exam #1 Flashcards
genome
all DNA/ genes
genes
parts of the genome; segments that code for proteins
how many nucleotides in each cell
3 billion
how many chromosomes in genome
46
how many genes in genome
20,000
how much of DNA are genes
only 2%
how long can DNA unravel to
2 meters
hierarchical structure of DNA
-DNA resides in chromosomes
-genes are nucleotides that get expressed in the real world
-nucleotides are multiple segments of DNA base pairs
-DNA is a combo of 4 possible amino acids
structure of a nucleotide
-phosphate (PO4): always attached to 5’ C
-deoxyribose sugar (ribose in RNA)
-base (only part that changes): always on 1’C
purines and structure
adenine and guanine; 2 rings
pyrimidines and structure
cytosine, thymine, and uracil in RNA; single rings
how to tell which is a deoxyribose and ribose
deoxy: no hydroxyl group on 2’ C
ribose: hydroxyl group on 2’ C
building a nucleotide process
-phosphate on 5’ adds to the 3’ C on the ext nucleotide
-H on 3’ OH group goes to water
-phosphodiester bond is formed between OH and COOH
adenine and thymine bond
2 hydrogen bonds
cytosine and guanine bond
3 hydrogen bonds; stronger bc need more energy to break
how to tell which is 5’ end and which is 3’ end
5’: contains phosphate group
3’: contains hydroxyl group
Avery, MacLeod, and McCarthy discovery of transforming principle
adding lethal bacteria to non-lethal bacteria caused mice to die
-cells lysed with RNAse and DNAse
ploidy
number of copies of chromosomes in the cell
what cells are haploid and what is this
sex cells (egg and sperm); half the number of chrom
diploid
double the number of chromosomes
reverse transcriptase
turns viral RNA into DNA, incorporates it into cells
retroviruses examples
-HIV
-SIV
-HTLV-1
-COVID-19
how can viruses cause cancer
activates genes to be expressed at higher rates than normal
linkage of genes
-on same chromosome: do not separate from each other in meiosis
-produces only two possible types of gametes
-3:1 ratio in Punnett square
segregation of genes
-2 genes on different chromosomes
-creates four possible types of gametes
-makes dihybrid pattern in Punnett
-9:3:3:1 ratio
unlinked genes
-2 genes on separate chromosomes or 2 alleles on same chromosome
-can recombine during meiosis
recombination of unlinked genes in meiosis
crossing over during meiosis 1, recombination in meiosis 2
-creates lots of variation
genotype
internally coded, inheritable info contained by an organism (genome)
phenotype
the outward physical manifestation; observable structure, function, or behavior of a living organism
silent mutations
changed codon codes for the same amino acid as normal
missense mutations
new amino acid created, can create a whole new protein that doesn’t have same function
-usually has largest effect
non-sense mutations
early stop codon created
how is protein structure related to function
-amino acid sequence dictates protein fold
-protein fold dictates protein function
-different sequences= different fold = different protein
how do mutations effect protein function
changes in DNA sequences (primary sequence) changes function
genetic code degeneracy
multiple nucleotides can code for the same codon
conservative mutation
change in the single amino acid doesn’t change the fold (same polarity)
problem substitution mutations and their effects
-functional: changes a critical amino acid, leads to changes in fold and thus change in function
-non-sense: results in stop codon and rest of protein isn’t made
sickle cell
substitution causes different amino acid to be made
deletion mutation
a single nucleotide or group of nucleotides is skipped
-causes reading frame shift and different protein is formed
insertion mutation
addition of unwanted nucleotide
-causes reading frame shift and different protein is formed
RFLP (restriction fragment length polymorphism)
-differences in DNA sequences detected bu restriction endonucleases
-RFLP/ band patten specific to clone/enzyme combo
-probe w/ labeled DNA sequence hybridizes fragments of digested DNA and revels unique blotting pattern to a specific genotype at specific locus
how is RFLP applied
-genetic testing: detects difference in homologous DNA sequences by looking at fragments made from specific RIs
-genome mapping
-genetic fingerprinting
-disease testing
-paternity testing
DNA sequencing
look at frequency of base amounts in DNA
-overlap show different versions of the gene
why do we need 3 nucleotides for a codon?
