Cloning and Sequencing the First HLA Gene by ProQuest


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									Copyright Ó 2010 by the Genetics Society of America
DOI: 10.1534/genetics.110.113852

                   Anecdotal, Historical and Critical Commentaries on Genetics
                               Cloning and Sequencing the First HLA Gene

                                                           Bertrand R. Jordan1
                                             Marseille-Nice Genopole, 13288 Marseille, Cedex 9, France

                This Perspectives article recounts the isolation and sequencing of the first human histocompatibility
             gene (HLA) in 1980–1981. At the time, general knowledge of the molecules of the immune system was
             already fairly extensive, and gene rearrangements in the immunoglobulin complex (discovered in 1976)
             had generated much excitement: HLA was quite obviously the next frontier. The author was able to use a
             homologous murine H-2 cDNA to identify putative human HLA genomic clones in a l-phage library and
             thus to isolate and sequence the first human histocompatibility gene. This personal account relates the
             steps that led to this result, describes the highly competitive international environment, and highlights
             the role of location, connections, and sheer luck in such an achievement. It also puts this work in
             perspective with a short description of the current knowledge of histocompatibility genes and, finally,
             presents some reflections on the meaning of ‘‘discovery.’’

B   Y 1980, when this story begins, much was already
     known about the immune system and its mole-
cules, although wide gaps remained in our knowledge.
                                                                            notably cytotoxic T cells directed against foreign cells
                                                                            (as in graft rejection) or against virus-infected autolo-
                                                                            gous cells, and ‘‘helper’’ T cells involved—in some still-
The major components, B and T lymphocytes, were                             mysterious fashion—in stimulating the production of
more or less understood, the former as producers of                         specific antibodies. Several types of cell-surface mole-
immunoglobulin antibodies and the latter as agents                          cules had been identified, among which were the class I
of cellular immune response. Very laborious protein                         histocompatibility antigens expressed on most cell types
studies had succeeded in determining the amino acid                         and recognized by cytotoxic T lymphocytes: HLA-A, -B,
sequence of a few immunoglobulin molecules. A major                         and -C in humans and H-2 K, D, and L in mice. These
puzzle, the existence of an apparently unlimited                            had originally been identified in the (artificial) context
repertoire of antibody specificities that seemed in-                         of graft rejection (hence their name), and their char-
compatible with the limited coding capacity of our                          acterization at the serological and functional level
genome, had just been solved (Gearhart 2004). The                           motivated the Nobel prize awarded in 1980 to Baruj
seminal article of Hozumi and Tonegawa (1976) had                           Benacerraf, Jean Dausset, and George Snell. HLA (or
shown, using techniques that now seem incredibly                            H-2) class II antigens, expressed mostly on B cells and
cumbersome, that the V and the C immunoglobulin                             involved in recognition by helper T cells, had also been
gene segments are separated in germline DNA and are                         defined. And the T-cell receptor, expressed on T cells
joined in differentiated DNA. This was the very first                        and responsible for these recognition events, would
gene rearrangement demonstrated in mammalian                                eventually be discovered—although this was still in the
DNA, and it led to intense activity over the succeeding                     future.
years that included cloning the numerous sequences                             The HLA (or the mouse H-2) genes and molecules
involved in the assembly of a functional immunoglob-                        raised many interesting questions that made them a
ulin gene and beginning to unravel the mechanisms                           prime target for molecular study, as detailed in Jean
and the molecules implicated in the execution and the                       Dausset’s Nobel Prize lecture (Dausset 1981). The class
regulation of these rearrangements (Honjo et al. 1981).                     I antigens, in particular, displayed a surprising level of
   T lymphocytes were also considered essential ele-                        polymorphism, first detected by serological analyses but
ments, but their complexity was only partially under-                       later documented by protein sequencing (Orr et al.
stood. They were known to include several classes,                          1979). In spite of their inherent difficulty (they required
                                                                            the purification of a transmembrane protein present
   Author e-mail:                               in low amounts at the cell surface), these studies had

Genetics 184: 879–886 (April 2010)
880                                                   B. R. Jordan

shown that the class I molecule consisted of three ex-        products with an HLA-specific antibody1 to detect the
tracellular domains followed by a transmembrane seg-          HLA mRNA-containing fractions. The partially purified
ment and a short cytoplasmic tail and that it was             HLA mRNA thus obtained was then copied into cDNA
associated at the cell surface with a smaller, nonpoly-       and cloned in a plasmid vector. Screening of this library
morphic protein called b-2 microglobulin. Interest-           was accomplished by a very laborious procedure: DNA
ingly, the first (outer) two extracellular domains, a-1        preparations from hundreds of individual cDNA clones
and -2, displayed extensive polymorphism while the            were individually immobilized on nitrocellulose filters,
third, a-3, appeared to be constant. Thus the situation  
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