RECOMBINANT DNA TECHNOLOGY (pp. 102-129)

 

I. DEFINITION OF RECOMBINANT DNA:

1. DNA molecules contructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate in a living cell

2. DNA molecules resulting from replication of a molecule described in 1. above.

II. GOALS OF RECOMBINANT DNA TECHNOLOGY

A. To ISOLATE and CHARACTERIZE a gene

B. To MAKE DESIRED ALTERATIONS in one or more isolated genes

C. To RETURN ALTERED GENES TO LIVING CELLS (or to multicellular animals or plants) to EXAMINE OR UTILIZE THEIR EXPRESSION

III. BASIC TOOLS OF RECOMBINANT DNA TECHNOLOGY:

A. NUCLEIC ACID ENZYMES: DNA polymerases, reverse transcriptase, DNA ligase, RESTRICTION ENDONUCLEASES, Table 3.2

B. BACTERIAL GENETICS: PLASMIDS, BACTERIOPHAGES (bacterial viruses)

a. Bacteriophages replicate via the LYTIC PHAGE CYCLE: the phage genome is injected into the cell, phage genes are expressed and phage proteins and DNA are made, progeny phage are packaged, and the cell is lysed, Fig. 5.37. Two genetically different phage that infect the same host cell may recombine during the lytic cycle, Fig. 5.28.

b. Some PHAGE can also replicate via the LYSOGENIC CYCLE. The phage genome is integrated into the host chromosome and becomes copied into chromosomes of all daughter bacteria like any gene. This "PROPHAGE" can be induced to enter the lytic cycle and kill its host by a variety of stresses like UV light. Fig. 5.37

c. Plasmids: Circular DNAs that replicate autonomously; (Fig. 3.22)

C. ANIMAL VIRUSES AND ANIMAL CELL CULTURE TECHNIQUES.

How Do We Know?

1. About restriction endonucleases? The phenomenon of "restriction" was described by geneticists working with bacteriophage (bacterial viruses), principally Werner Arber and colleagues. They noticed that, for example, l phage that was grown on the K strain of E. coli would only grow very poorly on E. coli B (0.01% plating efficiency relative to E. coli K). However, if one took phage from one of the few plaques that did survive on E. coli B, that phage would now grow well on the B strain, but would no longer grow well on E. coli K. So the E. coli B was said to restrict (prevent growth of) phage coming from lytic growth on E. coli K and also to modify any phage that did grow (allow it to grow on E. coli B in the future). Further analysis of mutants showed that one could genetically separate the properties of restriction and modification. Other investigators eventually showed that the different E. coli strains expressed two enzyme activities: 1., DNA methylases that put methyl groups on specific bases at certain DNA sequence recognition sites, this modifies the DNA, including that of the host chromosome, and 2., restriction endonucleases which cut non-methylated DNA into fragments. Subsequently, it was shown that an enormous variety of different restriction-modification systems, each with their own characteristic pair of nuclease and methylase for certain DNA recognition sequences, exist in different bacterial species.

IV. VECTORS: A VECTOR IS A DNA MOLECULE INTO WHICH FOREIGN DNA MAY BE INSERTED WHICH CAN THEN REPLICATE IN AN APPROPRIATE CELL.

A. Vectors must have one or more ORIGIN OF REPLICATION, and

B. One or more SITE into which the recombinant DNA can be inserted.

C. They often have convenient means by which cells with vectors can be SELECTED from those without: e.g., DRUG RESISTANCE GENES, Fig. 3.22.

D. Common vectors include PLASMIDS, VIRAL GENOMES, and (primarily in yeast) "ARTIFICIAL CHROMOSOMES". A SHUTTLE VECTOR is one that can replicate in 2 or more types of cell (e.g., E. coli and yeast, Fig. 3.34A).

V. JOINING DNA FRAGMENTS: The most common mechanism of JOINING the TARGET DNA into the vector DNA is with COHESIVE RESTRICTION ENZYME ENDS ("sticky ends") sealed by DNA LIGASE, Fig. 3.19.

VI. TARGET DNA: By "target DNA" we mean the DNA or gene that one wishes to isolate and use as described above. As specified in the definition of recombinant DNA, the target DNA must be joined to another DNA that can replicate (the vector). Target DNA can be of several forms including:

A. CHROMOSOMAL DNA ("GENOMIC DNA") isolated directly from cells or a tissue

B. COMPLEMENTARY DNA (cDNA) made in vitro (in the test tube) using the enzyme reverse transcriptase and isolated mRNA as template, Fig. 3.20

C. SYNTHETIC DNA (rarely), made by machines that rely on organic chemistry

How Do We Know?

