Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3

We report here the isolation and nucleotide sequence of tomato cDNA and genomic clones encoding a ubiquitin extension protein homologous to the yeast gene ubi3 . Sites similar to upstream activating sites commonly found in the promoters of yeast ribosomal genes were observed in the tomato promoter. The tomato ubi3 promoter also contained elements found in the rbcS promoter from pea. The transcription initiation site was determined to occur 66 bp upstream of the initiating Met. RFLP mapping revealed that the gene was located on chromosome 1, 23 cM from marker TG301. A ubi3 gene-specific probe hybridized to a single 800 nt transcript. Expression was reduced in heat-shocked plants and plants kept in the dark. Expression was highest in young leaves and immature green fruit and lowest in mature leaves and petals. We isolated the original cDNA clone using an antibody prepared against chloroplast polypeptides. Immunological studies did not detect ubiquitin or ubiquitin extension proteins in the chloroplast. However, higher-molecular-weight chloroplast proteins were detected with ubiquitin antisera suggesting that ubiquitin conjugates are transported into the chloroplast.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43430/1/11103_2004_Article_BF00028735.pd

Global gene expression during muscle hypertrophy induced by myostatin suppression and/or beta-adrenergic agonist

Ph.D. University of Hawaii at Manoa 2011.Includes bibliographical references.Skeletal muscle growth is important for animal agriculture, particularly for meat-producing animals, and for human health as well. A better understanding of the mechanisms regulating skeletal muscle growth is expected to contribute to improving the efficiency of meat animal production and alleviating human suffereings caused by muscle atrorphic conditions. Currently, two molecules have been shown to have dramatic effects on skeletal muscle mass: myostatin and β-adrenergic agonists. Myostatin (Mstn), a member of the TGF-β superfamily proteins, acts as a potent negative regulator of skeletal muscle growth. Suppression of Mstn by varying means has shown to increase skeletal muscle mass of animals. Administration of β-adrenergic agonist (BAA) such as clenbuterol (CL) induces dramatic increase in skeletal muscle mass or inhibits muscle atrophy. Although there have been numerous demonstrations of the muscle growth--promoting effect of BAA administration or Mstn suppression, the effect of a combination of BAA administration and Mstn inhibition on skeletal muscle growth has not been investigated. Similarly, very little is known about the molecular signaling pathways leading to muscle hypertrophy induced by the two stimuli and the genes that are commonly regulated by both Mstn and BAA administration. Therefore, the objectives of this study were 1) to investigate the combined effect of Mstn-suppression and BAA administration on skeletal muscle growth, 2) to examine the role of the Akt/mTOR pathway in the two muscle hypertrophic models, 3) to examine global changes in gene expression in skeletal muscle undergoing hypertrophy induced by chronic suppression of Mstn or BAA administration , and 4) to compare the changes in gene expression between these two muscle hypertrophic models. We used a transgenic mouse strain that overexpresses the Mstn-prodomain (Mstn-pro) and exhibits a significant increase in skeletal muscle mass regardless of age and sex. Clenbuterol (CL) was used as a BAA compound. Heterozygous Mstn-pro and wild-type littermates were produced and were given 0 or 20 ppm of CL in their drinking water. Phosphorylation of molecules involved in the Akt/mTOR pathway was examined by using the Western blot analysis. RNA samples of the gastrocnemius muscle in each group were subjected to microarray analysis using the Affymetrix GeneChip Mouse 430-2.0 platform. CL increased body and muscle mass of male and female mice in both genotypes, indincating that the muscle-hypertrophic effect of CL is additive to the effect of Mstn suppression. Levels of phosphorylated muscle 4E-BP1 and p70S6k, two downstream effectors of the mTOR pathway, were higher in Mstn-pro mice than in wild type mice. Levels of phosphorylated muscle Akt, an upstream effector of the mTOR pathway, were also higher in Mstn-pro mice than in wild type mice, indicating that the Akt/mTOR anabolic pathway is involved in the regulation of muscle mass by Mstn. CL increased the phosphorylation of Akt, 4E-BP1 and p70S6k in both genotypes, resulting in the highest phosphorylation levels of Akt, 4E-BP1 and p70S6k in CL-fed Mstn-pro mice. This result suggests that like Mstn, BAA also regulates muscle hypertrophy through the Akt/mTOR pathway, and the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy. Microarray analysis of global gene expression showed that Mstn suppression and CL administration induced significant changes in the mRNA abundance of various genes associated with muscle contraction, initiation of translation, transcription, and muscle hypertrophic signal pathway, suggesting that increased protein synthesis is partly responsible for the hypetrophy induced by Mstn and CL. Additionally, the alteration of Igf2 obsderved in Mstn suppressed mice, and the alterations of eIF4e, Acvr2b, FoxO and PTEN observed in mice treated with CL indicate that the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy

The Drosophila retinoblastoma binding protein 6 family member has two isoforms and is potentially involved in embryonic patterning.

