Bidirectional Expression Research Articles

Generation of a polyclonal fab phage display library to the human breast carcinoma cell line BT-20.

We have previously described a vector system for generating recombinant polyclonal antibody libraries. This system uses bidirectional phagemid and mammalian expression vectors to facilitate mass transfer of selected variable light and variable heavy (VL-VH) region gene pairs from the phagemid to the mammalian vector, to express polyclonal libraries of whole IgG antibodies. We report here the first stage of generating a polyclonal antibody library to the human breast carcinoma cell line BT-20, using this vector system. VL and VH region gene pairs were obtained from a mouse immunized with BT-20 cells, and cloned, in opposite transcriptional orientations, in the bidirectional phagemid vector, to produce an Fab phage display library. This library was selected by panning on BT-20 cells and shown to bind specifically to BT-20 cells. Such libraries, after suitable negative selection to eliminate major reactivities against normal tissue, could be transferred in mass to our bidirectional mammalian expression vector for production of libraries of chimeric antibodies with mouse V regions and human constant (C) regions. These polyclonal antibody libraries will mediate effector functions and are expected to be useful for breast cancer therapy, as well as diagnosis.

P53 mutants have selective dominant-negative effects on apoptosis but not growth arrest in human cancer cell lines.

A bidirectional expression vector that allowed equal transcription of cloned wild-type and mutant p53 cDNAs from the same vector was developed. The vector was transfected into CaLu 6 lung carcinoma cells or Saos-2 osteosarcoma cells. All p53 mutants examined were recessive to wild-type p53 transactivation of p21(WAF1/CIP1) but dominant-negative for transactivation of Bax. An examination of effects on growth arrest and apoptotic pathways indicated that all mutants were recessive to wild type for growth arrest but only three of seven mutants were dominant negative for induction of apoptosis.

Efficient control of tetracycline-responsive gene expression from an autoregulated bi-directional expression vector.

The tetracycline-responsive expression system is based on the ability of the chimeric tTA and rtTA transactivators to stimulate specifically transcription from a companion synthetic CMV* or TK* promoter element, and can provide tightly regulated gene expression that can be induced up to five orders of magnitude in cultured cells and transgenic mice. A major problem with the system is that high level expression of the tTA or rtTA transactivators causes cellular toxicity. Under conditions of prolonged expression this results in selective pressure against the stable incorporation of vectors expressing the tTA or rtTA transactivators, and makes the generation of stable cell lines and transgenic mice problematic. In this report we describe the development of a set of autoregulated bi-directional expression vectors in which the weaker TK* promoter is used to direct expression of the rtTA or tTA transactivator and the stronger CMV* element is used to direct cDNA expression. In this format the transactivator and response elements are encoded on the same vector, which simplifies the system and ensures that gene expression is effectively skewed in favor of the cDNA while maintaining a continuously low level of transactivator expression. We find that such an autoregulated system works equally well for both the tTA and rtTA transactivators, provided that they contain a nuclear localization signal. Similar to other versions of the tetracycline-responsive expression system, gene expression is tightly regulated and can be efficiently switched between the off and on expression states by doxycycline. In contrast with other tetracycline-responsive systems, however, expression of the rtTA and tTA transactivators from the autoregulated TK* promoter is low enough such that there is no cellular toxicity associated with either expression state. By incorporating a selectable marker into these vectors, all of the components required for using the system are now contained on a single plasmid construct, and we find that this format provides a more reliable and greatly simplified method for the generation of stable cell lines.

The nine genes of the Nocardia lactamdurans cephamycin cluster are transcribed into large mRNAs from three promoters, two of them located in a bidirectional promoter region.

The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Assessment of aryl hydrocarbon receptor complex interactions using pBEVY plasmids: expressionvectors with bi-directional promoters for use in Saccharomyces cerevisiae.

The pBEVY (bi-directional expression vectors for yeast) plasmids were designed with constitutive and galactose-induced bi-directional promoters to direct the expression of multiple proteins in Saccharomyces cerevisiae . Using human estrogen receptor as a test gene, relatively balanced expression levels from each side of a bi-directional promoter were observed. Expression of a functional heterodimeric transcription factor composed of human aryl hydrocarbon receptor (Ahr) and aryl hydrocarbon receptor nuclear translocator (Arnt) proteins was accomplished using a single pBEVY plasmid. Previous studies suggest that inhibitory cross-talk between the estrogen receptor and the Ahr/Arnt complex may occur and that Hsp90-Ahr complex formation is important for Ahr-mediated signal transduction. Evidence for functional interaction among these proteins was investigated using pBEVY plasmids in a yeast system. No inhibitory cross-talk was observed in signaling assays performed with yeast that co-expressed Ahr, Arnt and estrogen receptor. In contrast, Ahr/Arnt-mediated signal transduction was reduced by 80% in a temperature-sensitive Hsp90 strain grown under non-permissive conditions. We conclude that pBEVY plasmids facilitate the examination of multiple protein interactions in yeast model systems.

