To understand the interactions and functional role of each of the three mitochondrial NAD؉ -dependent isocitrate dehydrogenase (IDH) subunits (␣, ␤, and ␥), we have characterized human cDNAs encoding two ␤ isoforms (␤ 1 and ␤ 2 ) and the ␥ subunit. Analysis of deduced amino acid sequences revealed that ␤ 1 and ␤ 2 encode 349 and 354 amino acids, respectively, and the two isoforms only differ in the most carboxyl 28 amino acids. The ␥ cDNA encodes 354 amino acids and is almost identical to monkey IDH␥. Northern analyses revealed that the smaller ␤ 2 transcript (1.3 kilobases) is primarily expressed in heart and skeletal muscle, whereas the larger ␤ 1 mRNA (1.6 kilobases) is prevalent in nonmuscle tissues. Sequence analysis of the IDH␤ gene indicates that the difference in the C-terminal 28 amino acids between ␤ 1 and ␤ 2 proteins results from alternative splicing of a single transcript. Among the various combinations of human IDH subunits co-expressed in bacteria, ␣␤␥, ␣␤, and ␣␥ combinations exhibited significant amounts of IDH activity, whereas subunits produced alone and ␤␥ showed no detectable activity. These data suggest that the ␣ is the catalytic subunit and that at least one of the other two subunits plays an essential supporting role for activity. Substitution of ␤ 1 with ␤ 2 in the co-expression system lowered the pH optimum for IDH activity from 8.0 to 7.6. This difference in optimal pH was analogous to what was observed in mouse kidney and brain (␤ 1 prevalent; optimal pH 8.0) versus heart (␤ 2 prevalent; pH 7.6) mitochondria. Experiments with a specially designed splicing reporter construct stably transfected into HT1080 cells indicate that acidic conditions favor a splicing pattern responsible for the muscle-and heartspecific ␤ 2 isoform. Taken together, these data indicate a regulatory role of IDH␤ isoforms in determining the pH optimum for IDH activity through the tissue-specific alternative splicing.

Expression of major histocompatibility complex (MHC) class I genes exhibits unique tissue and developmental specificity. In an effort to study molecular mechanisms of MHC class I gene regulation, an in vitro transcription system has been established. In B cell nuclear extracts a template DNA containing the mouse H-2Ld promoter sequence accurately directed RNA polymerase II-dependent transcription of a G-free cassette. A conserved class I regulatory complex previously shown to moderately enhance promoter activity in vivo enhanced transcription in vitro by 2-3 fold. Much of this enhancement was accounted for by a 40 bp fragment within the complex, which was capable of activating a basal H-2Ld promoter in either orientation. Farther downstream, another element called site B was identified, which independently activated MHC class I transcription in vitro by 2-4 fold. Site B bound a specific nuclear factor(s) through an NF-1 binding site but not through a neighboring CCAAT site. The functional significance of site B in vivo was demonstrated in transfection experiments in which site B enhanced MHC class I promoter activity to a degree comparable to that seen in vitro. With the identification of the two upstream activators, MHC class I genes may serve as a model to study roles of sequence-specific DNA-binding proteins in transcription in vitro.

The thyroid hormone and retinoid X receptors form a heterodimer with each other and mediate thyroid hormone (T3)-dependent transcription. Retinoid X receptor, in addition, forms a homodimer and mediates 9-cis-retinoic aciddependent transcription. Here, recombinant thyroid hormone receptor and recombinant retinoid X receptor .8 expressed from baculovirus vectors have been studied for ligandmediated activation of transcription in vitro. We show that the two recombinant receptors, most likely as a heterodimer, cooperatively enhance transcription in vitro from a template containing functional T3 responsive elements. The enhancement was specific for the T3 responsive element and was greatest when T3 was added to the reaction (-14-fold increase). Albeit to a lesser degree, the two receptors also directed transcription in the absence of T3. Template competition experiments suggest that the two receptors enhance formation of the preinitiation complex and that activation by T3 occurs when the ligand binds the receptor prior to (or during), but not after, the formation of the preinitiation complex. Although 9-cis-retinoic acid had no effect on the T3-dependent transcription, this ligand activated transcription in vitro directed by recombinant retinoic X receptor 1A, most likely as a homodimer. This activation was observed when using nuclear extracts from embryonal carcinoma cells as a source of basal transcription factors, but not those from B lymphocytes. These results demonstrate that transcriptional activation mediated by T3 and 9-cis-retinoic acid can be reconstituted in vitro with the respective recombinant receptors.

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