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Gene
2002 May 01;2891-2:97-107. doi: 10.1016/s0378-1119(02)00480-8.
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Cloning and characterization of the cDNA and gene encoding Xenopus laevis osteocalcin.
Viegas CS
,
Pinto JP
,
Conceição N
,
Simes DC
,
Cancela ML
.
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A full length Xenopus laevis osteocalcin (bone Gla protein, BGP) has been cloned by a combination of reverse transcription and amplification by the polymerase chain reaction, sequenced, and found to encode a polypeptide with 101 amino acid residues, including a 52-residue prepro-region and a 49-residue mature protein. The N-terminal region of the mature Xenopus BGP (xBGP), as deduced from the cDNA, is in full agreement with the sequence of the BGP previously purified from Xenopus long bones. This cDNA was used to clone the xBGP gene and its promoter region. The xBGP gene spans 3727 bp from the site of transcription initiation corresponding to the 5'end of the cDNA to the site of insertion of the poly-A(+) tail, and it contains four exons. This structure is similar to the one obtained for both fish and mammalian BGP genes and indicates that the molecular organization of this gene has been conserved throughout vertebrate evolution. Also similar to other known vertebrate systems, xBGP gene expression is restricted to bone, with no signal for xBGP messenger RNA (mRNA) detected in all other tissues analyzed. The availability of the xBGP promoter will permit to analyze its regulation in a widely used non-mammalian model system for vertebrate development, taking advantage of the availability of sequences for various Xenopus steroid hormone receptors and transcription factors known to affect BGP expression in the mammalian system.
Fig. 1. Complete nucleotide sequence of the cDNA encoding Xenopus laevis BGP. Nucleotide 1 was assigned following determination of transcription start site and the 50 end obtained by 50 RACE experiments is signalled. Numerical positions in the nucleotide sequence are noted in the left side. Amino acid residues are numbered according to residue 1 of the mature protein and are shown above the respective sequence. The stop codon is indicated by asterisks and the polyadenylation signal is boxed. The codons for the Gla residues are underlined twice. Localization of XBGP3F, 4R, 7R, 8F, 8R and 9R oligonucleotides are denoted by horizontal arrows. The Pro/Mature cleavage site is signaled by a vertical arrow.
Fig. 2. Amino acid sequence alignment of mammalian, avian, fish and Xenopus BGPs. Sequences were aligned and conserved residues highlighted in grey. A box encloses the most conserved region of the protein and dashes indicate gaps in the sequence, introduced to increase homology. A square encloses amino acids corresponding to sites of intron insertion in the corresponding genes. Residues are numbered according to residue 1 of mature xBGP protein. A single asterisk signals the pre-pro and double asterisks mark the pro-mature border. Vertical arrows signal the g-carboxylated glutamic acid residues. Accession numbers for these sequences are: AF055576 for Xenopus; U10578 for chicken; X04141 for rat; X53699 for bovine; X53698 for human; AF048703 for Sparus; A42794 for bluegill; GEWF for swordfish; and S02208 for emu.
Fig. 3. Comparison of the migration profile of Xenopus BGP and Sparus aurata BGP on a 12% SDS-PAGE gel (NuPage, Invitrogen). Each lane was loaded with 10 mg of protein and the gel was stained with Coomassie Brilliant Blue as described in Section 2. 1 – S. aurata BGP; 2 – X. laevis BGP; and MWM – low range molecular weight markers from BioRad.
Fig. 4. Identification of transcription start site of the xBGP gene as determined by primer-extension. Total RNA isolated from Xenopus bones was annealed to a xBGP reverse primer, reverse-transcribed and subjected to RNase digestion, electrophoretic fractionation and autoradiography. The extension product is indicated in lane 1 and its size is shown on the right margin. A sequencing ladder (lanes G, A, T and C) was fractionated in the same gel for assignment of transcription start sites at single nucleotide resolution.
Fig. 5. Sequence of Xenopus laevis BGP gene 50-flanking region. Part of the sequence of the xBGP gene (11 to162) and its 50-flanking DNA (2938 to 21) are shown. The complete xBGP gene sequence can be found in the GenBank with accession number AY043179. The site of transcription initiation, designated as 11, is shown in bold and indicated with a vertical arrow. Nucleotides are numbered in the left margin. The beginning of the predicted amino acid sequence is shown above the displayed coding sequence starting with the first ATG. Localization of the XBGP10F primer is shown by an horizontal arrow under the sequence. Putative TATA and CAAT boxes are boxed, as well as two inverted CAAT boxes (ATTG). Putative SMAD-responsive (SBE), vitamin D-responsive (VDRE-LIKE), metal-responsive (METAL) AP2-responsive (AP-2) and CBFA1-responsive (1) elements are underlined.
Fig. 6. Analysis of the xBGP gene locus. (A) Genomic Southern analysis. Followingrestriction enzyme digestion with EcoRI and BglII, genomic DNA samples were electrophoresed on a 0.8% agarose gel, transferred to a nylon membrane (Nytran 1 ) and hybridized with xBGP cDNA, as described in Section 2. Size of the DNA marker (l/HindIII/EcoRI) is shown on the right margin. Positive hybridization signals obtained are marked by arrowheads. (B) Map of the xBGP gene and 50-flanking region. Localization of sites for restriction endonucleases used in the genomic Southern analysis are shown. The position of exons (boxes) and introns (lines between exons) is shown, sizes (in bp) of exonic sequences [coding (black boxes); non-coding (white boxes)] are indicated underneath the corresponding exons. Sizes of the introns are also shown. The 30 end of the gene corresponds to the site of insertion of the poly-A tail, 17 bp downstream from the polyadenylation site located in exon 4 (as deduced from the cDNA sequence). Restriction sites for the enzymes used in the genomic Southern analysis (EcoRI and BglII) and sizes of the corresponding DNA restriction fragments generated are shown.
Fig. 7. Analysis of tissue distribution of xBGP mRNA by Northern blot analysis. Total RNA was extracted from several tissues (gonad, muscle, kidney, heart, bone and liver) of Xenopus, size fractionated by denaturing agarose gel electrophoresis and transferred to a nylon membrane. RNA integrity was assessed by ethidium bromide staining of 28 and 18S ribosomal RNAs (top panel). Expression of xBGP was detected following hybridization with a 32P-labelled xBGP cDNA (bottom panel).
Fig. 8. Detection of xBGP mRNA by RT-PCR. Total RNA was extracted from whole froglet (65) and tissues (liver, heart, bone, kidney and muscle) of Xenopus and used to amplify xBGP mRNA by RT-PCR, using two specific primers (XBGP8F and XBGP4R), as described in Section 2. PCR products were size fractionated by agarose gel electrophoresis, transferred to a nylon membrane and hybridized with a 32P-labelled xBGP cDNA (bottom panel). To check for RNA integrity, the same RT reactions were used to amplify xODC mRNA using two specific primers based on the published sequence (top panel).
