C2005/F2401 '06 -- Lecture 1
7 -- Last Edited: 11/09/06 10:04 AM (Problems to do are indicated in red bold.)Handouts 17A &B -- not available on the web;
copies are in boxes outside 744 Mudd.
17A = Restriction enzymes, RFLP's, & Overview of Cloning; 17B = Making a
Library & Cloning Vehicle.
Note: References to texts are to Purves 7th ed. & Becker 6th ed. (refs. to previous ed. of each text, 6th & 5th respectively, are in parentheses). Recombinant DNA is covered in a different chapter in each edition. (This is true for both texts.)
I. Wrap up of Viral Genetics -- Continuation of IV-C from lecture 16.
C. Transduction & The Viral Life Cycle
7. Lysogenic Cycle
a. Integration. Some viruses can become part of the host chromosome by crossing over between the viral DNA and the bacterial DNA -- the process is parallel to the way a plasmid such as the F factor joins the bacterial chromosome. (Viruses, like plasmids, can pick up bacterial genes by the reverse of this process.)
b. Lysogeny. The integrated virus can remain dormant for long periods of time. This dormant state is known as lysogeny, and a bacterium with an integrated, dormant, virus is said to be lysogenic (capable of entering the lytic cycle). What keeps the virus from making viral proteins and entering the lytic cycle? A repressor protein made by the virus itself. (This repressor protein is not allosteric; it must be destroyed to to inactivated. The degradation of repressor protein allows the virus to leave the dormant state and enter the lytic cycle.) Jacob, Lwoff, & Monod received the Nobel Prize in Physiology in 1965 for figuring out how repressors control both operons and lysogeny.
c. Retroviruses. These are viruses (not necessarily of prokaryotes) that contain RNA in the viral particle. When the RNA enters the cell, it uses a special enzyme made by the virus, reverse transcriptase, to make a DNA copy of the RNA. (The reverse transcriptase is carried into the host inside the viral particle.) The DNA then inserts into the host chromosome and remains dormant, in much the same way as a lysogenic virus. HIV, the virus that causes AIDS, is a human retrovirus. Reverse transcriptase obtained from retroviruses is used in the lab as an important tool to make DNA copies of RNA. (Examples will be discussed next time.) For HIV life cycle see Purves 13.5. The 1975 Nobel Prize in Physiology was awarded to Dulbecco, Temin & Baltimore in 1975 for the discovery of reverse transcriptase in tumor causing viruses.
8. Viral Crosses -- If a cell is simultaneously infected with two variants (mutants) of the same virus, then crossing over and/or complementation can occur between the two viruses during the course of infection.
For an example, see problem 11-8. (For more problems involving compl. & recomb. in viruses, see 11-10 to 11-13.)
See last lecture, V-B-4 and handout 16B for more details on complementation (& how to distinguish complementation and recombination). See Becker fig. 20-18 for recombination in viruses. (Re-assortment may also occur in the case of flu virus, which has an RNA genome segmented into 8 pieces. See CDC page for more details. For life cycle of an RNA virus, see Purves 13.4)
II. Restriction Enzymes
A. Introduction: The idea/problem: The existence of plasmids and bacteriophages carrying bacterial genes inspired dreams of genetic engineering. Why not make new combinations to order? That way we could make plasmids with useful genes and add them to bacteria (or even human cells)! But how do you make recombinant plasmids in a laboratory? DNA is very, very long. How can you cut it into useful sized pieces, find the right pieces, stick them together, etc.?? The solution was discovered by pursuing a phenomenon known as restriction (described below), which seemed at the time to be of no practical consequence whatsoever.
B. Discovery of Restriction & Modification Enzymes See Becker Box 19B (16B) and/or Purves 16.1 (17.1)
1. The phenomenon: Some phages will not grow well on certain bacteria. The growth of the phages is said to be "restricted." But an occasional virus particle does manage to complete an infection and lyse a bacterium in spite of restriction. If it does, the progeny phage from the lysed cell grow just fine on that same type of bacteria -- they are no longer restricted. However, the sequence of bases in the DNA is the same before and after restriction! What's going on here? The solution holds the key to all genetic engineering and recombinant DNA technology, and the scientist who discovered it (Arber) received the Nobel Prize in 1978 along with two other scientists (Smith & Nathans) who extended Arber's work.
