Evolution of natural killer cell receptors: coexistence of functional Ly49 and KIR genes in baboons.

Natural killer (NK) cells represent an important first line of defense against viruses and malignancy [1]. NK cells express a variety of inhibitory and activating receptors that interact with classical major histocompatibility complex (MHC) class I molecules on potential target cells and determine the NK cell response [2-4]. Mouse NK receptors are encoded by the C-type lectin multigene family Ly49. However, in humans, a completely different family of receptors, the immunoglobulin-like killer inhibitory receptors (KIRs), performs the same function [2-4]. One Ly49-like gene, Ly49L, exists in humans but is incorrectly spliced and assumed to be nonfunctional [5, 6]. Mouse KIR-like genes have not been found, and evidence suggests that the primate KIRs amplified after rodents and primates diverged [7, 8]. Thus, two structurally dissimilar families, Ly49 and KIR, have evolved to play similar roles in mouse and human NK cells. This apparent example of functional convergent evolution raises several questions. It is unknown, for example, when the Ly49L gene became nonfunctional and if this event affected the functional evolution of the KIRs. The distribution of these gene families in different mammals is unstudied, and it is not known if any species uses both types of receptors. Here, we demonstrate that the Ly49L gene shows evidence of conservation in other mammals and that the human gene likely became nonfunctional 6-10 million years ago. Furthermore, we show that baboon lymphocytes express both full-length Ly49L transcripts and multiple KIR genes.

Identification and characterization of variant alleles of human acetyltransferase NAT1 with defective function using p-aminosalicylate as an in-vivo and in-vitro probe.

Although several variant alleles at the human NAT1 gene locus have been reported, their relationship to phenotypic variations in NAT1 function remains unclear. We have used in-vivo and invitro phenotyping tests, along with PCR-based cloning and heterologous expression, to investigate the extent of variation in NAT1 function and to characterize novel allelic variants at the NAT1 gene locus. The NAT1-selective substrate p-aminosalicylic acid (PAS) was used as a probe for NAT1 function. In-vivo PAS acetylation rates were estimated by determining the ratio of PAS to N-acetylated PAS (AcPAS) in urine and plasma following the oral ingestion of Nemasol Sodium. Excluding outliers, a 65-fold variation in the urinary AcPAS:PAS ratio was observed (n = 144), while a 5.6-fold variation in the plasma AcPAS:PAS ratio was seen in a subset (n = 19) of this sample. Urinary and plasma ratios correlated moderately (r = 0.74, p < 0.0005). One individual (case 244) had a marked impairment of PAS N-acetylation, with 10-fold lower urinary and plasma AcPAS:PAS ratios compared with other subjects. Biochemical investigations in whole blood lysates from case 244 suggested a NAT1 kinetic defect, with a 20-fold increased apparent K(m) for PAS and a 90-fold decreased Vmax for AcPAS formation. We subcloned, sequenced and expressed the protein-coding regions of the NAT1 alleles from case 244 and from seven other selected probands. Sequence analysis revealed the presence of two new variant alleles, designated as NAT1 x 14 and NAT1 x 15, in case 244, as well as one variant, NAT1 x 11, which has been observed in previous investigations. NAT1 x 14 contained a missense mutation (G560-->A) that is predicted to change a single amino acid (Arg187-->Gln), as well as two 3' non-coding region mutations (T1088-->A and C1095-->A) that have previously been observed in the NAT1 x 10 allelic variant. NAT1 x 15 had a single nonsense mutation (C559-->T; Arg187-->stop) and, thus, encodes a truncated protein. The activity of recombinant NAT1 14 mirrored the defective enzyme function in whole blood lysates from case 244, while NAT1 15 was completely inactive. Expressed NAT1 11, on the other hand, had identical activity to the wild type NAT1 4 allele, suggesting that the coding region mutations in this variant are functionally silent. The frequencies of NAT1 x 11, NAT1 x 14 and NAT1 x 15 were 0.021, 0.028 and 0.014 (n = 288 alleles), respectively, suggesting that they are relatively rare in our predominantly Caucasian sample.

Comparative analysis of the promoter regions and transcriptional start sites of mouse Ly49 genes.

Despite numerous studies on the function of Ly49 natural killer cell receptors in the mouse, relatively little is known about how these genes are regulated at the transcriptional level. In the present study, we sequenced and compared 800 bp of the promoter region of nine Ly49 genes from C57B1/6 mice. This comparison showed that there is a high degree of sequence identity between the genes, and also revealed a region which is conserved between the mouse genes and the human Ly49L gene, indicating a potential core promoter region. This analysis also found that Ly49B and H differ from the other genes in having long interspersed repetitive sequence in their promoter region which suggests a gene conversion or rearrangement involving these two genes. In addition, we performed 5' rapid amplification of cDNA ends on four Ly49 genes to localize transcriptional start sites. These experiments showed that the transcriptional initiation sites are heterogeneous for all of the genes examined, and that a large majority of Ly49G transcripts originate from the second exon as well as its first intron. Although potential TATA boxes have been previously identified for some of the genes, we did not find evidence that a majority of transcripts initiate at the expected distance downstream of these boxes. Our data suggest that differences in the location of transcriptional start sites contribute to the observed complexity in receptor repertoire patterns.

