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Genes encoding subunits of the mitochondrial ribosomal protein complex are distributed between the mitochondrial and nuclear genomes. In Arabidopsis thaliana only seven out of the nearly 70 genes coding for mitochondrial ribosomal subunit proteins are present in the mitochondrial genome. Nevertheless, these genes are co-ordinately expressed. Here, we present the first report of characterization of promoter of a plant mitochondrial ribosomal protein gene AtRps19 (At5g47320), a single copy nuclear gene in A. thaliana. Analysis of transgenic A. thaliana plants carrying seven different AtRps19 upstream fragments linked to the uidA reporter gene revealed that the 879 bp fragment containing the 5′ UTR and the intergenic region is capable of driving gene expression in most of the vegetative tissues. However, inclusion of 447 bp of the upstream gene sequences was essential for obtaining full expression in all tissues including anthers and pollen. Thus, we provide the first experimental proof of overlapping genes in plants. qRT-PCR analysis showed that uidA and native AtRps19 transcript levels were comparable in plants carrying the D4 (−676/+650) construct, indicating that the 1326 bp D4 fragment represents the native promoter of the AtRps19 gene. Comparison of AtRps19 promoter with the widely used CaMV35S promoter showed that AtRps19 promoter is capable of driving gene expression in all tissues including anthers and pollen but was less efficient than the CaMV35S promoter.

Sphingomyelin synthase 1 (SMS1) catalyses the biosynthesis of sphingomyelin in eukaryotic cells. We have previously determined the structure of the SGMS1 gene encoding this enzyme and a number of its alternative transcripts. Here, we describe a study of the expression of the full-length SMS1 protein and the sum of the alternative transcripts encoding this protein in human tissues. The SMS1 protein and mRNA levels in tissues differed significantly and were not correlated, implying the active post-transcriptional regulation of SMS1 protein expression. The putative truncated isoforms of the SMS1 protein, which are encoded by a number of alternative transcripts, were not detected by immunoblotting and thus may be absent or present in only small amounts.

Genetic studies have linked the evolutionary novel, anthropoid primate-specific gene locus G72/G30 in the etiology of schizophrenia and other psychiatric disorders. However, the function of the protein encoded by this locus, LG72, is currently controversially discussed. Some studies have suggested that LG72 binds to and regulates the activity of the peroxisomal enzyme D-amino-acid-oxidase, while others proposed an alternative role of this protein due to its mitochondrial location in vitro. Studies with transgenic mice expressing LG72 further suggested that high levels of LG72 lead to an impairment of mitochondrial functions with a concomitant increase in reactive oxygen species production. In the present study, we now performed extensive interaction analyses and identified the mitochondrial methionine-R-sulfoxide reductase B2 (MSRB2) as a specific interaction partner of LG72. MSRB2 belongs to the MSR protein family and functions in mitochondrial oxidative stress defense. Based on our results, we propose that LG72 is involved in the regulation of mitochondrial oxidative stress.

Acyl-CoA synthetase 5 (ACSL5), a mitochondrially localized enzyme, catalyzes the synthesis of long-chain fatty acid thioesters and is physiologically involved in pro-apoptotic sensing of enterocytes. The aim of the present study is to identify an ACSL5-dependent regulation of mitochondrially expressed proteins and the characterization of related pathways in normal and diseased human intestinal mucosa. Proteomics of isolated mitochondria from ACSL5 transfectants and CaCo2 controls were performed. ACSL5-dependent protein synthesis was verified with quantitative reverse transcription plus the polymerase chain reaction, Western blotting, short-interfering-RNA-mediated gene silencing and additional cell culture experiments. Lipid changes were analyzed with tandem mass spectrometry. ACSL5-related pathways were characterized in normal mucosa and sporadic adenocarcinomas of the human intestine. In CaCo2 cells transfected with ACSL5, mortalin (HSPA9) was about two-fold increased in mitochondria, whereas cytoplasmic mortalin levels were unchanged. Disturbance of acyl-CoA/sphingolipid metabolism, induced by ACSL5 over-expression, was characterized as crucial. ACSL5-related over-expression of mitochondrial mortalin was found in HEK293 and Lovo (wild-type TP53 [tumor protein p53]) and CaCo2 (p53-negative; TP53 mutated) cells but not in Colo320DM cells (mutated TP53). In normal human intestinal mucosa, an increasing gradient of both ACSL5 and mortalin from bottom to top was observed, whereas p53 (wild-type TP53) decreased. In sporadic intestinal adenocarcinomas with strong p53 immunostaining (mutated TP53), ACSL5-related mortalin expression was heterogeneous. ACSL5-induced mitochondrial mortalin expression is assumed to be a stress response to ACSL5-related changes in lipid metabolism and is regulated by the TP53 status. Uncoupling of ACSL5 and mitochondrial mortalin by mutated TP53 could be important in colorectal carcinogenesis.

Summary SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) with promising cancer prevention and therapeutic activity. Extensive preclinical testing documented lack of SHetA2 toxicity at doses 25 to 150 fold above effective doses. Knowledge of the SHetA2 molecular target(s) that mediate(s) the mechanism of SHetA2 action is critical to appropriate design of clinical trials and improved analogs. The aim of this study was to develop a method to identify SHetA2 binding proteins in cancer cells. A known metabolite of SHetA2 that has a hydroxyl group available for attachment was synthesized and conjugated to a linker for attachment to a magnetic microsphere. SHetA2-conjugated magnetic microspheres and unconjugated magnetic microspheres were separately incubated with aliquots of a whole cell protein extract from the A2780 human ovarian cancer cell line. After washing away non-specifically bound proteins with the protein extraction buffer, SHetA2-binding proteins were eluted with an excess of free SHetA2. In two independent experiments, an SDS gel band of about 72 kDa was present at differential levels in wells of eluent from SHetA2-microspheres in comparison to wells of eluent from unconjugated microspheres. Mass spectrometry analysis of the bands (QStar) and straight eluents (Orbitrap) identified mortalin (HSPA9) to be present in the eluent from SHetA2-microspheres and not in eluent from unconjugated microspheres. Co-immunoprecipitation experiments demonstrated that SHetA2 interfered with mortalin binding to p53 and p66 Src homologous-collagen homologue (p66shc) inside cancer cells. Mortalin and SHetA2 conflictingly regulate the same molecules involved in mitochondria-mediated intrinsic apoptosis. The results validate the power of this protocol for revealing drug targets. Electronic supplementary material The online version of this article (doi:10.1007/s10637-013-0041-x) contains supplementary material, which is available to authorized users.

