GENETICS

General Biology Lecture

CHROMOSOMAL PATTERNS OF INHERITANCE

DNA STRUCTURE AND FUNCTION

GENE ACTIVITY: HOW GENES WORK

REGULATION OF GENE ACTIVITY: GENE MUTATION

BIOTECHNOLOGY

Chapter 11 Mendelian Patterns of Inheritance

This chapter details Mendel’s discovery of the general laws of heredity including monohybrid and dihybrid crosses, human genetic disorders, and also introduces non-Mendelian genetics.

n 1866 the Austrian monk, Gregor Mendel, proposed what was to become the foundation of genetics. Mendel was completely unaware of our modern concept of the gene and that gene pairs are separated during the cellular event called meiosis.  But nonetheless, while working with garden peas Mendel was able to deduce that genes (he called them “factors”) occurred in pairs and that they “assorted” or “segregated” and then recombined during sexual reproduction.   We now know that assortment or segregation occurs during meiosis and recombination occurs during fertilization.   

Chapter Outline

11.1 Gregor Mendel

11.2 One-Trait Inheritance

In a figurative, if not real sense, genes occur in pairs in diploid organisms.  Of course the only time genes literally “pair up” is during synapsis of meiosis when the homologous chromosomes that carry the genes pair up.  A gene pair is represented by two alleles such as AA (homozygous dominant), aa (homozygous recessive), and Aa (heterozygous).²  A gene is a portion, segment, of the DNA molecule found in, or on, a chromosome.  We use the term allele when we wish to refer a specific form of a gene.  “A” and “a” for example represent different forms (alleles) of the same gene.    

Today it is convenient to use the Punnett-square method to predict inheritance based on these simple principles.  Restated differently, the principles behind the Punnett-square are these:  

Ex, Parent=AaBb when alleles A& a on one pair of homologous chromosomes and B & b are on another pair of homologous chromosomes, then the possible gamete genotypes are:Four different gametes =    AB,     ab,      Ab,   &      aB                             

The number of gamete genotypes is predicted by  2ⁿ where n=#heterozygous pairs   

11.3 Two-Trait Inheritance

11.4 Human Genetic Disorders

11.5 Beyond Mendelian Genetics

Comparison of traits between individuals for traits governed by a single gene pair reveals discontinuous variation, ex. tongue rolling, either you can or you can’t, only 2 phenotypes, no intermediates.  But most traits exhibit continuous variation- a range of phenotypes for a given trait, that is there are many possible phenotypes, not just two or three.  Continuous variation is a result of polygenic inheritance -  when two or more gene pairs control a single trait (see graphs on p. 190 & p. 210 depicting the variation in traits under polygenic control).

Skin color is another example of a trait under polygenic inheritance.  Darkness of skin color is determined by the production of the pigment melanin.  Consider this simplified example:  Two genes, A & B, contribute to melanin production.  Only the alleles represented by capital letters contribute to melanin production.  Each person has four alleles (two gene pairs) so the following are possible.

Genotype                                             Phenotype    
AABB                                                 Very dark    
AaBB or AABb                                   Dark                
AaBb  or aaBB or Aabb                      Medium             
Aabb or aaBb                                      light             
aabb                                                    very light

Continuous variation is exhibited in the variation range along the "continuum" from "very dark" to "very light"  

Chapter Outline

12.1 Chromosomal Inheritance

A. Chromosomal Theory of Inheritance

	Genes are located on chromosomes; behavior of chromosomes and genes is therefore similar during sexual reproduction.
	Chromosomes can be categorized as two types:

1. Autosomes are non-sex chromosomes that are the same number and kind between sexes.

2. Sex chromosomes determine if the individual is male or female.

	Sex chromosomes in the human female are XX; those of the male are XY.
	Males produce X-containing and Y-containing gametes; therefore males determine the sex of offspring.
	Besides genes that determine sex, sex chromosomes carry many genes for traits unrelated to sex.
	X-linked gene is any gene located on X chromosome; used to describe genes on X chromosome that are missing on the Y chromosome.

B. X-Linked Alleles

Work with fruit flies by Thomas Hunt Morgan (Columbia University) confirmed genes were on chromosomes.

1. Fruit flies are easily and inexpensively raised in common laboratory glassware.

2. Females only mate once and lay hundreds of eggs.

3. The fruit fly generation time is short, allowing rapid experiments.

Fruit flies have an XY system similar to the human system; therefore experiments are similar to the human situation.

1. Newly discovered mutant male fruit fly had white eyes.

2. Cross of the hybrids from the white-eyed male crossed with a dominant red-eyed female yielded the expected 3:1 red-to-white ratio; however, all of the white-eyed flies were males!

3. An allele for eye color on the X but not on the Y chromosome supports the results of this cross.

4. Behavior of this allele corresponds to the behavior of the chromosome; this confirmed the chromosomal theory of inheritance.

X-Linked Problems

1. X-linked alleles are designated as superscripts to the X chromosome.

2. Heterozygous females are carriers; they do not show the trait but can pass it on.

3. Males are never carriers but express the one allele on the X chromosome, whether it is dominant or recessive.

4. One form of color-blindness is X-linked recessive.

C. Human X-Linked Disorders

	More males have X-linked traits because recessive alleles on the X chromosome in males are expressed in males.
	Color Blindness

1. Color blindness can be an X-linked recessive disorder involving mutations of genes coding for green or red sensitive cone cells, resulting in an inability to perceive green or red, respectively; the pigment for blue-sensitive protein is autosomal.

2. About 8% of Caucasian men have red-green color blindness.

Muscular Dystrophy

1. Duchenne muscular dystrophy is the most common form and is characterized by wasting away of muscles, eventually leading to death; it affects one out of every 3,600 male births.

2. This X-linked recessive disease involves a mutant gene that fails to produce protein dystrophin.

3. Symptoms (e.g., waddling gait, toe walking, frequent falls, difficulty in rising) soon appear.

4. Muscle weakens until the individual is confined to wheelchair; death usually occurs by age 20.

5. Affected males are rarely fathers; the gene passes from carrier mother to carrier daughter.

6. Lack of dystrophin protein causes calcium ions to leak into muscle cells; this promotes action of an enzyme that dissolves muscle fibers.

7. As body attempts to repair tissue, fibrous tissue forms and cuts off blood supply.

8. A test now detects carriers of Duchenne muscular dystrophy; treatments are under research.

Hemophilia

1. About one in 10,000 males is a hemophiliac with impaired ability of blood to clot.

2. Hemophilia has two types: Hemophilia A is due to absence of clotting factor IX; Hemophilia B is due to absence of clotting factor VIII.

3. Hemophiliacs bleed externally after an injury and also suffer internal bleeding around joints.

4. Hemorrhages stop with transfusions of blood (or plasma) or concentrates of clotting protein.

5. Factor VIII is now available as a genetic engineering product.

6. Of Queen Victoria’s 26 offspring, five grandsons had hemophilia and four granddaughters were carriers.

Fragile X Syndrome

1. In this case, the X chromosome is nearly broken; most often found in males.

2. This affects one in 1,500 males and one in 2,500 females.

3. As children, they are often hyperactive or autistic with delayed or repetitive speech.

4. As adults, males usually have larger testes, unusually protruding ears, and other symptoms.

5. However, about one-fifth of males with fragile X do not show symptoms.

6. Fragile X passes from a symptomless male carrier to grandson.

7. It has been traced to excessive repeats of base triplet CGG (cytosine, guanine, guanine); up to 230 copies compared to normal 6–to–50 copies.

A. Linkage Groups

	Fruit flies have four pairs of chromosomes to hold thousands of genes; therefore each chromosome must hold many genes.
	All alleles on a chromosome form a linkage group that stays together except when crossing over.
	Crossing-over causes recombinant gametes and at fertilization, recombinant phenotypes.
	Linked alleles do not obey Mendel’s laws because they tend to go into the gametes together.

