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A quantitative and validated SAGE transcriptome reference for adult mouse heart.

Transcriptome comparisons facilitate the identification of developmental, aging, and disease-related genes. To quantify the functionally active genome of adult C57BL/6 mouse heart (AMH), we used serial analysis of gene expression (SAGE) to sequence a total of 88,860 tags or 23,941 unique tags. Over 66% of the unique tags matched either known genes or ESTs. Mitochondrial transcripts accounted for 18.7% of the total transcripts, whereas sarcomeric proteins accounted for 3.2% of all tags. After comparison of AMH expression profiles obtained by SAGE and cDNA arrays, we observed numerous quantitative discrepancies (for example: arrays, mt-Co1 > mt-Co2 > mt-Co3; SAGE, mt-Co1 >> mt-Co3 >or= mt-Co2). We carried out quantitative PCR analyses as an independent test of transcript abundance and determined that SAGE yielded quantitatively reliable data. These SAGE results thus represent the first quantitative expression profile of AMH and serve as a reliable transcriptome reference to identify dynamic changes in cardiac gene expression.

Animals↗

Isolation and characterization of Marek's disease virus (MDV) cDNAs mapping to the BamHI-I2, BamHI-Q2, and BamHI-L fragments of the MDV genome from lymphoblastoid cells transformed and persistently infected with MDV.

We have isolated and sequenced two cDNAs of sizes 2674 and 677 bp from a cDNA library derived from MKT-1, a lymphoblastoid cell line transformed and latently infected with Marek's disease virus (MDV) using probes corresponding to the right-hand end of the BamHI-I2 fragment of the MDV genome. The larger cDNA clone represents an abundant transcript, which extends from the right-hand end of BamHI-I2 to the adjacent BamHI-Q2 and BamHI-L fragments of the MDV genome and contains the Meq (MDV Eco-Q) open reading frame. The smaller cDNA clone represents a spliced transcript containing the putative DNA binding domain of Meq as well as sequences in the BamHI-L region. We prepared a polyclonal antibody against part of the protein sequence of Meq and detected a 44-kDa protein in MKT-1 cells and in cells lytically infected with MDV. In addition, riboprobes corresponding to sequences specific to each cDNA as well as shared sequences between cDNAs detected a number of transcripts in cells either lytically or latently infected with MDV. Our results indicate that the Meq transcriptional unit extends to the BamHI-L fragment and that the transcripts mapping to the right-hand end of the BamHI-I2 and adjacent BamHI-Q2 and BamHI-L fragments are not preferentially expressed during latency.

Amino Acid Sequence↗

Expression of the pea metallothionein-like gene PsMTA in Escherichia coli and Arabidopsis thaliana and analysis of trace metal ion accumulation: implications for PsMTA function.

The PsMTA gene from pea (Pisum sativum) shares similarity with metallothionein (MT) genes and related sequences have also been isolated from a number of other higher-plant species. The proteins encoded by these genes have not yet been purified from plants and their functions remain unclear although, by analogy to MT, roles in the metabolism and detoxification of metal ions have been proposed. By contrast, correlation between transcript abundance and Fe availability has led to an alternative proposal that these genes are involved in mechanisms of Fe efficiency. Phenotypic effects of constitutive PsMTA expression were examined in Escherichia coli and Arabidopsis thaliana. Copper accumulation by E. coli cells expressing recombinant PsMTA protein was approximately 8-fold greater than in control cells. No significant effects on the accumulation of Zn or Cd were detected. In segregating A. thaliana progeny, derived from a transgenic F1 parent containing the PsMTA gene under the control of a CaMV 35S promoter, 75% of individuals accumulated more Cu (several-fold in some plants) than untransformed, control plants. These data suggest that PsMTA protein binds Cu in planta and that uncoupled (constitutive) expression of the PsMTA gene causes enhanced Cu accumulation. Roots of P. sativum plants grown under conditions of low Fe availability showed elevated activity of root surface Fe(III) reductase and accumulated more Cu than roots of plants grown in an Fe-supplemented solution. Changes in the expression of MT-like genes, coincident with changes in Fe availability, are consistent with a role in Cu homoeostasis.

