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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.

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.

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.