GENOMICS 6,548-553 (1990) SHORT COMMUNICATION Localization of a Human T-Cell-Specific Gene, RANTES (D17S136E), to Chromosome 17ql1.2-q12 T. A. DONLON,* A. M. KRENSKY,t M. R. WALLACE,+ F. S. COLLINS,+ M. LOVETT,§ AND C. CLAYBERGERt Departments of *Pathology and tPediatrics, Stanford University Medical Center, Stanford, California 94305; Woward Hughes Medical Institute and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109; and §Genelabs Incorporated, Redwood City, California 94063 Received April 24, 1989; revised September 27, 1989 We report here the localization of the gene for a hu- man T-cell-specific molecule, designated RANTES, to human chromosome region 17q11.2-q12 by in situ hybridization and analysis of somatic cell hybrids us- ing a cDNA probe to the gene. We have recently shown that this gene, which encodes a small, secreted, pu- tative lymphokine, is a member of a larger gene family some of whose members reside on chromosome 4 but most of whose members have not to date been mapped. A secondary hybridization peak was noted on the re- gion of human chromosome 5q31-q34, which may represent the location of other members of the gene family. Interestingly, this latter region overlaps with the location of an extended linked cluster of growth factor and receptor genes, some of which may be co- regulated with members of the RANTES gene family. Ol@SOAcdemicPreas,Inc. T lymphocytes mediate many of their regulatory functions through a group of small, secreted molecules which are collectively referred to as lymphokines. Re- cently we have used a human cDNA library that was enriched by subtractive hybridization for sequences expressed by T but not B lymphocytes to isolate a gene, designated RANTES, which encodes a novel T-cell- specific molecule (Schall et al., 1988). This gene is ex- pressed by growth factor-dependent, antigen-specific T-cell clones that function as either helper or cytotoxic cells in vitro. However, RANTES is not expressed in unstimulated peripheral blood lymphocytes or in es- tablished T-cell tumor lines. RANTES expression is induced by antigen or mitogen stimulation of peripheral blood lymphocytes. The RANTES gene product is pre- dicted to be 10 kDa and, after cleavage of the signal peptide, approximately 8 kDa. Of the 68 residues, 4 are cysteines and there are no sites for N-linked glycos- ylation. There is significant homology (30-70%) be- tween the RANTES sequence and several other T-cell genes, suggesting that they constitute a family of small, secreted T-cell molecules. Here we report the chromosomal locations of the RANTES gene and discuss its relation to other lym- phokine genes. The cDNA clone for RANTES (Schall et al., 1988) was digested with EcoRI and ApaI and the 480-base insert, which corresponds to the 5’ end of the gene, was isolated by electrophoresis in low-melting-temperature agarose. The insert was labeled with 3H to a specific activity of 8.4 X 10’ cpm/pg (Feinberg and Vogelstein, 1983). In situ hybridization was performed on normal, 46,Xx chromosome preparations essentially as pre- viously described (Harper and Saunders, 1981). Briefly, the radiolabeled probe was hybridized overnight at 42°C at a final concentration of 10 pg/pl in 50% form- amide and 2X SSC and exposed to autoradiography for 8 days. Chromosome identification was performed us- ing simultaneous fluorescent R-banding/transmitted light as described (Donlon et al., 1983). Ten micrograms of DNA from each source was di- gested to completion with P&I, electrophoresed, and transferred to Hybond N nylon. The filter was hybrid- ized with 50 ng of oligo-labeled insert from the RANTES plasmid and washed to a final stringency of 0.1X SSC, 0.1% SDS at 65°C. The rat DNA is from hepatoma cell line PCTA-7A (Leach et al., 1989) and the mouse and human DNAs are of lymphocytic origin. Hybrid MH-22 contains one normal human chromo- some 17 as its only human DNA on a mouse back- ground, while P12.3B contains 17pter-q12 and SP-3 possesses17q11.2-qter, also on mouse backgrounds osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 548