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AP Biology Notes (15/16)
Genetics
Unit 3 (Chapters 13-20)
Overview
gene: the basic unit of heredity information (DNA)
chromosomes: cellular structures containing many genes
mitosis- duplication and transfer of chromosomes to new cells
-you need to review mitosis on your own!!
 meiosis- duplication and transfer of chromosomes to gametes ("sex cells") for the
creation of new individuals
Mendelian genetics: patterns in which different genes are inherited
population genetics: the types and numbers (%) of genes in a population
molecular genetics: nature of, processing and control of DNA



-
 Sexual Reproduction is the key to Diversity!
Sexual Reproduction the mixing of genes to produce new individuals
homologous chromosomes:
• most organisms have chromosomes in pairs (at some stage in life cycle)
- one comes from the male parent
- one comes from the female parent
• these pairs have genes coding for the same proteins in the same order
diploid: having chromosomes in pairs
- the common situation for most animals and higher plants
haploid: cells having only one of each chromosome
- prevalent part of the life cycle for some lower plants (eg: ferns)
- a necessary stage for sexual reproduction (gametes are haploid)
sexual recombination
• chromosome number must be reduced first
- gametes: haploid sex cells (sperm and egg)
- meiosis: process of forming gametes
• syngamy (fertilization): joining of gametes
• zygote: diploid fertilized egg
- this diploid cell now has pairs of chromosomes (homologous)
– one from each parent
• in most animals and higher plants:
- the zygote will continue to divide by mitosis
- to produce a multicellular, diploid individual
 Meiosis the formation of gametes for sexual reproduction
 Meiosis I
• Interphase and early prophase like mitosis
Prophase I
• homologous chromosomes (each with two chromatids) pair up
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• crossing over
- contact between chromatids of homologous chromosomes
- at places coding for the same genetic information
- forms chiasmata
- the chromosomes break apart at these sites
- and rejoin with the opposite partner
- allows for increased variety of genetic combinations
(all genes on same chromosome don't have to come from same grandparent)
Metaphase I
• homologous chromosomes line up on the equatorial plane; orientation to each pole
is random every time.
Anaphase I
• one of each homologous pair is drawn toward the opposite pole
- independent assortment (
recombination ):
– chromosomes from different parents
– shuffled randomly into gametes
– increasing genetic variability
Telophase I
• the cell now divides
• each cell now has one of each chromosome pair
(but each consisting of two chromatids)

Meiosis II
metaphase II: the paired chromatids line up on the equatorial plane; orientation is random
each time.
anaphase II: separate and move toward the spindle poles
telophase II: meiosis is completed
• cytokinesis completes the cell division
• there are now four cells from the original
• each with half the number of chromosomes (1/2 of 2n is n )
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In males, all 4 cells become sperm
In females, 1 cell will be an egg – other 3 will become polar bodies
each gamete now has:
— one chromosome from each original pair
— each randomly chosen from those contributed by both parents
— but is actually combination of both parent's due to crossing over
 Mendelian Genetics
 Gregor Mendel: Austrian monk (1822—1884), published 1856
- a very careful, patient man; well grounded in mathematics
- worked with controlled breeding of pea plants
- studied how certain traits were inherited over generations
Terms:
- P = parental: original pure breeding stock
- cross: breeding of parents with different characteristics
- mono-hybrid cross: involves only a single character pair
- F1 = first filial: first generation from a cross between parents
- F2 = second filial: offspring from crossing F1's
- dominant: traits which were expressed over others in the F1
- recessive: traits that "disappeared" in F1 (masked by dominant)
examples: round vs. wrinkled seeds; yellow vs. green seeds
• Mendel crossed purebred P’s with different traits
• for most pairs of traits— only one appeared in the F1 generation
• when F1's crossed with each other— the recessive trait reappeared !
– ratio of dominant to recessive usually about 3:1
Mendel’s hypotheses:
• traits caused by "discrete, heritable factors"
genes
• there are alternative forms for genes
alleles
• genes (traits) are inherited in pairs, one from each parent
• when pairs are different (in individual):
- one is expressed (dominant)
- the other is masked (recessive)
Law of segregation (2nd Law):
alleles of a gene isolate into different gametes during meiosis.
more terms:
-
-
alleles: genes coding for same trait but different expressions of it
example: seed surface— round or wrinkled,
seed color— yellow or green; hemoglobin— normal or sickle cell
phenotype: physical appearance due to a gene or genes (yellow etc.)
