The regulation of human immunodeficiency virus type 1 (HIV-1) gene expression involves a complex interplay between cellular transcription factors, chromatin-associated proviral DNA, and the virus-encoded transactivator protein, Tat. Here we show that Tat transactivates the integrated HIV-1 long terminal repeat (LTR), even in the absence of detectable basal promoter activity, and this transcriptional activation is accompanied by chromatin remodeling downstream of the transcription initiation site, as monitored by increased accessibility to restriction endonucleases. However, with an integrated promoter lacking both Sp1 and NF-B sites, Tat was unable to either activate transcription or induce changes in chromatin structure even when it was tethered to the HIV-1 core promoter upstream of the TATA box. Tat responsiveness was observed only when Sp1 or NF-B was bound to the promoter, implying that Tat functions subsequent to the formation of a specific transcription initiation complex. Unlike Tat, NF-B failed to stimulate the integrated transcriptionally silent HIV-1 promoter. Histone acetylation renders the inactive HIV-1 LTR responsive to NF-B, indicating that a suppressive chromatin structure must be remodeled prior to transcriptional activation by NF-B. Taken together, these results suggest that Sp1 and NF-B are required for the assembly of transcriptional complexes on the integrated viral promoter exhibiting a continuum of basal activities, all of which are fully responsive to Tat.

We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH : ubiquinone reductase (complex 1) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N . crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N . crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N . crassa.In a series of reports, Brody and coworkers [I-41 have demonstrated that mitochondria of Neurospora crassa and other microorganisms contain an acyl-carrier protein (ACP) with phosphopantetheine as prosthetic group. This mitochondrial ACP can mediate de novo fatty acid synthesis, independent of the fatty acid synthetase complex present in the cytoplasm [4]. The role of the mitochondrial synthetic pathway, however, remained unclear, and the authors considered the possibility that the pathway exists to satisfy special needs of lipids of the mitochondrion itself [4]. Antigenically similar proteins of similar apparent molecular masses were also found in the mitochondria of yeast, pea leaves and potato tubers, but not in animal mitochondria [3]. In order to isolate the ACP, the authors had to dissolve the N. crassa mitochondria in 6 M guanidine hydrochloride [2].The respiratory chain complex of NADH : ubiquinone oxidoreductase (complex I) in N . crassa mitochondria is an assembly of some 30 different subunits. Most of these are nuclear encoded and imported from the cytoplasm. In N . crassu, at least six (seven in mammals) are encoded and translated in the mitochondrion. Only a few subunits are directly involved in the binding of the substrates NADH and ubiquinone, the internal redox groups FMN and four (EPRdetectable) iron-sulfur clusters. The functions of the many additional subunits which do not appear to take part directly in electron transport remain largely unknown (for a review,

WC have cultivated the ccl mutant of Neurosporu crossa dcfectivc in cytosolic fatty acid synthesis with [2-"C_Imalonate and found radioactivity covalcntly attached to the mitochondrial acyl-carrier protein (ACP), a subunit of the respiratory chain NADH:ubiquinonc oxidorcductase. We purified the ACP by reverse-phase HPLC: the bound acyl groups were trans.cstcrilied to methylesters and analyzed by gas chromatography. The saturated C,, to CIb fatty acids and olcic acid were detected. De novo synthesis and desaturation of fatty acids at the ACP subunit of NADH:ubiquinonc oxidoreductase and 11se of the products of this mitochondrial synthetic pathway for cardiolipin synthesis is discussed.

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