CHAPTER 4 – BASIC TOOLS AND

TECHNIQUES OF DNA SCIENCE - PART 3

Moses Oh – Period 3

Review of Part 2

We left off discussing various processes of isolating and analyzing recombinant plasmids.

What were these processes and how did they work?

The First Recombinant DNA Molecules

Section 1: pg. 127-129

Recombinant DNA

In the early 1970s, the previously discussed techniques became used together in order to create the first recombinant DNA molecules.

What are recombinant DNA molecules?Recombinant DNA(rDNA) is artificial DNA

created by combining multiple sequences that naturally would not be found together.

Paul Berg’s Experiment, 1972

In 1972, Paul Berg and his team of researchers at Stanford University developed a method of joining DNA molecules called “tailing” (to be explained later), modeled after the sticky ends found at the chromosome ends of the bacteriophage λ.

Using the restriction enzyme EcoRI, Berg’s lab cut the circular SV40 chromosome as well as a plasmid derived from E. Coli. The enzyme cut each in one place, opening the DNA loops producing linear strands.

Using the terminal enzyme transferase, a “tail” of 50-100 adenine residues were added onto the ends of SV40 DNA, while another “tail” of thymine residues were added onto the ends of the E.Coli plasmid.

Looking at my diagram on the board, can anyone guess how this could create a whole, circular recombinant DNA molecule?

Berg’s Process of Making Recombinant DNA

Results of Berg’s Experiment and the Definition of “Tailing”

The poly- “A” tail and the poly- “T” tail both combined to create on whole piece of recombinant DNA.

Essentially, this is what the process of “tailing” is. By adding long strands of complementary bases to the desired pieces of DNA, the pieces are attached together.

What Did Berg Demonstrate Through His Experiment?

1. He showed that a restriction enzyme could be used to cut DNA in a predictable manner.

2. He showed that DNA fragments from different organisms can be joined together.

***What Berg did not do was to actually let his recombinant DNA express itself by inserting into an organism.***

Stanley Cohen and Annie Chang’s Experiment, 1973

Inspired by Berg’s research and the ideas he proved true, Stanley Cohen and Annie Chang, also from Stanford University, went a step further to prove that not only could recombinant DNA be created, but also could be maintained and replicated within E.Coli.

Cohen and Chang, in 1973, constructed a plasmid called pSC101 that contained several important features: 1. a unique restriction site, allowing for the plasmid to cut

at one single location by the restriction enzyme EcoRI 2. an origin of replication 3. a gene coding for resistance to the antibiotic

tetracycline. They then transformed the pSC101 plasmid into E.Coli cells. The

transformed cells then were grown on agar plates containing tetracycline. Can anyone guess what could’ve happened?

Results of the Cohen –Chang Experiment

When these bacteria were grown on the agar plates containing tetracycline, there were colonies that resisted the antibiotic and survived. They had proven that the gene could be expressed.

Notice, pSC101 itself was not recombinant DNA. It was fully synthesized but not produced by combining different pieces of different DNA.

Herb Boyer Joins Cohen and Chang, 1973

After the Cohen-Chang experiment, Herb Boyer from the University of California, San Francisco, constructed a second plasmid pSC102 in addition to the first, pSC101. pSC102 contained a gene coding for the resistance to the antibiotic kanamycin. Using another technique of creating recombinant DNA, developed by Boyer himself (to be explained later), they created rDNA containing both pSC101 and pSC102.

After the transformation the plasmid was inserted into E.Coli to be grown on agar plates which now contained both tetracycline and kanamycin.

How Did This Experiment Work?

If colonies both resistant to tetracycline and kanamycin grew, there were 2 possible reasons as to why this might have happened.1. Resistant E.Coli cells carried one copy of both

pSC101 and pSC102.2. Resistant E.Coli cells carried one new copy of one

recombinant plasmid containing genes coding for resistance to both antibiotics.

Colonies resistant to both antibiotics did appear, which leads us to ask the next question…

How Do We Know Which One Of Those Possibilities Is True?

Chang, Boyer, and Cohen used gel electrophoresis to confirm which one of their predictions became true.

Plasmid DNA from these colonies digested with EcoRI were run through the gel. Where two plasmids were present, the gel showed three bands, one from the pSC101 and two from the pSC102.