Arabidopsis↗

Identification of an essential Drosophila gene that is homologous to the translation initiation factor eIF-4A of yeast and mouse.

A gene encoding a protein homologous to the translation initiation factor eIF-4A in mouse has been identified in Drosophila melanogaster. The predicted amino acid sequence shows 73% identity with the mouse gene and 67% identity with a homologous protein from yeast. The single-copy Drosophila gene is located on chromosome arm 2L at 26A7-9. Several recessive lethal mutations have been isolated and genetically characterized. Northern blot hybridization shows two abundant transcripts of 1.75 kb and 1.9 kb throughout all developmental stages. Both transcripts are maternally provided to the oocyte.

Amino Acid Sequence↗

Characterization of the Brassica campestris mitochondrial gene for subunit six of NADH dehydrogenase: nad6 is present in the mitochondrion of a wide range of flowering plants.

We have isolated the Brassica campestris mitochondrial gene nad6, coding for subunit six of NADH dehydrogenase. The deduced amino-acid sequence of this gene shows considerable similarity to mitochondrially encoded NAD6 proteins of other organisms as well as to NAD6 proteins coded for by plant chloroplast DNAs. The B. campestris nad6 gene appears to lack introns and produces an abundant transcript which is comparable in size to a previously described, unidentified transcript (#18) mapped to the B. campestris mitochondrial genome. An alignment of NAD6 proteins (deduced from DNA sequences) suggests that B. campestris nad6 transcripts are edited. Southern-blot hybridization indicates that nad6 is present in the mitochondrial genome of all of a wide range of flowering plant species examined.

Amino Acid Sequence↗

Decrease in hypothalamic vasopressin mRNA poly(A) tail length following physiological stimulation.

1. The vasopressin mRNA in the adult male rat hypothalamus is modulated in two distinct ways by a dehydration stimulus. In addition to the well-established increase in transcript abundance, it has recently been demonstrated that the vasopressin mRNA poly(A) tail increases in length. 2. We have studied the ontogeny of poly(A) tail length modulation in neonates in response to milk deprivation and found that poly(A) tail length changes are age dependent. In neonates older than 12 days of age, the vasopressin mRNA poly(A) tail length increased with milk deprivation and this effect became more marked in older animals. However, in rats 5 to 9 days old, milk deprivation resulted in a detectable though not significant decrease in vasopressin mRNA poly(A) tail length. 3. As milk deprivation is a combination of dehydration and starvation, we investigated the effect of the latter stimulus in more mature animals. We found that starvation modifies the length of the vasopressin mRNA poly(A) tail in a manner opposite that due to dehydration. 4. Our data indicate a novel mode of regulation of the vasopressin mRNA, namely, poly(A) tail shortening. This system provides a model for future studies concerning the adaptive role of poly(A) tail length modulation in response to physiological stimuli.

Adaptation, Physiological↗

The cpc-2 gene of Neurospora crassa encodes a protein entirely composed of WD-repeat segments that is involved in general amino acid control and female fertility.

Phenotypic and molecular studies of the mutation U142 indicate that the cpc-2+ gene is required to activate general amino acid control under conditions of amino acid limitation in the vegetative growth phase, and for formation of protoperithecia in preparation for the sexual phase of the life cycle of Neurospora crassa. The cpc-2 gene was cloned by complementation of the cpc-2 mutation in a his-2ts bradytrophic background. Genomic and cDNA sequence analysis indicated a 1636 bp long open reading frame interrupted by four introns. The deduced 316 amino acid polypeptide reveals 70% positional identity over its full length with G-protein beta-subunit-related polypeptides found in humans, rat (RACK1), chicken, tobacco and Chlamydomonas. With the exception of RACK1 the function of these proteins is obscure. All are entirely made up of seven WD-repeats. Expression studies of cpc-2 revealed one abundant transcript in the wild type; in the mutant its level is drastically reduced. In mutant cells transformed with the complementing sequence, the transcript level, enzyme regulation and female fertility are restored. In the wild type the cpc-2 transcript is down-regulated under conditions of amino acid limitation. With cpc-2 a new element involved in general amino acid control has been identified, indicating a function for a WD-repeat protein that belongs to a class that is conserved throughout the evolution of eukaryotes.