Heat shock protein 70 (HSP70) is a predominant member of the HSP family of proteins which play a variety of functions in the cells and are responsible for cytoprotection under stress conditions. In this study, intronless gene of chicken HSP70 was amplified, cloned in E. coli and characterized. The HSP70 gene contains 1,905 bp ORF. Sequence analysis of the HSP70 gene using Mega 4 and DNA STAR softwares showed a high sequence homology among different species indicating that the gene is evolutionarily conserved. Synonymous substitution (dS) in the HSP70 gene was higher than non-synonymous substitution (dN), suggesting that the gene is not under positive selection and that no advantageous mutations had any significant role in its evolutionary adaptation. The predicted Swiss-model of HSP70 protein consisted of 3 α-helices, 19 β-turns, 7 G-turns and 4 hairpins. The G-factor score for the HSP70 protein model was −0.82 for dihedral bonds, −0.05 for covalent bonds and −0.45 overall, which suggest that the model obtained for HSP70 is a reliable one.

Armadillo (ARM) repeat proteins have tandem repeats of a degenerate sequence motif required for protein–protein interaction and are distributed widely in eukaryotes. In this study, we isolated and characterized a novel ARM repeat-like protein gene from the model legume Lotus japonicus that is differentially induced by abiotic stress. The gene, LjTDF-5, encodes a hypothetical protein of 369 aa with a protein signature "ARM-type fold". Three-dimensional protein structure was predicted to consist of 23 α-helices and no β-sheets by homology modeling. The LjTDF-5-homologous genes were distributed broadly in the plant kingdom and the C-terminal region, around 60 amino acids in length, was highly conserved in all of the homologs examined although any known functional domains or protein signatures in this region were not detected in silico analyses. Subcellular localization assays revealed that the sGFP-fused LjTDF-5 protein localized to the nuclei of onion epidermis cells, despite the protein not containing a typical nuclear localization signal. In quantitative real-time RT-PCR, the expression of LjTDF-5 was highly induced by 100 mM NaCl in the roots and by dehydration in the shoot, but not by abscisic acid (ABA, 10 μM). These results suggest that the ARM repeat-like protein LjTDF-5 functions in or around the nucleus in response to high-salt stress and dehydration in L. japonicus.

Neuroinflammation can contribute to neuronal dysfunction, death and several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, and multiple sclerosis. Lipopolysaccharide (LPS)-induced neuroinflammation severely affects neurons and can contribute to neuronal dysfunction and degeneration by causing the release of inflammatory and neurotoxic factors. We evaluated the long-term effects of treating differentiated SH-SY5Y cells with LPS to mimic LPS-induced neuroinflammation. Using matrix assisted laser desorption ionization-time of flight mass spectrometry and MetaCore pathway analysis software (GeneGo), the proteomic expression profiles of differentiated SH-SY 5Y cells after LPS treatment was studied to determine the inflammatory effects on the process of SH-SY5Y differentiation. Long-term LPS treatment resulted in the upregulation of phosphodiesterase 4B (PDE4B), slit robo GTPase (SRGAP2), transcription repressor E2F-6, vimentin, and 70 kDa heat shock protein 9 (Mortalin/HSPA9). Taken together, our results suggest that LPS-treated differentiation of SH-SY5Y cells can lend insight into the multiple pathways involved in neurological diseases.

Bridging the gap between functional genomics and traditional molecular cell biology is a challenge of the next decade. Here, we are aiming to find routines for targeted quantitation of protein silencing in response to RNAi based on complex cellular lysates. A workflow was established adapting siRNA treatment, processing the sample, generating isobaric iTRAQ®-reagent-labeled peptides, and analyzing the sample applying MRM-based peptide quantitation to verify protein silencing on a 4000 QTRAP LC/MS/MS mass spectrometer. Subsequently, eight targets were analyzed, mostly with two siRNA designs. Although transcript and protein silencing correlated, the downregulation on the protein level was less pronounced. A time-course analysis of the chaperon HSPA9/mortalin indicated a delayed kinetic of protein versus transcript silencing. Further, the analysis of the functional response on the example of HSD17B4, a multifunctional enzyme essential to generate precursors for cholesterol biosynthesis, confirmed that strong silencing on the transcript level accompanied by moderate reduction of protein is sufficient to generate a physiological significant response. Fifty percent protein silencing resulted in a 3.5-fold induction of low-density lipoprotein and therefore cholesterol uptake in human liver cells. The established routines pave the way for the development of targeted protein quantitation assays suitable for target and biomarker validation.

