Primary structure of a precursor for the delta-subunit of sweet potato mitochondrial F1-ATPase deduced from full length cDNA.

A cDNA library constructed from poly(A)-rich RNA of the sweet potato tuberous root using a newly developed plasmid vector carrying tac-SP6 promoters was used to identify full length cDNAs for the nuclear-encoded delta-subunit of mitochondrial F1-ATPase by oligonucleotide-hybridization selection. Selected clones contained cDNA insert which carry the entire coding capacity for the pre-delta-subunit, since the RNA transcribed in vitro from SP6 promoter on the vector directed the synthesis of pre-delta-subunit polypeptide in a wheat germ in vitro translation assay. The nucleotide sequence of one of these cDNAs indicates that it can code for the pre-delta-subunit of 244 amino acids of which 199 amino acids encode the mature subunit. The amino acid sequence of the mature delta-subunit shows similarities of about 18-25% amino acid positional identity with the delta-subunits of bacterial F1-ATPases, about 26% with the delta-subunit of chloroplast CF1-ATPase, and about 32-37% with oligomycin sensitivity conferring proteins of animal and fungal mitochondria. The N-terminal presequence of the precursor composed of maximum of 45 amino acids does not show any obvious sequence homology with either the transit peptide of the nuclear-encoded pre-delta-subunit of chloroplast CF1 or the presequence of the nuclear-encoded pre-oligomycin sensitivity conferring proteins. At least two types of the delta-subunit cDNAs with very similar structures were identified from the library, and the presence of multiple copies of the delta-subunit gene in the hexaploid genome of the sweet potato is also suggested by genomic Southern blot hybridization.     

Separation, amino-terminal sequence and cell-free synthesis of the smallest subunit of sweet potato cytochrome c oxidase

The smallest subunit (V) of sweet potato cytochrome c oxidase was separated into three polypeptides, Va, Vb and Vc with different molecular masses (7.4 kDa, 6.8 kDa and 6.2 kDa respectively) by highly resolving sodium dodecylsulfate polyacrylamide gel electrophoresis. Antibody against subunit V reacted specifically with the polypeptide Vc. When polyadenylated mRNA from sweet potato root tissue was translated in a wheat germ cell-free system, the smallest subunit (Vc) of the polypeptides was synthesized to the same size as the mature form, which suggests that the mature subunit retains the signal for import into mitochondria. Within the N-terminal first 25 amino acids there is a stretch of 16 non-polar residues, periodically linked by basic residues, which might form an amphiphilic helix as the targeting signal.     

Characterisation of three cDNA clones encoding different mRNAs for the precursor to the small subunit of wheat ribulosebisphosphate carboxylase.

We have isolated and sequenced three cDNA clones for the nuclear-encoded precursor to the small subunit of the chloroplast enzyme, ribulose-1,5-bisphosphate carboxylase of wheat. The nucleotide sequences of these clones are different, indicating that they are probably derived from three different mRNAs. This finding is consistent with the proposal that this polypeptide is encoded by a multigene family in wheat, in support of similar data reported by Broglie et al. (Bio/Technology 1:55-61, 1983). We deduce that the mature small subunit polypeptide is comprised of 128 amino acids and that its precursor contains an N-terminal transit peptide sequence. The sequences of both the mature small subunit and its transit peptide differ at several positions from those determined by Broglie et al, (1983) from a different wheat cultivar. Different wheat cultivars might therefore contain different small subunit polypeptides. A comparison of nucleotide and amino acid sequences of the small subunit from wheat, pea, soybean and spinach shows that these sequences are not highly conserved, particularly between monocotyledon and dicotyledon species.     

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

cDNA cloning and sequencing for the import precursor of subunit B in H(+)-ATP synthase from rat mitochondria

The nucleotide sequence of the import precursor of subunit b of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 1,124 nucleotides including a coding region for the import precursor of subunit b and noncoding regions of both the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame consisted of 256 and 214 amino acid residues with a molecular weight of 28,867 and 24,628, respectively. The presequence of 42 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M _r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.     