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-
-
genotype: which pair (or set) of genes is actually present
example: R = round (dominant)
r = wrinkled (recessive)
 organism with round phenotype could have genotype RR or Rr
homozygous: both genes are the same allele
heterozygous: the two genes are different alleles
Punnett square: keeping track of how alleles may be matched up
example: cross of purebred round with purebred wrinkled
genotypes being crossed
gametes produced
possible filial genotypes
-note that all possibilities are Rr (heterozygous)
Single Gene Crossings
(mono)-hybrid cross: If we cross the heterozygous F1 with itself: Rr x Rr
•
•
•
•
•
chances 3 out of 4 offspring will get an R gene
(but only 1/4 will be homozygous dominant)
1/4 will show recessive gene (1/2 will “carry”)
the ratio of R to r phenotypes will be 3:1 (with a large sample)
ratio of genotypes will be 1:2:1 (RR:Rr:rr)
test cross: to find genotype of individual with dominant phenotype
• dominant phenotype could be homozygous or heterozygous
• cross with homozygous recessive to find out:
RR
r
r
R
Rr
Rr
R
Rr
Rr
If homozygous:
• 100% dominant
Rr
r
r
r
rr
rr
R
Rr
Rr
If heterozygous:
• 50% dominant
• 50% recessive
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Law of independent assortment (1st Law): [Does not always hold true.]
Pairs of alleles sort independently of other genes during gamete formation.

Dihybrid cross: cross of individuals heterozygous for two different genes
RrYy x RrYy
With independent assortment:
(which genes will gametes receive?)
gametes = RY, Ry, rY, ry
Ways a phenotype may appear:
R Y : 9 combinations
Ry: 3
"
rY: 3
"
ry: 1
"
16 total
• 9/16 should appear dominant for both characteristics
• 3/16 dominant R, recessive y
• 3/16 recessive r, dominant Y
• 1/16 double recessive
Mendel observed these ratios with the traits he used
- but most trait pairs Mendel used were on separate chromosomes!
- linked
traits on same chromosome (usually) inherited together
If linked genes do recombine in different gametes, had to have
separated during crossing over (not common)
If genes on the same chromosome are far enough apart (not linked),
recombination can occur.
Segregation and independent assortment can be applied to genes
that are on different chromosomes (not linked).
crossing over
linked genes are on same chromosome but:
- linked genes may not assort independently
• during prophase I of meiosis chiasmata appear
- genes are exchanged between homologous chromosomes
- so the genetic material is reshuffled within chromosomes
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• this allows for more genetic variation
(in addition to recombination due to independent assortment)
• genes can be mapped on chromosomes
- the more crossing over, the farther between the genes
-----------------------------------------------------------------------------------------------------------Chi Square Statistical Analysis ~ Goodness of Fit Test
How well does an observed set of data fit into a hypothesized or expected set of data? In other
words, the Chi-square test measures how close your actual data collected follows a normal
distribution.
Ex. If you count the number of green M&M’s in a bag, does that percentage of green M&M’s in
the bag align with what the Mars Co. says with regards to how many green M&M’s are in one bag.
 Basic Computational Equation
 Oi = the frequencies observed
 Ei = the frequencies expected
 = the ‘sum of’
 Null Hypothesis:
The null hypothesis, (H0), is usually the hypothesis that sample observations result purely from
chance. In other words, the null hypothesis states that there is no statistical difference
between the observed and expected frequencies.
Ex. If you flip a coin 10 times you should get 5 heads and 5 tails.
 The point of the Chi-square test is to either accept or reject the Null Hypothesis.
Ex. If you flip a coin 10 times, the results would be anything other than 5 heads and 5 tails, due
to a possible problem with the coin (ex. weight distribution of the metal in the coin)
 Degree of Freedom:
In statistics, the number of degrees of freedom is the number of values in the final calculation
of a statistic that are free to vary.