Heat shock proteins (HSPs) are upregulated in response to stress and play a protective function in refolding of cellular proteins. In hypertension, the heart is a vital organ that requires examination and investigation, and primary induction of HSPs is predominantly effected. Hypertension results from osmotic imbalance during renin-angiotensin cycle inefficiency. Osmotic stress protein 94 (OSP94) is a stress protein induced upon osmotic imbalance. It is therefore necessary to analyze its precise role in the hypertensive heart. We have first reported the cloning and expression of human heart OSP94 followed by an analysis of gene sequence and protein homology. Directional cloning of OSP94 by PCR amplification yielded a 2.5 kb amplicon and was cloned into pET-15b. Site-directed mutagenesis was essentially followed. mRNA expression levels were evaluated in correlation with HSPs. Gene analysis indicated a 2520 bp sequence with an 838-amino acid protein complement. Protein homology revealed highly conserved sequence similarity among mammalian sequences. Structural predictions of OSP94 protein were also investigated. OSP94 is therefore recognized as a significant stress protein for investigations in hypertensive heart tissues and is a highly conserved protein in the HSP110 subfamily.

Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60-198) of Mtm1p plays an important role in localization of the protein to the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (beta-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial protein that may be playing an important role in activation of MnSOD1 in Arabidopsis.

EBV BARF1 gene encodes a secretory protein with transforming and mitogenic activities. In this report, the post-translational modification, folding, maturation and secretion of BARF1 are systematically studied by site-directed mutagenesis and overexpression of the protein in mammalian cells using the vaccinia/T7 system. The protein was shown to be post-translationally modified by N-linked glycosylation on the asparagine 95 residue. This modification was confirmed to be essential for the maturation and secretion of the protein. Analysis of the four cysteine residues by site-directed mutagenesis demonstrated that cysteine 146 and 201 were essential for proper folding and secretion of the protein. To search for human proteins involved in the maturation process of the protein, a yeast two-hybrid screening was carried out using the BARF1 sequence from amino acids 21-221 (BARF1Delta) as bait, leading to the identification of human hTid1 protein as a potential interacting protein. This interaction was subsequently confirmed by coimmunoprecipitation and dual immunofluorescent labeling of cells coexpressing BARF1 and hTid1, and the interaction domain in hTid1 was mapped to amino acids 149-320. Interestingly, coexpression of BARF1 with hTid1 demonstrated that hTid1 could promote secretion of BARF1, suggesting that hTid1 may act as a chaperone to facilitate the folding, processing and maturation of BARF1.

Proteins interacting with the promoter region of the cox1 gene have been isolated from maize mitochondria. Neither the poly[dIdC-dIdC] duplex nor KCl at concentrations up to 500 mM affect the binding, which indicates that the complex is highly specific. Moreover, no binding is observed with promoter regions of other mitochondrial genes (cox3 and rrn26). Mobility shift competition data confirm the assumption that the protein under study is specific to the cox1 promoter. The results of electrophoretic mobility shift assay (EMSA) combined with protein elution from polyacrylamide gel indicate that the DNA-protein complex contains three polypeptides with molecular weights of 60, 44, and 22 kDa. It has been shown that the core motif AAGTA is essential for DNA binding. It is proposed that the proteins under consideration play an important role in the transcription of the plant mitochondrial cox1 gene.

Protein phosphatase 3 (PPP3, formerly PP2B, Calcineurin), a serine/threonine protein phosphatase, is a heterodimer composed of one catalytic subunit (PPP3C, Calcineurin A) and one regulatory subunit (PPP3R, Calcineurin B). PPP3R, an EF-hand Ca2+ binding protein, contains four high-affinity EF-hand calcium-binding sites, indicating that PPP3 plays critical roles in many calcium-mediated signal transduction pathways. PPP3R has two isoforms, PPP3R1 (also known as PP2Bbeta1) and PPP3R2 (also known as PP2BB2). While PPP3R1 is ubiquitously expressed in different tissues, PPP3R2 is exclusively expressed in testis. PPP3R2 has only been identified in rat and mouse. Here we report a human homologue of PPP3R2, which is designated PPP3RL (PPP3R like protein). PPP3RL gene was predicated to encode 171 amino acid residues with four EF-hand calcium-binding domains and this putative protein shares 82.9% and 80.5% identity with PPP3R2 of rat and mouse, respectively. Our results show that PPP3RL gene localizes to human chromosome 9q22 and transcripts of PPP3RL gene are specifically expressed in the testis, moreover, this tissue-specific expression is due to demethylation of its promoter region in testis.

Mitochondrial genes for ribosomal proteins undergo relatively frequent transfer to the nucleus during plant evolution, and when migration is successful the mitochondrial copy becomes redundant and can be lost. We have examined the status of the mitochondrial rps19 gene for ribosomal protein S19 in closely related cereals. In oat, the mitochondrial rps19 reading frame is blocked by a premature termination codon and lacks abundant transcripts, whereas in the mitochondria of wheat and rye rps19 is a 5'-truncated pseudogene which is co-transcribed with the downstream nad4L gene. In barley and maize, rps19 sequences are completely absent from the mitochondrion. All five of these cereals differ from rice, in which an intact, transcriptionally active mitochondrial rps19 gene is found, and this is preceded by rpl2 in an organization reminiscent of that seen in bacteria. Based on EST sequence data for maize, barley and wheat, it can be inferred that a functional rps19 gene was transferred to the nucleus prior to the divergence of the maize and rice lineages (approximately 50 million years ago), and the present-day nuclear copies encode an N-terminal sequence related to the mitochondrial targeting signal of Hsp70 (heat shock protein) in cereals. Subsequent evolutionary events have included independent losses of the mitochondrial copies in the barley and maize lineages. In the rice lineage, on the other hand, the nuclear copy was lost. This is reflected in the persistence of the mitochondrial rps19 after a period during which rps19 genes coexisted in both compartments. These observations illustrate the dynamic nature of the location and structure of genes for mitochondrial ribosomal proteins in flowering plants.