The amino acid sequence of the 24-kDa subunit, an iron-sulfur protein, of rat liver mitochondrial NADH dehydrogenase deduced from cDNA sequence

Antiserum directed against bovine heart mitochondrial NADH dehydrogenase has been used to screen a rat liver cDNA expression library in lambda gt11. The insert cDNA of a positive clone was found to represent the 24-kDa subunit of NADH dehydrogenase by epitope selection using nitrocellulose filter containing the expressed proteins. The amino acid sequence deduced from the nucleotide sequence of the cloned cDNA indicated that the 24-kDa subunit is produced as a precursor with an amino-terminal extension, and that its mature form consists of 217 amino acid residues with a molecular weight of 23,933.     

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.     

Isolation and characterization of a complementary DNA clone coding for the E1 beta subunit of the bovine branched-chain alpha-ketoacid dehydrogenase complex: complete amino acid sequence of the precursor protein and its proteolytic processing

A 1.7-kb cDNA clone encoding the entire precursor of the E1 beta subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKDH) complex was isolated from a bovine liver cDNA library by screening with a mixture of synthetic oligonucleotide probes corresponding to the C-terminal five-residue sequence of the mature E1 beta subunit. A partial amino acid sequence was determined by Edman degradation of the intact subunit and the peptides generated by cleavage at the lysyl bonds. Nucleotide sequence analysis revealed that the isolated cDNA clone contained the 5'-untranslated sequence of 186 nucleotides, the translated sequence of 1176 nucleotides, and the 3'-untranslated sequence of 306 nucleotides with a poly(A) tail. A type AATAAA polyadenylation signal was located 17 nucleotides upstream of the start of a poly(A) tail. Comparison of the amino acid sequence predicted from the nucleotide sequence of the cDNA insert of the clone with the partial amino acid sequence of the mature BCKDH E1 beta subunit showed that the cDNA insert encodes for a 342 amino acid subunit with Mr 37,745 and that the subunit is synthesized as the precursor with a leader sequence of 50 amino acids and processed at the N-terminus. Northern blot analysis using the cDNA insert as a probe showed the presence of a 1.8-1.9-kb mRNA in bovine liver, suggesting that the insert covers nearly a full length of mRNA. Alignment of the deduced amino acid sequence of bovine BCKDH E1 beta with that of the human pyruvate dehydrogenase (PDH) complex E1 beta subunit revealed a high degree of sequence homology throughout the two enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Human F1-ATPase: molecular cloning of cDNA for the beta subunit

F1-ATPase is the major enzyme for ATP synthesis, and its beta subunit is the catalytic site. To date, no full-length cDNA for the eukaryotic F1 gene has been reported. Human F1 was studied because of its importance in medicine and cell biology. Here we report molecular cloning of a full-length cDNA for the human F1 beta subunit and purification of the human F1 beta subunit. The HeLa cell cDNA library constructed in an expression vector gamma gt11 was screened with antiserum against the yeast F1 beta subunit. One of the positive phage DNAs containing the human F1 beta gene and its flanking regions (1.8 kilobase pairs) was sequenced by the dideoxy chain termination method. The open reading frame started from a putative signal presequence, which was rich in both serine and arginine. There was a homologous segment in the signal presequence of human ornithine transcarbamoylase and that of F1 beta. The precursor of F1 beta was expressed in E. coli harboring a plasmid which had been constructed with T5 promotor and the F1 beta cDNA. Both the precursor and mature form of F1 beta were detected in HeLa cells in a pulse-chase experiment. The amino acid sequence of 480 residues (51,568.3 daltons) following the presequence was highly homologous with that of mature beef heart F1 beta (97.5%) and E. coli F1 beta (71.7%), but the codon usage in the human gene was very different from those of reported genes coding for F1 beta of other species.     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.