Progression through the somatic cell cycle requires the temporal regulation of cyclin gene expression and cyclin protein turnover. One of the best-characterized examples of this regulation is seen for the B-type cyclins. These cyclins and their catalytic component, cdc2, have been shown to mediate both the entry into and maintenance of mitosis. The cyclin B1 gene has been shown to be expressed between the late S and G 2 phases of the cell cycle, while the protein is degraded specifically at interphase via ubiquitination. To understand the molecular basis for transcriptional regulation of the cyclin B1 gene, we cloned the human cyclin B1 gene promoter region. Using a chloramphenicol acetyltransferase reporter system and both stable and transient assays, we have shown that the cyclin B1 gene promoter (extending to ؊3800 bp relative to the cap site) can confer G 2 -enhanced promoter activity. Further analysis revealed that an upstream stimulatory factor (USF)-binding site and its cognate transcription factor(s) are critical for expression from the cyclin B1 promoter in cycling HeLa cells. Interestingly, USF DNA-binding activity appears to be regulated in a G 2 -specific fashion, supporting the idea that USF may play some role in cyclin B1 gene activation. These studies suggest an important link between USF and the cyclin B1 gene, which in part explains how maturation promoting factor complex formation is regulated.Cyclins are a family of related proteins which are present at specific stages of the somatic cell cycle (58). They function as regulatory subunits for cyclin-dependent kinases (cdks), which phosphorylate key substrates that mediate cell cycle transit (37,43,56,58). Catalytic activation of the cdks requires sufficient accumulation of cyclin protein at particular stages of the cell cycle (41-43, 56). Dysregulation of cyclin gene expression through overexpression and/or unscheduled cdk activity results in inappropriate entry into the S or M phase and may be characteristic of some human cancer cells (7,20,26,30,31,38,45,53,60). The best-characterized cyclin-cdk complex is maturation-promoting factor, which consists of a B-type cyclin and cdc2 kinase (13,17,18,33,41,56). The B-type cyclins (B1, B2, and B3), as well as cyclin A, have been implicated in control of the G 2 /M transition (13,16,37,38,41,42,56,58). The interaction between B-type cyclins and cdc2 during the G 2 cell cycle phase is necessary for cdc2 kinase activation, and the resultant phosphorylations mediate structural changes crucial for the G 2 /M transition (12,16,37,43). Activation of the cdc2 kinase does not occur until sufficient cyclin B protein has been synthesized (56), whereas proteolysis of cyclin B via ubiquitination at the end of mitosis is critical for entry into interphase (19). The accumulation of cyclin B protein, as with many other cyclins, is correlated with nascent-gene expression. Cyclin B mRNA can be detected in late S phase, peaks in late G 2 phase, and cannot be detected in M, G 1 , or early S (41). Models that explain the ...

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable. I dentifying polymorphic sites in the genome is a basic aspect of molecular genetics and genomics. The process is needed for a variety of purposes, ranging from the development of polymorphic marker sets useful as a tool for genetic analysis of a chromosomal region or full genome scan, to the initial identification of variants or mutations in a newly discovered gene (1, 2). In most cases, the identity of base differences and their location relative to a gene or other polymorphic sites is either useful or required. Recent estimates of the number of singlenucleotide polymorphisms (SNPs) needed for whole genome association studies in humans vary from several thousand to several hundred thousand (1, 3); thus, efficient and cost-effective methods for identifying a large number of SNPs with the required characteristics of dense yet even spacing, and of known order over large uncharacterized regions of the genome, is of interest. A comparison of two methods to develop a densely ordered map of SNPs covering a 4-Mb region of the human genome was recently reported (4). In one approach, large-insert bacterial clones, bacterial artificial chromosomes (BACs) (5) and P1 artificial chromosomes (PACs) (6), spanning this region were fragmented and reconstructed in 2-kb plasmid libraries, which were then sequenced. This shotgun procedure is efficient in identifying SNPs; however, to approach a map of 30-kb average SNP spacing, bidirectional sequencing of approximately 500 randomly chosen subclones per 100 kb of genomic sequence was required. Multiple BAC and PAC clones mapping to the region...

In these studies, we show that NF-kappa B induces transcription from the human pro-IL-1 beta (IL-1 beta) gene. A recombinant plasmid pIL-1(-4000)-CAT, containing 4 kb of the IL-1 beta gene upstream regulatory sequence was transactivated by the p65 subunit of NF-kappa B or by treatment of the cells with a combination of NF-kappa B inducers including LPS, PMA, and dibutyryl cyclic AMP (L+P+C) in U937 cells. Coexpression of p65 with L+P+C treatment led to a synergistic response, whereas coexpression of the I kappa B alpha/MAD-3 protein, in place of p65, blocked L+P+C induction. A series of 5' deletion mutants of the IL-1 beta promoter were used to define two p65 response regions: region I located between -2800 to -2720 bp and region II located between -512 and -133 bp. Electrophoretic mobility shift assays confirmed that NF-kappa B-like proteins could bind to two consensus binding sites in region II. A site-specific mutation in only one of these NF-kappa B sites (-296/-286 bp) caused a specific loss of induction by p65 or L+P+C. A cyclic AMP response element (CRE) site (-2761/-2753 bp) in region I has been shown previously to be critical for L+P+C induction. Mutation of the CRE in an enhancerless test plasmid containing two copies of region I blocked transactivation by p65. Likewise, coexpression of I kappa B alpha inhibited CRE-dependent L+P+C induction of the wild-type counterpart. These data show that NF-kappa B regulates a nonconsensus CRE site in addition to the consensus binding site at -296/-286 bp and suggest that NF-kappa B may play multiple roles in the induction of IL-1 beta transcription.

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An intact cAMP response element (CRE) in the upstream regulatory sequence of IL-1 beta (-2755/-2762) has been shown to be essential for maintaining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL-1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dibutyryl cAMP treatment alone is capable of induction. Due to the critical nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling pathway(s) by determining whether CRE binding protein (CREB) and other CREB/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at this site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in electrophoretic gel mobility shift assays. These purification steps resulted in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREB protein in the complex at the CRE site. Supershift electrophoretic gel mobility shift assays and immunoprecipitation analysis provided further evidence that both CREB and ATF-1 are present in the complex. In addition, an increase in CREB phosphorylation was observed when THP-1 5A cells were treated with dibutyryl cAMP, LPS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level.