Characterisation of the immune microenvironment of primary breast cancer and brain metastasis reveals depleted T-cell response associated to ARG2 expression.

BACKGROUND: Immune checkpoint inhibition is an established treatment in programmed death-ligand 1 (PD-L1)-positive metastatic triple-negative (TN) breast cancer (BC). However, the immune landscape of breast cancer brain metastasis (BCBM) remains poorly defined. MATERIALS AND METHODS: The tumour-infiltrating lymphocytes (TILs) and the messenger RNA (mRNA) levels of 770 immune-related genes (NanoString™, nCounter™ Immuno-oncology IO360) were assessed in primary BCs and BCBMs. The prognostic role of ARG2 transcripts and protein expression in primary BCs and its association with outcome was determined. RESULTS: There was a significant reduction of TILs in the BCBMs in comparison to primary BCs. 11.5% of BCs presented a high immune infiltrate (hot), 46.2% were altered (immunosuppressed/excluded) and 34.6% were cold (no/low immune infiltrate). 3.8% of BCBMs were hot, 23.1% altered and 73.1% cold. One hundred and twelve immune-related genes including PD-L1 and CTLA4 were decreased in BCBM compared to the primary BCs (false discovery rate <0.01, log2 fold-change >1.5). These genes are involved in matrix remodelling and metastasis, cytokine-chemokine signalling, lymphoid compartment, antigen presentation and immune cell adhesion and migration. Immuno-modulators such as PD-L1 (CD274), CTLA4, TIGIT and CD276 (B7H3) were decreased in BCBMs. However, PD-L1 and CTLA4 expression was significantly higher in TN BCBMs (P = 0.01), with CTLA4 expression also high in human epidermal growth factor receptor 2-positive (P < 0.01) compared to estrogen receptor-positive BCBMs. ARG2 was one of four genes up-regulated in BCBMs. High ARG2 mRNA expression in primary BCs was associated with worse distant metastasis-free survival (P = 0.038), while ARG2 protein expression was associated with worse breast-brain metastasis-free (P = 0.027) and overall survival (P = 0.019). High transcript levels of ARG2 correlated to low levels of cytotoxic and T cells in both BC and BCBM (P < 0.01). CONCLUSION: This study highlights the immunological differences between primary BCs and BCBMs and the potential importance of ARG2 expression in T-cell depletion and clinical outcome.

Phase I/II trial of a dendritic cell vaccine transfected with DNA encoding melan A and gp100 for patients with metastatic melanoma.

This trial tested a dendritic cell (DC) therapeutic cancer vaccine in which antigen is loaded using a novel non-viral transfection method enabling the uptake of plasmid DNA condensed with a cationic peptide. Proof of principle required the demonstration of diverse T lymphocyte responses following vaccination, including multiple reactivities restricted through both major histocompatibility complex (MHC) class I and II. Patients with advanced melanoma were offered four cycles of vaccination with autologous DC expressing melan A and gp100. Disease response was measured using Response Evaluation Criteria in Solid Tumours. Circulating MHC class I- and II-restricted responses were measured against peptide and whole antigen targets using interferon-γ ELIspot and enzyme-linked immunosorbent assay assays, respectively. Responses were analyzed across the trial population and presented descriptively for some individuals. Twenty-five patients received at least one cycle. Vaccination was well tolerated. Three patients had reduction in disease volume. Across the trial population, vaccination resulted in an expansion of effector responses to both antigens, to the human leukocyte antigen A2-restricted modified epitope, melan A ELAGIGILTV, and to a panel of MHC class I- and II-restricted epitopes. Vaccination with mature DC non-virally transfected with DNA encoding antigen had biological effect causing tumour regression and inducing diverse T lymphocyte responses.

HOXC11-SRC-1 regulation of S100beta in cutaneous melanoma: new targets for the kinase inhibitor dasatinib.