2. Restriction enzymes. What causes restriction?
3. Modification enzymes. Why are bacteria "immune" to their own restriction enzymes? Why don't the restriction enzymes cut up the bacterium's own DNA?
4. How do rare virus particles escape restriction? If DNA is accidentally methylated, then it will be methylated every time it is replicated thereafter. The rare particles have DNA that was methylated accidentally. Therefore the DNA of the rare virus particles and their progeny are resistant to the restriction enzymes.
5. Modification in general: Modification of macromolecules by enzymatically adding or removing a small group or two is very wide spread, especially in eukaryotes. It is a common method of regulating and/or fine tuning macromolecular function permanently or temporarily (many modifications are reversible). For example, many enzymes are activated or inhibited by addition of phosphate groups; some sections of DNA may be kept "off" by addition of methyl groups. Many more examples will follow next semester.
C. Examples & Properties of Restriction Enzymes (Details on Handout 17A & Becker Box 18B [16B])
1. Restriction Sites are often palindromes (= read the same forwards and backwards)
| English examples: | "Madam I'm Adam" |
| "Able was I ere I saw Elba." | |
| DNA example: | 5' GAATTC 3' 3' CTTAAG 5' |
What do we mean by "reads the same backwards and forwards" for the DNA example? There are several ways to explain this :
a. By base sequences. The sequences on the two individual strands are the same if both sequences are read 5' to 3'.
b. By base pairs. The base pairs are the same, right to left and left to right as long as the "top" strand is always 5' to 3'.
c. By rotation. If you rotate the DNA 180 degrees, it looks exactly the same.
2. State of the ends. Cuts made by restriction enzymes can be staggered (generating so called "sticky ends") or blunt (see handout 17A or Becker Box 18B [16B] for examples)
3. Sites can sometimes be methylated -- this makes the sites resistant to cutting. (See Modification enzymes, above.)
4. There are a wide variety of restriction enzymes made by different bacteria. Therefore there are many different options for cutting up any given DNA. For example:
- Some enzymes recognize relative short sequences. For example, an enzyme may be a "4 cutter" = enzyme that recognizes a 4 base pair site. (See handout.) Short sites (sequences) are found more often, and enzymes that cut them produce many relatively short fragments.
- Some enzymes recognize longer sequences. Longer sites are found less often, and enzymes that cut them produce a smaller number of relatively long fragments.
D. Significance of Restriction enzymes = Essential tools for Recombinant DNA Analysis
1. Allows you to cut up DNA into manageable size pieces for manipulation and analysis.
a. Most DNA is very long. Without some sort of breakage, most DNA molecules are too big to handle.
b. Before this, all known DNases cut at random --> big mess (random collection of different sized pieces).
c. Restriction enzymes cut DNA into fixed size pieces. Pieces resulting from restriction enzyme digestion can be separated by size using gel electrophoresis. (Purves 16.2 (17.2) or Becker fig. 18-12 [16-12].) Principle is similar to SDS gel electrophoresis, except no SDS is used -- all nucleic acids are negatively charged and migrate to the positive pole -- smaller fragments travel farther. (Same procedure as used for analysis of PCR products.)
