Genetic Engineering III

Cloning of foreign DNA (in plasmid or phage vectors) is most easily carried out in E. Coli because the organism has been so thoroughly studied.

Subsequent manipulations often require vectors that can deliver a cloned segment into eukaryotic cells (e.g. - to understand the mechanism of gene expression in a eukaryotic cell). A number of vectors have been devised to satisfy this need. They often contain pieces of eukaryotic viruses to facilitate entry into the cell and expression or integration once in the cell itself.

Note that each small extrachromosomal genome (phage, plasmid, or eukaryotic virus) is found in nature within a particular species and replicates only within cells of its natural host or within cells of closely related species. The fundamental tool is, therefore, a two component system: a host-vector system.

Expression vectors allow you to express certain genes directly from their recombinant DNAs. A typical expression vector will have a promoter upstream of the DNA containing the sequence to be expressed. Usually the "gene" is a cDNA because if the gene contained introns the introns would not be removed in bacteria. In addition, the promoter that is used can be an inducible one, so that synthesis of the gene product can be regulated. For example, if you wanted to express human growth hormone, you cut the DNA with an appropriate restriction enzyme to isolate the growth hormone. Then open the vector with the same restriction enzyme, allowing you to place the growth hormone cDNA downstream of a lac promoter. In the presence of lactose, bacteria containing this construct will produce human growth hormone.

This approach has made available many reagents that were not available before such as tissue plasminogen activator for the treatment of heart attacks, erythropoietin for treatment of anemia, interferons for treatment of cancer and hepatitis infections, human insulin for diabetes, and many others to come.

Shuttle vectors allow DNA to be transferred between two different species. The shuttle vector has two origins of replication, allowing replication to occur in either system/host; it "shuttles" between two different species. Typically, one host is bacterial (e.g. E. Coli) and the other host is a eukaryotic organism (e.g. human). The bacterial host is used for all of the cloning steps and the eukaryotic host can be used to study the expression from that cloned gene or can be used to synthesize a product from the gene.

Shuttle vectors can be used to perform what has been called reverse genetics. It is possible to replace or alter the sequence of regulatory elements that control expression of a given gene -- then put gene back into their normal host cells to see how gene behavior has changed. This provides information on how the regulatory element might function and which are the important sequences within the regulatory element itself. Our understanding of promoters was brought about through this kind of study.

Another possibility is to put a regulatory element to be studied in front of a gene whose activity can be easily evaluated, such as the -galactosidase gene from the lac operon. There is a simple way to measure the level of -galactosidase in any cell -- the enzyme turns a colorless substrate called x-gal into a blue substance. Therefore, by adding this substrate to tissues, one can just measure the level of "blueness" to know the level of expression of the -galactosidase gene in that tissue. The -galactosidase gene is being used as a reporter gene in this case because it "reports" on the activity of the promoter by which it is controlled. Reporter genes provide a powerful way of determining in which tissues and under which circumstances specific promoters are active.

-galactosidase can be used in another way to measure the amount of transcription when the location of the promoter is unknown. Starting with the left end of the DNA being tested for the promoter, begin by deleting the DNA upstream of the -galactosidase gene, monitoring whether or not transcription occurs. If transcription still occurs, the promoter has been untouched. However, if transcription does not occur, part or all of the promoter has been deleted. The other end of the promoter can be tested by using restriction enzymes to delete fragments of the DNA.

One of the first things scientists do after isolating a specific fragment of DNA is to create a restriction map. A restriction map is simply a "map" of the locations different restriction enzyme cut along the length of the DNA. Restriction maps are useful in cloning of DNA fragments and for identifying specific regions in the cloned DNA (e.g. exons, introns, and promoters). Restriction maps can be created in sufficient detail that they can uniquely identify a piece of DNA as being different from any other piece of DNA.