 Figuring out Degree of Freedom: How many outcomes minus 1
Ex. roll dice: There are 6 possible outcomes (six sides to land on); Degree of Freedom would be
6-1=5

Critical Value: (use chart provided) - A value used to compute a margin of error
 Use the 0.05 column and the Degree of Freedom row.
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Using this column tells you that you can be 95% sure in accepting (or rejecting,) your Null
Hypothesis – depending on if you move to the right or left of 0.5 on chart.
Steps:
1.
2.
3.
4.

Calculate Chi square value
Calculate Degree of Freedom
Find Degree of Freedom on the Critical Value chart (0.05 column)
Compare your Chi square value to the value in this box on the chart
If your value is higher than Critical value = reject Null hypothesis
(Something more than chance is causing you to get higher than critical value; an
outside/other variable is affecting the experiment)
 If your value is lower than Critical value = accept Null hypothesis
(Observed data is within the range of the Expected data.)
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Other Factors in Heredity
dominance/recessiveness
• dominant allele produces a functional protein, the recessive does not
codominance: both alleles have effect
– both effects are seen in phenotype
◊ A, B and AB blood groups
• two different, functional proteins are produced
incomplete dominance:
Sometimes dominance is not observed: color in snapdragons
- CRCR = red, CWCW = white and CRCW = pink
- in a double heterozygous cross: 1 red : 2 pink : 1 white
◊ color in snapdragons, sickle cell disease
• double dose of some functional proteins
stronger effect
polygenic traits
continuous variation:
- some factors involve more than one gene
- so there is more than 2 or 3 phenotypes
◊ height, skin color, nose shape, etc.
multiple alleles:
- many genes have more than one or two alleles
◊ blood types: IA, IB, and i
*pleiotropy: some genes have multiple effects
*epistasis: some genes may hide the effects of others
 Human Genetics
 pedigrees: tracing genetic relationships in a “family tree”
carrier: heterozygote with recessive genetic defect
sickle–cell anemia, Tay–Sachs disease, cystic fibrosis, hemophilia
dominantly inherited disorders: some defects are dominant
Huntington’s disease
fetal testing:
amniocentesis: withdrawal by needle of amniotic fluid
chorionic villi sampling: removal of sample through cervix

Sex Linkage
• most chromosome pairs non–sex (= autosomes)
• in most animals one pair are sex chromosomes
- these appear different in different sexes
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◊ (in chickens: XX is a male
XY is a female hahahaha, but true!)
◊ (in grasshoppers: XX is a female Xo is a male) [o = no chromosome]
◊ in humans: XX is a female
Xy is a male
- Xo = Turner syndrome: moderately abnormal, sterile female
- XXy = Kleinfelter syndrome: decidedly abnormal, sterile male
• in males (human) the two sex chromosomes contain different genes so patterns of
inheritance are different
Inheritance of hemophilia, a sex linked trait in humans
- the gene for hemophilia is on X chromosome (X)
- if a man has the gene, he has the disease (Xy)
- if a woman has two of the genes, she has the disease (XX)
- if a woman has one X and one normal she is normal but a carrier
(also applies to red-green color blindness)
Thomas Hunt Morgan: genes associated with chromosomes
Drosophila : the common fruit fly
• the star of numerous experiments in genetics (including Morgan's)
• possess four pair of chromosomes
- one pair is sex chromosomes
- XX is female, Xy is male (just like humans)
*Note: notation is different in Drosophila (so many multiple alleles)
- lower case letter = mutant allele (usually recessive) [eg: v ]
- same letter with superscripted plus = wild type [eg: v+ ]
- y = y chromosome

sex–linked example in Drosophila :
v = vermilion eye color
v+ = wild type eye color (red)
cross: vv+ (female) x v+y (male)
males: 50% vermilion, 50% wild
females: 100% wild (but 50% are heterozygous)
**chromosomal alterations
chromosomal rearrangements: due to breaks in chromosome
- deletion: genes, gene or piece of gene is removed
- inversion: fragment is flipped end for end
- duplication: joins to homologous chromosome
- translocation: moved to another position
non–disjunction: failure of chromatids to separate in meiosis
• if gamete doesn’t receive copy = unviable (dead meat)
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• if gamete receives two copies:
- usually unviable
- sometimes produces abnormal individual
– trisomy: individual has three copies (2 + 1)
- extra gene products lead to unbalance = abnormality/death
• Down’s syndrome = trisomy 21 (3 copies of chromosome 21)
polyploidy: more than diploid set of chromosomes
- common in flowering plants (extremely rare in animals)
- must be same number of each chromosome
genomic imprinting
• genes may sometimes be selectively marked
• may occur selectively in one sex
• resulting in different effect depending upon from which parent
inherited
 Molecular Genetics Overview
proteins dictate structure and function of organisms (as enzymes)
DNA codes for type and activity of proteins
- replication: DNA must pass information to daughter cells
- transcription: DNA is read by a messenger mRNA
- translation: mRNA is template for protein synthesis
- control: when and to what extent are genes turned on

DNA as the information molecule ~ Discovery of the Role of DNA
• F. Griffith: (1928) heredity material is chemical in nature
transformation: uptake of new genetic information by bacteria
• Chargaff: (1947) proportions of N–bases unique to each species,
amounts of adenine = thymine, cytosine = guanine
• Hershey—Chase: (1952) DNA as genetic information
using radioactively bacteriophages (bacterial viruses)
labeled sulfur (in proteins only)
no uptake by bacteria
labeled phosphorus (in DNA only)
radioactive bacteria
• Wilkins—Franklin: x-ray crystallography of DNA
• James Watson and Francis Crick: (1953) the double helix structure
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
Structure of DNA REVIEW
-
basic unit = nucleotide
- joined in long strands:
P
+
pentose
P
phosphate
+
or
nitrogenous base
nucleotide
- deoxyribose is pentose sugar in DNA
- ribose is pentose sugar in RNA
pentose— P—pentose— —pentose—
P
—
P
• phosphate attached to 5'C of one sugar
is linked to 3'C of next sugar in chain
- so strand has 3' end and 5' end
• two strands joined by H-bonds between N-bases
• strands are joined "antiparallel"
5' end
5' carbon
- the 3' end of one strand is matched
with the 5' end of the other strand
P
3' carbon
3' end
• bases are always paired:
Adenine - Thymine
Thymine - Adenine
Cytosine - Guanine
Guanine - Cytosine
- these are complimentary pairs
• these strands form a double helix
Replication
DNA information must be duplicated for
- transmission to new offspring (meiosis)
- formation of new cells during growth (mitosis)
replication is semi-conservative: each strand copies a new partner
- Meselson—Stahl experiment with 15N DNA [Figures 15.7 & 15.8]
 Protein Synthesis
 Central Dogma
DNA
transcription
mRNA
translation
replication
(one gene, one polypeptide)
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P
protein
Transcription the formation of a messenger from the DNA gene
The flow of genetic information:
The Process of Transcription
The message in DNA (sequence of N-bases) is transferred to mRNA
• performed by the enzyme complex RNA polymerase
• process begins at special sites (promoter region)
• other transcription factors must also be attached at promoter
• DNA is split and partially unwound
• ribonucleotides are matched to a complimentary DNA base
- in RNA the base Uracil replaces the Thymine of DNA ( A-U & U-A )
• these are joined by phosphate bonds
• the new mRNA separates from the DNA
• DNA is rezippered and rewound behind the active region
• this RNA must be "trimmed" (in eukaroytes):
- some of it is "message" - exons [= expressed sequences]
- some of it is "garbage" - introns [= intervening sequences]
- exons are cut out and rejoined (without introns) to form mRNA
• final messenger is transported into the cytoplasm
- (same strand of DNA is not used for all genes on that chromosome)
Translation
the synthesis of proteins from mRNA
ribosomes: the sites of protein synthesis
— made in nucleus (nucleolus) from RNA (60%) and proteins
— composed of small (30s) and large (50s) subunits
— synthesis begins when 30s unit attaches to mRNA
— consists of two sites for tRNA attachment:
– P site: tRNA with growing peptide chain
– A site: for attachment of tRNA with new amino acid
codons: the basic information unit
– the nucleotides are the alphabet
– codons are the words
– mRNA contains the sentence for a protein molecule
— there are ~20 amino acids used by organisms
— 4 letters (A, C, G, U) need to be used in groups of 3 (a codon)
– 3 letter words necessary for minimum of 20 words (1/a.a.)
