Primary structure of rat liver elongation factor 2 deduced from the cDNA sequence
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A number of cDNA clones in summary encoding 700 amino acid residues from the N-end of rat liver elongation factor 2 (EF-2) and including 49 nucleotides of the 5'-untranslated mRNA region have been obtained. EF-2 cDNA clones were isolated from gradually constructed small (1000-5000 clones) specific cDNA libraries using the primer extension method for synthesis of the first cDNA chain. The complete primary structure of cDNA and protein EF-2 from rat liver was derived taking into account the primary structure of the 3'-terminal region EF-2 cDNA previously reported [(1986) Proc. Natl. Acad. Sci. USA 83, 4978-4982]. Comparison of this cDNA with hamster cDNA has shown that (i) the base sequences had a 89.7% homology while that of the 5'-untranslated region was 73%; (ii) there are two amino acid replacement in rat liver EF-2 as compared with hamster EF-2.Keywords:
Protein primary structure
Elongation factor
Primer (cosmetics)
Primer extension
In order to identify unknown encoding cDNAs of Cervus nioppon Temminck (sika deer), we constructed a cDNA library of uterus from Jilin-Shuangyang Cervus nippon Temminck using PCR cDNA library kit. PCR products of the library were cloned into pGEM-Teasy vectors and the cDNAs were sequenced and analyzed by nucleotide homology comparison against GenBank Database using the BLAST network service. The results showed that the cDNA library contained cDNA fragments of different lengths and a full length encoding cDNA highly homologous to human sentrin-1/SUMO-1 (small ubiquitin-related modifier 1) was identified. The cDNA was deposited in GenBank under the accession number AF 242526. These show that Cervus nippon Temminck-derived sentrin/SUMO gene has been discovered from PCR cDNA library of uterus from Cervus nippon Temminck.
Cervus
Homology
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To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes.Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers.A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed.The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.
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Vibrio alginolyticus is one of the main causative agents resulting in serious infectious diseases of Litopenaeus vannamei and other animals.Suppression subtractive hybridization(SSH) is a new and highly effective method for analysing differentially expressed or tissue-specific cDNA probes and libraries.It is applicable to diagnosis of disease,development,tissue specificity,or other differential expressing.Based on the user manual of Clontech PCR-SelectTM cDNA subtraction kit,a suppression subtraction cDNA libraries of Litopenaeus vannamei were made by a PCR method.Experimental shrimp were injected with 5×107 CFU live Vibrio alginolyticus and the control shrimp were injected with steriled normal saline.The mRNA from hemocytes of normal shrimp and vibrio-challenging shrimp must be isolated firstly.After mRNA was reverse transcribed into double strand cDNA,the two kinds of ds cDNA were denominated with Driver cDNA(normal shrimp) and Tester cDNA(vibrio challenging shrimp) separately.Then they were digested with Rsa I at the same time but only Tester ds cDNA were diluted and ligated to adaptor l and adaptor 2R in separate ligation reactions in the same total volume.Then an excess of driver cDNA was added to each tester cDNA for the first round of hybridization to enrich for differentially expressed sequences.Secondly,the two samples from the first hybridization were mixed together and freshly denatured driver DNA was added to each tester cDNA for differentially expressed sequences.Only the remaining normalized and subtracted ss tester cDNA were able to reassociate and form newly hybrids with different adaptors at their 5'-ends in the second hybridization.The new hybrids were preferentially amplified by PCR with a pair of primers,nested PCR primer 1 and nested PCR primer 2R.Litopenaeus vannamei β-actin gene was used as internal control to estimate the efficiency of subtractive successfully constructed.In this library,β-actin was subtracted significantly above 10 cycles,suggesting that the subtractive cDNA library was successfully constructed.The substracted products were cloned into the pMD-T 18 Vector.Then the recombinated plasmid was transformed into DH5α.PCR analysis showed that the inserts were 488bp equally in length.The subtracted cDNA library including 2000 clones and picked 600 clones to sequence randomly.The obtained 560 ESTs were assembled to 239 Unigenes(164 contigs and 75 singletons) with DNAMAN 5.2.2 software.Compared with sequences in unigene database of GeneBank with BLASTx and BLASTn algorithm,159 ESTs of them had comparatively clear results and the percent of them in acquired ESTs was 66.9%.The sequences of these ESTs were subjected to GO annotation.According to their physioligical function,they could be subdivided into 7 categories,36% were metabolism genes;15% were immune-related genes;8% were others genes;3% were regulation and signal transaction factors;2% were apoptotic-related proteins and antioxidant enzyme;1% were ribosomal proteins.The result showed that Litopenaeus vannamei,induced by Vibrio alginolyticus,could express serials of special genes.Analysis of the libraries indicates the PCR based suppression subtraction cDNA libraries is feasible used to discover the immune gene in shrimp.