Amino Acid Sequence↗

Mutational analysis of the Streptomyces scabies esterase signal peptide.

Ten site-directed mutations affecting the predicted 39-amino-acid signal peptide of the Streptomyces scabies esterase were used to examine start-codon usage and esterase secretion in S. lividans. The first of two in-frame AUG codons was preferred for translation initiation. Removal of 2 of the 4 positively charged amino acids at the amino terminus of the signal peptide reduced esterase expression more than 100-fold; however, deletion of all 4 charged residues reduced expression by only 2- to 5-fold. Deletion of 4 or 8 amino acids from the hydrophobic core of the signal peptide reduced esterase production more than 200-fold, and a signal peptide processing site deletion completely disrupted esterase expression. For all constructs in which a mutation in the signal sequence decreased esterase production, esterase mRNA levels were also reduced, suggesting that a defect in secretion or processing affected esterase transcript abundance.

Amino Acid Sequence↗

Isolation and characterisation of three moss-derived beta-tubulin promoters suitable for recombinant expression.

The moss Physcomitrella patens is an excellent tool to study plant gene-function relationships due to its high rate of homologous recombination (HR). It has also been shown to be very useful in the production of recombinant proteins which are secreted into a simple medium. Thus, there is a need for suitable promoters functional in this well established model organism. We isolated genomic flanking regions of the beta-tubulin gene family from Physcomitrella, concentrating on those family members showing high transcript abundance integrated over gametophytic tissues. Using a novel, fast and reliable quantification assay based on the transient expression and secretion of a recombinant human protein, three genomic upstream regions were characterised in serial deletion constructs. Expression rates were up to three times higher than those obtained with the 35S cauliflower mosaic virus (35S) promoter, which served as a reference.

5' Untranslated Regions↗

Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata.

Lignin peroxidase (LiP) is the first enzyme connected to oxidative breakdown of the aromatic plant heteropolymer lignin and related xenobiotics. However, this extracellular enzyme has been described in only a few species of wood-decaying basidiomycetous fungi. The white rot basidiomycete Phlebia radiata 79 readily produces a versatile set of lignin-oxidizing enzymes including lignin and manganese peroxidases (LiPs and MnPs) and laccases. Here we describe genomic and primary structure of two new LiP-encoding genes, Pr-lip1 and Pr-lip4, and genomic characterization for isozyme LiP3/LIII of P. radiata, encoded by the gene depicted Pr-lip3. Pr-lip1 and Pr-lip4 code for 370- and 361-amino-acid long proteins beginning with 26- and 24-amino-acid secretion pre-propeptides, respectively. Translated LiP1 and LiP4 share the highest protein sequence identity (74 and 86%) with P. radiata LiP3, and 70% identity with the one deduced LiP from Bjerkandera adusta. The three P. radiata LiP sequences form a coherent phylogenetic cluster, which is further supported by similarities within gene organization interrupted by 11-introns. To find out the significance of LiP upon fungal growth on natural lignocellulose, such as wood, we studied ligninolytic gene expression on hardwood (milled alder) and softwood (spruce chips). All the LiP-encoding genes were expressed on wood with predominance of Pr-lip3 transcript abundance, in particular on spruce wood chips, where also time-dependent expression of the multiple lip genes was observed.