– 3 letter words with 4 letter alphabet = 43 = 64 combinations
— 64 codons for 20 amino acids means redundancy:
• most amino acids have more than one codon, but there is no
ambiguity:
• each codon specifies only one amino acid
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— reading frame: grouping of bases into codon
A C T A G T T C G A A C T G A
- one choice of reading frame reads codons as ACT, AGT, TCG, AAC, etc.
- second choice gives CTA, GTT, CGA, ACT, etc.
- third choice yields TAG, TTC, GAA, CTG, etc.
— start codon = AUG (also codes for methionine)
– insures proper reading frame
— some codons are stop codons
tRNA: transfer RNA
— matches amino acids to their respective codons
— 4-leaf clover layout bent into L shape:
- one end binds to a specific amino acid (requires ATP)
• specific tRNA matched to specific amino acid by specific
enzymes (aminoacyl–tRNA synthetases)
- opposite end is anticodon: matches a specific codon
- other "leaves" probably aid in placement on the ribosome
The Process of Translation
Initiation:
• initiator tRNA—methionine binds to the mRNA + 30s subunit
- begins at start codon site on mRNA (AUG)
- requires protein initiation factors and GTP (similar to ATP)
- mRNA is read from 5’ to 3’ end
• 50s ribosome subunit then attaches
- initiator tRNA occupies P site of ribosome
cont.
Elongation:
• next codon paired with matching tRNA-amino acid at A site
• peptide bond is formed between the amino acids
• first amino acid is released from the tRNA (in P site )
- tRNA at A site now possess growing peptide chain
• (first) tRNA is released from the ribosome (vacates P site )
• {mRNA + tRNA + attached amino acid chain} shifts
- the {tRNA + a.a. chain} now occupies P site
• next codon (A site ) matched with another tRNA-amino acid
• peptide bond formed between
– a.a. attached to tRNA at P site and a.a. at A site
and a.a. chain released from tRNA of P site
• {tRNA + growing a.a. chain} shifts into P site
• cycle continues adding one amino acid at a time
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Termination:
-
• process continues until a stop codon is reached
• ribosome is released and separates into 30s and 50s
the newly forming protein is folded into its 3-D structure
initial methionine (start code) is cleaved from the protein
as the ribosome moves along the mRNA, others are free to follow
(increasing the efficiency of translation)
first few amino acids may signal ribosome attachment to E.R.
(signal mechanism)
wobble: 3rd letter of anticodon may match alternate letters in codon
Mutations
• base pair substitution: one nucleotide pair replaces another
missense:
- may result in no change of function
– especially in wobble position
– if amino acid is similar and not critical
. to determining 3–D shape or
. in domain of protein (a functional site)
- may result in change of function
– one amino acid substitution = sickle cell anemia
- occasionally leads to improved function
nonsense:
- early termination of translation
• insertions and deletions: addition or removal of base pairs
- results in frameshift mutation (unless in groups of three)
nonfunctional unless at unimportant end of protein chain
VIRUSES
structure:
• capsid: protein shell
– protection of genetic core
–
• nucleic acid core for directing more virus production
– DNA or RNA, single or double stranded
• some possess envelope: modified host cell membrane
– also assists entry into new cells
– found in some animal viruses only (ex: HIV)
reproduction:
lytic cycle: (typical of some bacteriophages ["phages"])
• attachment to specific host cell
• insertion of phage DNA (or RNA)
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• replication of phage DNA (using hosts enzymes & organelles)
• transcription of mRNA
- possible destruction of hosts DNA
• translation of phage proteins
• assembly of phages
• lysis of bacterial cell wall and release of new phages
lysogenic cycle:
• attachment to cell and insertion of DNA
• integration into host chromosome of viral DNA
(= provirus/prophage)
- codes for repressor protein, shuts other phage genes off
• replication of viral DNA along with cells mitotic cycle
- may change phenotype of cell (phage gene activity)
- may excise from chromosome and enter lytic cycle
RNA viruses (retroviruses):
• some viruses contain RNA as genetic material
• may be single or double stranded
• may act as template for more RNA or for DNA
– RNA
DNA = reverse transcription
– requires reverse transcriptase
HIV (a typical retrovirus, causes AIDS):
• viral envelope fuses with cell membrane
- CD4 receptor on cellular membrane binds to viral protein gp120 on envelope
possible variation is receptor–mediated endocytosis
• capsid is uncoated, DNA is free in cell
• viral RNA is transcribed to DNA