The combination of cytoplasmic male sterility (CMS) in one parent and a restorer gene ( Rf) to restore fertility in another are indispensable for the development of hybrid varieties. We have found a rice Rf-1 gene that restores BT-type CMS by applying a positional cloning strategy. Using linkage analysis in combination with 6,104 BC(1)F(3) progeny derived from a cross between two near-isogenic lines (NILs) differing only at the Rf-1 locus, we delimited the Rf-1 gene to a 22.4-kb region in the rice genome. Duplicate open reading frames ( Rf-1A and Rf-1B) with a pentatricopeptide (PPR) motif were found in this region. Since several insertions and/or deletions were found in the regions corresponding to both the Rf-1A and Rf-1B genes in the maintainer's allele, they may have lost their function. Rf-1A protein had a mitochondria-targeting signal, whereas Rf-1B did not. The Rf-1B gene encoded a shorter polypeptide that was determined by a premature stop codon. Based on the function of the Rf-1 gene, its product is expected to target mitochondria and may process the transcript from an atp6/orf79 region in the mitochondrial genome. Since the Rf-1A gene encodes a 791-amino acid protein with a signal targeting mitochondria and has 16 repeats of the PPR motif, we concluded that Rf-1A is the Rf-1 gene. Nine duplications of Rf-1A homologs were found around the Rf-1 locus in the Nipponbare genome. However, while some of them encoded proteins with the PPR motif, they do not restore BT-type CMS based on the lack of co-segregation with the restoration phenotype. These duplicates may have played diversified roles in RNA processing and/or recombination in mitochondria during the co-evolution of these genes and the mitochondrial genome.

A central problem in our understanding of mitochondrial (mt) function remains the question of how coordinate transcriptional control is accomplished between nucleus and mitochondria. Here, we report the initial characterization of a protein of previously unknown function, the product of the YMR030 W gene, that appears to mediate such coordinate gene expression. Expression of YMR030 W is glucose-repressible; a deletion mutant for this gene shows a severe growth defect on glycerol-, but not glucose- or ethanol-based medium. In that mutant, transcript levels from GUT1 and GUT2 are highly attenuated compared with those of the wild-type parent when both are grown on glycerol-based medium. Under the same growth conditions, transcripts from the mt OLI1 gene, which has one copy of a mt upstream activating sequence (UAS) in its 5'-flanking region, are attenuated in the DeltaYMR030 W mutant, but mRNA from the mt COX3 ( OXI2) gene, which lacks the mt UAS, are not. Some nuclear genes encoding mt-related proteins also show low transcript levels in the DeltaYMR030 W mutant in comparison with those of the wild-type parent strain during glycerol-based growth. Localization of the protein, via its expression fused to green fluorescent protein, indicates that it is present in both nucleus and mitochondria, supporting a respiration-related transcriptional role for this gene product in both cellular genetic compartments. Because of its role in both respiratory growth and mt function, we designate the YMR030 W coding sequence RSF1 (respiration factor 1).

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

During a large-scale screen of a human fetal brain cDNA library, a novel human gene GNB2L1 encoding a novel RACK (receptor of activated protein kinase C) protein was isolated and sequenced. The cDNA is 1142 bp long and has a predicted open reading frame encoding 316 aa. The predicted protein shows higher similarity to rat RACK1 and many RACK proteins of different organisms including Drosophila, C. elegans, mouse, rat, human, C. fasciculata, zebrafish, A. thaliana, S. cerevisiae and so on, suggesting it is conserved during evolution. The gene was mapped to human chromosome 5q35.3, the telomer position of chromosome 5q, in which the disease gene for early-onset primary congenital lymphedema was mapped. Also, 5q35.3 is a frequently reported location for cytogenetic and molecular abnormalities in renal cell carcinomas. The gene has 8 exons and 7 introns. It is expressed ubiquitously in many human tissues detected by northern blot analysis and RT-PCR.

A gene (rps2) coding for ribosomal protein S2 (RPS2) is present in the mitochondrial (mt) genome of several monocot plants, but absent from the mtDNA of dicots. Confirming that in dicot plants the corresponding gene has been transferred to the nucleus, a corresponding Arabidopsis thaliana nuclear gene was identified that codes for mitochondrial RPS2. As several yeast and mammalian genes coding for mt ribosomal proteins, the Arabidopsis RPS2 apparently has no N-terminal targeting sequence. In the maize mt genome, two rps2 genes were identified and both are transcribed, although at different levels. As in wheat and rice, the maize genes code for proteins with long C-terminal extensions, as compared to their bacterial counterparts. These extensions are not conserved in sequence. Using specific antibodies against one of the maize proteins we found that a large protein precursor is indeed synthesized, but it is apparently processed to give the mature RPS2 protein which is associated with the mitochondrial ribosome.

We report here a novel murine gene, Mrs3/4, encoding a putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing protein 3 and 4 (yMRS3&4) and its human counterpart hMRS3/4. By analyses of radiation hybrids, Mrs3/4 was mapped to mouse Chromosome (Chr) 19 between D19Mit66 and D19Mit24 with a distance of 22.4 cR to D19Mit66, a region that is syntenic to human Chromosome 10q24, where the human gene is located. Structural analysis shows that these proteins belong to the mitochondrial carrier family (MCF), which is characterized by three repeats with two transmembrane domains in each repeat. The murine Mrs3/4 gene has two splicing forms similar to the human homolog. The long form corresponds to the 341-amino acid (aa) protein with six transmembrane domains, and the short form corresponds to the 177-aa protein with three transmembrane domains. Both forms have well-conserved signature sequences of MCF. Targeting experiments showed that both forms have mitochondrial localization. Northern blot analyses showed that Mrs3/4 is ubiquitously expressed as a 1.8-kb transcript with a relative abundance in the heart, liver, kidney, and testis. In conclusion, the reported Mrs3/4 gene shows a strong conservation, mitochondrial protein localization, and a ubiquitous expression, which suggests that it has an important role in mammalian cells, most likely as an ion transporter across the mitochondrial inner membrane.

