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Mitochondrial transcription factor A (TFAM) has been shown to stimulate transcription from mitochondrial DNA promoters in vitro. In order to determine whether changes in TFAM levels also regulate RNA synthesis in situ, recombinant human precursor proteins were imported into the matrix of rat liver mitochondria. After uptake of wt-TFAM, incorporation of [alpha-32P]UTP into mitochondrial mRNAs as well as rRNAs was increased 2-fold (P < 0.05), whereas import of truncated TFAM lacking 25 amino acids at the C-terminus had no effect. Import of wt-TFAM into liver mitochondria from hypothyroid rats stimulated RNA synthesis up to 4-fold. We conclude that the rate of transcription is submaximal in freshly isolated rat liver mitochondria and that increasing intra-mitochondrial TFAM levels is sufficient for stimulation. The low transcription rate associated with the hypothyroid state observed in vivo as well as in organello seems to be a result of low TFAM levels, which can be recovered by treating animals with T3 in vivo or by importing TFAM in organello. Thus, this protein meets the criteria for being a key factor in regulating mitochondrial gene expression in vivo.

The biogenesis of giant mitochondria in flight muscle of Locusta migratoria (L.) was analyzed at the molecular level. During the 2 weeks between the beginning of the last larval stage and the imago capable of sustained flight, individual mitochondria have been shown to enlarge 30-fold and the fractional mitochondrial volume of muscle cells increases fourfold [Brosemer, R.W., Vogell, W. and Bücher, Th. (1963) Biochem. Z. 338, 854-910]. Within the same period, the activity of cytochrome c oxidase, containing subunits encoded on mitochondrial DNA, increased twofold. However, no significant change in mitochondrial DNA copy number, and even a threefold decrease in mitochondrial transcripts, was observed. Mitochondrial translation rate, measured in isolated organelles, was twofold higher in larval muscle, which can be explained only partly by the higher content of mitochondrial RNAs. Thus, rather unusually, in this system of mitochondrial differentiation, the mitochondrial biosynthetic capacity correlates with the rate of organelle biogenesis rather than the steady-state concentration of a marker enzyme. The copy number of mitochondrial DNA does not seem to play a major role in determining either mitochondrial transcript levels or functional mass.

We investigated the in vivo expression of myosin heavy chains (MHC) and myosin light chains (MLC) in the rat uterus during pregnancy and post partum (p. p.). According to their antigenic reactivity, we observed two smooth-muscle-specific MHC (SM-MHC) of 204 kDa and 200 kDa (SM1 and SM2 respectively) and one non-muscle-specific MHC band of 198 kDa (NM-MHC). Adult virgin female rats expressed SM1 and NM-MHC (65/35) but no SM2. During the first pregnancy NM-MHC expression decreased in favour of the SM1 form, the SM1/NM-MHC ratio being 82/12 after 20 days of pregnancy. SM2 is newly expressed in the first p. p. state, the SM1/SM2/NM-MHC ratio being 58/28/14 between 3 and 33 days p. p. During the second pregnancy, starting 34 days p. p., both SM2 and NM-MHC expression decreased, the SM1/SM2/NM-MHC ratio being 85/0/15 after 20 days of pregnancy. Two protein bands of approximately 154 kDa and 145 kDa (sodium dodecylsulphate electrophoresis), designated SMP1 and SMP2 respectively, with the same immunoreactivity as the SM-MHC were observed in vivo in the pregnant but not in the non-pregnant uterus. In addition, a Ca(2+)-independent protease, which uses MHC as substrate, is expressed in the pregnant but not in the non-pregnant rat uterus. Two isoforms of the 17-kDa MLC (LC17a and LC17b) exist in the rat uterus. Expression of the LC17a isoform increased during the first pregnancy from 46% in virgin rats to 65% in uteri of rats 20 days pregnant.(ABSTRACT TRUNCATED AT 250 WORDS)