MHC class I-like genes in cattle, MHCLA, with similarity to genes encoding NK cell stimulatory ligands

Abstract

A comparative genomics approach for mining databases of expressed sequence tags (ESTs) was used to identify two members of a novel MHC class I gene family in cattle. These paralogous genes, named MHC class I-like gene family A1 ( MHCLA1) and MHCLA2, were shown by phylogenetic analysis to be related to human and mouse genes encoding NK cell stimulatory ligands, ULBP, RAET, H60 and Raet-1. Radiation hybrid mapping placed cattle MHCLA1 on BTA9, which, on the basis of existing comparative mapping data, identified the ULBP, RAET1, H60 and Raet1 genes as homologues of the cattle MHCLA genes. However, the human and mouse orthologues of MHCLA1 and MHCLA2 could not be defined due to extensive sequence divergence from all known members of the ULBP1/ RAET1/H60/Raet1 gene family. The cattle MHCLA1 molecule is predicted to be missing an alpha(3) domain, similar to the human and mouse homologues. Like the human ULBP genes, MHCLA1 was found to be transcribed constitutively in a variety of fetal and adult tissues by RT-PCR. The patterns of hybridization obtained by Southern blotting using MHCLA1 as a probe and DNA from 14 species representing five mammalian orders suggests that the MHCLA genes evolved rapidly in the Cetartiodactyla. Previous findings demonstrating that ULBPs serve as ligands for the NK cell NKG2D stimulatory receptor, and that this interaction can be blocked by a human cytomegalovirus glycoprotein that binds to ULBPs, suggests that the extensive divergence found among the cattle, human and mouse MHCLA homologues is due to selection exerted by viral pathogens.

Discovery of eight novel divergent homologs expressed in cattle placenta

“…The advent of high-throughput DNA sequencing and associated genome databases permit a more global search for those genes that diversify through copy number and primary sequence. In the absence of complete genome sequence, EST collections offer a means to search for divergent homologs as well as transcripts and genes that are unique to a given species (38). In the present study, cattle placenta was chosen as a source of ESTs, because the placenta is among the most phenotypically divergent mammalian tissues and has been shown to express novel paralogs such as the PAG and TKDP genes (42,71).…”

“…1 and M. R. Band, R. E. Everts, Z. L. Liu, D. E. Morin, J. U. Peled, S. L. Rodriguez-Zas, and H. A. Lewin, unpublished data; NCBI GEO: GSE3029). Total RNA was isolated from a Hereford cow term placentome using an established protocol (38). Twenty micrograms of total RNA and titrated RNA spiking controls (5) were used as templates for the synthesis of aminoallyl-labeled cDNA (aa-cDNA) using an indirect labeling protocol (20).…”

“…Bacterial stocks containing cDNA clones with divergent 5Ј-ESTs were cultured and rearrayed using a Biorobot 8000 (Qiagen, Valencia, CA), and template plasmid DNA was isolated according to the manufacturer's protocol. Cycle sequencing and electrophoresis were performed using an established protocol (38), substituting an oligo(d)T 18V sequencing primer. PHRED (15) was used to remove low-quality sequence (PHRED score Ͻ20).…”

“…In the present study, eight new divergent homologs expressed in cattle placenta were identified using a subtractive bioinformatic approach previously developed for the identification of the cattle major histocompatibility complex class I-like gene family A genes (38). The combined approaches of sequence alignment and comparative mapping were used to establish homologous relationships.…”

Molecular cloning and characterization of a porcine UL16 binding protein (ULBP)-like cDNA

“…The DNA sequences encoding the ␣1 and ␣2 domains of PULBP, HULBP1-4, two cattle ULBP-like gene sequences (MHCLA1 and MHCLA2, Larson et al, 2003), and the natural ligand for the murine NKG2D receptor, Raet1, were used to examine the phylogenetic relationship of mammalian ULBPlike genes. A neighbor-joining tree is shown in Fig.…”