cDNA cloning and sequencing for the import precursor of coupling factor 6 in H+-ATP synthase from rat liver mitochondria

Higuti, Tomihiko; Osaka, Fumio; Yoshihara, Yutaka; Tsurumi, Chizuko; Kawamura, Yoshihiro; Tani, Isamu; Toda, Hirofumi; Kakuno, Tomisaburo; Sakiyama, Fumio; Tanaka, Keiji; Ichihara, Akira

doi:10.1016/0006-291x(90)90794-n

Cited by 14 publications

(8 citation statements)

References 33 publications

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“…As described previously [20], the probes used were a Xho I– Bgl II fragment of the cDNA for subunit b [21], an Acc I– Ava I fragment of cDNA for subunit d [22], a Hin dIII– Hin dIII fragment of cDNA for subunit e [23], a Hin dIII– Apa LI fragment of cDNA for OSCP [24], a Hin dIII– Apa LI fragment of cDNA for IF1 [24], a Hin dIII– Hin dIII fragment of cDNA for F6 [25], a Xho I– Eco RI fragment of cDNA for subunit c(P1) [24], and an Acc I– Eco O109I fragment of DNA for subunit c(P2) [24]. The probe for β‐subunit mRNA was a fragment of 634–925 bp of β‐subunit cDNA [26] that had been synthesized by a nested reverse transcription‐polymerase chain reaction method using four primers as described previously [20].…”

Section: Methodsmentioning

confidence: 99%

Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Himeda

Morokami

Arakaki

et al. 2000

European Journal of Biochemistry

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Little is known about the relationship between the stoichiometry of polypeptides of multisubunit enzyme complexes and the absolute amount of each transcript of the complexes in mammalian tissues. Here we showed that the absolute amounts of the transcripts of most subunits of rat H 1 -ATP synthase examined greatly differed in the different tissues, showing the following hierarchy of tissue-specificity: heart . kidney . brain < liver. However, surprisingly, there was no difference in the expression pattern of these in terms of the molar ratio of each transcript, indicating a nearly similar stoichiometric expression pattern irrespective of tissue or age of the rat. Therefore, the present finding clearly indicates that most of the transcripts of the 16 subunits of rat H 1 -ATP synthase were concertedly and synchronously expressed, having a constant expression pattern of the transcripts, irrespective of tissue or age of the rats. This is the first report of the absolute amounts of the transcripts of this multisubunit enzyme.Keywords: H 1 -ATP synthase; expression pattern; transcript; stoichiometry; synchronization.The mechanism of the transcriptional regulation of the genes involved in the biosynthesis and assembly of multisubunit mitochondrial enzyme complexes, e.g. Complex I, Complex III, Complex IV and H 1 -ATP synthase, is still obscure [1±4]. Of particular interest is the biogenesis of H 1 -ATP synthase, the world's smallest rotary motor [5±9], the key enzyme in oxidative phosphorylation and the one responsible for the production of most of the ATP in mammalian organisms. Mammalian H 1 -ATP synthase is a macromolecular supercomplex composed of 16 subunits [10±14] with a constant stoichiometry, 6 of which construct the catalytic site of ATP synthesis called F 1 (subunits a 3 , b 3 , g 1 , d 1 , and : 1 , and the loosely attached ATPase inhibitor protein IF1 1 ) [15,16] and 10 of which construct the energy-transduction part called F 0 (subunits a 1 , b 2 , c 10 , d 1 , e 2 , f unknown , g unknown , OSCP 1 , F6 2 , and A6L 1 ) [14,17±18a]. Subunit a and A6L are encoded by the mitochondrial genome, and all of the other subunits are separately encoded by nuclear chromosomes; however, the relationship between the stoichiometry of polypeptides of the complexes and the absolute amount of each transcript of this multisubunit enzyme complex in mammalian tissues is still entirely unknown.In the present work, we determined the absolute amount of nine species of mRNAs for eight nucleus-encoded subunits of H 1 -ATP synthase in the brain, liver, heart, and kidney tissues of 2-week-old to 90-week-old male Fischer 344 rats by using highly purified poly(A) 1 RNA and the mRNAs synthesized by an in vitro transcriptional system. M A T E R I A L S A N D M E T H O D S AnimalsMale Fischer 344 rats were obtained from Charles River Japan Inc (Atsugi, Japan), and were genetically identical because of approximately 300 matings between sister and brother. Purification of Poly(A) 1 RNAThe rats were decapitated, and tissues of brain, liver, h...

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Section: Methodsmentioning

confidence: 99%

Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Himeda

Morokami

Arakaki

et al. 2000

European Journal of Biochemistry

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Section: P-labeled Probe Dnasmentioning

confidence: 99%

“…As described previously [20], the probes used were a XhoI± BglII fragment of the cDNA for subunit b [21], an AccI±AvaI fragment of cDNA for subunit d [22], a HindIII±HindIII fragment of cDNA for subunit e [23], a HindIII±ApaLI fragment of cDNA for OSCP [24], a HindIII±ApaLI fragment of cDNA for IF1 [24], a HindIII±HindIII fragment of cDNA for F6 [25], a XhoI±EcoRI fragment of cDNA for subunit c(P1) [24], and an AccI±EcoO109I fragment of DNA for subunit c(P2) [24]. The probe for b-subunit mRNA was a fragment of 634±925 bp of b-subunit cDNA [26] that had been synthesized by a nested reverse transcription-polymerase chain reaction method using four primers as described previously [20].…”