B. Chromosome Mapping

	The percentage of recombinant phenotypes measures distance between genes to map the chromosomes.
	Crosses involving linked genes do not give same results as unlinked genes.
	A heterozygote forms only two types of gametes and produces offspring with only two phenotypes.

C. Linkage Data

	Linked genes indicate the distance between genes on the chromosomes.
	If 1% of crossing-over equals one map unit, then 6% recombinants reveal 6 map units between genes.
	If crosses are performed for three alleles on a chromosome, only one map order explains map units.
	Humans have few offspring and a long generation time, and it is not ethical to designate matings; therefore biochemical methods are used to map human chromosomes.

A. Mutations

	Changes in chromosomes or genes that pass to offspring if they occur in gametes.
	Mutations increase the amount of variation among offspring.
	Chromosomal mutations include changes in chromosome number and structure.

B. Polyploidy

	Eukaryotes with more than the 2n number of chromosomes are polyploids.
	Terms indicate how many sets of chromosomes are present: (triploids [3n], tetraploids [4n], etc.).
	Polyploidy does not increase variation in animals; judging from trisomies, it would be lethal.
	Polyploidy is a major evolutionary mechanism in plants; it is probably involved in 47% of flowering plants including major crops.
	Hybridization in plants can result in doubled number of chromosomes; an even number of chromosomes can undergo synapsis during meiosis; successful polyploidy results in a new species.

C. Monosomy and Trisomy

	Monosomy (2n – 1) occurs when an individual has only one of a particular type of chromosome.
	Trisomy (2n + 1)occurs when an individual has three of a particular type of chromosome.
	Nondisjunction is the failure of chromosomes to separate; it is more common during meiosis.
	Monosomy and trisomy occur in plants and animals; in autosomes of animals, it is generally lethal.
	Down syndrome is most common autosomal trisomy, involves chromosome 21.

1. Most often, Down syndrome results in three copies of chromosome 21 due to nondisjunction during gametogenesis.

2. In 23% of cases, the sperm had the extra chromosome 21.

3. In 5% of cases, there is translocation with chromosome 21 attached to chromosome 14; this translocation could have occurred generations earlier and is not age-related.

4. Chances of a woman having a Down syndrome child increase with age, starting at age 40.

5. Chorionic villi sampling testing or amniocentesis and karyotyping detects a Down syndrome child; however, risks for young women exceed likelihood of detection.

6. A Down syndrome child has tendency for leukemia, cataracts, faster aging, and mental retardation.

7. Gart gene, located on bottom third of chromosome 21, leads to high level of purines and is associated with mental retardation; future research may lead to suppression of this gene.

D. Changes in Sex Chromosome Number

	Nondisjunction during oogenesis can result in too few or too many X chromosomes; nondisjunction during spermatogenesis can result in missing or too many Y chromosomes
	Turner syndrome females have only one sex chromosome, an X.

1. Turner females are short, have a broad chest and folds of skin on back of neck.

2. Ovaries of Turner females never become functional; therefore, females do not undergo puberty.

3. They usually have normal intelligence and can lead fairly normal lives with hormone supplements.

Klinefelter syndrome males have one Y chromosome and two or more X chromosomes.

1. Affected individuals are sterile males; the testes and prostate are underdeveloped.

2. Individuals have large hands and feet, long arms and legs, and lack facial hair.

3. Presence of the Y chromosome drives male formation but more than two X chromosomes may result in mental retardation.

4. Barr body, usually only seen in female cell nuclei, is seen in this syndrome due to the two X chromosomes.

Poly-X females have three or more X chromosomes and extra Barr bodies in the nucleus.

1. There is no increased femininity; most lack any physical abnormalities.

2. XXX individuals are not mentally retarded but may have delayed motor and language development; XXXX females are usually tall and severely mentally retarded.

3. Many experience menstrual irregularities but many menstruate regularly and are fertile.

Jacobs syndrome (XXY) are males with two Y chromosomes instead of one.

1. This only results from nondisjunction during spermatogenesis.

2. Males are usually taller than average, suffer from persistent acne, and tend to have speech and reading problems.

3. Earlier claims that XYY individuals were likely to be aggressive were not correct.

A. Changes in Chromosomal Structure

Environmental factors including radiation, chemicals, and viruses, can cause chromosomes to break; if the broken ends do not rejoin in the same pattern, this causes a change in chromosomal structure.

B. Examples of Changes in Chromosomal Structure

	Deletion: a type of mutation in which an end of a chromosome breaks off or when two simultaneous breaks lead to the loss of a segment.
	Translocation: a chromosomal segment is removed from one chromosome and inserted into another, nonhomologous chromosome; In Down syndrome, 5% of cases are due to a translocation between chromosome 21 and 14, a factor that runs in the family of the father or mother.
	Duplication is the presence of a chromosomal segment more than once on the same chromosome.

1. A broken segment from one chromosome can simply attach to its homologue or unequal crossing-over may occur.

2. Duplication may also involve an inversion where a segment that has become separated from the chromosome is reinserted at the same place but in reverse; the position and sequence of genes are altered.

C. Human Syndromes

Deletion Syndromes

1. Williams syndrome occurs when chromosome 7 loses an end piece: children look like pixies, have poor academic skills but good verbal and musical skills; lack of elastin causes cardiovascular problems and skin aging.

Cri du chat syndrome is deletion in which an individual has a small head, is mentally retarded, has facial abnormalities, and abnormal glottis and larynx resulting in a cry resembling that of a cat.

Translocation Syndromes

1. If a translocation results in the normal amount of genetic material, the person will remain healthy; if a person inherits only one of the translocated chromosomes, that person may have only one allele or three alleles rather than the normal two.

2. In Alagille syndrome, chromosomes 2 and 20 exchange segments, causing a small deletion on chromosome 20 that may produce some abnormalities.

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Chapter 13 DNA: Structure and Functions

A. Early researchers knew that the genetic material must be:

	able to store information used to control both the development and the metabolic activities of cells;
	stable so it can be replicated accurately during cell division and be transmitted for generations; and
	able to undergo mutations providing genetic variability required for evolution.

B. Previous Knowledge About DNA

	Knowing the chemistry of DNA was essential to discovery that DNA is genetic material.
	In 1869, Swiss chemist Friedrich Miescher removed nuclei from pus cells and isolated DNA "nuclein"; it was rich in phosphorus and lacked sulfur.
	Nuclein was analyzed by other scientists who found that it contained an acid: nucleic acid.
	Two types of nucleic acids were soon discovered: DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
	In the early twentieth century, discovery that nucleic acids contain four types of nucleotides.

1. DNA was composed of repeating units, each of which always had just one of each of four different nucleotides (A, T, G, or C).

2. In this model, DNA could not vary between species and therefore could not be the genetic material; therefore some other protein component was expected to be the genetic material.

C. Transformation of Bacteria

	In 1931, bacteriologist Frederick Griffith experimented with Streptococcus pneumoniae (a pneumococcus) that causes pneumonia in mammals.
	Griffith injected mice with two strains of pneumococcus: an encapsulated (S) strain and a non-encapsulated (R) strain.

1. The S strain is virulent (the mice died); it has a mucous capsule and forms shiny colonies.

2. The R strain is not virulent (the mice lived); it has no capsule and forms dull colonies.

	In an effort to determine if the capsule alone was responsible for the virulence of the S strain, he injected mice with heat-killed S strain bacteria; the mice lived.
	Finally, he injected mice with a mixture of heat-killed S strain and live R strain bacteria.

1. The mice died and living S strain pneumococcus were recovered from their bodies.

2. Griffith concluded some substance necessary to synthesis of the capsule and, therefore, virulence must pass from dead S strain bacteria to living R strain bacteria so the R strain were transformed.

3. This change in phenotype of the R strain bacteria must be due to a change in their genotype, which suggested that the transforming substance may have passed from S strain to R strain.