1. About reverse transcriptase? (pp. 100-101). As will be discussed later in the course, there is a class of animal viruses that contain RNA rather than DNA as their genetic material and which cause tumors. These are the RNA tumor viruses (now called retroviruses); e.g. Rous sarcoma virus (RSV), a chicken tumor virus discovered by Peyton Rous early in the 1900's. Howard Temin noticed around 1960 that actinomycin D, a drug which binds DNA and inhibits RNA polymerase, inhibited RSV replication (but not that of other RNA viruses like influenza). He also reasoned that the tumorigenic property of the virus appeared to be transmitted to the progeny of infected cells in the same way as other genetic traits. He also was well aware of the way lysogenic bacteriophages could integrate into host chromosomes in bacteria. Temin proposed that RSV RNA was somehow copied into DNA after entering the host cell and that this DNA integrated into the host chromosome such that host cell RNA polymerase would copy new RSV RNA from this integrated copy. He called the integrated form a "provirus" after the "prophage" name used for lysogenic bacteriophage. Temin's proposal was not widely accepted (and was hard to prove with techniques of that time). For him to be correct, there must presumably be an enzyme which can copy RNA templates into DNA. This enzyme is called reverse transcriptase. About in 1970, both Temin and, independently, David Baltimore realized that RNA tumor viruses (like many RNA viruses) were likely to carry some of their replicative enzyme, reverse transcriptase, inside viral particles. Much like Kornberg did with DNA polymerase, they were able to design an assay for reverse transcriptase and prove biochemically that the enzyme did indeed exist in RSV particles. Subsequently, this enzyme has been enormously important in both cloning technology and in the understanding of viruses and of many transposable elements that use a reverse transcriptase step.

VII. RECOMBINANT DNA CLONING

A. "CLONING" is the isolation and growth of a single, genetically uniform cell and its offspring. Recombinant DNA cloning is the isolation of a cell line which contains (and replicates) a single, unique recombinant DNA from a pool (LIBRARY) of many different recombinant DNA’s, Fig. 3.33.

B. To clone (as with all isolation or purification techniques), you need two things: a SEPARATION METHOD and an ASSAY. The most common assay for a specific DNA is NUCLEIC ACID HYBRIDIZATION, Fig. 3.28. SEPARATION METHOD: With recombinant DNA technology one can use bacteria or bacteriophage to readily separate thousands or even millions of unique colonies or plaques in a clone bank or library.

How Do We Know?

1. About recombinant DNA? The idea of linking DNA molecules from different sources into one replicating DNA was apparently first proposed by Peter Lobban, a Stanford grad student working with Dale Kaiser. At about the same time, Paul Berg, a faculty member a Stanford began working with such a goal in mind. Both groups were successful in putting "foreign" DNA into l phage DNA in 1972, using techniques that are now somewhat outdated. (Berg's lab inserted DNA from the monkey virus SV40 into l DNA but did not try to grow the recombinant phage in E. coli due to hypothetical safety concerns.) Soon thereafter the labs of Stan Cohen (an expert on bacterial plasmids) and Herb Boyer (who was one of the first to purify a restriction enzyme, EcoRI) collaborated to generate recombinant DNAs by techniques much like those still used, and they were able to put their recombinant DNAs back into E. coli and get them to replicate. For example, DNA from plasmid A, encoding resistance to tetracycline, is isolated from one E. coli strain and DNA of plasmid B, encoding resistance to streptomycin, from another. Both DNAs are cut with EcoRI, the DNAs are incubated together with DNA ligase, and the mixture is transformed into non-resistant E. coli and spread on plates containing both drugs. A colony growing on this plate should contain a recombinant DNA plasmid that contains both drug resistance genes and, when isolated and cut with EcoRI, generates at least one DNA fragment identical to one from plasmid A and at least one other identical to one from plasmid B.

CONFUSION OVER "CLONING" (Sidelight)

Cloning an animal (like a frog) refers to using a single cell (like a germ cell or intestinal cell) and tricking it into believing it is a newly fertilized embryo (zygote) so it develops into a new copy or "CLONE" of the original animal (e.g., Dolly, the sheep). Note that in this case, one doesn't use anything smaller than an intact cell--there's no biochemistry involved. Note also how much easier it is for frogs, where the egg lives in water anyway. For mammals, the cell or cells can be grown briefly in vitro but usually must be implanted in a "surrogate" mother who provides the necessary incubator.