BACKGROUND: Cutaneous melanoma is an aggressive disease. S100beta is an established biomarker of disease progression; however, the mechanism of its regulation in melanoma is undefined. METHODS: Expression of HOXC11 and SRC-1 was examined by immunohistochemistry and immunofluorescence. Molecular and cellular techniques were used to investigate regulation of S100beta, including, western blot, qPCR, ChIP and migration assays. RESULTS: Expression levels of the transcription factor HOXC11 and its coactivator SRC-1 were significantly elevated in malignant melanoma in comparison with benign nevi (P<0.001 and P=0.017, respectively, n=80), and expression of HOXC11 and SRC-1 in the malignant tissue associated with each other (P<0.001). HOXC11 recruitment to the promoter of S100beta was observed in the primary melanoma cell line SKMel28. S100beta expression was found to be dependant on both HOXC11 and SRC-1. Treatment with the Src/Abl inhibitor, dasatinib, reduced HOXC11-SRC-1 interaction and prevented recruitment of HOXC11 to the S100beta promoter. Dasatinib inhibited both mRNA and protein levels of S100beta and reduced migration of the metastatic cell line MeWo. CONCLUSION: We have defined a signalling mechanism regulating S100beta in melanoma, which can be modulated by dasatinib. Profiling patients for expression of key markers of this network has the potential to increase the efficacy of dasatinib treatment.

Smoking initiation is followed by the early acquisition of epigenetic change in cervical epithelium: a longitudinal study.

BACKGROUND: To prove a causal link between an epigenetic change and an environmental or behavioural risk factor for a given disease, it is first necessary to show that the onset of exposure precedes the first detection of that epigenetic change in subjects who are still free of disease. METHODS: Towards this end, a cohort of women aged 15-19 years, recruited soon after they first had sexual intercourse, were used to provide sequential observations on the relationship between cigarette smoking and the detection in cervical cytological samples of methylated forms of CDKN2A (p16) using nested methylation-specific polymerase chain reaction. RESULTS: Among women who remained cytologically normal and who tested negative for human papillomavirus DNA in cervical smears during follow-up, those who first started to smoke during follow-up had an increased risk of acquiring CDKN2A methylation compared with never-smokers (odds ratio=3.67; 95% confidence interval 1.09-12.33; P=0.04). CONCLUSION: Smoking initiation is associated with the appearance of methylated forms of CDKN2A.

The association of an HPV16 oncogene variant with HLA-B7 has implications for vaccine design in cervical cancer.

HLA-restricted cytotoxic T-lymphocyte (CTL) recognition of human papillomavirus (HPV) oncogene products may be important in the control of the HPV infections associated with the development of cervical cancer. We have identified, in HLA-B7 individuals, a consistent variation in the HPV16 E6 oncoprotein sequence, which alters an HLA-B7 peptide binding epitope in a way likely to influence immune recognition by CTLs. These results illustrate a biologically relevant mechanism for escape from immune surveillance of HPV16 in HLA-B7 individuals. Thus, both HLA type and HPV16 strain variation need to be considered in the screening of at-risk individuals and for the rational design of anti-HPV vaccines.

Epstein-Barr virus (EBV)-associated lymphoproliferative disease in the SCID mouse model: implications for the pathogenesis of EBV-positive lymphomas in man.

When human peripheral blood lymphocytes (PBLs) from Epstein-Barr virus (EBV)-seropositive donors are injected intraperitoneally into SCID mice, EBV+ B cell tumors develop within weeks. A preliminary report (Mosier, D. E., R. J. Gulizia, S. M. Baird, D. D. Richman, D. B. Wilson, R. I. Fox, and T. J. Kipps, 1989. Blood. 74(Suppl. 1):52a) has suggested that such tumors resemble the EBV-positive malignancy, Burkitt's lymphoma. The present work shows that generally the human (hu) PBL-SCID tumors are distinct from Burkitt's lymphoma and instead resemble lymphoblastoid cell lines (LCLs) generated by EBV-infection of normal B cells in vitro in terms of: (a) their cell surface phenotype, with expression of B cell activation antigens and adhesion molecules, (b) normal karyotype, and (c) viral phenotype, with expression of all the transformation-associated EBV latent proteins and, in a minority of cells, productive cycle antigens. Indeed, in vitro-transformed LCLs also grow when inoculated into SCID mice, the frequency of tumor outgrowth correlating with the in vitro growth phenotype of the LCL which is itself determined by the identity of the transforming virus (i.e., type 1 or type 2 EBV). Histologically the PBL-derived hu-SCID tumors resemble the EBV+ large cell lymphomas that develop in immuno-suppressed patients and, like the human tumors, often present at multiple sites as individual monoclonal or oligoclonal foci. The remarkable efficiency of tumor development in the hu-SCID model suggests that lymphomagenesis involves direct outgrowth of EBV-transformed B cells without requirement for secondary genetic changes, and that selection on the basis of cell growth rate alone is sufficient to explain the monoclonal/oligoclonal nature of tumor foci. EBV+ large cell lymphoma of the immunosuppressed may arise in a similar way.

