Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.     

Escherichia coli genes expressed preferentially in an aquatic environment

We isolated genomic clones that contain the 5'-flanking region of the mouse activin beta A subunit gene. The nucleotide sequence determination of the 5'-flanking region of the gene and the comparison of that with the reported mouse cDNA structure identified the putative 5' regulatory region, a novel first exon and a part of the first intron of the gene within this region. The putative 5' regulatory region of the mouse activin beta A subunit gene directed the expression of CAT gene in transfected HT1080 cells. Successive deletions of this region demonstrated a 400-bp region that exerts a strong positive effect on promoter activity of the mouse activin beta A subunit gene.     

Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II

The insulin receptor gene is induced 8 to 10-fold during adipocyte differentiation. Plasmids containing the promoter, exon 1 and a portion of the first intron from either the mouse or human gene are able to modulate the expression of an insulin receptor/CAT gene 3 to 7-fold during differentiation. We have shown that several nuclear proteins from both preadipocyte and adipocyte nuclear extracts bind to two discrete sites within a 278-bp region in the 5' end of the first intron. Sequence comparison between the first intron of the human gene and the mouse gene shows two regions of sequence identity which correspond to the protein binding regions detected by DNase footprinting. One of these sites binds proteins that are enriched in adipocyte nuclear extracts and can be competed by adipose regulatory element, ARE6.     

The 5'-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus

The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5' noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions -131 to -100), which is highly conserved at the 5' end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncoding region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5'-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo.     

Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.     

Abnormal regulation of the Ke 6 gene, a new 17beta-hydroxysteroid dehydrogenase in the cpk mouse kidney

The function encoded by the Ke 6 gene has been recently determined to be 17beta-hydroxysteroid dehydrogenase. Previously, the abnormal expression of the Ke 6 gene has been intimately associated with development of recessive polycystic kidney disease. The Ke 6 gene is normally expressed at very high levels in the kidney and liver and is severely down regulated in all recessive murine models of polycystic kidney disease that have been examined to date. Here, we report a detailed examination of the promoter region of the Ke 6 gene in normal mouse kidney cells (CTA) and in cells derived from mouse kidneys homozygous for the cpk (congenital polycystic kidney) mutation, using transfection analysis and DNA-protein gel shift assays. The minimal promoter region, P1 (+1 to -96), and a putative enhancer site, P3 (-165 to -256), within the Ke 6 gene 5' flanking sequence have been identified. We have also identified another region, P2 (-97 to -165), that may be responsible for the lower promoter activity of the Ke 6 gene in cpk cells. Furthermore, absence of binding of a 38 kDa nuclear protein to a 16 bp sequence element (P1A) within the minimal promoter of the Ke 6 gene suggests that the P1A element could be responsible for the overall reduction in promoter function in cpk cells.