Biology Unit 2

The Cell Cycle

I. Organization of Cellular Genetic Material

-> CC chromosome structure and number 

II. The Cell Cycle 

-> SEQ, HD, CC steps of cell cycle 

I. Organization of Genetic Material

A. Introduction

• Cell theory - all cellular life is made of one or more cells

• Cells are the smallest unit of life 

• All cells come from other cells 

• Some cells are terminally differentiated 

  • Do not divide

  • Ex. Erythrocytes, neurons 

  • Most cells are going to divide at some point 

Cell Division = Cell reproduction

• Unicellular organisms - just reproduction 

• Multicellular organisms

  • Growth and development 

  • Renewal and repair

• During cell division 

  • You must allocate all parts of the existing cell into those two daughter cells

  • Each daughter cell must get a FULL copy 

B. Genetic Material

• Genome - a cells total genetic material 

  • Most of the genetic material is going to be in the nucleus 

  • Prokaryotes - usually 1 circular DNA molecule 

  • eukaryotes - usually >1 linear DNA molecules 

• Chromosome - molecule of DNA in a cell

  • Can be in eukaryotic and prokaryotic cells 

DNA Arrangement

• Nucleosomes: 8 histones with DNA wrapped around

  • Histones - DNA wrapped proteins, proteins that associate with DNA

• DNA is very precisely packaged because the amount of DNA is worth 2 meters of DNA in each cell 

• Adjacent nucleosomes linked by linker DNA

Chromatin

• DNA/protein complex in a dispersed state

• DNA in its lowest density of packaging

• DNA is usually in this state 

• When cell division is done, this is condensed and wrapped around itself to make structures called chromosomes 

• “Colored body” 

• DNA wrapped around proteins, highly organized

• Densely packed chromosomes are only present during cell division 

Genes

• Informational unit of DNA, parts that contain the information to make proteins 

• 100s or 1000s of genes per chromosome

• Each gene is at a specific place on a chromosome - Locus

• Arranged linearly on chromosomes

C. Chromosome Number

• Haploid (n) - having one complete set of chromosomes 

• Haploid cells have 1 of each chromosome 

• Gametes are haploid, some organisms are haploid for most of their lives 

• Diploid (2n) - when you have 2 complete sets of chromosomes in your cells 

  • 2 of same chromosomes - homologous chromosomes/pair 

  • Same length, centromere location, genes 

  • Somatic cells in humans are diploid 

Diploid Numbers 

• Humans 46

• More complex does not mean more chromosomes

• Haploid and diploid cells can undergo mitosis 

II. Phases of the Cell Cycle

A. Introduction

• Cells do not divide continuously 

  • Ex. For eukaryotes - interphase = non-dividing

  • M phase = dividing 

All Domains Divide

• Prokaryotes - binary fission 

B. Events of Interphase

• The time in between cell division where the cells are doing growth and synthesis 

  • Not a resting stage - metabolically active 

• Long - 90% of cell cycle 

• DNA is going to exist as chromatin, more diffused form of DNA 

• 3 phases of interphase

  • 1 G1 (gap 1)

    • Growth and normal development and functions

    • Preparation for S phase

    • No DNA synthesis 

    • Many cells spend most of their lives here 

  • S phase

    • DNA “synthesis” phase

    • Chromosomes duplicated - DNA and chromosomal protein synthesis 

      • Does not change ploidy 

  • G2 (Gap 2)

    • Usually shorter than G1 or S

    • DNA is still chromatin

    • High metabolic activities, preparation for mitosis 

    • Centrosome duplicate

      • Fibers that give the cell the shape get rearranged to help the cell divide 

Chromosomes After Replication

• 2 sister chromatids

  • Exact copies of DNA

  • Connected at the centromere 

    • Proteins attach to the centromere - kinetochore

      • Attach to microtubules

      • Move chromosomes during division 

• DNA is still chromatin at this stage 

C. Events of M Phase

• Shortest part of the cell cycle 

• Mitosis and cytokinesis

Mitosis

• Nuclear division of somatic cells 

  • Nonreproductive cells

• Continuous process

• Divides into 4 stages

  • Prophase, metaphase, anaphase, telophase 

1. Prophase

• 3 big things happen

  • Chromosomes condense

  • Mitotic spindle forms

    • Rearranging the cytoskeleton to make the spindle that sets the orientation of the cell and helps chromosomes move 

    • Made of microtubules 

  • Nucleus breaks down 

    • Allocate the DNA at the front of the process 

    • Disassemble to put them together later 

Mitotic Spindle

• Microtubules responsible for separation of chromosomes

• Microtubules - hollow rods of protein tubulin 

• Mitotic spindle oriented from pole to pole of cell 

2. Metaphase

• Chromosomes align at metaphase plate

  • Not a structure but a region

• Chromosomes get pushed to the center of the cell

• Kinetochores attach to spindle

  • As the spindle fibers orient themselves across the cell it pushes the chromosomes all into line 

3. Anaphase

• Sister chromatids separate, move to opposite poled

• Pulled nu kinetochores

• Separate - enzyme that separates sister chromatids

• After separation, each chromatid considered to be a chromosome 

4. Telophase

• Opposite of prophase

• Chromosomes start to de-cocndense

• Nuclear envelope reforms 

• New nuclei are identical to the parent nucleus of the original cell that divided 