Vibrio alginolyticus
Suppression subtractive hybridization
Litopenaeus
UniGene
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In order to rapidly clone the cellular immune function genes of the Tiplia on a large scale,a full-length cDNA library of the peripheral blood leucocytes from the Oreochromis niloticus that vaccinated of S.iniae vaccine by i.p was constructed by using SMART(switching mechanism at 5′ end of RNA transcript) techniques.The total RNA was extracted from peripheral blood leucocytes after vaccinated 3,5,and 7 days.The PowerscriptTM reverse transcriptase was used to synthesize and anchor the first-strand cDNA,and the long distance PCR(LD-PCR) method was used to amplify double-strand cDNA based on the SMART techniques for construction of a full-length cDNA library.The PCR products were digested by proteinase K.After digestion with Sfi I and size fractionation using CHROMA SPIN-400TM columns,cDNAs(500 bp) were ligated to the Sfi I digested,dephosphorylated pBluescript II SK vector.The ligation mixture was transformed into E.coil DH5a.The recombinant vectors were titered and the recombinant rate(blue/white) was determined,and 24 clones were picked randomly from the library for screening size of cDNA inserts through PCR reaction.By identification,the primary constructed cDNA library contained 1.021×106 independent clones.The titer of the cDNA library was estimated as 1.078×106/mL,the recombinant rate was above 94.71 %.The inserts varied from 0.7 to 3.0 kb with average size was about 1500 bp.24 clones were sequenced randomly-selected from each library and 18 contigs were acquired and analyzed by BLASTx.The results indicated that 15 contigs had the homologous gene information which 10 were full-length cDNA with 66.7 % of full-ratio.These results showed that the full-length cDNA library meet the requirement of a standard full-length cDNA library,and it could lay a strong basis for subsequent work of clone and selection of some immune functional genes.
Trizol
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In order to study the novel genes of peanut seeds,a full-length cDNA library from peanut seeds was constructed.mRNA from peanut seeds of different developmental stages was purified.Double strand cDNA was synthesized by SMART method,the ds cDNA fragments were ligated to the pDNR-LIB vector.The recombinant plasmids were transformed into the E.coli,a cDNA library of peanut seeds was successfully constructed.The full-length cDNA library was stocked after amplification,the titer of the cDNA library was estimated as 1.7×109 cfu/mL.PCR results showed that the inserts varied from 0.5 to 2.0kb with average size was larger than 1000bp.It indicated that this library could be used for full-length genes screening and cloning of low abundance genes.
