Quiz 1 Flashcards
What are the three components of genomics?
structural, functional, comparative
What is functional genomics?
transcriptome, proteome, epigenome
What is structural genomics?
physical nature of genomes
What is comparative genomics?
conservation of genomic content across species
What did Mendel discover?
the basis of inheritance
Who was mendel and what did he do?
-Augustinian monk worked with pea plants in the 1860s
Why did mendel work with pea plants?
1) Distinct binary traits - color, shape
2) Can either self or cross pollinate
3) Large numbers of offspring
4) Pure breeding lines
What is a phenotype?
observable trait
What did mendel call genes?
determinants
How did Mendel explain the proportions 1:2:1 of the F3 generation of a purebreeding round x pure breeding wrinkled cross?
Proportions can be explained by:
- each plant carries one or both determinants
- determinants are deposited randomly into gametes: sperm or egg
- one determinant per gamete
- gametes then combine at random
What are alleles?
alternative forms of a gene
What is a genotype?
a set of alleles for a particular individual
What is Aa called?
heterozygote “hybrid”
What is aa or AA called?
homozygote
What are mendel’s laws?
1) for a single trait a gene pair separates into gametes at random and combines at random
2) for multiple traits, different gene pairs separate at random into gametes and combine at random (shape gene pair is independent of color gene pair)
- Inheritance of genes can be explained by probabilities
What is penetrance?
- the percentage of individuals with a particular genotype who exhibit the phenotype associated with that genotype
- have a population that you know have the same genotype, but not all of them show the phenotype expected of that genotype
What is an example of penetrance in humans?
BRACA-1 mutation - all have genotype but not all develop breast cancer
What is expressivity?
- extent to which a given genotype is expressed at the phenotype level
- same genotype range of phenotypes
What is incomplete dominance?
phenotype of the heterozygote is intermediate to the two homozygous forms (pink Aa)
What is codominance?
heterozygote displays both of the homozygous phenotypes
What is linkage?
When genes are physically located on the same chromosome
What are chromosomes?
threadlike structures of nucleic acids and proteins found in the nucleus of most living things
What are some important characteristics of chromosomes?
they are observable and possess interesting behavior during cell divisions
Each species has a stable number of ______
chromosomes
How many chromosomes do human somatic cells have?
46 chromosomes, 22 pairs of autosomes, 1 pair of sex chromosomes this is the diploid number (2n), 1/2 of each pair comes from the mother and half from the father
How many chromosomes do sex cells have?
23 chromosomes, haploid number (n), unpaired chromosomes
What is mitosis?
nuclear division of somatic cells
What is the cell cycle?
- G1 phase (growth or gap)
- S phase (synthesis) DNA is replicated
- G2 phase (growth or gap)
- M phase (mitosis)
What makes up interphase?
G1,G2,S
What is the chromosome number in interphase? Prophase?
2n, 4n
What are two key things to remember about mitosis?
Replication of chromosomes, followed by segregation, produces two identical daughter cells
What is meiosis?
2 successive nuclear divisions that make sex cells
What are the four steps of meiosis?
1) DNA replication 2) Homologous chromosome pairing 3) Segregation 4) Segregation
What did Walter Sutton and Theodore Boven study/hypothesize?
- the behavior of Mendel’s particles parallels the behavior of chromosomes during meiosis
- Sutton-Boven Hypothesis - genes are physically located on chromosomes
How did Sutton and Boven defend their hypothesis? (What are three similarities between genes/alleles and the process of chromosome segregation?)
1) Genes and chromosomes both come in pairs (mom/dad and Aa)
2) Alleles of gene pairs and a member of a homologous pair of chromosomes both segregate equally into gametes
3) Different gene pairs and different sets of chromosome pairs act/segregate independently
What is the problem with the Sutton-Boven hypothesis?
correlation does NOT equal causation
What are Drosophila Melanogaster and who studied them?
fruit fly, Thomas Hunt Morgan 1900s
Why were Drosophila used for genetic studies?
easy to raise, cheap, produce lots of offspring, have observable traits
Explain the chromosome number of drosophila melanogaster.
- 4 pairs of chromosomes
- diploid = 8 chromosomes (4x2) (2n)
- haploid = 4 chromosomes (n)
Why dont white eye female fruit flies exist as commonly as males?
eye color trait is sex linked - physically located on the X chromosome
How do you determine litter size?
1 / probability of genotype or phenotype - if it is not an even number round up to find litter size
What are some charateristics of meiosis?
- separation of homologous pairs
- 4 daughter cells (haploid)
- 2 divisions
- germ cells, polar bodies
What are some characteristics of mitosis?
