Chapter 12 Genetic Engineering and the Molecules of Life

How close to “designer babies” are we?

The first draft of the human genome was completed in 2000

What have we learned from this?

What are stem cells?

What is recombinant DNA?

The Chemistry of Heredity

Deoxyribonucleic Acid (DNA) – the polymericmolecule that conveys genetic information in allspecies

Chromosomes – in humans, there are 46 double-stranded DNA molecules, which contain all ofan individual’s genetic information.These 46 chromosomes exist as 23 pairs, one setfrom each parent

Human genome – the approximately 30,000genes on the 46 chromosomes that code for allthe proteins that convey one or more hereditarytraits

Gene – a section of DNA that codes for aparticular protein

The Chemistry of Heredity

What are we made of?

12.1

• All genetic information is stored in the nucleus of the millions of cells in the body.

• Each nucleus contains chromosomes, 46 compact structures of intertwined molecules of DNA, and about 30,000 genes, components that convey one or more hereditary traits.

• DNA is a special template written in a molecular code on a tightly coiled thread that carries all genetic information.

12.1

DNA is made of fundamental chemical units, repeated over and over.

Each unit is composed of three parts: nitrogen-containing bases, the sugar deoxyribose, and phosphate groups.

Adenine (A), Guanine (G), Cytosine (C), and Thymine (T) are the bases.

What makes up DNA?

Nucleotides

A combination of a base, phosphate group, and a deoxyribose sugar is a nucleotide.

A covalent bond exists between the phosphate group and the sugar.

This nucleotide is an adenosine phosphate.

Any of the four bases can be used to form a nucleotide.

Another covalent bond is present between the ring nitrogen of the base and a ring carbon of the sugar.

12.1

A typical DNA molecule consists of thousands of nucleotides covalently bonded in a long chain.

The phosphate groups are responsible for linking each nucleotide.

12.1

What does a segment of DNA look like?

A phosphate group of one nucleotide reacts with an –OH group present on the deoxyribose ring of another nucleotide, forming and eliminating a H2O molecule.

This –OH group reacts with the phosphate group of another nucleotide

Chargaff’s RulesErwin Chargaff’s research showed that for all humans, the percentage of adenine in DNA is almost identical to the percentage of thymine.

Similarly, the percentages of guanine and cytosine are almost equal.

From this, Chargaff concluded that the bases always come in pairs; adenine is always associated with thymine and guanine is always associated with cytosine.

12.1

Thus, Chargaff’s rule states: %A = %T and %G = %C

Hydrogen bond – a weak bond-like interactionthat exists between a nitrogen or oxygen atomand a hydrogen atom directly bonded to anitrogen or oxygen atom

O HR

O

C

N H :N

R

HH

RH

. .

Nucleotide – combination of a base, adeoxyribose ring, and a phosphate group

The Double Helix of DNA

X-Ray Diffraction pattern of a hydrated DNA molecule taken in 1952.

This technique uses the fact that a molecule’s electrons diffract X-Rays at particular angles and the resulting pattern, like the one above, can be used to solve the structure of a crystal.

12.2

Rosalind Franklin- her data was used by Watson

and Crick (below)

The Double Helix of DNA

Using Rosalind Franklin’s X-ray diffraction data, Watson and Crick proposed a molecular model for DNA.

This model had a double strand of repeating nucleotides. Complementary base pairing (AT, CG) is held in place by hydrogen bonds (shown in red).

The nature of the base pairing required that the two strands be coiled in the shape of a double helix.

12.2

DNA Replication

12.2

The process by which copies of DNA are made is called replication.

The original DNA double helix partially unwinds and the two complementary portions separate.

Each of the strands serves as a template for the synthesis of a complementary strand.

The result is two complete and identical DNA molecules.

Ribonucleic acid – the polymeric moleculeconsisting of phosphate, the sugar ribose, andthe four bases cytosine (C), adenine (A), guanine(G), and uracil (U)

Messenger RNA (mRNA) – the single-strandedRNA molecule that transcribes the geneticinformation of a particular gene from the DNAdouble helix

Transfer RNA (tRNA) – the small RNAmolecule that carries a particular amino acidwhen the genetic information in mRNA istranslated into the correct amino acid sequenceof a particular protein. There is a differenttRNA for each of the 20 common amino acids.

12.3

Cracking the Chemical Code

The 3 billion base pairs in each human cell provide the blueprint for producing a human being.

The specific sequence of base pairing is important in conveying the mechanism of how genetic information is expressed.

The expression is seen through proteins.

Through directing the synthesis of proteins, DNA can control the characteristics of an individual, including inherited illnesses.

