Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states.

Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases.

Ligands presumed to label high affinity and low affinity ATP binding sites do not interact in an (alpha beta)2 diprotomer in duck nasal gland Na+,K+-ATPase, nor Do the sites coexist in native enzyme.

The interaction of ligands deemed to be ATP analogues with renal Na(+),K(+)-ATPase suggests that two ATP binding sites coexist on each functional unit. Previous studies in which fluorescein 5-isothiocyanate (FITC) was used to label the high affinity ATP site and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was used to probe the low affinity site suggested that the two sites coexist on the same alphabeta protomer. Other studies in which FITC labeled the high affinity site and erythrosin-5-isothiocyanate (ErITC) labeled the low affinity site led to the conclusion that the high and low affinity sites exist on separate interacting protomers in a functional diprotomer. We report here that at 100% inhibition of ATPase activity by FITC, each alphabeta protomer of duck nasal gland enzyme has a single bound FITC. Both TNP-ADP and ErITC interact with FITC-bound protomers, which unambiguously demonstrates that putative high and low affinity ATP sites coexist on the same protomer. In unlabeled nasal gland enzyme, TNP-ADP and ErITC inhibit both ATPase activity and p-nitrophenyl phosphatase activity, functions attributed to the putative high and low affinity ATP site, respectively, by interacting with a single site with characteristics of the high affinity ATP binding site. In FITC-labeled enzyme, TNP-ADP and ErITC inhibit p- nitrophenyl phosphatase activity but at much higher concentrations than with the unmodified enzyme. Low affinity sites do not exist on the unmodified enzyme but can be detected only after the high affinity site is modified by FITC.

Alphabeta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: formation of higher oligomers does not modify molecular activity.

The distance that separates alphabeta protomers of the Na(+), K(+)-ATPase in microsomes and in purified membranes prepared from duck nasal salt glands was estimated by measuring fluorescence resonance energy transfer between anthroylouabain bound to a population of alphabeta protomers and either N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain or 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain bound to the rest. Energy transfer between probes bound in the microsomal preparation was less than in the purified membranes. The efficiency of energy transfer between anthroylouabain and N-[7-nitrobenz-2-oxa-1, 3-diazol-4-yl]-6-aminohexyl ouabain was 29.2% in the microsomes compared with 62.6% in the purified preparation. Similar results were obtained with 5-(and-6)-carboxyfluorescein-6-aminohexyl ouabain as acceptor. We calculate that either the protomer bound probes were on the average 13 A farther apart in the microsomes than in the purified membranes, or that 53% of the protomers are monomeric in the microsome preparation. Microsomes prepared in the presence of phalloidin (a toxin that binds to F actin and stabilizes the actin-based cytoskeleton) showed less quench than those prepared in its absence. The data support the hypothesis that protomers are kept apart by their association with the cytoskeleton. The turnover rate while hydrolyzing ATP is the same in the microsomal and purified preparations; higher oligomer formation has no significant effect on the enzyme reaction mechanism.

Invasion and intracellular survival of Burkholderia cepacia.

Burkholderia cepacia has emerged as an important pulmonary pathogen in immunocompromised patients and in patients with cystic fibrosis (CF). Little is known about the virulence factors and pathogenesis of B. cepacia, although the persistent and sometimes invasive infections caused by B. cepacia suggest that the organism possesses mechanisms for both cellular invasion and evasion of the host immune response. In this study, cultured human cells were used to analyze the invasion and intracellular survival of B. cepacia J2315, a highly transmissible clinical isolate responsible for morbidity and mortality in CF patients. Quantitative invasion and intracellular growth assays demonstrated that B. cepacia J2315 was able to enter, survive, and replicate intracellularly in U937-derived macrophages and A549 pulmonary epithelial cells. Transmission electron microscopy of infected macrophages confirmed the presence of intracellular B. cepacia and showed that intracellular bacteria were contained within membrane-bound vacuoles. An environmental isolate of B. cepacia, strain J2540, was also examined for its ability to invade and survive intracellularly in cultured human cells. J2540 entered cultured macrophages with an invasion frequency similar to that of the clinical strain, but it was less invasive than the clinical strain in epithelial cells. In marked contrast to the clinical strain, the environmental isolate was unable to survive or replicate intracellularly in either cultured macrophages or epithelial cells. Invasion and intracellular survival may play important roles in the ability of virulent strains of B. cepacia to evade the host immune response and cause persistent infections in CF patients.

Role of polyamine structure in inhibition of K+-Cl- cotransport in human red cell ghosts.

