DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

Mapping and nucleotide sequence of the vaccinia virus gene that encodes a 14-kilodalton fusion protein.

A library of rabbit poxvirus DNA fragments contained in the expression cloning vector lambda gt11 was screened with monoclonal antibodies that react specifically against a 14-kilodalton envelope protein of vaccinia virus and rabbit poxvirus. The 14-kilodalton protein appears to play an important role in virus penetration at the level of cell fusion; it also elicits neutralizing antibodies, and it forms covalently linked trimers on the surface of virions and in infected cells (Rodriguez et al., J. Virol. 56:482-488, 1985; Rodriguez et al., J. Virol. 61:395-404, 1987). Two recombinant bacteriophages expressing beta-galactosidase fusion proteins were isolated. Restriction enzyme analysis and hybridization studies mapped the 14-kilodalton encoding sequences in the middle of vaccinia virus HindIII A DNA fragment. Nucleotide sequence analysis revealed an open reading frame (ATG) preceded by a characteristic TAA sequence of late genes. The sequence spans 330 nucleotides and codes for a protein with a molecular weight of 12,500 and an isoelectric point of 6.3. There are two small hydrophobic regions, one at the C terminus (11 amino acids) and the other at the N terminus (5 amino acids). The protein contains two cysteines for oligomer formation and one glycosylation site. Inspection of the deduced amino acid sequence of the 14-kilodalton protein revealed consensus sites with the hemagglutinin precursor of influenza A virus and with adenylate kinase and cytochrome c of various species.     

Mapping the gene expression universe

Methods to globally survey gene expression provide valuable insights into gene function during development. In particular, comprehensive in situ hybridization studies have demonstrated that gene expression patterns are extraordinarily diverse and new imaging techniques have been introduced to capture these patterns with higher resolution at the tissue, cellular, and subcellular levels. The analysis of massive image databases can be greatly facilitated by computer vision techniques once annotated image sets reach the crucial mass sufficient to train the computer in pattern recognition. Ultimately, genome-wide atlases of gene expression during development will record gene activity in living animals with at least cellular resolution and in the context of morphogenetic events. These emerging datasets will lead to great advances in the field of comparative genomics and revolutionize our ability to decipher and model developmental processes for a variety of organisms.     

NFkappaB negatively regulates interferon-induced gene expression and anti-influenza activity

Interferons (IFNs) are antiviral cytokines that selectively regulate gene expression through several signaling pathways including nuclear factor kappaB(NFkappaB). To investigate the specific role of NFkappaB in IFN signaling, we performed gene expression profiling after IFN treatment of embryonic fibroblasts derived from normal mice or mice with targeted deletion of NFkappaB p50 and p65 genes. Interestingly, several antiviral and immunomodulatory genes were induced higher by IFN in NFkappaB knock-out cells. Chromatin immunoprecipitation experiments demonstrated that NFkappaB was basally bound to the promoters of these genes, while IFN treatment resulted in the recruitment of STAT1 and STAT2 to these promoters. However, in NFkappaB knock-out cells IFN induced STAT binding as well as the binding of the IFN regulatory factor-1 (IRF1) to the IFN-stimulated gene (ISG) promoters. IRF1 binding closely correlated with enhanced gene induction. Moreover, NFkappaB suppressed both antiviral and immunomodulatory actions of IFN against influenza virus. Our results identify a novel negative regulatory role of NFkappaB in IFN-induced gene expression and biological activities and suggest that modulating NFkappaB activity may provide a new avenue for enhancing the therapeutic effectiveness of IFN.     

Nucleotide sequence of a DNA fragment that contains the abi gene of the ColIb plasmid

A 1989-bp PstI DNA fragment from the ColIb plasmid, which contains the abi gene that is necessary for the abortive response to infections by bacteriophage BF23 or T5, was sequenced. A candidate open reading frame for the abi gene has been suggested on the basis of a Shine-Dalgarno sequence appropriately placed ahead of its ATG initiation codon, a promoter upstream from the Shine-Dalgarno sequence, and a location compatible with deletion mapping. The polypeptide that would be coded by this open reading frame is 89 amino acids long and strongly hydrophobic. A promoter that could serve this open reading frame was detected by exonuclease III "footprinting" using RNA polymerase from uninfected Escherichia coli as the DNA-binding protein.     

DNA sequence of an amber replication mutant indicates that a 29 kd protein is the product of the F plasmid replication gene

Summary DNA sequencing shows that the mutational alteration resulting from an amber-suppressible replication-defective mutation of F plasmid is a single base pair change from C:G to T:A which yields an amber codon in the coding frame for a 29 kd polypeptide located in the minimal replication region. We thus identified the gene indispensable for F DNA replication as the coding frame which encodes a 29 kb polypeptide. We will designate this gene repA.Abbreviations kb kilobase pairs - bp base pair - kd kilodaltons     

DNA sequence of the F traALE region that includes the gene for F pilin

The complete sequence of a 1.4-kilobase PstI fragment containing the F transfer genes traA, -L, and -E is presented. The traA reading frame has been located both genetically and by comparing the primary structure of F pilin (the traA product) predicted by the DNA sequence to the amino acid composition and sequence of N- and C-terminal peptides isolated from purified F pilin. Taken together, these data show that there is a leader peptide of 51 amino acids and that F pilin contains 70 amino acids, giving molecular weights of 13,200 for F propilin and 7,200 for mature F pilin. Secondary structure predictions for F pilin revealed a reverse turn that precedes the sequence Ala-Met-Ala51, a classic signal peptidase cleavage site. The N-terminal alanine residue is blocked by an acetyl group as determined by 1H-nuclear magnetic resonance spectroscopy. The traL and traE genes encode proteins of molecular weights 10,350 and 21,200, respectively. According to DNA sequence predictions, these proteins do not contain signal peptide leader sequences. Secondary structure predictions for these proteins are in accord with traLp and traEp being membrane proteins in which hydrophobic regions capable of spanning the membrane are linked by sequences that form turns and carry positively charged residues capable of interacting with the membrane surface.