C4.6 - Genetic Research

Genomes

Dissecting Genomes

• genome: full DNA sequence of an organism
• 1st draft of the human genome was completed in June 2000
  • results were published in Nature and Science
• the DNA sequenced came from multiple individuals
• only 1.5–2% of the genome contains actual genes
  • other 98% includes repeats, regulatory regions, and viral DNA

Manipulating DNA

• most DNA molecules very long
  • must be cut into smaller pieces before analysis
  • “molecular scissors” to cut DNA: bacteria
• restriction enzymes: bacterial proteins that cut DNA wherever a particular nucleotide sequence occurs
  • restriction fragments: pieces of DNA cut out using restriction enzymes

Gel Electrophoresis

• once DNA cut, pieces sorted and analyzed using gel electrophoresis
• DNA gel (looks like gelatin slab) porous
• DNA fragments added to one end of gel
• electric current applied
• DNA negatively charged → move towards positive end
  • smaller fragments move faster
  • produces a band when done
• “DNA fingeprint”

DNA Sequencing

• DNA sequencing: determining the order of nucleotides in DNA
  • completing the human genome required hundreds of automated machines
  • machines use robotics to handle DNA
  • computer programs analyze sequencing data
• newer technologies are faster and more efficient
  • nucleotides are labeled with fluorescent dyes
  • DNA strands are separated and one is used as a template
  • complementary strand is built one nucleotide at a time
  • a flash of light signals which nucleotide is added
  • system records the sequence based on the light

Bioinformatics

• bioinformatics: science of handling and analyzing biological data
• involves creation, devel., and operation of
  • databases
  • and other computational tools
  • to collect, organize, and interpret data
• i.e. nucleotide sequences stored in searchable DBs
  • researches then use programs to recognize and align overlapping nucleotide sequences
• DNA sequences from Human Genome Project housed in GenBank (public access)

Some Completed Genomes

Species	Genome Size (Base Pairs)	Approximate Number of Genes
Haemophilus influenzae (bacterium)	1.8 million	1,700
Escherichia coli (bacterium)	4.6 million	4,400
Saccharomyces cerevisiae (yeast)	12 million	6,200
Caenorhabditis elegans (nematode)	96 million	19,000
Arabidopsis thaliana (mustard plant)	125 million	25,500
Drosophila melanogaster (fruit fly)	170 million	13,700
Oryza sativa (rice plant)	470 million	60,000
Mus musculus (mouse)	2.6 billion	30,000
Rattus norvegicus (lab rat)	2.8 billion	25,000
Macaca mulatta (macaque)	2.9 billion	22,000
Pan troglodytes (chimpanzee)	3.1 billion	22,000
Homo sapiens (human)	3.2 billion	21,000

Genomics

• genomics: study of genomes
• goal of genomics: map location of genes within genome
  • key identifier regions used to map genes; incl.
    • switches that turn genes on or off
    • start codons at beginning of genes
    • stop codons at end of genes

• coding regions of genes contain instructions for making proteins
  • i.e. alpha globulin gene has a coding region for hemoglobin
• noncoding regions help locate genes but their roles are not fully known
• gene mapping helps identify disease-related genes
  • i.e. Parkinson’s disease which affects brain cell function
  • symptoms include tremors, speech problems, and muscle rigidity
  • before the Human Genome Project, genetic links to Parkinson’s were unclear
  • researchers compared DNA of people with and without Parkinson’s
  • a mutation in a specific gene was found in many affected individuals
  • this gene is also linked to Alzheimer’s disease
  • other gene mutations connected to Parkinson’s have been found

Model Organisms

• cross-species comparisons help reveal gene functions
• similar gene sequences in different species suggest similar roles
• human genes can be studied by comparing them to known mouse genes
• genes related to smell in rats and song-learning in birds are similar to a human gene linked to Parkinson’s
• studying these animals gives clues about gene function in humans
• this knowledge supports the development of treatments and cures
• functional genomics focuses on understanding gene functions
• researchers use model organisms like mice to study gene inheritance
• common model organisms include E. coli, C. elegans, yeast, fruit flies, and mice
• mice are especially useful because their development and genetics are well understood
• mice and humans have similar genome sizes and gene counts
• most human genes have a matching gene in mice

Applying Genomics

• DNA chip: small glass wafer or slide spotted with an array of single-stranded DNA fragments
  • some spots, i.e. could contain DNA w/ cystic fibrosis causing mutations
• DNA chip process
  1. person provides DNA sample
  2. sample cut into pieces
  3. pieces tagged w/ fluorescent dye to make series of probes
  4. wherever there is match between probe and spot of DNA on chip, probe will bind
  5. fluorescent tag marks spot
• Uses
  • DNA chips can be made to span large regions of human genome
  • personalized health care
  • i.e. sequencing rice genome to make rice more nutritious

Genetic Engineering

Biotechnology

• biotechnology: use of organisms to make useful products
  • i.e. prod. of vaccines, antibiotics, hormones, food products, enzymes in detergents
• genetic engineering: intentional production of new genes and alternation of genomes by sub. / intro. of new genetic material
  • i.e. altering E. coli to make blue dye for jeans

Genetic Recombination

• 1940s: American scientists Joshua Lederberg and Edward Tatum demonstrated that 2 bacterial cells form a direct connection
  • transfer of DNA
  • viruses can also carry bacterial genes from one bacteria to another
• recombinant DNA technology: combination of genes from diff. sources into single DNA molecule