 

Cloning DNA from an animal or any other organism is taking a piece of the purified DNA from that organism and putting it into a bacterium (usually). One then isolates a single bacterial colony (or CLONE) which can be used to make millions and millions of copies of the single DNA fragment you wish to study. The common use of the term "cloning" for recombinant DNA cloning like that just described is an unfortunate coincidence of the way these bacteria were first made. Note that recombinant DNA cloning involves isolated, purified DNA; one works with only a small portion or fragment of the animal's genome; and that only new bacterial colonies are generated. In other words, if someone sent you DNA from a blue whale, you could "clone" blue whale DNA even though you've never seen a whale and have no intention ever to see one in your life.

VIII. ESSENTIAL TECHNIQUES IN WORKING WITH DNA

A. HYBRIDIZATION. Fig. 3.28. After DENATURING double stranded DNA, each strand can re-anneal with the other strand. However, each strand can also HYBRIDIZE with any added DNA or RNA (usually radioactively labeled) which also CONTAINS THE CORRECT COMPLEMENTARY BASE SEQUENCE. This is called the "HYBRIDIZATION PROBE". It can either be a synthetic DNA (made by organic chemistry) or isolated cloned DNA, usually radioactively labeled. To keep the original two DNA strands from reannealing, these are often attached to a solid support like a nitrocellulose membrane after DENATURATION.

B. GEL ELECTROPHORESIS, Fig. 3.16. DNA, being negatively charged moves towards the + POLE (anode) of an ELECTRICAL FIELD. Since all DNA has a uniform charge to mass ratio (-1 charge per base), DNA’s of all sizes move about equally through water. By using a GEL which SLOWS DOWN LARGER MOLECULES by sieving action, ONE CAN SEPARATE DNA FRAGMENTS BY SIZE AND CALCULATE THE SIZE OF EACH DNA. DNAs are detected by UV FLUORESCENCE when bound to the chemical, ethidium bromide. Most DNA fragments of interest are those generated by cutting a plasmid or viral DNA with RESTRICTION ENZYMES. By analyzing the number of fragments produced when a specific DNA is cut with each of several restriction enzymes, alone and in pairs, one can deduce the RESTRICTION MAP of the DNA. (Other methods are also used). Fig. 3.17.

C. SOUTHERN BLOTTING uses GEL ELECTROPHORESIS FOLLOWED BY HYBRIDIZATION to detect only the subset of DNA’s in a complex mixture or pattern which hybridize to a particular nucleic acid sequence (PROBE), Fig. 3.29.

D. SCREENING A CLONE LIBRARY BY HYBRIDIZATION uses HYBRIDIZATION to detect only those BACTERIAL COLONIES OR PLAQUES that contain a recombinant DNA insert which is COMPLEMENTARY (hybridizes to) to the PROBE. (This is much like Southern blotting except that instead of starting with a gel which is replicated onto a membrane filter, one starts with an agar plate containing bacterial colonies or phage plaques which is replicated onto the filter). Fig. 3.33

E. IN SITU HYBRIDIZATION detects a gene complementary to the hybridization probe in a KARYOTYPE OF MITOTIC CHROMOSOMES, Fig. 3.30., 4.29, 4.32.

F. DNA SEQUENCING. (2 types, we will only discuss "DIDEOXY"). A ddNTP is used to produce a LADDER of DNA fragments that terminate only AT THAT SPECIFIC NUCLEOTIDE (nucleotide "N"). By running 4 different ladders next to one another a pattern is generated from which the DNA SEQUENCE can be read, Fig. 3.25.

G. POLYMERASE CHAIN REACTION uses a pair of specific synthetic oligonucleotide primers and repeated cycles of (DNA replication in vitro followed by denaturation) to "AMPLIFY" a SPECIFIC DNA REGION (between the primer binding sites). Very little starting DNA is needed because the amplification is exponential. A DNA polymerase from a thermophilic bacterium is used which can withstand the heat of the repeated denaturation cycles, Fig. 3.27.

H. IN VITRO MUTAGENESIS. (Fig. 3.41) Using synthetic oligonucleotides and nucleic acid enzymes, one can alter any cloned gene in almost any way one wants (e.g., single bp change, deletion, insertion, gene fusion, etc.) So one can do "REVERSE OR BACKWARD GENETICS" in which you know the genotype first and then see what phenotype it produces in the organism!