Role of NRF-1 in bidirectional transcription of the human GPAT-AIRC purine biosynthesis locus.

GPAT and AIRC encode enzymes for steps one and six plus seven respectively in the pathway for de novo purine nucleotide synthesis in vertebrates. The human GPAT and AIRC genes are divergently transcribed from a 558 bp intergenic promoter region. Cis-acting sites and transcription factors important for bidirectional expression were identified. A cluster of sites between nt 215 and 260 are essential, although not sufficient, for expression of both genes. Two proteins from HepG2 cell nuclear extract, identified as NRF-1 and Sp1, bound to the promoter at sites within the 215-260 region. NRF-1 was required for stable binding of Sp1. Deletion of a 5'promoter region including nt 215-260 resulted in decreased expression of GPAT and AIRC in transfected HepG2 cells. The decreased expression was accounted for by point mutations in an NRF-1 site and either of two flanking sites for Sp1. These transcription factors account in part for the coordinated expression of human GPAT and AIRC.

The intergenic region between the divergently transcribed niiA and niaD genes of Aspergillus nidulans contains multiple NirA binding sites which act bidirectionally.

The niaD and niiA genes of Aspergillus nidulans, which code, respectively, for nitrate and nitrite reductases, are divergently transcribed, and their ATGs are separated by 1,200 bp. The genes are under the control of the positively acting NirA transcription factor, which mediates nitrate induction. The DNA binding domain of NirA was expressed as a fusion protein with the glutathione S-transferase of Schistosoma japonicum. Gel shift and footprint experiments have shown that in the intergenic region there are four binding sites for the NirA transcription factor. These sites can be represented by the nonpalindromic consensus 5'CTCCGHGG3'. Making use of a bidirectional expression vector, we have analyzed the role of each of the sites in niaD and niiA expression. The sites were numbered from the niiA side. It appeared that site 1 is necessary for the inducibility of niiA only, while sites 2, 3, and to a lesser extent 4 (which is nearer to and strongly affects niaD) act bidirectionally. The results also suggest that of the 10 binding sites for the AreA protein, which mediates nitrogen metabolite repression, those which are centrally located are physiologically important. The insertion of an unrelated upstream activating sequence into the intergenic region strongly affected the expression of both genes, irrespective of the orientation in which the element was inserted.

Conserved cis-elements bind a protein complex that regulates Drosophila ras2/rop bidirectional expression.

The Drosophila ras2 promoter region exhibits bidirectional activity, as has been demonstrated for the human c-Ha-ras1 and the mouse c-Ki-ras. Here we address a unique case of ras regulation, as Drosophila ras2 provides the only example to date in which the flanking gene (rop) and its product have been isolated. A linking mechanism of control suggests a mutual interaction between the two gene products. Our studies indicate that the Drosophila ras2 promoter region shares with the human c-Ha-ras1 promoter a CACCC box and an AP-1-like sequence. A 14 bp promoter fragment which holds a CACCC element is demonstrated to interact with a specific transcription factor (factor B). This CACCC promoter element represents a stretch of imperfect palindrome. We present evidence that this factor can form a complex with another specific DNA-binding protein (factor A). The binding sites (A + B) for these protein factors are essential for 95% expression of both genes flanking the promoter (ras2 and rop). Region A consists of four overlapping consensus sequences: a TATA-like element, a DSE-like motif (the core sequence of the serum response element), a DRE octamer, which has been shown to play a role in cell proliferation, and a 5 bp direct repeat representing the GATA consensus sequence. Factor A has a very weak affinity to the full promoter region, but when complexed with factor B binding efficiency is enhanced. We also show that alterations of DNA-protein binding specificities can be achieved by supplementing the growth media with different sera.

Expression of Human Carbonic Anhydrase in the Cyanobacterium Synechococcus PCC7942 Creates a High CO2-Requiring Phenotype

Active human carbonic anhydrase II (HCAII) protein was expressed in the cyanobacterium Synechococcus PCC7942 by means of transformation with the bidirectional expression vector, pCA. This expression was driven by the bacterial Tac promoter and was regulated by the IacIQ repressor protein, which was expressed from the same plasmid. Expression levels reached values of around 0.3% of total cell protein and this protein appeared to be entirely soluble in nature and located within the cytosol of the cell. The expression of this protein has dramatic effects on the photosynthetic physiology of the cell. Induction of expression of carbonic anhydrase (CA) activity in both high dissolved inorganic carbon (C(i)) and low C(i) grown cells leads the creation of a high C(i) requiring phenotype causing: (a) a dramatic increase in the K(0.5) (C(i)) for photosynthesis, (b) a loss of the ability to accumulate internal C(i), and (c) a decrease in the lag between the initial C(i) accumulation following illumination and the efflux of CO(2) from the cells. In addition, the effects of the expressed CA can largely be reversed by the carbonic anhydrase inhibitor ethoxyzolamide. As a result of the above findings, it is concluded that the CO(2) concentrating mechanism in Synechococcus PCC7942 is largely dependent on (a) the absence of CA activity from the cytosol, and (b) the specific localization of CA activity in the carboxysome. A theoretical model of photosynthesis and C(i) accumulation is developed in which the carboxysome plays a central role as both the site of CO(2) generation from HCO(3(-) ) and a resistance barrier to CO(2) efflux from the cell. There is good qualitative agreement between this model and the measured physiological effects of expressed cytosolic CA in Synechococcus cells.

