EF-1 Alpha Research Articles

Resolving Arthropod Phylogeny: Exploring Phylogenetic Signal within 41 kb of Protein-Coding Nuclear Gene Sequence

This study attempts to resolve relationships among and within the four basal arthropod lineages (Pancrustacea, Myriapoda, Euchelicerata, Pycnogonida) and to assess the widespread expectation that remaining phylogenetic problems will yield to increasing amounts of sequence data. Sixty-eight regions of 62 protein-coding nuclear genes (approximately 41 kilobases (kb)/taxon) were sequenced for 12 taxonomically diverse arthropod taxa and a tardigrade outgroup. Parsimony, likelihood, and Bayesian analyses of total nucleotide data generally strongly supported the monophyly of each of the basal lineages represented by more than one species. Other relationships within the Arthropoda were also supported, with support levels depending on method of analysis and inclusion/exclusion of synonymous changes. Removing third codon positions, where the assumption of base compositional homogeneity was rejected, altered the results. Removing the final class of synonymous mutations--first codon positions encoding leucine and arginine, which were also compositionally heterogeneous--yielded a data set that was consistent with a hypothesis of base compositional homogeneity. Furthermore, under such a data-exclusion regime, all 68 gene regions individually were consistent with base compositional homogeneity. Restricting likelihood analyses to nonsynonymous change recovered trees with strong support for the basal lineages but not for other groups that were variably supported with more inclusive data sets. In a further effort to increase phylogenetic signal, three types of data exploration were undertaken. (1) Individual genes were ranked by their average rate of nonsynonymous change, and three rate categories were assigned--fast, intermediate, and slow. Then, bootstrap analysis of each gene was performed separately to see which taxonomic groups received strong support. Five taxonomic groups were strongly supported independently by two or more genes, and these genes mostly belonged to the slow or intermediate categories, whereas groups supported only by a single gene region tended to be from genes of the fast category, arguing that fast genes provide a less consistent signal. (2) A sensitivity analysis was performed in which increasing numbers of genes were excluded, beginning with the fastest. The number of strongly supported nodes increased up to a point and then decreased slightly. Recovery of Hexapoda required removal of fast genes. Support for Mandibulata (Pancrustacea + Myriapoda) also increased, at times to "strong" levels, with removal of the fastest genes. (3) Concordance selection was evaluated by clustering genes according to their ability to recover Pancrustacea, Euchelicerata, or Myriapoda and analyzing the three clusters separately. All clusters of genes recovered the three concordance clades but were at times inconsistent in the relationships recovered among and within these clades, a result that indicates that the a priori concordance criteria may bias phylogenetic signal in unexpected ways. In a further attempt to increase support of taxonomic relationships, sequence data from 49 additional taxa for three slow genes (i.e., EF-1 alpha, EF-2, and Pol II) were combined with the various 13-taxon data sets. The 62-taxon analyses supported the results of the 13-taxon analyses and provided increased support for additional pancrustacean clades found in an earlier analysis including only EF-1 alpha, EF-2, and Pol II.

G2R Cre reporter transgenic zebrafish

The Cre/loxP site-specific recombination system has been widely used to manipulate DNA in vivo and to study gene function in the mouse by inducible transgenic and conditional gene targeting. To fully use this powerful genetic tool in a relatively new animal model, zebrafish, we generated reporter transgenic lines for easy detection of Cre recombinase activity in vivo. The transgenic fish lines, designated G2R, express two fluorescent protein genes, GFP and RFP, under the control of the ubiquitous promoter of the Xenopus EF1 alpha gene. The G2R animals change their color from green to red (G2R) after Cre-mediated recombination and are useful for development of cell type specific Cre transgenic lines and for cell lineage and fate mapping studies in zebrafish.

