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PubMedDate not supplied

Structural features of nonionic polyglycol polymer molecules responsible for the protective effect in sparged animal cell bioreactors.

The nonionic surfactant Pluronic F-68 polyol is commonly used to protect cultured animal cells from the detrimental effects of sparging. In this study we investigated the structural features of the Pluronic F-68 molecule responsible for this protective behavior. Poly(oxyethylene)-poly(oxypropylene) block copolymer polyols of various molecular weights and percentages of hydrophobe (poly(oxypropylene], including both Pluronic and reverse Pluronic polyols, were considered. The potential toxicity of these agents was examined in the absence of sparging (i.e., in spinner flasks) by using the attachment-independent Sf9 insect cell line as a model system. Each polyol resulted in one of three distinct types of behavior in these spinner flask experiments: (1) cells lysed at an exponential rate, (2) inhibition of cell growth (i.e., no net cell growth), or (3) uninhibited cell growth. It was then shown that all of the Pluronic and reverse Pluronic polyols that did not inhibit cell growth provided protection from sparging in the bioreactors used in this study; thus, finding a polyol that protected cells was synonymous with finding one that did not inhibit cell growth. The ability of these polyols to protect animal cells in sparged bioreactors was found to correlate well with the hydrophilic-lipophilic balance (HLB). Those polyols with the largest HLB values were found to be protective agents. These poly(oxyethylene)-poly(oxypropylene) polyols were also shown to be more effective protective agents than pure poly(oxyethylene); thus, the presence of the hydrophobe (poly(oxypropylene] is important in their ability to serve as protective agents.

Animals↗

PubMedDate not supplied

Microscopic visualization of insect cell-bubble interactions. I: Rising bubbles, air-medium interface, and the foam layer.

Through the use of microscopic, high-speed video technology, the interactions of two suspended insect cell lines, Trichoplusia ni (TN-368) and Spodoptera frugiperda (SF-9), with air and oxygen bubbles were studied. Events such as cell-bubble attachment, cell-bubble collision, cell transport into the foam layer, and trapping of cells in the foam layer are presented and discussed. Based on these observations and those in a companion paper (Chalmers, J. J.; Bavarian, F. Biotechnol. Prog. 1991, following paper in this issue) and the experimental and theoretical work of other researchers, several mechanisms of cell damage as a result of sparging are presented.

Air↗

PubMedDate not supplied

Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.

A novel insect cell line from Trichoplusia ni, BTI-Tn 5B1-4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein-Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28-fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5-fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment-dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1 L scales was optimized with respect to cell density using standard techniques for the growth of attachment-dependent cells. Roller bottles precoated with DEAE-based microcarriers and suspension cultures employing collagen-coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier-coated roller bottles or in suspension cultures using collagen-coated microcarriers at concentrations close to those observed in tissue culture flasks.

Antigens, Viral↗

PubMedDate not supplied

Insect cell hosts for baculovirus expression vectors contain endogenous exoglycosidase activity.

Four different insect cell lines that can be used as hosts for baculovirus infection were assayed for the presence of endogenous exoglycosidases. All four cell lines, derived from Spodoptera frugiperda, Trichoplusia ni, Bombyx mori, or Malacosoma disstria, contained N-acetyl-beta-glucosaminidase, N-acetyl-beta-galactosaminidase, beta-galactosidase, and sialidase activities. Exoglycosidase activities were found in cell lysates as well as cell-free supernatants from uninfected and wild-type baculovirus infected cells. Oligosaccharide analysis of cellular glycoproteins using lectins recognizing Gal beta 1, 3GalNAc, Gal beta 1, 4GlcNAc, and NeuAc alpha 2,6Gal demonstrated that only Gal beta 1,3GalNAc was present. The demonstration that these cells contain exoglycosidases raises the possibility that the oligosaccharides of baculovirus-expressed glycoproteins are subject to enzymatic degradation.

Acetylglucosaminidase↗

PubMedDate not supplied

Effect of culture conditions on the degree of sialylation of a recombinant glycoprotein expressed in insect cells.

