Transformation (based upon Griffith's discussion of a transforming principle) is the ability of a cell to pick up, and utilize, foreign DNA. All cells have the ability to pick up DNA, but transformation is the process by which that foreign DNA becomes incorporated into the cell's own DNA, affecting functioning, and being passed on to its progeny. How can you tell whether or not a property was picked up?
Selection allows you to identify which cells in an experiment picked up and utilized the gene, and which ones did not. If you control the environment, this is not difficult at all. Imagine a galactose filled tube where those cells which can't grow in galactose because they did not incorporate the galactose-metabolizing gene will die off. Once those cells are all dead, the ones left alive are the ones which incorporated the galactose metabolizing gene. This is an example of an experiment in which the biology does the work for you.
Conjugation is a process that is similar to sexual reproduction. Two bacterial cells form a conjugation bridge which connects them. The DNA from one of the bacteria moves across the bridge as it (the DNA) is replicated. The bridge is very fragile so many times the bridge breaks when only some of the DNA has been transferred. The transferred DNA can recombine with the original DNA (the cell with the transferred DNA is actually diploid for those genes that were transferred by conjugation). This recombination is carried out by enzymes and is similar to crossing-over in meiosis. When division occurs, the daughter cells will be possess this new DNA, and will thus be different from the parent cell (before the parent cell incorporated the foreign DNA).See figure 4.2 in text.
Another way to transfer information is through phage infection. The transfer of genetic information by this means is called transduction. When the phage attacks the cell, cuts up the cell's DNA, and replicates, the progeny phage sometimes pick up small fragments of the cell DNA instead of phage DNA, and integrates the fragment into the phage head. The progeny phage will then inject this cell DNA into another cell. These recipient cells would then have a diploid situation for this DNA segment and recombination can occur.
Another mechanism by which phage can transduce DNA is to start by integrating into the host DNA through cross-over to become a prophage. Sometimes when the prophage DNA excises itself it picks up part of the host DNA with it. This extra host DNA can be delivered into the next cell the progeny phage infects resulting, again, in a partial diploid situation and produce recombination. Figure 4.5 in the text shows a lambda phage infection of this sort, where an imprecise excision can cause cell DNA to be incorporated into the phage DNA.
When behaving in this way, the phage DNA is acting as a vector. A vector DNA is any DNA that is capable of carrying a foreign piece of DNA to a different location than its source location.
Specific DNA pieces can be isolated through molecular cloning. A petri dish is covered with bacteria. Phages are added, each of which will infect a cell. The infected cells will lyse, releasing progeny phage that will infect any nearby bacteria. The process is repeated and eventually the lysed cells will appear as a clear circle on the dish. This circle is called a plaque which is a clear area on a bacterial lawn that contains phage. Every single progeny phage in the plaque will be identical because they all were derived from the same parent phage - all the progeny phage are clones since they contain identical genomes.
Another kind of cloning is performed with bacterial cells. One cell is placed in a petri dish. This cell will eventually form a colony of identical cells through repeated rounds of division. All of the cells will have identical information as the parent cell - these cells are all clones. See figure 4.8 in text.
In order to learn more about a particular gene, it is possible to artificially insert the DNA containing that gene into another DNA and allow the biology to take over and replicate the gene. Linear phage DNA can be cleaved into two parts. A foreign DNA insert can then be added between the two phage ends. When the phage DNA is used to carry another foreign DNA in this way, the phage DNA is said to be a vector.
In cloning you start out with phage vector DNA and cut (cleave) it in the middle. Human DNA (or any foreign DNA) containing the gene you want to study is cut into many pieces and mixed with the phage DNA. The phage DNA picks up pieces of human DNA and inserts them between two phage arms. Each little segment of human DNA is cloned into a separate phage DNA. DNA ligase allows the phage DNA and the foreign DNA to be joined together. When this new recombinant DNA is put back into the cell, the particular gene that you are interested in learning more about will be replicated as the DNA is replicated.
The collection of all the recombinant DNA molecules containing phage which contains segments from the entire (human) genome is said to be a recombinant DNA library. In order to create this library, the recombinant DNA is incorporated into virus particles and grown in a Petri dish containing a lawn of bacteria. Individual plaques will develop corresponding to each phage. The phage in each plaque are identical and carry a specific piece of human DNA. (See text Figure 4.10). In this manner the whole human genome can be cloned. Human DNA consists of about 6 billion base pairs, which in the process of cleavage are divided into about 300,000 fragments of 20,000 basepairs each. Each of those 300,000 fragments is then cloned into a vector.
Plasmid DNAs are a small circular DNAs in prokaryotic cells that can range in size from 2000-40,000 base pairs. Antibiotic resistance genes are often encoded on plasmid DNAs. Antibiotics are naturally occurring substances that kill bacteria. Any cell which contains a plasmid with an antibiotic resistance gene will provide the cell with the ability to grow in the presence of that particular antibiotic. This is why antibiotics must be taken for a certain amount of time even if symptoms have disappeared, because the bacteria that was still alive would be resistant to the antibiotic (All of the non-resistant bacteria would have been killed) and would replicate itself, creating more resistant bacteria.
Plasmid DNA can also be used as a vector. For example ampicillin resistance plasmids allows the cell to grow in the presence of ampicillin. Foreign DNA can be inserted in the plasmid DNA in the same way that foreign DNA could be inserted into the phage DNA as was discussed above. see text Figure 4.11.
