Gene Regulation and Genomics

Differentiation

• As development progresses, cells becomes more specialized and restricted in the expression of their genetic content. This leads to many types of cells in a complex organism.

• However, differentiated cell may retain all their genetic potential.

• Salamanders can regenerate a lost limb.

• Tissue culture has allowed many plants to beproduced from a single plant.

Plant Tissue Culture

• This is BIG business, and is very important in the horticultural industry.

• Thirty years ago if an orchid breeder had a spectacular plant it might produce two or three asexual off-shoots per year. Each of these could be sold for, perhaps, $150-200. Sounds good?

• Today that same plant can be placed in tissue culture and the grower can produce 20,000 identical plants, sell them for $10 each.

• $200,000 vs. $300-400?? You choose.• The center for plant tissue culture in Florida is the area around

Apopka. • The process is simple in theory, but more complicated in practice.

What growth medium is required?; What part of the plant yields the best cells for tissue culture?; At what rate and how long are cells shaken in solution? Etc.

There’s a lot of information in that cell! How can we regulate its use?

• In eukaryotes the cells genes are located on chromosomes.

• A chromosome consists of DNA and histones. Histone proteins are a framework around which the DNA can be tightly coiled. The term for the bead-like DNA-histone complex that can be seen in electron micrographs is termed a nucleosome.

• The tight packing of the DNA in the chromosome helps to regulate gene expression by preventing transcription proteins from contacting the DNA. Only when portions of the DNA uncoil can transcription of information occur.

Alternative RNA splicing

• Remember: the DNA message that is copied contain both introns and exons.

• The introns are removed to produce a functional mRNA.

• If splicing occurs in different ways, different mRNAs are produced and their products will also be different.

• This means that alternative RNA splicing can allow a gene to code for several different peptide chains, depending upon how the information is spliced together.

A Cell is not a Simple Strucuture!

• After mRNA is produced other events will happen.– mRNA must be broken down. When this happens is regulated

by the cell.– When translation actually occurs can be controlled by the cell.– The peptide that is formed during translation may need to be

bent, folded, or chemically altered before it becomes functional.– Proteins may be destroyed by the cell after a set period of time.– These mechanisms enable the cell to control the amount and

the type of protein that is active in the cell at any given point in time.

Reproductive Cloning

• Reproductive cloning adds a nucleus from a donor cell to an egg cell, whose nucleus has bee removed.

• The result is a new animal exactly like the parent (i.e. Dolly , the sheep)

• The technique is not without problems , and its usefulness is still to be proven.

• There are great ethical concerns about human cloning, and most researchers are strongly opposed to it. (Would you really want another you, and would you be you if you did get cloned??) That’s a lot of you!

Therapeutic cloning

• Unlike reproductive cloning, therapeutic cloning has great medical potential.

• In this procedure stem cells (adult and/or embryonic) are cultured in the lab, and then induced to transform into specialized tissues.

• The use of embryonic stem cells has much promise, but much of this has been lost in the current moral and ethical debates.

• If one is truly opposed to embryonic stem cell research, one should also be ethically boound to forgo the use of any therapies or cures that result from it.

Signal Transduction Pathway

• How do cell know when to differentiate?• How are cellular activities regulated within an organism?• The processes is relatively straightforward:

– A signal molecule, produced elsewhere, attaches to a receptor protein on the cell membrane.

– This causes a cascade of reactions between relay proteins in the cell’s interior.

– The last relay molecule activates a transcription factor that causes the transcription of a specific gene in the DNA of the nucleus.

– This leads to the production of a protein that may act as an enzyme or structural element needed to effect a change in the cell.

Oncogenes

• Oncogenes are cancer causing genes, and usually trigger increased production of cell growth factors.

• Active oncogenes, along with defective tumor-supressor genes, can lead to the development of cancerous tumors.

• Usually cancer is the result of multiple mutations in a somatic cell.

• Many cells probably develop these mutations, but are detected by cells of the immune system and eliminated before that can give rise to a tumor.

• The next slides has two illustrations of steps in tumor development.

Tumor suppressor gene

• If a gene that inhibits cell division is defective, it can lead to the uncontrolled replication of cells and the development of a tumor.

• This is seen in the illustration below.

Carcinogens

• Many factors can cause DNA mutations that lead to tumor development. These factors are called carcinogens.

• Carcinogens can be physical factors, such as, X-rays, UV radiation, and radiation from radon (naturally occurring) or nuclear weapons (man-made).

• They can be chemical factors, like the chemicals in tobacco, or cleaning agents, such as benzene.

• Even some viruses can lead to cancer. Cervical cancer is one example. This is the reason for the development of a vaccine against sexually transmitted viruses.

• Avoidance of risk factors is one way to decrease the chance of developing cancer.