2. Forensics/ IDs & RFLPs
a. Inherited variations in base sequence lead to differences in places where DNA is cut. For example, if a sequence is GAATTC, EcoR1 will cut the DNA. If the sequence is changed to GGATTC, EcoR1 will not cut the DNA. So a change of A to G can "remove" a restriction site while a change of G to A can "add" a restriction site. (See handout 17A.) Note "addition" or "loss" of a restriction site does not mean the insertion or deletion of bases. An "addition" means a change in base sequence so a particular stretch of DNA is now recognized by a particular restriction enzyme; a loss means a change so that the DNA is no longer recognized by the restriction enzyme.
b. Detection. Differences in restriction sites (or variation in the lengths of the sequences between sites) are detected by cutting the DNA with restriction enzymes and running gels of pieces (to compare sizes).
c. RFLPs. When DNA from different individuals gives a different pattern of pieces, this is known as a RFLP = restriction fragment length polymorphism. (See handout 17A or Becker box 18C [16C] for examples.)
d. How does this compare to PCR fingerprinting? Note PCR method for DNA fingerprinting described previously picks up differences in the numbers of repeats in a particular region. This method picks up differences in the number of repeats between two fixed restriction sites and detects variations in sequences at possible restriction sites. (Also, to use PCR, you have to know enough about the DNA to make the correct primers.) Some uses of RFLPs will be explained in detail later, after human genetics. See Problem 13-3.
3. Splicing. Existence of "sticky ends" allows you to splice as well as cut DNA's using overlapping ends and ligase. See handout 17A, Becker fig.20-26 [18-26] or Purves 16.4 (17.4) Try Problems 13-1 & 13-2 parts B & C. Why is this helpful? See below.
III. Molecular (DNA) Cloning -- how do you isolate a gene and why bother?
A. What is DNA cloning? Means making a chimeric (recombinant) plasmid or virus and growing it up. For an overview, see Becker 20-27 [18-27] & handout 17A.
B. Why bother?
1. To get a lot of copies of a particular gene/DNA.
a. To look at gene structure. Allows you to examine structure of gene and/or its regulatory sequences. There are some big surprises from this, as will be discussed next time.
b. Why is cloning necessary nowadays if you have PCR? Can't do PCR if don't know flanking sequences & can't make primers -- have to clone the first time.
2. To get a lot of gene product (protein, not DNA). Set up conditions where gene will be transcribed and translated. This allows you to
a. Measure gene expression (how much product a gene makes) and its regulation. Some examples of this next term.
b. Produce useful amounts of a product that is otherwise unobtainable in large quantities (like growth hormone or erythropoietin (EPO). See Purves 16.14 (17.15) for info on a similar example (TPA).
3. For gene therapy -- to restore function to a defective cell. Want to add gene so it will remain and supply a missing product as needed.
C. Why you need a " cloning vehicle" or "vector." (See "fragment vs. plasmid" on handout 16B.) Need to add your gene of interest to a plasmid or modified virus so your gene can be replicated (using the origin of replication of the plasmid or virus). If you use a virus, it is usually modified, so that the DNA you are cloning replaces some of the viral genes.
D. How to actually make a chimera/library -- (Becker 20-27 [18-27] or Purves 16.7 (17.8)) Numbers 1-6 match steps on handout 17B.
1. Cut up all DNA. Cut up genomic DNA from say, humans, in pieces with some enzyme; cut cloning vehicle with same enzyme. (Generate matching sticky ends as on handout 17B.)
2.
Make recombinant plasmids (or viruses.) Mix fragments and cloning
vehicle (plasmid or virus), let sticky ends match up and add enzyme to ligate (Becker fig.
20-28b [18-28b]). This generates a
collection of plasmids (or viruses) with inserts. Each plasmid = one "book." The
entire collection of plasmids/books = "a library." Some plasmids
may lack an insert, as indicated in middle case on handout. (How to screen them out? See
below.)
3.
Get recombinant (chimeric) plasmids or viruses into cells.
Transform bacteria with recombinant plasmids (or infect
with recombinant viruses). Ideally, each bacterium gets one recombinant plasmid
or virus = gets one insert. (In cases shown on handout, cell on left has a
plasmid with an insert, middle cell has a plasmid with no insert, and cell on
left has no plasmid. You will screen out all but the left case.)