Co-infection of macrophages modulates interferon gamma and tumor necrosis factor-induced activation against intracellular pathogens.

Co-infection of macrophages (M phi) with Toxoplasma gondii and Mycobacterium avium-intracellulare complex (MAC) has been observed in patients with acquired immunodeficiency syndrome (AIDS). In this study we have demonstrated that co-infected murine M phi respond differently to cytokine stimulation than M phi infected with either of the microorganisms alone. Whereas treatment with interferon gamma (IFN-gamma) activated both single and co-infected groups of M phi to kill T. gondii, treatment with TNF did not influence the rate of MAC growth in co-infected M phi, in contrast with the inhibition of growth observed in MAC-infected M phi. These results suggest that in AIDS patients suffering infection with multiple intracellular pathogens, the ability of cytokines to stimulate microbicidal or static activity in mononuclear phagocytes can be impaired by the presence of more than one of the intracellular organisms.

Epstein-Barr virus and Hodgkin's disease: transcriptional analysis of virus latency in the malignant cells.

Epstein-Barr virus (EBV) is associated with a number of different human tumors and appears to play different pathogenetic roles in each case. Thus, immunoblastic B cell lymphomas of the immunosuppressed display the full pattern of EBV latent gene expression (expressing Epstein-Barr nuclear antigen [EBNA]1, 2, 3A, 3B, 3C, and -LP, and latent membrane protein [LMP]1, 2A, and 2B), just as do B lymphoblastoid cell lines transformed by the virus in vitro. In contrast, those EBV-associated tumors with a more complex, multistep pathogenesis show more restricted patterns of viral gene expression, limited in Burkitt's lymphoma to EBNA1 only and in nasopharyngeal carcinoma (NPC) to EBNA1 and LMP1, 2A, and 2B. Recent evidence has implicated EBV in the pathogenesis of another lymphoid tumor, Hodgkin's disease (HD), where the malignant Hodgkin's and Reed-Sternberg (HRS) cells are EBV genome positive in up to 50% of cases. Here we extend preliminary results on viral gene expression in HRS cells by adopting polymerase chain reaction-based and in situ hybridization assays capable of detecting specific EBV latent transcripts diagnostic of the different possible forms of EBV latency. We show that the transcriptional program of the virus in HRS cells is similar to that seen in NPC in several respects: (a) selective expression of EBNA1 mRNA from the BamHI F promoter; (b) downregulation of the BamHI C and W promoters and their associated EBNA mRNAs; (c) expression of LMP1 and, in most cases, LMP2A and 2B transcripts; and (d) expression of the "rightward-running" BamHI A transcripts once thought to be unique to NPC. This form of latency, consistently detected in EBV-positive HD irrespective of histological subtype, implies an active role for the virus in the pathogenesis of HD and also suggests that the tumor may remain sensitive to at least certain facets of the EBV-induced cytotoxic T cell response.

CD40 activation induces apoptosis in cultured human hepatocytes via induction of cell surface fas ligand expression and amplifies fas-mediated hepatocyte death during allograft rejection.

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.

A class III transcription factor composed of RNA.

It is generally assumed that the machinery that transcribes genes is composed entirely of polypeptides. However, in vitro transcription by silkworm RNA polymerase III requires a transcription factor that is not a polypeptide. This component, TFIIIR, is distinct from the previously identified transcription components: RNA polymerase III, and the accessory factors TFIIIA, TFIIIB, TFIIIC, and TFIIID. The newly discovered TFIIIR is a macromolecule that appears to be composed of RNA. It is resistant to heat, detergent, phenol, protease, and deoxyribonuclease, but it is sensitive to alkali and ribonuclease.

Epstein-Barr virus-encoded EBNA1 regulates cellular gene transcription and modulates the STAT1 and TGFbeta signaling pathways.

The Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all virus-associated tumors where it plays an essential role in the maintenance, replication and transcription of the EBV genome. Transcriptional profiling of EBNA1-expressing carcinoma cells demonstrated that EBNA1 also influences the expression of a range of cellular genes including those involved in translation, transcription and cell signaling. Of particular interest was the ability of EBNA1 to enhance expression of STAT1 and sensitize cells to interferon-induced STAT1 activation with resultant enhancement of major histocompatibility complex expression. A negative effect of EBNA1 on the expression of TGFbeta1-responsive betaig-h3 and PAI-1 genes was confirmed at the protein level in EBV-infected carcinoma cells. This effect resulted from the ability of EBNA1 to repress TGFbeta1-induced transcription via a reduction in the interaction of SMAD2 with SMAD4. More detailed analysis revealed that EBNA1 induces a lower steady-state level of SMAD2 protein as a consequence of increased protein turnover. These data show that EBNA1 can influence cellular gene transcription resulting in effects that may contribute to the development of EBV-associated tumors.

Protective activity of antibodies to exotoxin A and lipopolysaccharide at the onset of Pseudomonas aeruginosa septicemia in man.

Serum antibodies to exotoxin A and type-specific lipopolysaccharide were measured by passive hemagglutination in 52 patients with Pseudomonas aeruginosa septicemia. Their comparative protective activities were evaluated by relating the titers of each at the onset of bacteremia to subsequent outcome. High acute serum antitoxin and antilipopolysaccharide titers (log2 reciprocal mean titers greater than 5) were associated with survival (76% of 17 with high vs. 46% of 24 with low antitoxin titers, P = 0.05; 85% of 13 with high vs. 48% of 29 with low antilipopolysaccharide titers, P = 0.03). In contrast, neither antibody titer was significantly associated (P less than or equal to 0.05) with patients' age or sex, severity of underlying disease, presence of leukopenia, steroid or immunosuppressive therapy. Despite a correlation between acute titers of the two antibodies (r = 0.33, P = 0.06), they appeared to protect independently and additively. Whereas 75% of 8 patients with high antitoxin titers and only 38% of 16 with low titers survived with low antilipopolysaccharide titers (P = 0.10), 100% (6/6), 73% (8/11), and 38% (6/16) survived, respectively, when both, one, or neither antibody was present in high titer (P = 0.01). Furthermore, the association between high acute serum antitoxin titers and survival was more pronounced in patients with rapidly fatal underlying disease (P = 0.06) and leukopenia (P = 0.12) than in more favorable prognostic and immune categories. These data indicate that serum antibodies to exotoxin A and lipopolysaccharide are found in most patients with P. aeruginosa septicemia and both are protective. Both antibodies may have therapeutic or prophylactic potential, whereas serum antiexotoxin A antibodies may be particularly beneficial in compromised hosts.

Neutrophil function in gram-negative rod bacteremia. The interaction between phagocytic cells, infecting organisms, and humoral factors.

To assess the phagocytic and bactericidal function of neutrophils in the acute stages of gram-negative rod bacteremia, cells from 30 nonleukopenic patients were studied in a test system utilizing plasma obtained simultaneously with culture-positive blood, the autologous infecting strain, and two laboratory test strains of Staphylococcus aureus and Pseudomonas aeruginosa. Results were compared to those obtained with normal neutrophils and plasma. Patient and control plasma were simultaneously tested with each source of phagocytic cells to localize any abnormalities. Four patients had a defect against their infecting strain, 33% of the inoculum phagocytized and killed versus 80% by controls. In these cases differences were localized to the patients' plasma, as normal plasma tested with patients' cells reversed the defect. Thus, four patients had impaired opsonization when compared to normal controls, but we also observed that 11 of 30 bacteremic isolates, all Escherichia coli, showed absolute or relative resistance to phagocytosis in the patient and control assay system. No intrinsic granulocyte killing abnormalities were noted. There was poor correlation between results obtained with infecting strains compared to laboratory test organisms. We conclude that in patients without evidence of an inherited neutrophil bactericidal disorder, recurrent infection, or treatment with cytotoxic drugs, intrinsic bactericidal defects are uncommon at the onset of gram-negative bacteremia, and impaired opsonization is the most commonly encountered cause of neutrophil dysfunction.