 

IX. RETURNING RECOMBINANT DNA INTO A LIVING CELL

A. BACTERIA: TRANSFORMATION with pure DNA or "in vitro packaging". Fig. 3.18, 3.22

B. ANIMAL CELLS: TRANSFECTION with calcium phosphate-precipitated DNA; zapping with high electric field (ELECTROPORATION); animal virus vectors; microinjection into nucleus, (Fig. 3.35)

C. PLANT CELLS: ELECTROPORATION; particle gun; microinjection; T-DNA, (Fig. 3.39).

 

X. EXPRESSING A FOREIGN GENE FOR FUN OR PROFIT, Fig. 3.26

A variety of systems designed to express large amounts of a specific protein have been developed, mostly based on viruses or plasmids. At a minimum, an EXPRESSION SYSTEM must have a strong PROMOTER and produce a mRNA that is stable and effective in translation. Since patterns of POST-TRANSLATIONAL MODIFICATION of proteins (e.g., glycosylation) vary between different cell types, a eukaryotic protein made, for example, in bacteria might not be functional.

 

XI. TRANSGENIC ANIMALS AND PLANTS,

A. GERMLINE: TRANSGENE is integrated into the chromosome of a germ line cell and can be passed on to offspring as a Mendelian trait.

B. SOMATIC: TRANSGENE is integrated into chromosome(s) in some cells or tissues, but cannot be inherited by offspring (e.g., Gene Therapy) .

C. Among the most useful transgenics are TRANSGENIC MICE, which can be made by growing EMBRYONIC STEM (ES) CELLS in culture dishes (Fig. 3.38), transfecting them with DNA by standard procedures and then INJECTING THE ALTERED ES CELLS into a blastocyst stage (early) embryo. The product offspring is genetically CHIMERIC. If the chimerism extends to the germline, some of the offspring of the chimeric mouse will be GERMLINE TRANSGENICS. Transgenic mice can also be made by MICROINJECTING PURE DNA INTO THE PRONUCLEUS (Fig. 3.37) of a 1 cell embryo and reimplantation of the injected embryo. Some of these can survive and produce complete or chimeric transgenics. Other transgenic vertebrate species similar have been made, but with less routine success to date.

D. TRANSGENIC PLANTS (of some species, at least) are easier to produce because plants have a lot of TOTIPOTENT tissue (MERISTEM) that can be grown in culture and then induced to develop into a whole plant. One of the most common methods utilizes a natural gene transfer system called the Ti PLASMID OF AGROBACTERIUM TUMEFACIENS which transfers a piece of DNA called T-DNA into the genome of infected plants. By engineering the DNA of interest into the T-DNA region in the Agrobacterium, one can get the bacterium to transfer it into the genome of a plant cell or tissue for you, Fig. 3.39.

Food for thought questions

1. Explain how PCR resembles DNA cloning except without living cells being involved.

2. Assuming it were possible, would you object to using germline transgenic techniques to eliminate human genetic disease? Would you object to using somatic transgenic techniques to treat a genetic disease?

3. Hybridization is one of the most widely used and most powerful techniques in molecular biology. (Note how often it is a part of the techniques described above.) Explain how the double stranded and complementary nature of DNA is fundamental to hybridization.

IS "JURASSIC PARK" POSSIBLE?

ANSWER: NO!, BECAUSE DINOSAUR DNA IS TOO OLD

1. WHILE IT HAS BEEN POSSIBLE TO RECOVER "ANCIENT" DNA FROM MUMMIES, THE ICE MAN, FROZEN MAMMOTHS AND FLIES IN AMBER, DINOSAURS ARE MUCH OLDER AND THEIR DNA IS GONE (OTHER THAN ITS PROBABLE CONTINUED PRESENCE IN MODERN BIRDS).

ANCIENT DNA IS ONLY LITTLE, FUNCTIONLESS PIECES

2. THE TECHNIQUES USED IN #1. ABOVE RECOVER ONLY A VERY SMALL FRACTION OF THE ANIMALS DNA (MOST OFTEN MITOCHONDRIAL) AND THEN IN SMALL FRAGMENTS, NOT CHROMOSOMES.

NEED A CELL AS WELL AS DNA

3. EVEN IF WE COULD RECOVER THE COMPLETE GENOME OF SOME ANCIENT SPECIES, DNA ALONE CANNOT BUILD A NEW ORGANISM OR EVEN A CELL. YOU INHERIT NOT ONLY DNA BUT A CELL (FROM YOUR MOTHER). THAT CELL HAS STRUCTURES LIKE MITOCHONDRIA, ETC. (AND WHAT MIGHT BE CALLED POSITIONAL CONTEXT) THAT ARE EQUALLY ESSENTIAL TO LIFE.

Recombinant DNA: A Scenario.