Making Recombinant DNA

• plasmid: small circular DNA molecule separate from larger bacterial chromosome
  • bacteria share plasmids
• Steps for Making
  • plasmid removed from bacterial cell
  • desired gene inserted into plasmid
  • recombinant plasmid: modified plasmid w/ desired gene inserted
  • recombinant plasmid put back into bacterial cell

“Cutting and Pasting” DNA

• making a recombinant plasmid involves cutting and pasting DNA
• desired gene is cut from a longer DNA strand using restriction enzymes
• restriction enzymes make staggered cuts creating sticky ends
• sticky ends are single-stranded and can bind to complementary sequences
• sticky ends help join plasmid DNA with other DNA fragments
• DNA ligase pastes the sticky ends together to form a complete molecule
• Cloning Recombinant DNA
  • gene cloning: process of putting recombinant plasmid into bacterial cell to make copies of particular gene

Applications

Genetically Modified Organisms (GMOs)

• genetically modified organism: organism w/ genetic material that has been altered through genetic engineering
• transgenic: acquired genes from different type of organism
• i.e. GMO bacteria Caulobacter crescentus w/ surface molecules that bind heavy metals
  • remove heavy metals from drinking water

Producing Genetically Modified Plants

• recombinant DNA used to improve crop traits
• GM plants can have delayed ripening, better nutrition, stress tolerance, or disease resistance
• genetic engineering often replaces traditional breeding
• most useful when traits are controlled by few genes
• new plants can be grown from a single adult plant cell
• engineered cells can grow into full plants with new traits
• Agrobacterium plasmids used to insert genes into plant cells
• transgenic plants include tomato, potato, tobacco, soybean, walnut, apple
• DNA gun can insert genes into plants like corn

Pest-Resistant Crops

• i.e. incorporation of genes for bacterial toxin produced by Bacillus thuringiensis
• BT kills specific insects like corn borers and cotton bollworms
  • w/o harming humans or most other animals
  • upon ingestion, insect’s enzymes convert toxin into smth. that kills it
• other useful traits
  • tolerance to herbicides (weed killers)
  • resistance to viral infections

Plants as Factories

• GM plants used for making
  • pharmaceuticals
  • industrial products
  • biofuels
• i.e. GM safflowers (Charthamus tinctorius)
  • produces wide variety of products
  • like easily purifiable insulin
  • insulin extracted from oily seeds of plant

Producing Genetically Modified Animals

• more difficult than GM plants
• in vitro fertilization: fertilizing an egg in a test tube or laboratory dish, then transferring embryo to uterus (womb)
• many attempts needed before fertilized egg incorporates DNA from another source

Farm Animals and “Pharming”

• some goals: better-quality wool, leaner meat, fast-growing fish
• “pharming”: producing pharmaceuticals from transgenic animals
  • most cases: add gene for desired human protein to genome of farm mammal
  • added in such a way so that desired protein secreted via milk
  • good method when human gene not expressed well in bacteria

Animal Cloning

• entire genomes can be cloned, not just individual genes
• first cloned mammal was Dolly the sheep in 1996
• since then, many mammals have been cloned including goats, cows, pigs, and cats
• animal cloning uses nuclear transplantation
  • donor nucleus replaces nucleus of an unfertilized egg
  • egg develops into a clone with the same genome as the donor
• cloning from somatic cells has low success and health risks
• cloning can mass-produce animals with desired traits
• faster than traditional breeding
• useful for preserving traits in GM animals without further genetic changes

Process of cloning a sheep

Regulating Use of GMOs

• concerns exist about safety of GM crops and animals
• GM salmon might escape and compete with wild salmon
• GM crops could transfer genes to wild plants
• herbicide resistance could create hard-to-control super weeds
• GM plants sometimes found in non-GM fields
• pest-resistant GM crops not shown to pose major health or environmental risks
• strict regulations recommended for growing and using GM crops
• potential unknown risks to human health from GM products
• some consumers want clear labelling of GM foods
• pharmaceuticals from transgenic animals may differ from natural proteins
• GM proteins used in food or medicine must be tested for safety
• testing ensures GMOs don’t cause allergic or harmful reactions

Regulatory Agencies

• governments worldwide working to ensure biotech safety
• Canadian Food Inspection Agency (CFIA) regulates environmental release of novel plants in Ca.
• CFIA and Health Canada assess safety of biotech agricultural products
  • includes food for people and feed for livestock
• product developers must follow regulations and guidelines
• detailed product information must be submitted for evaluation

Genetic Technology in Medical Research

• model organisms used to study gene function and develop therapies
• genes can be added or deleted at specific genome locations
• knockout mice have a gene intentionally disabled
• knockout technique helps identify gene functions
• altered mice are bred to create lines with specific traits
• knockout mice used to study obesity by targeting the ob gene
• mice with mutated ob gene overeat and become obese
• ob gene codes for leptin, a protein that signals fullness to the brain
• leptin affects fat and glucose metabolism
• research on leptin supports development of treatments for obesity disorders
• knockout mice also help study inherited diseases
• used to test new drugs and therapies

Gene Therapy

• one form of treatment for one suffering from disease caused by missing / malfunctioning gene
• gene therapy: process of supplementing or replacing gene in order to treat medical condition
• gene therapy involves delivering functional genes to affected cells
  • i.e. viruses can be engineered to carry therapeutic genes
  • modified viruses insert the gene into the patient’s DNA
  • other delivery methods include transplants, injections, and inhalers
• gene therapy being studied for diseases like Huntington’s, skin cancer, and cystic fibrosis
• therapies are experimental and high-risk
• challenges include targeting the right cells and ensuring long-term effectiveness
• researchers aim to make delivery methods safe and reliable

Process of gene therapy using a virus