Strain-dependent expression of four structurally related rat Ly49 receptors; correlation with NK gene complex haplotype and NK alloreactivity

Natural killer (NK) cells from certain rat strains promptly kill MHC allogeneic lymphocytes in vivo, a rejection phenomenon termed allogeneic lymphocyte cytotoxicity (ALC). ALC can be reproduced in vitro, and is preferentially mediated by a subset of NK cells expressing the Ly49 stimulatory receptor 3 (Ly49s3) in PVG strain rats. Functional studies have suggested that Ly49s3 triggers NK cell alloreactivity, but its importance relative to other Ly49 receptors has not been investigated. In this study, we have characterized three rat Ly49 receptors with close sequence similarity to Ly49s3 in the extracellular region, i.e., Ly49s4, Ly49 inhibitory receptor 3 (Ly49i3), and Ly49i4. Similar to Ly49s3, Ly49s4 mediated cellular activation while Ly49i4 inhibited NK cytolytic function. Ly49s4, -i3, and -i4 all reacted with a previously described anti-Ly49s3 monoclonal antibody (mAb) (DAR13), but not a novel mAb (STOK6), which was shown to be specific for Ly49s3. Expression of these Ly49 receptors varied markedly between inbred strains, in patterns related to their NK gene complex (NKC) haplotype, and ability to mediate ALC. Three major groups of NKC haplotypes could be discerned by restriction fragment length polymorphism analysis. Ly49s3 was present in strains from one of the groups, which corresponded with the “high” ALC responders. Ly49s3 surface expression was also markedly reduced in the presence of its putative MHC class Ib ligand(s) in MHC congenic strains. These data support the notion that Ly49s3 functions as a triggering MHC receptor both in vitro and in vivo. MHC ligands for the other three Ly49 receptors remain to be determined.     

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. Received: 10 December 1997 · Revised: 28 February 1998     

Genomic organization and evolutionary analysis of <Emphasis Type="Italic">Ly49</Emphasis> genes encoding the rodent natural killer cell receptors: rapid evolution by repeated gene duplication

Ly49 genes regulate the cytotoxic activity of natural killer (NK) cells in rodents and provide important protection against virus-infected or tumor cells. About 15 Ly49 genes have been identified in mice, but only a few genes have been reported to date in rats. Here we studied all Ly49 genes in the entire rat genome sequence and identified 17 putative functional and 16 putative non-functional genes together with their genomic locations in a 1.8-Mb region of chromosome 4. Phylogenetic analysis of these genes indicated that the Ly49 gene family expanded rapidly in recent years, and this expansion was mediated by both tandem and genomic block duplication. The joint phylogenetic analysis of mouse and rat genes suggested that the most recent common ancestor of the two species had at least several Ly49 genes, but that the majority of current duplicate genes were generated after divergence of the two species. In both species Ly49 genes are apparently subject to birth-and-death evolution, but the birth and death rates of Ly49 genes are higher in rats than in mice. The rate of gene expansion in the Ly49 gene family in rats is one of the highest among all mammalian multigene families so far studied. The biochemical function of Ly49 genes is essentially the same as that of KIR genes in primates, but the molecular structures of the two groups of NK cell receptors are very different. A hypothesis was presented to explain the origin of the differential use of Ly49 and KIR genes in rodents and primates.     

Correlation of the cytotoxic activity of four different alkaloids, from Chelidonium majus (greater celandine), with their DNA intercalating properties and ability to induce breaks in the DNA of NK/Ly murine lymphoma cells

The purpose of this study was to examine the relationship between the DNA intercalating characteristics and the DNA damaging capacity of four alkaloids extracted from Chelidonium majus L, as well as their toxicity towards murine NK/Ly lymphoma cells. Chelerythrine, sanguinarine and coptisine were found to be intercalated into the DNA isolated from NK/Ly cells, meanwhile, chelidonine exhibited no affinity to DNA. Sanguinarine exhibited the greatest toxicity toward NK/Ly cells, and the toxicity of the other three decreased in descending order: chelerythrine, coptisine and chelidonine. Chelerythrine and sanguinarine caused DNA damage, illustrated by the formation of comets of the third class. Coptisine was less toxic than chelerythrine and sanguinarine, and affected the formation the same class of comets in higher concentration. The quantity of comets induced by chelidonine were negligible, a finding consistent with its inability to intercalate into DNA structure. The ability of four main alkaloids of Chelidonium majus L., to intercalate into DNA isolated from murine NK/Ly lymphoma cells, correlated with their ability to induce breaks in cellular DNA and with their toxic effect towards those cells.     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

EXPRESSION PROFILING REVEALS A POSITIVE REGULATION BY MPER2 ON CIRCADIAN RHYTHM OF CYTOTOXICITY RECEPTORS: LY49C AND NKG2D