Amino acid – the individual building blocks ofproteins. There are 20 common amino acids.

H2N C

RHO

OH

Protein – a polymer of amino acids with aparticular function. Proteins can be enzymes,hormones, or have other biological functions.

H2N

R1H HN

NH

O

O

OH

O

H

R3H

R2

12.3

Proteins are made of amino acids. The general formula for an amino acid includes four groups attached to a carbon atom: (1) a carboxylic acid group, -COOH; (2) an amine group, -NH2; (3) a hydrogen atom, -H; and (4) a side chain designated as R:

They differ from one another by the different R groups

There are 20 naturally occurring amino acids that make up proteins

Two amino acids can link together via a peptide bond:

12.3

Peptide bondThe two molecules join, expelling a molecule of water

The process may repeat itself over and over, creating a peptide chain.

Once incorporated into the peptide chain, the amino acids are known as amino acid residues.

12.3

Codons: How are they relevant in genetic expression?

The order of bases in DNA determines the order of amino acids in a protein.

Because there are 20 amino acids present in the proteins, the DNA code must contain 20 code “words”; each word represents a different amino acid.

The genetic code is written in groupings of three DNA bases, called codons.

The diagram shows possible codons, determined according to the base sequence of the nucleic acid strand. The expression of the genetic information is then seen through the specific proteins assigned.

DNA transcription and RNA translation

12.4

The primary structureof a protein is its linear sequence of amino acids and the location of any disulfide (-S-S-) bridges.

The sequence is characterized by the amino terminal or "N-terminal" (NH3

+) at one end; and the carboxyl terminal or "C-terminal" (COO-) at the other.

carboxylterminal

Tertiary structure of the enzyme, chymotrypsin

N-terminal

Protein Structure

Primary structure – the sequence of amino acidsin a protein from the first amino acid to the last

Secondary structure – the intermediate level oforganization that shows helical structure andchain linkages through disulfide (–S–S– ) bonds

Tertiary structure – the overall shape orconformation of the protein

12.4

Most proteins contain one or more stretches of amino acids that give rise to a characteristic three dimensional structure. The most common of these are the alpha helix and the beta conformation. The telephone cord illustrates the nature of the secondary structure of the protein.

Tertiary structure refers to the three-dimensional structure of the entire polypeptide.

Like this tangled-upphone cord.

Active Site – the region of the enzyme where thecatalytic reaction takes place

Substrate – the molecule or molecules whosereaction is catalyzed by the enzyme

Sickle-cell anemia is a hereditary disease, whichillustrates how small changes in a protein’sprimary structure can have a profoundlydeleterious effect on the protein’s function.When an individual with sickle-cell anemiaexperiences a low oxygen concentration in theblood (e.g. during strenuous exercise), some ofthe red blood cells convert into a rigid, sickle orcrescent-shaped form. Because these cells havelost their normal deformability, they clog tinycapillaries and cannot pass through tinyopenings in the spleen and other organs.

The property of sickling is caused by twomutations in the DNA sequence of the genecoding for the oxygen-transport protein,hemoglobin. Two amino acids that should beglutamic acid are replaced with valine instead.This substitution causes hemoglobin to convertto the sickled form at low oxygenconcentrations.

12.4

The function of a protein is dependent on its shape or three-dimensional structure.

Small changes in the primary structure can have dramatic effects on its properties.

Sickle cell anemia is an example of a condition that develops when red blood cells take on distorted shapes due to an error in the amino acid sequence.

Because these cells lose their normal shape, they cannot pass through tiny openings in the spleen and other organs.

Some of the sickled cells are destroyed and anemia results. Other sickled cells can clog organs so badly that the blood supply to them is reduced.

12.5

The Human Genome Project is the effort to map all the genes in the human organism.

On June 26, 2000, scientists announced that a rough draft of the project to decode the genetic makeup of humans had been completed.

The goal, to determine the sequence of all 3 billion base pairs in the entire genome, was completed for the approximately 30,000 genes found on the 46 human chromosomes.

This information might one day help to diagnose and cure diseases, understand human development, and trace our evolutionary roots.

This was a unique collaboration between government, private sector, and a philanthropic organization.

Human Genome Project – the determination ofthe sequence of all 3 billion base pairs in the 46human chromosomes, including that of theapproximately 30,000 genes. This project wascompleted in 2000.

Scientists are now trying to determine thefunction of all the proteins encoded by the30,000 genes.

Should we be concerned that employers orinsurance companies will use geneticinformation to discriminate against people whohave or have a hereditary predisposition tocertain diseases? Could this kind of informationbe used to try and create a race of “super”humans?

