CEACAM 6, a novel marker for the diagnosis of Barrett's esophagus.

Barrett's esophagus (BE) is a premalignant condition associated with the development of esophageal adenocarcinoma (EAC). Despite the low risk of progression to EAC, evidence highlights the notably poor survival rates of this malignancy. The mainstay form of diagnosis of BE is endoscopy and biopsy sampling. However, research emphasizes limitations with regards to the histological detection of BE and associated dysplasia. The aim of this study is to evaluate the clinical significance of CEACAM6 as a potential biomarker for the diagnosis of BE and beyond. Retrospective tissue samples were obtained from columnar lined esophagus without goblet cells (n = 27), BE (n = 18), BE associated dysplasia (n = 16), and EAC (n = 24). Standardized immunohistochemistry for CEACAM6 was performed followed by quantitative staining analysis. Statistical analysis across the BE spectrum for CEACAM6 was undertaken and a P value <0.05 was considered significant. CEACAM6 expression increased from columnar lined epithelium (CLE) to BE with a subsequent decrease to dysplasia and adenocarcinoma. The expression of CEACAM6 was significant from CLE to BE at p 0.001, CLE to dysplasia at p 0.001, BE to dysplasia at p 0.006, CLE to adenocarcinoma at p 0.001 and BE to adenocarcinoma at p 0.001. There was no significant difference in expression between dysplasia and adenocarcinoma (P = 0.15). Our findings highlight the increasing expression of CEACAM6 from CLE to BE with a subsequent decrease to dysplasia and adenocarcinoma. In view of this, we advocate the utilization of this marker for the enhanced diagnosis of BE and for the distinction of BE and dysplasia.

The cytomegalovirus gB/MF59 vaccine candidate induces antibodies against an antigenic domain controlling cell-to-cell spread.

Vaccination against human cytomegalovirus (CMV) infection remains high priority. A recombinant form of a protein essential for CMV entry, glycoprotein B (gB), demonstrated partial protection in a clinical trial (NCT00299260) when delivered with the MF59 adjuvant. Although the antibody titre against gB correlated with protection poor neutralising responses against the 5 known antigenic domains (AD) of gB were evident. Here, we show that vaccination of CMV seronegative patients induces an antibody response against a region of gB we term AD-6. Responses to the polypeptide AD-6 are detected in >70% of vaccine recipients yet in <5% of naturally infected people. An AD-6 antibody binds to gB and to infected cells but not the virion directly. Consistent with this, the AD-6 antibody is non-neutralising but, instead, prevents cell-cell spread of CMV in vitro. The discovery of AD-6 responses has the potential to explain part of the protection mediated by gB vaccines against CMV following transplantation.

Tailoring electron beams with high-frequency self-assembled magnetic charged particle micro optics.

Tunable electromagnets and corresponding devices, such as magnetic lenses or stigmators, are the backbone of high-energy charged particle optical instruments, such as electron microscopes, because they provide higher optical power, stability, and lower aberrations compared to their electric counterparts. However, electromagnets are typically macroscopic (super-)conducting coils, which cannot generate swiftly changing magnetic fields, require active cooling, and are structurally bulky, making them unsuitable for fast beam manipulation, multibeam instruments, and miniaturized applications. Here, we present an on-chip microsized magnetic charged particle optics realized via a self-assembling micro-origami process. These micro-electromagnets can generate alternating magnetic fields of about ±100 mT up to a hundred MHz, supplying sufficiently large optical power for a large number of charged particle optics applications. That particular includes fast spatiotemporal electron beam modulation such as electron beam deflection, focusing, and wave front shaping as required for stroboscopic imaging.

Ras and Raf pathways in epidermis development and carcinogenesis.