The mammalian circadian gene, mPer2, an indispensable component of the mammalian circadian clock, not only modulates endogenous circadian rhythms but also plays a crucial role in regulating innate immune function. Previously, we showed that mPer2 plays a crucial role in regulating cytotoxic response. To investigate the molecular mechanism for mPer2-controlled cytotoxic response, in the present study we conducted mRNA expression for 11 genes participating in cytotoxicity regulation in wild-type (WT) and mPer2 knockout (mPer2 ^{?;?;?/ ?;?;?}) mice bone marrow, that is, Dap-10, Ly49C, Ly49I, Rac1, Mapk1, Map2k1, Nkg2d, Shp-1, Pak1, Pik3ca, and Vav1. The mRNA levels of Ly49C (p?<?0.001), Ly49I (p?=?0.039), and Nkg2d (p?=?0.038) were significantly downregulated in mPer2 ^{?;?;?/ ?;?;?} mice. Time-dependence of expression profiling was then conducted for four core clock genes (Per1, Bmal1, Clock, Rev-erbα), and six out of these 11 cytotoxic regulation genes (Ly49C, Ly49I, Mapk1, Nkg2d, Shp-1, Pik3ca) in WT and mPer2 ^{?;?;?/ ?;?;?} entrained in light/dark (LD) or dark/dark (DD) cycles. Consistently, circadian oscillations were observed for Per1, Rev-erbα, Ly49C, and Nkg2d in WT mice under LD and DD cycles. However, these rhythmic expressions were either disrupted or dampened in mPer2 ^{?;?;?/ ?;?;?} mice. Comparison of gene expression between WT and mPer2 ^{?;?;?/ ?;?;?} mice showed that mPer2 knockout had systematically downregulated the mRNA expression of two cytotoxicity regulators, Ly49C and Nkg2d. FACS analysis further confirmed that the circadian expression of these genes was not due to the daily difference in cell numbers of NK, NKT, or T cells in bone marrow. Taken together, our results reveal that mPer2 is a critical clock component in modulating circadian rhythms in bone marrow. Furthermore, it implies that Ly49C and Nkg2d are two clock-controlled genes that may play an important role in mediating mPer2-controlled cytotoxic response. (Author correspondence: zsunusa@yahoo.com)     

TheCmv1Host Resistance Locus Is Closely Linked to theLy49Multigene Family within the Natural Killer Cell Gene Complex on Mouse Chromosome 6

Natural killer (NK) cells play important roles in controlling tumor cells and against a range of infectious organisms. Recent studies of mouse NK cell surface receptors, which may be involved in the specificity of NK cells, have shown that many of these molecules are encoded by theLy49andLy55(Nkrp1) multigene families that map to distal mouse chromosome 6. Also mapping to this NK cell gene complex (NKC) is the resistance locus,Cmv1,which is involved in genetically determined resistance to murine cytomegalovirus (MCMV). The aim of this study was to localizeCmv1more precisely in relation to other NKC loci by generating a high-resolution genetic map of the region. We have analyzed 1250 backcross mice comprising panels of 700 (BALB/c × C57BL/6J)F₁× BALB/c and 550 (A/J × C57BL/6J)F₁× A/J progeny. A total of 25 polymorphic genes or microsatellite markers were analyzed over a region of 10 map units fromD6Mit134toD6Mit59.TheCmv1phenotypes of mice recombinant in this interval were tested by infection with MCMV. The results obtained indicate that the functionally important NKC region is a tightly linked cluster of loci spanning at least 0.4 map units. Furthermore,Cmv1maps distal to, but very closely linked to, theLy49multigene family (<0.2 map units), suggesting that MCMV resistance may be conferred by MHC class I-specific NK cell receptors.     

Activation receptor-induced tolerance of mature NK cells in vivo requires signaling through the receptor and is reversible

NK cell responses are determined by signals received through activating and inhibitory cell surface receptors. Ly49H is an NK cell-specific activating receptor that accounts for the genetic resistance to murine CMV (MCMV). The Ly49H receptor has been shown to interact with two adaptor proteins (DAP12 and DAP10). In the context of MCMV infection, interaction of m157 (the MCMV-encoded ligand for Ly49H) with Ly49H results in activation of Ly49H-expressing NK cells. Chronic exposure of Ly49H with m157, however, induces tolerance in these same cells. The mechanism of this tolerance remains poorly understood. Using a transgenic mouse model, we demonstrate that induction of tolerance in Ly49H(+) NK cells by chronic exposure to m157, in vivo, requires signaling through the Ly49H adaptor protein DAP12, but not the DAP10 adaptor protein. Furthermore, mature Ly49H-expressing NK cells from wild-type mice can acquire a tolerant phenotype by 24 h posttransfer into a transgenic C57BL/6 mouse that expresses m157. The tolerant phenotype can be reversed, in vivo, if tolerant NK cells are transferred to mice that do not express the m157 protein. Thus, continuous activating receptor engagement can induce a transient tolerance in mature NK cells in vivo. These observations provide new insight into how activating receptor engagement shapes NK cell function and has important implications in how NK cells respond to tumors and during chronic viral infection.     

Differential core histone binding behavior: RNA polymerase I promoter region vs 5S rDNA positioning DNA sequences

In order to further characterize the previously observed disruptive effect of the RNA polymerase I promoter sequence (Pol I) from Acanthamoeba castellanii on tandemly repeated 5S rDNA positioning sequences from sea urchin (Lytechinus variegatus), we compared the histone-binding ability of the isolated 199-bp Pol I promoter region to that of the 208-bp 5S rDNA and that of nucleosome core particle sequences isolated from chicken erythocytes. We found the 5S rDNA positioning sequence to be more efficient at forming nucleosomes than the RNA polymerase I promoter sequence. Nevertheless, examination of the free-DNA half-depletion points during the titrations suggested that twice as much histone had bound to RNA polymerase I promoter sequence as to the 5S nucleosome-positioning or core particle sequences. DNA bending analysis suggested two potential DNA bending loci in the RNA polymerase I promoter, whereas only one such locus was predicted for the 5S positioning sequence. Such mixed bending signals on the RNA polymerase I promoter could favor non-nucleosomal deposition of histones on these sequences.