Functional role of antibody against "core" glycolipid of Enterobacteriaceae.

Antibodies against the "core" glycolipid of Enterobacteriaceae (2-keto, 3-deoxyoctonate-Lipid A) have been associated with protection against the sequelae of gram-negative rod bacteremia. To investigate the nature of this protection, dogs and rabbits were immunized with purified glycolipid prepared by phenol-chloroform-petroleum ether extraction of the "Re" mutant of Salmonella minnesota 595 and opsonophagocytic and bactericidal tests were carried out using lapine peritoneal granulocytes and serum factors. Whereas 1-4 mug/kg of glycolipid i.v. produced hypotension in dogs and 8 mug/kg i.v. was lethal, a rising dosage schedule of immunization with an average total dose of 1 mg/kg produced striking protection against shock and death against challenge with heterologous organisms. 20 control dogs, given approximately 10(10) live, serum-resistant Escherichia coli 0.85:H9 or Serratia marcescens 03 during a continuous intra-arterial pressure transducer recording, showed a drop in mean systolic pressure from 186 (+/- 6 SE) to 101 (+/- 12 SE) MM Hg and a fall in mean diastolic pressure from 118 (+/- 3 SE) to 64 (+/- 8 SE) mm Hg within 60-120 min. Minor pressure changes (average less than 12% of prechallenge levels) were seen in the same number of immunized dogs. In contrast, no significant difference was noted in the bloodstream clearance of these serum-resistant organisms over a period of 4-6 h between immunized and control dogs. Intravascular clearance was greater in animals immunized with the challenged strain or in glycolipid-immunized animals challenged with highly serum-sensitive E. coli 0.14:K7. Antibody against core glycolipid protected against the hemodynamic sequelae of bacillemia, augmented intravascular clearance of serum-sensitive organisms, and abrogated the pyrogenic response to enteric bacilli, but did not enhance clearance of serum-resistant organisms. Although canine and lapine antiserum against core glycolipid passively protected mice against a heterologous challenge, opsonophagocytic and bactericidal activity was at least 100-fold less than type-specific antiserum.

Enhanced survival in Pseudomonas aeruginosa septicemia associated with high levels of circulating antibody to Escherichia coli endotoxin core.

We studied the relationship between serum antibodies to the cross-reactive endotoxin core of Escherichia coli and survival following Pseudomonas aeruginosa septicemia. Core glycolipid was purified from the outer cell membrane of a uridine diphosphate galactose 4-epimerase-deficient rough mutant E. coli (J5 strain), characterized, and used as the antigen in a quantitative enzyme-linked immunosorbent assay (ELISA) to measure core-specific IgG and IgM antibodies. 43 patients with Pseudomonas septicemia, among whom there was a mortality of 42%, were evaluated. Core-specific antibody concentrations in acute sera ranged from 1 to 49 micrograms/ml in the case of IgG and from 1 to 200 micrograms/ml for IgM. Core-specific antibodies of both isotypes were higher in patients who survived compared with those who succumbed to their septicemias (mean, microgram/ml +/- SEM, 26 +/- 3 vs. 14 +/- 4, P = 0.005 for IgG, and 55 +/- 12 vs. 18 +/- 5, P = 0.009 for IgM). Although total IgG levels were also higher in acute sera from survivors compared with nonsurvivors (mean, mg/dl +/- SEM, 1,120 +/- 99 vs. 694 +/- 119, P = 0.004), total IgM levels were virtually identical in the two groups (146 +/- 23 vs. 148 +/- 48, P = 0.52). Conversely, patients with core-specific IgG levels greater than 10 micrograms/ml at the onset of septicemia had better survival than those with levels less than 10 micrograms/ml (79 vs. 14%, P less than 0.001), and patients with core-specific IgM levels greater than 30 micrograms/ml had better survival than those with levels less than 30 micrograms/ml (81 vs. 44%, P = 0.01). In comparison, patients with total IgG levels greater than 1,000 mg/dl also had better survival than those with levels less than 1,000 mg/dl (82 vs. 42%, P = 0.01), while those with total IgM levels greater than 150 mg/dl showed somewhat less improvement in survival compared with those with levels less than 150 mg/dl (71 vs. 50%, P = 0.12). Core-specific IgM was highly correlated with core-specific IgG (r = 0.52), but not with type-specific anti-lipopolysaccharide (r = 0.13) or anti-toxin A (r = 0.12) antibodies, or with total IgG (r = 0.28) or IgM (r = 0.31). In contrast, core-specific IgG correlated somewhat more closely with type-specific antibodies (r = 0.36), and with total IgG (r = 0.51) and IgM (r = 0.52). Stepwise linear discriminant analysis indicated that type-specific antibody levels were the best predictor of outcome, among those antibodies examined, followed by anti-core IgM. Although anti-core IgG, anti-toxin A, and total IgG levels all correlated individually with survival, none augmented the prognostic power of type-specific antibodies in combination with anti-core IgM, which together predicted outcome accurately 73.5% of the time. Host factors not significantly associated with anti-core antibody levels included rapidly fatal underlying disease, age, sex, leukopenia, and prior treatment with cytotoxic drugs. In contrast, prior steroid therapy was associated with low levels of both core-specific IgG and IgM (P < 0.05). These data suggest cross-protective activity against P. aeruginosa septicemia of naturally occurring antibodies to the endotoxin core of E. coli. Anti-core antibodies, particularly of the IgM isotype appear to augment the more specific protective immunity engendered by antibodies to the O-specific side chains of Pseudomonas lipopolysaccharides. This cross-protective immunity likely applies to other Gram-negative pathogens as well.

