Nucleotide sequence of a cDNA encoding murine CSF-1 (Macrophage-CSF)
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A human Colony Stimulating Factor-1 (CSF-1 or Macrophage-CSF) encoding cDNA was previously isolated from the pancreatic tumor cell line MIA-PACA-2 (I). This cDNA encodes a protein of 26 Kd molecular weight. To isolate a murine cDNA homologous to human M-CSF, a probe from the coding region of the human cDNA was prepared by nick-translation. A recotnbinant lambda phage library made from the mRNA of the murine fibroblast cell line. L929, was screened with the human M-CSF cDNA probe The cDNA isolated from the murine library has an open reading frame of 1656 nucleotides which encodes a protein of 552 amino acids. The first 32 amino acids encode a putative signal peptide which when cleaved yields a mature protein beginning with the amino acid sequence: Lys-Glu-Val-Ser. This deduced sequence is identical to the amino terminal residues reported for M-CSF as purified from L-cell supernatant? (2). The mature protein deduced from the cDNA sequence has a molecular weight of 57.261 d. All ten of the cysteine residues are found in approximately the amino-terminal half of the molecule. There are three potential relinked glycosylation sites (Asn-X-Thr/Ser) located at residues 154, 172, and 378. From the first amino acid encoded by the cDNA to residue 180 and from residue 476 to the cartxwy-terminal residue, approximately 80% homology is found between the murine and human cDNA's at both the nucleotide and amino acid levels. For comparison the deduced human M-CSF protein sequence is aligned below that of the murine sequence. The open reading frame in the murine cDNA encodes an additional 295 amino acids which are not found in the deduced amino-acid sequence of human M-CSF (I). However, higher molecular weight forms of human M-CSF mRNA have also been identified by Northern analysis of MIA-PACA-2 mRNA using the 26 Kd encoding cDNA as a probe (3). Furthermore, the murine M-CSF encoding cDNA we isolated was incorporated into an animal cell expression vector under SV40 early promoter transcrlptional control. When the vector was transfected Into the simian cell line, COS-7, murine M-CSF biological activity, as measured on mouse bone marrow cells, was observed in the transfected-culture supernalantsA cDNA library from peripheral immune organs of Merino sheep was constructed to screen genes related to Immunal Reaction and explore their functions.Total RNA were extracted from peripheral immune organs of Merino sheep and mRNA were purified to construct the cDNA library as template,according to the protocol of ZAP Express cDNA Synthesis Kit.Results showed that the cDNA library consisting of a primary contenet of 3.28×105 pfu and on amplified tite of 3.8×108 pfu/mL was constructed with the recombinant ratio of 97.7%.The average inserted fragments were about 1.5 kb with the majority ranging from 0.5 to 2.3 kb.It was concluded that the successfully constructed cDNA library has established a solid molecular basis for further study.
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The nucleotide sequence of the PHO5 gene of the yeast, Saccharomyces cerevisiae, which encodes repressible acid phosphatase (APase) was determined. Comparison of N-terminal amino acid sequence deduced from the nucleotide sequence with that of the purified repressible APase revealed the existence of a putative signal peptide in the precursor protein. The signal peptide was shown to contain 17 amino acid residues and its structural features were quite similar to those of higher eukaryotic and prokaryotic signal peptides. The nucleotide sequence of 5' and 3' noncoding flanking regions of the PHO5 gene are also discussed.
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A full length cDNA library of Hylarana latouchii skin tissue was constructed with the SMART technique.This cDNA library could be used to screen encoding gene of antimicrobial peptides and other functional genes.The titer of the cDNA library was 9.7×106 cfu/mL,and the recombination rate of it was 91%.Inserted fragments ranged was 0.3-1.5 kb.The titer of the amplified library was 1.9×109 cfu/mL.Successfully constructed cDNA library was a full length library with high quality.Two different cDNA sequences encoding antimicrobial peptide precursors named brevinin-2LT1 and brevinin-2LT2 were cloned by PCR from this cDNA library.All of the two antimicrobial peptides attributed to brevinin-2 family.