Growth, population dynamics, and diversity of Fusarium equiseti in ginseng fields

Fusarium equiseti is prevalent in ginseng soil, straw mulch and in ginseng root tissues and is the cause of a root surface discolouration on ginseng grown in British Columbia. Population levels of the fungus in ginseng fields ranged from 3.8 × 103 cfu g−1 soil to 1.4 × 104 cfu g−1 soil and were highest at 0–5 cm soil depths compared to 10–15 cm. Soil population levels were negatively correlated with S content in soil and positively correlated with Zn levels. Barley or wheat straw added to soil significantly increased population levels under laboratory conditions. Mycelial growth in culture was highest at 26–30°C and at pH 7.2–7.8. Samples of flowers and berries, and harvested seed, contained DNA of F. equiseti detected using a Fusarium-specific DNA array and the fungus was isolated from these tissues on agar medium. A high degree of genetic variation in the EF-1 alpha gene sequence was present among 52 isolates of F. equiseti which originated from ginseng fields. At least seven clades were identified. Inoculum dispersal from straw mulch used in ginseng gardens can result in seed contamination by the fungus. In addition, fungal growth near the soil surface under warm summer conditions can result in infection and crown discolouration of ginseng roots.

ASCR-004 Generation of genetically modified human embryonic stem cells expressing EGFP or ERFP regulated by EF1 alpha- and Oct-4 promoter

ASCR-004 Generation of genetically modified human embryonic stem cells expressing EGFP or ERFP regulated by EF1 alpha- and Oct-4 promoter

Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus.

BackgroundBrown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies.ResultsWe monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E) and protein degradation (ubiquitin, ubiquitin conjugating enzyme) or folding (cyclophilin), and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins) and its trafficking function (dynein), as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase). The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation.ConclusionComparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a) was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

Phanerochaete chrysosporium soluble proteome as a prelude for the analysis of heavy metal stress response

A 2-D reference map in pI range 3-10 was constructed for the soluble protein fraction of Phanerochaete chrysosporium growing vegetatively under standard conditions. Functional annotation could be made for 517 spots out of 720 that were subjected to MALDI-TOF-MS analysis, according to the specific accession numbers from the P. chrysosporium genomic database. Further analysis of the data revealed 314 distinct ORFs, 118 of which yielded multiple spots on the master gel. Functional classification of the proteins was made according to the eukaryote orthologous groups defined in the organism's genome website. The functional class of PTMs, protein turnover and chaperones was represented with the highest number (63) of the identified ORFs. Six proteins were assigned to the hypothetical proteins and 29 were predicted to have a signal peptide sequence. Subcellular localization predictions were also made for the identified proteins. Of the protein spots detected on the master gel, 380 were found to be probably phosphorylated and 96 of these matched to the identified proteins. The reference map was efficiently used in the identification of the proteins differentially expressed under cadmium and copper stress. Three new ribosomal proteins as well as zinc-containing alcohol dehydrogenase, glucose-6-phosphate isomerase, flavonol/cinnamoyl-CoA reductase, H+-transporting two-sector ATPase, ribosomal protein S7, ribosomal protein S21e, elongation factor EF-1 alpha subunit were demonstrated as the most strongly induced.

Coexpression of Vascular Endothelial Growth Factor and Interleukin-1 Receptor Antagonist for Improved Human Islet Survival and Function

Ex vivo gene therapy approaches can improve the outcome of islet transplantation for treating type I diabetes. We have recently shown improvement in islet survival and function following ex vivo infection of islets with a mixture of adenoviral vectors encoding human vascular endothelial growth factor (Adv-hVEGF) and human interleukin-1 receptor antagonist (Adv-hIL-1Ra). In this study, we constructed a bicistronic vector encoding these two genes (phVEGF-hIL-1Ra) by cloning hIL-1Ra under the cytomegalovirus (CMV) promoter and hVEGF under the elongation factor-1alpha (EF-1 alpha) promoter in pBudCE4.1 vector. There was dose and time dependent expression of hVEGF and hIL-1Ra at both mRNA and protein levels after transfection with human islets. Transfected islets were viable, as evidenced by insulin release upon glucose challenge. Coexpression of hVEGF and hIL-1Ra suppressed nitric oxide production, total caspases, apoptosis, and necrosis in the presence of inflammatory cytokine cocktail consisting of IL-1beta, TNFalpha, and IFNgamma. In conclusion, our results indicated that coexpression of growth factor and antiapoptic genes can improve islet survival and function.