Restriction enzymes recognize specific sequences on DNA, then cut the DNA at those sequences. There are about 2500 known restriction enzymes which have different specificities for cutting the DNA. A complete list is available in the REBase web site. see text Figure 4.12. Most of the sequences that are recognized by restriction enzymes are palindromic, meaning they read the same backward and forward. For example, the restriction enzyme BamH1 recognizes the DNA sequence GGATCC. The complementary strand of DNA, reading in the opposite direction, would also read GGATCC. This palindromic quality makes it easier for the restriction enzyme to recognize the sequence, since it is present on both strands of DNA.
Sometimes the restriction enzyme cuts straight through the DNA, cutting both strands at the same location. Most of the time, however, restriction enzymes cleave the DNA in a staggered cut - leaving a few nucleotides of single stranded DNA extending from the cut site. For example, the BamH1 restriction enzyme cuts both strands of DNA between the adjacent G's, leaving "sticky ends" of DNA on each strand:
5'-----G GATCC-------------CCTAG G-----5'These sticky ends allow separate DNA molecules to get together. The short sticky ends actually can base pair between two different DNAs to align the two DNA molecules. Any two DNAs cut with the same restriction enzyme will have the same sticky ends and therefore can be joined. It is this ability provided by restriction enzymes that allows most of recombinant DNA techniques to work.
BamH1 will recognize this sequence and cut DNA approximately once in every 4096 basepairs (46). If a plasmid is cut by a restriction enzyme, it will also have sticky ends and could base pair with pieces of human DNA. In this way it is possible to create a plasmid library similar to the way it is done with phage vectors.
Once the DNA has been cut into pieces by restriction enzymes, different sized DNA molecules can be separated by means of gel electrophoresis. The DNA is pushed by an electric field through a gel (a three dimensional matrix of fibers that are all tangled together - the texture of a gel is just like Jell-O, another gel). The big DNA molecules move slowly because they have trouble getting through the net. The small molecules, however move right through. Intermediate sized molecules move at an intermediate rate. Thus the different sizes are separated and can appear as discrete bands on the gel. This is useful to isolate a particular piece of DNA.
To create a human library using plasmid DNA vectors involves transforming cells with DNA and then selecting those cells in the population that have picked up the recombinant DNAs. To generate the recombinant DNA library, you mix together linearized plasmid DNAs and human DNAs and incubate the mixture under conditions that will facilitate forming recombinant DNAs. After incubating, the mixture will contain some recombinant DNA molecules, some non-recombined plasmid DNA and some plain old human DNA. This mixture is then incubated with E. coli under conditions that favor the bacteria picking up DNA from their environment.
From this mixture of bacteria containing different DNAs, you want to isolate only those that have picked up recombinant DNA molecules. The trick one can use is to utilize a plasmid that contains two antibiotic resistance genes: an ampicillin resistance gene, and a tetracycline resistance gene - the insertion point for the foreign DNA is in the middle of the tetracycline gene. The four possible different kinds of bacteria in this mixture can then be represented as having the following antibiotic resistance traits:
The idea now is to select only those bacteria containing recombinant DNA. Ampicillin will kill any non-resistant bacteria. Tetracycline will stop non-resistant bacteria from growing but will not kill them. To perform the selection do the following:
If you want to find a particular gene within your library you go through a process called screening the library. For example, let's detail the process used to find the globin gene. First, the phage library is spread out on a series of petri dishes containing bacterial lawns. Plaques are allowed to form, each plaque representing a specific fragment of cloned DNA. A nitrocellulose disc is then overlaid on each petri dish and then carefully peeled off from the surface of the dish. This yields a replica of the phage plaques in the exact same location as the original petri dish.
The disc is treated to remove proteins, to denature the DNA and then to anchor the DNA to the filter. Next a radioactively-labeled nucleic acid from red blood cells (a radioactive probe) is hybridized to each filter disc. The probe (globin mRNA) will hybridize only to its complementary DNA sequence on the filter. The unbound probe is removed and the disc is exposed to x-ray film.
Only the DNA from the plaque that has hybridized to the probe will expose the x-ray film. This allows the particular clone containing the desired gene to be identified and then grown up by picking phage from the original petri dish from which the filter was made. see text Figure 5.3.
If you are just interested in the mRNA sequences, it is possible to clone them alone. An enzyme called reverse transcriptase is used to make a complementary DNA by synthesizing DNA starting from the poly(A) tail on the mRNA. This complementary DNA (cDNA) runs the along the mRNA and creates a double stranded molecule that has one DNA strand (the cDNA) and one RNA strand (the mRNA) paired to each other. Through a number of enzymatic and chemical steps the RNA is replaced by DNA in this hybrid molecule, resulting in a double stranded cDNA molecule (a "complementary DNA")
The advantage of using this approach is that one can create a cDNA library of only those sequences that are expressed in a given tissue. Only those genes which are expressed will produce mRNA. It is possible to create tissue specific cDNA libraries: a liver cDNA library, kidney cDNA library, tumor cDNA library. By studying the composition of the cDNA library it is possible to gain some insight into gene expression in different tissues.
Another useful feature of cDNA clones is that they do not contain any introns, only exons, since they were made from an mRNA template. All introns are removed during RNA processing. By comparing the sequence of a cDNA clone with the sequence of the corresponding genomic clone, it is possible to determine the location and size of all of the introns in a gene. Finally cDNAs can be used in expression vectors.
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 b-galactosidase gene from the lac operon. There is a simple way to measure the level of b-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 b-galactosidase gene in that tissue. The b-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.
b-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 b-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.