1. K+-Cl- cotransport in human red cell ghosts is inhibited by divalent inorganic cations, soluble polycations and amphipathic organic cations. These findings suggest a common mechanism of inhibition, namely, binding of the cations to negative charges at the surface of a hydrophobic structure. 2. We have characterized the inhibitory capacity of a number of polyamines in order to obtain information about the nature of the charges with which they interact. Neomycin inhibited swelling-stimulated cotransport. The diquaternary amines dimethonium and decamethonium were relatively ineffective inhibitors. These compounds are thought to shield negative charges, but not bind to them. 3. Comparison of a homologous series of polyamines indicated that primary amines were better inhibitors than secondary amines, that inhibition increased with the charge of the polyamine, and that inhibition increased as the distance separating the amines increased. 4. The results indicate that the negative charges to which polycations bind are multiple and mobile. Since they must be associated with a hydrophobic environment, it is likely that they are negatively charged phospholipids located in the inner leaflet of the bilayer membrane. 5. Heating red cells or ghosts to 49 C denatures spectrin. Heating markedly increased K+ uptake in swollen ghosts but not in shrunken ghosts. The increase in uptake was reversed when swollen ghosts were shrunk even though denaturation of spectrin was not reversed. Polyamines, which inhibited swelling-activated K+ uptake in control ghosts, similarly inhibited the increased uptake in heated ghosts. 6. We speculate that spectrin, which is closely associated with the inner bilayer leaflet, shields negative charges in a volume-dependent manner and so regulates volume-sensitive K+ transport.

Membrane channel formation by antimicrobial protegrins.

Protegrins are small, arginine- and cysteine-rich, beta-sheet peptides with potent activity against bacteria, fungi, and certain enveloped viruses. We report that protegrins form weakly anion-selective channels in planar phospholipid bilayers, induce potassium leakage from liposomes and form moderately cation-selective channels in planar lipid membranes that contain bacterial lipopolysaccharide. The disruption of microbial membranes may be a central attribute related to the host defense properties of protegrins.

Preparation of Na+,K+-ATPase with near maximal specific activity and phosphorylation capacity: evidence that the reaction mechanism involves all of the sites.

The phosphorylation capacity of Na+,K+-ATPase preparations in common use is much less than expected on the basis of the molecular weight of the enzyme deduced from cDNA sequences. This has led to the popularity of half-of-the-sites or flip-flop models for the enzyme reaction mechanism. We have prepared Na+,K+-ATPase from nasal salt glands of salt-adapted ducks which has a phosphorylation capacity and specific activity near the theoretical maxima. Preparations with specific activities of >60 micromol (mg of protein)-1 min-1 at 37 degrees C had phosphorylation capacities of >60 nmol/mg of protein, and the rate of turnover of the enzyme was 9690 min-1, within the range reported for the enzyme from other sources. The fraction of the maximal specific activity of the enzyme compared well with the fraction of the protein on SDS-PAGE which was alpha and beta chains, especially at the highest specific activity which indicates that all of the alphabeta protomers are active. The gels of the most reactive preparations contained only alpha and beta chains, but less active preparations contained a number of extraneous proteins. The major contaminant was actin. The preparation did not contain any protein which migrated in the molecular weight range of the gamma subunit. The subunit composition of the enzyme was alpha1 and beta1 only. This is the first report of a pure, homogeneous, fully active preparation of the protein. Reaction models which incorporate a half-of-the-sites or flip-flop mechanism do not apply to this enzyme.

Evaluation of an intervention to change attitudes toward date rape.

The prevalence of date rape among college students is a major concern. Although much research has been done on risk factors for date rape, few researchers have specifically described interventions for the various stages of developing a date-rape prevention program. Previous programs have often relied on educational videos that feature a "typical" date-rape scenario, a format that some researchers suggest may have a negative effect on the way people engage in aggressive sexual behavior. A less violent theatrical production based on social learning theory and risk-factor reduction that resulted in a significant improvement in attitudes related to date rape among both male and female students at an elite Texas university is described.

Role of the DR2 repeat array in the regulation of the ICP34.5 gene promoter of herpes simplex virus type 1 during productive infection.