D. DNA: The Transforming Substance

	Oswald Avery and his coworkers reported that the transforming substance was DNA.
	Purified DNA is capable of bringing about the transformation; their evidence included the following:

1. DNA from S strain pneumococcus causes R strain bacteria to be transformed.

2. Enzymes that degrade proteins cannot prevent transformation, nor do enzymes that digest RNA.

3. Digestion of the transforming substance with enzyme that digests DNA prevents transformation.

4. Molecular weight of the transforming substance is great enough for some genetic variability

Their experimental results demonstrated DNA is genetic material and DNA controls biosynthetic properties of a cell.

E. Transformation Experiments Today

	Transformation experiments today are common in high schools and research labs.
	Transformation occurs whenever organisms receive foreign DNA and receive a new trait.
	Modern experiments with bacteria show some can take up DNA to gain penicillin resistance.

F. Reproduction of Viruses

	Bacteriophage is a virus that infects bacteria; it consists only of a protein coat surrounding a nucleic acid core.
	Bacteriophage T2 is a virus that infects the bacterium Escherichia coli (E. coli), a species of intensely studied bacteria that normally lives within the human gut.
	In 1952, Alfred Hershey and Martha Chase used bacteriophage T2 in their experiments.

1. The purpose of their experiments was to see which of the bacteriophage components—the protein coat or the DNA—entered bacterial cells and directed reproduction of the virus.

2. In two separate experiments, they labeled the protein coat with ³⁵S and the DNA with ³²P.

3. Viral coats are sheared away from bacterial cells; they are separated by centrifugation.

4. Results: radioactive ³²P alone is taken up by bacterial host and incorporated in virus reproduction.

5. This result reinforced the notion that DNA (and not the protein) is the genetic material.

	In the 1940s, Erwin Chargaff analyzed the base content of DNA using new chemical techniques.
	It was known DNA contained four different nucleotides:

1. two with purine bases, adenine (A) and guanine (G); a purine is a type of nitrogen-containing base having a double-ring structure.

2. two with pyrimidine bases, thymine (T) and cytosine (C); a pyrimidine is a type of nitrogen-containing base having a single-ring structure.

	The results of his analysis proved DNA does have the variability necessary to code genetic material.
	Chargaff discovered that for a species, DNA has the constancy required of genetic material.
	This constancy is given in Chargaff’s rules:

1. The amount of A, T, G, and C in DNA varies from species to species.

2. In each species, the amount of A = T and the amount of G = C.

The tetranucleotide hypothesis (proposing DNA was repeating units of one of four bases) was disproved; each species had its own constant base composition.

B. Variation in Base Sequence

	The variability is staggering; a human chromosome contains about 140 million base pairs.
	Since any of the four possible nucleotides can be present at each nucleotide position, the total number of possible nucleotide sequences is 4140 x 106 = 4140,000,000.

C. Diffraction Data

	Rosalind Franklin, a student at King’s College, produced X-ray diffraction photographs.
	Franklin’s work provided evidence that DNA had the following features:

1. DNA is a helix.

2. One part of the helix is repeated.

C. The Watson and Crick Model

	American James Watson joined with Francis H. C. Crick in England to work on structure of DNA.
	Watson and Crick received the Nobel Prize in 1962 for their model of DNA.
	Using information generated by Chargaff and Franklin, Watson and Crick built a model of DNA as a double helix; sugar-phosphate molecules were on the outside, paired bases were on the inside.
	Their model was consistent with both Chargaff’s rules and the dimensions of the DNA polymer provided by Franklin’s photograph of X-ray diffraction of DNA.
	Complementary base pairing is the paired relationship between purines and pyrimidines in DNA, such that A is hydrogen-bonded to T and G is hydrogen-bonded to C.

DNA Anatomy: http://www.johnkyrk.com/DNAanatomy.html

A. DNA replication is the process of copying a DNA molecule.

	Unwinding: old strands of the parent DNA molecule are unwound as weak hydrogen bonds between the paired bases are unzipped and broken by the enzyme helicase.
	Complementary base pairing: free nucleotides present in the nucleus bind with complementary bases on unzipped portions of the two strands of DNA; this process is catalyzed by DNA polymerase.
	Joining: complimentary nucleotides bond to each other to form new strands; each daughter DNA molecule contains an old strand and a new strand; this process is also catalyzed by DNA polymerase.
	DNA replication must occur before a cell can divide; in cancer, drugs with molecules similar to the four nucleotides are used to stop replication.

B. Replication is Semiconservative

	DNA replication is semiconservative because each daughter double helix has one parental strand and one new strand.
	In 1958, Matthew Meselson and Franklin Stahl confirmed a model of DNA replication.

1. They grew bacteria in a medium with heavy nitrogen (¹⁵N), then switched to light nitrogen (¹⁴N).

2. The density of DNA following replication is intermediate as measured by centrifugation of molecules.

3. After one division, only hybrid DNA molecules were in the cells.

4. After two divisions, half the DNA molecules were light and half were hybrid.

These were exactly the results to be expected if DNA replication is semiconservative.

C. Prokaryotic Versus Eukaryotic Replication

Prokaryotic Replication

1. Bacteria have a single loop of DNA that must replicate before the cell divides.

2. Replication in prokaryotes may be bidirectional from one point of origin or in only one direction.

3. Replication only proceeds in one direction, from 5' to 3'.

4. Bacterial cells are able to replicate their DNA at a rate of about 10⁶ base pairs per minute.

5. Bacterial cells can complete DNA replication in 40 minutes; eukaryotes take hours.

Eukaryotic Replication

1. Replication in eukaryotes starts at many points of origin and spreads with many replication bubbles—places where the DNA strands are separating and replication is occurring.

2. Replication forks are the V-shape ends of the replication bubbles; the sites of DNA replication.

3. Eukaryotes replicate their DNA at a slower 500–5,000 base pairs per minute.

4. Eukaryotes take hours to complete DNA replication.

D. Replication Errors

	A genetic mutation is a permanent change in the sequence of bases.
	Base changes during replication are one way mutations occur.
	A mismatched nucleotide may occur once per 100,000 base pairs, causing a pause in replication.
	DNA repair enzymes perform a proofreading function and reduce the error rate to one per billion base pairs.
	Incorrect base pairs that survive the proofreading process contribute to gene mutations.

A. Investigators Recognize Gene Activity

English physician Sir Archibald Garrod introduced phrase inborn error of metabolism.

1. Garrod proposed that inherited defects could be caused by the lack of a particular enzyme.

2. Knowing that enzymes are proteins, Garrod suggested a link between genes and proteins in the early 1900s.

B. Genes Specify Enzymes

	In 1940, George Beadle and Edward Tatum X-rayed spores of red bread mold, Neurospora crassa.
	They discovered some resulting cultures lacked a particular enzyme for growth on medium.
	They found that a single gene was mutated, which resulted in the lack of a single enzyme.
	They proposed the one gene–one enzyme hypothesis: one gene specifies the synthesis of one enzyme.

C. Genes Specify a Polypeptide

	Linus Pauling and Harvey Itano compared hemoglobin in red blood cells of persons with sickle-cell disease and normal individuals.
	They discovered that the chemical properties of chain of sickle-cell hemoglobin differed from normal hemoglobin by using electrophoresis to separate molecules by weight and charge.
	Vernon Ingram showed the biochemical change to chain of sickle-cell hemoglobin is due to the substitution of a nonpolar amino acid valine for the negatively charged amino acid glutamate.
	Pauling and Itano formulated the one gene–one polypeptide hypothesis: each gene specifies one polypeptide of a protein, a molecule that may contain one or more different polypeptides.

D. From DNA to RNA to Protein

	Genetics treats a gene as any of the particles of inheritance on a chromosome.
	To a molecular geneticist, a gene is a sequence of DNA nucleotide bases that codes for a product.
	DNA is restricted to nucleus; protein synthesis occurs at ribosomes in the cytoplasm.
	Ribonucleic acid (RNA) is found in both regions and was likely intermediary in protein synthesis.

