Housekeeping Na,K-ATPase alpha 1 subunit gene promoter is composed of multiple cis elements to which common and cell type-specific factors bind.

Na,K-ATPase alpha 1 subunit gene (ATP1A1) is one of the housekeeping genes involved in homeostasis of Na+ and K+ in all animal cells. We identified and characterized the cis-acting elements that regulate the expression of ATP1A1. The region between -155 and -49 was determined as a positive regulatory region in five cultured cell lines of different tissue origins (MDCK, B103, L6, 3Y1, and HepG2). The region was divided into three subregions: from -120 to -106 (including the Sp1 binding site), from -102 to -61, and from -58 to -49 (including an Sp1 consensus sequence). Cell type-specific factors binding to the middle subregion (from -102 to -61) were detected by gel retardation analysis, using nuclear extracts prepared from MDCK and B103 cells. Two gel retardation complexes were formed in the B103 nuclear extract, and three were formed in the MDCK nuclear extract. DNA binding regions of these factors were located at -88 to -69 and differed from each other in DNase I footprinting experiments. These factors also showed different binding characteristics in gel retardation competition and methylation interference experiments. The identified cis element was named the ATP1A1 regulatory element. The core sequence of this element is found in several other genes involved in cellular energy metabolism, suggesting that the sequence is a common regulatory element responsive to the state of energy metabolism.     

A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region.

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.     

Characterization of the promoter activity of a poxvirus conserved element

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.     

Nucleotide sequence of a staphylococcal plasmid gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin-like antibiotics

The nucleotide sequence of a 1883 bp fragment isolated from a resistance plasmid harbored by a Staphylococcus aureus clinical isolate and carrying the gene, vgb, encoding a hydrolase inactivating the B components of virginiamycin family has been determined. The sequence contains one open reading frame which extends from the ATG codon at nt 641 to a TGA codon at nt 1537 and which potentially codes for a protein of 33.035 Da. This value is in agreement with the apparent size (33 kDa) of the protein observed, in minicell extracts. Inactivation of the B components of the virginiamycin antibiotics as well as resistance to these antibiotics were expressed in a virginiamycin sensitive mutant of Escherichia coli recipient containing the gene on a high copy number plasmid.     

Interaction of cell and virus proteins with DNA sequences encompassing the promoter/regulatory and leader regions of the herpes simplex virus thymidine kinase gene

During the course of a productive infection with herpes simplex virus (HSV), gene expression is coordinately regulated in a cascade fashion. Three major kinetic classes of genes, termed alpha, beta, and gamma, are sequentially activated. The mechanism responsible for repression and subsequent activation of beta and gamma genes is not known. A mobility-shift electrophoresis assay was used to examine DNA fragments containing the promoter/regulatory and the mRNA leader regions of the thymidine kinase gene (TK, a model beta gene) for their ability to bind proteins present in nuclear extracts prepared from uninfected and infected cells. Specific complexes unique to each extract were formed. Using a monoclonal antibody specific for ICP4 (the major regulatory protein of HSV) we demonstrated that this protein is present in the complexes formed between probes encompassing either the promoter/regulatory or leader sequence DNAs and proteins in infected-cell extracts. These complexes formed despite the lack of a high affinity binding site for ICP4 in either of these regions. The stability of complexes formed in infected-cell extracts with DNA probes containing the promoter/regulatory, leader region, and a high affinity ICP4-binding site were compared by dissociation analysis. The relative kd(obs) for these DNA-protein complexes was in the order: TK-leader region much greater than TK-promoter/regulatory region greater than or equal to high affinity ICP4-binding site. Cu+/1,10-phenanthroline footprinting revealed that infected-cell complexes which form on a probe containing a high affinity ICP4-binding site generate a protection pattern, whereas those formed on a probe containing the TK-leader sequence do not. In contrast, complexes formed with the latter probe in extracts from uninfected cells are kinetically stable and refractile to cleavage. A model for activation of the TK gene which incorporates these results is presented.     

Binding sites for two novel phosphoproteins, 3AF5 and 3AF3, are required for rbcS-3A expression.

