Chapter 11 The Control of Gene Expression Life

Chapter 11: The Control of Gene Expression Life 1402: Principles of Biology

l http: //www. pbs. org/wgbh/nova/tech/labmeat. html

1. Cellular Differentiation during embryonic development 46 23 23 a. a zygote is the first cell of an organism with a full compliment of DNA

1. Cellular Differentiation during embryonic development b. the zygote undergoes mitosis giving rise to genetically identical daughter cells

2. Cellular Differentiation during embryonic development c. with each mitotic division during development, the daughter cells inherit identical copies of DNA; therefore, every diploid cell in an organism is genetically identical unless mutations occur

2. Cellular Differentiation during embryonic development d. cellular differentiation is the process by which genes are turned on and off, not changed, during embryonic development

2. Cellular Differentiation during embryonic development e. cellular differentiation results in different tissue types in a single organism

Turning Genes On and Off #49(Gene expression walkthrough) l http: //www. youtube. com/watch? v=d. Si. SHR w. R 49 k l #50(turning off genes with the flick) l http: //www. youtube. com/watch? v=Ck. R 53 X 8 vks. Y l

3. The lac operon a. Collection of genes that regulate protein synthesis depending upon the cell’s needs b. Described as found in E. coli

3. The lac operon c. Operon is turned off in the absence of lactose d. Operon is turned on in the presence of lactose

4. Cloning

4. Cloning a. A clone is a cell or organism that is genetically identical; e. g. , identical twins which result from separation of an embryo before differentiation occurs b. Variable success has been realized in attempts to clone organisms, even mammals c. Both ethical and physical barriers exist when cloning of humans is considered

4. Cloning d. Basic process of cloning 1. nucleus is removed from an egg 2. nucleus is removed from an adult somatic cell and injected into the egg which had its nucleus removed 3. the resulting cell is then grown in culture to produce a blastocyst; i. e. , an early embryo consisting of a ball of app. 200 cells 4. the blastocyst can then be used to produce an entire organism (reproductive cloning) or used to provide embryonic stem cells which can be grown in culture (therapeutic use)

l Dolly the sheep l (July 5, 1996 – Feb 14, 2003)

5. Nuclear transplantation a. the process of cloning described above results from nuclear transplantation; i. e. , a nucleus from a differentiated adult cell is transplanted into a non-differentiated egg cell b. nuclear transplantation experiments have resulted in clones of plants and animals

5. Nuclear transplantation c. the resulting clones are genetically identical to the donor parent regardless of the adult cell type used as a donor d. differentiated adult cells must all have the same DNA if clones result from nuclear transplantation regardless of the donor cell type

5. Nuclear transplantation e. if the genome were different among different adult cell types, clones from these different cells would not be the same

6. Application of reproductive cloning a. Dolly is a sheep that was cloned from an adult cell b. Since Dolly, reproductive cloning has been used to establish clones for scientific, medical, and agricultural uses

6. Application of reproductive cloning c. Scientists have cloned pigs with a gene "knocked out" that codes for a protein making their hearts activate our immune system --- What's the application?

6. Application of reproductive cloning d. Scientists have cloned farm animals with specific sets of desirable traits e. Scientists are cloning mammals that are genetically engineered to synthesis valuable drugs

7. Stem cells video #85 (what can stem cells do) a. stem cells are cells that have not yet differentiated b. in the appropriate conditions, stem cells can be stimulated to differentiate into specific cell types; e. g. , nerve cells, muscles cells, etc. thus providing a source of tissue for therapeutic use

Stem Cells http: //video. pbs. org/video/1506726820/ http: //video. pbs. org/video/1511335379/ l http: //www. pbs. org/wgbh/nova/body/replac ing-body-parts. html

7. Stem cells c. growth factors can turn on and turn off particular genes and thus determine the fate of a particular cell d. embryonic stem cells are taken from early embryos and have the potential to differentiate into any cell type

7. Stem cells e. adults have stem cells; e. g. , bone marrow cells differentiate into different blood cell types and epidermal germ cells differentiate into different skin cell types f. although adult stem cells are typically more difficult to work with, they provide a potential source of stem cells without destroying embryos