Amino Acid Sequence↗

Light Regulation of Gibberellin Biosynthesis and Mode of Action.

Some phenotypic effects produced in plants by light are very similar to those induced by hormones. In this review, the light-gibberellin (GA) interaction in germination, de-etiolation, stem growth, and tuber formation (process regulated by GAs) are discussed. Germination of lettuce and Arabidopsis seeds depends on red irradiation (R), which enhances the expression of GA 3-oxidase genes (GA3ox) and leads to an increase in active GA content. De-etiolation of pea seedling alters the expression of GA20ox and GA3ox genes and induces a rapid decrease of GA1 content. Stem growth of green plants is also affected by diverse light irradiation characteristics. Low light intensity increases stem elongation and active GA content in pea and Brassica. Photoperiod controls active GA levels in long-day rosette (spinach and Silene) and in woody plants (Salix and hybrid aspen) by regulating different steps of GA biosynthesis, mainly through transcript levels of GA20ox and GA3ox genes. Light modulation of stem elongation in light-grown plants is controlled by phytochrome, which modifies GA biosynthesis and catabolism (tobacco, potato, cowpea, Arabidopsis) and GA-response (pea, cucumber, Arabidopsis). In Arabidopsis and tobacco, ATH1 (a gene encoding an homeotic transcription factor) is a positive mediator of a phyB-specific signal transduction cascade controlling GA levels by regulating the expression of GA20ox and GA3ox. Tuber formation in potato is controlled by photoperiod (through phyB) and GAs. Inductive short-day conditions alter the diurnal rhythm of GA20ox transcript abundance, and increases the expression of a new protein (PHOR1) that plays a role in the photoperiod-GA interaction.

Journal Article↗

Jasmonate and salicylate induce the expression of pathogenesis-related-protein genes and increase resistance to chilling injury in tomato fruit.

Treatment of tomato (Lycopersicon esculentum L. cv. Beefstake) fruit with low concentrations of (0.01 mM) methyl jasmonate (MeJA) or methyl salicylate (MeSA) substantially enhanced their resistance to chilling temperature and decreased the incidence of decay during low-temperature storage. While studying the expression of pathogenesis-related (PR) protein genes, different accumulation patterns of PR-protein mRNAs in tomato fruit were observed. MeJA substantially increased the accumulation of PR-2b transcripts encoding intracellular beta-1,3-glucanase and enhanced the mRNA levels of PR-2a and PR-3b encoding extracellular beta-1,3-glucanase and intracellular chitinase, respectively. MeSA substantially increased accumulation of PR-2b and PR-3a mRNAs and slightly increased PR-3b mRNA accumulation. Chilling temperature did not appreciably enhance the accumulation of PR-protein mRNAs in untreated fruit. However, the accumulation of PR-3b mRNAs in MeSA-treated fruit was enhanced following low-temperature storage. Transcript abundance of catalase genes also was investigated in different pretreated tomatoes. The accumulation of cat1 mRNA was increased substantially by MeJA, while it was reduced by MeSA treatment. These results suggest that the pre-treatment of tomato fruit with MeSA or MeJA induces the synthesis of some stress proteins, such as PR proteins, which leads to increased chilling tolerance and resistance to pathogens, thereby decreasing the incidence of decay.

Acetates↗

Salt-induced expression of NADP-dependent isocitrate dehydrogenase and ferredoxin-dependent glutamate synthase in Mesembryanthemum crystallinum.

