The promoter regions of most plastid transcription units have been reported to consist of prokaryotic -10 and -35-like consensus sequences. However, a few promoters with no homology to the consensus elements have also been characterized. A novel class of non-consensus plastid promoters--designated as non consensus type II (NC-II) promoters--that effect low-level constitutive expression of respective genes in photosynthetic as well as non-photosynthetic plastids is described in this paper. The abundance of NC-II promoter-derived transcripts remains unaltered even when light-grown seedlings are shifted to the dark. In contrast, transcripts from -10 and -35-like elements containing consensus type (CT) promoters accumulate to high levels in chloroplasts as compared with non-photosynthetic plastids of roots and cultured cells. Moreover, accumulation of these transcripts is greatly affected by light. The inhibition of plastid protein synthesis has no apparent effect on the abundance of the NC-II transcripts whereas levels of CT transcripts are greatly reduced. In vivo tagetitoxin (a plastid transcription inhibitor) treatment also reduces the levels of CT transcripts with no apparent inhibitory effect on the accumulation of NC-II transcripts. The accumulation of transcripts from both classes of promoters, however, is reduced when cytoplasmic protein synthesis is inhibited by in vivo treatment with cycloheximide. The results are suggestive of the possible existence of at least two distinctive systems for the synthesis and/or maintenance of plastid transcripts which differentiate between two classes of transcripts in a promoter-type specific manner.

Macrophages secrete interferon (IFN), as well as other cytokines, following lipopolysaccharide (LPS) stimulation. The interferon regulatory factors (IRFs) comprise a family of DNA-binding proteins that have been implicated in the transcriptional regulation of IFN and certain IFN-inducible genes. We therefore characterized basal and LPS-inducible levels of IRF-1, IRF-2, and interferon consensus sequence binding protein (ICSBP) mRNA in LPS-responsive macrophages and compared the expression of these genes in macrophages that typify two murine models of LPS hyporesponsiveness. In the first model, the LPS-hyporesponsive phenotype of the C3H/HeJ mouse is genetically determined and maps to the Lps locus on mouse chromosome 4. In the second model, normally LPS-responsive macrophages acquire a transient LPS-hyporesponsive phenotype following a prior exposure to LPS, a phenomenon referred to as \"endotoxin tolerance.\" Using reverse transcription PCR, we detected basal levels of IRF-1 mRNA in LPS-responsive (Lpsn) macrophages that were approximately 15 times higher than those found in LPS-hyporesponsive (Lpsd) macrophages. Conversely, Lpsd macrophages expressed basal levels of IRF-2 mRNA that were approximately 18 times higher than those expressed in Lpsn macrophages. LPS stimulation resulted in a dose- and time-dependent accumulation of IRF-1, IRF-2, and ICSBP mRNA only in Lpsn macrophages. Cycloheximide inhibited the accumulation of LPS-stimulated IRF-2 and ICSBP mRNA, but not IRF-1 mRNA, thus designating IRF-1 an immediate-early, LPS-inducible gene. Finally, macrophages rendered tolerant to endotoxin expressed elevated but nonmaximal mRNA levels for all three transcription factors that are not reinduced upon secondary challenge with LPS. Thus, the IRFs may represent yet an additional molecular pathway in the complex response to LPS.

Interferon (IFN) induces transcription of major histocompatibility class I genes by way of the conserved cis-acting regulatory element, termed the IFN consensus sequence (ICS). Binding of nuclear factors to the ICS was studied in gel mobility shift assays with the 5\' upstream region of the murine H-2Ld gene. We found that the ICS binds a constitutive nuclear factor present in lymphocytes and fibroblasts regardless of IFN treatment. Within 1 hr after IFN treatment, new ICS binding activity was induced, which consisted of at least two binding activities distinguished by their requirement for de novo protein synthesis. Methylation interference and competition experiments showed that both constitutive and induced factors bind to the same approximately equal to 10-base-pair binding site within the ICS. Site-directed mutagenesis of H-2Ld-chloramphenicol acetyltransferase fusion genes showed that mutations in the binding site, but not in other regions of the ICS, abolish transcriptional activation of class I genes by IFN, providing evidence that specific binding of nuclear factors to the ICS is an essential requirement for transcriptional induction. Finally, we show that IFN-inducible genes of various species share a sequence motif that is capable of competing for the nuclear factors identified here. We propose that specific protein binding to the conserved motif represents a basic mechanism of IFN-mediated transcriptional induction of a number of genes.

By utilizing chimeric genes constructed from 5\'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.

The promoter regions of many interferon-inducible genes share a short DNA sequence motif, termed the interferon consensus sequence (ICS) to which several regulatory proteins bind. A murine cDNA which encodes an ICS binding protein has been reported (M-ICSBP). The cloning of the human homologue of ICSBP (H-ICSBP) is described. H-ICSBP shares high sequence homology with its murine cognate. The derived sequence of H-ICSBP reveals restricted homology within the first 120 amino acids to three other interferon regulatory factors, IRF-1, IRF-2, and ISGF3 gamma. Truncated ICSBP lacking the first 33 amino-terminal amino acids fails to bind to the ICS, indicating that at least part of the DNA binding domain is located within the well conserved amino terminus. H-ICSBP is expressed exclusively in cell lines of hematopoietic origin. The results of transient transfection assays carried out either in hematopoietic or nonhematopoietic cells suggest that ICSBP acts as a negative regulatory factor on ICS-containing promoters. Furthermore, either interferon-gamma (IFN-gamma) or IFN-beta can alleviate the repression mediated by ICSBP. Therefore, ICSBP may be involved in maintaining submaximal transcriptional activity of IFN-inducible genes in hematopoietic cells. IFN treatment would then alleviate repression allowing maximal transcriptional activity of these genes.