Stem cells Video #88 (stem cells) l Video #86 (stem cell therapy injections) l Vidoe #87 (ask the young scientist) l

8. Cancer a. cancer can be caused by mutations in genes that control cell growth b. genes that cause cancer are called oncogenes, these are usually mutated genes that produce growth factor to stimulate cell division

8. Cancer c. genes that may become cancer causing are protooncogenes d. tumor suppressor genes inhibit cell growth, mutations of these genes can also cause cancer

8. Cancer e. carcinogen – “cancer generator” factors in the environment that can cause cancer

CHAPTER 12 DNA Technology and the Human Genome

l Video: 97(Genetic engineering will change)

1. Cloning of genes through genetic engineering a. genetic engineering = direct manipulation of genes for practical purposes l b. gene cloning = making identical copies of genes (fig 12. 1) l

1. Cloning of genes through genetic engineering c. recombinant DNA = joining of two different sequences of DNA l d. plasmid = small, circular DNA molecule separate from the larger bacterial DNA l

1. l Cloning of genes through genetic engineering e. vector = virus or cell that transfers DNA to another cell

2. Restriction enzymes (fig 12. 2) a. enzyme = a substance, usually a protein, that catalyzes (facilitates) a reaction l b. restriction enzyme = an enzyme that recognizes a specific sequence of DNA and digests (cuts) the DNA at that recognition site l

2. Restriction enzymes AATTC G GAATTC l c. the resultant pieces of DNA after digestion with a restriction fragment are termed restriction fragments (if a linear strand of DNA has two recognition sites, digestion will produce three restriction fragments)

2. Restriction enzymes TGATCGTATCGATGCTAGCACATTATCGATGCTAGCACA TGAGCTAGCATCGATC A TGAGCTAGCATCGATAA ATCGTTCGATGCTAGCACATTATCGATGCTAGCACA TGAGCTAGCATCGATAATGAGCTAGCATCGAACGATCA l d. the cut ends of DNA are “sticky”; i. e. , they will join together (anneal) with other “sticky” ends cut with the same restriction enzyme

2. Restriction enzymes ATCGATGCTAGCATCGATAA TGATCGATGCTAGCACATT A TGAGCTAGCATCGATC TCGATGCTAGCACATT TGAGCTAGCATCGAAA l d. the cut ends of DNA are “sticky”; i. e. , they will join together (anneal) with other “sticky” ends cut with the same restriction enzyme

l http: //www. check 123. com/videos/11203 genetic-engineering-gmo-gfp

2. Restriction enzymes l e. this is how the human insulin gene is removed from human DNA and spliced into the bacterial DNA

2. Restriction enzymes l f. restriction enzymes are also used in forensic science to establish a DNA fingerprint = a set of restriction fragments unique to an individual

2. Restriction enzymes (fig 12. 2) Bacterium Bacterial chromosome l Plasmid g. Overview of the technology behind gene cloning using bacterial plasmids (fig 12. 3)

1 Bacterium Plasmid isolated 2 3 Gene Bacterial chromosome Plasmid inserted into plasmid Recombinant DNA (plasmid) Cell containing gene of interest DNA isolated Gene of interest DNA 4 • 1. gene of interest; e. g. , human insulin gene, is removed from host DNA and inserted into bacterial plasmid DNA thus making recombinant DNA Figure 12. 3

1 Bacterium Plasmid isolated 2 3 Gene Bacterial chromosome Plasmid DNA isolated inserted into plasmid Recombinant DNA (plasmid) 4 Cell containing gene of interest Gene of interest DNA Plasmid put into bacterial cell Recombinant bacterium – 2. recombinant plasmid reinserted into bacterium thus producing a recombinant bacterium Figure 12. 3

1 Bacterium Plasmid isolated 2 3 Gene Bacterial chromosome DNA isolated inserted into plasmid Plasmid Recombinant DNA (plasmid) 4 Cell containing gene of interest Gene of interest DNA Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Clones of cell 3. as recombinant bacterium reproduces, gene of interest is cloned; i. e. , the bacterium replicates the gene of interest along with its own DNA prior to each mitotic division

