Construction of a Full-Length Murine Proα2(I) Collagen cDNA by the Polymerase Chain Reaction

1991 ◽  
Vol 97 (6) ◽  
pp. 980-984 ◽  
Author(s):  
Charlotte L Phillips ◽  
Laura W Lever ◽  
Sheldon R Pinnell ◽  
Leigh D Quarles ◽  
Richard J Wenstrup
Keyword(s):  
Polymerase Chain Reaction ◽  
Full Length ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Full-Length Sequence of Mouse Acupuncture-Induced 1-L (Aig1l) Gene Including Its Transcriptional Start Site

2011 ◽  
Vol 2011 ◽  
pp. 1-7
Author(s):  
Mika Ohta ◽  
Aki Sugano ◽  
Shuji Goto ◽  
Surini Yusoff ◽  
Yushi Hirota ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Polymerase Chain Reaction ◽  
Tissue Distribution ◽  
Cdna Sequence ◽  
Transcriptional Start Site ◽  
Full Length ◽  
Start Site ◽  
Chain Reaction ◽  
Polymerase Chain

We have been investigating the molecular efficacy of electroacupuncture (EA), which is one type of acupuncture therapy. In our previous molecular biological study of acupuncture, we found an EA-induced gene, named acupuncture-induced 1-L (Aig1l), in mouse skeletal muscle. The aims of this study consisted of identification of the full-length cDNA sequence ofAig1lincluding the transcriptional start site, determination of the tissue distribution ofAig1land analysis of the effect of EA onAig1lgene expression. We determined the complete cDNA sequence including the transcriptional start site via cDNA cloning with the cap site hunting method. We then analyzed the tissue distribution ofAig1lby means of northern blot analysis and real-time quantitative polymerase chain reaction. We used the semiquantitative reverse transcriptase-polymerase chain reaction to examine the effect of EA onAig1lgene expression. Our results showed that the complete cDNA sequence ofAig1lwas 6073 bp long, and the putative protein consisted of 962 amino acids. All seven tissues that we analyzed expressed theAig1lgene. In skeletal muscle, EA induced expression of theAig1lgene, with high expression observed after 3 hours of EA. Our findings thus suggest that theAig1lgene may play a key role in the molecular mechanisms of EA efficacy.

scholarly journals miR-206 Promotes Cancer Progression by Targeting Full-Length Neurokinin-1 Receptor in Breast Cancer

2019 ◽  
Vol 18 ◽  
pp. 153303381987516 ◽  
Author(s):  
Yu Zhou ◽  
Meng Wang ◽  
Yingna Tong ◽  
Xiaobin Liu ◽  
Lufang Zhang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Cancer Cell ◽  
Breast Cancer Cell ◽  
Full Length ◽  
Luciferase Reporter ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Neurokinin 1

Substance P plays a pivotal role in human cancer development and progression by binding to its receptor, neurokinin-1. Neurokinin-1 has 2 isoforms: full-length neurokinin-1 and truncated neurokinin-1, the latter lacking the cytoplasmic terminal 96-amino acid residues of the full-length protein. We have identified 3 candidate miR-206 target sites within the 3′-untranslated region of the full-length neurokinin-1 gene from bioinformatics database searches. In the present study, real-time quantitative polymerase chain reaction was performed to quantify the expression of miR-206, and the expression of neurokinin-1 and full-length neurokinin-1 was detected by immunohistochemistry in 82 clinical cases of breast cancer and paired adjacent normal tissues. The miR-206 target gene was demonstrated by using a dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and Western blotting. Transwell migration and invasion, colony formation, and proliferation assays were performed to evaluate the effects of miR-206 expression on various aspects of breast cancer cell behavior in vitro. We showed that miR-206 expression is upregulated in breast cancer cell lines and breast cancer tissues when compared to that in adjacent normal tissues, and full-length neurokinin-1 expression inversely correlates with Tumor Lymph Node Metastasis (TNM) stage and lymph node metastasis. Western blotting, quantitative real-time polymerase chain reaction, and dual-luciferase reporter assays demonstrated that miR-206 binds the 3′-untranslated region of full-length neurokinin-1 messenger RNA, regulating protein expression. We showed that the overexpression of miR-206 promotes breast cancer cell invasion, migration, proliferation, and colony formation in vitro. The present study furthers the current understanding of the mechanisms underlying breast cancer pathogenesis and may be useful for the development of novel targeted therapies.

scholarly journals 52-kD SS-A/Ro: genomic structure and identification of an alternatively spliced transcript encoding a novel leucine zipper-minus autoantigen expressed in fetal and adult heart.

