Evolution of mammalian CD1: marsupial CD1 is not orthologous to the eutherian isoforms and is a pseudogene in the opossum Monodelphis domestica

Abstract

CD1 is a member of the major histocompatibility complex (MHC) class I family of proteins that present lipid antigens to T cells and natural killer (NK) T cells; it is found in both eutherian mammals and birds. In eutherians, duplication of the CD1 gene has resulted in multiple isoforms. A marsupial CD1 homologue was identified in a set of expressed sequence tags from the thymus of the bandicoot Isoodon macrourus. Southern blot and genomic sequence analyses revealed that CD1 is a single copy gene in both I. macrourus and a distantly related marsupial, the opossum Monodelphis domestica, which is currently the only marsupial species for which a whole genome sequence is available. We found that the opossum CD1 is located in a genomic region with a high degree of conserved synteny to the chromosomal regions containing human and mouse CD1. A phylogenetic analysis of mammalian CD1 revealed that marsupial CD1 is not orthologous to the eutherian CD1 isoforms, consistent with the latter having emerged by duplication after the separation of marsupials and eutherians 170-180 million years ago. The I. macrourus CD1 gene is actively transcribed and appears to encode a functional protein. In contrast, transcription of the M. domestica CD1 was not detected in any tissue and the predicted CD1 gene sequence contains a number of deletions that appear to render the locus a pseudogene.

Figure 2

Determination of the number of CD1 loci in I. macrourus by Southern blot analysis. Genomic DNA was digested with the indicated restriction enzymes and probed with fragments corresponding to exon 4 of the I. macrourus CD1.

Figure 1

Alignment of the deduced amino acid sequence from the bandicoot and opossum CD1 sequences with human CD1 isoforms. The boundaries between the domains are indicated. Dashes indicate identity and gaps are indicated by dots. Conserved cysteines in the α2 and α3 domains, potential N-linked glycosylation sites and the YXXZ motif in the cytoplasmic tail are shaded. Structural features of the protein are indicated below the sequences as follows: A, A′ pocket; C, C′ pocket; F, F′ pocket; T, tunnel.

Figure 3

Comparative gene maps of the region surrounding the CD1 loci on mouse chromosomes 1 and 3, human chromosome 1, and opossum chromosome 2. The mouse and human gene maps were adapted from the Ensembl annotation.

Figure 4

Phylogenetic analysis based on nucleotide alignments of exons 2, 3 and 4 of marsupial CD1 sequences with CD1 and MHC class I sequences from representative vertebrate species. Branch support is indicated as the percentage out of 1000 bootstrap replicates and is shown where support is greater than 50%.

Figure 5

Expression of the CD1 gene in bandicoot and opossum thymus and spleen tissues. RT-PCR was performed on mRNA from bandicoot thymus (T, lane 2) and spleen (S, lane 3) and opossum thymus (T, lane 6) and spleen (S, lane 7). Actin was used as a positive control for the RT-PCR in all samples. In addition, bandicoot (lane 4) and opossum (lane 8) genomic DNA were included using identical PCR conditions as for the cDNA in the RT-PCR as another positive control for the CD1 primers.