Switching Mechanism At 5 ' End Of RNA Transcript Research Articles

The proteins interacting with C-terminal of μ receptor are identified by bacterial two-hybrid system from brain cDNA library in morphine-dependent rats.

Opioid addiction is associated with long-term adaptive changes in the brain that involve protein expression. The carboxyl-terminal of the μ opioid receptor (MOR-C) is important for receptor signal transduction under opioid treatment. However, the proteins that interact with MOR-C after chronic morphine exposure remain unknown. The brain cDNA library of chronic morphine treatment rats was screened using rat MOR-C to investigate the regulator of opioids dependence in the present study. The brain cDNA library from chronic morphine-dependent rats was constructed using the SMART (Switching Mechanism At 5' end of RNA Transcript) technique. Bacterial two-hybrid system was used to screening the rat MOR-C interacting proteins from the cDNA library. RT-qPCR and immunoblotting were used to determine the variation of MOR-C interacting proteins in rat brain after chronic morphine treatment. Column overlay assays, immunocytochemistry and coimmunoprecipitation were used to demonstrate the interaction of MOR-C and p75NTR-associated cell death executor (NADE). 21 positive proteins, including 19 known proteins were screened to interact with rat MOR-C. Expression of several of these proteins was altered in specific rat brain regions after chronic morphine treatment. Among these proteins, NADE was confirmed to interact with rat MOR-C by in vitro protein-protein binding and coimmunoprecipitation in Chinese hamster ovary (CHO) cells and rat brain with or without chronic morphine treatment. Understanding the rat MOR-C interacting proteins and the proteins variation under chronic morphine treatment may be critical for determining the pathophysiological basis of opioid tolerance and addiction.

Four methods of preparing mRNA 5' end libraries using the Illumina sequencing platform.

BackgroundThe 5′ untranslated regions of mRNA play an important role in their translation.ResultsHere, we describe the development of four methods of profiling mRNA 5′ ends using the Illumina sequencing platform; the first method utilizes SMART (Switching Mechanism At 5′ end of RNA Transcript) technology, while the second involves replacing the 5′ cap structure with RNA oligomers via ligation. The third and fourth methods are modifications of SMART, and involve enriching mRNA molecules with (nuclear transcripts) and without (mitochondrial transcripts) 5′ end cap structures, respectively. Libraries prepared using SMART technology gave more reproducible results, but the ligation method was advantageous in that it only sequenced mRNAs with a cap structure at the 5′ end.ConclusionsThese methods are suitable for global mapping of mRNA 5′ ends, both with and without cap structures, at a single molecule resolution. In addition, comparison of the present results obtained using different methods revealed the presence of abundant messenger RNAs without a cap structure.

Development of Normalized Cdna Library from Fusarium Wilt Infected Roots of a Tolerant Banana Genotype 'Calcutta-4' Musa acuminata ssp. burmannicoides

Management of the most devasting disease, Fusarium wilt of banana, caused by the fungus Fusarium oxysporum f. sp., cubense, is a challenge to the plant pathologist and the banana grower. Currently, genomics is providing the way for understanding plant defense mechanism, having acquired an important place in crop improvement. To identify the relevant genes and to understand the defense mechanism induced during Fusarium wilt infection, a normalized cDNA library was constructed from infected root samples of a tolerant banana genotype, Musa acuminata spp. burmannicoides 'Calcutta-4', by duplex specific nuclease (DSN) based normalization, using the SMART (switching mechanism at 5' end of RNA transcript) full-length cDNA construction method. Sequencing and analysis of 600 clones revealed 392 non-redundant clones. In all, of 88% of the sequences were annotated using Musa genome database, and the remaining 12% were identified as novel loci not annotated. We observed several resistance genes, ROS scavenging genes and genes involved in ubiquitin-proteosome pathway in this study. These genes may have a possible role against Foc infection. These sequences would enrich the EST data developed against specific stress, which is an indispensable tool for predicting functional genes and understanding the defense mechanism.

Rapid sequencing of influenza A virus vRNA, cRNA and mRNA non-coding regions

Characterizing the genomic sequences of influenza A viruses is important for pathophysiological and evolutionary studies. Noncoding regions (NCR) of influenza A virus have been shown to play critical roles in replication and transcription but their sequences are infrequently determined. In this study, a method employing poly(A) addition and SMART (switching mechanism at 5′ end of RNA transcript) technology is described for directly determining and discriminating both NCR ends of viral RNA (vRNA), complementary RNA (cRNA), or NCR and cap sequences from viral mRNA. This modified method may also be used to characterize the NCRs of influenza A virus samples in which the RNA has been degraded.