The epidermis is the outermost layer of the body and protects it from environmental insults. This crucial function is sustained by a continuous process of self-renewal involving the carefully balanced proliferation and differentiation of progenitor cells constantly replacing the mature cells at the surface of the epidermis. Genetic changes in the signalling pathways controlling keratinocyte proliferation and differentiation disrupt this balance and lead to pathological changes including carcinogenesis. This review discusses the role of Ras, an oncogene critically involved in the development of skin neoplasia, and its downstream effector Raf in epidermal homeostasis and tumourigenesis. In particular, we will focus on the recently established role of Raf-1 as the decisive element that, by restraining keratinocyte differentiation, allows the development and maintenance of Ras-driven tumours.

The myelin protein MAL affects peripheral nerve myelination: a new player influencing p75 neurotrophin receptor expression.

The myelin and lymphocyte protein (MAL) is a raft-associated membrane protein predominantly expressed by oligodendrocytes and Schwann cells. Here we show that MAL regulates myelination in the peripheral nervous system. In mice overexpressing MAL, myelination was retarded and fibers were hypomyelinated, whereas myelination in MAL knockout mice was accelerated. This was not due to impaired Schwann cell proliferation, differentiation or axonal sorting. We found that the expression level of p75 neurotrophin receptor mRNA and protein was strongly reduced in developing sciatic nerves in MAL-overexpressing mice. This reduction is well correlated with the observed alterations in myelination initiation, speed of myelination and alterations in Remak bundle development. Our results suggest a functional role for MAL in peripheral myelination by influencing the expression of membrane components that mediate axon-glia interaction during ensheathment and myelin wrapping.

Simultaneous characterization of phospho-proteins and cell cycle in activated T cell subsets.

Multi-colour flow cytometry is the only technological platform that can analyse the highly complex cellular composition of the immune system in parallel and at a single cell resolution. Analysis of the T cell compartment, in particular, requires the simultaneous measurement of multiple markers in order to account for lineage, phenotype and function. Flow cytometry also enables the analysis of intracellular signalling events. By combining the expression of surface markers, intracellular cytokines, phosphorylated versus unphosphorylated kinases, cell proliferation and DNA profile, mechanistic and kinetic information of subset-specific signalling may be obtained: this has not previously been achieved. Here we present a protocol which permits all of these aspects to be explored simultaneously. By comparing basic procedures previously described we were able to optimise different variables, including the choice of antibody/fluorochrome pairs, permeabilisation, fixation and labelling time, to obtain the best DNA staining of different cell types. We applied this method to study subset-specific signalling related to cytokine production and DNA synthesis in T cells responding to specific antigens.

Expression of the SNT-1/FRS2 phosphotyrosine binding domain inhibits activation of MAP kinase and PI3-kinase pathways and antiestrogen resistant growth induced by FGF-1 in human breast carcinoma cells.

Fibroblast growth factor (FGF) signaling can bypass the requirement for estrogen receptor (ER) activation in the growth of ER-positive (ER+) breast cancer cells. Fibroblast growth factor-1 stimulation leads to phosphorylation of the adaptor protein Suc1-associated neurotrophic factor-induced tyrosine-phosphorylated target (SNT-1) on C-terminal tyrosine residues, whereas it is constitutively bound through its N-terminal phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). By expressing the PTB domain of SNT-1 (SNT-1 PTB) in an inducible manner in an ER+ breast carcinoma line, ML20, we asked whether we could uncouple FGFR activation from its downstream signaling components and abrogate FGF-1-induced antiestrogen-resistant growth. Induction of SNT-1 PTB resulted in a significant decrease of FGF-1-dependent tyrosine phosphorylation of endogenous SNT-1, strong inhibition of complex formation between SNT-1, Gab-1 and Sos-1, and reduced activation of Ras, mitogen-activated protein kinase (MAP kinase), and Akt. SNT-1 PTB also inhibited the phosphorylation of p70S6K on Thr421/Ser424 and Ser411, which may result from the abrogation of MAP kinase activity. Moreover, we also observed a decreased phosphorylation of the MAP kinase-independent site Thr389. This may reflect both inhibition of PI-3 kinase pathways and mammalian target of rapamycin (mTOR)-dependent signaling, as the phosphorylation of Thr389 site was sensitive to treatment with the PI3-K and mTOR inhibitors, LY294002 and rapamycin, respectively. Collectively these results suggest that SNT-1 plays a pivotal role in FGF-dependent activation of the Ras-MAP kinase, PI-3 kinase, and mTOR pathways in these cells. Fibroblast growth factor-1 dependent colony formation of ML20 cells in media containing the pure antiestrogen ICI 182,780 was also markedly inhibited upon induction of SNT-1 PTB, suggesting that blockade of FGFR-SNT-1 interactions might abrogate FGF-mediated antiestrogen resistance in breast cancers.