However, some colonies showed that there was one plasmid containing only two bands, one for pSC101 and pSC102. This new plasmid, named pSC105, proved that a rDNA molecule was successfully created and introduced into living bacterial cells.

Boyer – Cohen – Chang, 1973

Boyer’s Newly Developed Technology for Creating rDNA

Boyer discovered, previously to his Chang-Cohen collaboration, that the “tailing” method devised by Berg was unneccessary. He proved that EcoRI, when cutting the DNA, already created its own “sticky” complementary pairs, rendering the “tailing” method as useless. This method is what he used to create the rDNA of pSC101 and pSC102.

Cohen – Boyer Experiments

Shortly after the previous experiment, Cohen and Boyer both collaborated to produce rDNA containing DNA from two different species.

They spliced a gene from the frog Xenopus laevis into plasmid pSC101 and transformed the DNA into E.Coli.

The results also turned out positively for them; some of the colonies resistant to tetracycline were found to contain plasmid with the frog gene inserted.

Cohen – Boyer Frog Experiment

Summary of Section 1 Berg and his team first developed a

recombinant DNA molecule. Chang and Cohen, as well as Boyer, who later

joined the team, proved together that rDNA can be expressed, replicated, and maintained in a living organism.

Cohen and Boyer, who teamed up afterwards, showed that rDNA composed of DNA pieces from different species could still be maintained and expressed in a living organism.

Introduction To Model Systems

Section 2 – pg 130

Origins of Using Model Systems

Dutch microbiologist Albert Jan Kluyver suggests use of model systems in 1926 when he proposed the “unity of biochemistry.”

His theory of the “unity of biochemistry” emphasized the similarity of enzymatic reactions found between many species.

“Basic life processes are conserved between species.”

Various Model Systems, Their Uses, and Their Characteristics

Section 3 – pgs 131-140

Escherichia Coli In the 1920s and 1930s, studied demonstrating the

uniformity of biosynthetic pathways in microorganisms. Microorganisms quickly became a favorite for experimenting, mostly because they are easy and inexpensive to maintain, and culturing them is relatively easy.

E. Coli, discovered in 1855, as well as other bacteria, are key models for studying metabolism (the combination of catabolism and anabolism). Anabolism is a multi-step process where several enzymes work to produce a final product.

E. Coli Metabolic Pathway

Catabolism and Anabolism

Yeast E.Coli are prokaryotes, but yeast are single-celled

eukaryotes, making it easier to study processes that are different from eukaryotes and prokaryotes.

Yeast cells can grow rapidly using inexpensive nutrients, and large numbers of colonies can be produced from a small culture.

Two types of yeast are commonly used: Saccharomyces cerevisae Schizosaccharomyces pombe

Discoveries Made Through Yeast

Unlike bacteria, S. cerevisae, one of the two major species of yeast, has multiple chromosomes and multiple origins of replication.

Using this to his advantage, Bruce Stillman of the Cold Spring Harbor Laboratory discovered the first eukaryotic origin of replication.

This discovery became very important in understanding DNA replication in all eukaryotes. Even to this day, yeast is used in order to find out more about eukaryotic DNA replication.

How is Yeast Used to Study DNA Replication?

Nematode Worm (Caenorhabditis elegans)

In the 1960s, Sydney Brenner of the Medical Research Council Laboratory in Cambridge, England introduced the nematode worm as a model system.

C. elegans are small and easy to maintain, and also has a quick growth time (3 days from egg to reproducing adult). Since C. elegans is also multicellular organism, scientists can use them to view complex interactions between cells.

C. elegans is also transparent, allowing scinetists to identify every cell in the organism and making it easier for them to trace each cell throughout the life of the worm.

Genome Sequence of C. elegans

C. elegans was also the first multicellular eukaryote to have its genome sequenced.

Fruit Fly (Drosophilus melanogaster)

Fruit flies became an attractive model system for scientists to use because mutations were easily and quickly visible. Also, many fruit flies can be stuffed into a little space and fed with relatively little food.

There have been many examples of how fruit flies have been demonstrated to be great model systems. One well-known experiment includes Christiane Nusslein-Volhard and her collaborator Eric Wieschaus. After mutagenitizing a random large number of flies with a chemical that would damage about half the Drosophila genes at random, they observed the embryos resulting from the genetic crosses between the mutagenitized strains.