Generation and analysis of cDNA library from lipopolysaccharide-stimulated gastropod abalone (Haliotis diversicolor supertexta) hemocytes

A cDNA Library of lipopolysaccharide-stimulated gastropod abalone hemocytes was constructed using switching mechanism at 5' end of RNA transcript (SMART) approach. 300 random clones were selected and sequenced. 180 ESTs was gained. After assembling, we obtained 24 contigs and 119 singlets finally, some of which were immune- related genes. These immune-related genes include cytidine deaminase, ferritin, nonmuscle myosin essential light chain, cytochrome c oxidase subunit I, CD63 antigen-like protein and lysosomal-associated transmembrane protein. This study may contribute to the understanding of the immune mechanism of gastropod abalone Haliotis diversicolor supertexta . Key words: Haliotis diversicolor supertexta, cDNA Library, gene expression, immune mechanism.

Improved Method for Analysis of RNA Present in Long-Term Preserved Thyroid Cancer Tissue of Atomic Bomb Survivors

Since many thyroid cancer tissue samples from atomic bomb (A-bomb) survivors have been preserved for several decades as unbuffered formalin-fixed, paraffin-embedded specimens, molecular oncological analysis of such archival specimens is indispensable for clarifying the mechanisms of thyroid carcinogenesis in A-bomb survivors. Although RET gene rearrangements are the most important targets, it is a difficult task to examine all of the 13 known types of RET gene rearrangements with the use of the limited quantity of RNA that has been extracted from invaluable paraffin-embedded tissue specimens of A-bomb survivors. In this study, we established an improved 5' rapid amplification of cDNA ends (RACE) method using a small amount of RNA extracted from archival thyroid cancer tissue specimens. Three archival thyroid cancer tissue specimens from three different patients were used as in-house controls to determine the conditions for an improved switching mechanism at 5' end of RNA transcript (SMART) RACE method; one tissue specimen with RET/PTC1 rearrangement and one with RET/PTC3 rearrangement were used as positive samples. One other specimen, used as a negative sample, revealed no detectable expression of the RET gene tyrosine kinase domain. We established a 5' RACE method using an amount of RNA as small as 10 ng extracted from long-term preserved, unbuffered formalin-fixed, paraffin-embedded thyroid cancer tissue by application of SMART technology. This improved SMART RACE method not only identified common RET gene rearrangements, but also isolated a clone containing a 93-bp insert of rare RTE/PTC8 in RNA extracted from formalin-fixed, paraffin-embedded thyroid cancer specimens from one A-bomb survivor who had been exposed to a high radiation dose. In addition, in the papillary thyroid cancer of another high-dose A-bomb survivor, this method detected one novel type of RET gene rearrangement whose partner gene is acyl coenzyme A binding domain 5, located on chromosome 10p. We conclude that our improved SMART RACE method is expected to prove useful in molecular analyses using archival formalin-fixed, paraffin-embedded tissue samples of limited quantity.

Establishment and Identification of a Normalized Full-Length cDNA Library of CCRI 36

Short season cotton (SSC) breeding plays an important role in solving the competition for more growing area between cotton and food crops. To explore the premature mechanism of SSC, to clone genes that have a close relation with premature flowering, and to speed process of premature cultivar breeding, we established a normalized full-length cDNA library using the flower and bud of CCRI 36 by DSN (duplex-specific nuclease)-normalization method combined with SMART (switching mechanism at 5' end of RNA transcript) technique. The titer of un-amplified cDNA library was about 1.7×106 cfu mL-1. The average size of cDNA inserts was 1 200 bp with a recombination rate of 100%. The abundance of transcripts Histon3 and UBQ7 decreased obviously in normalized cDNA library comparing with that in non-normalized samples detected by Virtual Northern Blot. Meanhile, protein genes associated with flower development were obtained on the basis of the positive signal of cDNA library by PCR. These results indicated that the normalized full-length cDNA library has been established successfully, which is convenient for further study on the molecular mechanism and gene cloning of flower development.