Previous analyses using transient transfection assays indicated that the promoter for the gene encoding the herpes simplex virus type 1 (HSV-1) neurovirulence protein ICP34.5 can be divided into an essential core region of approximately 80 bp and two potent upstream silencer domains corresponding to the DR2 and DR6 repeat arrays. In order to examine the potential role of transcriptional silencing during productive HSV-1 infection, recombinant viruses were generated in which wild-type or mutant ICP34.5 promoters controlling the expression of a chloramphenicol acetyltransferase reporter gene were inserted into the thymidine kinase gene of the viral genome. The intact promoter in the virus HSV-delta1CAT exhibited delayed-early kinetics of expression that were comparable to those of the ICP34.5 gene promoter at its native site in the genome. Deletion of the core promoter domain eliminated promoter activity in the virus HSV-delta5CAT, indicating that this region was required for expression not only in transient transfections assays but also in the context of the viral genome. However, deletion of the DR2 repeat array from the ICP34.5 promoter in the virus HSV-delta7CAT was found to increase promoter activity only minimally at late times, and even to reduce activity at early times. Thus, in marked contrast to its behaviour in transient expression assays, the DR2 repeat array does not appear to act as a transcriptional silencer in the context of the HSV-1 genome during productive infection.

DNA replication promotes high-frequency homologous recombination during Autographa californica multiple nuclear polyhedrosis virus infection.

The relative ease with which foreign genes can be incorporated into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) indicates that a highly efficient recombinational process exists within infected cells. However, it is unclear whether this is due to marker transfer mediated by host cell enzymes or recombination events promoted by AcMNPV itself. To address the latter possibility, a pair of inverted repeat IS50 elements derived from the bacterial transposon Tn5 was inserted into the polyhedrin gene locus of the AcMNPV genome. Inversion of Tn5 sequences arising from recombination between its IS50 repeats could be readily detected in this virus, indicating that AcMNPV DNA undergoes high-frequency recombination during infection. To further characterize this process, a transient recombination assay was developed and used to identify the cis- and trans-acting requirements for Tn5 inversion in AcMNPV. A transfected Tn5-containing plasmid was found to undergo the same sequence inversion events seen in the viral genome, but only if it also contained a putative AcMNPV origin of replication (homologous region 2) in cis and was replicated by AcMNPV gene products supplied in trans. Taken together, these results indicated that recombination events which occur in infected cells were strictly dependent upon AcMNPV-mediated DNA replication. Direct support for this hypothesis was provided by the observation that the minimal set of AcMNPV genes that was essential for plasmid DNA replication also promoted recombination events leading to Tn5 inversion in the absence of any other viral function. Finally, using a panel of deletion mutants of the IS50 elements in Tn5, sequence inversion was shown to be the result of homologous rather than site-specific recombination, since it occurred independently of a discrete sequence within the transposon. These results demonstrate that the AcMNPV DNA replication machinery exhibits a strong propensity to promote homologous recombination events during infection and is likely to play a role in the high frequency of marker transfer observed in this virus.

Replication of simian virus 40 origin-containing DNA during infection with a recombinant Autographa californica multiple nuclear polyhedrosis virus expressing large T antigen.

Autographica californica multiple nuclear polyhedrosis virus (AcMNPV) has been shown to encode many of the enzymes involved in the replication of its own DNA. Although the AcMNPV genome contains multiple sets of reiterated sequences that are thought to function as origins of DNA replication, no initiator protein has yet been identified in the set of viral replication enzymes. In this study, the ability of a heterologous origin initiator system to promote DNA replication in AcMNPV-infected cells was examined. A recombinant AcMNPV that expressed the simian virus 40 (SV40) large T antigen was surprisingly found to induce the efficient replication of a transfected plasmid containing an SV40 origin. This replication was subsequently found to involve three essential components: (i) T antigen, since replication of SV40 origin-containing plasmids was not induced by wild-type AcMNPV which did not express this protein; (ii) an intact SV40 core origin, since deletion of specific functional motifs within the origin resulted in a loss of replicative abilities; and (iii) one or more AcMNPV-encoded proteins, since viral superinfection was required for plasmid amplification. Characterization of the replicated DNA revealed that it existed as a high-molecular-weight concatemer and underwent significant levels of homologous recombination between inverted repeat sequences. These properties were consistent with an AcMNPV-directed mode of DNA synthesis rather than that of SV40 and suggested that T antigen-SV40 origin complexes may be capable of initiating DNA replication reactions that can be completed by AcMNPV-encoded enzymes.

The a sequence is dispensable for isomerization of the herpes simplex virus type 1 genome.