Chimeric 3-hydroxy-3-methylglutaryl coenzyme A reductase-dihydrofolate reductase genes display bidirectional expression and unidirectional regulation in stably transfected cells.

We have constructed hybrid dihydrofolate reductase (DHFR) genes which are controlled by the sterol-responsive hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase promoter. Stable transfection frequencies of these chimeric templates into a DHFR-deficient Chinese hamster cell line indicate that the HMG CoA reductase promoter fragment confers DHFR transformation irrespective of its orientation relative to a downstream murine DHFR cDNA. Sterol-regulated levels of DHFR RNA and protein are detected from hybrid genes which carry a properly oriented promoter fragment. Constructions which invert this HMG CoA reductase promoter, however, generate DHFR RNA levels which do not respond to sterols. In the context of these transfected fusion genes, we present evidence of divergent opposite-strand transcription initiating from the HMG CoA reductase 5' fragment. In contrast, the endogenous HMG CoA reductase promoter region shows no apparent evidence of such bidirectional activity.

Chimeric 3-hydroxy-3-methylglutaryl coenzyme A reductase-dihydrofolate reductase genes display bidirectional expression and unidirectional regulation in stably transfected cells.

We have constructed hybrid dihydrofolate reductase (DHFR) genes which are controlled by the sterol-responsive hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase promoter. Stable transfection frequencies of these chimeric templates into a DHFR-deficient Chinese hamster cell line indicate that the HMG CoA reductase promoter fragment confers DHFR transformation irrespective of its orientation relative to a downstream murine DHFR cDNA. Sterol-regulated levels of DHFR RNA and protein are detected from hybrid genes which carry a properly oriented promoter fragment. Constructions which invert this HMG CoA reductase promoter, however, generate DHFR RNA levels which do not respond to sterols. In the context of these transfected fusion genes, we present evidence of divergent opposite-strand transcription initiating from the HMG CoA reductase 5' fragment. In contrast, the endogenous HMG CoA reductase promoter region shows no apparent evidence of such bidirectional activity.

Construction and application of plasmids containing bidirectional promoters of vaccinia virus.

A bidirectional expression vector containing both the 11KD late promoter (p11) and the presumptive 25KD early promoter (p25) was constructed. These bidirectional vectors have been applied to the expression of hepatitis B surface antigen by using one of the promoters for beta-galactosidase as the marker gene and the other one for hepatitis B surface antigen as the target gene.

69 THE HPRT PROMOTER

The human HPRT promoter, like that of other “house keeping” genes, lacks the TATA box transcriptional signal which is commonly found 20-30 nucleotides 5′ to the RNA start sites of most other characterized genes. Since several viral genes contain regions upstream from the TATA box that can be inverted without impairing transcription, we decided to study the effect of orientation on HPRT promoter activity. Recombinant plasmids containing the human HPRT promoter in either orientation upstream from the firefly luciferase (lux) reporter gene were transfected into HeLa cells and luciferase activity was assayed after 48 hours. Efficient expression of luciferase was detected, independent of the orientation of the HPRT promoter. Studies with other plasmids containing the HPRT promoter flanked by both the luciferase and the chloramphenicol acetyltransferase (CAT) genes demonstrated that the HPRT promoter could function bidirectionally, and drive expression of two reporter genes simultaneously. We are currently examining bidirectional activity of other promoters and determining the degree of bidirectional expression from the HPRT promoter in vivo.

Bidirectional reflectance spectroscopy: 2. Experiments and observations

Certain aspects of the theoretical bidirectional reflectance expression derived by Hapke [this issue] were studied experimentally. The theoretical expression satisfactorily describes the measured angular scattering and spectral properties of a cobalt glass powder whose absorptance and reflectance vary greatly with wavelength. The theoretical expression for the spectral single‐scattering efficiency for irregular particles derived in paper I is shown to describe the measured efficiency of the cobalt glass particles. The predicted approximately linear dependence of the bidirectional espat‐function W on the effective particle absorption optical thickness αDe is shown experimentally to be valid for αDe at least as high as 3. The theoretical reflectance function also describes measured distributions of intensity across the surfaces of planets, except near the sunlit limb, where the macroscopic roughness influences the brightness.

Bidirectional Scattering of Electromagnetic Waves from Rough Surfaces

A general bidirectional expression is derived from the Stratton-Chu-Silver integral for the scattering of electromagnetic waves from rough surfaces. This expression is then expanded in a power series of surface slope terms for the special case of scattering in the plane of incidence. The results of this expansion are then compared with other models and closed-form solutions are obtained for comparison with experiment.