Functional analysis of 5' and 3' regions of the closely related Ly49c and j genes.

The Ly49 multigene family consists of at least 14 closely related genes located in the natural killer (NK) gene complex on mouse Chromosome 6. Reverse transcriptase (RT)-PCR on single NK cells has shown that Ly49c is expressed on approximately 50% of NK cells, whereas the closely related Ly49j gene is expressed on 5-8% of NK cells. In this study, we examined three regions to determine whether they contain cis-acting elements involved in regulating the expression of these two closely related Ly49 genes within NK cells. Luciferase reporter assays in EL-4 cells suggested that the 5' regions of Ly49c and j contain promoter elements and repressor sequences. In addition, luciferase assays suggest that Ly49j also contains an active promoter in the first intron, although the transcripts produced from this promoter appear to be severely truncated. Finally, comparisons of the 3' noncoding regions of Ly49c and j revealed that the sequence of Ly49j diverges completely from Ly49c 130 bp downstream of the termination codon. The polyadenylation signal for Ly49j is located downstream of the Ly49c poly(A) site, which results in a much longer 3' untranslated region (UTR). When the Ly49j 3'UTR was used to provide the polyadenylation signal for the green fluorescent protein (GFP) reporter gene, GFP expression was reduced twofold. These results suggest that both internal promoters, repressors, and 3' regions play a role in regulating Ly49 gene expression.

Localization of five new Ly49 genes, including three closely related to Ly49c.

Nine genes belonging to the mouse Ly49 multigene family of natural killer cell receptors have been identified to date. Two of these genes, Ly49h and i, are very closely related to the well characterized Ly49c gene in the carbohydrate recognition domain. Here we show by Southern blotting that at least two additional new sequences exist in C57BL/6 mice that are also closely related to Ly49c in the carbohydrate recognition domain. Furthermore, in contrast to Ly49a, extensive variation in the arrangement and number of Ly49c-related genes in different mouse strains was observed. To characterize and localize the new Ly49c-related genes in C57BL/6 mice, we isolated and mapped genomic P1 clones hybridizing to an Ly49C exon 7 probe. Locations and the relative order of all Ly49 genes found within the clones was determined. We also used polymerase chain reaction to sequence exons 2, 4, and 7 from all genes. In this manner, we identified five new potential Ly49 genes which have been tentatively termed Ly49j-n. Ly49j, k, and n belong to the Ly49c-related subfamily, whereas Ly49l and Ly49m are most similar to Ly49d and g, respectively. Interestingly, the members of the Ly49c-related subfamily are not clustered as a unit but are interspersed among other Ly49 genes. These results illustrate the complex nature of the Ly49 gene family and should aid in the understanding of functions, such as the mediation of hybrid resistance, in which Ly49c-related genes play a role.

Expression analysis of new Ly49 genes: most transcripts of Ly49j lack the transmembrane domain.

Five new Ly49 genes, named Ly49j-n, have recently been identified in C57BL/6 mice. This study examined the expression of three of these new genes, Ly49j, k, and n. To determine whether the Ly49j, k, and n genes were transcribed, gene-specific primers were used to amplify cDNA clones for each gene from C57BL/6 interleukin-2-activated natural killer (NK) cell cDNA. A full-length cDNA for Ly49j was detected which encodes a 267 amino acid protein and shares approximately 96% nucleotide identity with Ly49c and i. COS cells transfected with the Ly49j cDNA were shown to react with the monoclonal antibody 8H7, suggesting that the gene likely encodes a functional protein. Many different sized Ly49k and n transcripts were observed, although it is likely that they do not encode functional proteins due to missing exons or severe truncations in the open reading frames. Interestingly, the most abundant Ly49j transcript detected was shown to lack exon 3, which encodes the transmembrane domain. Similar studies performed on the same source of NK cell cDNA using Ly49c- and i-specific primers revealed the presence of transmembrane-less Ly49i transcripts, although at a much lower frequency than observed for Ly49j. We also detected Ly49g and h transcripts lacking the transmembrane domain. Despite the absence of the transmembrane region, the resulting Ly49 transcripts maintain their open reading frames, and therefore could potentially encode cytoplasmic proteins with a role in NK cell function.

Ly49 and CD94/NKG2: developmentally regulated expression and evolution.

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.