need to encode for 20 different amino acids, and if we only have 1 or 2 per codon, its not enough combos
why do codons need to be a consistent length
don’t know how to adjust the reading frame, and can’t tell where one stops and ends
gel electrophoresis
larger= travels slower, stays closer to the top (negative side)
why is DNA attracted to the positive electrode in gel
DNA is negatively charged due to its phosphate group, so negative attracts to positive
Southern blot general use and process
-after gel: use a DNA probe to detect what fragments contain the gene you are studying
1. DNA digested by RIs, fragments separated by size in gel and then denatured
2. filter/ membrane added to gel to transfer fragments to and fragments are blotted on nitrocellulose paper
3. replica of gel made and is incubated with labeled, complementary radioactive probe
4. probe attaches to the DNA fragments with the complementary sequence (only sticks to where the sequence matches)
5. look at results: any matching fragments shows the presence of that allele (called autoradiography)
similarities and differences between prok and euk
prok: oldest, small and simple, no nucleus, no organelles, single celled, circular DNA
euk: larger and more complex, nucleus, organelles, single or multi celled
both: DNA, ribosomes, cytoplasm, plasma membrane, linear DNA
restriction endonucleases
-cleave DNA at specific, palindromic sequences, generates single stranded bits of DNA and allows recombinant DNA to form
-fragments made can then be sorted by size in gel
-ex: EcoRI
recombinant DNA
-molecular cloning: insert DNA fragment into a DNA molecule (vector)
-can then be grown to amplify the plasmid
parts of vectors and functions
-origin of replication (ori): tells bacteria to replicate plasmid
-antibiotic resistance gene (Amp^r): allows people to select transformed bacteria by incubation and use its specific antibiotic
-cloning site: 1 or more RI recognition sites; used to insert DNA fragment of interest
steps of PCR
- denaturation
- annealing
- extension
what happens in denaturation of PCR?
heat increased, H bonds between strands break
what happens in annealing of PCR?
-RNA primers added
-forward: binds on left of bottom (antisense strand)
-reverse: binds on right of top (sense strand)
-temperature lowered so that primers can bind to template
what happens in extension of PCR?
-temp raised a little more
-Taq polymerase uses primers to make complementary strands to each template strand
-moves 5’-3’
-two new DNA molecules form
how is DNA packaged
-chromosome: at metaphase, has two copies of one chromosome
-uncoils into chromatin
structure and functions of parts of euchromatin diagram
-nucleosome: contains 4 histones; DNA wraps around
-histone H1: under the nucleosome; clamps it all together
-linker DNA: in between nucleosomes (and nonhistone proteins); nuclease cuts every 200 bp
how do DNA polymerases add nuc
add to the 3’ hydroxyl group of the growing chain
DNA replication: leading strand
DNA poly adds; synthesis occurs towards the fork
general process of DNA replication
- helicase unwinds DNA (coupled with ATP)
- single stranded binding proteins bind to single stranded DNA and stabilize it
- leading (strand with 3’ end unwound): RNA primers added, and DNA polymerase adds nucleotides from the primer towards the replication fork
- lagging (strand with 5’ end unwound): RNA primer made by primate and added, extended by DNA polymerase to form an Okazaki fragment; fragments added close to the fork but are attached moving away from fork
how are RNA primers removed in prokaryotes
DNA polymerase 1: acts as exonuclease and removes RNA from 5’ ends of Okazaki fragments
how are RNA primers removed and gaps filled in eukaryotes
RNAse H removes primers; gaps filled by DNA polymerase sigma and joined by DNA ligase
euchromatin
decondensed, transcriptionally active chromatin
-uncoiled
heterochromatin
condensed, transcriptionally inactive chromatin
-more coiled
phases of the cell cycle
G1: cell growth
S: DNA replication
G2: prep for mitosis
M: mitosis/ cell division
G0
where in the cell cycle do you find euchromatin and heterochromatin
euchromatin: interphase
heterochromatin: mitosis