Recombinant DNA

Recombinant DNA is used to produce humaninsulin for diabetics. Insulin is a small protein(51 amino acids) that helps the body metabolizeglucose (blood sugar).

Plasmid – a ring of DNA that bacteria have

Vector – a plasmid with a foreign gene insertedinto it

Clone – a collection of cells or moleculesidentical to an original cell or molecule

Uses of Recombinant DNA

Recombinant DNA is used to produce vaccinesagainst viruses and bacteria by inserting DNAencoding for a viral or bacterial protein into avector. The protein is expressed and thenisolated and purified. This protein is then usedto in a vaccine to stimulate the immune systemto produce antibodies against it. A subsequentinfection by the virus or bacteria leads to a rapidresponse by the immune system.

Transgenic plants (and animals) can be createdwith some desirable property. Desirableproperties for agricultural crops includeresistance to certain pests and diseases, synthesisof specific nutrients, and resistance to particularherbicides. The latter property means that aminimal amount of herbicide can be applied peracre to clear weeds and improve crop yield.

12.6

A representation of genetic engineering

12.6

The mythical creaturechimera represents a combination of a lion, a goat, and a serpent.

Recombinant DNA is sometimes referred to asa chimera.

Current world population (approaching 7 billion) owes much to NormanBorlaug (1914-2009; Distinguished Professor of InternationalAgriculture at Texas A&M; winner of the 1970 Nobel Peace Prize).Borlaug is credited with saving hundreds of millions of lives bydeveloping advanced crop breeding and agricultural practices for use incountries suffering from drought-induced famine. For example, Mexico,which imported 60% of its wheat in the 1940’s, was able to become self-sufficient by the mid-1950’s despite an ever increasing population.

The green revolution of the 1950’s and 1960’s allowed the world foodsupply to keep pace with explosive population growth.

The United Nations’ Food & AgricultureOrganization (FAO) estimate that by 2030, itwill be necessary to increase the current grainsupply by 30% to feed a projected globalpopulation of 8.3 billion.

A combination of rapidly increasing globalpopulation and climate change will severelychallenge the world’s farmers to meet this goal.

A further complication is that as per capitaincome climbs in China and some other middleincome countries, demand for food (particularlymeat) increases faster than population. Meatproduction means diverting some grains intoanimal feed.

Agricultural research is focusing on developinggenetically engineered drought-tolerant anddisease-resistant crops to meet the challenge ofincreasing the global food supply. Geneticengineering allows genes from other species tobe inserted into important food crops. It is a stepbeyond simply crossbreeding different varietiesof the same plant species to develop new strainswith desired characteristics.

Genetically-Engineered Agriculture Transgenic Plants

12.7

Frankenfood?

Controversial: protestors at the 2005 WTO meeting in China

Virus resistant transgenic rice

In Europe, there is widespread publicapprehension towards genetically modified(GM) crops. One poll found that over 80% ofEuropeans view GM foods as “bad”. Even inthe US, a majority (55%) disapproved of GMfood products. New EU regulations requirelabeling and traceability of all food and animalfeed containing more than 0.5% of GMingredients. Some polls indicate that Americanswould like labeling, but it has not yet become amajor issue. Americans have historically placeda considerably greater degree of trust in theregulatory oversight of the US Dept. ofAgriculture than Europeans do in theircounterpart agencies.

In 1998, the US exported $63 million worth ofcorn to the EU, but the exports decreased to$12.5 million in 2002

At the end of 2002, EU ministers agreed to newlabeling controls for GM foods, which will haveto carry a special harmless DNA sequence (aDNA bar code) identifying the origin of thecrops, making is easier to spot contaminatedcrops and withdraw them from the food chainshould problems arise.

US Agricultural Department officials argue thatsince the US does not require labeling, Europeshould not require labeling either. They claimmandatory labeling is a trade barrier since itcould imply there is something wrong with GMfood.

Is the official US complaint to the World TradeOrganization (WTO) regarding the EU banjustified? The complaint was also filed byArgentina, Canada, Egypt, Australia, NewZealand, Mexico, Chile, Colombia, El Salvador,Honduras, Peru, and Uruguay.

Are individual consumers’ fears of GM foodsjustified?

American farmers lost market share in certaincountries after changing to GM crops because ofskeptical consumers. Some famine-threatenedAfrican countries (e.g. Zambia, Zimbabwe, andMozambique) have refused to accept US aidbecause it contains GM food.

Recombinant DNA used to restore sight tochildrenwith congenital blindness

A research team at the Univ. of PennsylvaniaSchool of Medicine created a vector (agenetically engineered virus) to carry anormal version of a gene called RPE65, thatis mutated in one form of Leber’s congenitalamaurosis (LCA), a genetic disease thatprogressively damages the retinas leavingmany patients totally blind in their twentiesor thirties.