Cytokinesis

• Part of M phase

• Cytoplasmic division -. 2 cells, each with 1 nucleus

• Generally overlaps with telophase 

• Nucleus reverts to interphase condition during/after cytokinesis 

• Animal cells have a cleavage furrow - cell pinches

• Plant cells have a cell late - new cell wall in between

Meiosis

1. Introduction to Heredity

CC Sexual and asexual reproduction 

2. Life Cycles

SEQ, CC sexual life cycles

3. Four Stages of Meiosis

SEQ, CC, HD process of meiosis 

1. Introduction to Heredity

Heredity - the transmission of traits from one generation to the next

Variation - differences between individuals

Genetics - the study of heredity and hereditary variation 

Gametes - reproductive cells that transmit genes from one generation to the next 

1. Asexual reproduction

• When a single parent produces offspring

  • Ex. mitosis

• Unicellular organisms split into two cells 

• Multicellular uses budding or fragmentation

• Asexual repro in eukaryotes 

  • Mitotic division

    • 1 diploid (2n) parents -> 2 diploid offspring

    • 1 haploid (n) parent -> 2 haploid offspring 

    • Produces clones - offspring genetically identical to parent

• Advantages

  • Faster 

  • Requires less energy

  • Safer than sexual reproduction

  • A lot of offspring 

  • If you are well adapted to your environment then you do not have to change your offspring to be different than you 

    • Can make clones

2. Sexual Reproduction

• Fusion of 2 gametes to form a zygote

• Gamete (n) + gamete (n) -> fertilization -> zygote (2n) 

  • Gametes are usually from different parents (but not always) 

• Offspring is not genetically identical to parents 

• Disadvantages 

  • Slow process 

  • Requires more energy 

  • Dangerous

    • Predation and disease 

  • Often fewer offspring 

  • Well adapted? - offspring only get half of your genes (genome dilution) 

• Advantage of sexual reproduction - genetic variation 

  • Generating new combinations of genes 

  • Better able to respond to change or stress 

• Why variation is good

  • If we have a low variation and a new predator comes which the population cannot hide from, everyone on susceptible to being eaten by the predator

  • A population with high variation can use different strategies and some individuals may be out of luck, but others of the same population may be able to deal with the same predator

• If gametes have the same number of chromosomes as parents

  • Chromosome number doubles when they fuse

  • Solution: meiosis 

    • Reduction division, reduces the ploidy 

    • Cell divides twice

    • 1 diploid (2n) cell -> 4 haploid (n) cells 

3. Chromosomes in heredity

• karyotype - orderly display of chromosomes

  • Mitotic chromosomes stained 

• Human karyotype 

  • Somatic cells - 46 chromosomes arranged in 23 pairs

    • 22 of the pairs are autosomes (non sex chromosomes)

    • The 23rd pair are the sex chromosomes, X and Y

      • Determine sex, females are XX, males are XY 

• Homologous Chromosomes - same length, centromere position, staining pattern but from different sources 

  • Same genes in the same places, sequencing not exactly the same 

II. Life Cycles

Life cycle - sequence of stages from generation to generation 

• All sexual life cycles will contain Fertilization and meiosis

  • Alternate from diploid -> haploid to haploid -> diploid 

  • Timing varies 

1. Human life cycle 

• Meiosis -> gametes -> fertilization -> zygote -> mitosis 

2. Variety in Sexual life Cycles

• Somethings only have gametes in the first stage while some only have zygotes 

• Only 2n cells can undergo meiosis, n and 2n cells can go through mitosis 

III. Meiosis

Reduction division, 4 stages and involves 2 cell divisions

• Interphase

• Meiosis I

• Interkinesis

• Meiosis II

1. Interphase

• Chromosome and centrioles duplicate

• Each chromosome now 2 sister chromatids (still chromatin) 

• Humans: 2n = 46, 92  chromatids enter meiosis 

2. Meiosis I (and cytokinesis)

• Where ploidy is reduced 

• First meiotic division - homologous chromosome separate 

• Prophase I

  • Includes crossing over

    • Homologous recombination

  • Synapsis - homologous chromosomes from parent cells pair up

    • Results in tetrad - 2 homologous chromosome (4 chromatids) held together 

  • homologous recombination 

    • Enzymes break, swap, and rejoin DNA

    • Exchange occurs between non sister chromatids 

    • Important source of genetic diversity 

    • Generates new combinations of alleles 

  • Crossing over at gene level

  • Also during prophase

    • Chromatin condenses

    • Nuclear envelope goes away

    • Spindle forms centromeres of homologous chromosomes start to separate but the tetrad itself stays together 

  • At the end of prophase

    • 46 chromosomes 

    • 23 tetrads

    • 92 chromatids

• Metaphase I

  • Tetrads align at metaphase plate

  • Homologous chromosomes orient towards opposite poles

  • Both sister kinetochores of one chromosome -> spindle for same pole

  • Kinetochores of homologous chromosomes -> spindle for opposite poles 

• Anaphase I

  • Tetrads have separated and the chromosomes begin to move towards the poles

  • Disjunction - homologous chromosomes separate 

  • Chromosomes act independently

  • Direction depends on orientation of tetrad 

  • Nondisjunction - when tetrads go to the same pole because they failed to separate 

• Telophase I

  • Chromosomes may decondense 

  • Nuclear envelope forms

  • Cytokinesis occurs 

  • Results in 2 haploid cells, with duplicated chromosomes (sister chromatids still together) 

