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. 2004 Mar 2;101(9):3077-82.
doi: 10.1073/pnas.0308532100. Epub 2004 Feb 18.

The SPANX Gene Family of Cancer/Testis-Specific Antigens: Rapid Evolution and Amplification in African Great Apes and Hominids

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The SPANX Gene Family of Cancer/Testis-Specific Antigens: Rapid Evolution and Amplification in African Great Apes and Hominids

Natalay Kouprina et al. Proc Natl Acad Sci U S A. .
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Abstract

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes comprise a gene family with five known members (SPANX-A1, -A2, -B, -C, and -D), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human SPANX. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes, SPANX-N, which are present in all primates as well as in mouse and rat. In humans, four SPANX-N genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to SPANX-A/D, human SPANX-N genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of SPANX is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that SPANX-N is the ancestral form, from which the SPANX-A/D subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the SPANX genes evolved much faster than the intron and the 5' untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the SPANX family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.

Figures

Fig. 1.
Fig. 1.
PCR amplification of members of the SPANX family from chimpanzee (African great apes), orangutan (great apes), rhesus macaque (Old World monkey), and tamarin (New World monkey). Oligonucleotides were designed within the promoter and 3′ uncoding regions. The double upper bands for rhesus macaque and tamarin are presumably due to polymorphism in paralogs.
Fig. 2.
Fig. 2.
Location of the SPANX family genes on human chromosome X. Five members of the SPANX-A/D subfamily, SPANX-A1, -A2, -B, -C, and -D, are clustered within an ≈800-kb region at Xq27.2. Four members of the SPANX-N subfamily, SPANX-N1 (positions 142995930-143005820), -N2 (positions 141495326-141490625), -N3 (positions 141297635-141291834), and -N4 (positions 140806882-140816198), are located ≈2 Mb apart from SPANX-A/D (University of California, Santa Cruz, July 2003, http://genome.ucsc.edu). SPANX-N5 (positions 51791606-51793934) is located on the short arm of chromosome at Xp11. SPANX genes reside within ≈20-kb blocks, which are duplicated retaining a high homology across the chromosome.
Fig. 3.
Fig. 3.
Multiple alignment of the SPANX protein sequences. Consensus includes amino acid residues that are conserved in both rodent sequences and in the majority of sequences in each of the two primate subfamilies; consensus_prim consists of residues represented in the majority of the sequences in each primate family but not in rodents. h, hydrophobic residues; S, small residues; +, positively charged residues. Residues that conform to the consensus are shaded. In the predicted secondary structure: E, extended conformation (β-strand), and H, α-helix; the rest of the protein is predicted to consist of loops and unstructured regions. Residues that comprise predicted nuclear localization signal are show in bold type. The upper line corresponds to the N-terminal duplication of rat SPANX.
Fig. 4.
Fig. 4.
RT-PCR analysis of the SPANX-N gene subfamily. (a) cDNA was prepared from a panel of human tissue mRNAs. Oligonucleotides were designed within exons 1 and 2 to amplify putative transcripts. A 264-bp band of the expected size was observed only in testis and the melanoma cell line LOX IMVI. Two members of the human SPANX-N subfamily, SPANX-N2 and -N3, were revealed by cloning and sequencing of PCR products. (b) cDNA was prepared from a panel of mouse tissue mRNAs. Oligonucleotides were designed within exons 1 and 2 to amplify a putative transcript. A 264-bp band of the expected size was observed only in testis. Control PCRs were carried out with the same samples by using actin-specific primers.
Fig. 5.
Fig. 5.
A hypothetical evolutionary scenario for the SPANX gene family. The expansion of SPANX genes is superimposed on the tree of primate evolution.
Fig. 6.
Fig. 6.
Isolation of the syntenic genomic fragments containing the LDOC1 gene from chimpanzee, bonobo, and gorilla by TAR cloning. (a) TAR vector containing a 3′ hook specific to a human unique sequence (ORF-X) that is ≈16 kb upstream of SPANX-C and a 5′ hook specific to the human LDOC1 gene. Recombination between genomic DNA and the vector leads to cloning the syntenic regions of the species. CEN corresponds to the yeast chromosome VI centromere, HIS3 is a yeast selectable marker, and DG1 and DG2 are diagnostic primers. (b) Comparison of sequences revealed that TAR clones from chimpanzee, bonobo, and gorilla do not contain the SPANX-C gene along with the duplication where it was inserted. The clone from gorilla contains a 3.4-kb insertion that corresponds to a hypothetical gene consisting of eight exons on human chromosome 3.

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