- reverse transcriptase present with RNA in capsid
• viral DNA synthesizes complementary strand
• integrates into host chromosome as provirus
- may remain latent for extended periods of time
• transcription of viral RNA
- new viral cores and mRNA for:
• translation into protein
- for capsids,
- for integration into cell membrane and
- reverse transcriptase
• packaging of RNA and reverse transcriptase into capsids
• budding off membrane to form envelope, leaving cell
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Bacterial Genetics
one chromosome: circular with few associated proteins
plasmids: smaller rings of circular DNA with accessory genes
recombination in bacteria:
transformation: uptake of foreign DNA from surroundings
transduction: transfer of DNA by phages
- part of host DNA packaged into new “virus” or
- some of host chromosome excised and packaged with prophage
conjugation: transfer of DNA through pilli (cytoplasmic bridges)
- F+ factor carried on plasmid
– can transfer to other cells
- Hfr cells: F+ plasmid integrated into chromosome
– can transfer to other cells with some of chromosome
- leads to crossing over and recombination
[can be used to map bacterial chromosomes]
transposons: transposable elements (“jumping genes”)
- inverted repeats at each end + code for transposase
– can be clipped and inserted into various loci
- used as vector in biotechnology
Control of Protein Production in Prokaryotes
How much of a given protein do we want synthesized?
Regulation may be by differential:
1. transcription
2. processing (cutting & pasting exons)
3. stability of mRNA in the cytoplasm
4. translation
Transcriptional control of mRNA production
structural genes ("SG"): code for enzymes, structural proteins
regulatory genes: control the output of structural genes
the lac operon of E. coli : [prokaryote example of inducible operon]
• SG's for related enzymes often adjacent on chromosome
- lactose metabolism uses three such enzymes
• operator gene next to 3 SG's for lactose metabolism
• promoter gene adjacent to operator
- the operator and promoter do not code for mRNA)
regulator
promoter operator
Op
SG 1
SG 2
• this is the lac operon [ P + Op + SG1 + SG2 + SG3 ]
◊ RNApolymerase attaches to promoter
◊ synthesis of all three SG's commences
• a regulatory gene is located close by
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SG 3
◊ this produces a repressor protein
◊ which attaches to the operator
◊ blocking RNApolymerase, preventing transcription
• in the presence of lactose:
◊ lactose binds to the repressor protein
◊ preventing it from binding to the operator
◊ so transcription can continue unblocked
• this is an inducible operon:
– it is induced to transcribe
– by the presence of a molecule (lactose)
– requiring enzymes for its metabolism
positive feedback with catabolite activator protein (CAP)
• lac operon can also be accelerated (= )
• as glucose is depleted
cyclic AMP (cAMP) accumulates
— cAMP forms complex with CAP
binds to promoter
— facilitates attachment of RNA polymerase
— increases rate of transcription
• minimizes waste of energy making lactose enzymes if glucose present
the tryp operon of E. coli : [ example of repressible operon]
• if tryptophan is present, no need for synthesis
◊ tryptophan (the corepressor) binds to the repressor protein
◊ which is then able to bind to the operator
◊ blocking RNApolymerase, preventing transcription
• in absence of tryptophan
◊ the repressor protein alone is unable to bind to operator
◊ transcription is free to proceed
• this is a repressible operon
- when a needed substance appears in environment
- structural genes for its synthesis are no longer transcribed
– saving the cell energy and materials
Levels of Gene Control in Eukaryotes: (much more complicated)
chromosome structure
DNA is associated with proteins called histones
- nucleosome: DNA is wrapped twice around histones
- chromatin: nucleosomes ordered in tight spiral
- chromatin fibers folded in “looped domains”
- more folding and spiraling into final chromosome
chromosomal
• packing and unpacking in chromosomes
• amplification: genes replicated many times
max. mRNA
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• methylation: CH3 added to N–bases
reduction of transcription
transcriptional
• activation and repression by regulatory proteins
posttranscriptional
• splicing mRNA
• transport to cytoplasm
• degradation (mRNA may last from minutes to weeks)
translational
• initiation factors
Recombinant DNA technology
. introduction of genes from one species into another
. custom tailoring of specific nucleotide sequences
• gene of interest: gene we are interested in moving
• restriction enzymes break DNA at specific sequences
Bam HI
GGAT CC
CCT AGG
Hin dIII
e.g.