1 Bacterium Plasmid isolated 2 3 Gene Bacterial chromosome DNA isolated inserted into plasmid Plasmid Recombinant DNA (plasmid) 4 Cell containing gene of interest Gene of interest DNA Plasmid put into bacterial cell Recombinant bacterium 5 Cell multiplies with gene of interest Copies of gene Gene for pest resistance inserted into plants Copies of protein Clones of cell Gene used to alter bacteria for cleaning up toxic waste Protein used to make snow form at higher temperature Protein used to dissolve blood clots in heart attack therapy 4. bacteria now make human insulin for practical purposes

l Video #89(what is crispr)

3. Forensics and DNA technology: a. Back ground l 1. everyone has a unique sequence of DNA (genome); although we are all human, our nucleotide sequences vary considerably

3. Forensics and DNA technology: 2. since each person’s genome differs, a given restriction enzyme will digest each genome differently and produce restriction fragments of different sizes and number (the odds of two people having the same DNA fingerprint are slim and next to none, one in a billion!)

3. Forensics and DNA technology: b. Crime scene analysis 1. a suspect’s DNA can be compared to crime scene DNA to determine if the suspect was present (DNA fingerprinting at work)

3. Forensics and DNA technology: 2. some DNA sample; e. g. , blood, semen, or hair, that is found at some crime scene is compared with a DNA sample from a suspect by digestion with the same restriction enzymes

Restriction enzymes CK A B A+B M A B 10 kb A 8 kb 2 kb B 7 kb 3 kb A 5 kb + 3 kb B 2 kb 3. digested DNA (restriction fragments) are “run” on an electrophoresis gel (fig 12. 12) which separates the fragments based on size and charge; i. e. , the smaller fragments travel further in the gel and opposite charges attract

3. Forensics and DNA technology: Defendant’s blood Blood from defendant’s clothes Victim’s blood 4. DNA fingerprints of the suspect and crime scene sample are compared, and, if found identical, a match is made

Epigenetics refers to external modifications to DNA that turn genes "on" or "off. " http: //www. pbs. org/wgbh/nova/body/epigen etics. html (Video 51)(epigenetics) http: //www. youtube. com/watch? v=kp 1 b. ZE Ugq. VI

Bio. Clue

Mr. Bodie is dead! l He was found murdered in the library of the Bodie mansion.

Murder Weapon? #1 l #2 Near his body you find a candlestick and a lead pipe, both test positive for blood using phenolphthalein. You collect a sample from the candlestick and label it sample #1 and a sample from the pipe labeled #2

l There were signs of a struggle and a suspicious trail of blood drops leading away from the scene. A sample of the blood drops, and Mr. Bodie’s blood, have already undergone analysis and you are awaiting the results.

#5 Suspects #4 #6 #3 Prof. Grape Mrs Pheasant l Col Ketchup Miss Violet Four people beside you were in the mansion at the time of the murder, Misses Pheasant, Professor Grape, Miss Violet and Colonel Ketchup

DNA l • Analyze samples collected with Highlightthe all. DNA of the GCG sequences using the restriction enzymes CT and AT the probe. with the probe GCG. GCAGCATCGTAGCATGCTAGCTAGCTGACTGCGCATCGAT GCAGCATCGTAGCATGCTAGCTAGCTGACTG CGCATCGAT GCATGCTAGCTAGTCGACTGAGGCGCGCTATAGCGATACGCATA GCATGCTAGCTAGTCGACTGAG GCGCGCTATAGCGATACGCATA GCAGATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC GCAGATGCA GCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC

DNA l Now, find all of the sequences where the restriction enzyme AT will cut the fragment. GCAGCAT|CGTAGCAT|GCTAGCTAGCTGACTG GCAGCATCGTAGCATGCTAGCTAGCTGACTG CGC ATCGAT AT|CGAT| GCATGCTAGCTAGTCGACTGAG GC AT|GCTAGCTAGTCGACTGAGGCG GCGCGCTATA CGCTAT|A GCG ATACGCATA AT|ACGCAT|A GCAGATGCA GCAG AT|GCAGCG GCGGATCGAGCTAGCATCGA GAT|CGAGCTAGCAT|CGA GCGGCG CGGATA CGGAT|GCG ATCGAGC AT|CGAGC