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

Silkworm TFIIIB binds both constitutive and silk gland-specific tRNA Ala promoters but protects only the constitutive promoter from DNase I cleavage.

We have identified a complex between TFIIIB and the upstream promoter of silkworm tRNA Ala genes that is detectable by gel retardation and DNase I footprinting. Formation of this complex depends on the integrity of previously identified upstream promoter elements and on the presence of other silkworm transcription factors, either TFIIID or a fraction that contains both TFIIIC and TFIIID. We have used this complex to compare the interactions of TFIIIB with two kinds of tRNA Ala genes whose different in vitro transcription properties are conferred by the upstream segments of their promoters. These are the tRNA C Ala genes, which are transcribed constitutively, and the tRNA SG Ala genes, which are transcribed only in the silk gland. We find that TFIIIB binds tRNA SG Ala genes with lower affinity than it binds tRNA C Ala genes. In addition, the TFIIIB complex formed on tRNA SG Ala genes differ qualitatively from those formed on tRNA C Ala genes. Both the transcriptional activity of tRNA SG Ala complexes and the ability of the complexes to protect upstream DNA from DNase I digestion are reduced.

TFIIIR is an isoleucine tRNA.

Promoter-specific transcription by silkworm RNA polymerase III is dependent on several transcription factors (TFs) in addition to the polymerase itself. The activities present in silk gland nuclear extracts that are necessary to reconstitute transcription from class III genes in vitro have been resolved into several partially purified components. These include TFIIIR, which is unusual because it is composed of RNA. Here, we identify the RNA that provides TFIIIR activity as silkworm tRNA(IleIAU). This conclusion is based on copurification of tRNA(IleIAU) with TFIIIR activity, TFIIIR activity in synthetic tRNA(Ile), and hybrid selection of TFIIIR activity by antisense tRNA(IleIAU). We have tested the ability of a variety of other tRNAs to stimulate transcription and find that TFIIIR activity is highly specific to silkworm tRNA(IleIAU).

tRNA(IleIAU) (TFIIIR) plays an indirect role in silkworm class III transcription in vitro and inhibits low-frequency DNA cleavage.

tRNA(IleIAU) provides an activity, originally called TFIIIR, necessary to reconstitute transcription by silkworm RNA polymerase III in vitro from partially purified components. Here we report studies on the role of tRNA(IleIAU) in in vitro transcription. We show that tRNA(IleIAU) does not act positively but, rather, is required to prevent the action of a transcriptional inhibitor. We also show that the presence of tRNA(IleIAU) in transcription reaction mixtures prevents low-frequency DNA cleavage by the TFIIIB fraction. Studies on the mechanism of transcriptional inhibition suggest that this DNA cleavage could cause transcriptional inhibition through trans-inactivation of transcription machinery. The ability to block DNA cleavage, like the ability to facilitate transcription, is highly specific to silkworm tRNA(IleIAU).