How were the first genes isolated from genomic or cDNA libraries without already knowing a hybridization probe?

Question

Let us go back in time to around 1975. It is my understanding that at that point (or at least by the end of the decade), both genomic and cDNA libraries had been created for a few organisms, e.g. rabbits. I'm interested in the logic of techniques that first used these libraries to isolate genes encoding for particular proteins. Note: I'm not interested in how we'd isolate a gene today; I want to understand the history.

First, consider the following thought experiment. Say it's around 1975. The one assignment for my Cal Tech biology class is a doozy: it goes as follows. The instructor (Tom Maniastis?) hands me a protein along with the cells from which the protein was isolated as well as genomic and cDNA libraries of the organism that makes the protein; the challenge is to isolate the gene encoding for the protein. I do not know anything about the protein except that the product I've been given is pure.

Of course, the basic idea is simple: isolate the gene by screening the libraries with a radioactive nucleic acid probe and using a hybridization assay. But alas, I do not have a complementary probe: all I have is the protein! What do I do?

Here is one solution I can imagine. (I don't know whether it was possible around 1975, much less whether it actually happened.) I could try to isolate the mRNA that codes for my protein (but how to do that?), then radiolabel it and use the radiolabelled mRNA as a probe in a hybridization assay on the cDNA library (not the genomic library, since the unprocessed DNA could have noncoding regions).

1. Would this have worked?
2. Did anyone actually do this?
3. If yes to either (1) or (2), how could one figure out which mRNA coded for the protein?

The question, in essence, is how to get to DNA, given only a protein, circa 1975.

Answer 1

The following is based on a chapter on Cloning DNA in the 10th edition of The Biochemistry of the Nucleic Acids (RLP Adams et al.) published by Chapman & Hall in 1986. (This is probably more pertinent than the 11th and final edition, which was published in 1992 and had a more contemporary treatment.) Apologies for the fact that it will be difficult to find. It should be available from some university libraries and through inter-library loan systems in certain countries. Section A.7.3 gives a taste of the methodology used at that time.

Protein properties that could be used as the basis of a cloning strategy

In attempting to clone DNA for a particular protein for which there were no pre-existing clones to use as hybridization probes, one had to consider the properties of the protein one is able to exploit. These might include:

• Amino acid sequence
• Antibodies to the protein
• Biological activity
• Particular physical properties, such as size

(Clearly if one had no such ‘handle’ to the protein one was not in a position to try to clone its cDNA or gene.)

Three general approaches

There seemed to be three general approaches:

• No expression
• Indirect expression
• Direct expression

The approach that one might take was also governed by whether the protein is particularly abundant in a specific tissue, when one might expect less sophisticated more labour-intensive approaches to stand a chance of success.

Another point to emphasize is that, at least with eukaryotes, one would look for a cDNA clone first, and only try for a genomic clone after one had this to employ as a hybridization probe.

1. ‘No expression’ approaches

In the no expression approaches one relied on the amino acid sequence to identify the cloned DNA. One such approach was to use hybridization probes against stretches of amino acids, the codons of which had limited redundancy (Met and Trp have a single codon, and some other amino acids only two) using low stringency hybridization conditions. This required luck. Alternatively one could sequence random clones and hope to find one that correlated with the amino acid sequence. At that time DNA sequencing was slow and labour intensive, so that this was only applicable to proteins that were relatively abundant in the tissue from which the libarary was prepared.

2. Direct expression approaches

Direct expression generally entailed using a bacterial expression vector which produced a fusion of a bacterial protein with a part of the eukaryotic protein. (Statistically only one in six of clones would have the correct reading frame in a fusion protein.). This approach was favoured if one had an antibody, which did not require a complete protein.

3. Indirect expression approaches

By indirect expression was meant using immobilized cloned cDNA to select corresponding mRNAs (‘hybrid selection’) which could be expressed in a cell-free system. This was appropriate if the distinguishing characteristic required a full-length protein, as in the case of biological activity or size.

Answer 2

Specific to the insulin gene, the following paper is, as far as I can tell, the first to isolate it:

Ullrich A, Shine J, Chirgwin J, Pictet R, Tischer E, Rutter WJ, Goodman HM. 1977. Rat insulin genes: construction of plasmids containing the coding sequences. Science 196:1313-1319.

The took advantage of the fact that insulin is produced by β cells in the pancreas and so are enriched in insulin mRNA. After poly(A) RNA was isolated and reverse transcribed, the major band observed upon electrophoresis of the resulting cDNA was expected to belong to the insulin gene:

It seemed likely that the [major] fragments were derived from insulin cDNA because of their prominence in the total cDNA preparation. Therefore, these fragments as well as the complete cDNA preparations were used in the cloning experiments.

Thus they cloned this cDNA, sequenced it and discovered that it contained the entire proinsulin coding sequence:

Since the mRNA contains a terminal poly(A) sequence, the [cloned] DNA strand containing 3' terminal poly(dA) is of the same sense. This strand determines an amino acid sequence which exactly corresponds to the entire coding region for rat proinsulin I and 13 out of 23 amino acids of the prepeptide sequence.

With this sequence determined and because of its high degree of conservation, the homologous human gene could be isolated by hybridizing with rat insulin cDNA:

Bell GI, Swain WF, Pictet R, Cordell B, Goodman HM, Rutter WJ. 1979. Nucleotide sequence of a cDNA clone encoding human preproinsulin. Nature 282:525-527.