Genomics, the study of whole sets of genes

• DNA technology has lead to the development of the field of genomic research. This research has many potential applications in the areas of medicine, agriculture, forensic science, and production of products for industrial and pharmacological uses.

• With the development of these lines of research, a host of legal, ethical, social, and environmental issues have arisen. It may take years, if not decades, to resolve many of the concerns that individuals are voicing today.

• None the less, DNA technology and genomics is one of the most exciting areas of modern biology.

Recombinant DNA Technology

• Plasmids are circular DNA molecules found in bacteria which can be used to insert genes into bacterial cells.

• The genes that are inserted can come from many sources other than bacteria.

• This technology has allowed the insertion of genes into bacteria, which can then be grown in great quantity, yield large amounts of the product for which that gene codes.

• This procedure can also yield large quantities of cells that carry gene that chaanges something aboutr the cell in which it is found.

• A diagram of this process can be seen in the next slide.

Using plasmids to copy genes and make proteins

Restriction enzymes

• To get a piece of DNA containing the gene to be studied, the DNA is cut into pieces using restriction enzymes.

• These enzymes recognize short segments of DNA and cut the DNA at those points.

• Because the cuts are staggered, there are unattached bases at the ends (sticky ends)

• New DNA pieces can attach to the sticky ends, and then DNA ligase covalently bonds the pieces together.

• The process of inserting and cloning a gene can be seen in the illustration to the right.

• Note that once the plastid has a gene inserted into its DNA, and the plastid is put into a bacterium, tremendous numbers of bacteria containing the gene can be produced in a short period of time.

How to make a clean gene!

• Remember, when you copy a DNA gene it consists of introns and exons. You need to remove the introns to get a ‘clean’ piece of mRNA.

• To get a gene to clone, one must isolate the ‘clean’ mRNA, and then use reverse transcriptase to make a DNA copy of the mRNA.

• After the RNA is removed, a complementary stand of DNA is formed. The result is double-stranded cDNA.

Uses of Recombinant DNA Technology

• Both bacterial cells and mammalian cells have been used to generate useful products. Mammalian cells can be genetically engineered so that the desired protein is secreted in the animal’s milk. The product can then be isolated from the milk and used.

Gel Electrophoresis

• One technique for studying DNA uses a thin gel to separate DNA based on size and electrical charge. A DNA sample is introduced into a well in the gel, and a high voltage current causes the molecule to migrate through the gel. The finished gels can be stained and photographed. Samples can be compared using the migration patterns that are present.

DNA Fingerprints

• The use of DNA evidence in forensic science has become very widespread.

• The chance of two individuals having the same DNA pattern is extremely small if enough markers are used.

• DNA matches have enabled the identification of the victims of tragedies such as, plane crashes, 9-11, ethnic cleansing.

• Innocent persons on death row have been freed due to DNA evidence which proved their innocence.

Gene Therapy

• There has been much hope for effective gene therapy. It could allow the body to compensate for genes that are defective or non-functional.

• At present success has been very limited, and some clinical trials have been discontinued due to unforeseen problems.

• Will gene therapy place a significant role in the future of medicine? Only time will tell.

Polymerase Chain Reaction (PCR)

• This technique has allowed researchers to take a minute amount of DNA and, from it, produce a large quantity of DNA for analysis.

• Using a series of enzymes and cycles of heating and cooling, new copies of DNA are created. A complete cycle only takes a few minutes, therefore in a few hours the number of DNA molecules produced is tremendous. (calculate how many you would have after 20 cycles)

Human Genome Project

• No one in today’s world should leave a basic biology course and not be aware of the Human Genome Project, its purpose and its potential for mankind.

• Purpose: map out the entire nucleotide sequence of the DNA of the human chromosome.

• This was completed years ahead of schedule due to the rapid development of techniques in sequencing.

• Potential: insight into human development; insight into evolutionary relationships; and .

• better diagnosis and treatment of many of our commonest and most debilitating diseases.

• Go to the links provided in this lesson to explore the Human Genome Project and the whole field of genomics.

Pros and Cons of Genetically Modified Organisms

• Since the advent of the first genetically modified crops, questions of

environmental impact and risks to health have surfaced repeatedly.• The EU (European Union) has a very strong bias against GM crops.

There is the concern that transgenic crops could endanger individuals with food allergies. You could consume a food not knowing that it contained an allergen that could trigger a severe and dangerous reaction.

• China has made wide use of GM crops. If you need to feed 1,300,000,000 people, high yield may be more important than the slight chance of mortality due to food allergy.

• There are many other concerns and benefits that we as a society will have to carefully weight as we move into this uncharted era GM plants and animals.

This represents the information known to be located on chromosomes #9 and #4. The graphics are courtesy of the U.S. Dept. of Energy, Genome

Management Information System of the Oak Ridge National Lab