1995 ◽  
Vol 182 (4) ◽  
pp. 983-992 ◽  
Author(s):  
E K Chan ◽  
F Di Donato ◽  
J C Hamel ◽  
C E Tseng ◽  
J P Buyon
Keyword(s):  
Polymerase Chain Reaction ◽  
Cdna Clone ◽  
Leucine Zipper ◽  
Full Length ◽  
Neonatal Lupus ◽  
Chain Reaction ◽  
Beta Form ◽  
Alternatively Spliced ◽  
Polymerase Chain ◽  
Exon 4

The 52-kD SS-A/Ro protein is one of the antigenic targets strongly associated with the autoimmune response in mothers whose children have manifestations of neonatal lupus. In addition to the cDNA clone we previously reported for the full-length 52-kD SS-A/Ro protein, an interesting MOLT-4 cDNA clone, p52-2, was found to have an internal deletion of 231 nucleotides including the domain encoding the leucine zipper motif. To further investigate the nature of this deletion, genomic DNA clones were isolated from a lambda FIXII library. The complete gene for the full-length 52-kD protein (alpha form, 52 alpha) spans 10 kb of DNA and is composed of seven exons. Exon 1 contains only the 5' untranslated sequence, while the translation initiation codon is located 3 kb downstream in exon 2, which also encodes the three zinc finger motifs. Exon 4 encodes amino acids 168-245, including the coiled coil/leucine zipper domain. Exon 7 is the longest and encodes the rfp-like domain and the 3' untranslated region. The cDNA p52-2 can now be accounted for as a product of alternative messenger RNA (mRNA) derived from the splicing of exon 3 to exon 5, skipping exon 4, which results in a smaller protein (52 beta) with a predicted molecular weight of 45,000. An initial approach to identifying this alternatively spliced form in the human heart used a ribonuclease protection assay. Using an RNA probe corresponding to bases 674-964 of the full-length cDNA, two protected mRNA fragments were identified, a 290-bp fragment corresponding to expression of 52 alpha and a smaller fragment of 144 bp, the predicted size of 52 beta. Using reverse transcription followed by polymerase chain reaction, cDNAs from a 16-wk fetal heart, 24-wk heart, and adult heart were amplified with primers flanking exon 4. Two polymerase chain reaction products were observed in each tissue, one 1.0 kb likely representing 52 alpha and a second 0.78 kb, consistent with 52 beta. The 0.78-kb fragment identified in the 16-wk heart was cloned, and DNA sequencing confirmed the 52 beta type. Immunoprecipitation of in vitro-translated 35S-labeled 52 beta form was performed to evaluate the antigenicity of this novel form of 52-kD SS-A/Ro. 26 (87%) of 30 sera tested from mothers whose children were known to have neonatal lupus immunoprecipitated the 52 beta form.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction

Genomics ◽  
1989 ◽  
Vol 4 (2) ◽  
pp. 198-205 ◽  
Author(s):  
Ruud Jansen ◽  
Frantisek Kalousek ◽  
Wayne A. Fenton ◽  
Leon E. Rosenberg ◽  
Fred D. Ledley
Keyword(s):  
Polymerase Chain Reaction ◽  
Cdna Library ◽  
Full Length ◽  
Chain Reaction ◽  
Polymerase Chain

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes

1990 ◽  
Vol 10 (1) ◽  
pp. 68-74 ◽  
Author(s):  
R Dornburg ◽  
H M Temin
Keyword(s):  
Polymerase Chain Reaction ◽  
Full Length ◽  
Target Cells ◽  
Hygromycin B ◽  
Direct Repeats ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Cis Acting ◽  
Processed Pseudogenes ◽  
Polymerase Chain

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

Identification of the full-length Hs1pro-1 coding sequence and preliminary evaluation of soybean cyst nematode resistance in soybean transformed with Hs1pro-1 cDNA

2007 ◽  
Vol 85 (4) ◽  
pp. 437-441 ◽  
Author(s):  
Michael D. McLean ◽  
Gordon J. Hoover ◽  
Bonnie Bancroft ◽  
Amina Makhmoudova ◽  
Shawn M. Clark ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Soybean Cyst Nematode ◽  
Nematode Resistance ◽  
Cyst Nematode ◽  
Full Length ◽  
Preliminary Evaluation ◽  
Chain Reaction ◽  
Coding Sequence ◽  
Scn Resistance ◽  
Polymerase Chain

The Hs1pro-1 gene reportedly confers resistance to the beet cyst nematode in wild beet and sugar beet. Here, we tested the hypothesis that Hs1pro-1 confers resistance in soybean against the soybean cyst nematode (SCN). The full-length Hs1pro-1 coding sequence, which encodes a predicted polypeptide of 490 amino acids, was first acquired then expressed in ‘Westag’ soybean using a constitutive octopine synthase – mannopine synthase promoter. Thirty T0 lines that successfully expressed the Hs1pro-1 gene, as indicated by both polymerase chain reaction and reverse transcriptase – polymerase chain reaction analyses, were generated. Bioassay of the T1 progeny from these lines revealed that only five T0 lines grew normally and exhibited a high degree of SCN resistance. On average, these T1 transgenic progeny were about 70% more resistant to SCN than susceptible control cultivars. These preliminary data suggest that Hs1pro-1 is a promising candidate for genetically engineering SCN resistance in elite, locally adapted soybean cultivars.

scholarly journals cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

1990 ◽  
Vol 10 (1) ◽  
pp. 68-74 ◽  
Author(s):  
R Dornburg ◽  
H M Temin
Keyword(s):  
Polymerase Chain Reaction ◽  
Full Length ◽  
Target Cells ◽  
Hygromycin B ◽  
Direct Repeats ◽  
Chromosomal Dna ◽  
Chain Reaction ◽  
Cis Acting ◽  
Processed Pseudogenes ◽  
Polymerase Chain

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.

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