IDENTIFICATION OF TISSUE-SPECIFIC cDNAs:

Tissue specific cDNAs are identified by using the technique of differential hybridization. Differential hybridization is a technique used to clone a family of genes that are related by a common mechanism of regulation. This strategy was used to clone a family of genes that are rapidly induced when quiescent cells are stimulated to grow by the addition of serum (which contains growth factors). The genes themselves can be structurally different, but they are all expressed under the same cellular conditions. Examples include genes expressed in specific tissues, genes expressed at specific stages of the cell cycle, and genes regulated by a growth factor. The basis of this technique relies simply on producing two cell populations, one in which the genes are, and the other in which the genes are not, expressed. No specific information about an individual gene is required. Figure shows the experimental approach used to isolates a family of genes whose expression is stimulated by addition of serum to quiescent cells. STEPS FOR CLONING FACTOR-REGULATED DIFFERENTIAL HYBRIDISATION: GENES BY

Poly(A) RNA was isolated from (1) cultures of quiescent cells and from (2) cells stimulated with serum for 3 hours. RNA from the stimulated cells was used to produce a cDNA library in a lambda phage vector, which was plated out. Phages from the plaques produced were transferred to duplicate filter replicas.

Radioactively labeled probes were generated from the mRNA of quiescent cells and stimulated cells by synthesizing the cDNA strands in the presence of 32P-labeled dNTPs.

One set of filters was hybridized to each cDNA probe.

 The resulting autoradiographs were carefully compared to identify the position of phage plaques that hybridized to the probe from stimulated cells but not to the probe from quiescent cells. These phages carried cDNAs from genes turned on in the serum-stimulated cells.

ISOLATION OF VERY RARE cDNAs:

Further, one of the drawbacks being that this method of differential hybridization for identifying tissue specific cDNAs works well in practice for genes that are highly expressed in the stimulated cells. However, mRNAs of low abundance are difficult to isolate by differential screening. The use of absorbed probes and subtracted libraries improve the chances of cloning rare cDNAs. Both of these tricks were used in the very difficult cloning of cDNAs encoding the subunits of the T-cell receptor. The strategy assumed that the genes encoding the T-cell receptor were expressed only in T-cell receptor and not in B-cells. STEPS FOR MAKING SUBTRACTED LIBRARIES AND PROBES FOR ISOLATION OF RARE cDNAs:

Poly(A) RNA was prepared both from T cells and from B cells, which were closely related to T cells but do not express the T-cell receptor.

Using oligo(dT) primers, researchers synthesized the cDNA strands from the T-cell RNA. The RNA was removed from the resulting RNA-DNA hybrids by alkaline hydrolysis.

What was left was single-stranded cDNA complementary to T-cell mRNA. The cDNA was incubated with a large excess of B-cell mRNA under conditions that favored DNA-RNA hybridization. All the T-cell cDNAs for genes also expressed in B cells (about 98 percent) hybridized to B cell RNA.

T-cell-specific cDNAs remained single-stranded.

The mixture was passed over a column of hydroxylapatite, which retained the double-stranded molecules and let the single-stranded cDNAs pass through.

 The T-cell specific cDNA recovered from the column was converted to double stranded cDNA and cloned into a lambda phage vector. This produced a library of 5000 clones highly enriched for T-cell specific cDNAs.

To screen the library, 32P-labeled T-cell cDNA was subtracted with B-cell mRNA in the same fashion and used a probe. Thirty-five clones reacted with this probe. Among them were clones that encoded the T-cell receptor.