Previous annotation of the Class III region of the human Major Histocompatibility Complex (MHC) depicts NG36 as an independent gene, which lies immediately centromeric to the G9a gene. However, data presented in this report show that in human and mouse cells the NG36 and G9a genes are predominantly expressed within a single approximately 3.9-kbp transcript. Thus, the human NG36/G9a gene contains 28 exons (4 exons from the NG36 gene and 24 exons from the G9a gene), spans 17.938 kb, and encodes a 1210-amino acid polypeptide. In addition, a splice variant (NG36G9a-SPI), which lacks exon 10, was found to be coexpressed together with the full-length NG36/G9a transcript in both human and mouse cells. To aid functional characterization of the novel NG36/G9a gene-product, T7-epitope-tagged versions of the complete NG36/G9a protein or the G9a region alone (amino acids 210 to 1210) was transiently expressed in mammalian cells. Surprisingly, the sub-cellular distribution of the NG36/G9a-T7 and G9a-T7 proteins was found to be quite distinct. Whereas the G9a-T7 protein was observed in both the cytoplasm and the nucleus, the NG36/G9a-T7 protein was extensively concentrated within the nucleus. Also, the G9a-T7 protein frequently appeared marginalized at the nuclear periphery, while the NG36/G9a-T7 protein was generally found throughout the nucleoplasm. As such, it would appear that the NG36 domain plays a key role in controlling the sub-cellular distribution of the NG36/G9a protein.

The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 andsdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.

Optic atrophy type 1 (OPA1, MIM 165500) is a dominantly inherited optic neuropathy occurring in 1 in 50,000 individuals that features progressive loss in visual acuity leading, in many cases, to legal blindness. Phenotypic variations and loss of retinal ganglion cells, as found in Leber hereditary optic neuropathy (LHON), have suggested possible mitochondrial impairment. The OPA1 gene has been localized to 3q28-q29 (refs 13-19). We describe here a nuclear gene, OPA1, that maps within the candidate region and encodes a dynamin-related protein localized to mitochondria. We found four different OPA1 mutations, including frameshift and missense mutations, to segregate with the disease, demonstrating a role for mitochondria in retinal ganglion cell pathophysiology.

The pea mitochondrial genome contains a truncated rps7 gene lacking ca. 40 codons at its 5' terminus. This single-copy sequence is immediately downstream of and slightly overlapping an actively transcribed and edited reading frame of 744 bp (designated ccb248) homologous to the bacterial helC gene which encodes a subunit of the ABC-type heme transporter involved in cytochrome c biogenesis. This region of mitochondrial DNA appears recombinogenic, and the carboxy-termini of helC-type proteins are predicted to vary in sequence and length among plants. Sequences corresponding to the 5' coding region of rps7 were not detected elsewhere in the pea mitochondrial genome using wheat rps7 probes, and only a very short internal rps7 segment was observed in soybean mitochondrial DNA. The presence of rps7-homologous sequences in the nuclear genomes of pea and soybean is consistent with the recent transfer of a functional mitochondrial rps7 gene to the nucleus in certain plant lineages.

The genomic organization of the human 51-kDa subunit gene (NDUFV1) on human Chromosome (Chr) 11q13 was determined. The NDUFV1 gene consists of 10 exons. Exon 1 encodes for the 20-amino-acids-long import sequence, and exon 1 through 10 codes for the 444-amino-acids-long mature protein. The protein sequence is highly conserved between human and bovine. Northern blotting analysis showed that the NDUFV1 gene expression varies widely among tissues and that in testis a unique mRNA species is present. In comparison with the other complex I flavoproteins, the expression of the 51-kDa gene in pancreatic tissue is high.

A gene coding for a protein that shows homologies to prokaryotic ribosomal protein S2 is present in the mitochondrial (mt) genome of wheat (Triticum aestivum). The wheat gene is transcribed as a single mRNA which is edited by C-to-U conversions at seven positions, all resulting in alteration of the encoded amino acid. Homologous gene sequences are also present in the mt genomes of rice and maize, but we failed to identify the corresponding sequences in the mtDNA of all dicotyledonous species tested; in these species the mitochondrial RPS2 is probably encoded in the nucleus. The protein sequence deduced from the wheat rps2 gene sequence has a long C-terminal extension when compared to other prokaryotic RPS2 sequences. This extension presents no similarity with any known sequence and is not conserved in the maize or rice mitochondrial rps2 gene. Most probably, after translation, this peptide extension is processed by a specific peptidase to give rise to the mature wheat mitochondrial RPS2.

The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28 degrees C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S. cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.

A cDNA (VUpur5) encoding phosphoribosyl aminoimidazole (AIR) synthetase, the fifth enzyme of the de novo purine biosynthesis pathway has been isolated from a cowpea nodule cDNA library. It encodes a 388 amino acid protein with a predicted molecular mass of 40.4 kDa. The deduced amino acid sequence has significant homology with AIR synthetase from other organisms. AIR synthetase is present in both mitochondria and plastids of cowpea nodules [7]. A signal sequence encoded by the VUpur5 cDNA has properties associated with plastid transit sequences but there is no consensus cleavage site as would be expected for a plastid targeted protein. Although the signal sequence does not have the structural features of a mitochondrial targeted protein, it has a mitochondrial cleavage site motif (RX/XS) close to the predicted N-terminus of the mature protein. Southern analysis suggests that AIR synthetase is encoded by a single gene raising questions as to how the product of this gene is targeted to the two organelles. VUpur5 is expressed at much higher levels in nodules compared to other cowpea tissues and the gene is active before nitrogen fixation begins. These results suggest that products of nitrogen fixation do not play a role in the initial induction of gene expression. VUpur5 was expressed in Escherichia coli and the recombinant protein used to raise antibodies. These antibodies recognize two forms of AIR synthetase which differ in molecular size. Both forms are present in mitochondria, although the larger protein is more abundant. Only the smaller protein was detected in plastids.

The nad7 gene, encoding subunit 7 of NADH dehydrogenase, is mitochondrially encoded in seed plants. In the liverwort, Marchantia polymorpha, only a pseudogene is located in the mitochondrial genome. We have now identified the functional nad7 gene copy in the nuclear genome of Marchantia, coding for a polypeptide of 468 amino acids. The nuclear-encoded nad7 has lost the two group II introns present in the mitochondrial pseudogene copy. Instead, a typical nuclear intron is found to split an exon encoding the presumptive mitochondrial targeting signal peptide and the mature subunit 7 of NADH dehydrogenase. These results suggest that RNA-mediated gene transfer from the mitochondrial into the nuclear genome occurs not only in seed plants but also in bryophytes.

Northern analyses and reverse transcription-polymerase chain-reaction (RT-PCR) experiments, followed by PCR amplification product sequencing, were performed on total mitochondrial (mt) RNAs from wheat seedlings and tissue cultures. It was shown that the rps13 gene, which encodes ribosomal protein S13, and the atp6 gene, which encodes subunit 6 of the ATP synthase complex, were co-transcribed. However, rps13 transcripts were virtually undetectable in seedlings under conditions where atp6 transcripts appeared abundant. In addition, markedly higher steady state transcript levels were observed in tissue culture. Expression of the mitochondrial rps13 gene was confirmed by showing that its transcripts were edited. Slight differences between editing patterns of tissue-culture and whole-plant transcripts were found. Taken together, these results suggest that in vitro culture could disturb the post-transcriptional regulation of gene expression.

The Saccharomyces cerevisiae open reading frame YDL202w has been characterised in the course of the EUROFAN yeast genome analysis program. Disruption of YDL202w causes a respiratory deficient phenotype accompanied by a loss of mitochondrial DNA. This phenotype is usually found in mutants defective in mitochondrial replication or gene expression. YDL202w has the potential to encode a soluble protein of 249 amino acids. It shows significant similarities to the ribosomal protein L10 from various bacteria and to a previously determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal protein L11. The predicted amino-acid sequence of YDL202w starts with a stretch which has neither any correspondence in the bacterial sequences nor in the protein isolated from mitochondrial ribosomes. Furthermore, this stretch matches the requirements for a signal sequence for mitochondrial protein import. A mitochondrial location of the YDL202w gene product was proven by use of a carboxy terminally HA-tagged version. These findings clearly indicate that YDL202w encodes this mitochondrial ribosomal protein (YmL11).

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc 1 complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc 1 complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc 1 complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc 1 complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc 1 complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc 1 complex; however, this association did not lead to enzymatic activity of the bc 1 complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.

The intrinsic 28.5-kDa iron-sulfur protein of complex I in the mitochondrial respiratory chain is encoded in the nucleus in animals and fungi, but specified by a mitochondrial gene in trypanosomes. In plants, the homologous protein is now found to be encoded by a single-copy nuclear gene in Arabidopsis thaliana and by two nuclear genes in potato. The cysteine motifs involved in binding two iron-sulfur clusters are conserved in the plant protein sequences. The locations of the seven introns, with sizes between 60 and 1700 nucleotides, are identical in the A. thaliana and the two potato genes, while their primary sequences diverge considerably. The A + T contents of the intron sequences range between 61% and 73%, as is characteristic for dicot plants, but are in some instances not higher than in the adjacent exons. Here, differences in T content may instead serve to discriminate exons and introns. In potato, both genes are expressed, with the highest levels found in flowers. Sequence similarities between the homologous nuclear and mitochondrial genes indicate that the nuclear forms in animals and plants originate from the endosymbiont genome.

Characterizing the spatial organization of the human mitochondrial proteome will enhance our understanding of mitochondrial functions at the molecular level and provide key insight into protein-disease associations. However, the sub-organellar location and possible association with mitochondrial diseases are not annotated for most mitochondrial proteins. Here, we characterized the functional and spatial organization of mitochondrial proteins by assessing their position in the Mitochondrial Protein Functional (MPF) network. Network position was assigned to the MPF network and facilitated the determination of sub-organellar location and functional organization of mitochondrial proteins. Moreover, network position successfully identified candidate disease genes of several mitochondrial disorders. Thus, our data support the use of network position as a novel method to explore the molecular function and pathogenesis of mitochondrial proteins.

SSM4 was isolated as a suppressor of rna14-1, a mutant involved in nuclear mRNA maturation. In order to isolate genes interacting with SSM4, we have searched for mutants that are syntheticlethal in association with an SSM4 deletion. Among the mutants obtained, one, named sls1-1, shows a pet- phenotype. We have cloned and sequenced this gene. It encodes a protein with a calculated molecular mass of 73 kDa. This protein contains a mitochondrial targeting presequence but does not show homology with other known proteins. Deletion of SLS1 does not affect cell viability on glucose but is lethal on a non-fermentable medium. The Sls1p protein does not appear to be involved in mitochondrial DNA replication, transcription, or in RNA splicing maturation or stability. We have also tagged this protein and localized it in mitochondria. Treatment with alkaline carbonate does not extract this protein from mitochondria, suggesting strongly that it is a mitochondrial integral membrane protein. Thus, the SLS1 gene, encodes a mitochondrial integral membrane protein and is paradoxically synlethal in association with a deletion of the SSM4 gene, which encodes an integral nuclear membrane protein.