Effects of dietary cholesterol on cholesterol and bile acid homeostasis in patients with cholesterol gallstones.

We examined changes in cholesterol and bile acid metabolism produced by dietary cholesterol in gallstone subjects and matched controls. Healthy women were recruited and, after confirming the presence or absence of radiolucent gallstones, they were studied on regular diets and again on the same diet supplemented with five eggs daily for 15-18 d. Studies included plasma lipids, lipoproteins and apolipoproteins, dietary records, cholesterol absorption, cholesterol synthesis, plasma clearance of chylomicron remnants, biliary lipid composition, and secretion and bile acid kinetics. On low cholesterol, gallstone subjects absorbed a slightly lower fraction of dietary cholesterol, synthesized more cholesterol, and had smaller bile acid pools and faster fractional turnover rate (FTR) of bile acids. On high cholesterol, the fraction of cholesterol absorbed decreased in both groups and cholesterol synthesis decreased, especially in the gallstone group. Biliary cholesterol secretion increased in the gallstone group only. FTR of bile acids did not change in either group. Bile acid synthesis and pool tended to increase (P = NS) in the controls, but in gallstone subjects, synthesis and pool size decreased. We concluded that in gallstone subjects cholesterol and bile acid homeostasis is significantly altered, and that increasing dietary cholesterol increases biliary cholesterol secretion and decreases bile acid synthesis and pool, changes associated with cholesterol gallstone formation.

Defective histamine release in chronic urticaria.

Histamine release from peripheral blood leukocytes challenged with anti-human IgE was studied in patients with chronic urticaria and nonatopic controls. 19 of 23 controls, but only 6 of 20 patients, released over 20% of the total available leukocyte histamine. The response to anti-IgE concentrations of 1.66, 0.33, 0.066, and 0.013 mug antibody N/ml was significantly lower in patients than in controls. Serum IgE levels were significantly higher in the patients but total histamine content of about 10(7) leukocytes was not. Deuterium oxide (D2O) greatly increased histamine release (in both groups), indicating that the anti-IgE interacted with the basophils of urticaria patients. Passive sensitization of leukocytes with biologically active IgE was achieved in both patients and control subjects whose cells responded to anti-IgE, but was not achieved in either patients or control subjects whose cells were nonresponsive to anti-IgE challenge. 125I-anti-IgE autoradiographic studies revealed no obvious quantitative abnormality in the amount of basophil-bound IgE in chronic urticaria patients. Ionophore stimulation of aliquots of the same leukocytes used for anti-IgE challenge demonstrated that the urticaria patients' basophils were capable of releasing normal amounts of histamine. Leukocyte cyclic AMP levels in the two groups were not significantly different either in base-line levels or in responsiveness to stimulation with isoproterenol. These data indicate that chronic urticaria patients have a (acquired?) defect in leukocyte histamine release that occurs after the anti-IgE-IgE interaction, but before the actual (second-stage) release process, and that is comparable to the phenomenon of desensitization.

Mechanisms of gallstone formation in women. Effects of exogenous estrogen (Premarin) and dietary cholesterol on hepatic lipid metabolism.