In this way, they were able to identify more than 100 genes of the fruit fly that affect the normal formation of the body, which led to our current understanding of how organisms specify a body axis.

Part of the Nusslein-Volhard/Wieschaus

Experiment, 1980

Zebrafish (Danio rerio) In the late 1980s, Nusslein-Volhard turned her attention

to yet another model system – the zebrafish, a model organism pioneered by Charles Kimmel of the University of Oregon. Although the fruit fly was an outstanding model organism, it wasn’t one of the higher organisms, meaning they weren’t closely related to humans. The zebrafish addressed all these concerns but also possessed most of the qualities of the fruit fly. Their eggs are also clear, which enabled Nusslein-Volhard and her collaborators to yet again identify mutants of the zebrafish , this time about 1200 mutants.

Nusslein-Volhard’s Experiments with Zebrafish

Amphibians Amphibian embryos have been used as model systems

because the eggs are large, which makes them ideal for experimental manipulation.

Hilde Mangold, in a laboratory run by Hans Spemann of the Kaiser Wilhelm Institute, Germany, experimented with the embryo of an amphibian. She transplanted small regions of one embryo to a second embryo. One specific region transplanted resulted in a two-headed embryo with a completely separate, different axis opposite the normal one.

Hilde Mangold’s Experiments, 1920s

Chicken Eggs (Gallus gallus)

Chicken embryos are sometimes used because the relatively large size and accessibility of the chicken embryo is easy for scientists to use for observing growth and differentiation. Manipulating the embryo is as easy as cutting a small hole in the shell, manipulating, and taping over the hole.

Chicken embryos are also evolutionarily closer to humans, and the external development of chicken embryos makes them easier to access than internally developing embryos.

Mouse Mice have been used as a model system because they

are vertebrates and mammals, making them more like humans than any of the other organisms so far. However, they reproduce slowly and are more expensive to maintain.

Many laboratory strains of mice have been inbred to have a homogeneous genome. By starting with common mice, researchers can easily tell which changes have occurred. Mice are also good to use because their genes can be easily manipulated.

Human Cell Culture Human cell cultures are used as an ethical and noncontroversial

solution to experimenting with and studying human cells. The source of these cultures are derived from human blood or tissues, and once the samples are prepared to be grown, they are grown on plates or in suspension.

However there are disadvantages to using these cultures. One is that when they are grown, they can only grow for a limited amount of time (several generations) and then stop. To get around this scientists have devised a way to make these cultures “immortalized”, but immortal cultures behave like cancer cells and as a result often have abnormal genomes such as polyploidy or aneuploidy. Although there are severe drawbacks to this model system, much about cell function and disease has been learnt throughout these cultures.

Plants The disadvantages of working with plants are

numerous: Research on plants only marginally helps toward human

advances especially in medicine. Reliable transformation of plants (such as gene transfer) is more

difficult with plants because of the cell walls. Plant genomes are insanely biggger than the mammalian

genome; the haploid corn genome is about 5 times larger that the human genome.

Plant genomes are also intensely more complicated; polyploid chromosomes confuse genetic analysis, and high proportions of repetitive DNA complicate sequencing.

Barbara McClintock (1980s) and Robert Martiensen (mid 1990s)

Examining plants were much more difficult than it is now until Barbara McClintock introduced the Ac/Ds transposon system which in essence became a tool to produce new mutations in corn.

Expanding upon McClintock’s idea, Robert Martiensen from the Cold Spring Harbor Laboratory modified her method so that the system would work for the Arabidopsis thaliana, the now model plant in genomic research.

Arabidopsis thaliana Used as a Model System

The A. thaliana is a popular plant for research because:It is a wild type plantA life cycle lasts 8 weeks, which is relatively shortEasy to grow and smallEasy to transform (by spraying flowers with a

solution of tumor bacterium containing the transgene)

Self-fertile, facilitating screening for rare mutants or transgenes.

Review of Section 2

Model SystemsUnderlying idea that serves as the basis for

using model systems (or organisms)?

Review of Section 3 Examples of Model Systems (reasons for use and how

is it used?) E. Coli Yeast Nematode Worm (C. elegans) Fruit Fly (D. melanogaster) Zebrafish (D. rerio) Amphibians (in particular X. laevis) Chicken (G. gallus) Mouse Human Cell Cultures (or cell cultures in general) Plants (in particular corn (Z. mays) and A. thaliana)