Ubiquitin-conjugating enzymes (E2s) are essential components of the post-translational protein ubiquitination pathway, mediating the transfer of activated ubiquitin to substrate proteins. We have identified a human gene, UBE2L3, localized on Chromosome (Chr) 22q11. 2-13.1, encoding an E2 almost identical to that encoded by the recently described human L-UBC (UBE2L1) gene present on Chr 14q24.3. Using chromosome-specific vectorette PCR, we have determined the intron/exon structure of UBE2L3. In contrast to the intronless UBE2L1 gene, the coding sequence of UBE2L3 is interrupted by three large introns. UBE2L3-derived mRNA appears to be the predominant species in most tissues rather than the transcript from UBE2L1 or another homologous gene UBE2L2, which maps to Chr 12q12. We also present additional evidence that these genes are members of a larger multigene family. The primary sequence of the protein encoded by UBE2L3 is identical to partial peptide sequence derived from the rabbit E2 'E2-F1,' suggesting that we have identified the human homolog of this protein. This latter E2 has been demonstrated to participate in transcription factor NF-kappaB maturation, c-fos degradation, and human papilloma virus-mediated p53 degradation in vitro.

Mitochondrial transcription factor A (mtTFA) is a key regulator of mammalian mitochondrial DNA transcription. We report here that a testis-specific isoform of mouse mtTFA lacks the mitochondrial targeting sequence and is present in the nucleus of spermatocytes and elongating spermatids, thus representing the first reported mammalian gene encoding protein isoforms targeted for the mitochondria or the nucleus. The presence of the mitochondrial transcriptional activator in the nucleus raises the possibility of a role for this protein in both genetic systems. Mutations in the nuclear mtTFA gene may therefore exhibit phenotypic consequences due to altered function in either or both genetic compartments.

The most common adult form of muscular dystrophy, myotonic dystrophy, is due to a triplet repeat (CTG) expansion in the 3' untranslated region of the myotonic dystrophy gene. Although this gene is known to encode a protein kinase, the mechanism by which a defect in this gene results in a disease state is not understood. To gain insight into this mechanism, the yeast two hybrid system was utilized to identify proteins which interact with myotonic dystrophy protein kinase. Eight positive clones were identified that interact specifically with the myotonic dystrophy protein kinase. One clone, which encodes a novel protein interacting with myotonic dystrophy protein kinase both in vivo in yeast and in vitro, was characterized further. The gene encoding this protein may represent a member of a small gene family, and the protein (95 amino acids) exhibits a high degree of homology to an snRNP protein, D1. This novel protein may be a member of the signal transduction pathway which is responsible for the manifestation of this disease.

DDX3 is a highly conserved member of ATP-dependent DEAD-box RNA helicases with multiple functions in RNA metabolism and cellular signaling. Here, we describe a novel function for DDX3 in regulating the mitochondrial stress response in the parasitic protozoan Leishmania. We show that genetic inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species (ROS) associated with a defect in hydrogen peroxide detoxification. Upon stress, ROS production is greatly enhanced, causing mitochondrial membrane potential loss, mitochondrial fragmentation, and cell death. Importantly, this phenotype is exacerbated upon oxidative stress in parasites forced to use the mitochondrial oxidative respiratory machinery. Furthermore, we show that in the absence of DDX3, levels of major components of the unfolded protein response as well as of polyubiquitinated proteins increase in the parasite, particularly in the mitochondrion, as an indicator of mitochondrial protein damage. Consistent with these findings, immunoprecipitation and mass-spectrometry studies revealed potential interactions of DDX3 with key components of the cellular stress response, particularly the antioxidant response, the unfolded protein response, and the AAA-ATPase p97/VCP/Cdc48, which is essential in mitochondrial protein quality control by driving proteosomal degradation of polyubiquitinated proteins. Complementation studies using DDX3 deletion mutants lacking conserved motifs within the helicase core support that binding of DDX3 to ATP is essential for DDX3’s function in mitochondrial proteostasis. As a result of the inability of DDX3-depleted Leishmania to recover from ROS damage and to survive various stresses in the host macrophage, parasite intracellular development was impaired. Collectively, these observations support a central role for the Leishmania DDX3 homolog in preventing ROS-mediated damage and in maintaining mitochondrial protein quality control.

The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153-278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho- or rho0 cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.

Multidrug-resistant cancer cells frequently overexpress the 110-kD LRP protein (originally named Lung Resistance-related Protein). LRP overexpression has been found to predict a poor response to chemotherapy in acute myeloid leukaemia and ovarian carcinoma. We describe the cloning and chromosome localization of the gene coding for this novel protein. The deduced LRP amino acid sequence shows 87.7% identity with the 104-kD rat major vault protein. Vaults are multi-subunit structures that may be involved in nucleo-cytoplasmic transport. The LRP gene is located on chromosome 16, close to the genes coding for multidrug resistance-associated protein and protein kinase C-beta, and may mediate drug resistance, perhaps via a transport process.