NADP-specific isocitrate dehydrogenase is a key cytosolic enzyme that links C and N metabolism by supplying C skeletons for primary N assimilation in plants. We report the characterization of the transcript Mc-ICDH1 encoding an NADP-dependent isocitrate dehydrogenase (NADP-ICDH, EC 1.1.1.42) from the facultative halophyte Mesembryanthemum crystallinum L., focussing on salt-dependent regulation of the enzyme. The activity of NADP-ICDH in plants adapted to high salinity increased in leaves and decreased in roots. By transcript analyses and Western-type hybridizations, expression of Mc-ICDH1 was found to be stimulated in leaves in salt-adapted M. crystallinum. By immunocytological analyses, NADP-ICDH proteins were localized to most cell types with strongest expression in epidermal cells and in the vascular tissue. In leaves of salt-adapted plants, signal intensities increased in mesophyll cells. In contrast to Mc-ICDH1, the activity and transcript abundance of ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1), which is the key enzyme of N assimilation and biosynthesis of amino acids, decreased in leaves in response to salt stress. The physiological roles of NADP-ICDH and Fd-GOGAT in the adaptation of plants to high salinity are discussed.

Adaptation, Physiological↗

Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don.

Madagascar periwinkle, Catharanthus roseus (L.) G. Don, a medicinally important plant, produces anticancer dimeric alkaloids, vinblastine and vincristine, in the leaves and accumulates antihypertensive alkaloids, ajmalicine and serpentine, in the roots. This plant grows wild in distant tropical and sub-tropical geographical locations with different agro-climates and shows wide variations in morphological and alkaloid yield-related traits. In order to understand the correlation between the expression of terpenoid indole alkaloid (TIA) pathway genes and accumulation of related alkaloids, six different genetic resources of C. roseus, including the medicinal cultivars Nirmal, Prabal, Dhawal, the mutants gsr-3 and gsr-6, and one horticultural variety, Pacifica blush, were studied. The expression profiles of one early and two late TIA biosynthetic pathway genes, namely, strictosidine synthase, desacetoxyvindoline 4-hydroxylase and deacetyl vindoline 4-O-acetyl transferase were analyzed in these plants. A positive correlation between transcript abundance and accumulation of related alkaloids was observed in the different genetic resources. The potential of these TIA biosynthetic pathway genes for use in screening of high-yielding C. roseus germplasm has been discussed.

Acetyltransferases↗

Characterization of a beta-carotene hydroxylase of Adonis aestivalis and its expression in Arabidopsis thaliana.

Carotenoids are plant secondary metabolites that comprise two main groups: carotenes and xanthophylls. The latter group includes zeaxanthin which is synthesized by beta-carotene hydroxylase catalyzing the hydroxylation of the beta-rings of beta-carotene molecules. To develop tools to alter carotenoid biosynthesis in plants, we isolated a cDNA clone encoding a candidate beta-carotene hydroxylase, CrtH1, from the flower petals of Adonis aestivalis. CrtH1 protein has homology to beta-carotene hydroxylases from other organisms, and possesses the four histidine motifs conserved in this family of enzymes. Sequence analysis predicted the presence of a putative plastid transit peptide at the amino terminus and four transmembrane helical regions. Southern-blot analysis showed CrtH1 to be encoded by a multicopy gene family with at least three members in A. aestivalis. Analysis of CrtH1 transcript abundance by Northern blotting indicates it is highly expressed in flower petals, roots and stems, with relatively low expression in leaves and developing seeds. CrtH1 was able to catalyze the formation of zeaxanthin and its intermediate precursor beta-cryptoxanthin from beta-carotene in functional assays conducted in E. coli. Expression of CrtH1 in Arabidopsis thaliana wild type and a mutant deficient for endogenous beta-carotene hydroxylases enhanced the biosynthesis of violaxanthin in the seeds.

Adonis↗

Fate of mitochondrially located S19 ribosomal protein genes after transfer of a functional copy to the nucleus in cereals.

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.

Amino Acid Sequence↗

Arabidopsis-rice-wheat gene orthologues for Na+ transport and transcript analysis in wheat-L. elongatum aneuploids under salt stress.