Secreted human placental alkaline phosphatase (SEAP) was produced in a nonengineered Trichoplusia ni insect cell line, Tn-4s, using a recombinant Autographa californica baculovirus expression vector. The effect of culture conditions on SEAP specific yield and glycosylation was studied. When cultured in the high aspect ratio vessel (HARV) or in tissue culture flasks (T-flasks), baculovirus-infected Tn-4s cells produced high levels of SEAP (13 and 23 U/10(6) cells, respectively; 4 days postinfection), but in those conditions SEAP possessed only high mannose, paucimannosidic, and hybrid structures. In spinner flasks, lower SEAP yields were obtained (<4 U/10(6) cells, 3 days postinfection), but in such cultures, sialylation of SEAP could be achieved. Several spinner-flask culture conditions were tested and resulted in different SEAP specific yields and levels of sialylation. The highest level of sialylation (9%) was obtained in the culture with the lowest agitation rate and lowest yield (1.2 U/10(6) cells), suggesting a limiting capacity of the Tn-4s cells to process glycoproteins to sialylation. High specific yield, low passage number Tn5B1-4 cells did not produce SEAP with complex glycosylation when cultured in a low agitation rate spinner-flask. On the basis of these results, we propose that the Golgi apparatus has a limited capacity for processing proteins to complex glycosylation and sialylation and that this capacity is easily overwhelmed by high levels of foreign protein productivity. Selected media additives such as Pluronic F-68, dextran sulfate (MW 12 500) and a lipids premix did not allow improvement of the specific yield of sialylated SEAP when supplemented to spinner-flask cultures.

Alkaline Phosphatase↗

PubMedDate not supplied

Production of a sialylated N-linked glycoprotein in insect cells: role of glycosidases and effect of harvest time on glycosylation.

Using a nonengineered Trichoplusia ni insect cell line, Tn-4s, infected with an Autographa californica recombinant baculovirus, 20% sialylation of human secreted placental alkaline phosphatase (SEAP) was observed. In contrast to this level of sialylation, intermediate complex forms with terminal galactose or N-acetylglucosamine were found in low proportions (<3% and <1%, respectively). We tested whether time of harvest or degradation of intermediate complex forms is responsible for this distribution of glycoforms. Spinner-flask cultures were infected with the SEAP baculovirus expression vector, and the cultures were harvested 48, 72, and 96 h post-infection. Structural analysis revealed that the glycoform distribution of SEAP was very similar at the different times of harvest, indicating that the cellular machinery was not significantly affected by the progress of infection and that the glycoforms obtained were stable. High levels of beta-galactosidase and N-acetylglucosaminidase activity were detected throughout infection. In contrast, sialidase activity was below detection level both in cell extracts and in supernatants. These levels of glycosidases activities raise the possibility that intermediate complex glycoforms may be degraded while sialylated forms should not experience significant degradation in this cell line. However, culture in the presence of extracellular beta-galactosidase and N-acetylglucosaminidase inhibitors did not significantly improve glycosylation, suggesting that extracellular degradation processes are not taking place. Instead, results suggest that the intracellular machinery of the Tn-4s cells tends to either shunt the glycans to paucimannosidic forms or drive them completely to sialylation.

Acetylglucosaminidase↗

PubMedDate not supplied

Insights into the central metabolism of Spodoptera frugiperda (Sf-9) and Trichoplusia ni BTI-Tn-5B1-4 (Tn-5) insect cells by radiolabeling studies.

The insect cell baculovirus expression vector system (BEVS) is one of the most commonly used expression systems for recombinant protein production. This system is also widely used for the production of recombinant virus and virus-like particles. Although several published reports exist on recombinant protein expression using insect cells, information dealing with their metabolism in vitro is relatively scarce. In this work we have analyzed the metabolism of glucose and glutamine, the main carbon and/or energy compounds, of the two most commonly used insect cell lines, Spodoptera frugiperda (Sf-9) and the Trichoplusia ni BTI-Tn-5B1-4 (Tn-5). Radiolabeled substrates have been used to determine the flux of glucose carbon entering the tricarboxylic acid cycle (TCA) and the pentose phosphate (PP) pathway by direct measurement of 14CO2 produced. The percentage of total glucose metabolized to CO2 via the TCA cycle was higher in the case of the Sf-9 (2.7%) compared to Tn-5 (0.6%) cells, while the percentage of glucose that is metabolized via the PP pathway was comparable at 14% and 16% for the two cell lines, respectively. For both cell lines, the remaining 83% of glucose is metabolized through other pathways generating, for example, lactate, alanine, etc. The percentage of glutamine oxidized in the TCA cycle was approximately 5-fold higher in the case of the Tn-5 (26.1%) as compared to the Sf-9 cells (4.6%). Furthermore, the changes in the metabolic fluxes of glucose and glutamine in Tn-5-PYC cells, which have been engineered to express a cytosolic pyruvate carboxylase, have been studied and compared to the unmodified cells Tn-5. As a result of this metabolic engineering, significant increase in the percentage of glucose oxidized in the TCA cycle (3.2%) as well as in the flux through the PP pathway (34%) of the Tn-5-PYC were observed.