Intraspecific diversity of species of thePseudallescheria boydiicomplex

In order to establish intraspecific diversity of Pseudallescheria boydii and Scedosporium apiospermum, and to develop tools for their identification, variability within P. boydii and related species was investigated at different levels of diversity. Sequences of the D1/D2 region of large subunit (LSU) and of the internal transcribed spacer (ITS) region of ribosomal DNA (rDNA) gene were analyzed for a set of 57 strains, as well as partial sequences of the elongation factor 1-alpha (EF 1-alpha). Incongruence among 3 locus lineages was detected by partition homogeneity test (PHT). The maximum parsimony (MP) tree of the combined sequence data set, with the exception of strain CBS 499.90, formed 3 clades with high bootstrap support, corresponding to previously described nuclear DNA (nDNA)/DNA reassociation groups. These groups are known to differ slightly in predilection and temperature relations. Using Structure software, population genetic analysis revealed 3 clusters within the complex on the basis of multi-locus genotype data. Strain distribution in the clusters was concordant with that in the 3 clades of combined multi-locus MP tree. Recombination among individuals of a clade in evolutional history was found in 2 of the 3 clades. There was population differentiation among the 3 clades. Restriction fragment length polymorphism (RFLP) analysis of the intergenic spacer (IGS) region of rDNA gene was added to further characterize subspecific entities. When the IGS regions of 22 strains were digested with the restriction endonucleases Hae III and Mbo I, seven and five distinct patterns were detected, respectively. This subtyping did not reveal any correspondence with geographic origin or clinical appearance. Though we need more evidence to locate the 3 clades of the P. boydii complex at species or population level, the sequence of the D1/D2 region is sufficiently variable for identification of taxa belonging to the P. boydii complex.

Systematics and mating systems of two fungal pathogens of opium poppy: the heterothallicCrivellia papaveraceawith aBrachycladium penicillatumasexual state and a homothallic species with aBrachycladium papaverisasexual state

This paper presents a systematic revision of the fungal opium poppy (Papaver somniferum L.) pathogens formerly known as Pleospora papaveracea (de Not.) Sacc., along with allied asexual states formerly placed in Dendryphion . The revision is based on analysis of phylogenetic relationships, comparative morphology, and analysis of mating systems. Using morphology, 18S and ITS rDNA, we established that these species belong to the Alternaria group rather than to Pleospora, a conclusion supported by the Shimodaira–Hasegawa test. For these fungi, we erect the new genus Crivellia , with Crivellia papaveracea as type. ITS rDNA analyses suggested with moderate support Alternaria brassicicola (Schw.) Wiltshire, Alternaria japonica Yoshii, and Ulocladium alternariae (Cooke) Simmons as Crivellia’s closest relatives. Combined ITS, partial GPD and EF-1 alpha analyses confirmed earlier studies that show that asexual isolates in the Crivellia lineage of poppy pathogens represent two closely related species. Because Dendryphion was determined to be polyphyletic, the former genus Brachycladium was resurrected for B. penicillatum Corda and B. papaveris (K. Sawada) Shoemaker & Inderbitzin, the Crivellia asexual states that had been in Dendryphion. Molecular and morphological comparison with isolates from field-collected ascomata and morphological comparison with the type specimen of P. papaveracea indicated that B. penicillatum , and not B. papaveris , is the anamorph of C. papaveracea. The mycelia from single conidium or single ascospore isolates, including mycelia from 14 single ascospores from one field-collected C. papaveracea ascoma, either have a MAT1-1 or MAT1-2 gene and are thus heterothallic. In contrast, each single-conidium isolate of B. papaveris has an incomplete MAT1-2 gene fused to a MAT1-1 region and is inferred to be homothallic. We speculate that ancestral MAT fusion might have led to speciation in Crivellia.

