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RT6 is a glycosyl phosphatidylinositol-anchored cell membrane protein, whose expression is restricted to peripheral T cells and intraepithelial lymphocytes. It has attracted interest as a T cell differentiation marker and activation antigen in rats. The only known protein to which RT6 shows significant homology is a recently cloned mono(ADP-ribosyl)transferase of rabbit skeletal muscle which is distantly related also to certain bacterial toxins. Intriguingly, whereas the rat carries a single copy RT6 gene with two known highly divergent alleles, the mouse carries two closely linked, functional RT6 genes that show approximately 85% sequence identity. We have now cloned and sequenced the homologues of the RT6 genes from humans of distinct ethnic backgrounds and of the chimpanzee. Surprisingly, in each case, three premature in-frame stop codons preclude expression of the single copy RT6 gene as a cell surface protein. Otherwise, the RT6 genes of human and chimpanzee exhibit high structural conservation to their rodent counterparts. RNA expression analyses indicate that the RT6 gene is not transcriptionally active in human T cells or any other human tissue analyzed so far. To our knowledge, RT6 represents the first mammalian membrane protein identified that has been lost universally in the human and chimpanzee species due to gene inactivation.

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.

RT6 is a T cell membrane protein that has attracted interest because a defect in RT6 expression is associated with susceptibility to autoimmune type I diabetes in DP-BB rats and NOD mice. Using PCR screening of human/rodent somatic cell hybrids and fluorescence in situ hybridization, we have determined that the gene for the human RT6 homologue is located at 11q13, centromeric to the gene for tyrosinase (TYR, albino locus) and telomeric to that for fibroblast growth factor 4 (FGF4). The data suggest that the human RT6 gene constitutes a new linkage group with TYR and the gene for olfactory marker protein (OMP) on 11q, which has a counterpart in mouse chromosome 7. Thus, in the human, the RT6 locus is dissociated from the hemoglobin beta chain locus (HBB) and its neighboring conserved linkage group at 11p15, in contrast to the mouse, in which RT6 shows a tighter linkage to Hbb than to Tyr. The results support the conclusion that there has been considerable intrachromosomal reshuffling of linked genes since the divergence of primates and rodents.

Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designatedART3andART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32–41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and byin situhybridization, we have mapped the two genes to human chromosomes 4p14–p15.1 and 12q13.2–q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the “tip of an iceberg,” i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation.

We have screened different mouse strains - including strains with enhanced susceptibility for autoimmune diseases - for deviations of Rt6 gene expression by RT-PCR. Most strains expressed varying amounts of Rt6-1 and Rt6-2. NZW mice, however, do not show any detectable Rt6-2 gene transcripts. BxSB mice show a near complete absence of Rt6-1 gene transcripts. Southern blot and sequence analyses revealed that NZW mice have suffered a deletion of the Rt6.2 gene while the Rt6-1 gene of BxSB mice has been inactivated by a premature stop codon. Thus, these mouse strains represent natural Rt6-2 and Rt6-1 single-gene ’knock-out’s, respectively. Since the NZW mouse does not show any gross immunological abnormalities, loss of the Rt6-2 gene by itself is not associated with any obvious immunological phenotype. However, crosses between NZW and certain other mouse strains, e.g. (NZW x NWB)F1 and (NZW x SB)F1 animals, develop a systemic autoimmune disease reminiscent of human lupus erythematosus. Moreover, the BxSB mouse strain is considered to be an independent model for the same disease. It will be of interest to determine whether these spontaneous Rt6 gene defects constitute part of the polygenetic contribution to autoimmune disease in these animals.

This report describes the localization of the human and mouse genes for muscle ecto mono (ADProbosyl)transferase to human chromosome 11p15 and mouse chromosome 7 using polymerase chain reaction of somatic cell hybrids. Mono ADP-ribosylation is the mechanism by which bacterial toxins interfere with protein biosynthesis in human host cells. 11 refs., 1 fig.

RT6 is an enzymatically active GPI-anchored membrane protein that was originally discovered in the rat as a peripheral T cell alloantigen. It has attracted interest as an activation antigen and because defective RT6-expression coincides with increased susceptibility for autoimmune type I diabetes in the BB rat. Southern blot analyses indicate that the rat carries a single copy RT6 gene whereas the mouse carries a duplication of the homologous locus. We had previously cloned and sequenced a RT6 -homologous cDNA from BALB/c mouse spleen. We now report the cloning and characterization of a second RT6 -homologue from BALB/c and 129/Sv mice. The two mouse Rt6 genes (designated Rt6 -1 and Rt6 -2) encode similar open reading frames that are disrupted by conserved introns. The nucleotide sequences of the Rt6 -1 and Rt6 -2 coding regions show 87% sequence identity, the deduced amino acid sequences 79% identity. The amino acid sequences reveal significant similarity to recently cloned ADP-ribosylating ectoenzymes from rabbit and human skeletal muscle as well as chicken bone marrow cells. RT-PCR analyses reveal that the two Rt6 genes are differentially expressed in distinct inbred mouse strains and that their transcripts are properly processed .Western blot analyses demonstrate that the respective gene products are released from cells by treatment with PI-PLC. The results further show that both mouse Rt6 genes are translated into GPI-anchored cell surface molecules and that Rt6 gene expression is restricted to peripheral lymphoid tissues.

Rt6 is a T cell-restricted GPI-anchored membrane protein and a member of the family of mono(ADP-ribosyl)transferases. One of the two murine Rt6 genes is deleted in NZW mice. This finding is reminiscent of the deletion of one of the TCR beta genes in the same mouse strain and it is an intriguing possibility that these gene deletions arose by a common genetic mechanism. The Rt6 locus retained by the NZW mouse (designated Rt6-1) is polymorphic among inbred strains of laboratory mice. The NZW mouse shows several strain-specific restriction fragment length variants in this Rt6 locus and five amino acid substitutions occur in the predicted native Rt6 polypeptide of the NZW mouse relative to the corresponding polypeptides of NZB and BALB/c mice. Whereas transcript levels of the two Rt6 genes appear to be normal in spleen and intestine of NZB mice, the corresponding tissues of NZW mice show reduced levels of transcripts from the Rt6 locus retained in this mouse strain. Moreover, reduced levels of Rt6 mRNA also occur in spleen and intestine of (NZB x NZW)F1 hybrid animals, indicating that F1 animals have inherited a dominant factor from the genetic background of the NZW mouse, resulting in low levels of Rt6 expression. It is conceivable that the alterations in the Rt6 genes of the NZW mouse and/or the factor(s) affecting defective Rt6 expression constitute part of the genetic contribution of the NZW mouse to the autoimmune lupus-like disease in (NZB x NZW)F1 animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Minor H (histocompatibility) loci govern immune responses causing slow skin-graft rejection and graft versus host disease in major histocompatibility complex (MHC)-compatible animals. It is generally assumed that minor H antigens represent allelic variants of normal proteins and that peptides bearing the allelic amino acid substitutions are presented by MHC molecules in the context of which they may be recognized by H-allotype-specific T cells. 24 refs., 1 fig., 2 tabs.

A genomic probe of the human RT6 gene detects a frequent biallelic BglII polymorphism. Allele A has a frequency of 63%, whereas that of allele B is 37%. This restriction fragment length polymorphism provides the first known genetic marker for this gene.