Identification of nuclear genes encoding mitochondrial proteins: isolation of a collection of D. melanogaster cDNAs homologous to sequences in the Human Gene Index database

As a first step towards using cross-species comparison to complete the inventory of the nuclear genes that encode mitochondrial polypeptides, and ultimately to understand their function through systematic molecular and genetic analysis in a model organism of choice, we report here the characterization of 41 Drosophila melanogaster cDNAs. These cDNAs were isolated by screening an ovarian expression library with antibodies against mitochondrial proteins and identify 17 novel Drosophila genes. The deduced amino acid sequences encoded by the majority of these cDNAs turned out to show significant homology to mitochondrial proteins previously identified in other species. Among others, ORFs putatively encoding six different subunits of ATP synthase and three NADH:ubiquinone reductase subunits were detected. By in situ hybridization, all cDNAs were mapped to single bands on polytene chromosomes, thus identifying candidate Drosophila genes required for mitochondrial biogenesis and maintenance. A search of the Human Gene Index database made it possible in most cases to align the entire Drosophila coding sequence with a human consensus sequence, suggesting that the cDNAs originate from insect counterparts of expressed mammalian genes. Our experimental strategy represents an efficient approach to the identification and interspecies comparison of genes encoding products targeted to the mitochondrion. Received: 13 July 1998 / Accepted: 12 October 1998     

Amplification of transcription of separate mitochondrial genes in human and monkey B-cell non-Hodgkin's lymphoma

Mitochondrial genes that are overexpressed in human and monkey B-cell non-Hodgkin lymphomas (B-NHLs) were sought via subtraction hybridization, cloning, and differential screening of the resulting cDNA libraries. The cDNAs of mitochondrial genes made an appreciable proportion of all lymphoma-specific cDNAs. Lymphomogenesis was associated with overexpression of a mitochondrial gene set which varied with lymphoma type and always included NADHIV. A possible association between overexpression of certain mitochondrial genes and cell malignant transformation is discussed.     

Cloning and characterization of five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var.deliciosa)

Five cDNAs for genes differentially expressed during fruit development of kiwifruit (Actinidia deliciosa var.deliciosa cv. Hayward) were isolated from a library made from young fruit, 8–10 days after anthesis. One gene (pKIWI503) has low levels of expression in young fruit but is induced late in fruit development and during fruit ripening, and has some homology to plant metallothionein-like proteins. The other four genes are highly expressed in young fruit with reduced expression in the later stages of fruit development. pKIWI504 has strong homology to plant metallothionein-like proteins and pKIWI505 exhibits homology to the -subunit of the mitochondrial ATP synthase gene. The two other genes (pKIWI501 and 502) encode proteins with no significant homology to other known sequences.     

Identification of age-dependently expressed genes in mouse liver by differential display–PCR analysis

The aim of this study was to identify genes expressed in an age-dependent manner in mouse (Mus musculus) liver. To search for age-dependently expressed genes, we used a fluorescence differential display–PCR (FDD–PCR) technique on total RNA extracted from mouse livers collected at seven different developmental stages. All differentially expressed cDNAs detected by FDD–PCR were reamplified, subcloned and sequenced, and six genes were confirmed to show age-dependent expression by quantitative real-time PCR analysis. Nucleotide sequence analyses showed that four of them had high homology with known genes (mitochondrial DNA, cytosolic aldehyde dehydrogenase, cell division cycle 2-like 5 and complement component 8 alpha polypeptide), and two with expressed sequence tags of unknown genes. The FDD–PCR technique was effective for detecting novel age-dependently expressed genes, and also for newly characterizing individual expression patterns of known genes. The age-dependent expression patterns of known genes revealed in this study may provide an opportunity to investigate the unknown physiological roles of the proteins they encode.     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Two bovine genes for mitochondrial ADP/ATP translocase expressed differences in various tissues

Two different bovine cDNAs have been characterized that encode closely related homologues of the mitochondrial membrane carrier protein ADP/ATP translocase. One of them codes for the protein that has been characterized previously from bovine heart mitochondria, and the other codes for a protein that differs from it in 33 amino acids out of 297. Including the base substitutions required to bring about these changes in amino acid sequence, the coding regions of the cDNAs differ at 184 positions. In addition, they are extensively diverged in their 3' noncoding sequences, which differ greatly in both length and sequence, and these segments of the cDNAs have been used as hybridization probes to demonstrate that the expression of the two genes giving rise to the two proteins is very different in various bovine tissues. Expression of one gene predominates in heart muscle and that of the other in intestine. Hybridization experiments with digests of genomic DNA have shown the presence of numerous sequences related to the two cDNAs in both the bovine and human genomes. Some of these probably arise from pseudogenes, but three expressed genes have been detected in the human genome. The study of the regulation of the expression of these genes may help to illuminate the basis of tissue-specific human mitochondrial diseases which arise because of defects in mitochondrial enzymes only in the affected tissue and not in other tissues of the same individual.     

