98 Annual AACHT Meeting, 1984

and probed with DR 13 and DQ/3 cDNA probes. Only rare DNA polymorphisms were seen between these HTCs, which did not appear to correlate well with Dw type. This is in contrast to the multiple DNA polymorphisms easily detected with virtually any restriction enzyme between different DR types. These studies demonstrate that polymorphism (serological or protein) can be found on the DR molecule which correlate with Dw type suggesting that "D" determinants may be situated on the DR molecule. The possible evolutionary implications of the relative infrequency of DNA polymorphism associated with Dw as opposed to DR, which suggests a recent evolution of Dw types, will be discussed.

CLASS II DNA POLYMORPHISM AMONG DR4 VARIANTS. Susan H. Hsu, Wilma B. Bias, Nancy Delaney, Robert Christy, and Ru Chih C. Huang; The Johns Hopkins University. Baltimore. MD

We reported in 1981 two new DR4-associated HLA-D homozygous typing cells, DAm4.1 and DAm4.2 (Hum Immunol 2:165, 1981). At the 9th International Workshop these two specificities were formally designated as Dwl 3 and Dwl4, respectively. Using cDNA probes for DRc~,/3; DCa,/3; and SBa,/3 and cells from their own HTC repertoire, we have studied the molecular genetic basis for this serologically indistinguishable but cellularly distinct HLA-D cross-reacting group that includes Dw4, Dwl0, Dw13, and Dw14. Genomic DNA from each HTC was digested at 37°C for 18 hr with the following restriction endonucleases: BamHI, BlglI, EcoR1, and HindlII. The restricted DNA was electrophoresed on 1% agarose gel. Genomic blots were prehybridized and then hybridized (in 3xSSC containing 0.1% SDS, 0.02% PVP, 0.02% ficoll and 0.02M sodium phosphate, pH 6.8) with radiolabeled probes (at approximately 1 × 10 s CPM//~g DNA) at 65°C for 24 hr. Preliminary analysis of the blots revealed that some DR4 variants share similar restriction fragment length polymorphism (RFLP) patterns. However, distinctive restriction fragments which characterize each var- iant were also obtained. For example, when probing with DR/3 cDNA, Dw13 appeared to have a distinct RFLP pattern when digested with EcoR1; Dwl4 had a unique 6.4 kb BamHI band not found in the other three HTCs, while Dw4 cells lacked a 12.0 kb HindlII band which was present in the other cells. When the DC/3 probe was used, a unique 9.2 kb HindlIl band was observed in Dw14 cells. Data from other probes and enzyme digests will be presented. As expected from 2D gel electrophoretic patterns of membrane-bound antigens, these pre- liminary data indicate that the genes encoding individual antigens of the DR4 cross-reacting group may differ in nucleotide sequence in either DR or DC genomic DNA.

Ia POLYMORPHISM DETECTED BY ALLOREACTIVE T CELL CLONES AND RESTRIC- TION FRAGMENT MAPPING. J. Silver, H. C. Chang, S. Goyert, A. Zeevi, and R.J. Duquesnoy; Hospital for Joint Diseases. New York. NY

We have previously described two HLA-DR2 associated alloreactive T cell clones generated from MLR cultures between two unrelated individuals, both typing as DR2. These clones could distinguish two cellular subtypes of the serologically defined DR2, one associated primarily with B27 and the other primarily asso- ciated with B7 and B18. Both DR2 associated cellular subtypes were positive for DQw 1. Conventional monomorphic DR monoclonal antibodies (mAb) failed to inhibit these T cell clones, whereas other mAb with additional class II spec-

Abstracts 99

ificities showed significant effects. To analyze the molecular basis of these two DR2 associated cellular subtypes, we have performed restriction fragment poly- morphism analysis using several class II cDNA probes. These studies were con- ducted on EBV transformed B lymphoblastoid cell lines generated from an in- formative family and unrelated donors expressing different DR2 associated cellular haplotypes. The results showed major differences in DQa and DQ/3 genes sug- gesting that the DR2 associated cellular subtypes may reflect polymorphism of the DQ subregion. Studies are in progress to isolate these genes and determine the molecular basis of the polymorphism detected by these T cell clones.

THE USE OF NONRADIOACTIVE DNA PROBES FOR HLA DNA TYPING: A NOVEL METHOD EMPLOYING BIOTINYLATED PSORALEN TO LABEL PROBES FOR HYBRID- IZATION. Ed Sheldon, Corey Levenson, Carolyn Flugstad, Bob Watson, and Henry Erlich; Cetus Corporation. Emeryville, CA

Genetic polymorphism in the HLA region has been analyzed using specific HLA class I and class II DNA sequences as probes to reveal restriction fragment length polymorphism (RFLP) in Southern blot experiments. These studies, which have revealed correlations of RFLP patterns with serotype and disease susceptibility, were performed with ~2P-labeled probes. Here, we report the development of a novel procedure for labeling DNA probes with biotinylated psoralen and the use of such probes for HLA RFLP DNA typing. Our procedure for constructing nonradioactive probes was based on a method for labeling the DNA of the single- stranded phage M 13. Cloned cDNA sequences encoding the alpha and beta chains of the DR, DQ(DC), and DP(SB) were inserted into M13 phage vectors. Each M 13 clone was made partially double-stranded, leaving the hybridizing regions single-stranded. Next the double-stranded regions were labeled selectively with biotin groups using biotinylated psoralen that intercalates into double-stranded regions and can be cross-linked to DNA by UV irradiation. To use biotinylated psoralen labeled probes, human DNA was digested with a restriction endonu- clease, electrophoresed through agarose using a minigel apparatus, and blotted to a Genetran membrane. Hybridization was performed using the same protocol used with radioactive probes. After hybridization, the presence of biotinylated probe was detected with a streptavidin-enzyme complex. After streptavidin bound the biotin groups on the probe, detection was accomplished by adding the sub- strate which is converted to a colored precipitate by the enzyme. As a result of this reaction, hybridization was seen as a band of stain. Using this procedure, experiments have been performed to detect polymorphic class II loci. In Southern blot experiments, using overnight hybridization and overnight detection with the streptavidin enzyme complex, single copy class II genes have been detected in 2.5 p.g of human DNA. In conclusion, we have shown the feasibility of using DNA probes labeled with biotinylated psoralen to determine HLA class II poly- morphisms. These nonradioactive probes, which have sensitivity comparable to radioactive ones, can be stored indefinitely, and used without special precautions, offer the potential for simplifying HLA typing at the DNA level.

EXPRESSION OF TRANSFECTED GENES 1N T CELL TUMOR LINES. Kevin G. Becker, Susan H. Hsu, and Wilma B. Bias; The Johns Hopkins University School of Medicine. Baltimore. MD

The creation of stable cell lines having defined characteristics has been a goal of many laboratories. We present what we believe to be the first successful intro-