E. Types of RNA

	Like DNA, RNA is a polymer of nucleotides.
	Unlike DNA, RNA is single-stranded (not a double helix), contains the sugar ribose, and contains the base uracil instead of thymine.
	There are three major classes of RNA.

1. Messenger RNA (mRNA) takes a message from DNA in nucleus to ribosomes in cytoplasm.

2. Ribosomal RNA (rRNA) and proteins make up ribosomes where proteins are synthesized.

3. Transfer RNA (tRNA) transfers a particular amino acid to a ribosome.

F. The Required Steps

	DNA undergoes transcription to mRNA, which is translated to a protein.
	DNA is a template for RNA formation during transcription.
	Transcription is the first step in gene expression; it is the process whereby a DNA strand serves as a template for the formation of mRNA.
	During translation, an mRNA transcript directs the sequence of amino acids in a polypeptide.

A. Sequence of Bases in DNA

	The central dogma of molecular biology states that the sequence of nucleotides in DNA specifies the order of amino acids in a polypeptide.
	The genetic code is a triplet code comprised of 64 three-base code words (codons).
	A codon consists of 3 nucleotide bases of DNA.
	Four nucleotides based on 3-unit codons allows up to 64 different amino acids to the specified.

B. Finding the Genetic Code

	In 1961, Marshall Nirenberg and J. Heinrich Matthei found that an enzyme that could be used to construct synthetic RNA in a cell-free system; they showed the codon UUU coded for phenylalanine.
	By translating just three nucleotides at a time, they assigned an amino acid to each of the RNA codons, and discovered important properties of the genetic code.
	The code is degenerate: there are 64 triplets to code for 20 naturally occurring amino acids and this robustness protects against potentially harmful mutations.
	The genetic code is unambiguous; each triplet codon has only one meaning.
	The code has start and stop signals: there is one start codon and three stop codons.

C. The Code Is Universal

	The few exceptions to universality of the genetic code suggests the code dates back to the very first organisms and that all organisms are related.
	Once the code was established, changes would be very disruptive.

	Transcription is the first step required for gene expression and takes place in the nucleus of eukaryotic cells.
	mRNA formation usually leads to a polypeptide gene product; however, tRNA and rRNA are also transcribed from DNA templates and are products themselves.
	Enzymes called RNA polymerases are involved in transcription.

Transcription Animation: http://www.johnkyrk.com/DNAtranscription.html

DNA Transcription: http://www.fed.cuhk.edu.hk/~johnson/teaching/genetics/animations/transcription.htm

B. Messenger RNA is Formed

	Next, a segment of the DNA helix unwinds and unzips.
	As RNA polymerase moves along the template strand of the DNA, complementary RNA nucleotides pair with DNA nucleotides of the strand.
	Transcription begins when RNA polymerase attaches to a promoter on DNA.
	RNA polymerase is an enzyme that speeds formation of RNA from a DNA template.
	A promoter is region of DNA region defines the start of the gene, the direction of transcription, and the strand copied.
	RNA polymerase joins the RNA nucleotides together in the 5' —> 3' direction.
	Transcription begins when RNA polymerase attaches to a region of DNA called a promoter; a promoter defines the start of a gene, the direction of transcription, and the strand transcribed.
	The RNA/DNA association is not as stable as DNA helix; therefore, only the newest portion of the RNA molecule associated with RNA polymerase is bound to DNA; the rest dangles off to side.
	Elongation of mRNA continues until RNA polymerase comes to a DNA terminator.
	The terminator causes RNA polymerase to stop transcribing DNA and to release mRNA transcript.
	RNA polymerase molecules work to produce mRNA from same DNA molecule at same time.
	Cells produce thousands of copies of same mRNA molecule and many copies of coded protein in a shorter period of time than if a single copy of DNA were used to direct protein synthesis.

C. Messenger RNA is Processed

	In eukaryotes, newly formed primary mRNA transcript is processed before leaving the nucleus.
	Primary mRNA transcript is the immediate product of transcription; it contains exons and introns.
	The ends of the mRNA molecule are altered: a cap is put on the 5' end and a poly-A tail is put on the 3' end.

1. The "cap" is a modified guanine (G) that tells a ribosome where to attach to begin translation.

2. The "poly-A tail" consists of a 150–200 adenine (A) nucleotide chain that facilitates transport of mRNA out of the nucleus and inhibits degradation of mRNA by hydrolytic enzymes.

Portions of the primary mRNA transcript, called introns, are removed.

1. An exon is a portion of DNA code in primary mRNA transcript eventually expressed as result of polypeptide synthesis.

2. An intron is a non-coding segment of DNA removed by spliceosomes before the mRNA leaves nucleus.

Spliceosomes are a complex that contains several kinds of ribonucleoproteins.

1. Spliceosomes cut the primary mRNA transcript and then rejoin adjacent exons.

2. Spliceosomes may allow the same DNA to be divided differently and produce different products.

	The role of introns is being investigated; introns may divide a gene into regions that can be joined in different combinations for different products: the thyroid and pituitary glands process same primary mRNA transcript that produces different products.
	Investigators have found that the simpler the eukaryote, the less likely that introns will be present.
	An intron has been discovered in the gene for a tRNA molecule in the cyanobacterium Anabaena; this particular intron is "self-splicing" (it has capability of splicing itself out of an RNA transcript).
	Ribozymes are RNAs with an enzymatic function restricted to cleaving RNA at specific locations.

1. RNA could have served as both genetic material and as first enzymes in early life forms.

2. This hypothesis eliminates dilemma of which came first, DNA or protein; RNA came first.

	Translation takes place in cytoplasm of eukaryotic cells.
	Translation is the second step by which gene expression leads to protein synthesis.
	One language (nucleic acids) is translated into another language (protein).

B. The Role of Transfer RNA

	Transfer RNA (tRNA) molecules transfer amino acids to the ribosomes.
	tRNA is a single-stranded ribonucleic acid that doubles back on itself to create regions where complementary bases are hydrogen-bonded to one another.
	At the 3' end it binds to amino acid; at other end it has an anticodon that binds to mRNA codon; an anticodon is group of nucleotides on tRNA that is complementary to the codon on mRNA.
	There is at least one tRNA molecule for each of the 20 amino acids found in proteins.
	There are fewer tRNAs than codons because some tRNAs pair with more than one codon; if an anticodon contains a U in the third position, it will pair with either an A or G–this is called the wobble effect.
	The tRNA synthetases are amino acid-activating enzymes that recognize which amino acid should join which tRNA molecule, and then catalyze ATP-requiring reactions joining them.
	Amino acid–tRNA complex forms, then travels in the cytoplasm to a ribosome for protein synthesis.

C. The Role of Ribosomal RNA

	Ribosomal RNA (rRNA) is produced from a DNA template in the nucleolus of nucleus.
	The rRNA is packaged with a variety of proteins into ribosomal subunits, one larger than the other.
	Subunits move separately through nuclear envelope pores into the cytoplasm where they combine when translation begins.
	Ribosomes can float free in cytosol or attach to endoplasmic reticulum.
	Prokaryotic cells contain about 10,000 ribosomes; eukaryotic cells contain many times more.
	Ribosomes have a binding site for mRNA and binding sites for two transfer RNA (tRNA) molecules.
	They facilitate complementary base pairing between tRNA anti-codons and mRNA codons; one protein is an enzyme that joins amino acids together by means of a peptide bond.
	A ribosome moves down the mRNA molecule, new tRNAs arrive, the amino acids join, and a polypeptide forms.
	Translation terminates once the polypeptide is formed; the ribosome then dissociates into its two subunits.
	Polyribosomes are clusters of several ribosomes synthesizing the same protein.
	To get from a polypeptide to a function protein requires correct bending and twisting; chaperone molecules make sure that the final protein develops the correct shape.
	Some proteins contain more than one polypeptide; they must be joined to achieve the final three-dimensional shape.

