Kinetoplast DNA minicircles of trypanosomatids encode for a protein product

The major constituent of the trypanosomal kinetoplast DNA network are several thousand duplex DNA minicircles whose biological function is still unknown. The coding capacity and expression of these DNA minicircles, was studied in the trypanosomatid Crithidia fasciculata. Kinetoplast DNA minicircle fragments inserted into bacterial plasmid vectors were expressed in the bacterial cell. Sera elicited in rabbits, by immunization with the translational products of kinetoplast DNA minicircles in E. coli, reacted specifically with Crithidia fasciculata cellular antigens. It is inferred that kinetoplast DNA minicircles contain long open reading frames of nucleotides which are expressed in the trypanosomatid cell.     

Trypanosoma brucei minicircles encode multiple guide RNAs which can direct editing of extensively overlapping sequences.

Small guide RNAs (gRNAs) may direct RNA editing in kinetoplastid mitochondria. We have characterized multiple gRNA genes from Trypanosoma brucei (EATRO 164), that can specify up to 30% of the editing of the COIII, ND7, ND8, and A6 mRNAs and we have also found that the non-translated region of edited COIII mRNA of strain (EATRO 164) differs from that of another strain. Several of the gRNAs specify overlapping regions of the same mRNA often specifying sequence beyond that required for an anchor duplex with the next gRNA. Some gRNAs have different sequence but specify identical editing of the same region of mRNA. These data indicate a complex gRNA population and consequent complex pattern of editing in T. brucei.     

Species-specific expression of cytochrome c oxidase subunit III mRNA

1. A cDNA probe encoding cytochrome c oxidase subunit III cloned from rat liver mitochondria was used to quantify mRNA levels in rat, mouse and rabbit tissues. This was compared to its phenotypic expression, using enzyme activity. 2. Enzyme activities were highest in mouse, intermediate in rat, and lowest in rabbit tissues. 3. Subunit III mRNA levels were easily quantified in rat, but could not be accurately measured in rabbit or mouse tissues despite high cytochrome c oxidase activities. 4. Significant subunit III sequence divergence must exist, among these species. Caution should be exercised in quantifying the expression of this mitochondrial gene.     

Alternative editing of cytochrome c oxidase III mRNA in trypanosome mitochondria generates protein diversity

Trypanosomes use RNA editing to produce most functional mitochondrial messenger RNA. Precise insertion and deletion of hundreds of uridines is necessary to make full-length cytochrome c oxidase III (COXIII) mRNA. We show that COXIII mRNA can be alternatively edited by a mechanism using an alternative guide RNA to make a stable mRNA. This alternatively edited mRNA is translated to produce a unique protein that fractionates with mitochondrial membranes and colocalizes with mitochondrial proteins in situ. Alternative RNA editing represents a previously unknown mechanism generating protein diversity and, as such, represents an important function for RNA editing.     

Partially edited mRNAs for cytochrome b and subunit III of cytochrome oxidase from Leishmania tarentolae mitochondria: RNA editing intermediates

Partially edited mRNAs were selected by the polymerase chain reaction and sequenced. In the case of cytochrome b, 102 out of 106 clones displayed patterns of editing that were consistent with a strictly progressive 3' to 5' editing process, as predicted by the guide RNA model of RNA editing. In the case of cytochrome oxidase subunit III (COIII), 177 out of 304 clones displayed strictly progressive 3' to 5' patterns of editing. However, the remaining 127 COIII clones displayed unexpected patterns in which upstream editing preceded downstream editing, uridines were inserted at sites not normally edited, and purine residues were deleted. We suggest that many of these RNAs are produced by normal 3' to 5' editing of the COIII mRNA with incorrect guide RNA molecules.     

Activation of guide RNA-directed editing of a cytochrome b mRNA

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created only after the addition or deletion of specific uridines. Although in vitro systems have been valuable in characterizing the editing mechanism, only a limited number of mRNAs are accurately edited in vitro. We demonstrate here that in vitro editing of cytochrome b mRNA is inhibited by an A-U sequence present on both the 5'-untranslated sequence and on a cytochrome b guide RNA. Mutation of the sequence on the guide RNA stimulates directed editing and results in the loss of binding to at least one component within the editing extract. Mutation of the sequence on the mRNA increases the accuracy of the editing. Evidence is provided that suggests the A-U sequence interacts with the editing machinery both in vitro and in vivo.     

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.     

Kinetoplast DNA minicircles of Leishmania donovani express a protein product

We describe an unprecedented finding of an open reading frame present in the variable region in one of the minicircle sequence classes of a human pathogenic strain of Leishmania donovani (MHOM/IN/90/RMRI 68) which is transcribed and translated. The encoded protein showed homologies to known transport proteins.     

