XB-ART-36068Gene August 15, 2007; 398 (1-2): 94-102.
Gene expression and tissue distribution of cytoglobin and myoglobin in the Amphibia and Reptilia: possible compensation of myoglobin with cytoglobin in skeletal muscle cells of anurans that lack the myoglobin gene.
Cytoglobin (Cygb), a recently discovered vertebrate cytoplasmic heme-binding globin, is considered to be in a clade with vertebrate myoglobin (Mb), which is exclusively distributed in the cytoplasm of cardiac and skeletal muscles as an oxygen storage protein. GenBank databases (NCBI and JGI) and gene synteny analyses showed the absence of the Mb gene (mb) in two anuran amphibians, Xenopus laevis and X. tropicalis. Here we conducted comparative studies on the gene expression and tissue distribution of Cygb and Mb in anuran and reptilian tissues. Cygb and Mb genes were cloned from a reptile, iguana (Iguana iguana). Two types of cygb (cygb-1 and -2) were cloned, with lengths of 1066 and 1034 bp, and 196 and 193 amino acid residues, respectively. Their nucleotide and amino acid sequence identities were 90 and 87%, respectively. The Mb gene covered 1416 bp with an open reading frame of 465 bp, giving rise to a 154 amino acid protein. The distal ligand-binding histidine at E7, the proximal heme-binding histidine at F8, and the phenylalanine residue at CD1 were conserved in Mb and Cygb. The nucleotide and amino acid sequence identity of I. iguana cygb-1 and cygb-2 against X. laevis cygb were approximately 67% and 65%, respectively. RT-PCR demonstrated that X. laevis cygb was uniquely expressed in the heart and skeletal muscles, and faintly in the liver and spleen, which was quite contrasted with Iguana and the other vertebrates, where mb is exclusively expressed in the heart and skeletal muscles. Immunohistochemical analyses showed the distribution of Cygb in the cytoplasm of skeletal muscle cells. Interestingly, Cygb in the heart was localized in the nuclei. Considering the absence of mb in the Anura, we hypothesize that Cygb in muscle cells of anurans compensates for the lack of Mb for the storage and intracellular transportation of oxygen.
PubMed ID: 17560742
Article link: Gene
Species referenced: Xenopus laevis
Genes referenced: aanat commd5 crebbp cygb eef1a1 esyt1 evpl foxred2 gabarapl2 hmox1 ift27 jmjd6 mcm5 myh9 prpsap1 rasd2 rbfox2 rhbdf2 rpl8 sphk1 tlcd4 tom1 zic1 znf250
Antibodies: Cygb Ab1
Article Images: [+] show captions
|Fig. 1. Comparison of iguana Mb, Cygb and sperm whale Mb amino acid sequences. The secondary structure of the sperm whale Mb is superimposed in the upper row, with a-helices designated A through H. The strictly conserved amino acid sequences are shaded. Dashed lines indicate insertions in the best alignments. The amino acid sequence of sperm whale Mb is from GenBank (accession no. BAF03579).|
|Fig. 2. Neighbor-joining trees of selected animals based on amino acid sequences. A. Cygb phylogenetic tree. B. Mb phylogenetic tree. Trees were reconstructed in MEGA3.1 using the JTT model. The statistical confidence of each branch was estimated by bootstrapping with 1000 replications. Numbers on branches indicate bootstrap support. Bootstrap probabilities higher than 50% are shown. The scale bar is equal to 0.1 JTT distance.|
|Fig. 3. Comparison of syntenic regions of cygb and mb among vertebrates. A. Genes flanking cygb. B. Genes flanking mb. Conserved genes have the same number. chr, chromosome; un, unkown. Each numbered box represents one gene. For example, box 1 and box + 2 represent prpsap1 and mbc2, respectively. Names or GenBank accession numbers in A are: box 1, prpsap1; 2, sphk1; 3, ube20; 4, aanat; 5, rhbdf2; 6, stggacnac2; 7, stggacnac1; 8, mxr7; 9, ptdsr; 10, LOC124512; + 1, LOC417357; + 2, mbc2; 1′, ZGC:153218; 2′, LOC561406; 3′, LOC554770; 4′, LOC561211; 5′, LOC561133; 6′, tmem56; 7′, ZGC:101015; 8′, LOC560643; 9′, LOC560405; 10′, LOC560332; 1″, WU:fk32g07; 2″, junbl; 3″, WZ12713; 4″, ZL5479; 5″, evpl; 6″, FB77d07; 7″, ZSNP208; 8″, ZC12P12; 9″, FB95e10; 10″, WZ11449; 1″′, LOC571619; 2″′, LOC571572; 3″′, ZGC:152745; 4″′, LOC571541; 5″′, LOC571536; 6″′, LOC571469; 7″′, LOC571446; 8″′, LOC571436; 9″′, LOC571429; 10″′, LOC571417. Names or GenBank accession numbers in B are: box 1, tom1; 2, hmox1; 3, mcm5; 4, LOC730090; 5, rasd2; 6, LOC284912; 7, apol6; 8, mrps16p3; 9, apol5; 10, rbm9; 1′, zfp7; 2′, commd5; 3′, rpl8; 4′, zfp647; 5′, 1110038F14Rik; 8′, 9130022K13Rik; 9′, BC020489; 10′, BC085284; 7″, myh9; 8″, foxred2; 9″, LOC427909; 10″, rabl4; 1″′, LOC566653; 2″′, gabarapl2; 3″′, LOC573573; 4″′, LOC566776; 5″′, pane1; 6″′, LOC566914; 7″′, LOC566984; 8″′, LOC567048; 9″′, cbp-a; 10″′, LOC567164.|
|Fig. 4. Expression of cygb and mb in tissues. A. cygb and mb expression patterns in iguana tissues. B. cygb expression patterns in the corresponding tissues of X. laevis. Iguana gapdh and Xenopus ef-1 alpha were used as internal controls in A and B, respectively.|
|Fig. 5. Distribution of Cygb in X. laevis adult tissues. Serial sections were prepared from liver (A through D), skeletal muscle (E through H), heart (I through L), brain (M through P), and retina (Q through T), stained with H&E (A, E, I, M, Q), Hoechst 33342 (B, F, J, N, R), anti-Cygb antibodies (C, G, K, O, S), or normal rabbit IgG (D, H, L, P, T). In Hoechst 33342 stains, nuclei are blue and immunosignals in anti-Cygb antibody stains are green. Cygb showed clear localization in the cytoplasm in skeletal muscle (G), and nuclear localization in the heart (K) and the brain (O). In the retina (S), Cygb was abundantly distributed in photoreceptor inner segments, indicated by the white arrow. IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars indicate 50 μm.|
|Fig. 6. Distribution of Cygb in X. laevis larval tissues. Serial sections were prepared from liver (A through D), skeletal muscle (E through H), heart (I through L), brain (M through P), and retina (Q through T), and stained with H&E (A, E, I, M, Q), Hoechst 33342 (B, F, J, N, R), anti-Cygb antibodies (C, G, K, O, S) or normal rabbit IgG (D, H, L, P, T). Cygb showed clear localization in the cytoplasm in skeletal muscle (G), and nuclear localization in heart (K) and brain (O). In the retina (S), Cygb was abundantly distributed in the outer nuclear layer (ONL), inner nuclear layer (INL) and ganglion cell layer (GCL), indicated by white arrows. Scale bars indicate 50 μm.|