D. Translation Requires Three Steps

	During translation, mRNA codons base-pair with tRNA anti-codons carrying specific amino acids.
	Codon order determines the order of tRNA molecules and the sequence of amino acids in polypeptides.
	Protein synthesis involves chain initiation, chain elongation, and chain termination.
	Enzymes are required for all three steps; energy is needed for the first two steps.
	Chain Initiation

Translation Animation: http://www.johnkyrk.com/DNAtranslation.html

1. A small ribosomal subunit attaches to mRNA in the vicinity of the start codon: a base triplet (AUG).

2. First or initiator tRNA pairs with this codon; then the large ribosomal subunit joins to the small subunit.

3. Each ribosome contains three binding sites: the P (for peptide) site, the A (for amino acid) site, and the E (for exit) site.

4. The initiator tRNA binds to the P site although it carries one amino acid, methionine.

5. The A site is for next tRNA carrying the next amino acid.

6. The E site is for discharged tRNAs.

7. Initiation factor proteins are required to bring the necessary translation components (the small ribosomal subunit, mRNA, initiator tRNA, and large ribosomal subunit) together.

Chain Elongation

1. tRNA with attached polypeptide is at the P site and a tRNA—amino acid complex is just arriving at the A site.

2. Proteins called elongation factors facilitate complementary base pairing between the tRNA anticodon and the mRNA codon.

3. The polypeptide is transferred and attached by a peptide bond to the newly arrived amino acid.

4. This reaction is catalyzed by a ribozyme, which is part of the larger subunit.

5. The tRNA molecule in the P site leaves.

6. Translocation occurs with mRNA, along with peptide-bearing tRNA, moves to the P site and spent tRNA moves from the P site to the E site.

7. As the ribosome moves forward three nucleotides, there is a new codon now located at the empty A site.

8. The complete cycle is rapidly repeated, about 15 times per second in Escherichia coli.

Chain Termination

1. Termination of polypeptide synthesis occurs at a stop codon that does not code for amino acid.

2. The polypeptide is enzymatically cleaved from the last tRNA by a release factor.

3. The tRNA and polypeptide leave the ribosome, which dissociates into its two subunits.

Definition of a Gene and a Genetic Mutation

1. Originally a gene was defined as a locus on the chromosome; this allowed us to solve genetic problems.

2. The one gene—one polypeptide concept connected inborn errors of metabolism with a sequence of DNA bases.

3. A gene could also be defined as a sequence of DNA bases coding for a single polypeptide or a single RNA.

4. These concepts can allow us to define a mutation as a permanent change in the sequence of DNA bases.

E. Protein Synthesis and the Eukaryotic Cell

	If a polypeptide is to enter the rough ER, the signal peptide is recognized by signal-recognition particles (SRP) which bring it to a receptor protein in the ER membrane.
	After the polypeptide enters the lumen of the ER, it is folded and further processed by addition of sugars, phosphates, or lipids.
	Transport vesicles carry the proteins between organelles and to the plasma membrane.

Protein Synthesis Animation: http://www.lewport.wnyric.org/jwanamaker/animations/Protein Synthesis - long.html

Transcribe and Translate a Gene: http://gslc.genetics.utah.edu/units/basics/transcribe/

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CHAPTER 15: REGULATION OF GENE ACTIVITY:

GENE MUTATION

This chapter addresses operons, levels of control of gene expression in eukaryotic cells, and cancer as a failure in genetic control.

Chapter Outline

15.1 Prokaryotic Regulation

A. The Operon Model

	Bacteria do not require the same enzymes all the time; they produce just those enzymes needed at the moment.
	In 1961, French microbiologists Francois Jacob and Jacques Monod proposed the operon model to explain regulation of gene expression in prokaryotes; they received a Nobel prize for this.

1. In the operon model, several genes code for an enzyme in the same metabolic pathway and are located in a sequence on a chromosome; expression of structural genes is controlled by the same regulatory genes.

2. An operon is the structural and regulatory genes that function as a single unit; it includes the following:

a) A regulator gene located outside the operon codes for a repressor protein molecule that controls whether the operon is active or not.

b) A promotor is the sequence of DNA where RNA polymerase attaches when a gene is transcribed.

c) An operator is a short sequence of DNA where an active repressor binds, preventing RNA polymerase from attaching to the promotor and transcription therefore does not occur.

d) Structural genes are one to several genes coding for enzymes of a metabolic pathway that are transcribed as a unit.

B. The trp Operon

	Jacob and Monod found some operons in E. coli usually exist in the on rather than the off condition.
	This prokaryotic cell (E. coli) produces five enzymes as part of the metabolic pathway to synthesize the amino acid tryptophan.
	If tryptophan is already present in medium, these enzymes are not needed and the operon is turned off by the following method.

1. In the trp operon, the regulator codes for a repressor that usually is unable to attach to the operator.

2. The repressor has a binding site for tryptophan (if tryptophan is present, it binds to the repressor).

3. This changes the shape of the repressor that now binds to the operator.

	The entire unit is called a repressible operon; tryptophan is the corepressor.
	Repressible operons are involved in anabolic pathways that synthesize substances needed by cells.

C. The lac Operon

	If E. coli is denied glucose and given lactose instead, it makes three enzymes to metabolize lactose.
	These three enzymes are encoded by three genes.

1. One gene codes for $-galactosidase that breaks lactose to glucose and galactose.

2. A second gene codes for a permease that facilitates entry of lactose into the cell.

3. A third gene codes for enzyme transacetylase, which is an accessory in lactose metabolism.

	The three genes are adjacent on a chromosome and under control of one promoter and one operator.
	The regulator gene codes for a lac operon repressor protein that binds to the operator and prevents transcription of the three genes.
	When E. coli is switched to medium containing an allolactose, this lactose binds to the repressor and the repressor undergoes a change in shape that prevents it from binding to the operator.
	Because the repressor is unable to bind to the operator, the promoter is able to bind to RNA polymerase, which carries out transcription and produces the three enzymes.
	An inducer is any substance (lactose in the case of the lac operon) that can bind to a particular repressor protein, preventing the repressor from binding to a particular operator, consequently permitting RNA polymerase to bind to the promoter and causing transcription of structural genes.

D. Further Control of the lac Operon

	Since E. coli prefers to break down glucose, how does E. coli know how to turn on when glucose is absent?
	When glucose is absent, cyclic AMP (cAMP) accumulates; cAMP has only one phosphate group and attaches to ribose at two locations.

1. CAP is a catabolite activator protein (CAP) in the cytoplasm.

2. When cAMP binds to CAP, the complex attaches to a CAP binding site next to the lac promoter.

3. When CAP binds to DNA, DNA bends, exposing the promoter to RNA polymerase.

4. Only then does RNA polymerase bind to the promoter; this allows expression of the lac operon structural genes.

When glucose is present, there is little cAMP in the cell.

1. CAP is inactive and the lactose operon does not function maximally.

2. CAP affects other operons when glucose is absent.

3. This encourages metabolism of lactose and provides a backup system for when glucose is absent.

Negative Versus Positive Control

1. Active repressors shut down the activity of an operon; they are negative control.

2. CAP is example of positive control; when the molecule is active, it promotes the activity of the operon.

3. Use of both positive and negative controls allows cell to fine-tune its control of metabolism.

4. If both glucose and lactose are present, the cell preferentially metabolizes glucose.

15.2 Eukaryotic Regulation

A. Expression of Genes

	Different cells in the human body turn on different genes that code for different protein products.
	Eukaryotes have a four levels of regulatory mechanisms to control gene expression; two in the nucleus and two in the cytoplasm.
	There levels of control modify the amount of gene product.

1. Transcriptional control in nucleus determines which structural genes are transcribed and rate of transcription; it includes organization of chromatin and transcription factors initiating transcription.

2. Posttranscriptional control occurs in nucleus after DNA is transcribed and preliminary mRNA forms.

a) This may involve differential processing of preliminary mRNA before it leaves the nucleus.

b) The speed that mature mRNA leaves nucleus affects ultimate amount of gene product.