Once a restriction map is produced, and it sufficiently shows the details of DNA, the next step is to discover where on the cloned DNA, the gene lies. It is also necessary to figure out if what is found is the entire gene sequence or only a fragment of it. We can do this through DNA blotting followed by hybridization with a probe. In this protocol, the cloned DNA is digested with a specific restriction enzyme or with a set of restriction enzymes and then the different restriction fragments are separated by size using gel electrophoresis. After being separated, the fragments are transferred directly from the gel onto a nitrocellulose filter, producing a replica on the filter of the pattern of bands on the gel. In a process similar to that for screening a library, the filter is treated to denature the DNA and then to anchor the DNA to the filter. This filter is then hybridized with a radioactive probe containing the sequence of interest. For example, an experiment might use a radioactive pure mRNA to hybridize to a gene for that mRNA. In this case, only those restriction fragments containing exon sequence will hybridize. The hybridized regions on the nitrocellulose filter can be detected by exposing x-ray film.

Genomic blotting is a technique in which the starting material for the electrophoresis gel is whole genomic DNA rather than just the DNA from a cloned fragment. In this case, the probe will hybridize only to the restriction fragment(s) from the total genomic digest that contains its complementary sequence. Different individuals might have slight differences in their genes and this difference will be detected in genomic blotting of this type. For example, two individuals will both have insulin genes, but the two genes might have slightly different sequences either in the gene itself or in the flanking regions to the gene. This could lead to different restriction fragment sizes for different alleles of the same gene.

This difference can be used in forensics to identify individuals and can be used by scientists to identify similar genes from different species.

RFLP mapping is based on the fact that allelic DNA segments on homologous chromosomes frequently have slightly different sequences. For example, there may be a single change, a small deletion/insertion of a sequence, a slight rearrangement of a sequence, or a slight difference in number of repeat genes. Such differences in sequence are detectable by DNA blotting because different length restriction fragments will be produced. These differences in restriction patterns are called restriction fragment length polymorphisms or RFLPs.

The ras gene's RFLP patterns can be observed. It has been implicated in a number of different cancers. (figure 7.7 from text)

RFLPs are common -- even in two non-mutant alleles. Thousands of specific RFLP sites have been identified so far. RFLPs can occur in coding or non-coding regions. Changes in DNA sequence in a coding region might not affect the information because of redundancy in the genetic code. The RFLPs (segments of DNA) are inherited in a Mendelian fashion. Thus, each RFLP probe is equivalent to a specific site on the DNA for genetic analysis. By relying on RFLPs, which are plentiful, instead of mutations, which are rare, geneticists are beginning to fill in the previously sparse human genetic map. The closer together two RFLP markers are along a chromosome, the less likely there will be crossing over between the two positions -- just like two genes. Using this kind of information and starting with many candidate RFLPs, an RFLP map of the human genome is being constructed.

For example, the cystic fibrosis gene and RFLP markers have been observed. In this case, the appearance of the disease correlated very closely with some specific RFLP marker sites. Starting with those "close" RFLP sites, scientists then "walked" along the chromosome until they found the gene which is defective in cystic fibrosis. Once the gene was identified, the protein product could be identified by cloning the cDNA for the gene and synthesizing the product in E. Coli. After the protein was identified, its function (and malfunction) could be studied and a treatment devised. It is hopeful now that a treatment can be found for this previously untreatable disease. Other diseases are being tackled in the same way.

Because there are several thousand RFLPs known, each individual in a population is likely to be different from every other individual at many of these sites. The set of RFLPs that an individual has is a kind of fingerprint of the individual's DNA -- a DNA fingerprint. This has implications in forensic and paternity cases.

If enough starting tissue is available, 20-30 different RFLPs can be ascertained from an individual sample (e.g. blood, hair, semen). If there are 4 alleles at each RFLP then 20 different RFLPs present 4²⁰ (=10¹²) different possible combinations. Therefore, a match at each of 20 locations will occur by chance once every 10¹² individuals. (This is about 200x the population of the Earth.) Some RFLP locations may have hundreds of different alleles.

Some probes can be used to hybridize restriction fragments containing repeated sequences found at numerous locations in the genome. Each individual will have a different number of repeats of a given sequence and therefore a different length restriction fragment (the RE does not cut within the repeat) at each location hybridizing with the probe. So, in DNA fingerprinting which uses repeated sequence RFLP, if an individual matches at all RFLPs, it is a fairly convincing identification of a specific person. However, a single different RFLP is enough to rule out a specific individual.