Our aim was to define mechanisms whereby conjugated estrogens (Premarin, exogenous estrogen; Ayerst Laboratories, New York) increase the risk of developing cholesterol gallstones and to determine the role, if any, of dietary cholesterol. We studied gallbladder motor function, biliary lipid composition and secretion, cholesterol absorption, cholesterol synthesis and esterification by peripheral blood mononuclear cells, the clearance of chylomicron remnants, and bile acid kinetics in 29 anovulatory women. 13 were studied on both a low (443 +/- 119 mumol/d) and high (2,021 +/- 262 mumol/d) cholesterol diet. Premarin increased the lithogenic index of bile (P less than 0.05), increased biliary cholesterol secretion (P less than 0.005), lowered chenodeoxycholate (CDCA) pool (P less than 0.001) and synthesis (P less than 0.05), altered biliary bile acid composition [( CA + DCA]/CDCA increases, P less than 0.005), stimulated cholesterol esterification (P less than 0.03), and enhanced the clearance of chylomicron remnants (P = 0.07). Increases in dietary cholesterol stimulated the biliary secretion of cholesterol (P = 0.07), bile acid (P less than 0.05), phospholipid (P = 0.07), and as a result, did not alter lithogenic index. The reduction in CDCA pool and synthesis by Premarin was reversed by increasing dietary cholesterol. Off Premarin, only 24% of the increase in cholesterol entering the body in the diet was recovered as biliary cholesterol or newly synthesized bile acid. On Premarin, 68% of this increase in cholesterol was recovered as these biliary lipids. We conclude that Premarin increases biliary cholesterol by enhancing hepatic lipoprotein uptake and inhibiting bile acid synthesis. These actions of Premarin divert dietary cholesterol into bile.

Fecal excretion of bile acids: a new technique for studying bile acid kinetics in patients with ileal resection.

The fecal elimination and enterohepatic circulation of bile acid was studied in 11 patients. 10 patients with varying degrees of ileal disease or resection and 1 patient with pancreatic insufficiency and no ileal disease. A new technique was employed which involved the nearly simultaneous administration of cholic acid-(14)C and a nonabsorbable marker. (51)CrCl(3). Each individual stool specimen was collected for 36-96 hr and analyzed separately. Assay of the radioactivity of each isotope allowed the accurate determination of an excretion rate for both cholic acid and (51)Cr. The difference between these rates was used to calculate an absorption coefficient for cholic acid. In addition, bile acid concentration measured by the steroid dehydrogenase technique, and the water content of each stool was determined. THE PATIENTS WERE DIVIDED INTO GROUPS DEPENDING UPON HOW MUCH SMALL INTESTINE WAS RESECTED OR DISEASED: six patients with less than 100 cm of ileal resection or disease (group A), and five patients with more than 100 cm of ileal disease or resection (group B). The (51)Cr excretion rate was similar in the two groups, but cholic acid-(24)C excretion rates were significantly more rapid in group B than in group A. The cholic acid absorption coefficient was essentially normal in the patient with pancreatic insufficiency, moderately decreased in group A patients, and extremely low or zero in group B patients. It was inversely related to the length of intestine diseased or resected. Daily fecal bile acid excretion was normal to twice normal in group A patients and 2-8 times normal in group B patients. In all patients with ileal disease or resection, there was a direct correlation between fecal bile acid, fecal mass, and fecal water. Each millimole of additional bile acid in the stool was associated with an increase in stool water of 11 moles (P < 0.01). These studies show that the kinetics of bile acids in the enterohepatic circulation can be accurately studied in patients with extensive ileal resection. The regular relationship between fecal bile acid and fecal mass and water suggests, but does not prove, a critical role of bile acid in determining stool water.

Protein starvation and the small intestine. 3. Incorporation of orally and intraperitoneally administered 1-leucine 4,5-3H into intestinal mucosal protein of protein-deprived rats.