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AIM: To construct human myeloma cell cDNA expression library. METHODS: Total RNA were extracted from human myeloma cell line HMy2 and mRNA were purified with Oligotex mRNA kit. 1 st and 2 nd strands of cDNA were synthesized through reverse transcription. After blunting the cDNA termini, the cDNA fragments were connected with Eco RI adapters, and the end of Eco RI adapters was phosphorylated. The cDNA smaller than 400 bp were removed by Sephacryl S400 spin column,the remaining fragments were ligated with the dephophrylated arms of λ gt11. The recombinants were packaged in vitro, and a small portion of packaged phage was used to infect E. coli Y1090 for titration. RESULTS: The HMy2 cell line cDNA library consisting of 1.5 ×10 6 recombinant bacteriophages was constructed for the first time. The average exogenous insert of the recombinants was about 1.4 kb. CONCLUSION: The constructed cDNA library can be used to screen target clones.
Insert (composites)
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The cDNA expression library of Clonochis sinensis metacercaria was constructed for selecting stage specific diagnosis antigen gene or vaccine candidate gene.Total RNA of leucocytes was then extracted,pooled together and mRNA was isolated with a column of oligo(dT) cellulose.And cDNA was synthesized by Stratagene cDNA library protocol.The first and second strand of cDNA were synthesized using Stratagene Uni-ZAP XR cDNA synthesis kit,then ligated to EcoR I adapters and digested with Xho Ⅰ.After size fractionated with CHROMA Spin-400 column cDNA were ligated into Uni-ZAP vector and packaged in vitro to construct cDNA libraries.It was found that the size of the primary library was 9.15×105 PFU with a recombination rate of 99.5% and the titer of the amplified library was 1.6×1010 PFU/mL.cDNA library for Clonochis sinensis Metacercaria has been constructed.
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Purpose To construct a cDNA library by using mRNA from Gloydius saxatilis venom gland, to clone and sequence analysis defibrase from the cDNA library. Methods Total RNA was isolated from venom gland of G. saxatilis , and mRNA was purified by using mRNA isolation kit. Full length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure. Lately cDAN was linked to vector pBluescript-sk. The recombinant cDNA was transformed into E.coli DH5 α. It detected and amplified the cDNA of defibrase gene in the venom gland of G. saxatilis using the hybridization in situ method. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined . Results The capacity of cDNA library of venom gland was above the 2.0×106 . Its open reading frame is composed of 777 nucleotides and codes a pre-zymogen of 258 amino acids. It contains 12 cysteine residues. Conclusion The capacity of cDNA library of venom gland was above the general level of cDNA library. The constructed cDNA library of G. saxatilis venom gland would be a helpful platform to detect new target genes and further gene manipulate. The sequence analysis indicates that the deduced amino acid sequence of the defibrase shares more than 86% identity with the Thrombin-like enzyme genes of other snakes in the gene bank.
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AIM: To elucidate molecular mechanism of chemopreventive efficacies of garlic against human gastric cancer (HGC).METHODS: HGC cell line BGC823 was treated with Allitridi (a kind of garlic extract) and Allitridi-treated and parental BGC823 cDNA libraries were constructed respectively by using λ λ λ λ λZAP II vector.cDNA Representational Difference Analysis (cDNA RDA) was performed using BamH I cuttingsite and abundant cDNA messages provided by the libraries.Northern blot analysis was applied to identify the obtained difference products.RESULTS: Two specific cDNA fragments were obtained and characterized to be derived from homo sapiens folate receptorα α α α α (FRα α α α α) gene and calcyclin gene respectively.Northern blot results showed a 4-fold increase in FRα α α α α gene expression level and 9-fold increase in calcyclin mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION:The method of cDNA RDA based on cDNA libraries combines the high specificity of cDNA RDA with abundant cDNA messages in cDNA library; this expands the application of cDNA library and increases the specificity of cDNA RDA.Up-regulation of FRα α α α α gene and calcyclin gene expressions induced by Allitridi provide valuable molecular evidence for the efficacy garlic in treating HGC as well as other diseases.
Northern blot
Representational difference analysis
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cDNA library is the pool of cDNA fragments which are ligated with vectors,and cDNA fragments are reverse-transferred from total mRNAs of some organism at certain period.cDNA library has the important significance in the research on the differential gene expressing of different tissues and growth-periods.In the paper the kind of cDNA library construction was simply summarized and the application of cDNA library in the study on plant resistance was described.
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