Nuclear-encoded protein-coding genes of the agarophyte Gracilaria verrucosa (Gracilariales,Rhodophyta)

Single-copy nuclear genes encoding cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC), triosephosphate isomerase (TPI1) and polyubiquitin (UBI6R), plastid-localized GAPDH (GapA), and mitochondrial aconitase (m-ACN), together with their corresponding cDNAs, have been cloned or PCR-amplified from Gracilaria verrucosa, sequenced, and characterized by genomic Southern hybridization. Three spliceosomal introns were found, one each near the 5 end of GapA, GapC, and m-ACN. Codon usage is biased, with third-position A's underrepresented. Nucleotide compositions of the genes are balanced except in TPI1. Upstream promoter structure, downstream poly(A) processing signals, intron splice junctions, and one translation initiation site were analyzed. Poly(A) processing features of these red algal genes resemble those of green plants more than those of animals.     

Seed-specific,developmentally regulated genes of peanut

Four cDNAs of seed-specific and developmentally regulated peanut (Arachis hypogaea L.) genes were identified by differential screening of a peanut-seed cDNA library using cDNA probes constructed from mRNAs isolated from immature and mature stages of the seed. Northern analysis, probed with the four cloned cDNAs, indicated that the genes represented by two cDNAs were expressed abundantly early in seed development, while another two were abundantly expressed later at the cell-expansion stages of seed development. These four genes did not show expression in roots, pegs or leaves. However, one of the early expressed genes was seed coat-specific. One of the clones, Psc11, had significant sequence similarity to subtilisin-like genes in Arabidopsis and soybean. Clones Psc32 and Psc33 had significant similarity to the peanut allergen genes Ara h II and Ara h 6, respectively. The sequence of clone Psc12 was unique and did not show significant similarity to any sequence in the databases. One of the four seed-specific clones showed restriction fragment length polymorphism (RFLP) among peanut lines representing the four peanut botanical varieties. These findings indicate that polymorphism exists in peanut seed-storage genes. This contrasts with other genes previously used for genetic mapping of cultivated peanut. Received: 1 September 2000 / Accepted: 4 May 2001     

Pine genes regulated by the necrotrophic pathogen <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

A targeted genomics approach was used to construct a cDNA array of potential pathogen-regulated genes for investigating host–pathogen interactions in pine trees (Pinus species). This array, containing a nonredundant set of 311 cDNAs, was assembled by combining smaller sets of cDNAs generated by differential display or suppression subtraction hybridization using a variety of pathogen treatments and elicitors. The array was probed to identify host genes regulated by Fusarium circinatum, a necrotrophic fungus that incites pitch canker disease on pine stems. A set of 29 cDNAs were induced during the disease state. Notably, cDNAs on the array that were derived from experiments with fusiform rust, incited by Cronartium quercuum f. sp. fusiforme (a biotrophic fungus) were unregulated by Fusarium. The results imply distinct genetic responses in pine to diseases incited by necrotrophs and biotrophs. This cDNA collection expands the genomics toolkit for understanding interactions between conifers and their microbial associates in forest ecosystems.     

Up-regulation and down-regulation of genes expressed in cocultures of rat Sertoli cells and germ cells

To better understand the molecular interactions between somatic and germ cells in the mammalian testis, we have begun to analyze with mRNA differential display changes in gene expression induced by coculturing rat Sertoli cells and germ cells. We have identified 10 cDNAs that are either down-regulated or up-regulated in cocultures of germ cells and Sertoli cells. Three genes expressed in Sertoli cells and three genes expressed in germ cells were down-regulated in Sertoli cell-germ cell cocultures, whereas four genes were up-regulated in the cocultures. Northern blot analysis was used to establish the expression pattern of the mRNAs encoded by the cDNAs and to define the sizes of the differentially expressed mRNAs. Sequence analysis of the cDNAs and computer searches against the GenBank and EMBL DNA databases were used to relate the ten cDNAs to known genes. Of the three Sertoli cell cDNAs, one appeared identical to transferrin, while the other two shared regions of similarity to an endoplasmic reticulum stress protein and to a pro-α₂ XI collagen, respectively. The three germ cell cDNAs shared sequences with fibronectin, with a basic fibroblast growth factor receptor and with an IgG gamma 2b, respectively. The four cDNAs that were up-regulated in the Sertoli-germ cell cocultures showed similarity to an isoform of casein kinase 1δ, to an epidermal growth factor, to a statin-related protein, and to an integral membrane glycoprotein. These data demonstrate that a number of specific genes are up- and down-regulated when germ cells and Sertoli cells are cocultured, and suggest these genes are important in cell to cell communication during spermatogenesis. Mol. Reprod. Dev. 47:380–389, 1997. © 1997 Wiley-Liss, Inc.     