The herpes simplex virus type 1 (HSV-1) genome consists of two components, L (long) and S (short), that invert relative to each other during productive infection to generate four equimolar isomeric forms of viral DNA. Recent studies have indicated that this genome isomerization is the result of DNA replication-mediated homologous recombination between the large inverted repeat sequences that exist in the genome, rather than site-specific recombination through the terminal repeat a sequences present at the L-S junctions. However, there has never been an unequivocal demonstration of the dispensability of the latter element for this process using a recombinant virus whose genome lacks a sequences at its L-S junctions. This is because the genetic manipulations required to generate such a viral mutant are not possible using simple marker transfer, since the cleavage and encapsidation signals of the a sequence represent essential cis-acting elements which cannot be deleted outright from the viral DNA. To circumvent this problem, a simple two-step strategy was devised by which essential cis-acting sites like the a sequence can be readily deleted from their natural loci in large viral DNA genomes. This method involved initial duplication of the element at a neutral site in the viral DNA and subsequent deletion of the element from its native site. By using this approach, the a sequence at the L-S junction was rendered dispensable for virus replication through the insertion of a second copy into the thymidine kinase (TK) gene of the viral DNA; the original copies at the L-S junctions were then successfully deleted from this virus by conventional marker transfer. The final recombinant virus, HSV-1::L-S(delta)a, was found to be capable of undergoing normal levels of genome isomerization on the basis of the presence of equimolar concentrations of restriction fragments unique to each of the four isomeric forms of the viral DNA. Interestingly, only two of these genomic isomers could be packaged into virions. This restriction was the result of inversion of the L component during isomerization, which prevented two of the four isomers from having the cleavage and encapsidation signals of the a sequence in the TK gene in a packageable orientation. This phenomenon was exploited as a means of directly measuring the kinetics of HSV-1::L-S(delta)a genome isomerization. Following infection with virions containing just the two packaged genomic isomers, all four isomers were readily detected at a stage in infection coincident with the onset of DNA replication, indicating that the loss of the a sequence at the L-S junction had no adverse effect on the frequency of isomerization events in this virus. These results therefore validate the homologous recombination model of HSV-1 genome isomerization by directly demonstrating that the a sequence at the L-S junction is dispensable for this process. The strategy used to remove the a sequence from the HSV-1 genome in this work should be broadly applicable to studies of essential cis-acting elements in other large viral DNA molecules.

Calcium stimulation of procoagulant activity in human erythrocytes. ATP dependence and the effects of modifiers of stimulation and recovery.

The human erythrocyte membrane is generally considered to have no procoagulant activity. The normal membrane is characterized as having an asymmetric distribution of phospholipid species such that negatively charged and aminophospholipids are predominantly located on the inner leaflet of the membrane bilayer. Elevation of cytoplasmic Ca2+ in erythrocytes produces an assortment of biochemical and structural responses that include diminished phospholipid asymmetry and an elevation in procoagulant activity. Maintenance of the normal asymmetric distribution of phospholipid species is believed to be largely mediated by a phospholipid translocase mechanism. We have utilized a recently developed single-step kinetic assay of procoagulant activity to investigate the mechanisms of Ca2+ stimulation of procoagulant activity and recovery from the procoagulant state upon removal of Ca2+. This study demonstrated that stimulation of procoagulant activity by elevated cytoplasmic Ca2+ is greatly diminished in ATP-depleted erythrocytes. Phospholipid translocase inhibitors failed to fully inhibit recovery from the procoagulant state after removal of Ca2+. The data indicate that recovery of endogenous lipid from a procoagulant cofiguration may not be entirely mediated by the phospholipid translocase. Additionally, the data are inconsistent with the phospholipid translocase mediating the Ca(2+)-induced elevation of procoagulant activity, although the involvement of other protein(s) is indicated.

Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response.

Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.

Sterol 27-hydroxylase: high levels of activity in vascular endothelium.

Sterol 27-hydroxylase activity in bovine aortic endothelial (BAE) cells in culture has been compared with that in HepG2 cells and in Chinese hamster ovary (CHO) cells using identical culture conditions. The total enzyme activity of BAE cells (3.0 nmol/72 h per mg cell protein) was comparable with that of HepG2 cells (4.0 nmol/72 h per mg protein) and both values were significantly greater than that in CHO cells (0.002 nmol/72 h per mg protein). The enzyme was identified in the mitochondria extracted from BAE cells by Western blotting using an antibody of proven specificity, and its metabolites 27-hydroxycholesterol and 3 beta-hydroxy-5-cholestenoic acid were identified by mass spectrum analysis. The presence of the enzyme in endothelium provides a mechanism for preventing accumulation of intracellular cholesterol by initiating a pathway of bile acid synthesis different from that initiated by 7 alpha-hydroxylation of cholesterol in the liver.

Cloning and expression of an equine herpesvirus 1 origin-binding protein.