Previous studies of boxes II (-151 to -138) and III (-125 to -114), binding sites for the nuclear factor GT-1 within the -166 deleted promoter of the ribulose-1,5-bisphosphate carboxylase-3A (rbcS-3A) gene, suggested that GT-1 might act in concert with an additional protein to confer light-responsive rbcS-3A expression. In this work, S1 analysis of RNA isolated from transgenic tobacco plants carrying mutant rbcS-3A constructs led to the identification of two short sequences located at the 5' and 3' ends of box III that are required for expression. These two sequences serve as binding sites for two novel proteins, 3AF5 and 3AF3. Gel shift studies using tetramerized binding sites for both 3AF5 and 3AF3 showed that complexes with faster mobilities were formed using nuclear extracts prepared from dark-adapted plants compared with those from light-grown tobacco plants. Phosphatase treatment of extracts from light-grown plants resulted in the formation of complexes with faster mobility. Although the binding of 3AF3 to its target site is dependent upon phosphorylation, the binding of 3AF5 does not appear to be affected by its phosphorylation state. These results suggest that the phosphorylated forms of both 3AF5 and 3AF3 are required for -166 rbcS-3A expression but that the mechanisms differ by which phosphorylation regulates the activities of 3AF5 and 3AF3.     

Rapid Communication: A cis-Acting Element, Tα-1, in the Upstream Region of Rod α-Transducin Gene that Binds a Developmentally Regulated Retina-Specific Nuclear Factor

Abstract: The G protein transducin (T) is an integral component of the signal transduction pathway in photoreceptors. We have identified a cis -acting element, Ta-1, in the upstream region of the mouse rod a-T (T_rα) gene that may be important for tissue-specific expression. Tα-1 binds a retina-specific nuclear factor of apparent molecular mass of 90 kDa. Binding to the Tα-1 site is developmentally regulated and peaks between postnatal days 6 and 9. This corresponds to the time of rod photoreceptor maturation and the rise in T_rα gene expression. The sequence of Tα-1 shows homology with RET-1, a cis -acting element in the proximal promoter of opsin gene that binds a distinct retina-specific factor. Tα-1 and RET-1 sequences may have been derived from a prototype Tα-1/RET-1 sequence, evolved to confer photoreceptor specificity on retina-specific genes.     

Identification of hepatocyte growth factor activator inhibitor-1B as a potential physiological inhibitor of prostasin

Prostasin is a trypsin-like serine protease that is glycosylphosphatidylinositol-anchored to the epithelial cell surface, from where it can be released in a soluble form. We undertook a co-expression search using the Genesis Enterprise System Database from Gene Logic to identify prostasin inhibitors, on the assumption that prostasin and its natural inhibitors may have a similar gene expression pattern. We found the expression profile of prostasin in normal human tissues to correlate highly with hepatocyte growth factor activator inhibitor-1B (HAI-1B) and its splice variant HAI-1. Soluble HAI-1B (sHAI-1B), comprising the entire extracellular domain, formed a 1:1 complex with purified prostasin in protein binding assays and inhibited prostasin enzymatic activity with an IC(50) of 66 +/- 15 nM. Two sHAI-1B mutants with inactivated N- and C-terminal Kunitz domains (KD1 and KD2) were used to show that the interaction of sHAI-1B with prostasin is mediated by KD1. In agreement, KD1 (Thr(246)-Val(303)) alone potently inhibited prostasin activity (IC(50) = 4.7 +/- 0.5 nM). Furthermore, prostasin was isolated with two major HAI-1/1B fragments (40 and 58 kDa) from OVCAR3 cell medium, demonstrating that prostasin.HAI-1/1B complexes are formed naturally. Moreover, when prostasin and HAI-1B were co-expressed in Chinese hamster ovary cells, complexes of prostasin with HAI-1B were detected on the cell membrane as well as in the culture medium, suggesting that preformed complexes were shed from the cell surface. The identification of HAI-1B as a potential physiological regulator of prostasin function, as described herein, may further the investigation of the role of prostasin in normal physiology and cancer.