Weanling rats were fed diets which contained either no protein or 27% protein. In one experiment after 23-35 days both groups were given l-leucine-4,5-(3)H either intragastrically or intraperitoneally and then sacrificed 24 hr later. In a second experiment animals were given these diets for 21 days and sacrificed 3, 6, or 12 hr after either intragastric or intraperitoneal administration of the labeled leucine. In both experiments the intestinal mucosa of proximal and distal segments of the small intestine was scraped, weighed, the protein concentration measured, and the specific activty of the mucosal protein was determined. The wet weight of the mucosa and the protein concentration of the mucosa were significantly greater in the control animals than in the protein-depleted animals. The mucosal protein per 100 g of body weight was the same in the protein-deprived and the control groups. The specific activity of the intestinal mucosal protein was higer in the protein-deprived animals than in the control animals. In the protein-deprived animals the proximal segment incorporated more radioactive amino acid into mucosal protein than did the distal segment at 3, 6, 12, and 24 hr after the amino acid was given by mouth. A similar difference was found between the proximal and distal segments of the control animals 6 hr after oral adminisstration of l-leucine-(3)H. On the other hand, when the l-leucine-(3)H was given intraperitoneally to both groups of animals there was no difference between proximal and distal small intestine. These findings suggest that intestinal mucosal protein can be synthesized directly from intraluminal amino acids, especially during protein deprivation, and that endogenous intraluminal protein might be important in the nutrition of the small intestinal mucosa.

Bile salt malabsorption in regional ileitis, ileal resection and mannitol-induced diarrhea.

Fecal bile salt excretion was studied in healthy volunteers, patients with regional ileitis, and patients with ileal resection. 10 muc of carboxyl-(14)C-cholic acid was given orally. Stools and urine were collected daily for 5-10 days, the bile salts extracted, and the radioactivity assayed. Urinary excretion was negligible. All patients with ileal resection excreted bile salts in the feces significantly faster than controls, and five of the six excreted 50% of the radioactivity within 24 hr. Their mean intestinal transit time was 5.6 hr compared to 26 hr for the controls. Two of the three patients with regional ileitis excreted bile salts almost as rapidly as patients with ileal resection. Vitamin B(12) absorption was also defective in those patients, but the intestinal transit time was not decreased. To study the effect of rapid intestinal transit on bile salt excretion, four of the control subjects were given orally 1200 ml of 10% mannitol for 7 days, and the labeled cholic acid excretion rate was again studied. The mean intestinal transit time was markedly shortened, mild steatorrhea developed, and the fecal bile salt excretion rate increased slightly. It is concluded that ileal resection and ileal disease are major factors and rapid intestinal transit is a minor factor in causing excessive fecal bile salt loss. The relevance of bile salt wastage to lipid malabsorption is unknown because of insufficient information about compensatory jejunal absorption, maximum rate of hepatic bile salt synthesis, and the minimum necessary intraluminal concentration of conjugated bile salt.

Gallbladder and small intestinal regulation of biliary lipid secretion during intraduodenal infusion of standard stimuli.