Equine herpesvirus 1 (EHV-1) is an important pathogen of horses and is closely related to several important human pathogens, herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and varicella-zoster virus. The EHV-1 genome contains open reading frames similar in sequence to the HSV-1 replication genes. PCR was used to clone EHV-1 gene 53, which is similar in sequence to the HSV-1 UL9 gene. The gene 53 product has regions of striking similarity to the HSV-1 UL9 and VZV gene 51 products. In vitro transcription and translation of this gene generated a protein of 87 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further characterization of this protein was accomplished through the use of gel shift analysis. The in vitro-synthesized protein bound sequence specifically to EHV-1 OriS as well as HSV-1 OriS. A site was used in gel shift analysis to show that the EHV-1 origin-binding protein bound to the same consensus site as the HSV-1 origin-binding protein, 5'-CGTTCGCACTT-3'. Using a nuclear extract of EHV-1-infected RK13 cells, we have identified an activity that interacts similarly with this consensus site. In gel shift assays, the retarded band arising from the nuclear extract migrated similarly to the retarded band arising from in vitro-translated EHV-1 gene 53. An N-terminal deletion of EHV-1 gene 53 was also created, expressed in vitro, and used in gel shift assays to localize the DNA-binding domain. Results of these experiments indicated that amino acids 1 to 499 were dispensable for binding and that the C-terminal fragment (amino acids 500 to 888) recognized the same consensus site as did the wild-type protein. Thus, the product of EHV-1 gene 53 is an origin-binding protein with a high degree of similarity to the HSV-1 and varicella-zoster virus origin-binding proteins and possibly serves as the initiator of DNA replication in EHV-1.

Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy.

Conversion to mucoidy, caused by the overproduction of the exopolysaccharide alginate in laboratory and cystic fibrosis strains of Pseudomonas aeruginosa, can occur via frameshift or nonsense mutations in the second gene of the algU mucA mucB cluster. The first gene of the cluster, algU, encodes a putative alternative sigma factor required for algD transcription. The algD gene encodes a critical alginate biosynthetic enzyme and is invariably activated in mucoid P. aeruginosa cells. To investigate the function of the genes controlling conversion to mucoidy, the wild-type algU mucA mucB cluster from the standard genetic strain PAO1 was used to reconstitute algD transcription in Escherichia coli. Transcription of an algD-lacZ chromosomal fusion in E. coli was detected upon introduction of plasmid-borne algU mucA mucB. Moreover, insertional inactivation of either mucA or mucB resulted in further stimulation of transcriptional activity from the algD promoter. This activation was dependent on algU, since a double algU mucA mutation abrogated transcription of algD. These experiments suggest that the phenotypic manifestations of muc mutations, i.e., increased algD expression and mucoid phenotype, depend on the presence of an active algU gene and that this regulator and the factors encoded by the downstream genes interact. Further support for these conclusions came from the investigations of the mechanism of reversion to nonmucoidy in P. aeruginosa, a phenomenon frequently referred to as the instability of mucoid phenotype. Spontaneous nonmucoid derivatives of the mucoid strain PAO578 carrying the mucA22 mutation were examined for the presence of alterations within the algU mucA mucB locus. Point mutations which inactivated algU were detected in some, but not all, nonmucoid revertants. No reversion of the original mucA22 mutation (a deletion of one C) was observed in any of the investigated strains. This observation suggests that the process of conversion to nonmucoidy ban be explained, at least partially, by second-site suppressor mutations and that a fraction of such mutations occurs in algU.

Overlapping domains on the p53 protein regulate its transcriptional activation and repression functions.

Wild-type p53 has been shown to inhibit transcription from several viral and cellular promoters without known p53-binding sites, while transactivating promoters with p53-binding sites. Using a series of N- and C-terminal p53 deletion mutants and wild-type p53, we have defined the domains on p53 responsible for its transcriptional functions. To test transcriptional activation by p53 we have used a promoter-chloramphenicol acetyltransferase (CAT) construct containing synthetic p53-binding sites. To check transcriptional inhibition by p53 we have used a human cytomegalovirus immediate-early promoter construct, CMV-CAT. Using transient transfection-transcription assays in Saos-2 cells, we determined that the p53 transcriptional activation and repression domains overlap at the N-terminus. This suggests the possibility that the same transcriptional machinery is involved in both functions. A C-terminal deletion up to amino acid 327 (del 393-327) eliminated repression of CMV-CAT, while preserving the transactivation function to a large extent. Using gluteraldehyde cross-linking experiments, we observed that the mutant del 393-327, which is transactivation-competent, but repression-defective, could not oligomerize. Thus, oligomerization of p53 is not required for transactivation, but may be essential for repression. Interestingly, transactivation by the oligomerization-defective mutant could be inhibited by cotransfection with a plasmid expressing the transforming mutant p53-175H.