Co-linear organization of Xenopus laevis and mouse mitochondrial genomes

Cloned fragments of Xenopus laevis mitochondrial DNA and Pleurodeles waltlii mitochondrial cDNAs have been hybridized together and with mouse mtDNA. In the three cases cross-hybridization was observed. The overall organization of the X. laevis fragments appeared to be co-linear with the mouse mtDNA, most sequences being conserved except for the D-loop and the URF6 regions. The use of mouse mtDNA has enabled us to identified several mitochondrial genes in X. laevis and P. waltlii.     

Mitochondrial unfolded protein response gene Clpp is required to maintain ovarian follicular reserve during aging,for oocyte competence,and development of pre‐implantation embryos

Caseinolytic peptidase P mediates degradation of unfolded mitochondrial proteins and activates mitochondrial unfolded protein response (mtUPR) to maintain protein homeostasis. Clpp^?/? female mice generate a lower number of mature oocytes and two‐cell embryos, and no blastocysts. Clpp^?/? oocytes have smaller mitochondria, with lower aspect ratio (length/width), and decreased expression of genes that promote fusion. A 4‐fold increase in atretic follicles at 3 months, and reduced number of primordial follicles at 6–12 months are observed in Clpp^?/? ovaries. This is associated with upregulation of p‐S6, p‐S6K, p‐4EBP1 and p‐AKT473, p‐mTOR2481 consistent with mTORC1 and mTORC2 activation, respectively, and Clpp^?/? oocyte competence is partially rescued by mTOR inhibitor rapamycin. Our findings demonstrate that CLPP is required for oocyte and embryo development and oocyte mitochondrial function and dynamics. Absence of CLPP results in mTOR pathway activation, and accelerated depletion of ovarian follicular reserve.     

cDNA cloning and partial sequencing of homeobox genes in Dugesia japonica

In the freshwater planarian Dugesia japonica, four types of cDNAs of homeobox-containing genes have been isolated by screening a cDNA library using a homeobox guessmer. Partial sequencing analysis of two types of cDNAs revealed that one was a homolog of Dth2 which is a homeobox gene in Dugesia tigrina and another was similar to Distal-less gene in Drosophila. This suggests that planarians have many homeobox genes.     

The AMPK-PPARGC1A pathway is required for antimicrobial host defense through activation of autophagy

AMP-activated protein kinase (AMPK) is a crucial energy sensor and plays a key role in integration of cellular functions to maintain homeostasis. Despite this, it is largely unknown whether targeting the AMPK pathway can be used as a therapeutic strategy for infectious diseases. Herein, we show that AMPK activation robustly induces antibacterial autophagy, which contributes to antimicrobial defense against Mycobacterium tuberculosis (Mtb). AMPK activation led to inhibition of Mtb-induced phosphorylation of the mechanistic target of rapamycin (MTOR) in macrophages. In addition, AMPK activation increased the genes involved in oxidative phosphorylation, mitochondrial ATP production, and biogenesis in Mtb-infected macrophages. Notably, peroxisome proliferator-activated receptor-gamma, coactivator 1α (PPARGC1A) was required for AMPK-mediated antimicrobial activity, as well as enhancement of mitochondrial function and biogenesis, in macrophages. Further, the AMPK-PPARGC1A pathway was involved in the upregulation of multiple autophagy-related genes via CCAAT/enhancer binding protein (C/EBP), β (CEBPB). PPARGC1A knockdown inhibited the AMPK-mediated induction of autophagy and impaired the fusion of phagosomes with MAP1LC3B (LC3B) autophagosomes in Mtb-infected macrophages. The link between autophagy, mitochondrial function, and antimicrobial activity was further demonstrated by studying LysMCre-mediated knockout of atg7, demonstrating mitochondrial ultrastructural defects and dysfunction, as well as blockade of antimicrobial activity against mycobacteria. Collectively, our results identify the AMPK-PPARGC1A axis as contributing to autophagy activation leading to an antimicrobial response, as a novel host defense mechanism.     