  • At end of telophase I in humans

    • Chromosomes - 23, ploidy reduces

    • Sister chromatids - 46

    • Tetrads - 0, all homologous chromosomes separated 

3. Interkinesis

• In Between the first and second divisions 

• Usually short, interphase like

• No S phase, no DNA synthesis occurs 

4. Meiosis II

• 2nd meiotic division 

• Chromatids separate into daughter cells

• Very similar to mitosis

• Stages

  • Prophase II

  • Metaphase II

  • Anaphase II

  • Telophase II 

• Ploidy does not change 

Genetics

1. Gregor Mendel and his experiments

-> SEQ Mendel’s work

-> CC competing hypotheses

-> CC Principles of heredity

2. Genetic Crosses

-> SEQ, CC, APPLY Genetic crosses

3. Using probability in genetics

-> APPLY probability rules 

Vocabulary

• Gene - unit of heredity information 

• Allele - alternative versions of genes

• Character - observable, heritable feature 

• Trait - (or character state): detectable valiant of a character 

• Genotype - the genetic makeup, what alleles are present

• Phenotype - observable physical traits

Gene -> character -> hair color

Allele -> trait (character state) -> red hair

1. Gregor Mendel

1. Background 

• Austrian monk 

• Cultivated pea plants, was able to determine the basic rules of inheritance in eukaryotes 

• Published his work in the 1860’s, basically nobody cared

• In the early 1900’s his work was rediscovered

• Foundation of genetics 

Experimental organism - garden pea

• Model organism 

• Good for studying genetics 

• Inexpensive, easy to obtain

• Lots of identifiable traits 

• Easy to grow, short generation time 

• Many varieties available 

Applied Quantitative Methods

• Mendel used the scientific method, kept track of what he did and the numbers

  • Documented protocols

Developed true breeding lines

• True breeding - always express same trait after self-fertilization, no exceptions

  • All express same phenotype

• 2 years of true breeding before starting experiments

• Important to make sure that the alleles were the same for the trait in question 

2. Testing “Blending Inheritance”

Blending inheritance hypothesis - gametes contain sampling of fluids from parents bodies

Fuse during reproduction, fluids blend and offspring is intermediate, prevailing idea in mid-1800s

Experimental Crosses

• Mating 2 organisms to see what the offspring's phenotype will be 

• P generation - parental generation 

• F1 generation - 1st filial generation (latin filius - son)

• F2 generation - 2nd filial generation 

• Crossed true breeding plants (P generation) with contrasting traits 

Prediction

• If blending is accurate the f1 phenotype should be intermediate between Phenotypes 

Mendel's Experiments

• Start with true breeding purple flower and true breeding white flower

• Mate those together and mate the F1 generation with each other

Observations

• F1 generation always represents one parent, other parent is absent, in F2 the traits that were missing come back 

• 3:1 ratio of one phenotype to the other phenotype in the F2 generation 

Conclusion

• There was no intermediate phenotype

• Lost phenotypes reappear

• Blending of fluids cannot explain either observation

• Reject blending hypothesis

3. Mendel's Model

Alternative hypothesis: particulate inheritance

Characters determined by “heritable factors”

• Each character determined by 2 factors - 1 from each parent 

• Heritable factors = genes

4 components of Mendel's Model

1. Alleles - alternative version of a gene

2. 2 “factors” for each character

   1. Diploid individuals inherent 2 copies of each gene

   2. 1 from each parent 

   3. May be identical (as in true breeding lines, may be different) 

   4. Found on homologous chromosomes 

3. Dominance

   1. If 2 alleles differ 

   2. Dominant allele determine phenotype

   3. Recessive allele has no noticeable effect on phenotype

4. Principles of heredity

   1. “Mendel's laws” 

   2. Law of segregation

   3. Law of independent assortment 

Law of Segregation

• Refers to the separation of alleles during gamete formation

• Correctly identified anaphase 1 

• Example

  • Seed color

  • Dominant yellow allele : Y

  • Recessive allele : y

  • Homozygous: 2 of the same allele for a particular gene

  • Heterozygous: 2 different alleles for a particular gene

Law of Independent Assortment

• Genes on different chromosomes assort independently during gamete formation

• Due to random orientation of tetrads during metaphase I

• Results in genetic recombination - new combination of alleles in offspring

• This is the 2nd mechanism for increasing genetic variation in sexual reproduction

• Independent assortment + crossing over = lots of new variation  

II. Genetic Crosses

1. Introduction

• A method for predicting the genotype of offspring 

• Following the behavior of alleles 

• When you do a cross you determine what gametes are possible in each parent based on the parent genotype 

• Combine those gametes to figure out every possible combination in the  offspring

• Calculate the frequency of each possible genotype 

2. Monohybrid Cross

• An individual who is heterozygous for one character

• Cross between heterozygotes 

• P: true breeding

  • 1 homozygous dominant, 1 homozygous recessive

  • P = YY x yy

  • Offsprings have to be heterozygous

    • Frequency 1 = 100%

III. Using Probability to Determine Outcomes

A. Introduction to probability in Genetics 

• Laws of segregation and independent assortment reflect basic rules of probability 

• Genetic ratios expressed in terms of probability

  • Fractions

  • Decimal

  • Percentages

B. Multiplication Rule

• Allows for the prediction of combined probabilities of independent events

• Independent events - occurrence of one does not affect the probability that the other will occur 

  • States as P(event 1) and P(Event 2), ‘and” means multiply the separate probabilities 

• Multiplication rule example 

  • Mothers genotype: Aa

  • Fathers Genotype: Aa

  • Probability that 1st child will be homozygous recessive?