A gene in Paracoccus for subunit III of cytochrome oxidase

The region of Paracoccus denitrificans chromosome where the genes coding for cytochrome oxidase (cytochrome aa3) subunits are located has been cloned. DNA sequencing revealed an open reading frame that codes for a protein homologous to the subunit III of the eukaryotic, mitochondrial enzyme. This subunit is absent from the isolated Paracoccus oxidase. It now seems that it is part of the native enzyme in the bacterial cytoplasmic membrane. This may explain the observed discrepancies in the function of the isolated enzyme.     

Hormonal regulation of cytochrome oxidase subunit messenger RNAs in rat Sertoli cells.

Using differential hybridization to screen a rat Sertoli cell cDNA library for hormonally regulated gene products, we isolated a clone, designated 13-10, which contained a 1.0-kilobase insert and hybridized to a 1.7-kilobase message in total testis, Sertoli cells, and peritubular cells. This mRNA was decreased relative to untreated control levels in total testicular RNA from hypophysectomized rats, but was increased by FSH treatment begun on the day of hypophysectomy. FSH caused a transient rise in 13-10 mRNA at 24 h in cultured Sertoli cells. There was no comparable rise in beta-actin RNA or the RNA/DNA ratio at this time, suggesting that the effect on 13-10 was specific. Testosterone had no effect at any time interval studied. The 13-10 mRNA was not increased in peritubular cells treated in vitro with FSH or testosterone. Sequence analysis of 13-10 revealed more than 99% homology with a portion of the sequence of rat liver cytochrome oxidase subunit I (COX I). The clone included 58% of the open reading frame of COX I as well as that for the adjacent Ser-tRNA. COX I is a mitochondrial gene, and Southern analysis confirmed 13-10 sequence in testicular mitochondrial DNA. In addition to FSH, forskolin and (Bu)2cAMP also increased COX I steady state mRNA in Sertoli cells (3.8-, 4.1-, and 9.2-fold, respectively). (Bu)2cAMP increased mRNA for other mitochondrial gene products, COX subunit II and 16S rRNA (6.9- and 5.4-fold, respectively), whereas the smaller effects elicited by forskolin and FSH were not statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetoplast DNA minicircles of Trypanosoma brucei share regions of sequence homology

Kinetoplast DNA (kDNA) of Trypanosoma brucei consists of massive networks of 10,000 or more interlocked molecules of maxicircle DNA (about 23 kb each) and minicircle DNA (1.1 kb each). Individual minicircle DNA molecules were released from the network by digestion with HaeIII, HpaII, AluI, HhaI, PstI, or HindIII and cloned in E. coli via the plasmid pBR322 and the poly(dG):poly(dC) tailing technique or the DNA ligase technique. The cloned minicircle DNA molecules were compared (i) by two types of filter hybridization, (ii) by renaturation kinetics, and (iii) by heteroduplex analysis. The sequence complexity of total network kDNA is about 300 times that of a single cloned minicircle kDNA molecule. The filter hybridizations and heteroduplex analyses suggest that minicircle molecules possess sequences in common with each other. The renaturation kinetics indicates that these homologous regions comprise about one-fourth of the 1.1-kb minicircle molecule. Therefore each minicircle molecule appears to have about one-fourth of its sequence in common with a large percentage of the total minicircle population and the remaining three-fourths in common with about 1 out of 300 minicircle molecules.     

Monomerization of cytochrome oxidase may be essential for the removal of subunit III

1. Incubation of cytochrome oxidase, under conditions used as initial steps in treatment to remove subunit III, causes at least partial monomerization of the enzyme. 2. The extent of removal of subunit III by anion-exchange fast protein liquid chromatography (FPLC) is much increased if the enzyme is fully monomerized before it is applied to the column. 3. Subunit III is incompletely removed by chymotrypsin treatment. A digestion product of subunit III migrating in SDS-PAGE like subunit IV, is detected with specific antibodies. The amount of this product is reduced when monomerization is increased by raising the detergent/protein ratio. 4. The results suggest that monomerization facilitates removal of subunit III and exposes it to further chymotrypsin digestion. We propose that subunit III is at least in part located in the junction between the monomers in the cytochrome oxidase dimer.     

Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase.

COIII is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COIII gene from Paracoccus denitrificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blue-shifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COII complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIII gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COIII and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intermediates of the assembly pathway or dissociation products of a labile COI + COII complex and its conformational variants.     

Properties and reconstitution of a cytochrome oxidase deficient in subunit III

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.