4. Grow up the "library." Grow up the cells that received plasmids; screen out those that got no plasmid (or no virus).
a. How? Set up conditions so that only cells with plasmids will grow -- details in E-3 below. Handout shows a Petri dish with 6 colonies = 6 clones of bacteria with plasmids. You know all these clones contain plasmids because only cells with plasmids will grow under these conditions. Remaining steps will allow you to figure out which colonies have plasmids with inserts, and which ones are useful.
b. What makes it a "library?" Each colony or plaque = clone = descendants of one cell with the same recombinant plasmid (or virus). Each cell (or virus) in a clone has a copy of the same 'book' in the library = carries same added sequence (same insert). The entire collection of clones with different inserts = collection of 'books' = a library.
5. Replica Plating. How to weed out all the clones that carry plasmids without inserts. A way to identify colonies that do NOT grow under some particular conditions (and still recover viable cells). More details below.
6. Find the right clone. This is the only hard part. Often you want to find 1 out of more than 50,000 possibilities! Step 6 shows one way to find the right clone -- by replica plating; next time we'll discuss another way -- by colony hybridization. To understand how you eliminate the clones you don't want and find the clone you want, you need to know some more details on the cloning vehicle.
E. Details to know about plasmid/virus = cloning vehicle. See handout 17B (right half) or Becker fig 20-28a [18-28a] or Purves 16.5 (17.5) -- How to get a vehicle with an insert.
1. Why you want 1 recognition site for each restriction enzyme per plasmid. You want 1 insertion site on each plasmid (for each dif. enzyme). There may be many different sequences recognized by dif. restriction enzymes, but only one site for each.
2. Why sites for many dif. enzymes -- so you can cut up DNAs in different ways and insert any of the fragments in the same plasmid.
3. Why drug resistant -- need a selectable "marker" on the plasmid -- gene that confers growth only to cells that got the plasmid. Then cells without the added "selectable marker" (that is, without a plasmid) won't grow under some condition. For example, the "selectable marker" can be a gene that confers resistance to the antibiotic tetracycline. (Tet-R) This allows you to select for cells that got a plasmid and against cells that got no plasmid at all. Cells without plasmids will be sensitive to the drug and won't grow in the presence of antibiotic. Only cells that have received a plasmid with the Tet-R gene will be drug resistant and will grow in the presence of antibiotic. (So Petri dish with 6 colonies shown at end of step 4 would have tetracycline in the medium to eliminate growth of cells without plasmids.)
4. Why usually double drug resistant or has second selectable marker (See Purves 16.6 (17.6) or handout 17B) -- so you can detect cells that received a plasmid without an insert. The selectable marker (= active gene) confers some property that is easily detectable, for example, growth in the presence of an antibiotic or formation of blue colonies. If there is an insert in the 'selectable marker', that gene will be inactivated, and the cells will not have the corresponding property -- they will not grow, or not turn blue, etc. Conversely, if there is no DNA fragment inserted in the plasmid, the selectable marker gene will continue to function, and the cells will have the appropriate property -- they will continue to grow (plus antibiotic) or turn blue, etc. Examples of selectable markers:
a. Drug resistance. In the plasmid shown on the handout, the second selectable marker is the gene for ampicillin (Amp) resistance. If the plasmid has no insert, it confers Amp resistance. But if the plasmid has an insert, Amp resistance is lost. You want to identify the cells that got a plasmid, but are NOT Amp resistant.
b. LacZ. Another common selectable marker is the lacZ gene. Normal cells take up and cleave an analog of lactose; the product turns the colonies blue. Cells with an inactivated lacZ gene do not split the analog and remain colorless. (See Becker fig. 20-28 & accompanying text).