    • P(recessive in egg) = 0.5

    • P(recessive in sperm) = -0.5

    • -> 0.25

C. Addition rule

• Predicts combines probabilities of mutually exclusive events

• Mutually exclusive events - cannot occur simultaneously 

• States as P(event 1) or P(Event 2), “or” means to add the separate probabilities 

Chromosomes

I. Chromosomal Theory of Inheritance 

-> SEQ Morgan’s experiments

II. Inheritance Patterns on Sex Chromosomes 

-> CC, HD, sex linkage 

I. Chromosomal Theory of Inheritance 

1. Genes have specific loci on chromosome 

2. Chromosomes undergo segregation and independent assortment 

• Chromosomes rather than independent genes that experience mendels laws

A. Thomas Morgan and D. Melongaster

• Early 20th century, embryologist 

• 1860’s-1900’s: cytologist - chromosomes behave like Mendels “heritable factors” 

• Originally a skeptic of Mendel’s work and chromosome theory

• Accidentally ended the debate about evolution

• First experimental support showing that genes are transmitted on chromosomes 

Drosophila Melanogaster

• Fruit fly - Morgan’s model organism 

• Advantages

  • A lot of offspring 

  • Short generation time - 2 weeks, and based on temperature 

  • 4 pairs of chromosomes - 3 pairs of autosomes, 1 pair of sex chromosomes 

Describing Traits

• Wild type - most common phenotype for a character in natural populations

  • Ex. Red eyes

  • Wild type does not mean dominant 

• Mutant phenotype - one or more alternatives to wild type 

  • Ex. White eyes 

Notation

• Conventions differ, each system has different ones 

• Drosophila, symbol base on 1st observed mutant

  • Allele for white eyes = w

  • Allele for red eyes = w+

B. Correlating Allelic and Chromosome Behavior 

• P: red-eyed female and x white-eyed male

• F1: all offspring have red eyes

  • Red eyes are dominant over white eyes 

• F2: 3:1 phenotype ratio 

  • All white eye flies are male

Sex Determination Explains Ratio

• Y chromosome does not = males

• Y chromosome -> makes sperm

• Ratio of X chromosomes to sets of autosomes (A) determines sex 

  • XX:AA -> 1:1 -> Female

  • XY:AA -> 1:2 -> male 

  • XO:AA -> 1:2 -> Sterile male 

  • XXX:AAA -> 1:1 -> Female 

Why no white-eyed F2 Females? 

• Eye-color gene locates on X chromosome 

• No corresponding locus on the Y chromosome

Eye-color monohybrid Cross 

Morgans Findings

• Specific gene carries on a specific chromosome

• Unique inheritance patterns for genes on sex chromosomes 

• Strong support for chromosome theory of inheritance 

II. Inheritance Patterns in Sex Chromosomes 

A. Sex chromosomes 

• 1 pair heteromorphic 

• They act like homologues chromosomes during meiosis even though they are structurally different

• X and Y chromosome contain sex determining genes but also genes that are separate from sex determination

  • Sex-linked genes (x-linked or y-linked)

Human Sex Determination

• Males - XY -> heterogametic 

  • Half of sperm get an X, half get a Y

  • Y chromosome has genes fro male development 

• Females - XX -> homogametic 

  • All eggs have an X

  • Female phenotype due to absence of Y

B. X-Linked Genes

• Many genes, many required 

• X-linked traits - controlled by genes on the X chromosome and because we have this different pattern between males and females there is a unique inheritance pattern 

Inheritance of Sex Chromosomes

• XX* x XY

X* x

C. X Inactivation 

• Genes on X chromosome expressed in females and males

• Females - 2 copies

• Males - 1 copy

• Do females make twice the proteins? No

• Genes are tightly regulated in terms of levels of expression 

• Compensate for having 2 X chromosomes by inactivating one of them 

• 1 X chromosome inactivated during development

  • Barr body - most dense form of DNA

  • DNA +protein, along inside of nuclear envelope 

• Involves modification of DNA and histone proteins

• Inactivation is random - different X in different cells 

All About DNA 

1. DNA is the genetic material

-> SEQ, CC, DNA experiments

2. DNA structure

-> CC, HD, DNA structure

3. DNA replication

-> SEQ, HD semi-conservative DNA replication 

1. DNA is the genetic material 

• DNA - deoxyribonucleic acid

  • Monomers - 4 nucleotides 

  • 20 amino acids 

1. Fredrick Griffith 

• Work published in 1928

• Bacteriologist and embryologist 

• Working on pneumonia vaccine 

• First person to show evidence against the protein hypothesis for inheritance 

• Model organism - streptococcus pneumoniae 

Griffith's experiment 

• Take smooth bacteria and inject it into mice

  • They get pneumonia and die

• Take rough bacteria and inject it into the mice (cannot make you sick)

  • The mice survive

• Boil smooth bacteria to kill it and inject it into mice

  • The mice survive 

• Combine rough bacteria that are harmless and smooth that are dead and inject into the mouse 

  • The mice will die

    • Can isolate living smooth pathogenic bacteria out of the mice 

    • First demonstration of transformation  

      • Where bacteria take up material from the environment and change their genotype or phenotype 

        • The rough bacteria was taking up DNA from the dead smooth bacteria 

2. Avery, MacLeod, McCart  (1944)

• Wanted to figure out what was doing the transformation in the Griffith experiment

• Took the cells from the smooth colony and lysed them (broke them apart into components)

• Then separated the components and sorted them out

• Evaluated each component on its ability to transform 

  • Found that lipids, carbs, and proteins did not result in smooth, did not transform the rough

  • The DNA turned the rough into smooth

    • Only DNA can transform the genetic material 

3. Hershey and Chase (1952)

• Used bacteriophage T2 

  • Virus that infects bacteria 

  • 2 main components; DNA (or RNA) and protein coat surrounding the genome 

  • When they infect the host cell part of the virus goes into the cell and part of the virus stays out of the cell 

    • Finding out which part stays inside the cell tells us what inject the genetic material

• Experimental design

  • Grow 1 virus population with radioactive sulfur

    • Proteins contain sulfur while DNA does not 

    • Proteins radiolabeled - has radioactive isotopes and we can track the location by looking for the radioactivity

  • Grow second radioactive phosphorus

    • DNA contains phosphorus, proteins do not

    • Follow viruses through the life cycle and see what radioactivity ends up inside the host cell to determine if proteins or DNA are responsible for heredity 

  • Allow each to infect bacteria

  • One the viruses infect the bacteria, separate the infected bacteria cells from the bits of the viruses that get left outside

  • Determine where the radioactivity is and you can now tell which components of the virus infect those cells

    • Phosphorous was in the infected cells, DNA infected the host cell 

    • Radiolabeled sulfur was in the separated viruses

II. DNA structure

1. Nucleotides

• Monomers of nucleic acids

• Parts

  • Single phosphate group

  • Sugar 

  • Nitrogenous base 

    • Variable, makes each of the 4 variants different from each other

• 4 nitrogenous bases in DNA

  • Purines: 2 rings

    • Adenine 

    • Guanine 

  • Pyrimidines: 1 ring

    • Cytosine

    • Thymine 

• DNA strand

  • Double helix 

2. Determining DNA structure

• Published in 1953 by James Watson and Drancis Crick

• Erwin Chargaff 

  • Interested in chemical composition of DNA

    • 2 consistent observations

      • Total purine content (A+G) was always equal to the pyrimidine content (C+T)

      • Amount of A = T, C=G 

        • But A+T does not have to equal C+G 

        • Be able to use these rules to calculate nucleotide contect

          • 30% guanine = 20% thymine because this means there is also 30% C and there is 40% left to be divided between A and T 

• Rosalind Franklin

  • DNA is double helix, bases on the inside 

  • Diameter is consistent 

  • Distance between turns 

  • Nucleotides per turn 

• DNA structure - Watson and Crick Model

  • Used information from Franklin and Chargaff

  • Explained how DNA could carry information and replicated

3. Features of Watson-Crick Model

1. Double helix - 2 strands of DNA wound around each other 

   1. ONE DNA double helix = ONE DNA molecule 

   2. Unduplicated chromosome = 1 DNA molecule 

   3. Duplicated chromosome = 2 DNA molecules 

2. Each strand has…

   1. Sugar-phosphate backbone

      1. Alternating sugar and phosphate group

      2. Outside of helix, not variable 

   2. nitrogenous bases

      1. Inside of helix

      2. Variable - 4 bases

      3. Information in sequence 

3. Strands held together by H bonds

   1. A&T - 2 hydrogen bonds

   2. G&C - 3 hydrogen bonds 

4. Strands are antiparallel 

   1. Run in opposite directions 

   2. Carbons in sugar numbered 1-5 

      1. 3’ bonds downwards and 5’ bonds upwards

      2. Phosphate on each 5’ C

      3. Hydroxyl on each 3” C

5. Base pairing rules 

   1. Always purine pyrimidine

      1. A -> T (2 H bonds)

      2. G -> C (3 H Bonds)

Strands are Complementary

• If you know the sequence in one strand in a double helix, you can determine the other strand 

III. DNA replication 

1. Semi-conservative replication 

• Proposed by watson and crick, confirmed by Meselson and Stahl (1958) 

• Each new DNA molecule is half new half old, one helix old one helix new 

2 parts to DNA replication

2. Initiation

• Start with a double helix and must unwind the DNA into two single stranded parts to make a copy 

• Proteins required

  • DNA helicase - breaks the H bonds and opens up the double helix 

  • Single strand binding proteins (SSB) - DNA molecule, once the helicase come through, blocks the nucleotides from bonding and reforming H bonds 

  • Topoisomerases - prevent supercoiling, DNA packed too tightly and enzymes cannot get in to replicated 

  • All occurs at the origin of replication 

Requires RNA primer

• Short piece or RNA

• Made by primase - enzyme that can start a new strand 

• DNA polymerase builds on primer 

• RNA primer -> elongation 

  • Primer will ultimately be removed and replaced with DNA 

3. Elongation

• Use nucleoside triphosphates 

• 5’ phosphate attaches to previously existing 3’ hydroxyl 

  • Always added to the 3’ end exposed hydroxyl group

• Occurs in the 5’ to 3’ direction 

• Forms new phosphodiester bond 

• Release pyrophosphate 

• Elongated strand by a single nucleotide

Involves DNA Polymerase

• Adds one nucleotide at a time to the 3’ hydroxyl 

• Can only add to an existing strand, not start a new strand 

• Requires RNA primer 

Elongation occurs at Replication Fork

• 2 different processes 

  • Leading strand synthesis: 5’-3’ towards replication fork

    • Elongates smoothly and continuously 

  • Lagging strand synthesis: 5’-3’ away from replication fork 

    • Elongates in short discontinuous segments

Gene Expression 

1. Relationship between Genes and Proteins

-> SEQ information flow in cells

-> CC RNA types

2. Transcription

-> SEQ, HD eukaryotic transcription

3. Translation 

-> SEQ, HD eukaryotic translation

1. Relationship between genes and proteins

1. Flow of genetic information

• Proposed by Francis Crick in 1956 

• DNA -> RNA -> Protein

  • Unidirectional set of processes 

• Processes 

  • Transcription: DNA -> RNA

    • Use DNA gene sequence as a template to synthesize a complementary strand of RNA 

  • Transaction 

    • Use the RNA made to direct the sequence of amino acids in a newly synthesized protein

      • Carried out by ribosomes, information in RNA used to make polypeptide

2. RNA Composition and Types

• Ribonucleic acid 

• DNA vs RNA

  • Sugar: Deoxyribose - Ribose 

  • Nucleotide Bases: A, T, C, G - A, U, G, C

  • Strandedness (not universal rule): Double stranded - Single Stranded 

• Ribose

  • Ribonucleic acid vs Deoxyribonucleic acid

    • Deoxyribose is missing a hydroxyl group

    • RNA is less stable than DNA because oxygen is very electronegative and is more likely to react with things 

      • DNA is better for storage because of this 

• Uracil (U)

  • Pyrimidine base - U instead of T in RNA

  • Complementary to adenine, forms two H bonds

    • Performs the same way as thymine in regards to base pairing 

3 Main Kinds of RNA

• mRNA - messenger

• rRNA - ribosomal

• tRNA - transfer 

3. Genetic Code 

• Tells the order of amino acids

• Mechanism for A U C G -> 20 different amino acids

• Read 3 bases at a time 

  • Each group of 3 specifies a single amino acid

Codons

• Sequence of 3 bases -> information for 1 amino acid 

• 4 bases = 64 combination 

  • Only 20 amino acids 

  • We still use all 64 combinations 

Characteristics of the Genetic Code

• Unambiguous - each codon codes for just 1 amino acid 

• Redundant - most amino acids called by more than 1 codon

  • Ex.

    • CCC, CCA, CCU, CCG all mean proline 

    • AAA, AAG both mean lysine 

The Genetic Code 

• Wobble hypothesis 

  • If there are multiple codons for one amino acid, the overlap helps there not be dangerous effects if there is a mutation

• AUG - start codon 

• UAA, UAG, UGA - stop codons

  • Neither have cytosine 

Reading Frame

• Codons must be grouped correctly 

• Frameshift mutation - single base deletion or insertion, results in frame shift and changes every amino acid from that point forward  

II. Transcription 

• First part of gene expression

• Synthesis of an RNA that is complementary to the DNA in the gene sequence

• 3 stages

1. Initiation 

• DNA serves as template for RNA synthesis 

• One DNA strand is transcribed (template strand) 

• DNA read in 3’-5’ direction

• RNA synthesized in 5’-3’ direction

• Other strand not transcribed 

• Components 

  • Promoter - specific DNA sequence on transcribed strand that says “start here”

    • Promoter itself is not transcribed to RNA 

  • RNA polymerase

    • Does the job of helicase, primase, and polymerase

    • Enzyme that makes new RNA strand 

    • Binds promoter, unwinds helix, begins transcription

2. Elongation 

• RNA synthesis, anti-parallel to template strand

• Uses RNA nucleoside triphosphates 

RNA Synthesis 

• 3’ T A C T G A 5’

• 5’ A U G A C U 3’ 

• Direction —-----> 

3. Termination

• After the gene, you have a sequence in the DNA that says stop transcription and causes polymerase to let go of the DNA

• RNA transcript releases

• Can be helped by a protein that attaches to the DNA and polymerase bumps into the protein and falls of

4. mRNA modification

• Transcription -> Pre-mRNA - not useful in protein synthesis (translation)

• Need to add stuff to each each

  • 5’ end add 5’ cap, nucleotides 

    • Export from nucleus, ribosome recruitment

  • 3’ end add poly-A tail

    • Protection, longer the tale the longer the RNA will last 

• Need to remove stuff 

  • Exons and introns 

RNA Splicing

• Get rid of the introns

  • The regions of the gene in your DNA that do not code for proteins, do not have amino acid sequence

• The exons do code for proteins and have an amino acid sequence 

• Splicing - process of removing introns 

• All of this happens in the nucleus, cannot leave nucleus if there is no cap, tail, and still have introns 

  • mature-mRNA 

III. Translation 

• Use mRNA to make a polypeptide

• Occurs in ribosomes either floating in cell or attached to ER

  • Most occurs with the rough ER 

1. Components 

• Transfer RNA

  • Single stranded RNA ~80 nucleotides

  • Brings amino acids into the ribosomes by matching the sequence 

  • Many variants where the sequences of each variant are a little different, makes each variant specific so its amino acid 

    • Tells how it's going to match up with each codon in the RNA 

  • Anticodon - binds to codons during translation

    • 3 specific bases on loop of tRNA, complementary to specific codon in the mRNA 

    • Each anticodon is associated with a specific amino acid

    • 1 amino acid for each anticodon 

• Ribosome

  • Big structures made of protein and ribosomal RNA 

  • Small subunit

  • Large subunit

  • Within the ribosome - 3 sites

    • A, P, E

  • As the mRNA feeds through the ribosome at each step of the cycle there is one codon each in the A site, P site, and E site

  • rRNA - enzymatic 

    • Catalyze the reactions of forming peptide bonds 

    • Transcribed from DNA - not translated to protein 

    • Example of ribozyme 

2. Translation Initiation

• Small ribosomal subunit binds mRNA and first tRNA 

  • tRNA that matches the start codon

  • Once you are aligned to the first codon at the p site, then the large subunit and proteins called initiation factors 

    • With large subunit -> translation initiation complex 

3. Translation Elongation 

• Repeating cycle 

• A site unoccupied, bring in tRNA with an anticodon that matches the codon in the A site

• Form a new peptide bond, transfer the polypeptide chain that is attached to the P site in the middle

  • Break the bond between tRNA and polypeptide and attach that amino acid to a new amino acid

• Translocation - shift the mRNA within the ribosome by one codon, transfer new polypeptide to the P site, a site shifts to E site, leaves

• GTP is used for energy instead of ATP (energetically equivalent) 

• Endergonic, storing potential energy in peptide bond 

4. Translation Termination

• Occurs when there is a stop codon at the A site

  • Does Not match anticodon on tRNA

  • Release factors (small proteins) match the stop codon at the A site 

  • Cannot form new peptide bond 

  • Polypeptide released 

• Ribosomes tries to make peptide bond formation but to translocation anyways so the release factor is in the p site, it is not stable and everything dissociates 

• Everything comes apart: polypeptide, mRNA, tRNA, ribosomal subunits 

• We can take polypeptide (most likely in the rough ER) and do the modifications in the endomembrane system 

I. Prokaryotic Gene Regulation 

• Mainly at transcription level

• DNA -> RNA

A. Lac Operon Model - The Basics

• In E. coli 

• Normal gut bacteria, eats what you eat

• Human drinks milk

  • E. coli digests lactose 

Vocabulary

• Induction - turning on gene expression

• Inducer - compound that stimulates gene expression 

• Inducible gene - gene activated by inducer

  • Default state is off and not expressing genes, when the inducer is present the lac operon is induced and the genes are expressed 

• constitutive expression - genes are always expressed and turned on, preparing DNA

• Operon - a complex in DNA consisting of groups of gene with related functions and regulatory DNA sequences

  • Only found in prokaryotes, mostly 

Ex. Lac operon contains

• Promoter - RNA polymerase binding site, not transcribed 

• Operator - on/off switch sequence for gene expression

• 3 genes - lacZ, lazY, and lacA 

B. Negative Regulation of lac Operon

• Negative is the yes/no on/off switch, are these genes expressed at all

• Governed by the presence or absence of lactose itself 

• No lactose -> operon OFF, no expression 

  • Negative regulation active 

• With lactose -> operon expressed

  • Negative regulation inactive 

• Works via a repressor

  • Separate gene from the lac operon that makes the repressor protein and prevents the expression of the lac operon (turns the operon off) 

  • Always want repressor to be available 

  • Not a part of the operon 

Lac Repressor Activity

• No lactose - repressor protein binds to the operator

  • Negative regulation is in effect 

• RNA polymerase is the enzyme that expresses the genes, it can still bind to the promoter but it cannot move downstream to transcribe the genes because the repressor protein is blocking the pathway 

Lactose Affects lac Expression

• Lactose enters the cell, converted to allolactose (lactose isomer)

• Binds 2nd site on repressor protein 

• Repressor is inactivated and cannot bind to operator 

• Allolactose in this system is the inducer, turns the genes on 

  • Induces the lac operon by inactivating the repressor protein and allows transcription to occur 

• Results in low level of lac operon expression

What is Transcribed? 

• 1 long mRNA with all 3 genes 

• RNA translated to 3 different polypeptides 

• Each gene has own start and stop codons

After Transcription 

• No nucleus, dealing with bacteria 

• Cotranslation process

  • As the mRNA is being synthesized, it's also being translate at the same time, incomplete mRNA 

C. Positive Control of lac Operon

• Positive control - regulation by an activator that binds DNA to stimulate transcription, HIGH expression when active

• E. coli will eat lactose but prefer glucose 

• Lac Promoter is inefficient 

  • Low affinity for RNA polymerase 

    • RNApol binds to promoter, but then might start transcription or might release

    • Match is not great 

• Solution - CAP

  • Catabolite activator protein

  • Binds to the promoter and stabilizes the RNA polymerase so that it always starts transcription 

  • Does not work on its own, only active when its bound to another molecule

    • cAMP - cyclic AMP (adenosine monophosphate) 

      • AMP is a signaling molecule in cells that indicates cellular stress 

      • Indicates lack of food 

• Effects of CAP-cAMP

  • Low glucose -> cAMP increases 

  • cap -cAMp complex that binds to the CAP binding site on the promoter next to RNApol binding site 

    • Bends double helix

    • Change in shape increases RNApol binding 

      • Increases rate of transcription 

  • All if you run out of glucose 

  • = positive control 

4 Situations

• High glucose/no lactose - no CAP-cAMP, repressed operon 

  • No expression 

• No glucose/no lactose - CAP-cAMP present, repressed operon 

  • No expression 

• High glucose/high lactose - no CAP-cAMP. Operon induced 

  • Low level of expression

• No glucose/high lactose - CAP-cAMP present, operon induced 

  • High levels of expression 

II. Eukaryotic gene regulation

1. Introduction

• Eukaryotic cells also respond to environment 

• Multicellular - allows for specialization and organization

• Achievednthrough differential gene expression 

• Does not only regulate transcription, regulates every step of the process 

2. Regulation of Chromatin Structure

• 2 forms

  • Euchromatin 

    • Loosely packed, genes can be active

    • Not densely coiled, enzymes can get in and do transcription

  • Heterochromatin

    • Densely packed, no gene expression

    • When the DNA is coiled up around itself enzymes cannot get in to initiate transcription 

    • Suppresses gene expression via DNA methylation 

      • Methyl groups are hydrophobic, want to be coiled up together 

      • Methylating DNA - down regulating gene expression 

3. Regulation of Transcription Initiation

• Transcription factors - proteins that bind to DNA and either increase or decrease level of transcription

  • Enhancers and silencers 

4. Post transcriptional Regulation 

• Length of poly A tail affects level of expression

• Longer the tail, the longer it takes the enzymes to get to the coding sequence 

• Short poly A tail - mRNA breaks down quickly and cannot be used as mu

Alternative splicing

• When we take out the introns and keep the exons 

• Different polypeptides from same gene 

5. Post Translational Regulation

• Proteins get modified when they go through the ER and Golgi

• Polypeptides often processed -> final protein

• Must be folded correctly 

• Often chemical modifications

DNA Technology

1. PCR

-> SEQ, HD, PCR Gel Electrophoresis

2. DNA Sequencing

-> SEQ, HD dideoxy sequencing 

3. Applications

-> HD, APPLY DNA technology 

• Techniques for sequencing and manipulating DNA 

  • Nucleic acid hybridization

    • Template strand -> complementary strand (base pairing) 

1. PCR (polymerase chain reaction)

1. Introduction to PCR

• Method for producing many copies (billions) of desired DNA sequence in short time 

• Important when studying genes - any specific gene is a tiny component of total DNA (millionth)

• Exons in the human genome are only 1.5% of the 3 million base pairs, a single gene is 1/1000th of that percent

• The amount of DNA from an individual that you care about is only a small fraction of the content, need to amplify the part of the gene that you care about

  • PCR is used to amplify and make copies of the target region so that we can study and manipulate it 

PCR components

• Template DNA - sample of DNA that contains the target region you want to amplify 

• dNTPs - deoxyribonucleotide triphosphates (A, T, C, and G) 

• Primers - similar to DNA replication , DNA primers that are 15-20 bp long, primers are designed to be complementary to the flanking sequences of the DNA, effective replication to copy target region in both directions 

• Taq polymerase - DNA polymerase from Themus aquaticus, bacteria from hot springs in yellowstone 

  • Stable at very high temperatures

2. The process of PCR

• Cycle of 3 steps repeated 30-40 times

  • Denaturation

    • Heat to ~90 degrees C, breaks hydrogen bonds in the DNA double helix 

    • Goes from one double stranded DNA to two single stranded DNA molecules 

      • Similar to helicase function 

  • Annealing

    • Cool down the temperature to 40-65 degrees C

    • Allows the primers to bind to the complementary sites on the template DNA strand

      • Similar to primase function 

  • Extension 

    • ~72 degrees C

    • Taq polymerase replicated DNA from 3’ end of primers 

    • Polymerase elongates from primers 

      • Similar to semi conservative DNA replication 

      • Using primers we designed instead of primers made  by an enzyme 

      • DNA polymerase still required 

    • Take the results and use it as the template for the next round 

• Amount of target DNA is doubled every cycle 

• Done in thermal cycler - machine that can precisely modulate temperature of mixture 

  • Aka PCR machine 

• Extremely sensitive to conditions and contamination 

3. Gel Electrophoresis

• Used to conform PCR results 

• Separates the nucleic acids by size 

Agarose Gel

• Polysaccharide gel

• Porous 

• Pass electrical current through gel, DNA moves from - to + 

  • Passes to + because phosphate groups are acidic and hydrophilic, DNA as a molecule is negatively charged 

  • Constant ratio between charge of DNA molecule and the length of the DNA molecule 

Uses

• Confirm PCR results 

• DNA sequencing 

• DNA profiling

• Disease diagnosis 

II. DNA sequencing 

1. Introduction 

• Many DNA sequencing techniques

  • Almost all of them involve complementary base pairing and hybridization

• Divided into 2 main groups: “old” and “next gen” 

Technique: Dideoxyribonucleotide Chain Termination Sequencing 

• First automated sequencing method 

• Frederick Sanger - nobel prize, 1980 

• Can sequence DNA fragments up to ~700 bp

• SAME process as PCR, a few differences 

2. Ingredients

• Target DNA - what you want to sequence, start with PCR to have billions of copies

• Primer - (only one in this process) designed to base par with 3’ end of template strand 

  • Only want the new strand to tell us what the sequence is, only one new strand is made each cycle 

• DNA polymerase - taq polymerase (same as PCR) 

• Nucleoside triphosphates - 4 deoxynucleotides 

• 4 dideoxyribonucleotides - very low percent of our reaction mixture, each base labeled with different fluorescence 

  • A, T, C, G all fluoresce with a different wavelength 

Deoxyribose 

• 3rd sugar we see in our nucleotides 

• Inability to form a phosphodiester bond

3. Process

• Target DNA denatured

• Primers annealed

• Complementary strand synthesized

• Dideoxyribonucleotides randomly incorporated

  • Incorporated a fraction of the time 

• Elongation stops at dideoxy nucleotide 

Why Does the Chain Terminate? 

• No hydroxyl group, cannot add another group

• Nitrogenous base is labeled so we can tell whether it is an A, T, C, or G

Results in:

• Many strands synthesized 

• Every strand is a different length 

• Each labeled with fluorescence at LAST base in line, we can only read the last base 

• Color of label indicates last base in fragment - separate by size get complete sequence 

Next step: Polyacrylamide Gel 

• One long gel column

• DNA fragments migrate through it and go past a detector 

• Whatever nucleotide is at the end of the sequence the detector will read the wavelength and tell you what nucleotide it is

• Go from smallest to largest 

4. Uses of Sequences

• Evolutionary relationships

• Determine loci or function

• Disease diagnosis 

• Impress reviewers when you want to publish a paper

• Others 

III. Applications

1. Species diversity

• Wanna know exactly how diverse an ecosystem is 

• Can be difficult to get accurate measure 

• DNA bar-coding = uses sequence of same small region of genome to identify 

2. Gene Therapy

• Used to treat a type of genetic blindness

• Uses retrovirus to insert “correct” genes into cells of the retina 

• CRISPR