Honora Biosci, a recent MSU graduate, was just hired by Eels Be Gone, a chemical company that make lampricides, chemicals that kill sea lampreys. Honora was called in to see her boss, Dr. Leah K. Trout, who said: "Unlike vertebrates, lampreys and a few other primitive chordates make a hemoglobin consisting of only one type of globin polypeptide, instead of the usual two, a - and b -globin. Perhaps this means that the structure of lamprey hemoglobin is different in a way such that we can design a chemical that will bind to and inactivate lamprey hemoglobin, but not harm fish, humans, etc. Unfortunately, we can't get enough lamprey hemoglobin to crystalize, but we do have a sample of lamprey DNA (giving a tube to Honora). You need to use recombinant DNA techniques to determine the sequence and structure of lamprey globin."

1. Honora went to her computer and obtained all the known a - and b -globin gene DNA (or protein) sequences, along with those for myoglobin, a related, single chain oxygen-binding protein found in muscle. She had the computer align all the sequences to indicate regions of identity. Although each sequence had one or more differences from the others, there were a few regions in which the sequence was the same (or nearly so) in all globin genes. Honora reasoned that these regions would also have that sequence in lamprey.

2. Honora selected two identical regions separated by 400 base pairs (bp). She had small (15-20 nucleotide) DNA oligonucleotide primers made, one identical to the sequence on one DNA strand of one region and the other identical to the sequence of the opposite strand of the second region (think about how and why she chose primer sequences in this way). These were used with the lamprey DNA in a PCR reaction.

3. Honora analyzed the PCR products by gel electrophoresis, noticing one DNA product that was 400 bp long (plus the length of the primers) and purifying it from the gel. She took it to Dr. Trout, who said "I hear PCR reactions often generate artifacts. Prove to me that this DNA is from a globin gene and from lampreys."

4. Honora first used dideoxy sequence analysis on her PCR product. As she expected, the full sequence aligned well with the other globin genes, but was somewhat different from all other known globin sequences.

5. Honora also radioactively labeled some of her PCR product (either by including radioactive nucleotide precursors in a PCR reaction or in another type of in vitro DNA synthesis reaction). She also digested the lamprey DNA in a few different reactions with a different restriction enzyme for each. She electrophoresed each reaction on a separate lane of a gel, and then made a Southern blot of this gel. She hybridized her blot with the radioactive PCR product, washed it at high temperature such that only exactly matching DNAs were likely to remain double stranded and bound to the blot, and exposed it to film. She found one or two different sized bands in each lane. This suggested (not final proof) that the PCR product did indeed come from lamprey DNA and, furthermore, that lamprey DNA probably contained only about one or two globin genes. Dr Trout said: "Good job, but we need the full sequence of the lamprey globin gene, including regions at the ends that may be different enough from other globins that PCR won't work for you for the ends.

6. Honora decided to isolate the full lamprey globin gene from a recombinant DNA library constructed in bacteriophage l . She cut her lamprey DNA to fragments of about 15-20 kb (1 kb = 1000 bp) with a restriction enzyme, ligated the fragments into DNA of a l vector, packaged the DNA in vitro into phage heads, and generated a phage library stock of about 1010 phage/ml derived from about 105 independent recombinant phage (each independent phage could contain a different lamprey DNA fragment). She then screened her library by hybridization. She plated out about 4 x 105 phage on bacterial lawns on agar plates and prepared filter replicas of each plate. The filters were hybridized together to her radioactive PCR product, much as with her Southern blot. After washing and exposing to film, Honora found a few replicate plaques that hybridized, and she went on to purify one of these by one or two further rounds of hybridization as before. The purified l recombinant was grown and used to make large quantities of clone DNA for further tests.

7. First, Honora digested her recombinant phage DNA in separate restriction digests followed by running each on the lane of a gel. In each lane, she saw several DNA fragments, some of which she knew came from the l DNA and others which must be from the lamprey DNA insert. She could sometimes guess which ones might contain the lamprey globin gene based on their identical sizes to fragments seen in her earlier Southern blot, and she could confirm this by doing a similar Southern blot of this gel.

8. Since the likely gene-containing fragments were 2 to 5 kb, whereas the whole phage DNA was 50 kb long, Honora decided to subclone the globin gene fragment. She cut the phage DNA with one or more restriction enzymes and ligated the desired fragments into a plasmid or phage vector designed for use in DNA sequence analysis. She then determined the complete sequence of the lamprey globin gene (about 2000 bp or so), again by the dideoxy technique (there are now machines to do this) and took it to Dr. Trout.

What do you think Dr. Trout asked Honora to do next to determine the structure of lamprey globin protein?