Animals↗

PubMedDate not supplied

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell suspension culture system: from shake flasks to a 20-L bioreactor.

Production of recombinant adeno-associated viral vectors using a baculovirus/insect cell system at various scales is presented. Shake flask studies were conducted to assess conditions to be used in bioreactors. Two insect cell lines, Trichoplusia ni (H5) and Spodoptera frugiperda (Sf9), were compared for their ability to produce rAAV-2 after infection with recombinant baculoviruses coding for the essential components of the vector. The effect of varying the ratio between individual baculoviruses and the effect of the overall multiplicity of infection (MOI), as well as the cell density at infection, were also examined. Infectious rAAV-2 particles were proportionally produced when increasing the individual MOI of BacRep virus up to 1.6. When equal amounts of each virus were used, a leveling effect occurred beyond an overall MOI of 5 and a maximum titer was obtained. Increasing the cell density at infection resulted in higher yields when infecting the cells in fresh medium; however, for the production of bioactive particles, an optimal peak cell density of approximately 1 x 10(6) cells/mL was observed without medium exchange. Infection in 3- and 20-L bioreactors was done at an overall MOI of 5 with a ratio of the three baculoviruses equal to 1:1:1. Under these conditions and infecting the cells in fresh medium, a total of approximately 2.2 x 10(12) infectious viral particles (bioactive particles) or 2.6 x 10(15) viral particles were produced in a 3-L bioreactor. Without replacing the medium at infection, similar titers were produced in 20 L. Our data demonstrates the feasibility of rAAV-2 production by BEVS at various scales in bioreactors and indicates that further optimization is required for production at high cell densities.

Animals↗

PubMedDate not supplied

Effects of long-term passaging of BTI-Tn5B1-4 insect cells on growth and recombinant protein production.

The BTI-Tn-5B1-4 insect cell line from Trichopulsi ni has enabled better secretion and higher productivity of recombinant proteins in the baculovirus expression system. An early passage stock of this cell line is compared here to the commercially available "High Five" cell line stock. The early passage Tn5B1-4 cells grew to over 11 x 10(6) cells/mL and expressed 2.5-fold more recombinant secreted alkaline phosphatase (SEAP) than the High Five cells. While the specific productivity on a cell number basis was only slightly higher, the early passage Tn5B1-4 cells maintained this productivity at higher cell densities, increasing their volumetric productivity to 50 microg/mL under elevated oxygen conditions. We propose that the difference in these two stocks of cells is the length of time they have been cultured, 130 total passages for the early passage versus 360 passages for the High Five cells. This conclusion is supported by the facts that the early passage Tn5B1-4 cells are smaller in diameter by 4 micron, smaller in cellular protein content (206 versus 270 microg of protein/10(6) cells), have an increased propensity to clump in suspension culture, and are less susceptible to dispersion of these clumps by dextran sulfate compared to the High Five cells.

Alkaline Phosphatase↗

PubMed2001

Effects of temperature on the susceptibility of insect cells to infection by baculoviruses.