- separation of sister chromatids
- 2 daughter cells (diploid)
- 1 division
- somatic cells
What was Griffiths experiment?
- (1928)
- bacteria Streptococcus pnemonaie
- S-strain: smooth and lethal R-strain: rough and non-lethal
- infect mouse with S strain - die
- infect mice with heat killed S strain - live
- infect mice with heat killed S strain and live R strain - die
- concept of transformation
What was Avery, Macleod, and McCarthy’s experiment?
(1944)
-showed that DNA is the active component of the transforming principle that converted R bacteria into lethal bacteria when mixed with the S strain
What was the Hershey and Chase experiement?
(1952)
- used radiolabelling to show that when bacterial culture is infected with bacteriophages (a virus) DNA is the major component that enters the cells
- labelled the sulphur in protein and phosphorus in DNA
- agitated in blender - found radioactive phosphorus in cell (indicating the presence of DNA)
What is DNA
- deoxyribonucleic acid
- linear, unbranched polymer made up of four chemically distinct subunits (nucleotides)
What makes up a nucleotide?
sugar (deoxyribose), nitrogenous base (1’), phosphate group (5’)
What are the two groups of nitrogenous bases?
Purines - Adenine and Guanine (2 rings)
Pyrimidines - Cytosine and Thymine (1 ring)
What are nucleotides connected by?
- phosphodiester bonds
- new nucleotides must be added to the 3’ OH terminus
- 5’ -> 3’ is the directionality of DNA
What did Watson and Crick already know when they began to solve the structure of DNA?
1) DNA was composed of multiple polynucleotides
2) base ratio - chargaffs rule
What strategy did Watson and Crick use to study the structure of DNA? Who’s image did they use?
X-Ray diffraction- crystallize your model of interest
- Rosalind Franklin - photo 51
What structure did Watson and Crick suggest?
double helix
Describe the structure of DNA
- Double helix
- two strands are antiparallel
- electrostatic interactions
- base stacking stabilizes the molecule
What were three models for the replication of DNA?
conservative - both strands completely replicated
semi-conservative - one strand replicated using one strand as a model to form a new DNA molecule with one new and one old DNA strand
dispersive- random segments replicated
What was the Meselson and Stahl experiment?
- used a heavy isotope of N (N15) in E. Coli bacteria
- grew the cells on lighter nitrogen for 1 or 2 generations
- isolated DNA and ran on a cesium gradient to identify the weights of the new and old DNA strands
- after 1 gen - medium band (ruled out conservative)
- after 2 gen - 2 bands (ruled out dispersive)
How is DNA replication controlled?
tightly controlled, must occur with high fidelity to prevent introducing mutations
What is an origin of replication?
- where replication of DNA starts, well defined in prokaryotes and yeast but less so in higher Eukaryotes
What enzymes are required for DNA replication?
Topoisomerase, DNA polymerase, Helicase, Ligase, single stranded binding proteins
What is the function of Topoisomerase?
- nicks, cuts 1 DNA strand - different type of topoisomerase cuts both strands (initiates DNA replication)
- to relieve the torsional strain in DNA during DNA replication and in RNA during transcription
What is the function of DNA polymerase?
- Enzyme that synthesizes and degrades DNA polynucleotides
- adds nucleotides 5’->3’
- Also has exonuclease activity (proof reading) 3’->5’
- 5’ -> 3’ exonuclease activity (used for removing primer)
What are the components of a DNA molecule during DNA replication?
Replication fork, leading strand, lagging strand
Describe the process of DNA replication. (Leading strand)
1) DNA uncoils/unwinds (topoisomerase)
2) Helicase - disrupts hydrogen bonds between nucleotides (unzips)
3) Single stranded binding proteins bind and prevent the two strands from binding back together
4) Primer (30 bp RNA sequence) - synthesized by RNA polymerase or primase
5) DNA polymerase adds nucleotides 5’-3’ (then replaces the primer)
How is DNA replication different on the lagging strand?
- DNA polymerase must work backwards as the replication fork is unzipped
- short DNA fragments called okazaki fragments are created and DNA ligase catalyzes the phosphodiester bond between the 2 nucleotides
Why can’t DNA fully unzip to avoid the problem of the lagging strand?
because DNA strands are very long and have a relatively strong bond
What other enzyme is required when synthesizing okasaki fragments in Eukaryotes?
flap endonuclease (FEN1) cuts at the branching point between okazaki fragments in Eukaryotes
What problem does the lagging strand cause in the long run? How is this combatted?
- Lagging strand - no room for last okazaki fragment - ends of chromosomes get shorter - lose genetic information
- Telomerase
What is telomerase?