primase and difference in euk
makes RNA primer complementary to lagging strand
-in complex with polymerase alpha in eukaryotes
DNA ligase
creates phosphodiester bonds between short fragments of nucleotides
DNA polymerase 1 in prokaryotes
removes RNA primers and replaces it with DNA
lagging strand process
primase synthesizes RNA primer and attaches to the complementary sequence, DNA polymerase extends it and creates Okazaki fragment, DNA poly sigma removes RNA primers and ligase fills the gaps
-process continues in general direction towards the replication fork
DNA polymerase 3 in prok and euk
-prok: major replication polymerase; synthesizes off of primers to make leading strand continuously and lagging strand Okazaki fragments
-euk: proofreading
topoisomerase
cuts DNA strands ahead of the replication fork and allows the DNA to rotate freely and unwind, and then rejoins it (in euk)
-prevents twists and kinks
sliding clamp proteins
load polymerase onto DNA at the primer-template junction; maintains stable association of enzyme DNA polymerase with template DNA so that synthesis proceeds for long distances
replisome
the whole replication complex
errors of DNA replication through DNA poly 3 vs base selection/ H bonding of base pairs
DNA poly 3 much more error prone
proofreading activity of DNA poly 3
-detects if nucleotides will be matches or not (5’-3’)
-edits 3’-5’ (exonuclease): when incorrect nucleotide is attached and elongation is blocked, its removed by DNA poly via hydrolysis and moved to its exonuclease domain from the polymerase domain, and then new nucleotide that’s a correct match can come in
3 levels of insuring accuracy of DNA replication
-polymerase selectivity
-proofreading
-mismatch repair
origin of replication in euk and prok
-prok: a DNA element, a specific sequence of nucleotides that are identified (recognized by protein complex) as the site to start replication
-euk: multiple ori’s
ORC
origin recognition complex
ORC in yeast
-ori: ARs = ACS consensus sequence + elements B1-3
-ORC binds to ACS and B1, and then recruits helices and other proteins to start replication
reverse transcriptase
takes RNA and makes DNA
telomerase
a reverse transcriptase; allows ends of chromosomes to replicate
telomerase process
-binds to 3’ end of telomere sequence along with RNA template
-catalyzes the addition of bases and restores the telomere length (transcribes off the RNA)
-DNA polymerase extends and seals the DNA strands
where was Taq polymerase originally found
hot springs; stable at very high temperatures
deamination damage and type of repair used
amino group is let off of a nucleotide that is supposed to be added, and a different nucleotide is formed and added instead
-ex: cytosine -> uracil
-base-excision repair
base-excision repair
-used for deamination
-DNA glycosylase cuts out the mismatch and DNA poly adds correct one, then ligase joins them together
UV light DNA damage and type of repair used (and caveat)
-adjacent thymines on the same strand create cyclobutane ring, creating thymine dimers (can be other pyrimidines but thymine is most common)
-photoreactivation
-not present in humans, only in prok and some euk
reaction with carcinogen DNA damage and type of repair used
-base reacts, addition of bulky group
-nucleotide excision repair
photoreactivation
enzyme (photolyase) activated by light and cleaves thymine dimer to make 2 thymines
nucleotide excision repair
damage seen by RAD (protein complex), DNA unwound by helicase and cut by nuclease, gap filled with DNA polymerase, sealed with ligase
repair random damage of DNA (mismatch)
(in euk): 1. MutS and MutL bind to the mismatch
2. direct excision of the mismatch
3. replication continues
repair of double stranded breaks
-homologous recombination: detected by protein complex, resection by nuclease/ helicase, D-loop formation and strand invasion, strand extension by DNA polymerase
BRCA and DSB repair
involved in detection of DSB, and can cause cancer if mutated and break is not detected/ fixed
how can polymerases tell which strand is template vs which is copy
template strand is methylated
what are the non-coding regions and what are their purposes?
