Cells in mammalian tissues are primarily aerobic and therefore highly dependent upon a continuous supply of oxygen. A primary mechanisms by which mammals respond to reduced O 2 (hypoxia) is hyperventilation, which enhances the delivery of O 2 to cells by increasing arterial O 2 tension. The hyperventilation that occurs during hypoxia is mediated by the O 2 -sensitive (type I) cells in the carotid body. The carotid body is located bilaterally at the bifurcation of the carotid artery and releases dopamine in response to a reduction in arterial O 2 tension. The activity of tyrosine hydroxylase (EC 1.14.16.2, TH), 1 the rate-limiting enzyme in the biosynthesis of dopamine, is enhanced in the carotid body (3) and adrenal gland (4) during hypoxia. We reported recently that hypoxia enhances TH gene expression in the rat carotid body (1) and stimulates both the rate of TH gene transcription and TH mRNA stability in pheochromocytoma (PC12) cells (2). We use the PC12 clonal cell line as a model system to study the molecular genetic mechanisms that regulate TH gene expression during hypoxia. Transcriptional studies using nested deletions of the proximal promoter region of the TH gene linked to a reporter gene revealed that the O 2 -responsive sequences reside within a fragment that extends from Ϫ272 to ϩ 27 bases, relative to transcription start site (2).In the present study experiments were undertaken to further characterize the sequences and trans-acting protein factors that regulate the rate of transcription of TH gene during hypoxia. We report here that the O 2 responsiveness of the TH gene is mediated by a short fragment of the proximal promoter that contains the AP1 and HIF-1 (hypoxia-induced factor binding site) sequences. Findings from immunological studies revealed that c-Fos and JunB bind to the AP1 element during hypoxia. In addition, we report that mutation of the AP1 element abolished the transcription response to hypoxia. EXPERIMENTAL PROCEDURES Materials[␣-32 P]dATP (Ͼ3000 Ci/mmol) and [ 14 C]chloramphenicol (50 -60 mCi/mmol) were purchased from Amersham Corp. Klenow, G-25, and G-50 purification spin columns were purchased from Boehringer Mannheim. pCAT plasmids were originally obtained from Promega. Lipofectin reagent was purchased from Bio-Rad, and all cell culture reagents were obtained from Life Technologies, Inc. Antibodies for supershift assays, shift-Western analysis, and immunoblotting assays were purchased from Santa Cruz Biotechnology. Immunoblotting assays were performed using the ECL kit from Amersham. MethodsCell Culture-Rat pheochromocytoma (PC12) cells were maintained in Dulbecco's modified Eagle's medium/F-12 containing 15 mM Hepes buffer, L-glutamine, 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 g/ml). Cells were grown on culture dishes to 90% confluence in a strict environment (21% O 2 , 5% CO 2 , remainder N 2 at 37°C). Medium was changed twice weekly. Hypoxia exposures were carried out in an O 2 regulated incubator by exposing cells to 5% or 10% O 2 , 5% CO 2 , r...

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (R).i.; (Chevallier-Greco, A., et al., (1986) EMBO J., 5, 3243-9; Countryman, and Miller, G. (1985) Proc. Natl. Acad. Sci. USA, 82, 4085-4089). We have previously demonstrated that these proteins cooperatively activate the EBV early promoter BMRF1 in lymphoid cells but not in epithelial cells. Although cooperative transactivation by these proteins has been demonstrated with a number of EBV promoters, the mechanism of this interaction is not well understood. We now show that the cooperative activation of the BMRF1 promoter by Z-plus-R requires an intact R binding site and at least one functional Z response element (ZRE). Despite the presence of an R binding site, the BMRF1 promoter is only moderately responsive to R alone in either HeLa or Jurkat cells. Efficient activation of the BMRF1 promoter by Z alone in HeLa cells requires two ZREs (located at -59 and -106), whereas two additional Z binding sites (located at -42 and -170) contribute very little to Z-induced activation. In the absence of ZREs, Z acted as a repressor of R-induced transactivation. These observations, along with observations made by other investigators (Giot, J.F. et al., (1991) Nucleic Acids Res., 19, 1251-8), suggest that Z-plus-R cooperative activation is dependent upon 1) direct binding by R and Z to responsive promoter elements and 2) contributions by cell-specific factors.

Immunocompromised patients, particularly those with AIDS, develop progressive multifocal leukoencephalopathy (PML) due to central nervous system infection with JC virus (JCV). It is unknown whether JCV infection in the central nervous system can occur in the absence of PML symptoms. To address this question, autopsy specimens from patients with AIDS were examined. The brains of a group of patients without AIDS or central nervous system disease were also examined. JCV DNA was detected by the polymerase chain reaction in brain tissue from 4 (31%) of 13 human immunodeficiency virus (HIV)-positive patients. JCV was also detected in 1 elderly HIV-negative patient but not in the 11 other control brains. JCV was not detected in 22 myocardial specimens obtained at autopsy from HIV-negative patients nor 10 peripheral blood specimens from HIV-positive patients. The presence of JCV in brains of patients without clinically evident PML suggests that JCV may be present in the central nervous system without clinical disease.