Cloning (programming)
Library
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The objective of the present study was to construct a cDNA library for male adult of Ascaris suum with SMART(switching mechanism at 5′end of RNA transcript) technique using Creator~(TM) SMART~(TM) cDNA library construction kit.The total RNA was extracted from A.suum male adult using TriPure isolation reagent and mRNA was purified using Poly(A)Purist~(TM) kit.Single-strand cDNA was synthesized using PowerScript~(TM) reverse transcriptase,and then double-strand cDNA was synthesized and amplified by long-distance PCR(LD-PCR).The PCR products were digested by proteinase K and purified.After digestion with SfiⅠ and size fractionation(using) CHROMA SPIN-400TM columns,SMART cDNA was ligated to the Sfi I-digested,dephosphorylated pDNR-LIB vector.The ligation mixture was transformed into E.coli DH5α by electroporation.The constructed cDNA library contained 7.26×10~5 independent clones.The recombination rate was 96.7%.The average cDNA insert size was 1 kb.After the library was amplified,its capacity was 6.359×10~9 cfu/mL.A cDNA library for A.suum male adult was successfully constructed using SMART technology,which provides foundation for the screening and isolation of male-specific genes from A.suum.
Ascaris suum
Trizol
Insert (composites)
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Objective: To construct RACE cDNA library of Hydra magnipapillata.Methods: Total RNA was isolated from Hydra magnipapillata,and purified mRNA from total RNA was used to construct RACE cDNA library with the SMART cDNA library con-struction kit.In order to identify the cDNA library,the polymerase chain reaction(PCR) primers for 5' RACE,3' RACE and the full-length cDNAs of actin gene were designed based on the pupative cDNA sequence of actin from GenBank.Results: Agarose gel elec-trophoresis showed that the lengths of full-length cDNAs in this library were pooled mainly between 500 and 2 000 base pairs.By RACE PCR,amplified products were obtained with all the gene-specific primers and adaptor primers.Conclusion: The quality of the RACE cD-NA library was high and appropriate for cloning the full-length cDNAs of functional genes in Hydra magnipapillata.
Lernaean Hydra
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Aim To establish a new technique for rapid isolation full length cDNA sequence of human novel gene from cDNA library using hot start PCR. Methods The library vector specific primers and gene specific primers have been designed for performing hot start PCR to attain the full length cDNA of differential expressed sequence tag(FRG4) of U937 foam cell formation induced by ox LDL from human fetal liver cDNA library. The products of PCR are cloned to pGEM T vector and sequenced. Results We have successfully attained the full length cDNA sequence of FRG4. Conclusion This technique is a rapid and efficient method for isolating full length cDNA sequence of human novel gene from cDNA library.
Sequence (biology)
Primer (cosmetics)
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mRNA of Flammulina velutipes was extracted with Quick Prep mRNA Purification Kit, after synthesis of double-strand cDNA molecules using Time Saver cDNA Synthesis Kit, EcoRⅠ/NotⅠadaptors were added to the end of ds cDNA, phosphorylated by T4 polynucleoted kinase. Then the cDNA was ligated with λZAP Express Predigested Vector and packaged to transfect E.Coli XL1-Blue MRF? The results revealed that the tilter of the cDNA library was 4.0×106 pfu/ml, the amplified cDNA library is 2×1010 pfu/ml, the recombinant rate reached to 90%, and the length of inserts were between 0.3-3.5 kb. A candidate cDNA related with flammulin gene was screened by western blotting in situ.
Flammulina
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Total RNA was extracted from laticifers of Hevea brasiliensis,and total RNA was used to purify mRNA with MACHERY-NAGEL Purification of poly( A) RNA kit. The first strand cDNA was synthesized by reverse transcription of mRNA with SMART oligo-dT technique,and LD-PCR was performed to synthesize double strand cDNA. The double strand cDNA was then purified by running through a CHROMA SPINTMTE-400 column of clontech to select with ds cDNA molecules 200 bp. Purified ds cDNA and lineared pGADT7-Rec were cotransformed into Y187 yeast strain to construct a yeast two-hybrid cDNA library of Hevea brasiliensis laticifers. The detection showed that the library contained 2. 8 × 107independent clones,and that the titer of library was 4. 92 × 107·mL- 1. The sizes of most inserts ranged from 500 to 2 000 bp in this library,and the recombination rate was 96%. These results showed that the library was suitable for yeast mating and can be used to screen interaction proteins.
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