We have determined the nucleotide sequence of a 5159 base-pair (bp) region of the Chlamydomonas reinhardtii plastome containing three photoelectron transport genes, psbF, psbL and petG, and an unusual open reading frame, ORF712. The photosynthetic genes have an unprecedented arrangement, psbF and psbL are located in close proximity to petG, and are not grouped with two other genes of the cytochrome b559 locus, psbE and ORF42. ORF712, located adjacent to psbL, has homology at its 5'- and 3'-ends to the ribosomal protein rps3 gene, but contains a central 437 residue domain that lacks similarity to any other known sequence. These sequences add to the growing body of evidence that the chloroplast genome of C. reinhardtii has a significantly different gene arrangement to its counterpart in plants. The structure of ORF712 also provides another example of a phenomenon we have discovered with C. reinhardtii RNA polymerase genes (Fong and Surzycki 1992); namely, that the algal plastome contains chimeric genes in which reading frames with homology to known genes are juxtaposed in-frame with long coding regions of unknown identity.

cDNAs encoding the bovine immunodeficiency virus (BIV) transactivator gene (tat) were cloned from virally infected cells and characterized. BIV expresses two distinct tat mRNAs composed of three exons that are derived by alternative splicing. The BIV tat mRNA splice variants encode Tat proteins of 103 (Tat103) and 108 (Tat108) amino acids. The Tat103 coding region is specified only by exon 2, while that of Tat108 is specified by a truncated exon 2 and the first 30 nt of exon 3. Thus, the first 98 amino acids of each Tat are identical, and have amino terminal, cysteine-rich, conserved core, basic, and carboxyl-terminal domains similar to Tats encoded by primate lentiviruses. BIV-infected bovine cells express a 14-kDa phosphorylated Tat protein identical in size to recombinant Tat expressed in bacteria. BIV Tat was shown to localize exclusively in the nucleoli of virally infected and Tat-expressing cells. Reporter gene assays indicated that Tat103 and Tat108 can strongly transactivate the BIV long terminal repeat (LTR) in virally permissive canine Cf2Th and nonpermissive HeLa and mouse NIH 3T3 cells, but not in permissive lapine EREp cells. However, an intact BIV tat gene is required for viral replication in both Cf2Th and EREp cells. Strong LTR activation by BIV Tat requires a TAR (transactivation responsive) element delimited by viral nt +1 to +31 and the Tat basic domain. BIV Tat strongly cross-transactivates the HIV-1 LTR in a TAR-dependent manner in Cf2Th, but not in EREp, HeLa, or NIH 3T3 cells. In contrast, strong, TAR-dependent cross-transactivation of the BIV LTR by HIV-1 Tat could not be demonstrated in any of these cell types. In Cf2Th cells Tat108 effects a moderately stronger transactivation of the BIV LTR than Tat103, indicative of a functional difference in BIV Tat proteins encoded by the mRNA splice variants. The present studies demonstrate that BIV Tat parallels the primate lentiviral Tats in structure and biochemistry but is not interchangeable with the latter.

Nucleotide sequence analysis of a 17043 base-pair (bp) region of the Chlamydomonas reinhardtii plastome indicates the presence of three open reading frames (ORFs) similar to RNA polymerase subunit genes. Two, termed rpoB1 and rpoB2, are homologous to the 5'- and 3'-halves of the Escherichia coli beta subunit gene, respectively. A third, termed rpoC2, is similar to the 3'-half of the bacterial beta' subunit gene. These genes exhibit several unusual features: (1) all three represent chimeric structures in which RNA polymerase gene sequences are juxtaposed in-frame with long sequences of unknown identity; (2) unlike their counterparts in plants and eubacteria, rpoB1 and rpoB2 are separated from rpoC2 by a long (7 kilobase-pair, kbp) region containing genes unrelated to RNA polymerase; (3) DNA homologous to the 5' half of rpoC (termed rpoC1 in other species) is not present at the 5' end of rpoC2 and could not be detected in C. reinhardtii chloroplast DNA. RNA expression could not be detected for any of the RNA polymerase genes, suggesting that they are pseudogenes or genes expressed at stages of the C. reinhardtii life-cycle not investigated. The three genes are flanked by GC-rich repeat elements. We suggest that repeat DNA-mediated chloroplast recombination events may have contributed to their unusual arrangement.

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Functional cis-acting regulatory elements in the bovine immunodeficiency virus (BIV) long terminal repeat (LTR) were identified by deletion mapping and nuclear protein gel shift analysis using three BIV-infectible cell lines, Cf2Th, BLAC-20, and EREp. Deletion mapping studies indicated that putative NF-kappa B, GRE, AP-4, AP-1, CAAT, and ATF/CRE transcription factor elements positively contribute to LTR-directed gene expression in each cell line both in the presence and absence of the viral transactivator Tat. Sp1 and overlapping AP-3 and retroviral core enhancer elements had variable effects on LTR-directed gene expression depending on cell type and presence or absence of Tat. In addition, a sequence spanning the LTR U5 region and the untranslated viral leader was strongly repressive in all cell lines. Tat transactivated the LTR 25-fold over basal levels in a TAR-dependent manner in Cf2Th cells. In contrast, Tat transactivated the LTR only 2.5-fold over basal levels in EREp and BLAC-20 cells in a TAR-independent manner. Probes for putative NF-kappa B, GRE, Sp1, AP-4, AP-1, overlapping AP-3 and retroviral core enhancer, and juxtaposed CAAT and ATF-CRE elements specifically bound nuclear proteins from these three cell lines and HeLa cells, with the stoichiometry of binding being cell-type dependent. Probes for AP-4, AP-1, and juxtaposed CAAT and ATF/CRE elements exhibited greater protein binding with extracts from virally infected cells than with extracts from uninfected cells, suggesting that viral infection can modulate nuclear factor binding. The present studies indicate that several transcription factor elements in the BIV LTR have functional roles and that cell type can strongly determine the role they play in gene expression.