- Telomerase is an enzyme that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes
- Telomerase has reverse transcriptase activity and can synthesize DNA from an RNA template
What is telomerase made of?
- made of protein and RNA subunits
- RNA - primer
- Protein - reverse transcriptase converts RNA to DNA
What is a telomere?
end of chromosomes, possesses DNA sequences that are repeats TTAGGG
What cells in telomerase active in?
- telomerase is very active in dividing cells
- as cells age telomerase activity goes down
- cancer cells reactivate telomerase
How was it discovered that genes encode for proteins?
- it was known that DNA was located in the nucleus and proteins in the cytoplasm
- proposed that a messenger exists
Why was RNA proposed to be the messenger from DNA to protein?
- identified as a candidate through the pulse chase experiment
- pulse - radioactive RNA precursor (newly synthesized RNA marked by radioactivity)
- chase- non radioactive RNA precursors
What is RNA?
- a polynucleotide, contains a phosphate group, ribose not deoxyribose, RNA uses uracil in place of thymine
- single stranded and much less stable than DNA
What does the process of transcription do?
converts DNA sequence to an RNA molecule
How does transcription occur?
RNA polymerase adds nucleotides to a template strand, RNA polymerase does not need a primer and adds nucleotides in the 5’-3’ direction
What is a short RNA sequence called?
a transcript - a few thousand base pairs
What occurs to RNA following transcription?
splicing - introns are removed and exons are kept
addition of a 7 methyl guanosine 5’ cap
addition of a poly-A tail -> polyadenylation
Where does post-transcriptional splicing of RNA occur?
in eukaryotes but not prokaryotes
What four groups are attached to the carbon in an amino acid?
R, protonate amine, carboxyl, and H
What links together amino acids?
peptide bonds - loss of water molecule
Who identified that 3 nucleotides code for one amino acid?
Marshall Nirenberg and Heinrich Matthaei
used custom mRNA sequences and bacteria protein making machinery and saw what protein was produced
What is degeneracy in transcription?
multiple codons encode for a particular amino acid
What is mRNA sequence punctuation?
- Initiation codon AUG -> methionine
- Termination codons -> UAA, UAG, UGA
What is translation?
process by which mRNA is converted into an amino acid sequence
What are ribosomes?
structures located in the cytoplasm and consist of rRNA and protein
What is tRNA?
(transfer) adapter RNAs which form a hairpin structure - charged with an individual amino acid, contain an anticodon that binds to the complementary mRNA codons
What are the steps of translation?
Initiation - ribosome finds initiation codon AUG
Elongation - addition of amino acids
Termination - termination codons
What are the two strands of DNA called in the process of transcription?
sense or coding strand and antisense or template strand
What is a silent mutation?
change in DNA sequence that doesnt alter the amino acid sequence
What is a missense mutation?
a change in the DNA sequence that alters one amino acid in the sequence
What is a nonsense mutation?
a change in the DNA sequence that results in an early stop codon
What is a frameshift mutation?
addition or deletion of nucleotides in the DNA sequence
What are some examples of groups of amino acids?
small uncharged, large aromatic rings, negatively charged, positively charged, non-polar
What is the primary structure of amino acids?
chain of amino acids
What is the secondary structure of amino acids?
alpha helix and beta sheet (antiparallel) stabilized by hydrogen bonds - weak lectrostatic interactions
What are some examples of tertiary structure of proteins?
hydrogen bonds, disulfide bonds, hydrophobic effects
What are some roles of proteins in the cell?
enzymes, structure, movement, transport of materials, regulation, protection, storage
What is another random type of RNA?
ncRNA - short or long
never translated, nc = non-coding
What is a gene?
a discrete sequence of DNA that is transcribe and translated into a protein
How was the coronavirus vaccine created?
- Using mRNA
- mRNAs are relatively easy to make
- mRNAs are unstable: must be kept cold, but leave the body quickly
How many base pairs in the genome composed of?
3 billion base pairs
How do we analyze our genome?
Recombinant DNA, mapping, and DNA sequencing
What is recombinant DNA?
DNA sequences that have been formed artificially by combination from different organisms
What is molecular cloning?
enzymes that are naturally occuring are used by cells from replication, repair, recombination, and defense
What are the uses of molecular cloning?
- make copies of DNA molecules (cloning)
- cut DNA molecules into shorter fragments
- join together into combinations that don’t exist in nature
- characterize gene structure and function
What are the enzymes used in recombinant DNA manipulation?