-telomeres: protects ends of DNA
-promoter: tells enzymes for DNA transcription where to start
-regulators: tells RNA transcriptase to enhance (make lots of copies) certain parts
differences between RNA and DNA
-RNA: single stranded, less stable, uracil, has second OH group on 2’ C and one on 3’ C
-DNA: double stranded, more stable, thymine, only one OH group on 3’ C
what would happen if telomeres were too short
ends of DNA wouldn’t be protected and over time the DNA is eaten away
what would happen if telomeres were too long
telomere sequences are repetitive, so the longer the telomeres remain, the more chances for mutations and thus cancer
how to tell where the forward and reverse primers are
forward: same sequence as the beginning of the sense (5’ on left) strand
reverse: same sequence as the end of the antisense (5’ on right) strand
what happens in first cycle of PCR
forward and reverse primers bind to their template strands and transcribe all the way to the end of the DNA, left with two strands that end at same place but one is slightly shorter
what happens in second cycle of PCR
forward and reverse primers bind to templates and the new strands made in first cycle, primers bound to original make two strands with one shorter than the other, and primers bound to the new strands make 2 strands of same length (shortened, only contains the parts that were within the primer region)
what do you need to have lots of in order to run many cycles of PCR
free nucleotides and forward/ reverse primers
how many copies of DNA are made per PCR cycle
1: 2 copies
2: 4 copies
3: 8 copies
4: 16 copies
what gave us fundamental knowledge about mutation, genetic linkage, and the relationships between genes and chromosomes?
Drosophila
bonds linking polysaccharides
glycosidic
what type of cell are animal and most higher plant cells
diploid
RNA functions in cells
-catalyst or enzymes
-regulator of gene expression
-carrier of info from the nucleus to the cytoplasm
what did Meselson and Stahl’s experiment do and show
-grew cells with different nitrogen isotopes, transferred DNA from one isotope to other and made new DNA, the DNA was made of a mix of the light and heavy isotopes
-showed that replication is semiconservative
type of bonds between nucleotides
phosphodiester
when do sugars cyclize
if they contain five or more carbons
oligosaccharide
a few sugars joined together
meiosis products
formation of eggs and sperm: one member of each chromosome pair is transmitted to each progeny cell
complementary sequence to 5’-TCAAGG-3’
5’-CCTTGA-3’
bonds between complementary base pairs
hydrogen
in mitosis, where do highly condensed chromosomes, consisting of two sister chromatids, join
centromere
what is the purpose of PCR
amplify specific fragments of DNA in vitro
kinetochores
sites of spindle fiber attachment to chromosomes
DNA in euk cells is wrapped around histones to form ___________
nucleosomes
telomeres
chromosome end structures required for complete replication of linear chromosomes
nucleic acid hybridization
formation of a double-stranded molecule from the interaction of two complementary sequence single-stranded molecules
what is the mechanism that DNA polymerases use to synthesize DNA
add dNTPs to a hydroxyl group on the growing polynucleotide chain hydrogen-bonded to a strand of DNA
proliferating cell nuclear antigen (PCNA)
a sliding clamp protein
how far apart are nucleosomes spaced
200 bp apart
homology directed repair
-used to repair double stranded breaks
-greater accuracy compared to non-homologous end joining
-mediated recombination of homologous chromosomes during meiosis
difference between a phosphodiester and ester bond
phosphodiester: the whole bond between the 5’ and 3’ of two nucleotides
ester: bond between the 3’ C and oxygen of phosphate; also between 5’ C and oxygen of phosphate
polymerases that synthesize leading and lagging strands in eukaryotes
-leading: DNA polymerase epsilon
-lagging: DNA polymerase sigma
