DNA technology Ch. 10 Cloning genes Into bacteria Into plants Into animals

Recombinant bacteria Ch 10 p. 281

Human Growth Hormone (hGH) cloned into bacteria (1980s) Pituitary dwarfism mutation in hGH gene hGH protein is produced by pituitary gland Pre-1985 hGH from cadaver brains Drawbacks? Today hGH cloned 26 inches tall

Cloning steps: 1. isolate hGH mRNA from normal human

2. reverse transcribe to cDNA How is cDNA different from DNA?

3. ligate hGH cDNA into plasmid vector

4. transform bacteria  grow bacteria and they express hGH 5. purify protein

Other cloned drugs made by bacteria Human insulin for diabetics1987 Factor VIII for hemophiliacs1993 Interferon for chemotherapy1993 EPO for anemia1992 FSH for fertility clinics1996 TPA to prevent blood clots1996 Hepatitis B vaccine bGH to increase milk yield in cows

Advantages of r bacteria/drugs Clean Worlds supply in one lab Cheap ?

When rBST injected into cows  digestive systems are more efficient at converting feed to milk. 8 gallons of milk/day/cow  studies have found no differences in milk from rBST-supplemented cows. National Institutes of Health, the Congressional Office of Technology Assessment and the drug-regulatory agencies of Britain, Canada and the European Union, Department of Health and Human Services Traces of BST are found in milk from all cows, supplemented or not. rBST is broken down in the human gut, does not enter blood stream Even if injected into the human bloodstream, has no biological activity. What do you think?

Affect of high human growth hormone in childhood ….. Pituitary tumor

Cloning into plants (GM) pg 282 Transgenic plants Plants acquire new genetic trait by direct introduction of gene We have been modifying plant genes by breeding for 1000’s of years

Getting a gene into a plant embryo A natural system: Agrobacterium tumefaciens bacteria infects plants  crown gall disease (tumor) at wound sites Agrobacterium tumefaciens cells attached to a plant cell. From Genome News Network and Martha Hawes. Genome News Network and Martha Hawes

1. Engineer Ti plasmid Remove tumor inducing genes Add gene of interest

2. transform agrobacterium with Ti plasmid Ti plasmid is 200kb

3. wound plant and infect with agrobacterium

4. gene of interest transferred to plant cell Infection stimulates excision of 30 kb region of Ti called T-DNA  insertion into chromosome ssT-DNA ~20kb excised

5. grow explant = transgenic plant Agrobacterium  transgenic plantAgrobacterium  transgenic plant Virigina Tech

Why is this referred to as horizontal gene transfer? How could this be used to introduce engineered genes into a plant cell?

Last year, 12 million farmers in 23 countries grew million acres (114.3 million hectares) of GM crops genetically modified foods in US do not require labeling to notify consumers.

GM plants 2007

genetically modified plants iron-storage gene from wild soybeans and vitamin E has been enhanced by the use of genes from rice and rock cress Resistance gene to potato beetle have been approved in the US and in Canada. Fungus chitinase gene Nicotine-free GM tobacco Osmotin gene to tolerate salt stress

Benefits Increased crop yield Resistance to drought, freezing Decreased use of pesticide Decreased use of herbicides Increased nutrition Increased shelf life Can remove allergens Drawbacks increased seed costs pesticide resistant bugs new allergens harmful to insects? may spread to other plants

Bacillus thuringiensis (Bt) produce a protein crystal toxic to insects. The toxin genes are on a plasmid The proteins bind to receptors in insect's intestine. Humans do not have these receptors…………………….

Bt transgenic corn Ch. 10 pg. 284 Corn engineered with gene that codes for a Bt toxin More on Bt cornMore on Bt corn Univ. MN

Non engineered cotton Bt cotton Effect of insect infestation on cotton

Mustard plant genetically engineered to remove selenium from soil Can then grind and use in selenium deficient soils

Golden Rice Oryza sativa 2012! Vitamin A deficiency Leading cause of childhood blindness (500,000 new cases /year)

Engineer rice to produce genes needed for  carotene in endosperm (phytoene synthase and phytoene desaturase) Tissue specific expression cassette One serving of golden rice supplies enough beta-carotene to meet 10 percent of the daily requirement for Vitamin A Promoter--- transit sequence--- gene--- -

Do we need legislation for labeling of GM foods? Should GM genes, plants, animals, be patented?