The human retinoblastoma binding protein 6 (RBBP6) is implicated in esophageal, lung, hepatocellular and colon cancers. Furthermore, RBBP6 was identified as a strong marker for colon cancer prognosis and as a predisposing factor in familial myeloproliferative neoplasms. Functionally, the mammalian protein interacts with p53 and enhances the activity of Mdm2, the prototypical negative regulator of p53. However, since RBBP6 (known as PACT in mice) exists in multiple isoforms and pact−/− mice exhibit a more severe phenotype than mdm2−/− mutants, it must possess some Mdm2-independent functions. The function of the invertebrate homologue is poorly understood. This is complicated by the absence of the Mdm2 gene in both Drosophila and Caenorhabditis elegans. We have experimentally identified the promoter region of Snama, the Drosophila homologue, analyzed potential transcription factor binding sites and confirmed the existence of an additional isoform. Using band shift and co-immunoprecipitation assays combined with mass spectrometry, we found evidence that this gene may be regulated by, amongst others, DREF, which regulates hundreds of genes related to cell proliferation. The potential transcription factors for Snama fall into distinct functional groups, including anteroposterior embryonic patterning and nucleic acid metabolism. Significantly, previous work in mice shows that pact−/− induces an anteroposterior phenotype in embryos when rescued by simultaneous deletion of p53. Taken together, these observations indicate the significance of RBBP6 proteins in carcinogenesis and in developmental defects.SP201

Mammalian ribosomal protein S3a genes and intron-encoded small nucleolar RNAs U73 and U82

http://www.ester.ee/record=b4330778~S58*es

Translational machinery of the chaetognath Spadella cephaloptera: a transcriptomic approach to the analysis of cytosolic ribosomal protein genes and their expression

<p>Abstract</p> <p>Background</p> <p>Chaetognaths, or arrow worms, are small marine, bilaterally symmetrical metazoans. The objective of this study was to analyse ribosomal protein (RP) coding sequences from a published collection of expressed sequence tags (ESTs) from a chaetognath (<it>Spadella cephaloptera</it>) and to use them in phylogenetic studies.</p> <p>Results</p> <p>This analysis has allowed us to determine the complete primary structures of 23 out of 32 RPs from the small ribosomal subunit (SSU) and 32 out of 47 RPs from the large ribosomal subunit (LSU). Ten proteins are partially determined and 14 proteins are missing. Phylogenetic analyses of concatenated RPs from six animals (chaetognath, echinoderm, mammalian, insect, mollusc and sponge) and one fungal taxa do not resolve the chaetognath phylogenetic position, although each mega-sequence comprises approximately 5,000 amino acid residues. This is probably due to the extremely biased base composition and to the high evolutionary rates in chaetognaths. However, the analysis of chaetognath RP genes revealed three unique features in the animal Kingdom. First, whereas generally in animals one RP appeared to have a single type of mRNA, two or more genes are generally transcribed for one RP type in chaetognath. Second, cDNAs with complete 5'-ends encoding a given protein sequence can be divided in two sub-groups according to a short region in their 5'-ends: two novel and highly conserved elements have been identified (5'-TAATTGAGTAGTTT-3' and 5'-TATTAAGTACTAC-3') which could correspond to different transcription factor binding sites on paralog RP genes. And, third, the overall number of deduced paralogous RPs is very high compared to those published for other animals.</p> <p>Conclusion</p> <p>These results suggest that in chaetognaths the deleterious effects of the presence of paralogous RPs, such as apoptosis or cancer are avoided, and also that in each protein family, some of the members could have tissue-specific and extra-ribosomal functions. These results are congruent with the hypotheses of an allopolyploid origin of this phylum and of a ribosome heterogeneity.</p

The salivary gland transcriptome of the neotropical malaria vector Anopheles darlingi reveals accelerated evolution of genes relevant to hematophagy

BackgroundMosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).ResultsA total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.ConclusionComparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi