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Unit 2: Molecular Genetics - Part 2 (8.5 hours) |
Students will explain the concepts of gene and gene expression and the roles of DNA, RNA, and chromosomes in cellular metabolism, growth, and division, and demonstrate an awareness of the universality of the genetic code. Laboratory activities and conceptual models will be used to explain processes within the nucleus. Descriptions will be given of some of the theoretical issues surrounding scientific research into genetic continuity; the general impact and philosophical implications of the knowledge gained; and some of the issues raised by related technological applications.
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Ontario
Curriculum objectives: |
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Text: Biology 12, Nelson |
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Lesson One |
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Summary:(U3) |
Homework: |
GENETICS
Genetics is the study of hereditary information with respect to its coding and method of transmission from one generation to the next
Gene: a specific amount
of DNA coding for a specific protein...3% of human DNA constitutes our
25,000 genes
-the letter chosen to represent the gene is best if capital and small case
letters are easily distinguished
Allele: alternate form of
the same gene
e.g.
the gene for hair colour has brown and blonde alleles
Dominant: When two different alleles are present the one that is expressed
is dominant
e.g.
brown is dominant to blonde, indicated with capital letter (B)
-dominant
alleles are not always the most common trait
Recessive: When two different alleles are present the
one that is not expressed is recessive
e.g.
blonde is recessive to brown, indicated with a lower case letter (b)
Homozygous: when an organism has two identical alleles for one gene
e.g.
BB (dominant) or bb (recessive)
Heterozygous: when an organism has two different alleles for one gene
e.g.
Bb (the dominant trait is expressed)
Genotype: the symbolic representation of the alleles for the trait
e.g.
BB, Bb, bb
Phenotype: the physical result of the genotype
e.g.
Brown, Blonde
Gametes: cells resulting from meiosis and are haploid (one set of
chromosomes) e.g. sperm and egg
Somatic cells are regular body cells and are diploid (two sets of
chromosomes)
Zygote: fertilized egg, diploid because it is formed by the fusion of
two haploid cells
Progeny: another term for offspring
Diploid Number: a double set of chromosomes (normal number found in somatic
cells)
e.g.
humans 2n# = 46, chimps 2n# = 48, fruit fly 2n# = 8
Haploid Number: a single set of chromosomes (normal number found in
gametes)
e.g.
humans 1n# = 23, chimps 1n# = 24, fruit fly 1n#=4
Monohybrid Cross: a mating looking at one gene and two alleles
e.g. BB X bb uses the gene for hair colour, alleles are brown and blonde
Dihybrid Cross: a mating looking at two genes and four alleles
e.g.
BBRR X bbrr uses the genes for hair colour and eye colour and the alleles are
brown and blonde hair, brown and blue eyes
Parental (P) Cross: a mating of any two parents
First Filial (F1)Generation: offspring of P cross
F1 cross: mating of first filial generation
(not done in humans too often...generally frowned upon because the danger of
two rare deleterious recessives combining is high in siblings)
Second Filial (F2) Generation: progeny of F1
cross
Genome: set of genes found in an organism (2 copies of genome per somatic
cell...100 trillion cells in body)
Reproduction: the production of new organisms from old ones
Asexual Reproduction: one organisms produces more of the same (done by
mitosis) with identical DNA
Sexual Reproduction: a new organism is produced by the union of
two gametes
-gametes are produced by meiosis
Mitosis: one cell produced
two cells with identical DNA
-in
unicellular organisms, mitosis occurs as a means of reproduction
-in
multicellular organisms, mitosis is needed for growth and replacement of dead
cells
-during
interphase the size of chromosomes doubles (but not the number) by
semiconservative replication
-then
Prophase, Metaphase, Anaphase and Telophase occur in which the chromosomes
split into two cells
-each new cell gets one copy of each double helix
-the DNA doubles again in the next interphase
-the chromosome number remains constant
Meiosis: one diploid cell produces four haploid cells called gametes
-during
interphase, the size of chromosomes doubles
-during
Prophase I, pairs of homologous chromosomes join (bivalents) and then separate into two
cells during the rest of meoisis I
-each
of these two cells now has the haploid number of chromosomes
-the chromosomes split into two new cells each during meosis II, each new cell
getting one copy of the double helix
-this results in the production of four haploid cells
Work on monohybrid crosses review by doing practice crosses
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Lesson Two |
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Summary:(R1,R2) |
Homework: |
GENETIC VARIATION
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Read pages
259-263, and answer the following: |
Discuss moral questions of Human Genome Project
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Lesson Three |
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Summary:(D4) |
Homework: |
HISTORY OF GENETICS
1850’s: Gregor
Mendel
-studied
genetics in peas
-found
“genes” come in pairs of traits or alleles, one copy from each parent
Principle
of Dominance: one allele may be dominant (mask) the other allele (tall peas
are dominant to short)
Principle
of Segregation: one allele from each pair goes randomly into gametes (50%
chance of passing on an allele)
Principle
of Independent Assortment: genes often behave independently from other
genes (pea colour and texture are not related)
early
1900’s: Mendel’s work rediscovered
1902: Walter Sutton (U.S.) studied grasshoppers and Theodor Boveri
(Germany) studied sea urchins independently
-they found cells in produced in mitosis had twice as may chromosomes as those
produced by meiosis
-they
found that chromosomes behave indepentently and segregate during meiosis similar to the
behaviour of Mendel’s genes
Sutton-Boveri Theory: genes are on chromosomes
1907: Thomas
Hunt Morgan (U.S.) worked with fruit flies trying to disprove the
Sutton-Boveri theory
Why
fruit flies? Short life span, cheap, reproduce quickly, many offspring, small and have many
obvious traits
-looked for mutants/variations and found white eyed male fruit flies and no
white eyed females
-this indicated gene for eye colour was on the X chromosome showing
one gene was on one chromosome
-this proved the Sutton-Boveri theory was correct
-Morgan used crossovers during meiosis to map 2000 genes on 4 fruit fly
chromosomes
(Nobel Prize: 1933)
GENETIC VARIATION IN SEXUAL REPRODUCTION
Sexual
Reproduction produces variation by the following methods:
independent assortment:
-homologous chromosomes separate independent of other homologous
chromosomes
-the probablilty of any getting one chromosome is 0.5, any two chromosomes is (0.5)(0.5)=0.25 and
so on.
-the
chance of a human getting the same 23 chromosomes is therefore very small (many different
sperm and egg are made by meoisis)
Fertilization/hybridization:
-zygotes are the result of a fusion of sperm and egg
-these gametes are often from different organisms with different alleles
resulting in many possible combinations
Crossing
over:
-during Prophase I of meiosis, synapsis of homologous chromosomes may occur
-in synapsis DNA may be randomly exchanged between these chromosomes
-this
creates new combinations of alleles on chromosomes not present in either parent
B______________D
B______________D
crosses over with
b______________d
b______________d
Where
B represents purple flowers, b represents red flowers and D
represents long pollen, d represents round pollen
These
genes for flower colour and pollen type are linked (both on the same
chromosome)
Crossover
occurs as follows between two homologous chromosomes
B______________D
B______ ______D
X
b______ ______d
b______________d
Producing four chromosomes which each go to a separate gamete
B______________D
b______________D
B______________d
b______________d
The two
middle chromosomes are formed as a result of this crossover
-as
these crossovers are random events, the chance of a crossover between two genes is greater they are
far apart
-there is less chance of a crossover if the genes are closer together
Geneticists use this information to map genes on chromosomes, measuring the
distance between genes in
percentage crossovers
Do practice sheet on mapping genes from SBI 3U
Do chromosome mapping activity
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Lesson Four |
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Summary: (U4) |
Homework: |
MUTATIONS
-sexually
reproducing organisms use crossing over, independent assortment and fertilization to
acquire variations in the genome
-all living organisms may acquire variations by mutations
Mutations are any alteration of genetic information
CAUSES OF MUTATIONS
natural causes:
-accidents during semiconservative replication occur at a rate of 1 base pair
per 109 base pair replications.
ultra-violet
light:
-U.V. light is produced by the sun
-this high energy radiation is not strong enough to cause electrons to be
removed from atoms
-the energy absorbed by the DNA
causes adjacent thymine molecules to form a link called a thymine dimer
-thymine dimers damage the DNA (these thymine bases no longer pair with
adenine)
high
energy radiation/ ionizing radiation:
-ionizing ratiation is produced by x-rays, cosmic rays, or radioactive material making alpha, beta,
and gamma rays
-this radiation causes electrons to become so energized that they are
stripped from their atoms
-molecules containing these atoms form "free radicals" as a result of
the electron loss
-free radicals are atoms or groups of atoms which may react with DNA causing potential
damage
chemical
mutagens:
-some chemicals react with DNA
(i)deaminating
agents (e.g. HNO2) remove amino groups from bases, causing them
to form different pairings
e.g. C without amino pairs with A...CG becomes TA
A without amino pairs with C...AT becomes GC
G without amino pairs with nothing...GC becomes missing pair
(ii)alkylating agents (e.g. mustard gas...used in chemotherapy) add hydrocarbon
groups (e.g. CH3) to bases
e.g. G with C2H5 pairs with T..GC becomes AT
alkylated DNA (with methyl CH3 joining cytosine) prevents
transcription
alkylation occurs in a
promoters and is a major part of gene control
cancer cells and new embryos
have little methylation of promoter regions
(iii)intercalating agents (e.g. proflavin) are flat molecules which that insert between
bases
intercalation causes
loss of base pairs when the DNA replicates
viruses:
-some viruses have "reverse transcriptase", an enzyme that allows
these viruses to convert their RNA into DNA
-these enzymes are useless to humans but allow virus DNA to become incorporated
into chromosomes
-these
viruses are called retroviruses (e.g. HIV) and 1.3% of the human genome is made
of inactivated retroviruses
Review sheet on dihybrid crosses, incomplete dominance (roan cattle) and codominance (blood type)
TYPES OF MUTATIONS
point/gene
mutations:
-mutations that alter one or two base pairs but do not affect the entire chromosome
(cannot be viewed with karyotype)
(a) base pair substitutions: one base pair
replaces another, with two possible results
(i) missense mutation: a base pair substitution results in the translation
of a new amino acid to replace an old one
e.g. GAA (codes glu) becomes GUA (codes val)
-this change of glu to val in hemoglobin can result in altered hemoglobin characteristic of
sickle cell anemia
-in sickle cell anemia the altered hemoglobin is no longer able to carry oxygen
(ii) nonsense/chain termination mutation: a base pair substitution
results in the translation of a stop codon to replace an amino acid
e.g UAU (codes for tyr) becomes UAA (stop)
-this change of tyr to stop in hemoglobin can result in shortened hemoglobin
characteristic of thalassemia
-in thalassemia the altered hemoglobin is no longer able to carry oxygen
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Lesson Five |
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Summary: (U4) |
Homework: |
TYPES OF MUTATIONS (continued)
(iii)silent
mutation: a base pair substitution in which one amino acid is not altered,
just the codon
-both codons result in the production of the same amino acid
(b)frameshift
mutations: one/two base pairs are deleted or added/inserted
-this
results in all the base pairs in the codon shifting, altering all codons past
this point
-the next gene is not always altered
These
point/gene mutations are often repaired (all but 1 in 109 base pairs
are copied correctly)
DNA polymerase I and DNA polymerase Ill repair DNA by moving up and down
recently replicated DNA
(see notes on DNA replication in the first unit)
chromosomal mutations:
-mutations that are visible alterations in the chromosome involving large
sections of DNA
(a)deletions: a large section of DNA is lost from a chromosome
e.g.
Cri-du-chat involves loss of part of chromosome 5, resulting in brain damage,
altered voice box)
(b)inversions: a large section of DNA is flipped and returned to the chromosome
-this damages the gene at the place the DNA is cut, but otherwise
does not affect the genes
-these chromosomes do not crossover during meiosis, and are used in many genetic
crosses as stable "balancer" chromosomes
(c)translocations:
the transfer of a large section of DNA from one chromosome to
another
-if this occurs on non-homologous chromosomes, altered gametes may result
e.g.
familial/inherited Down syndrome is the result in a translocation of part of
chromosome 21 onto chromosome 14
(d)changes in chromosome number: whole chromosomes may be gained or lost
-the cause is often nondisjunction, which is when sister chromatids (mitosis)
or homologous
chromosomes
(meiosis) stick together
-the result is a cell with monosomy (single copy) for that chromosome or
trisomy (three copies) of
that chromosome
e.g.
Down syndrome is the result of nondisjuction resulting in trisomy 21, three
copies of chromosome 21
Worksheet on point/gene mutations...
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Assume you have a piece of DNA with the following structure: Complete the following chart:
1.
Under each mutation, list a potential chemical cause. Additional Questions: Page 263, #5,6,7 |
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Lesson Six |
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Summary: (U3,U5) |
Homework: |
GENE CONTROL
lac
Operon:
1961: Francois Jacob and Jacques Monod proposed the "lac
operon" to explain gene regulation in E. coli bacteria
-a
sequence of DNA contains a promoter
-the promoter is the site at which RNA polymerase joins DNA
-the
promoter is followed by an operator sequence
-the operator sequence is followed by three genes that produce enzymes to digest
lactose
-a
repressor protein lands on the operator, preventing the RNA polymerase from transcribing
the genes
-when
lactose is present (needing digestion) some lactose binds to the repressor
-this
changes the shape of the repressor, and the repressor breaks free of the
operator
-now
the genes to produce enzymes can be transcribed and the enymes made to break
lactose down
-when
lactose is broken down the repressor is able to bind again and the enzyme genes
are turned off
-this
system allows trascription to occur when lactose is present, and stops
transcription when lactose is
absent
-the E. coli does not waste energy
(Nobel Prize: 1965)
trp
Operon:
"trp
operon" is another method of gene regulation in bacteria
-a
sequence of DNA contains a promoter followed by an operator sequence
-the operator is followed by five genes that produce enzymes allowing the production of tryptophan
-when
tryptophan is produced some of it binds to another molecule creating a corepressor
-the corepressor sticks to the operator, blocking RNA polymerase and
turning off transcription
-this
allows the product of genes to turn off the genes when present
-when
tryptophan is used up the corepressor comes free, and transcription begins
again
TYPES OF MUTATIONS (continued)
A third
type of mutation (point and chromosomal being the first two):
transposition:
- individual pieces of DNA may move from place to place on chromosomes, jumping
from one to the next
-1
in 700 human mutations are caused by these (1 in 10 mouse mutations as well)
1951:Barbara
McClintock discovered transposons while studying corn
-a promoter moved around from place to place in corn chromosomes,
changing the colour of the corn
-she
proposed existence of repressors in gene regulation as well
-McClintock studied corn because it was economically important and provided
good statistics
-she
created some of the first mapped chromosomes using crossovers
-she also showed chromosomal mutations caused by X-rays
(Nobel Prize: 1983)
Transposons
cause the following effects:
(a)
Insertional inactivation
-a transposon lands in the middle of a gene and inactivate the gene
(b)
Gene mobilization
-transposons contain genes which may alter their function with new locations
(c)
Transposable promoters
-a promoter moves around in front of genes turning on genes (and off
those it leaves)
-this was found by
McClintock
in the genes for corn colour
-trasposable promoters also occur in trypanosomes resulting in different
identity markers being
produced
-altered identity markers results in difficulty for the immune
system fighting the protazoa, resulting in fatal sleeping sickness
GENETIC ENGINEERING
Genetic
engineering is human manipulation of DNA
-this often involves transfer of DNA between eukaryotic and prokaryotic cells
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Prokaryotes |
Eukaryotes |
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-no internal membranes (no nucleus) |
-have internal membranes (nucleus) |
Introns:
-introns are noncoding regions of gene cut out of primary mRNA to make
secondary mRNA
-introns
are not present in prokaryotes
Exons:
-exons are coding region of a gene
e.g.
Human hemoglobin gene has 1356 base pairs
-this gene should be able to code for 452 amino acids
-all but 432 of these 1356 base pairs are introns, resulting in a protein of only 144 amino
acids
Transposons are important in genetic engineering as they demonstrated DNA can move from location to location on chromosomes
Plasmids:
-plasmids are small circular pieces of DNA
-some
plasmids contain fertility genes (F-plasmids/transfer plasmids) resulting in the formation of
a conjugation bridge/pili
-the pili allows the plasmid to be transferred from one bacteria to the next
-this transfer allows DNA to be moved from cell to cell (transformation)
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Lesson Seven |
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Summary: (U5,U6) |
Homework: |
GENETIC ENGINEERING (continued)
Fertility plasmids are circular pieces of DNA which contain a fertility F gene allowing them to move from cell to cell
Restriction
Enzymes (Endonuclease) cut DNA at specific base pairs/restriction sites
-these
enzymes were discovered in bacteria which use them to break apart virus DNA
-the
enzymes often cut at regions with twofold rotational symmetry
(the top read forwards is the same as the bottom read backwards)
e.g. EcoR1 cuts -GAATTC- to form
-G
AATTC-
-CTTAAG-
-CTTAA
G-
-this
cut results in two ends which are staggered
-these ends are called "sticky ends" because they
can rejoin with anything else cut with the same enzyme
Recombinant
DNA is DNA created by joining DNA from two organisms (a CHIMERA)
Cloning a gene is making multiple copies of a gene for one organism
inside another organism
CLONING A GENE
1.
Locate the gene you wish to clone on a chromosome
-many
techniques are used but this course only deals with gene walking (using
crossovers to find genes)...see lesson 3
2.
Decide which restriction enzyme to use. Your enzyme must cut:
(a)
on either side of the gene to be cloned
e.g. animal chromosome ---CGGCCG-GENE-CGGCCG---
---GCCGGC-GENE-GCCGGC---
-this gene can be cut on either side by the enzyme Cfr1 (which cuts at GCCGGC)
(b) on a bacterial plasmid with the TcR and F genes (TcR is the gene for tetracycline resistance)
3. Cut the animal chromosome and bacterial chromosomes with the restriction enzyme(s)
---C
GGCCG-GENE-C
GGCCG---
---GCCGG
C-GENE-GCCGG
C---
-this is the cut animal chromosome (cut with Cfr1)
GGCCG-F-TcR-C
C-F-TcR-GCCGG
-this is the cut bacterial chromosome (cut with Cfr1)
4. Mix
the cut DNA and join (ligate) sticky ends with DNA ligase
-some
of the animal gene may join the bacterial chromosomes
5. Mix
the ligated DNA with bacteria that is not tetracycline resistant
-add
tetracycline.
-those bacteria that have taken up the plasmid will be able to survive and
grow into colonies on agar
6. Check
the colonies for production of the gene product
-discard other colonies (those remaining have the animal gene carried by the plasmid)
7. Grow the
bacteria in large amounts, harvesting and purifying the animal gene product
(e.g. human growth hormone, insulin)
This experiment was first done in 1973 by Stanley Cohen and Herbert Boyer, moving a gene for toad rRNA into bacteria
Work on genetics questions
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Lesson Eight |
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Summary:(U6) |
Homework: |
GENE CLONING ASSIGNMENT
-compete
Gene Cloning Assignment with models
-quiz next class on genetic terms
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Lesson Nine |
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Summary: (D4) |
Homework: |
-do quiz on Genetic Terms
POLYMERASE CHAIN REACTION
Polymerase
Chain Reaction (PCR) allows production of multiple copies of a DNA fragment
-the
double stranded DNA fragment desired is “melted” with high temperature into two single
strands of DNA
-a
piece of DNA primer is annealed to each single strand at low temperature
-a
DNA polymerase is added to turn each single strand into a double strand
-the
process is repeated numerous times
GEL ELECTROPHORESIS
Gel
Electrophoresis allows different fragments of DNA to be separated
-DNA
has a negative charge
-DNA
is cut with restriction enzymes and added to wells on a gel of agarose
-electricity
is added with a positive charge at the other end of the gel
-DNA
fragments migrate towards this positive charge with smaller fragments migrating quicker
-the
DNA is then stained and the fragments compared
-do DNA murder activity
Other
Information:
-97%
of human DNA is not genes
-part
of the remaining DNA consists of inactivated retroviruses, transposons, inactive genes,
repeating sections of DNA (microsatellites)
-methylation
occurs in prokaryotes on restriction enzyme sites preventing the action of
restriction enzymes (sites now look different)
-methylation
in eukaryotic cells is involved in control of gene expression
Do DNA microviewer
DNA
Sequencing
1977:
Fredrick Sanger used DNA sequencing to sequence a bacteriophage genome of 5386
base pairs
Technique:
1)
DNA to be sequenced is melted into single strands to provide a template
2)
A short radioactive primer is added to the single stranded DNAs using DNA polymerase
3)
Four test tubes have these primed single stranded DNAs added
4)
Into each of the four test tubes four deoxynucleotide triphosphates are added
in high concentration
(dGTP, dCTP, dATP, dTTP)
5)
Into each of the four test tubes a lower concentration of one of the four
dideoxynucleotides (radioactively labeled)
One test tube gets ddGTP, ddCTP, ddATP or ddTTP.
Dideoxynucleotides:
deoxynucleotide triphosphates with the OH (hydroxyl) group missing from carbon
3' of the deoxyribose
This
means that when DNA is assembled in a 5'-3' direction NO additional nucleotides
will be added after the dideoxy-nucleotide as the 3' end is unable to bind
6)
DNA polymerase is the added to each test tube to begin the assembly of the complementary strand
on the single stranded DNAs
7)
When a dideoxy-nucleotide is added the DNA production ends. As the concentration of
dideoxy-nucleotides is low compared to regular nucleotides the
DNA
sequence is completely made in many cases, but stops at each dideoxynucleotide
in some cases. Results produce different size fragments depending on length sequenced.
e.g.
for a test tube with ddGTP most are:
5'-ATG GGC CTC GAC-3'
also
made: 5'-ATG-3'
5'-ATG G-3'
5'-ATG GG-3'
5'-ATG GGC CTC G-3'
8)
The four test tubes are run on a gel using electrophoresis and the gel is
stained.
9)
The DNA sequence is then read.
eg.
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ddATP |
ddCTP |
ddGTP |
ddTTP |
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The
modern technique uses one test tube with fluorescent stains of four different
colours on the four different dideoxynucleotides.
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Lesson Ten |
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Summary: (R2) |
Homework: |
GENETIC ENGINEERING VIDEO
View video on genetic engineering
-test on genetics next class (lessons 1-10)
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Lesson Eleven |
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Summary: |
Homework: |
TEST ON LESSONS 1- 10 OF GENETICS
-test on genetics