mRNA species continued in an well-ordered manner. Other state changes, resulting from nutrient and metabolic starvation, or inhibition of oxidative phosphorylation, in addition to the staurosporine treatments, also failed to disintegrate these ordered activities. The steady-state levels of specific mRNA underwent certain changes in these conditions, however, without maintaining any proportional relationships with the staurosporine concentrations applied or the ATP levels in the cell. These results thus led us to propose that the internal energy or a certain intrinsic property of the participating components, rather than the physiological state of the cell, acts as the dominant force in maintaining order and stability of genetic regulatory functions in a cell. Kinetic analyses under different energy states of the cell also supported the hypothesis, and further demonstrated the autoregulatory nature of the genetic order establishment. All of these results suggest a process of molecular self-organization as the fundamental principle for genetic regulation in a cellular system.

Regulation of c-jun gene expression in endothelial cells by the protein kinase inhibitor staurosporine.

The proto-oncogene c-jun, a member of the family of immediate-early genes, is transcriptionally induced in different cell types by a variety of stimuli, including mitogens, tumor promoters, growth factors. We show here that the protein kinase inhibitor staurosporine, which inhibits both the serine-threonine and tyrosine specific protein kinases, also causes differential regulation of the c-jun gene in endothelial cells. Increasing concentrations of staurosporine modulated the steady-state levels of c-jun mRNA in bovine aortic endothelial (BAE) cells in a multiphasic manner. The half-life of c-jun mRNA did not change significantly under these conditions, suggesting that the modulations in the mRNA levels were caused primarily by differential transcriptional activity of the gene. The expression of c-jun gene is believed to be regulated by its own product, the JUN protein, which constitutes a major component of the inducible transcription factor AP-1. In order to test whether the differential regulation of c-jun gene was caused by the differential activation (or inactivation) of the AP-1 transcription factor, the DNA-binding activity of this transcription factor in staurosporine-treated cells was measured. Gelshift analysis with a synthetic oligonucleotide probe showed modest effects of staurosporine on the DNA-binding activity of the transcription factor AP-1. The changes observed in the DNA-binding activity of AP-1 did not parallel the changes observed in the steady-state levels of c-jun mRNA. Similarly, the expression of an AP-1 dependent reporter gene construct was regulated in a fashion entirely different from the c-jun gene during the same protein kinase inhibitory conditions. These results suggest the existence of an alternative pathway that regulates the c-jun gene expression in endothelial cells independent of both the protein kinase and AP-1 transcription factor activation steps.

Hypoxia induces AP-1-regulated genes and AP-1 transcription factor binding in human endothelial and other cell types.

Hypoxia results in differential expression of specific genes in certain cell types. In endothelial cells, hypoxia activates several genes that are known to be inducible by transcription factor AP-1, including endothelin-1 and platelet-derived growth factor-B (PDGF-B). In this study we demonstrated that other AP-1-inducible genes are activated by hypoxia in these cells, including collagenase IV and c-jun, and sought to correlate the activation of genes by hypoxia with the activation of transcription factor AP-1. Depending upon the type of cell studied, hypoxic exposure resulted in the induction of AP-1 transcription factor DNA-binding activity with wide variations in levels of binding. The magnitude of activation of transcription factor AP-1 by hypoxia did not always strictly correlate with the level of induction of AP-1-inducible genes. This finding indicates a requirement for additional mechanisms of controlling transcription beyond the simple activation of AP-1 factor DNA-binding activity for the activation of AP-1-inducible genes during hypoxia. Hypoxia has been reported to lower the intracellular redox potential. The effect of redox state changes on AP-1 transcription factor activity and on the activation of AP-1-inducible genes was also studied. PDTC, a potent reducing agent, activated the AP-1 transcription factor in HeLa cells, and also resulted in increased accumulation of c-jun mRNA in these cells. In contrast to PDTC-mediated activation of the AP-1 transcription factor and the subsequent induction of the AP-1-regulated c-jun gene, hypoxic activation of AP-1 transcription factor binding to its cognate DNA sequence did not activate the c-jun gene in HeLa cells, thus documenting distinct differences in signals generated by the reducing intracellular microenvironments created by hypoxia and PDTC. These results demonstrate the induction of AP-1 transcription factor activity by hypoxic environments, but suggest that additional factors or cell-specific signals are involved in the regulation of hypoxia-induced genes.

Activation of B-cells by sIgM cross-linking induces accumulation of CD5 mRNA.

The surface membrane molecule CD5 is expressed on mature T cells and on the B-1a subpopulation of B cells. These CD5 positive B cells express an antibody repertoire with a relatively high frequency of self-reactivity. There is uncertainty about the origins of CD5 B cells and the reasons for this are reviewed. Recent reports which relate to the lineage/selection debate are discussed. For instance, an increase in the frequency of CD5 B cells is a feature of several genetically determined polysystem autoimmune syndromes. In the case of motheaten (me, mev) the pathogenesis of this increase in CD5 B cells is not yet understood, even though the mutation has been mapped to the Hematopoietic cell protein-tyrosine phosphatase (Hcph) gene. Another mutation which affects B cell development, X-linked immunodeficiency (xid), encodes a point mutation in a B cell cytoplasmic tyrosine kinase. Expression of xid in otherwise normal mice causes a lack of CD5 B cells and a shift in the antibody repertoire. Interestingly, expression of both xid and motheaten results in an amelioration of autoantibody production. Evidence is presented that in B cells regulation of expression of CD5 can occur at the level of mRNA and that cross-linking of sIgM can induce the accumulation of CD5 mRNA. The overall concept advanced is that cells expressing natural autoantibodies are triggered via sIgM ligation to become CD5 B cells.

Regulatory elements within the agropine synthase promoter of T-DNA.

DNA sequences involved in the expression of the agropine synthase gene (ags) of T-DNA were identified by analysis of transcriptional activity of promoter mutants in crown gall tumors of sunflower. Precise quantification of activity was achieved using a homologous reference gene as an internal standard. Analysis of 5'-deletion mutants demonstrated the requirement of 314 base pairs of upstream DNA sequences for optimal activity. Five regions involved in transcriptional regulation were identified in the 5'-flanking sequences between positions -74 and -314. Four of these regions make a positive contribution to promoter activity, and the fifth exerts a negative influence. The TATA motif (-26 to -33) and the TATA proximal domain (-74 to -105), which contains two sequences similar to the mammalian CCAAT box, are the major determinants of promoter activity. The two TATA distal domains A and B are separated by a negative element (-166 to -205) which may attenuate promoter strength by distancing the TATA distal domain B (-206 to -314 base pairs) from downstream components of the promoter. The TATA distal domain B contains the a/b repeat first described in the nopaline synthase (nos) promoter and was unable to support transcription in the absence of elements within the TATA proximal domain.