Three insect cell lines were tested for susceptibility to baculovirus infection by use of a typical endpoint assay procedure. Cell lines from Spodoptera frugiperda (IPLB-Sf21AE), Lymantria dispar (IPLB-LdEIta), and Heliothis virescens (IPLB-HvE6s) in 96-well tissue culture plates were each infected with dilutions of extra cellular virus suspensions of the Autographa californica nucleopolyhedrovirus (AcMNPV). In addition, the L. dispar and H. virescens cells were also infected with L. dispar nucleopolyhedrovirus, and Helicoverpa zea nucleopolyhedrovirus, respectively. Each cell/virus combination was incubated at three temperatures: 22, 27 and 32 degrees C and wells were scored for positive infection (presence of occlusion bodies in cell nuclei) at 2 to 4 day intervals for up to 4 weeks. The resulting data were analyzed by the Spearman-Kärber method, providing virus titers for each combination of virus, cell line, and temperature. The results were categorized by accuracy (assuming the highest titer achieved was the most accurate) and by rapidity of maximum titer. AcMNPV reached the highest titer in each line at 22 degrees C although equivalent titers were reached with both AcMNPV and HzSNPV in the HvE6a line at all three temperatures. This line actually reported about 100-fold less AcMNPV than the other two lines with the same virus sample. Alternatively, the Sf21AE and LdEIta lines reached 10-fold higher titers at the lowest temperature as compared with the higher temperatures, although also at a slower rate.

Animals↗

PubMed1999

Biphasic modulation of GABA(A) receptor binding by steroids suggests functional correlates.

Neuroactive steroids and other positive modulators of GABA(A) receptors showed regional variation in both the efficacy and potency for modulation of [35S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA(A) receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [35S]TBPS binding was sensitive to the presence of the gamma2 subunit and the nature of the alpha subunit (alpha1 beta2 gamma2S > alpha1 beta2, alpha6 beta2, alpha6 beta2 gamma2S, and alpha6 beta2 delta). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.

Androstanes↗

PubMed2005

Baculovirus as versatile vectors for protein expression in insect and mammalian cells.

Today, many thousands of recombinant proteins, ranging from cytosolic enzymes to membrane-bound proteins, have been successfully produced in baculovirus-infected insect cells. Yet, in addition to its value in producing recombinant proteins in insect cells and larvae, this viral vector system continues to evolve in new and unexpected ways. This is exemplified by the development of engineered insect cell lines to mimic mammalian cell glycosylation of expressed proteins, baculovirus display strategies and the application of the virus as a mammalian-cell gene delivery vector. Novel vector design and cell engineering approaches will serve to further enhance the value of baculovirus technology.

Animals↗

PubMedDate not supplied

Cytotoxic cyplasin of the sea hare, Aaplysia punctata, cDNA cloning, and expression of bioactive recombinants in insect cells.

A 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a prefer ential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparent at nanomolar concentrations in a cell type-dependent manner. In contrast, injection of micromolar concentrations into mice is tolerated without apparent negative consequences. Microsequencing of the 56-kDa protein released a peptide sequence whose corresponding nucleotide sequence was used as probe to screen A. punctata RNA-based cDNA and to select cDNA clones encoding polypeptides comprising the target peptide. Two closely related cDNA were detected. The cDNA encoding a polypeptide 558 aa in length was considered to reflect a bonafide clone encoding the cytotoxic protein. Its protein-coding section was recloned in vectors suitable for expression in Escherichia coli, in mammalian cells, and in insect cells, respectively. The E. coli-expressed polypeptide was biologically inactive. Transfected mammalian cells expressed a cytotoxic factor and died thereof as if treated with the genuine cytotoxic protein. In contrast, transfected insect cells, which proved to be much less sensitive when treated with the genuine protein, expressed the cytotoxic factor and continued to proliferate, allowing to establish stable insect cell lines expressing sufficient amounts of the cytotoxic factor for further characterization.

Amino Acid Sequence↗

PubMed2001

Expression and characterization of recombinant human alpha-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system.

The human alpha-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4 unit/l (1 mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were k(cat)=0.4 s(-1) and K(m)=0.87 mM for the SFT3 secreted by Tn cells, and k(cat)=0.09 s(-1) and K(m)=0.76 mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.

Amino Acid Sequence↗

PubMed2005

Mannosamine supplementation extends the N-acetylglucosaminylation of recombinant human secreted alkaline phosphatase produced in Trichoplusia ni (cabbage looper) insect cell cultures.

