Mechanical strain regulation of the Chicken glypican-4 gene ...

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Rapid amplification of cDNA ends (RACE) Rapid amplification of cDNA ends (RACE), to complete the 3’ region of the chicken GPC- 4, was performed with the SMART(TM) RACE cDNA amplification kit (Clontech) according to the manufacturer’s manual. In brief, the first strand cDNA was synthesized from chicken ESG mRNA by means of the MMLV reverse transcriptase with the aid of SMART II oligonucleotide and 3'-RACE cDNA synthesis primers, at 42 0 C for 60 min. PCR was then carried out with a 5’ gene-specific primer – 5’- GCTGGAGGGGCCTTTTAACATTGAGTC-3’ – synthesized according to the cloned 969-bp fragment and the mix of two universal adapter primers provided with the kit. Cycling conditions were: five cycles of 94 0 C for 15 s and 72 0 C for 3 min; five cycles of 94 0 C for 15 s, 70 0 C for 30 s and 72 0 C for 3 min; and 30 cycles of 94 0 C for 15 s, 68 0 C for 30 s and 72 0 C for 3 min. A nested PCR reaction was carried out under similar conditions, with the kit primer (Nested Universal Primer) and a specific nested GPC-4 5’ primer (5'- GAACAGCATGCAAGTGTCTCA-3'). Figure 1 describes the strategy in the assembly of the chicken GPC-4. The obtained PCR product was separated on 1% agarose and purified, cloned into the pGEM-T easy vector and sequenced from both directions with T7, SP6 and specific primers. The cDNA and deduced amino acid sequences were compared with the DNA and protein databases at the National Center for Biotechnology Information. Preparation of riboprobe and in situ hybridization. A fragment of chicken GPC-4 cDNA (646bp) was synthesized by reverse transcription (RT)-PCR from the chicken ESG (see under "RT-PCR"). This fragment shared no homology with any other known chicken cDNA and was subcloned into the pGEM-T Easy cloning vector to produce a template for riboprobe synthesis. The sense and antisense riboprobes were synthesized by in vitro transcription with T7 and SP6 RNA polymerase, respectively, in the presence of linearized plasmid DNA and DIG RNA labeling mix (Boehringer Mannheim, Ottweiler, Germany). Serial 5-µm sections of the ESG or magnum were hybridized with a digoxigenin-labeled chicken GPC-4 probe as described previously (25). No hybridization was observed with the sense riboprobe, which was used as a negative control. Hybridization with avian �-actin probe was performed in order to rule out the possibility that changes in GPC-4 expression were due to variation in tissue processing and/or RNA preservation. 8
RESULTS Identification of the chicken GPC-4 gene in chicken ESG RNA fingerprinting was used to identify genes involved in the eggshell calcification. Differential screening was performed between cDNA derived from ESG biopsies from two different states of the daily egg cycle: 1- no egg resides in the ESG, no calcification and no mechanical strain; 2- the egg resides in the ESG, at the peaks of eggshell calcification and of mechanical strain. We isolated and sequenced 14 clones that appeared to be up-regulated at the time of eggshell calcification. One of these clones – a 382-bp cDNA sequence – showed 80% similarity to the mouse K-glypican (GenBank, accession number X83577). This sequence was extended to both the 3’ and 5’ directions, resulting in a complete 3’ region of the gene, and a 5’ region with a missing part estimated to comprise the first 71 bp of the open reading frame, according to the mammalian homologues (Fig. 2). The amplified PCR fragments were subcloned, and 11 randomly selected subclones were analysed by sequencing, the obtained sequence was additionally verified by RT-PCR. Analysis of the obtained sequence revealed 77 and 78% similarity to the mouse and human GPC-4 genes, respectively, with two stretches showing 92% homology. Multiple alignment of the predicted protein product of the GPC- 4 gene showed 74 and 75% identity to the amino acid sequences of mouse and human GPC-4, respectively. Of most importance is the conservancy within the sequence of motifs that is characteristic of all members of the glypican family; these motifs includ the glypican signature region and the GPI binding site (Fig.2). Out of the 13 cystein residues known to be conserved among all mammalian glypicans, 12 were found to be conserved also in the avian GPC-4. One cystein residue at position 224 corresponding to the mouse sequence was replaced by glycine as confirmed by RT-PCR analysis of cDNA obtained from different avian tissues. Temporal and spatial expression of the chicken GPC-4 gene during eggshell formation Temporal and spatial localization of the avian GPC-4 in the ESG during the eggshell formation cycle was performed by in situ hybridization (Fig. 3). Very low levels of expression of the GPC-4 gene was observed prior to the entry of the egg into the ESG, while the egg was still located in the magnum or isthmus (Fig. 3A), or 1 h after the egg 9
Page 1 and 2: AJP-Regu Articles in PresS. Publish
Page 3 and 4: Abbreviations ESG - avian eggshell
Page 5 and 6: about 10% of the total body calcium
Page 7: for 1 min, 60°C for 1 min and 68°
Page 11 and 12: The expression of the GPC-4 gene in
Page 13 and 14: to become part of the assembly of p
Page 15 and 16: 9. Clark EA, King WG, Brugge JS, Sy
Page 17 and 18: 30. Numaguchi K, Eguchi S, Yamakawa
Page 19 and 20: LEGENDS TO FIGURES Fig. 1. The asse
Page 21 and 22: 1. 2. 3. 4. 81 bp PCR 482 bp PCR 96
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