DNA This leaves the following fragments. l The ones that are not marked with the probe will not appear in the gel. l GCAGCAT AT CG CGAT AT CGTAGCAT AT GCTAGCTAGCTGACTGCGCAT CG CGAT AT GC GCAT AT GCTAGCTAGTCGACTGAGGCGCGCTAT AGCGAT ACGCAT AT AGCAGAT AT GCAGCGGAT CGAGCTAGCAT AT CGAGCGGCGCGGAT AGCGAT CGAGCd

DNA l Count the bases in each of the marked fragments GCTAGCTAGCTGACTGCGCAT - 29 AT-35 GCTAGCTAGTCGACTGAGGCGCGCTAT AT-6 AGCGAT AT-9 GCAGCGGAT CGAGCGGCGCGGAT AT-14 AT-6 AGCGAT

DNA If fragment has an odd number of bases, round up to the next even number. GCTAGCTAGCTGACTGCGCAT - 30 29 GCTAGCTAGTCGACTGAGGCGCGCTAT-36 35 AGCGAT-6 GCAGCGGAT-10 9 CGAGCGGCGCGGAT-14 AGCGAT-6

DNA l Now, find all of the sequences where the restriction enzyme CT will cut the fragment. GCAGCATCGTAGCATGCTAGCTAGCTGACTGCGCATCGAT GCAGCATCGTAGCATGCTAGCTAGCTGACTG GCATGCTAGCTAGTCGACTGAG GCATG CTAGCTAGTCGACTGAGGCGCG CGCTATAGCGATACGCATA GCAGATGCAGCGGATCGAGCTAGCATCGAGCGGCGCGGATAGCGATCGAGC

GCAGCATCGTAGCATGCT GCAGCATCGTAGCATG CT AGCT AG CT GACT GA CT GCGCATCGATGCT AGCT AG CT AGTCGA CT GAGGCGCGCT ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT AGCATCGAGCGGCGCGGATAGCGATCGAGC

GCGCATCGATGCT - 17 GAGGCGCGCT - 10 ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36 AGCATCGAGCGGCGCGGATAGCGATCGAGC -30

GCGCATCGATGCT - 18 17 GAGGCGCGCT -10 ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36 AGCATCGAGCGGCGCGGATAGCGATCGAGC - 30

DNA If fragment has an odd number of bases, round up to the next even number. GCGCATCGATGCT - 18 GAGGCGCGCT -10 ATAGCGATACGCATAGCAGATGCAGCGGATCGAGCT -36 AGCATCGAGCGGCGCGGATAGCGATCGAGC - 30

4. PCR Polymerase Chain Reaction is a series of chemical reactions that copy small DNA samples so that there is enough material to be analyzed

4. PCR a. Denaturation – heating the DNA to 94 C for 1 minute separates the 2 strands

4. PCR b. Annealing – DNA is cooled to 54 C and primers are added that select the strand to be replicated

4. PCR c. Extension – DNA is heated to 72 C the polymerase replicates the strand

4. PCR d. This process is repeated and the sample grows exponentially, doubling every cycle

Human Genome Project Sequencing the Genetic code of a human l Completed in 2003 l Humans ~ 31, 000 genes l

Rapid Sequencing

Rapid Sequencing “Shotgun sequencing” l Sequencing many genes at the same time l Computers look for overlapping strands and put them in order l CTGTAGCTGATGTCGTAGCTGATCGATGCTGA ATACGTAGGCATGCTGT

Rapid Sequencing “Shotgun sequencing” l Sequencing many genes at the same time l Computers look for overlapping strands and put them in order l CTGTAGCTGATGTCGTAGCTGATCGATGCTGA ATACGTAGGCATGCTGT

Rapid Sequencing “Shotgun sequencing” l Sequencing many genes at the same time l Computers look for overlapping strands and put them in order l ATACGTAGGCATGCTGTAGCTGATGTCGTAGCTGATCGATGCTGA