3. Translational control occurs in cytoplasm after mRNA leaves nucleus but before there is a protein product.

a) The life expectancy of mRNA molecules can vary, as well as their ability to bind ribosomes.

b) Some mRNAs may need additional changes before they are translated at all.

4. Posttranslational control takes place in the cytoplasm after protein synthesis.

a) Polypeptide products may undergo additional changes before they are biologically functional.

b) A functional enzyme is subject to feedback control; binding of an end product can change the shape of an enzyme so it no longer carries out its reaction.

D. Transcriptional Control

Organization of Chromatin

1. During interphase, some chromatin is highly compact, darkly stained, and genetically inactive heterochromatin.

2. The rest is diffuse lightly colored euchromatin thought to be genetically active.

3. Barr Bodies

a) Since human males have only one X chromosome, it might be supposed that they produce half the gene product of a female with two X chromosomes.

b) However, females have a darkly staining Barr body that is condensed at the side of the nucleus that is the inactive chromatin of the second X chromosome.

c) Which X chromosome is condensed is determined by chance.

d) Body of heterozygous females is mosaic; half her cells express alleles on one X chromosome and half of her cells express the alleles on the other X chromosome.

e) Female gonads do not show Barr bodies; X chromosomes are both needed in development.

f) Only one active X chromosome in female zygote means that a lower gene product is normal.

g) Other examples of this mosaic effect include: ocular albinism, Duchenne muscular dystrophy, and female calico cat coat color.

4. Euchromatin activity is related to the extent nucleosomes are coiled and condensed.

a) A nucleosome is a bead-like unit made of a segment of DNA wound around complex of histone proteins.

b) Nucleosomes contain five primary histones: H1, H2A, H2B, H3 and H4.

c) When DNA is transcribed, activators called remodeling proteins are able to push aside the histone portion so transcription can begin.

5. Lampbrush chromosomes in egg cells of vertebrates present many loops for mRNA synthesis.

6. Polytene chromosome puffs in larval insects are made of many duplicated sister chromatids.

7. Use of radioactive uridine label for RNA shows DNA is being actively transcribed at puffs.

8. Gene amplification is replication of a gene so there are many copies; Xenopus frog germ cells increase nucleoli and therefore copies of rRNA genes by 1,000-fold.

Transcription Factors

1. Transcription is controlled by DNA-binding proteins called transcription factors; operons have not been found in eukaryotic cells.

2. Each cell contains different transcription factors; different combinations regulate activity of gene.

3. A group of transcription factors binds to a promoter adjacent to a gene; then the complex attracts and binds RNA polymerase but transcription may still not begin.

4. As well as DNA sequences, enhancers are involved in controlling transcription in eukaryotes.

a) Enhancers are regions where factors that help regulate transcription of the gene can bind.

b) Enhancers can be quite a distance from the promoter.

c) A hairpin loop in the DNA brings the factor attached to an enhancer into contact with transcription factors and RNA polymerase at promotor; this enables transcription to begin.

5. Transcription factors are always present in cell and most likely they have to be activated in some way (e.g., regulatory pathways involving kinases or phosphatases) before they bind to DNA.

C. Posttranscriptional Control

	Posttranscriptional control begins once there is an mRNA transcript.
	Messenger RNA molecules are processed before they leave the nucleus and enter the cytoplasm.
	Differential excision of introns and splicing of mRNA can vary the type of mRNA that leaves nucleus.

1. The hypothalamus and thyroid glands produce calcitonin but the mRNA that leaves the nucleus is not same in both types of cells.

2. Radioactive labeling shows they vary because of a difference in mRNA splicing.

3. Evidence of different patterns of mRNA splicing is found in cells that produce neurotransmitters, muscle regulatory proteins, and antibodies.

	Speed of transport of mRNA from nucleus into cytoplasm affects the amount of gene product realized per unit of time.
	There is difference in the length of time it takes various mRNA molecules to pass through nuclear pores.

D. Translational Control

Masking of mRNA

1. Frog eggs contain mRNA "masked messengers" that are not translated until fertilization occurs.

2. When fertilization occurs, they unmask and there is rapid gene product synthesis.

Life of mRNA

1. The longer an active mRNA molecule remains in the cytoplasm, the more product is produced.

2. Mature mammal red blood cells eject their nucleus but synthesize hemoglobin for several months; the mRNAs must persist during this time.

3. Ribonucleases are enzymes associated with ribosomes that degrade mRNA.

4. Mature mRNA has non-coding segments at 3' cap and 5' poly-A tail ends; differences in these segments influence how long the mRNA avoids being degraded.

Influence of Hormones

1. Prolactin promotes milk production by affecting the length of time mRNA persists and is translated.

2. Estrogen interferes with action of ribonuclease to prolong vitellin production in amphibian cells.

E. Posttranslational Control

Degradation of the Protein Product

1. Some proteins are not active after synthesis; the polypeptide product has to undergo additional changes before it is biologically functional.

2. Bovine proinsulin is inactive when first produced; a single long polypeptide folds into a three-dimensional structure, a sequence of 30 amino acids is removed from the middle, and the two polypeptide chains are bonded together by disulfide bonds resulting in an active protein.

Degradation of a Protein

1. Many proteins are short-lived in cells and degraded or destroyed so they are no longer active.

2. Giant protein complexes call proteasomes carry out this task. .

3. One example is cyclins that control the cell cycle; they are only temporarily present..

15.3 Genetic Mutations

A. A genetic mutation is a permanent change in the sequence of bases in DNA; mutations range from no effect to total inactivity.

B. Effect of Mutations on Protein Activity

Point mutations change a single nucleotide and therefore change a single specific codon.

1. They range in effect depending on the particular codon change.

2. Changes to codons that have same effect have no effect; UAU to UAC both code tyrosine.

3. A change from UAC to UAG (a stop codon) results in a shorter protein, and a change from UAC to CAC incorporates histidine instead of tyrosine.

4. Sickle cell disease results from a single base change in DNA where the $ chain of hemoglobin contains valine instead of glutamate at one location and the resulting distorted hemoglobin causes blood cells to clog vessels and die sooner.

Frameshift Mutations

1. Reading frame depends on the sequence of codons from the starting point: THE CAT ATE THE RAT.

2. If C is deleted, the reading frame is shifted: THE ATA TET HER AT.

3. Frameshift mutations occur when one or more nucleotides are inserted or deleted from DNA.

4. The result of a frameshift mutation is a new sequence of codons and nonfunctional proteins.

Nonfunctional Proteins

1. A single nonfunctioning protein can cause dramatic effects.

2. PKU results when a person cannot convert phenylalanine and it builds up in the system.

3. A faulty code for an enzyme in the same pathway results in an albino individual.

4. The human transposon Alu is responsible for hemophilia when it places a premature stop codon in the gene for clotting factor IX.

5. Cystic fibrosis is due to inheriting a faulty code for a chloride transport protein in plasma membrane.

6. Androgen insensitivity is due to a faulty receptor for male sex hormones; body cells cannot respond to testosterone and develop as a female although all of the body cells are XY.

C. Carcinogenesis

	Researchers have identified many proto-oncogenes whose mutation to an oncogene cause increased growth and lead to a tumor.
	The ras family of genes are the most common oncogenes implicated in human cancers.
	Alteration of one nucleotide pair converts a normal functioning ras proto-oncogene to an oncogene.
	A major tumor-suppressor gene p53 is more frequently mutated in human cancers than any other known gene.

1. The p53 protein acts as a transcription factor to turn on the expression of genes whose products are cell cycle inhibitors.

2. The p53 can also stimulate apoptosis, programmed cell death.

D. Cause of Mutations

	Some mutations are spontaneous, others are due to environmental mutagens.
	Mutations due to replication errors are very rare
	DNA polymerase constantly monitors, proofreads a new strand against the old, and repairs any irregularities, reducing mistakes to one out of every one billion nucleotide pairs replicated.
	Environmental mutagens are environmental substances that increase the chances of mutation.