A spectrum of genes expressed during early stages of rice panicle and flower development

To unravel gene expression patterns during rice inflorescence development, particularly at early stages of panicle and floral organ specification, we have characterized random cloned cDNAs from developmental-stage-specific libraries. cDNA libraries were constructed from rice panicles at the stage of branching and flower primordia specification or from panicles undergoing floral organogenesis. Partial sequence analysis and expression patterns of some of these random cDNA clones from these two rice panicle libraries are presented. Sequence comparisons with known DNA sequences in databases reveal that approximately sixtyeight per cent of these expressed rice genes show varying degrees of similarity to genes in other species with assigned functions. In contrast, thirtytwo per cent represent uncharacterized genes. cDNAs reported here code for potential rice homologues of housekeeping molecules, regulators of gene expression, and signal transduction molecules. They comprise both single-copy and multicopy genes, and genes expressed differentially, both spatially and temporally, during rice plant development. New rice cDNAs requiring specific mention are those with similarity toCOP1, a regulator of photomorphogenesis inArabidopsis; sequence-specific DNA binding plant proteins like AP2-domain-containing factors; genes that specify positional information in shoot meristems like leucine-rich-repeat-containing receptor kinases; regulators of chromatin structure like Polycomb domain protein; and also proteins induced by abiotic stresses.     

Molecular analysis of de novo pyrimidine synthesis in solanaceous species

The de novo synthesis of pyrimidine nucleotides in plants has been analysed on a molecular level with special focus on cDNA cloning and structure analysis of all genes involved and their expression pattern during development. The exhaustive cloning of all cDNAs resulted from screening with heterologous cDNAs or by using complementation strategies with Escherichia coli mutants and subsequent enzyme activity measurements. Southern hybridization and comparison with the Arabidopsis genome reveals plant specific aspects and a simple genomic organization of pyrimidine synthesis in plants, which is superimposed by the postulated, complex subcellular compartmentalization. Northern hybridization evinces coordinated expression of all genes under developmental control during tobacco leaf growth.     

Identification of cold acclimation-responsive Rhododendron genes for lipid metabolism, membrane transport and lignin biosynthesis: importance of moderately abundant ESTs in genomic studies

We have previously analysed expressed sequence tags (ESTs) from non-acclimated (NA) and cold-acclimated (CA) Rhododendron leaves, and identified highly abundant complementary DNAs (cDNAs) possibly involved in cold acclimation. A potentially significant, but relatively unexplored, application of these EST data sets is the study of moderately abundant cDNAs, such as those picked only 1-3 times from each Rhododendron EST library containing approximately 430 ESTs. Using statistical tests and Northern blots, we established that the probability of differential expression of moderately abundant cDNAs based on the EST data is, indeed, a reasonably accurate predictor of their 'true' upregulation or downregulation as 11 out of 13 cDNAs (85%) studied fit this criterion. The analyses also revealed four aspects of cold acclimation in Rhododendron leaf tissues. Firstly, the concomitant upregulation of long-chain acyl-coenzyme A (acyl-CoA) synthetase, CTP:cholinephosphate cytidylyltransferase and delta-12 fatty acid desaturase in CA leaf tissues suggests that phospholipid biosynthesis and desaturation are important components of cold hardening in Rhododendron. Secondly, upregulation of plastidic nicotinamide adenine dinucleotide phosphatemalic enzyme (NADP-ME) in CA tissues suggests that malate is an important source of acetyl-CoA used for fatty acid biosynthesis during cold acclimation. Thirdly, down-regulation of plasma membrane intrinsic protein (PIP)2-1 aquaporin and upregulation of gated outward rectifying K+ channel (GORK) in CA tissues may be associated with the protection of overwintering leaves from freeze-induced cellular dehydration. Fourthly, upregulation of coumarate 3-hydroxylase may be associated with cell wall thickening in CA tissues. Physiological implications of these results, which reveal potentially novel regulations of cold acclimation in overwintering woody evergreens, are discussed. This work highlights the importance of also investigating low/moderately abundant ESTs (in addition to highly abundant ones) in genomic studies, in that it offers an effective strategy for identifying stress-related genes, especially when large-scale cDNA sequencing/microarray studies are not possible.