•
•
•
•
•
•
AAG C TT
TT CG AA
the restriction enzyme Bam HI will cleave
wherever the 6 base pair sequence is found
- these enzymes evolved as bacterial defense to viruses
(bacterial DNA protected by methylation at same sequences)
sticky ends: unpaired bases at end of restricted fragment
- will pair with like sticky ends
ligases: join DNA fragments (@ backbone) and repair breaks in DNA
gel electrophoresis: used to separate DNA fragments by length
- DNA (with - charge) drawn to + pole through thick agarose gel
vectors for transporting DNA into target cell:
- insertion into viruses
– some virus DNA sections can be replaced and maintain function
– certain viruses can insert DNA into host (target) DNA (prophages)
- insertion into plasmids
– can be broken by restriction enzymes
– and desired DNA (same sticky ends) inserted
– then introduced back into bacteria (transformation)
- transferred to all progeny during division
markers often included with desired gene:
– such as an antibiotic resistance gene
– so the colony with the new gene can be selected
source of genes:
- gene library (shotgun):
– all genome fragments put in vectors, transformed and cloned
- copy DNA: uses mRNA to make DNA (with reverse transcriptase)
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– useful for placing eukaryote genes in bacteria
(prokaryotes lack exon splicing machinery)
- probe: length of lab synthesized DNA
– used to find presence of gene or mRNA (complimentary)
Recombinant DNA Technology: An Example Using Bacteria
• isolate the gene of interest
• select a suitable vector
— such as a plasmid for bacteria
— including marker gene(s)
• restrict both DNA's with same enzyme (so sticky ends will match)
— choose one that cuts gene of interest fragment close to gene
— and cuts plasmid once
• insert gene of interest into plasmid (incubate and ligate)
• insert into bacteria
• incubate & culture bacteria
• use phenotype of marker gene to select for recombinant colonies
• isolate and cultivate successful recombinants
— this is cloning the gene of interest
sequencing genes The Sanger method
from the man who first deciphered a protein: Frederic Sanger
◊ extract and clone single stranded DNA of desired gene
◊ divide into four reaction mixtures
- each contains the s.s. DNA template
- plus all four nucleotide triphosphates (ATP, GTP, CTP & TTP)
- and all enzymes for synthesis
◊ each reaction mixture contains a different dideoxynucleotide
- this will terminate new strand at that point
- since incorporation is random, different lengths will result
– but all lengths in a mixture end in same nucleotide
◊ gel electrophoresis is run on all four mixtures (side by side)
◊ fragment length (position) is compared to column (mixture)
- each sequential fragment length shows in only one column
- corresponding with a specific nucleotide (at termination)
locating genes
• radioactive (or otherwise labeled) probes
• hybridization
• Southern blotting
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Applications of recombinant DNA technology
Agriculture
• developing resistant plant strains
- toxin production to insects
- resistance to herbicides and insecticides
• increasing growth and nutritive value of plants and animals
- adding lysine producing enzymes to corn
- growth factors in animals
• increasing yields in crops
- incorporation of nitrogen fixation
- increase of growing season (ice minus bacteria)
• post harvest properties
- tomatoes that can be picked when ripe and won’t rot (Calgene)
Medicine
• pharmaceuticals
- using bacteria to mass produce important proteins
– insulin
– human growth hormone
- vaccines
– possibility of more effectiveness, greater safety
• diagnosis
- testing for genetic diseases
- detection of carriers (consultation)
• gene therapy
- repairing genetic disorders
– remove stem cells from afflicted individual
– insert working version of defective genes
– replace into individual
Research
• ability to produce proteins in quantity
• detection of genes with transient expression
• information on evolutionary relationships
• understanding development
Society
• DNA fingerprinting in forensic investigations
- one hair may identify a rape suspect, paternity
- rights of suspects to sample seizures???
• patenting of new organisms (and offspring)???
• environmental effects of released engineered organisms???
• germ line experiments: creation of new humans???
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