1. Common mutagens are radiation and organic chemicals.

2. Cancer is a genetic disorder caused by a failure in the regulation of gene activity.

3. Carcinogens are mutagens that increase the chances of cancer.

4. X rays and gamma rays are ionizing radiation that creates free radicals, ionized atoms with unpaired electrons.

5. Ultraviolet (UV) radiation is easily absorbed by pyrimidines in DNA.

a) Where two thymine molecules are near each other, UV may bond them together as thymine dimers.

b) Usually dimers are removed from damaged DNA by special enzymes called repair enzymes.

6. Lack of repair enzymes produces xeroderma pigmentosum with a higher incidence of skin cancer.

7. Some organic chemicals act directly on DNA.

a) 5-bromouracil pairs with thymine but rearranges to a form that pairs with cytosine at the next DNA replication: an A—T pair becomes a G—C pair.

b) Chemicals may add hydrocarbon groups or remove amino groups from DNA bases.

c) Tobacco smoke contains a number of chemical carcinogens.

Transposons

1. Transposons are specific DNA sequences that can move within and between chromosomes.

2. Such "jumping genes" were first detected in corn and are now recognized in bacteria, fruit flies, and other organisms.

3. Charcot-Marie-Tooth disease is a rare human disorder where muscles and nerves of legs and feet wither away; caused by a transposon also found in fruit flies.

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Chapter 16 Biotechnology and Genomics

Gene cloning, biotechnology products, the polymerase chain reaction, DNA fingerprinting, transgenic organisms, gene therapy, and the Human Genome Project are detailed.

Chapter Outline

16.1 Cloning of a Gene

A. Cloning

	Cloning is the production of identical copies through some asexual means.
	An underground stem or root sends up new shoots that are clones of the parent plant.
	Members of a bacterial colony on a petri dish are clones because they all came from division of the same cell.
	Human identical twins are clones; the original single embryo separate to become two individuals.
	Gene cloning is production of many identical copies of the same gene.
	If the inserted gene is replicated and expressed, we can recover the cloned gene or protein product.
	Cloned genes have many research purposes: determining the base sequence between normal and mutated genes, altering the phenotype, etc.
	Humans can be treated with gene therapy; alteration of other organisms forms transgenic organisms.

B. Recombinant DNA Technology

Means of inserting DNA into live cells:

            1.  Inserting genes into bacteria (ex. human insulin gene)
                        1^st. isolate target (human) gene w/restriction enzyme and isolate bacterial plasmids
                        2^nd  treat plasmid w/restriction enzyme to cut plasmid open making place for human gene to be inserted
                        3^rd  combine human gene to plasmid with DNA ligase [an enzyme that promotes N-base pairing thus sealing the
                               two strands of DNA].  This 3rd step produces a recombinant plasmid (draw)
                        4^th  recombinant plasmids mixed with living bacteria which undergo transformation and thereby
                              acquire genetically engineered genes.

            2.  Inserting genes into eukaryotes
                              ·        use mechanical injection - microneedles or micropellet guns
                              ·        use a vector    
                                               ex. genetically engineered viruses can carry genes into animal and plant cells
                                               ex. genetically engineered bacteria can insert genes into plant cells

 

	Recombinant DNA (rDNA) contains DNA from two different sources. Click on http://library.thinkquest.org/24355/data/details/media/recombinantanim.html for animation
	To make rDNA, technician selects a vector.
	A vector is a plasmid or a virus used to transfer foreign genetic material into a cell.
	A plasmid is a small accessory ring of DNA in the cytoplasm of bacteria.
	Plasmids were discovered in research on reproduction of intestinal bacteria Escherichia coli.
	Introduction of foreign DNA into vector DNA to produce rDNA requires two enzymes.

1. Restriction enzyme is a bacterial enzyme that stops viral reproduction by cleaving viral DNA.

2. The restriction enzyme is used to cut DNA at specific points during production of rDNA.

3. It is called a restriction enzyme because it restricts growth of viruses but it acts a molecular scissors to cleave any piece of DNA at a specific site.

Restriction enzymes cleave vector (plasmid) and foreign (human) DNA.

1. Cleaving DNA makes DNA fragments ending in short single-stranded segments with "sticky ends."

2. The "sticky ends" allow insertion of foreign DNA into vector DNA.

The foreign gene is sealed into the vector DNA by DNA ligase.

1. Treated cells take up plasmids, and then bacteria and plasmids reproduce.

2. Eventually, there are many copies of the plasmid and many copies of the foreign gene.

3. When DNA splicing is complete, an rDNA (recombinant DNA) molecule is formed.

If the human gene is to express itself in a bacterium, the gene must be accompanied by the regulatory regions unique to bacteria and meet other requirements.

1.The gene cannot contain introns because bacteria do not have introns.

2. An enzyme called reverse transcriptase can be used to make a DNA copy of mRNA.

3. This DNA molecule is called complementary DNA (cDNA) and does not contain introns.

4. A laboratory DNA synthesizer can produce small pieces of DNA without introns.

C. The Polymerase Chain Reaction

PCR Animations: http://www.dnalc.org/vshockwave/pcrwhole.dcr and http://library.thinkquest.org/24355/data/details/media/polymeraseanim.html

	PCR can create millions of copies of a single gene or a specific piece of DNA in a test tube.
	PCR is very specific—the targeted DNA sequence can be less than one part in a million of the total DNA sample; therefore a single gene can be amplified using PCR.
	The polymerase chain reaction (PCR) uses the enzyme DNA polymerase to carry out multiple replications (a chain reaction) of target DNA.
	PCR automation is possible because heat-resistant DNA polymerase from Thermus aquaticus, which grows in hot springs, is an enzyme that withstands the temperature necessary to separate double-stranded DNA.

D. Analyzing DNA Segments

	Mitochondria DNA sequences in modern living populations can decipher the evolutionary history of human populations.
	DNA fingerprinting is the technique of using DNA fragment lengths, resulting from restriction enzyme cleavage and amplified by PCR, to identify particular individuals.

1. DNA is treated with restriction enzymes to cut it into different sized fragments.

2. During gel electrophoresis, fragments separate according to length, resulting in a pattern of bands.

3. DNA fingerprinting can identify deceased individuals from skeletal remains, perpetrators of crimes from blood or semen samples, and genetic makeup of long-dead individuals or extinct organisms.

PCR amplification and DNA analysis is used to:

1. detect viral infections, genetic disorders, and cancer;

2. determine the nucleotide sequence of human genes: the Human Genome Project; and

3. associate samples with DNA of parents because it is inherited.

Plasmid Cloning: http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html

16.2 Biotechnology Products

A. Transgenic Organisms

Transgenic organisms - organisms that have had a foreign gene inserted into them.
(the gene which was inserted may be from same or different species)

	Genetically engineered organisms can produce biotechnology products.
	Organisms that have had a foreign gene inserted into them are transgenic.

 Mass produce human proteins needed to treat human disorders. ex. gene for human blood clotting factor put into pigs, transgenic pigs (genetically engineered pigs) then produce a human blood clotting protein. ex. gene for human insulin put into bacteria, transgenic bacteria then produce human insulin. ex. gene for human growth hormone put in mice (the growth hormone extracted from mice urine  

5. Improve Livestock and Crops – ex. Roundup Ready cotton and Bt corn: check out this web site from the University of Kentucky).

 

B. Transgenic Bacteria

Bacteria are grown in large vats called bioreactors.

1. Foreign genes are inserted and the product is harvested.

2.  Products on the market include: insulin, hepatitis B vaccine, t-PA, and human growth hormone.

Transgenic bacteria have been produced to protect and improve the health of plants.

1. Frost-minus bacteria protect the vegetative parts of plants from frost damage.

2. Root-colonizing bacteria receive genes from bacteria for insect toxin, protecting the roots.

3. Bacteria that colonize corn roots can be endowed with genes for insect toxin.

Transgenic bacteria can degrade substances.

