NICK
I'm a biologist. I'm in the biology department.
GEORGE
You're the one! You're the one's going to make all that trouble ... making everyone the same, rearranging the chromozones, or whatever it is. Isn't that right?
NICK (with that small smile)
Not exactly: chromosomes.
GEORGE
Biology, hunh?
I read somewhere that science fiction is really not fiction at all ... that you people are rearranging my genes, so that everyone will be like everyone else. Now I won't have that! It would be a shame. I mean ... look at me! Is it really such a good idea ... if everyone was forty something and looked fifty-five?
-Edward Albee, Who's Afraid of Virginia Woolfe?1962

"In the year 6565, ain't gonna need no husband, won't need no wife.
You'll pick your son, pick your daughter too
from the bottom of a long glass tube.
Woh - woh."
-Rick Evans (Zager and Evans), Zerland Music, 1969
In the Year 2525 (Exordium & Terminus)

Assignment
Audesirk, Audesirk & Byers Chapter 13, Selections from Chapters 4, 9, 11 & 40

Today's musical selection
Wierd Al Yankovic - I think I'm a clone now

Clone

The word "clone" has three meanings:
(1) Grow an organism from a somatic cell's nucleus
(2) Chop off a gene and grow it in another cell
and
(3) (as a noun) The group of cells descended from one cell (not discussed further in this outline)

Dolly, etc

One of the best known to the lay public started when a sheep named Dolly was made from a nucleus from another sheep. Text box "carbon copies" (pp. 202-204, Chapter 11) discusses similarities with any asexual reproduction used in agriculture.

1950's work on amphibians - Since all nuclei should have all the genes, any nucleus should work to make whole organism, here taken out of an intestine cell. But not all cells work, so put it in an egg where it is certain that the nucleus already there has been destroyed.

Figure E-11-1
1997 work to make sheep Dolly. Nuclear transfer by removal followed by cell fusion. Need surogate mother.
Cloning has been extremely controversial, and human cloning was banned. Some scientists hoping to advance medical treatments would like to distinguish "theraputic cloning" from cloning to produce a person genetically identical with the donor. Some think the issue would be simplified by use of the term "nuclear transplantation."

Review

Figure 9-1
Transformation in bacteria

Figure 9-2
Molecular mechanism of transformation

Figure 4-20
Bacterial cells have circular chromosome plus plasmids

Gene cloning

Figure 13-1
In lay terms, cloning is chopping a gene at both ends, putting it in and growing it up in bacteria. Plasmids are important. This allows manufacture of proteins of interest, and examples are given: growth hormone and a clot dissolving enzyme. Also, bacteria can be altered with inserted genes so that they do useful things like clean up oil spills. Crops can also be altered like inserting resistance to pests.

Figure 13-9
restriction nucleases often cut at "palindromes"
"restriction" restrict phage infections in bacteria
"Able was I ere I saw Elba" Napoleon "Madam I'm Adam" first sentence
Eco R1 E. coli
staggered cuts with single stranded cohesive ends

The1978 Nobel prize in Physiology and Medicine was awarded jointly to: WERNER ARBER , DANIEL NATHANS and HAMILTON O. SMITH for the discovery of restriction enzymes and their application to problems of molecular genetics

Figure 13-10
(How to put Bt [a gene from Bacillus thuringiensis] into plants using Agrobacterium tumefaciens for insect resistance)
If the same restriction enzyme is used to cut a piece of DNA with the gene of interest and the plasmid, they will have complementary sticky ends and will stick together.

PCR

It's a lot of trouble to clone a gene.

Figure E9-7
DNA polymerase works on single stranded DNA.

The1993 Nobel Prize in Chemistry was shared and awarded in part to KARY B. MULLIS for his invention of the polymerase chain reaction (PCR) method.

Figure 13-3
One strand is sense strand, the other is the antisense strand, and they are antiparallel.
If you know sequence at beginning and end of the gene, you make one primer for the sense strand at the beginning of the gene and another for the antisense strand at the end of the gene.
These will copy gene and then continue beyond in the first round, which is a small problem.
But on next round, going the other way stops at the end of the gene.

Figure E13-1
If 98oC to denature, cool to 60oC for polymerase to add to prime, that would require new polymerase at each cooling, but Thermus aquaticus from Yellowstone hot spring polymerase.
It all becomes very automated, and here's a picture I shot of one of the Biology Department's PCR machines.