A major limitation of the insect cell-baculovirus expression vector system is its poor capacity to perform complex glycosylation, since the glycoproteins produced are usually only of the high-mannose and paucimannose types. Nonetheless, recent evidence indicates that, under various conditions, some insect cell lines are capable of producing complex-type glycans. In the present study, we assessed the effects of supplementation with ManN (mannosamine) ManNAc (N-acetylmannosamine) and Cyt (cytidine) on the glycosylation of recombinant human secreted alkaline phosphatase produced by suspension cultures of Trichoplusia ni (cabbage looper) BTI-Tn5B1-4 cells. Addition of ManN in the range 5-20 mM resulted in a 10-fold increase of the terminal GlcNAc (N-acetylglucosamine) associated with the recombinant protein produced after baculovirus infection. Such an increase yielded a maximum of 12.5% hybrid glycans having terminal GlcNAc with respect to total N-linked glycans. In contrast, no changes in the glycan composition associated with recombinant human secreted alkaline phosphatase were observed on supplementation with up to 20 mM ManNAc or up to 1.5 mM Cyt. N-acetyl-beta-glucosaminidase activity in cell extracts was decreased on incubation with 20 mM ManN, but not with 20 mM ManNAc or 1.5 mM Cyt, indicating that the increased proportion of hybrid glycans obtained on the addition of ManN could be a result of N-acetyl-beta-glucosaminidase inhibition.

Alkaline Phosphatase↗

PubMed1988

Activation of insect cell adenylate cyclase by Bacillus thuringiensis delta-endotoxins and melittin. Toxicity is independent of cyclic AMP.

Insecticidal Bacillus thuringiensis (Bt) delta-endotoxins are cytolytic to a range of insect cell lines in vitro. Addition of Bt var. aizawai or var. israelensis toxins to Mamestra brassicae (cabbage moth) cells in vitro increased intracellular cyclic AMP, which was paralleled by activation of adenylate cyclase in isolated membranes. Var. kurstaki toxin, which does not lyse M. brassicae cells, had no effect on cyclic AMP concentrations in intact cells, but was able to stimulate adenylate cyclase in membrane preparations. In contrast, the bee-venom toxin melittin, which is also cytolytic, increased intracellular cyclic AMP in whole cells, but inhibited adenylate cyclase in isolated membranes. Octopamine and forskolin also increased cyclic AMP in cells, but were not cytolytic. When added to cells at concentrations exceeding their LC90 (concentration causing 90% cell death), melittin and var. israelensis toxins caused cell lysis without a concomitant increase in intracellular cyclic AMP. Taken together, these results suggest that activation of adenylate cyclase by cytolytic toxins is a secondary effect (related perhaps to interactions of these toxins with membrane lipids) and is neither necessary nor sufficient for cytolysis.

Adenylyl Cyclases↗

PubMed1990

Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1.

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.

Animals↗

PubMed1998

Transcription of the juvenile hormone esterase gene under the control of both an initiator and AT-rich motif.

The binding of transcription factors to the core promoter of the juvenile hormone esterase gene was functionally characterized using both a cell-free in vitro transcription functional assay and a cell transfection assay. A core JHE promoter (-61 to +28 bp relative to transcription start site) supported faithful transcription from the in vivo transcription start site. The nuclear extracts from the Sf9 insect cell line that provided transcription from that template also bound to that template as a probe in gel-mobility shift assays. Deletion or transversion of the initiator-binding motif (-1 to +4 bp) abolished detectable transcription either in vitro or in transfected cells. An AT-rich motif (ATATAT; -28 to -23 bp) serves another transcription factor-binding site. Mutation of the AT-rich motif to a canonical TATA-box preserved transcription, while either its deletion or complete transversion abolished or significantly reduced detectable transcriptional activity. These results indicate that, under these conditions, the functional operation of this core promoter approaches that of a composite promoter in which both the TATA- and initiator-binding protein complexes are necessary, even for basal transcription. On the other hand, these debilitating mutations to either the TATA box or initiator motif did not prevent the ability of the corresponding gel-shift competitive probes to compete with the wild-type promoter for binding by the transcription factors. Even a double transversion of both the AT-rich motif and the initiator-binding motif was able to competitively displace the protein complex that bound to the labelled wild-type probe. These data strongly indicate the presence of (an) additional core-promoter-associated transcription factor(s) (that is not the 'downstream element') that contact(s) the AT-binding complex and/or initiator-binding factor with sufficient avidity to remove them from binding to the competing wild-type promoter sequence.

Animals↗