1. Bacteria selected for ability to degrade oil can be improved by genetic engineering.

2. Bacteria can be bio-filters to prevent airborne chemical pollutants from being vented into the air.

3. Bacteria can also remove sulfur from coal before it is burned and help clean up toxic dumps.

4. Bacteria can also be given"suicide genes" that caused them to die after they have done their job.

Transgenic bacteria can produce chemical products.

1. We can manipulate genes coding for enzymes to catalyze synthesis of valuable chemicals.

2. Phenylalanine used in aspartame sweetener can be grown by engineered bacteria.

Transgenic bacteria process minerals.

1. Many major mining companies already use bacteria to obtain various metals.

2. Genetically engineered "bio-leaching" bacteria extract copper, uranium, and gold from low-grade ore.

C. Transgenic Plants

	Plant cells that have had the cell wall removed are called protoplasts.
	Electric current makes tiny holes in the plasma membrane through which genetic material enters.
	The protoplasts then go on to develop into mature plants.
	Foreign genes now give cotton, corn, and potato strains ability to produce an insect toxin and soybeans are now resistant to a common herbicide.
	Plants are being engineered to produce human proteins including hormones, clotting factors, and antibodies in their seeds; antibodies made by corn deliver radioisotopes to tumor cells and a soybean engineered antibody can treat genital herpes.
	Mouse-eared cress has been engineered to produce a biodegradable plastic in cell granules.

D. Transgenic Animals

Animal use requires methods to insert genes into eggs of animals.

1. It is possible to micro-inject foreign genes into eggs by hand.

2. Vortex mixing places eggs in an agitator with DNA and silicon-carbide needles that make tiny holes through which the DNA can enter.

3. Using this technique, many types of animal eggs have been injected with bovine growth hormone (bGH) to produce larger fishes, cows, pigs, rabbits, and sheep.

Gene pharming is the use of transgenic farm animals to produce pharmaceuticals; the product is obtainable from the milk of females.

1. Genes for therapeutic proteins are inserted into animal’s DNA; animal’s milk produces proteins.

2. Drugs obtained through gene pharming are planned for the treatment of cystic fibrosis, cancer, blood diseases, and other disorders.

E. Cloning Transgenic Animals

	For many years, it was believed that adult vertebrate animals could not be cloned; the cloning of Dolly in 1997 demonstrated this can be cone.
	Cloning of an adult vertebrate would require that all genes of an adult cell be turned on again.
	Cloning of mammals involves injecting a 2n nucleus adult cell into an enucleated egg.
	The cloned eggs begin development in vitro and are then returned to host mothers until the clones are born.

Conceiving a Clone: http://library.thinkquest.org/24355/data/details/media/willadsenanim.html

Roslin Technique of Cloning: http://library.thinkquest.org/24355/data/details/media/roslinanim.html

 

F Animal Organs as Biotechnology Products

	It may be possible to use genetically engineered pigs to serve as a source of organs for human transplant.
	Scientists are learning how to stimulate human cells to construct organs in the laboratory.

16.3 The Human Genome Project

A. The Human Genome Project had two goals: (1) to map the sequence of base pairs along our chromosomes and (2) to construct a map of the genes on all human chromosomes.

	The first task is completed; it took 15 years to learn the sequence of the three billion base pairs along the length of our chromosomes.
	The International Human Genome Sequencing Consortium was supported by public funds; Celera Genomics was funded by pharmaceutical industry.
	There is little difference between the sequence of our bases and other organisms whose DNA sequences are known.
	We share a large number of genes with simpler organisms; perhaps our uniqueness is due to regulation of these genes.

B. The Genetic Map

	A genetic map will locate each gene along each chromosome.
	With the base map completed, the chromosomal genetic map should be completed faster.
	The total number of human genes appears to be far lower than expected–perhaps only 30,000.
	With a roundworm possessing 20,000 genes, either more genes are yet to be found or each gene could code for three proteins by sing different combinations of exons.
	A genetic map of a chromosome could help tailor medical treatments to an individual.
	Gene therapy genes could be inserted into an egg before it is fertilized.
	Such potentials raise many ethical issues.

16.4 Gene Therapy

 Gene therapy - the transfer of normal genes/alleles into tissues of one w/genetic disorder = “a partial cure”
     Examples of human genetic disorders currently or soon to be treated with gene therapy:

Cystic fibrosis - lung tissue given gene that thins mucus (this gene therapy has had limited success). v Immune disorders (e.g. Bubble Boy) - white blood cells given gene that empowers them to fight infection.

Sickle cell anemia - Bone marrow stem cells divide to produce blood cells. To cure one with sickle cell anemia bone marrow stem cells need to be genetically corrected, i.e., only bone marrow cells need to receive the normal hemoglobin gene. Bioengineers recently claimed they  have cured sickle cell anemia in mice in this way and that a cure for sickle cell anemia will be forthcoming in three years.

 

A. Gene Therapy Inserts Healthy Genes

	This includes procedures to give patient healthy genes to make up for a faulty gene.
	Gene therapy also includes the use of genes to treat genetic disorders and various human illnesses.
	There are ex vivo (outside body) and in vivo (inside body) methods of gene therapy.

B. Ex Vivo Gene Therapy

Children with severe combined immunodeficiency (SCID) underwent ex vivo gene therapy.

1. Lacking the enzyme ADA involved in maturation of T and B cells, they faced life-threatening infections.

2. Bone marrow stem cells are removed, infected with a retrovirus that carries a normal gene for the enzyme ADA, and returned.

3. Use of bone marrow stem cells allows them to divide and produce more cells with same genes.

4. Patients who undergo this procedure show significant improvement.

Gene therapy trials include treatment of familial hypercholesterolemia where liver cells lack a receptor for removing cholesterol from blood.

1. High levels of blood cholesterol make the patient subject to fatal heart attacks when young.

2. A small portion of the liver is surgically removed and infected with retrovirus with normal gene for receptor.

3. This has lowered cholesterol levels following the procedure.

C. In Vivo Gene Therapy

Cystic fibrosis patients lack a gene for trans-membrane chloride ion carriers; patients die from respiratory tract infections.

1. Liposomes, microscopic vesicles that form when lipoproteins are in solution, are coated with healthy cystic fibrosis genes and sprayed into a patient’s nostrils.

2. Various methods of delivery are being tested for effectiveness.

	A gene for vascular endothelial growth factor (VEGF) can be injected alone or within a virus into the heart to stimulate branching of coronary blood vessels.
	Another strategy is to make cancer cells more vulnerable, and normal cells more resistant, to chemotherapy.
	Injecting a retrovirus containing a normal p53 gene–that promotes apoptosis–into tumors may stop the growth of tumors.

Chromatography and Gel Electrophoresis: http://www.lewport.wnyric.org/jwanamaker/animations/Chrom&Elpho.html

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CHAPTER RELATED WEBSITES

Genetics Glossary: http://www.genome.gov/glossary.cfm

                    Paternity Testing: http://www.sumanasinc.com/webcontent/anisamples/dynamicillustrations/paternitytesting.html

                    DNA Timeline: http://www.dnai.org/timeline/index.html

                    Human Genome: http://www.dnai.org/c/index.html

                    Human Genome Landmarks: http://www.ornl.gov/sci/techresources/Human_Genome/posters/chromosome/chooser.shtml

                    DNA Code: http://www.dnai.org/a/index.html

                    DNA Manipulation: http://www.dnai.org/b/index.html

                    Human Genome Sequencing: http://www.ncbi.nih.gov/mapview/map_search.cgi

                    Genetics Practice Problems: http://biology.clc.uc.edu/courses/bio105/geneprob.htm

                                                               http://web.mit.edu/esgbio/www/mg/problems.html

                                                                http://www.ksu.edu/biology/pob/genetics/intro.htm

EXTRA CREDIT ASSIGNMENT: http://www.ornl.gov/sci/techresources/Human_Genome/publicat/genechoice/1_martin.html