Section: Preparation Of 32 P-labeled Probe Dnasmentioning

confidence: 99%

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Himeda¹,

Morokami²,

Arakaki³

et al. 2000

Eur J Biochem

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Little is known about the relationship between the stoichiometry of polypeptides of multisubunit enzyme complexes and the absolute amount of each transcript of the complexes in mammalian tissues. Here we showed that the absolute amounts of the transcripts of most subunits of rat H 1 -ATP synthase examined greatly differed in the different tissues, showing the following hierarchy of tissue-specificity: heart . kidney . brain < liver. However, surprisingly, there was no difference in the expression pattern of these in terms of the molar ratio of each transcript, indicating a nearly similar stoichiometric expression pattern irrespective of tissue or age of the rat. Therefore, the present finding clearly indicates that most of the transcripts of the 16 subunits of rat H 1 -ATP synthase were concertedly and synchronously expressed, having a constant expression pattern of the transcripts, irrespective of tissue or age of the rats. This is the first report of the absolute amounts of the transcripts of this multisubunit enzyme.Keywords: H 1 -ATP synthase; expression pattern; transcript; stoichiometry; synchronization.The mechanism of the transcriptional regulation of the genes involved in the biosynthesis and assembly of multisubunit mitochondrial enzyme complexes, e.g. [14,17±18a]. Subunit a and A6L are encoded by the mitochondrial genome, and all of the other subunits are separately encoded by nuclear chromosomes; however, the relationship between the stoichiometry of polypeptides of the complexes and the absolute amount of each transcript of this multisubunit enzyme complex in mammalian tissues is still entirely unknown.In the present work, we determined the absolute amount of nine species of mRNAs for eight nucleus-encoded subunits of H 1 -ATP synthase in the brain, liver, heart, and kidney tissues of 2-week-old to 90-week-old male Fischer 344 rats by using highly purified poly(A) 1 RNA and the mRNAs synthesized by an in vitro transcriptional system. M A T E R I A L S A N D M E T H O D S AnimalsMale Fischer 344 rats were obtained from Charles River Japan Inc (Atsugi, Japan), and were genetically identical because of approximately 300 matings between sister and brother. Purification of Poly(A)1 RNAThe rats were decapitated, and tissues of brain, liver, heart, and kidney were rapidly removed. Total RNA from these tissues was isolated by the guanidinium thiocyanate method [19], followed by centrifugation at 120 000 g for 24 h in 5.7 m CsCl solution and then extraction with acid phenol. The poly(A) 1 RNA was purified with Oligotex-dT30 (Takara) according to the manufacturer's instructions, and the purification steps with Oligotex-dT30 were repeated twice. The poly(A) 1 RNA eluted from the 2nd Oligotex-dT30 was extracted with acid phenol, precipitated with 70% ethanol, dried, and dissolved in diethyl pyrocarbonate-treated water. The amount of poly(A) 1 RNA was determined from the absorbance at 260 nm (one unit of absorbance at 260 nm 40 mg´mL 21). The A260/A280 ratios were in the range 1.8±2.0. Contamination by ribosoma...

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“…1, the probes used were a XhoI-BglII fragment of cDNA for subunit b (13), an AccI-AvaII fragment of cDNA for subunit d (17), a HindIII-HindIII fragment of cDNA for subunit e (15), a HindIII-ApaLI fragment of cDNA for OSCP (16), a HindIII-ApaLI fragment of cDNA for IF1 (16), a HindIII-HindIII fragment of cDNA for F 6 (14), a XhoI-EcoRI fragment of cDNA for subunit c(P1) (16), and an AccI-EcoO1091 fragment of cDNA for subunit c(P2) (16). The probe for ␤-subunit mRNA was a fragment of 634 -925 bp of ␤-subunit cDNA (21) that was synthesized by a nested reverse transcription-polymerase chain reaction method using four primers (outer primers: 5Ј-ATAAGGTTGTGGATCTGCTGGCC-3Ј and 5Ј-AACTCAGCAATAGCACGGGACAGCA-3Ј; inner primers: 5Ј-GGGATATCGCGTTGGTATATGGGCAGATGA-3Ј and 5Ј-GGGCGGC-CGCACATAGATAGCCTGCACTGAG-3Ј).…”

Section: Purification Of Poly(a)mentioning

confidence: 99%

“…We then determined the primary sequences of the synthase subunits by the protein sequence and cDNA cloning techniques (13)(14)(15)(16)(17).…”

mentioning

confidence: 99%

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

Sangawa

Himeda

Shibata

et al. 1997

Journal of Biological Chemistry

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cDNA cloning and sequencing for the import precursor of coupling factor 6 in H+-ATP synthase from rat liver mitochondria

Cited by 14 publications

References 33 publications

Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

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cDNA cloning and sequencing for the import precursor of coupling factor 6 in H+-ATP synthase from rat liver mitochondria

Cited by 14 publications

References 33 publications

Synchronized transcriptional gene expression of H+‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H+‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H+-ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

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Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages

Synchronized transcriptional gene expression of H⁺‐ATP synthase subunits in different tissues of Fischer 344 rats of different ages