DNA polymerase, nucleases, end modification enzymes, and ligases
What are nucleases?
enzymes that degrade DNA molecules by breaking the phosphodiester bond
What are ligases?
enzymes that join together DNA molecules by synthesizing the phosphodiester bond to link two polypeptides
What are end modification enzymes?
make changes to the ends of DNA molecules
What are restriction Endonucleases? What are 2 examples?
- restriction endonucleases create internal cuts in DNA sequences - enzymes bind DNA at a specific site and make a double stranded cut
- EcoRI - cuts DNA at a palindromic 6 nucleotide sequence
- EcoRV cuts through double stranded DNA
What are two things to note about restriction endonucleases?
- the same sticky ends can be produced by different enzymes
- 5’ and 3’ overhangs may both occur depending on the enzyme used
What is ligation?
take two DNA fragments that have been cut by the same restriction enzymes and incubate them with the enzyme ligase
What is gel electrophoresis?
movement of charged molecules in an electric field, separates DNA fragments by size -> smaller travel farther
What is DNA charged?
negatively charged and linear
What type of gel is used in gel electrophoresis?
agarose or polyacrylamide
What are two other components of gel electrophoresis?
DNA Ladder, ethidium bromide- compound that binds DNA and reacts with UV light
What is PCR?
- Polymerase chain reaction
- amplify specific DNA sequences many, many times, uses the principles of DNA replication
What do in vitro and in vivo mean?
- in vitro - in a test tube
- in vivo - in a cell
What are the components of PCR?
DNA polymerase, DNA template, nucleotides, primer (forward and reverse) , buffer
What are the steps of PCR?
Denaturation - separate DNA strands 94C
Annealing - primers anneal to the single stranded DNA 50C
Synthesis - DNA polymerase synthesizes new strands 72C
Repeat steps 1-3 30-40 times
What is PCR machine also called?
a thermocycler
What is the short product in PCR?
the DNA sequence between the primers becomes the main template and is amplified exponentially
How much DNA does PCR produce?
130 million copies, goes from nanograms of template DNA to Micrograms of amplified DNA
Describe the polymerase used in PCR.
polymerase comes from a thermophilic micro organism Thermus aquaticus (Taq)
Describe the primers used in PCR.
Pair of primers: forward and reverse 20-30bp, direct polymerase to a certain site in template DNA, must be designed
What is quantitative PCR?
reports how much product you have at each cycle, uses dye that binds double stranded DNA, if it hits the threshold sooner you know the reaction started with more target DNA
What are constraits to PCR?
- must know the DNA sequence at the boundaries
- cant amplify sequences greater than 40kb
What are some used of PCR?
screen DNA for mutations, genetic profiling, recovering DNA sequences from ancient humans, COVID-19 test
What is an example of a cloning vector?
a plasmid, small circular DNA molecule made from bacteria
What are some components of a plasmid?
Origin of replication, amp resistance gene, cluster of unique restriction sites
What are some examples of plasmids?
pUC8
What are the steps in the process of cloning?
- cut DNA and plasmid with BamH1
- ligate - animal gene is inserted into the plasmid
- recombinant plasmid is transformed into the bacterium
- replicated inside the bacteria (grown on plate)
- take bacterial colony and grow it in liquid culture w/amp
- harvest bacterial cells and isolate plasmid
What is transformation?
bacterial cells take up naked DNA
What is the purpose of ampicillin in a plate?
selects for bacterial cells with plasmid
What is the size limit for plasmids?
DNA sequences >10kb in a plasmid, plasmid will no longer carry it
What is an alternative cloning vector to bacterial cells?
virus - lambda bacteriophage - infects bacteria
How many kb of DNA can bacteriophages incorporate into their genome?
up to 18kb of DNA
Describe cloning using a bacteriophage.
- the lambda genome is linear, but at its ends it had cos sites - 12 nucleotide long single strands of DNA that are complementary
- infection is visualized as a plaque on a plate: a zone of clearing in a lawn of bacteria
What are 5 other vectors in cloning?
- Cosmid - special plasmid that has cos sites - like the viral genome (44kb)
- Bacterial Artificial Chromosome (BACs) - F plasmid (300kb) - used extensively in HGP
- P1 bacteriophage - similar to lambda (110kb)
- P1 derived artificial chromosome - P1 vector + BAC (300kb)
- Fosmid - F plasmid and cos sites (44kb)
How large is the human genome?
3 billion base pairs
In general, how was the human genome studied?
- fractionated and put each piece into its own separate vector -> clone library
- the more the vector can accomodate DNA, the fewer vectors are required to sequence the entire genome
What are cloning techniques useful for?
sequencing genomes, interrogating gene function