Animal studies with mice and dogs had shownthat visual improvement was age-dependent, sothe research team hypothesized that youngerpatients would receive the greatest benefit. Aclinical trial with five children and seven adultsranging in age from 8 to 44, received injectionsof therapeutic genes into their retinas.

As expected, the greatest improvement occurredin the children, all of whom are now able tonavigate a low-light obstacle course. Beforethey received the gene therapy, the patients hadgreat difficulty avoiding barriers, especially indim light. Not all the adults performed better onthe obstacle course and those who did, showedmore modest improvements than did thechildren.

The clinical benefits have persisted for nearlytwo years after the first injections withtherapeutic genes were given. Although none ofthe patients attained normal eyesight, six of thetwelve test subjects improved enough that theyare no longer classified as legally blind.

Gene therapies for other retinal diseases, such asage-related macular degeneration may also bedeveloped.

These results are based on nearly twenty yearsof research and animal studies with mice anddogs. Is it justified?

Cloning Mammals and Humans

In 1996, Dolly the sheep was born – the firstcloned mammal. Dolly was created by atechnique called nuclear transfer. The nucleus(contains the chromosomes) from an adult cellwas placed in a donor egg from another sheepwhose nucleus had been removed. The nucleusand donor egg were fused with an electrical jolt.The DNA then initiated the growth of theembryo, which was then implanted into asurrogate sheep’s uterus. Since then, severalother mammalian species have been successfullycloned.

Dolly is an example of “reproductive cloning”,in which an embryo is transferred to agestational carrier in the hopes that a pregnancywill result and be carried full term.

Cloning Humans and Mammals

12.8

Cloning Humans and Mammals

12.8

Dolly, the cloned sheep

Snuppy, the cloned dog, next to his “father”

“Therapeutic cloning” refers to harvesting stemcells from 3- to 5-day-old embryos to establishstem cell lines. Scientists hope to induce thesestem cells to differentiate into variousspecialized cells.

In 2004, a team of scientists led by Woo SukHwang and Shin Yong Moon of Seoul NationalUniversity reported that they had successfullycloned human cells to generate embryonic stemcells. In 2006, Hwang admitted the data hadbeen fabricated and resigned from his universityposition.

Would blastocysts created in this manner beextensions of the people whose DNA was usedto create them or would they be separate, uniquebeings in the same way that identical twins areunique, even though they share the same geneticblueprint?

Results from Stem Cell Research

Scientists at the Burnham Institute for Medical Research in La Jolla, CA,have programmed embryonic stem cells into becoming nerve cells whentransplanted into the brains of mice. None of the mice formed tumors,which have been a major setback in previous attempts at stem celltransplantation.

This research is a first step toward developing new treatments for stroke,Alzheimer’s, and other neurological conditions.

The Food & Drug Administration (FDA) approved a phase I clinical trialfor the transplantation of a human embryonic stem cell-derived cellpopulation into spinal cord-injured individuals on January 23, 2009.

Eight to ten paraplegics who had had their injuries no longer than twoweeks before the trial begins, will be selected since the neural stem cellsmust be injected before scar tissue forms. These first trials are mainly totest for the safety of the procedures. Based on earlier results with mice,researchers say the restoration of myelin sheaths (insulation around nervecells) and an increase in mobility is probable. The injections are notexpected to fully restore mobility.

Human embryonic stem cells could be used asmodels for human genetic diseases. The relativeinaccessibility of human tissue is an obstacle toresearch in these areas. This approach could bevery valuable in studying cystic fibrosis orfragile-X syndrome or other genetic diseaseswhere no reliable animal model exists.

Embryos with a genetic disease could beidentified by prenatal genetic diagnosis (PGD)and used to establish a stem cell line featuringthe genetic disorder.

The National Institutes of Health (NIH) announced the approval of thirteen new

human embryonic stem cell lines for NIH funding on Dec. 2. 2009.

Where do we go from here?

Is saving a human life worth the cost of a potential human life?

12.8

What We Should Know from Ch. 10 and 12

Be able to write structural formulas and line-angle drawings.

Be able to draw isomers.

Recognize functional groups.

Recognize enantiomers and chiral carbons.

Understand the structure of DNA and how it istranslated into a protein sequence.

Understand how the base pairs are held togetherby hydrogen bonds.

Understand what recombinant DNA is and howit works. Be able to give examples ofrecombinant DNA technology.

Understand the polymerase chain reaction andbe able to give examples of this method.

Understand cloning via nuclear transfer.