Lophopyrum elongatum is a wild relative of wheat that provides a source of novel genes for improvement of the salt tolerance of bread wheat. Improved Na(+) 'exclusion' is associated with salt tolerance in a wheat-L. elongatum amphiploid, in which a large proportion (ca. 50%) of the improved regulation of leaf Na(+) concentrations is controlled by chromosome 3E. In this study, genes that might control Na(+) accumulation, such as for transporters responsible for Na(+) entry (HKT1) and exit (SOS1) from cells, or compartmentalisation within vacuoles (NHX1, NHX5, AVP1, AVP2) in the model plant, Arabidopsis thaliana, were targeted for comparative analyses in wheat. Putative rice orthologues were identified and characterised as a means to bridge the large evolutionary distance between genomes from the model dicot and the more complex grass species. Wheat orthologues were identified through BLAST searching to identify either FL-cDNAs or ESTs and were subsequently used to design primers to amplify genomic DNA. The probable orthologous status of the wheat genes was confirmed through demonstration of similar intron-exon structure with their counterparts in Arabidopsis and rice. The majority of exons for Arabidopsis, rice and wheat orthologues of NHX1, NHX5 and SOS1 were conserved except for those at the amino and carboxy terminal ends. However, additional exons were identified in the predicted NHX1 and SOS1 genes of rice and wheat, as compared with Arabidopsis, indicating gene rearrangement events during evolution from a common ancestor. Nullisomic-tetrasomic, deletion and addition lines in wheat were used to assign gene sequences to chromosome regions in wheat and L. elongatum. Most sequences were assigned to homoeologous chromosomes, however, in some instances, such as for SOS1, genes were mapped to other unpredicted locations. Differential transcript abundance under salt stress indicated a complex pattern of expression for wheat orthologues that may regulate Na(+) accumulation in wheat lines containing chromosomes from L. elongatum. The identification of wheat orthologues to well characterized Arabidopsis genes, map locations and gene expression profiles increases our knowledge on the complex mechanisms regulating Na(+) transport in wheat and wheat-L. elongatum lines under salt stress.

Acclimatization↗

Role of two different glyceraldehyde-3-phosphate dehydrogenases in controlling the reversible Embden-Meyerhof-Parnas pathway in Thermoproteus tenax: regulation on protein and transcript level.

The hyperthermophilic archaeum Thermoproteus tenax uses a variant of the Embden-Meyerhof-Parnas (EMP) pathway as the main route for carbohydrate metabolism. This variant is characterized by a reversible nonallosteric PPi-dependent phosphofructokinase and two glyceraldehyde-3-phosphate dehydrogenases differing in cosubstrate specificity, phosphate dependence, and allosteric behavior. Although the nonphosphorylating NAD+-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPN; E.C. 1.2.1.8) fulfills exclusively catabolic purposes, the phosphorylating NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP+-GAPDH; E.C. 1.2.1.13) exhibits anabolic features. The gene encoding the NADP+-GAPDH was cloned, sequenced, and expressed in Escherichia coli. The deduced protein sequence displayed 47%-53% sequence identity to archaeal phosphorylating GAPDHs. The kinetic parameters of the NADP+-GAPDH showed a clear preference for the reductive reaction with a 5-fold-higher specific activity in the reductive reaction as compared to the oxidative reaction and a 20-fold-lower Km for 1,3-bisphosphoglycerate as compared to glyceraldehyde-3-phosphate. Contrary to GAPN, the enzyme is not allosterically regulated. The coding gene overlaps by 1 bp with a preceding open reading frame coding for 3-phosphoglycerate kinase (PGK; E.C. 2.7.2.3). Northern analyses identified mono- and bicistronic messages of both genes in an equimolar ratio. Transcript levels and specific activity of NADP+-GAPDH and PGK were 3- to 4-fold higher under autotrophic conditions as compared to heterotrophic conditions, whereas transcript abundance and specific activity of GAPN remained constant in autotrophically and heterotrophically grown cells. The different regulation of the two counteracting glyceraldehyde-3-phosphate dehydrogenases is discussed with respect to the flux control of the T. tenax-specific EMP variant.

Amino Acid Sequence↗