Conserved regulatory elements in the promoter region of the N-CAM gene.

Genomic clones containing 5'-flanking sequences, the first exon, and the entire first intron from the chicken N-CAM gene were characterized by restriction mapping and DNA sequencing. A > 600-bp segment that includes the first exon is very G + C-rich and contains a large proportion of CpG dinucleotides, suggesting that it represents a CpG island. SP-1 and AP-1 consensus elements are present, but no TATA- or CCAAT-like elements were found within 300 bp upstream of the first exon. Comparison of the chicken promoter region sequence with similar regions of the human, rat, and mouse N-CAM genes revealed that some potential regulatory elements including a "purine box" seen in mouse and rat N-CAM genes, one of two homeodomain binding regions seen in mammalian N-CAM genes, and several potential SP-1 sites are not conserved within this region. In contrast, high CpG content, a homeodomain binding sequence, an SP-1 element, an octomer element, and an AP-1 element are conserved in all four genes. The first intron of the chicken gene is 38 kb, substantially smaller than the corresponding intron from mammalian N-CAM genes. Together with previous studies, this work completes the cloning of the chicken N-CAM gene, which contains at least 26 exons distributed over 85 kb.     

Structure of the chicken interferon-γ gene,and comparison to mammalian homologues

The sequence of the chicken interferon-γ (ifn-γ) gene was determined, one of the first non-mammalian cytokine gene structures to be elucidated. Initial genomic clones were amplified from chicken genomic DNA and were used to isolate a cosmid clone covering the entire gene for sequencing. The exon:intron structure of chicken ifn-γ is very similar to those of its mammalian homologues, with the exception of the third intron, which is markedly shorter in the chicken. The first exon contains both 5′ UTR and signal sequence and the first 22 aa of the mature protein. The remainder of the coding region lies in exons 2–4. Exon 4 also encodes the stop codon and the 3′ UTR, including two possible polyadenylation signals. A number of potential regulatory sequences similar to those found in mammals have been identified, in the promoter, in each intron and in the 3′ UTR. In the promoter, these include the TATAATA- and CCAT-boxes, a consensus GATA motif in the reverse orientation and a potential NF-κB binding site. Other regulatory elements identified in the promoters of mammalian ifn-γ genes are absent. Internal to the gene structure, regulatory sequences identified include elements found in the DNase I hypersensitivity region of the first intron of the human ifn-γ gene and several potential NF-κB binding sites. The 3′ UTR contains an AT-rich sequence, including nine repeats of the `instability' motif ATTTA. As in mammals, chicken ifn-γ is a single copy gene. The gene is highly conserved, with no polymorphisms yet identified using either RFLP or SSCP in the coding region. However, promoter sequence polymorphisms between different inbred lines of chickens have been identified, with possible links to disease resistance.     

Differential roles for NF-kappa B in endotoxin and oxygen induction of interleukin-8 in the macrophage

The alveolar macrophage is an important source of interleukin (IL)-8 during pulmonary injury. The IL-8 gene promoter sequence contains nuclear factor (NF)-kappa B, NF-IL6, and activator protein (AP)-1 binding sequences. These sites may have differing regulatory roles in hyperoxia-exposed macrophages than in those stimulated by bacterial lipopolysaccharide (LPS). U-937 and THP-1 macrophage-like cells were exposed to air-5% CO2 or 95% O2-5% CO2, with or without 1.0 microg/ml of LPS, and transfected with an IL-8 promoter-reporter containing NF-kappa B, NF-IL6, or AP-1 mutations. Hyperoxia and LPS caused additive increases in IL-8 production by U-937 cells, whereas THP-1 cells responded only to LPS. An NF-kappa B mutation ablated baseline and O2- and LPS-stimulated reporter activity in both cell lines, whereas NF-IL6 mutations had little effect. An AP-1 mutation had an intermediate effect. LPS, but not hyperoxia, stimulated nuclear translocation of NF-kappa B in both cell lines. Pharmacological blockade of NF-kappa B nuclear translocation ablated LPS-, but not hyperoxia-, stimulated IL-8 production. Although an intact promoter NF-kappa B site is crucial to macrophage IL-8 production, only LPS-stimulated production appears to require additional nuclear translocation of NF-kappa B.     

cDNA and structural organization of the gene Pole1 for the mouse DNA polymerase epsilon catalytic subunit.

The cDNA and the gene for the mouse DNA polymerase epsilon catalytic subunit were cloned. The deduced protein sequence shows remarkable evolutionary conservation in DNA polymerase epsilon family. However, several conserved elements involved in template-primer binding differ from those of other class B polymerases. This is likely to reflect a distinctive function of the enzyme. The gene that was assigned to chromosome 5 region E3-E5, consists of 49 exons and has a non-conforming splice site in the junction of exon and intron 13. A CpG island covers the promoter region which contains several putative consensus elements critical for S phase upregulated and serum responsive promoters.     

Modulation of exon skipping by high-affinity hnRNP A1-binding sites and by intron elements that repress splice site utilization

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5' splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5' splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3' splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.     

Promoter analyses of SCC antigen genes

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5'-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5'-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5'-flanking region, the analysis of the -337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.