5. How do you check for an inactive version of the selectable marker? How do you identify cells that don't grow because a selectable marker isn't working? Use replica plating to selective media. (Steps 5 & 6 on 17B.)
a. How do you make replicas? Using sterile velvet or the equivalent you transfer bacteria from the original Petri dish to a new dish. (You gently press the velvet on the old dish, and then peel it off and press it down gently on a new dish. The new dish contains bacteria arranged in the same pattern as on the original. This process can be repeated to make several replicas on different dishes.) Click here for pictures of the apparatus
b. What good are replicas? In new Petri dishes, can have conditions that select for (allow growth of) cells with different properties. (Add Amp, or Tet, or leave out trp, etc.) Can test for ability to grow under some set of conditions or ability NOT to grow. In step 5, you want to detect cells that don't grow because they have an inactive version of the gene for Amp resistance. If replica of clone does NOT grow on selective media (with Amp) then that clone has a gene with an insert. If replica of clone DOES grow, that clone got a plasmid without an insert (and you don't want that clone). After you see what grows (or doesn't) with Amp present, you can go back to the original plate and recover the right clones -- the ones that had inserts. Click here for sample results. (Note: in sample result, insert was in Tet-R gene, not the Amp-R gene.)
Step 6 represents replica plating to different selective media. Step 5 on handout is replica plating to a Petri dish with ampicillin; step 6 is replicating to a Petri dish without trp. (Additional replicas are possible.) Left and right picture are included to show how you identify colonies that are Amp-resistant (have no insert) or able to make trp (trp+) respectively.
6. If vehicle (often a virus) is used for gene therapy, there are other considerations. How big a piece you can insert, how immunogenetic vehicle is, how to target added gene to right cells, whether DNA will insert into genome or not, etc. (For the latest development in gene therapy, click here.)
Try Problem 13-4, parts A-D. If you want more practice on cloning, try 13-7, parts A & B, and 13-8 A-C.
IV. How to find the Right Clone -- the one with the insert you want
How to find out if the gene you are looking for is there and/or working? (You
may want to be sure protein
is made, or may be sufficient just to show DNA is present.) Need to find a cell with some
selectable property because it has (or is using) gene of interest. What will that property
be? Can check at any step DNA (genotype) →
RNA →
protein →
job →
phenotype.
For an example, let's suppose you are looking for the human
gene that codes for the enzyme tryptophan synthetase. This enzyme was previously
discussed as part of the trp operon in bacteria. TS = last enzyme in synthesis
of trp. For the sake of this example, let's suppose that humans actually make
this enzyme. (I doubt if they really do, since humans can not synthesize trp.)
A. DNA (can be detected by hybridization) -- minimal requirements for detection -- DNA has to be there, but doesn't have to "do anything." Need not be transcribed, translated etc. You do need a probe (something to hybridize to DNA) to detect the DNA.
B. RNA from DNA (detected by hybridization with probe as above). To make RNA requires correct transcription signals but detection of RNA has advantages of amplification -- cell can make many copies of RNA from the one and only copy of DNA. RNA is usually easier to find than DNA (more copies to hybridize to probe).
C. Protein (detected using antibodies) -- Detection relies on structure, not function. Requires proper processing of mRNA (not yet discussed) and correct translation signals. (But protein need not work properly to be detected by antibodies.)D. Job (measure function of protein -- usually enzymatic activity). Have to supply substrate. Requires all of above (transcription, translation, etc.) plus proper folding and/or modification sometimes. Note function of a protein is sometimes hard to measure if protein is not an enzyme or requires binding to membrane. etc. in order to work
E. Phenotype (usually measure growth under certain conditions). If you start with a drug-sensitive or TS- bacterium (trp-) and add a plasmid, is bacterium now drug resistant or trp+ etc? Requires that protein be made and function well inside a cell, not just in a test tube. Added DNA must supply a new function or replace (complement) a missing or defective function. Note this one is "selectable" in terms of growth/no growth in many cases. (Replica plating on different selective media can be used to find out if clones are drug resistant, trp+ etc.)
Important: properties near the top of list require the least in terms of function, but properties near bottom are easiest to measure and/or most significant.
Next Time: Wrap up of genetic engineering -- blots &
probes; and then on to Eukaryotic Cell
Division. How does one eukaryotic cell make two?
© Copyright 2006
Deborah Mowshowitz
and Lawrence Chasin Department of Biological Sciences Columbia University New York, NY.