Session 10 -- Page 1
July 17, 2000

Introduction to genetic engineering

Genetic engineering arose in the 1970's because biologists of the time wanted desperately to understand how genes worked. In particular, they wanted to know how genes were organized, how transcription was turned off and on, and how the action of many genes was coordinated. They wanted to find out the regulatory signals that allowed some genes to be expressed at certain times, in specific cells, and in varying amounts during an organism's lifetime?

Almost all of the previous information about these matters had come from indirect experiments carried out for more than a half century by several generations of geneticists. Most of the genetic data that had been collected over the years were often based on beautifully designed and imaginative experiments, but they needed confirmation by complementary, and more direct, biochemical studies.

To get these confirmations, the most straight-forward approach would have been to isolate specific genes, modify the regions of these genes that were suspected to be involved in the gene's control, and then determine what effect these changes had on the genes' functions. The difficulty with this idea is that it requires the researcher to have specific genes in hand. It was suspected at the time, and later confirmed, that each gene is regulated individually. So, for example, to modify a hemoglobin gene, one must isolate the hemoglobin gene away from the tens of thousands of others, and have it in sufficient quantity to carry out experiments on it. Unfortunately, except for a few very special cases, no genes had been purified before 1970. It's important to understand why it was hard to purify genes.

Genes are chemically similar
Biochemists find it difficult to purify specific genes for several reasons. First, DNA is a relatively simple polymer. As we have seen, it consists of only four kinds of units joined together like circus elephants in a line. All of these four units are chemically rather similar. After they join together in long chains, the enormously long molecules that form have nearly identical chemical properties regardless of the arrangement and composition of their components. This makes specific regions of these DNA's -- genes -- almost impossible to separate on the basis of their chemical behavior.

Contrast this situation with proteins, the other large polymers that we've encountered. They are composed of 20 different kinds of units (amino acids) many with quite different chemical properties. Different proteins have been separated from one another for decades (James Sumner was the first to crystallize an enzyme -- urease -- in 1926) because of the distinct chemical and physical behaviors that these different amino acids contribute.

Large pieces of DNA are very fragile
A second problem derives from the enormous size of DNA. Each chromosome consists of a single molecule of double-stranded DNA that is extremely long. The individual chromosomes of humans, for example, consist of hundreds of millions of bases. They are so long that if these molecules were to be strung out (most of the time they are folded back on themselves many times) to their maximum size, they would be several inches long. In fact, the total length of all the DNA in all the chromosomes in each dipoid human cell is about two meters (more than two yards, for those who are metrically challenged). Because of their long length but very thin diameter, these molecules are extremely fragile when removed from the cell. Gently shaking the container in which they are collected will shatter them into many smaller pieces. And, unfortunately for the biochemist, the DNA breaks randomly, not conveniently at the ends of genes.

Consequently, when biochemists extracted DNA from a piece of tissue of higher organisms containing millions of cells, they were left with a mish-mash of pieces of many millions of molecules randomly broken into different sizes. Some of the pieces contained one or more whole genes, and some had pieces where one or more genes were broken. Since every piece was broken randomly, each piece was different. Since all these pieces had similar chemical properties, biochemists couldn't figure out how to isolate the specific piece that they wanted in any quantity.

The three steps of gene cloning
Gene cloning was invented in the early 1970's in an effort to remedy this situation. Gene cloning is essentally an amplification procedure that uses biological methods in the place of of chemical or physical techniques to increase the amount of specific sections of DNA. Instead of dealing with a woeful mess of DNA pieces of all sizes and origin, one piece is replicated by this method.

I'm going to discuss a cloning procedure in which the goal is to obtain a particular gene -- for example, a hemoglobin gene -- from the DNA of a higher organism such as a human. Gene cloning can take other forms, some of which we'll discuss later.

There are four major steps in the process.

1. First, the DNA of the organism containing the gene (or, for that matter, any sequence of nucleotides) of interest must be broken into smaller pieces that are convenient to manipulate. These pieces of DNA, one which is the object of the cloning procedure, are called passenger DNA.
2. Second, the pieces of passenger DNA must be joined to a second piece of DNA that can replicate itself and any attached passenger. This second DNA is often called a vector or cloning vehicle. The result of the joining is a hybrid molecule, a chimera, a recombinant DNA, consisting of two kinds of DNA coupled to one another on a single piece (often a closed ring) of nucleic acid.
3. Third, the joined passenger and vector must be introduced into a living cell. The cell serves as a biological copying machine, making many exact copies of the recombinant molecule.