The Arabidopsis AGL3 gene was previously identified on the basis of sequence similarity to the floral homeotic gene AGAMOUS (AG), which encodes a protein with a conserved MADS domain that is also found in human and yeast transcription factors (SRF and MCM1, respectively). Analysis of newly isolated full-length cDNA clones as well as genomic clones indicates that AGL3 is indeed a MADS-box gene with a general intron-exon structure similar to other plant MADS-box genes. However, unlike the others, which are expressed specifically in flowers, AGL3 is expressed in all above-ground vegetative organs, as well as in flowers, but not in roots. Furthermore, since AGL3 is MADS-domain protein, it is likely that it is also a DNA-binding protein regulating transcription. To characterize AGL3 as a DNA-binding protein in vitro, we expressed the AGL3 protein in Escherichia coli, and characterized its DNA-binding properties. We show that AGL3 binds to sequences which resemble the target sequences of SRF and MCM1, and have determined the consensus sequence to which AGL3 binds using random oligonucleotides. These results suggest that AGL3 is a widely distributed DNA-binding protein, which may be involved the transcriptional regulation of genes in many cells.

cDNA clones of a tomato protein, called Lycopersicum esculentum putative Mg(2+)-dependent ATPase (LeMA-1), were isolated from a cDNA library. Sequence comparison of the tomato protein with other genes in the database revealed that the protein is highly homologous to a human protein called TBP-1 and a yeast Tat-binding-analogue protein YTA1A. All three proteins belong to the recently discovered protein family of putative Mg(2+)-dependent ATPases and form within this family a subgroup of proteins involved in controlled protein degradation and possibly also in transcriptional regulation. Expression of the mRNA of LeMA-1 could be monitored in several plant tissues. LeMA-1 is the first member of this subgroup of proteins isolated from plants.

The single-stranded DNA binding protein, or gene V protein (gVp), encoded by gene V of the filamentous bacteriophage M13 is a multifunctional protein that not only regulates viral DNA replication but also gene expression at the level of mRNA translation. It furthermore is implicated as a scaffolding and/or chaperone protein during the phage assembly process at the hostcell membrane. The protein is 87 amino acids long and its biological functional entity is a homodimer. In this manuscript a short description of the life cycle of filamentous phages is presented and our current knowledge of the major functional and structural properties and characteristics of gene V protein are reviewed. In addition models of the superhelical complexes gVp forms with ssDNA are described and their (possible) biological meaning in the infection process are discussed. Finally it is described that the 'DNA binding loop' of gVp is a recurring motif in many ssDNA binding proteins and that the fold of gVp is shared by a large family of evolutionarily conserved gene regulatory proteins.

The family of hsp70 (70 kilodalton heat shock protein) molecular chaperones plays an essential and diverse role in cellular physiology, Hsp70 proteins appear to elicit their effects by interacting with polypeptides that present domains which exhibit non-native conformations at distinct stages during their life in the cell. In this paper we review work pertaining to the functions of hsp70 proteins in chaperoning mitochondrial protein biogenesis. Hsp70 proteins function in protein synthesis, protein translocation across mitochondrial membranes, protein folding and finally the delivery of misfolded proteins to proteolytic enzymes in the mitochondrial matrix.

The S10 ribosomal protein gene (rps10), which has not been previously reported in any angiosperm mitochondrial genome, was identified by sequence analysis in the potato mitochondrial DNA. This gene is found downstream of a truncated non-functional apocytochrome b (cob) pseudogene, and is expressed as multiple transcripts ranging in size from 0.8 to 5.0 kb. Southern hybridization analysis indicates that rps10-homologous sequences are not present in the wheat mitochondrial genome. Sequence analysis of a single-copy region of the pea mitochondrial genome located upstream of cox1 [11] shows that a non-functional rps10 pseudogene is present in this species. These results suggest that the functional genes coding for wheat and pea mitochondrial RPS10 polypeptides have been translocated to the nucleus.

A mitochondrial protein, able to specifically bind two double-stranded homologous sequences of seaurchin mitochondrial DNA, has been partially purified from Paracentrotus lividus eggs. This protein, present at a low concentration, is a polypeptide of 40 kDa. One of the binding sequences, located in the main non-coding region, contains the replication origin of the mitochondrial DNA H-strand. By a combination of band-shift, DNase footprinting, and modification interference analyses with homologous and heterologous probes we identified YCYYATCAN(A/T)RC as the minimum sequence required for the binding. The protein also shows a single-stranded DNA-binding activity, as it is able to specifically interact with one of the strands of the binding sites. These features are consistent with a function of the protein in the modulation of sea-urchin mitochondrial DNA replication during the developmental stages.

Using low-stringency hybridization and polymerase chain reaction (PCR)-based DNA amplification, we have isolated three Drosophila melanogaster genes that encode troponin-C isoforms and one specifying a protein that is closely related to calmodulin. Two of the troponin-C genes, located within the 47D and 73F subdivisions of chromosomes 2 and 3, respectively, encode very closely related isoforms. That specified by the 47D gene accumulates almost exclusively in larval muscles, while that encoded by the 73F gene is present in both larvae and adults. The third gene, located within the 41C subdivision of chromosome 2, encodes a more distantly related troponin-C isoform that accumulates only within adults. The gene that encodes the calmodulin-related protein is located within the 97A subdivision of chromosome three. The protein encoded by this gene has a different primary sequence from that of conventional calmodulin, which is specified by a gene located within the 49A subdivision of chromosome 2. Our report is the first to describe insect troponin-C isoforms and further avails genetic methods for investigating the in vivo functions of the troponin-C/myosin light-chain/calmodulin protein superfamily.

The nuclear gene PET112 was originally identified by a mutation (pet112-1) that specifically blocked accumulation of cytochrome c oxidase subunit II. The mutation causes a post-transcriptional defect since the level of COX2 mRNA in the mutant is the same as in the wild-type. However, PET112 does not have a function similar to that of PET111, a COX2 mRNA-specific translational activator: while pet111 mutations are suppressed by chimeric COX2 mRNAs bearing 5' leaders of other mitochondrial mRNAs, pet112-1 is not. The PET112 gene was isolated and shown to code a protein of 541 residues (62 kDa) with no significant homology to known amino-acid sequences. By hybridization to defined genomic clones the gene was mapped to chromosome II between cdc25 and ils1. Disruption of the PET112 open reading frame destabilized the mitochondrial genome, causing cells to become rho-. This finding suggests that PET112 has an important general function in mitochondrial gene expression, probably in translation.