The gallbladder and small intestine are reservoirs for the bile acid pool during its enterohepatic circulation and, as such, may regulate biliary secretion of bile acid. During studies of biliary bile acid secretion, a stimulus to gallbladder contraction is continuously infused into the duodenum. Under these conditions, it is assumed that the gallbladder is tonically contracted and that the rate of bile acid secretion into the duodenum equals the hepatic bile acid secretion rate. However, secretion rates vary by as much as 100%, depending upon which of two standard stimuli is used. Therefore, we studied the role of gallbladder emptying and small intestinal transit in determining biliary lipid secretion rate and composition during infusion of these stimuli in five healthy subjects. Each subject was studied with a liquid formula containing 40% of calories as fat, and with an amino acid solution for 10 h. Bile acid, phospholipid, cholesterol, and markers were measured in duodenal bile and hourly secretion rates were calculated by marker dilution technique. Real-time gallbladder sonographs and serum pancreatic polypeptide levels were obtained every 30 min. Small bowel transit time was estimated levels were obtained every 30 min. Small bowel transit time was estimated by the breath hydrogen response after giving lactulose intraduodenally.During liquid formula infusion, gallbladder emptying was more complete, small intestinal transit was faster, and pancreatic polypeptide levels were higher. Secretion rates of all lipids were greater and molar percent cholesterol was lower. For the combined data from both infusions, the secretory relationships of cholesterol to bile acid, cholesterol to phospholipid, and phospholipid to bile acid were curvilinear. We conclude that more complete gallbladder emptying and faster intestinal transit increase the enterohepatic cycling of bile acids and lower the molar percent cholesterol of bile. Some of the fluctuation observed in biliary lipid secretion rates, especially during amino acid infusion, is due to gallbladder refilling and emptying.

Biliary lipids, bile acids, and gallbladder function in the human female. Effects of pregnancy and the ovulatory cycle.

To study the events that might lead to an increased risk of cholesterol gallstones, we examined biliary lipid composition and secretion and bile acid composition and kinetics at different stages of pregnancy or ovulation in young, nonobese, healthy women. Lipid composition and bile acid distribution were determined in duodenal fluid obtained in the fasting state and after stimulation of the gallbladder. Biliary lipid secretion was measured by the marker-perfusion technique. Bile acid kinetics were determined with cholic and chenodeoxycholic acids labeled with carbon13, by measuring the relative abundance of 13C in duodenal bile acids for 4--5 d. In a subset of patients we measured gallbladder storage and emptying during the kinetic study. The phase of the ovulatory cycle had no effects, but there were significant changes during pregnancy. The lithogenic or cholesterol saturation index of fasting hepatic and gallbladder bile increased during the second and third trimesters. The mean secretion rate of biliary lipids was not altered, but in the last two-thirds of pregnancy, cholesterol secretion increased in relation to bile acid and phospholipid secretion. There was a progressive decrease in the percentage of chenodeoxycholic acid and a similar increase in the percentage of cholic acid. The pool size of each major bile acid increased in the first trimester. Chenodeoxycholic acid and deoxycholic acid pools, but not cholic acid pools, subsequently decreased. The fractional turnover rate of both primary bile acids was slower during pregnancy. The synthesis rate of chenodeoxycholic but not cholic acid decreased in a linear manner during the first 20 wk of pregnancy. The rate of enterohepatic cycling of the bile acid pool was reduced throughout pregnancy. The volume of the fasting gallbladder and the residual volume after a physiologically stimulated contraction were directly correlated with bile acid pool size. The residual volume was also directly related to total bile acid synthesis.

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.

Adenovirus E1a proteins repress expression from polyomavirus early and late promoters.

We have examined the effects of the E1a products of adenovirus types 5 and 12 on the expression of polyomavirus early and late promoters. In cotransfection experiments in HeLa cells, plasmids expressing the E1a region of adenovirus type 5 or 12 repressed both the early and late promoters of polyomavirus, and deletion analysis indicates that the polyomavirus enhancers were the target of the E1a repression. With mutants lacking enhancer sequences, the polyomavirus early promoter but not the late promoter was trans-activated by E1a. Chimeric mutant plasmids with deletions in the regulatory region that contained either the A enhancer or the B enhancer were repressed to the same extent, indicating that E1a can repress both elements. Polyomavirus variant plasmids with rearrangements in the regulatory region conferring activity in embryonal carcinoma stem cells were repressed by E1a as was the wild type, suggesting that the repressor function is quite general. We discuss a model in which the influence of E1a on the transcriptional activity of a gene is the sum of positive and negative effects on promoter and enhancer elements and discuss possible mechanisms of negative regulation of enhancer function.