The amplified DNA fragment was digested with BamHI and EcoRI, cloned into pcDNA3.1/His C (Invitrogen), and designated pcNAT. of CREB (pCREB) rapidly activates and, ultimately, to the termination of mRNA synthesis (14, 40). AANAT mRNA degradation is possibly involved in its circadian oscillation patterns (6, 42). Here, we elucidate the molecular mechanisms of dynamic AANAT mRNA degradation, and propose a biological role in the circadian rhythmicity of clock-controlled genes. MATERIALS AND METHODS Plasmids. In the absence of convenient restriction sites for cloning, standard PCR techniques were employed to amplify the desired sequences. The coding region of AANAT mRNA as a reporter gene was obtained by reverse transcription (RT)-PCR. The AANAT level decreases rapidly, with a half-life of 3.5 min, after sudden exposure to light during the night (16). Since AANAT is unstable in the absence of protein kinase A activation (29), it is an accurate reporter of the remaining mRNA level. In KRCA-0008 brief, total RNA prepared from nocturnal pineal glands was subjected to reverse transcription with oligo(dT) (Roche) as a primer. Amplification of cDNA was performed with polymerase (SolGent) and a primer pair specific for the AANAT coding region (5 primer 5-CGGGATCCATGTTGAGCATCCACCCCCTG-3 KRCA-0008 and 3 primer 5-AAGAATTCCTC-AGCAGCCACTGTTCCTCC-3; accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”U38306″,”term_id”:”1244550″U38306 [flanking sequences for cloning purposes are underlined]). The amplified DNA fragment was digested with BamHI and EcoRI, cloned into pcDNA3.1/His C (Invitrogen), and designated pcNAT. Rat AANAT 3UTR (3N578) was obtained for in vitro binding assays between 3UTRs and cell extracts, as described above. In brief, cDNA was amplified with two primers specific for 3N578 (5 primer 5-GGGAATTCCCAAGCTGCGCACTTGGC-3 and 3 primer 5-AATCTAGAGGGAACATAGCTGCTT-3). The amplified DNA fragment was digested with EcoRI and XbaI, cloned into pBluescript SK(+) (pSK) (Stratagene), and designated pSK-3N578. Where necessary, DNA with 5- or 3-protruding ends was treated with T4 DNA polymerase (Roche) or Klenow (Roche) to obtain blunt ends. pSK-3N578 was treated with KpnI-EcoRI-T4 DNA polymerase to remove a 48-nucleotide (nt) region between the KpnI site flanking the T7 promoter and EcoRI site flanking the 3UTR region, and it was self-ligated to create pSK-3N578. To construct the various deletion mutants, pSK-3N336, pSK-3n223 as shown in Fig. ?Fig.2A,2A, and various regions of AANAT 3UTR were amplified by using standard PCR techniques with pSK-3N578 KRCA-0008 as a template. The sequences of the amplified fragments are shown in Fig. ?Fig.1A.1A. All of the PCR fragments are flanked by an EcoRI site at one end and an XbaI site at the other end. Following EcoRI-XbaI digestion, fragments were introduced into the corresponding restriction sites of plasmid pSK. Sense strands were transcribed with T7 RNA polymerase (Roche). To generate chimeric reporter plasmids, as shown in Fig. ?Fig.2A,2A, various deletion mutants of AANAT KRCA-0008 3UTR were amplified by Sstr1 PCR and introduced into the EcoRI-XbaI site of pcNAT. For the construction of pcNAT-b3UTR, AANAT 3UTR was amplified from bovine AANAT 10-1B cDNA (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”NM_177509″,”term_id”:”31343555″NM_177509) kindly provided by C. M. Craft (8) and introduced into the EcoRI-XbaI site of plasmid pcNAT, as described above. To create pFlag-hnRNP R, a full-length coding sequence of rat hnRNP R was obtained by PCR of a rat fetal brain cDNA library (Clontech), with primers based on the published sequences of human and mouse hnRNP Rs (23, 44). The coding region of hnRNP R was amplified with two primers specific for hnRNP R cDNA (5 primer 5-AAAGCTTATGGCTAATCAGGTGAA-3 and 3 primer 5-AATCTAGACTACTTCCACTGTTGC-3; accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AF441128″,”term_id”:”17066598″AF441128). The amplified DNA fragment was digested with HindIII and XbaI, and cloned into pFlag-CMV2 (Sigma). Rat hnRNP R cDNA has been newly cloned and deposited in GenBank with accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AY184814″,”term_id”:”27903508″AY184814. For the construction of pchnRNP L, plasmid pTM/hnRNP L (21) was treated with EcoRI and XhoI. The DNA fragment was inserted into the EcoRI= ?0.83, = 3.8, = 1.6, and = 5.0, where may be converted to the period by the relationship = 12 ? , and represents zeitgeber time. For RQL, individual curves were fit for each = 0.75, = 0.17, = 1.2, and = 4.8; for hnRNP L, = 1.1, = 2.2, = 1.4, and.
- T-cell epitopes are peptides derived from antigens and identified by the T-cell receptor (TCR) when bound to MHC molecules displayed within the cell surface of APCs
- Cloning of gene fragments encoding diagnostic antigens
- Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope)
- Spleens were harvested in 1 (C) or 2 wpi (B, C) and cells were analyzed by movement cytometry in comparison to na?ve mice
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