The second step of the cloning process seems counter-intuitive. The goal is to purify a piece of DNA, yet one adds a "contaminating" DNA, in the form of the vector, to the passenger. As we'll see, vecotr DNA is critical to the cloning process.

The process is called molecular cloning because the hybrid DNA molecule replicates within the living cell without further recombination. In addition, the cell in which the recombinant molecule finds itself multiplies many times asexually forming a colony. Each cell in colony is an exact duplicate of every other, and each carries many molecules of one particular recombinant DNA molecule.

The fourth step

4. Once a colony has grown with a hybrid DNA contained within it, the essence of molecular cloning has been achieved. Subsequently, all the genetic engineer has to do is break open the cells, isolate the recombinant DNA, and begin to work with it. However, most of the time the work has just begun. That's because a typical genetic engineering experiment produces many colonies of cells, each of which may harbor a different passenger/vector complex. Thus the last step of molecular cloning: The identification of the colony that carries the particular passenger of interest.

How can recombinant molecules be used?
In the following sessions, each of the steps of molecular cloning will be described in more detail. But before getting to them, I think it worthwhile to discuss briefly how purified recombinant DNA molecules can be utilized. More specific examples are presented in later sessions.

• The passenger DNA can be sequenced. That is, a researcher can figure out the order of the successive bases in a piece of DNA. Determining the sequence of thousands or even tens of thousands of bases is now carried out, more or less routinely, in even the smallest of molecular biology and genetic engineering laboratories. In the last year or two, with the advent of automation and new techniques, the rapidity of DNA sequencing has increased by an order of magnitude or more. The human genome sequencing initiative proposes to take advantage of these advances to determine the DNA sequence of the three billion or so bases of the 23 human chromosomes. Besides new biochemical techniques, very large sequencing projects will require sophisticated computer programs to keep the enormous amount of data in a usable form. I will discuss the impact of computers on genetic engineering later in the semester.
• The sequence of any protein can be changed. In principle, any enzyme -- or for that matter, any protein -- can be altered by modifying the DNA that codes for it. Prior to recombinant DNA technology, proteins could be modified by chemical treatment, but that was often done with great difficulty, in low yield, and the range of changes that could be introduced was small. Now, virtually any aspect of a protein's primary sequence can be easily refashioned as long as the gene that codes for that protein has been cloned. By making these changes, genetic engineers dream of producing enzymes that can catalyze reactions that are unknown in nature or modifying enzymes to make them more stable or more efficient. Unfortunately, not enough is understood about the relationship of the primary structure of proteins to their function to take full advantage of the changes that the technology allows. Being able to accurately predict the structural consequences of making changes to a protein's amino acid sequence is one of the major questions that genetic engineers are applying to themselves to in the next few years.
• The function of genes can be deduced. Molecular cloning has had an enormous impact on this area. The usual method of analysis is to change the DNA sequence of a particular gene and to determine what effect the change has on gene function. By this means, a scientist can reason backward and figure out what any given sequence does. In particular, alterations in DNA combined with transformation (the ability to introduce DNA molecules into living organisms) has already yielded exciting insights into how genes are regulated and controlled.
• The passenger DNA can be used to synthesize a protein of interest, often in large quantities. The interleukins, blood clotting factors, growth hormones, and growth factors are among the proteins that have made their way from the recombinant technology workbench to money-making products. This area has been the most visible manifestation of genetic engineering, and many more products are in the pipeline.
• A gene can be introduced into an organism that is lacking or deficient in a particular function. The old dream of gene therapy has become real. On the farm, transgenic animals and plants are playing an increasingly important role. For humans, whether it will prove to be a widely used tool or even one that will be practical in the near future is another matter. If gene therapy can be done in humans, we will have to wrestle with the ethical problems that this ability raises, especially if we mean to tamper with genes in the germ line. We'll discuss some of these ethical later in the course .
• A cloned DNA sequence can be used as a diagnostic tool. Prospective parents can be checked for a wide range of genetic disturbances. Embryos can be assayed for the absence or presence of genetic disease and undesirable traits. Paternity, maternity, and genetic relatedness can be established with unprecedented assurance. In the crime laboratory, DNA "fingerprinting" may complement the more familiar kind. The ethical and social problems that these techniques will introduce are enormous. We'll take time to discuss some of them later.

Summary

1. The first step in molecular cloning is to cut the passenger DNA into pieces of convenient size.
2. The passenger is then joined to a vector DNA, forming a hybrid (or recombinant DNA) molecule.
3. The recombinant DNA is then introduced into a convenient host.
4. Finally, the clone that harbors the desired recombinant DNA molecule must be identified.