The TCR 3Chain Gene Was Cloned by Use of Subtractive Hybridization

In order to identify and isolate the TCR genes, S. M. Hedrick and M. M. Davis sought to isolate mRNA that encodes the a and (3 chains from a TH-cell clone. This was no easy task because the receptor mRNA represents only a minor fraction of the total cell mRNA. By contrast, in the plasma cell, immunoglobulin is a major secreted cell product, and mRNAs encoding the heavy and light chains are abundant and easy to purify.

The successful scheme of Hedrick and Davis assumed that the TCR mRNA—like the mRNAs that encode other integral membrane proteins—would be associated with membrane-bound polyribosomes rather than with free cytoplasmic ri-bosomes. They therefore isolated the membrane-bound polyribosomal mRNA from a TH-cell clone and used reverse transcriptase to synthesize 32P-labeled cDNA probes (Figure 9-2). Because only 3% of lymphocyte mRNA is in the membrane-bound polyribosomal fraction, this step eliminated 97% of the cell mRNA.

Hedrick and Davis next used a technique called DNA sub-tractive hybridization to remove from their preparation the [32P] cDNA that was not unique to T cells. Their rationale for this step was based on earlier measurements by Davis showing that 98% of the genes expressed in lymphocytes are common to B cells and T cells. The 2% of the expressed genes that

Killing

TCR Viral peptide A

Self MHC

Killing

Nonself MHC

No killing

Viral peptide 1

Viral peptide 1

Self MHC

No killing

Viral peptide A

Viral peptide A

Nonself MHC

No killing

TH-cell clone

B cell

TH-cell clone mRNA

B cell

mRNA

97% in free 3% in cytoplasmic membrane-bound polyribosomes polyribosomes mRNA

32p_

mRNA

Reverse transcriptase

Hybridize

Reverse transcriptase

Hybridize

Separate on hydroxyapatite column cDNAs specific to T cells

Separate on hydroxyapatite column cDNAs specific to T cells

10 different cDNA clones

Hybrids with cDNAs common to T cells and B cells

Use as probes in Southern blots of genomic DNA

Use as probes in Southern blots of genomic DNA

Hybrids with cDNAs common to T cells and B cells

is unique to T cells should include the genes encoding the T-cell receptor. Therefore, by hybridizing B-cell mRNA with their TH-cell [32P]cDNA, they were able to remove, or subtract, all the cDNA that was common to B cells and T cells. The unhybridized [32P]cDNA remaining after this step presumably represented the expressed polyribosomal mRNA that was unique to the TH-cell clone, including the mRNA encoding its T-cell receptor.

Cloning of the unhybridized [32P]cDNA generated a library from which 10 different cDNA clones were identified. To determine which of these T-cell-specific cDNA clones

FIGURE 9-2

Production and identification of a cDNA clone encoding the T-cell receptor. The flow chart outlines the procedure used by S. Hedrick and M. Davis to obtain [32P]cDNA clones corresponding to T-cell-specific mRNAs. The technique of DNA subtractive hybridization enabled them to isolate [32P]cDNA unique to the T cell. The labeled TH-cell cDNA clones were used as probes (inset) in Southern-blot analyses of genomic DNA from liver cells, B-lymphoma cells, and six different TH-cell clones (a-f). Probing with cDNA clone 1 produced a distinct blot pattern for each T-cell clone, whereas probing with cDNA clone 2 did not. Assuming that liver cells and B cells contained unrearranged germ-line TCR DNA, and that each of the T-cell clones contained different rearranged TCR genes, the results using cDNA clone 1 as the probe identified clone 1 as the T-cell-receptor gene. The cDNA of clone 2 identified the gene for another T-cell membrane molecule encoded by DNA that does not undergo rearrangement. [Based on S. Hedrick et al., 1984, Nature 308:149.]

represented the T-cell receptor, all were used as probes to look for genes that rearranged in mature T cells. This approach was based on the assumption that, since the ap T-cell receptor appeared to have constant and variable regions, its genes should undergo DNA rearrangements like those observed in the Ig genes of B cells. The two investigators tested DNA from T cells, B cells, liver cells, and macrophages by Southern-blot analysis using the 10 [32P]cDNA probes to identify unique T-cell genomic DNA sequences. One clone showed bands indicating DNA rearrangement in T cells but not in the other cell types. This cDNA probe identified six different patterns for the DNA from six different mature T-cell lines (see Figure 9-2 inset, upper panel). These different patterns presumably represented rearranged TCR genes. Such results would be expected if rearranged TCR genes occur only in mature T cells. The observation that each of the six T-cell lines showed different Southern-blot patterns was consistent with the predicted differences in TCR specificity in each T-cell line.

The cDNA clone 1 identified by the Southern-blot analyses shown in Figure 9-2 has all the hallmarks of a putative TCR gene: it represents a gene sequence that rearranges, is expressed as a membrane-bound protein, and is expressed only in T cells. This cDNA clone was found to encode the p chain of the T-cell receptor. Later, cDNA clones were identified encoding the a chain, the 7 chain, and finally the 8 chain. These findings opened the way to understanding the T-cell receptor and made possible subsequent structural and functional studies.

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