DNA profiles

Figure 13-5
Gel electrophoresis

Figure 13-11
(Example of diagnosing sickle cell anemia)
restriction fragment length polymorphisms (RFLP)
In general, a marker is something like fruit fly eye color (something that has a phenotype (trait)), that can be used for genetic mapping but RFLPs can be markers too. This is the older method behind DNA fingerprinting

Figure 13-7
Now short tandem repeats (STRs) provide most unequivocal profiling
"...a perfect match...means that there is less than one chance in a trillion..." (recall scientific method)

Figure 13-12
Microarray - test for expression of all the genes (supplies are expensive)

Issues

Figure (Chapter 13 opener)
Chapter 13 case study (guilty or innocent) p. 251 and revisited p. 271. Case of Earl Ruffin in prison 21 years before DNA evidence showed him innocent

Figure E40-3
(look ahead to p. 831 box on Hi-tech reproduction)
Now cloning is controversial. In the 1970's, in vitro fertilization was controversial.

Agriculture

Figure E13-4
GMO stands for "genetically modified organisms." In this country, environmental groups such as Greenpeace, have spearheaded the opposition to GMO. At the economic level, the US, a major agricultural exporter and with big biotech firms, opposes trade sanctions and labeling requirements. At the level of the world trade organization, (WTO), especially the European union (EU), there is consumer anxiety, witnessed by terms such as "Frankenfoods." Clearly, there can be wonderful achievements, such as the recent expression of beta-carotene in rice; in many third world areas, rice is so preponderant in the diet that people suffer from vitamin A deficiency. But there are dangers such as the possibility that sterility alleles will transfer from patented crop to other plants.

Questions used in 2007 relating to this outline

Nuclear transplantation was used
(a) for in vitro fertilization.
(b) to transform R-strain bacteria with S-strain DNA.
*(c) to clone Dolly.
(d) to clone a gene.
(e) to create DNA arrays.

Golden rice with beta carotene
(a) was crossed with green rice by Mendel.
(b) solves the problem of vitamin B deficiency.
(c) was a product of stem cell research.
*(d) should be good for vision.
(e) arose by sympatric speciation.

Gel electrophoresis can separate globin alleles by
(a) color.
(b) histology.
(c) symmetry.
(d) hydrophobicity.
*(e) length.

What is a danger of genetically modified agriculture mentioned in class?
*(a) Sterility alleles might be spread.
(b) It led to the Irish potato famine.
(c) Autotrophs would go extinct.
(d) Genetically modified plants lack a vascular system.
(e) Genetically modified plants are susceptible to penicillin.

Sticky ends refer to
*(a) DNA cut by restriction endonucleases.
(b) Gram stining.
(c) implantation into a surrogate mother.
(d) Creutzfeld-Jacob disease (CJD).
(e) transfer RNA (tRNA).

In the polymerase chain reaction (PCR)
(a) theraputic cloning is achieved.
(b) an enzyme is used to cut a gene and a plasmid.
(c) a gene is inserted into a plant using Agrobacterium tumefaciens.
(d) a protein is mass produced using bacteria.
*(e) a defined length of DNA is amplified.

RFLPs (restriction fragment length polymorphisms) can be used for
(a) in vitro fertilization.
*(b) genetic profiling.
(c) achieve conjugation in bacteria.
(d) copying DNA to another copy after everything had been heated to denature DNA.
(e) preventing the evolution of antibiotic resistant bacteria.

One, two or three bands on a gel representing homozygous sickle globin, homozygous normal and heterozygous respectively
(a) allowed Earl Ruffin to be freed after 21 years in prison.
*(b) were created when one enzyme cut different DNA sequences to different numbers and lengths.
(c) would be of little value without a surrogate mother.
(d) represent the amino acid sequence of alpha and beta chains of hemoglobin.
(e) allowed insertion of a Bacillus thuringiensis gene into a bacterium.

What property of DNA allowed more unequivocal identification of people by DNA fingerprinting?
*(a) short tandem repeats (STRs)
(b) divergence
(c) heterozygosity
(d) poly-A tails
(e) dominance


Questions used in 2002 relating to this outline (and other outlines)

A disease-causing allele of a gene can be mapped to a "marker" that is a nearby alteration in DNA sequence called a(n)
(a) autoradiogram.
(b) PCR primer.
(c) G protein.
(d) hybridization.
*(e) RFLP (restriction fragment length polymorphism).

Which of the following has plasmids?
(a) HIV
(b) flu
*(c) E. coli
(d) small pox
(e) T4

PCR is used to
(a) determine the sizes of different restriction fragments.
(b) insert a new gene in gene therapy
*(c) make multiple copies of a gene of interest.
(d) identify protein in a blot.
(e) get rid of p53.

Which would most likely be a line from Weird Al Yankovic's song "I think I'm a clone?"
(a) "You people are rearranging my genes."
(b) "The basic secret of life is something about protein."
(c) "The gingham dog and the calico cat side by side on the table sat."
(d) "Northern blot, Southern blot, Western blot, all around the blot blot."
*(e) "They took a donor's body cell and fertilized a human egg."

This page was last updated 8/7/08

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