3G vector–primer plasmid for constructing full-length-enriched cDNA libraries

We designed a 3G vector–primer plasmid for the generation of full-length-enriched complementary DNA (cDNA) libraries. By employing the terminal transferase activity of reverse transcriptase and the modified strand replacement method, this plasmid (assembled with a polydT end and a deoxyguanosine [dG] end) combines priming full-length cDNA strand synthesis and directional cDNA cloning. As a result, the number of steps involved in cDNA library preparation is decreased while simplifying downstream gene manipulation, sequencing, and subcloning. The 3G vector–primer plasmid method yields fully represented plasmid primed libraries that are equivalent to those made by the SMART (switching mechanism at 5′ end of RNA transcript) approach.

Expression analysis of light-regulated genes isolated from a full-length-enriched cDNA library of Onosma paniculatum cell cultures

Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.

Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5 end of RNA transcript (SMART) approach. This library had a plaque liter of 1 × 10 9 pfu mL −1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.

Cloning of Full-Length cDNA of Teleost Corticotropin-Releasing Hormone Precursor by Improved Inverse PCR

We designed a new inverse PCR protocol combined with switching mechanism at 5' end of RNA transcript (SMART) technology, and applied it to the cloning of teleost corticotropin-releasing hormone precursor cDNA. Due to the advantages of both techniques, this method can efficiently amplify the complete 5'- and 3'-ends of cDNA in a single reaction, and might prove to be an alternative to the conventional rapid amplification of cDNA ends (RACE) approaches.

Study on construction of cDNA libraries from rat normal liver and regeneration liver with smart technique

The aim of the study is to construct cDNA libraries from the normal liver and regeneration liver of rat by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze their quality. The total RNA was separated from the normal liver and regeneration liver of rat and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained sfi IB site) while the SMART oligonucleotide (contained sfi IA site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction, enzyme. After cDNA size fractionation through Chroma Spin 400 column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packagedin vitro. The unamplified rat normal liver cDNA library consists of 1.3×10(7) pfu/ml, and regeneration liver cDNA library consists of 1.6×10(7) pfu/ml in which the percentage of recombinant clones both are about 100%. Through testing, the high quality cDNA libraries containing full-length cDNA of rat normal liver and regeneration liver have been constructed. The titer of the amplified cDNA library is 4.5×10(10) pfu/ml and 3.6×10(10) pfu/ml. the average exogenous inserts of the recombinants both are about 1.5 kb. These results show that the normal liver and regeneration liver of rat cDNA libraries both have an excellent quality and lay solid foundation to study liver functions and the mechanism of liver regeneration.

Study on construction of cDNA library of the treated changliver cell and quality analysis

The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 10(10) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. These results showed that the Changliver cell cDNA library had an excellent quality and lay foundation for screening whole long cDNA of related genes.

Detecting herbivore‐specific transcriptional responses in plants with multiple DDRT‐PCR and subtractive library procedures

Differential display‐reverse transcriptase PCR (DDRT‐PCR) and subtractive hybridization with magnetic beads (SHMB) procedures were modified to compare the transcriptional responses of the postfire desert annual, Nicotiana attenuata when it is attacked by its two most abundant herbivores: the voracious Lepidopteran caterpillars of Manduca sexta and the piercing/sucking nymphs and adults of the mirid bug, Tupiocoris notatus. The relative merits of the two procedures were compared. DDRT‐PCR requires less starting material, allows for comparisons of multiple herbivores, and identifies both down‐ and up‐regulated responses, but is more laborious than SHMB. SHMB produced a greater proportion of known sequences (43.8 versus 35.6%), but the sequences were not significantly longer than those obtained by DDRT‐PCR, suggesting that the SMART (switching mechanism at 5′ end of RNA transcript) modification of the SHMB procedure did not produce the desired result. Both procedures produced apparent false positives and microarray‐based verification of differential expression would be a powerful approach to identify novel genes involved in ecological interactions. During the N. attenuata–T. notatus interaction, the expression of several photosynthesis‐related genes (cytochrome f, psaE, rubisco, rubisco activase, two aldolases, and phosphoglycerate kinase) was altered, which suggests that reprogramming of photosynthesis contributes to the mechanisms mediating tolerance to mirid damage. In addition, T. notatus‐induced changes in the expression of transcripts coding for a rhamnosyl transferase, a thionin, and an S‐adenosylmethionine‐decarboxylase are particularly interesting in the context of what is known about defence against herbivores in N. attenuata.