Biodiversity and Biogeography of Fusarium Species from Northeastern North American Asparagus Fields Based on Microbiological and Molecular Approaches

Sixteen Fusarium species were recovered from 52 asparagus commercial fields, representing all major ecological (edaphic and climatic) area of asparagus production in the province of Québec, eastern Canada. This study extends our understanding of the geographic range of these species. It also provides climatological and edaphic properties linked to community changes and adaptations. Fusarium oxysporum and F. proliferatum were omnipresent and abundant in all five ecological area under study, whereas F. redolens was less frequently found. Species of Fusarium that produce carmine red pigmentation on potato dextrose agar, i.e., F. acuminatum, F. avenaceum, etc., were common at the northern limit of asparagus production. Abundance of red Fusarium species corresponded with a low isolation frequency of F. proliferatum. Nevertheless, F. proliferatum had a high recovery rate throughout Québec asparagus growing areas, under climatic conditions as cold as those of northern Europe where this species is uncommon in asparagus fields. In the light of these results, redefinition of the geographical distribution of F. proliferatum in asparagus fields is proposed. Intraspecific molecular differences in F. proliferatum and F. oxysporum were detected in the EF-1 alpha sequences and compared with well-characterized strains of North America.

Gene analysis of Cryptosporidium parvum HNJ-1 strain isolated in Japan

We analyzed genetically Cryptosporidium parvum HNJ-1 strain, which is the Japanese reference strain isolated from human in Japan. DNA sequences of genes for thrombospondin-related adhesive protein of Cryptosporidium-1 and Cryptosporidium-2 (TRAP-C1, TRAP-C2), heat shock protein 70 (HSP70), oocyst wall protein (COWP), beta-tubulin, alpha-tubulin, polythreonine-region (Poly-T), elongation factor 1 alpha (EF-1 alpha), and 18S rRNA of this strain were determined. They showed high rate of homology to published sequences of genotype 2 strains, which were considered to be infective to both humans and animals. However, HNJ-1 had synonymous and non-synonymous substitutions in the nucleotide sequence of TRAP-C1 and beta-tubulin among HNJ-1 and published sequences of genotype 2 strains. These results implied that HNJ-1 strain was an unique subpopulation of genotype 2 strain of C. parvum.

Phylogenetic analysis of EF-1 alpha gene sequences from isolates of Microdochium nivale leads to elevation of varieties majus and nivale to species status

Degenerate PCR primers were designed based on EF-1 alpha (EF-1alpha) gene sequences of several filamentous fungi retrieved from sequence databases. These primers were used to isolate a partial sequence, approximately 830 bp in length of the EF-1alpha from isolates of Microdochium nivale obtained from various geographic locations across the world. Two distinct groups of isolates were evident among those isolates examined. Sequence homology for comparisons within group was 99.7% for group A and 99.8% for group B. Primers specific to either group A or group B sequences were designed and tested on isolates from around the world. Comparisons were made with primers previously reported for the two varieties of M. nivale and revealed that Group A type isolates correlated with M. nivale var. majus and group B isolates with M. nivale var. nivale. The primers from this study and those previously reported were in agreement for all isolates with the exception of one isolate (NRRL 3289) which failed to amplify with previously published M. nivale primers. Sequence analysis of NRRL 3289 suggested that it was an isolate of M. nivale var. nivale as indicated by the EF-1alpha based primers developed in this study. This study provides sequence based phylogenetic evidence of two species and when taken together with biological differences reported, leads to the recognition of M. majus comb. nov. (syn. Fusarium nivale var. majus). Descriptions of the two species are provided.

A PCR-denaturing gradient gel electrophoresis approach to assess Fusarium diversity in asparagus

In North America, asparagus ( Asparagus officinalis) production suffers from a crown and root rot disease mainly caused by Fusarium oxysporum f. sp. asparagi and F. proliferatum. Many other Fusarium species are also found in asparagus fields, whereas accurate detection and identification of these organisms, especially when processing numerous samples, is usually difficult and time consuming. In this study, a PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess Fusarium species diversity in asparagus plant samples. Fusarium-specific PCR primers targeting a partial region of the translation elongation factor-1 alpha (EF-1 alpha) gene were designed, and their specificity was tested against genomic DNA extracted from a large collection of closely and distantly related organisms isolated from multiple environments. Amplicons of 450 bp were obtained from all Fusarium isolates, while no PCR product was obtained from non-Fusarium organisms. The ability of DGGE to discriminate between Fusarium taxa was tested over 19 different Fusarium species represented by 39 isolates, including most species previously reported from asparagus fields worldwide. The technique was effective to visually discriminate between the majority of Fusarium species and/or isolates tested in pure culture, while a further sequencing step permitted to distinguish between the few species showing similar migration patterns. Total genomic DNA was extracted from field-grown asparagus plants naturally infested with different Fusarium species, submitted to PCR amplification, DGGE analysis and sequencing. The two to four bands observed for each plant sample were all affiliated with F. oxysporum, F. proliferatum or F. solani, clearly supporting the reliability, sensitivity and specificity of this approach for the study of Fusarium diversity from asparagus plants samples.

Transient expression in mammalian cells of transgenes transcribed from theCauliflower mosaic virus35S promoter

Gene constructs containing the Cauliflower mosaic virus (CaMV) 35S promoter and a sequence coding either for a green fluorescent protein (GFP) or for firefly luciferase were transfected into Chinese hamster ovary (CHO) cells. Both reporter genes were expressed to significant levels. The 35S promoter was 40 times less active than the human eF1 alpha promoter, which is known to be one of the most potent promoters in mammalian cells. The 35S promoter must therefore be considered to be a promoter of significant potency in mammalian cells. RT-PCR analysis suggested that transcription initiation in CHO cells occurred between the TATA box and the transcription start site of the 35S promoter that function in plant cells. Further analysis by 5'RACE confirmed that transcription was initiated in CHO cells at different sites located essentially between the TATA box and the plant transcription start site, showing that 35S promoter activity in animal cells is due to the presence of promoter elements that are functional in mammalian cells, but that are not those used in plants. The data reported here raise the possibility that genes controlled by the 35S promoter, which is commonly used in transgenic plants, have the potential for expression in animal cells.

Systematic reappraisal of Coniella and Pilidiella, with specific reference to species occurring on Eucalyptus and Vitis in South Africa

The genus Pilidiella, including its teleomorphs in Schizoparme, has a cosmopolitan distribution and is associated with disease symptoms on many plants. In the past, conidial pigmentation has been used as a character to separate Pilidiella (hyaline to pale brown conidia) from Coniella (dark brown conidia). In recent years, however, the two genera have been regarded as synonymous, the older name Coniella having priority. To address the generic question, sequences of the internal transcribed spacer region (ITS1, ITS2), 5.8S gene, large subunit (LSU) and elongation factor 1-alpha gene (EF 1-alpha) were analysed to compare the type species of Pilidiella and Coniella. All three gene regions supported the separation of Coniella from Pilidiella, with the majority of taxa residing in Pilidiella. Pilidiella is characterised by having species with hyaline to pale brown conidia (avg. length:width > 1.5), in contrast to the dark brown conidia of Coniella (avg. length:width < or = 1.5). Pilidiella diplodiella, which is a pathogen associated with white rot of grapevines, was shown to be an older name for C. petrakii. To delineate species in the P. diplodiella species complex, isolates were also compared based on histone (H3) gene sequences. Analyses derived from these sequence data separated P. diplodiella from a newly described species, P. diplodiopsis. The new species P. eucalyptorum sp. nov. is proposed for isolates formerly treated as C. fragariae and associated with leaf spots of Eucalyptus spp. This species clustered basal to Pilidiella, and may represent yet a third genus within this complex. Pilidiella destruens sp. nov. is newly described as anamorph of Schizoparme destruens, which is associated with twig dieback of Eucalyptus spp. in Hawaii. A key based on morphological characteristics is provided to separate the taxa treated in this study.

Kinectin Anchors the Translation Elongation Factor-1δ to the Endoplasmic Reticulum

Kinectin has been proposed to be a membrane anchor for kinesin on intracellular organelles. A kinectin isoform that lacks a major portion of the kinesin-binding domain does not bind kinesin but interacts with another resident of the endoplasmic reticulum, the translation elongation factor-1 delta (EF-1 delta). This was shown by yeast two-hybrid analysis and a number of in vitro and in vivo assays. EF-1 delta provides the guanine nucleotide exchange activities on EF-1 alpha during elongation step of protein synthesis. The minimal EF-1 delta-binding domain on kinectin resides within a conserved region present in all the kinectin isoforms. Overexpression of the kinectin fragments in vivo disrupted the intracellular localization of EF-1 delta proteins. This report provides evidence of an alternative kinectin function as the membrane anchor for EF-1 delta on the endoplasmic reticulum and provides clues to the EF-1 complex assembly and anchorage on the endoplasmic reticulum.

Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells.

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

Characterisation of a Non-canonical Genetic Code in the Oxymonad Streblomastix strix

The genetic code is one of the most highly conserved characters in living organisms. Only a small number of genomes have evolved slight variations on the code, and these non-canonical codes are instrumental in understanding the selective pressures maintaining the code. Here, we describe a new case of a non-canonical genetic code from the oxymonad flagellate Streblomastix strix. We have sequenced four protein-coding genes from S.strix and found that the canonical stop codons TAA and TAG encode the amino acid glutamine. These codons are retained in S.strix mRNAs, and the legitimate termination codons of all genes examined were found to be TGA, supporting the prediction that this should be the only true stop codon in this genome. Only four other lineages of eukaryotes are known to have evolved non-canonical nuclear genetic codes, and our phylogenetic analyses of α-tubulin, β-tubulin, elongation factor-1 α (EF-1 alpha), heat-shock protein 90 (HSP90), and small subunit rRNA all confirm that the variant code in S.strix evolved independently of any other known variant. The independent origin of each of these codes is particularly interesting because the code found in S.strix, where TAA and TAG encode glutamine, has evolved in three of the four other nuclear lineages with variant codes, but this code has never evolved in a prokaryote or a prokaryote-derived organelle. The distribution of non-canonical codes is probably the result of a combination of differences in translation termination, tRNAs, and tRNA synthetases, such that the eukaryotic machinery preferentially allows changes involving TAA and TAG.

Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice ( Oryza sativa L.)

The mitochondrial elongation factor EF-Tu (tufM) in rice (Oryza sativa L.) was isolated and characterized. The rice tufM cDNA clone contained 1,726 nucleotides and coded for a 453 amino acid protein including a putative mitochondrial transit peptide of 64 amino acid residues. This coding region was composed of 12 exons and 11 introns. The deduced amino acid sequence showed 62% and 88% identities with rice chloroplast EF-Tu (tufA) and Arabidopsis mitochondrial EF-Tu, respectively. As previously observed for the rice tufA gene, the tufM gene is likely present as one copy in rice. The mitochondrial EF-Tu gene was differentially expressed during flower development, and the other translational EF-Tu genes (chloroplast EF-Tu and cytosolic EF-1 alpha) were also distinctly expressed in a temporal manner. Phylogenetic analysis of the rice tufM gene showed that the mitochondrial tufA homologue of Reclinomonas was more closely related to the mitochondrial tufM genes of flowering plants than fungal and other mitochondrial tuf genes. In addition, the tufM encoded an N-terminal extension showing significant similarity to that of rps14 (or sdhB), which is also a nuclear-encoded rice mitochondrial gene.

Identification of differentially expressed genes associated with androgen-independent growth of prostate cancer.

The human prostate cancer xenograft, CWR22, similar to most human prostate cancers, regresses after castration and recurs several months after the removal of androgen. Genes uniquely associated with proliferation were identified by comparison of tumors that exist in androgen absence but differ in proliferative capacity. cDNA libraries from CWR22 tumors from 20-day castrate mice (proliferation undetectable) and recurrent CWR22 tumors (proliferation rate similar to androgen-dependent CWR22) were compared to evaluate the possibility that proliferation is triggered by either gain of function or loss of suppression. Differentially expressed genes were evaluated further for their temporal association with the onset of cellular proliferation using northern and western analysis and immunohistochemistry of a series of CWR22 tumors that spanned the transition from androgen-dependent to recurrent growth. Subtractive hybridization identified 11 candidate genes from among 1,057 clones examined. Northern analysis confirmed differential expression of 8 genes. Western analysis revealed an association between tomoregulin, translation elongation factor-1 alpha (EF-1 alpha), Mxi-1, and thioredoxin-binding protein 2/vitamin D up-regulated protein, and the onset of recurrent growth. Immunohistochemistry revealed expression of tomoregulin, EF-1 alpha, Mxi-1, and thioredoxin reductase-1 coincidental with the onset of cellular proliferation on day 120 after castration. One or more of these genes may represent an appropriate target to prevent, delay or treat recurrent prostate cancer.