Exam 2: Chapters 5-9 Flashcards
Linkage, recombination, DNA structure, replication
What is special about the female X chromosomes?
they silence an X chromosome
- inactivate during embryonic development “Barr body”
- Barr body: so condensed, inaccessible for making proteins
- ie calico cat: orange or black on X chromosomes, silencing not affect homozygous genes, heterozygous leads to calico (some orange silenced, some black silenced)
What is linkage? What does it affect?
- Genes within 50 map units of each other have a decreased likelihood of being separated by recombination
- Affects equal percentage of gamete formation
- Affects 9:3:3:1 ratio
What are the types of gametes?
“parental types” gene combination from a certain parent
“recombinant types” gene combination because of recombination
- percentage decreases with linkage
What are syntenic genes?
genes on the same chromosomes
What is the “+” notation?
normal or wild type
What is a testcross parent?
a parent with either both recessive or both mutated alleles
How does gene location affect type of chromosome?
As genes get closer % parental types increases, % of recombinant type decreases.
- if gametes all have equal change then the genes are greater than 50 map units apart
What happened in 1909 concerning crossing over?
observed chiasmata at chromosomes during prophase of meiosis I (microscope help progression of crossing over)
How did Thomas Hunt Morgan influence crossing-over?
suggest chiasmata were sites of chromosome breakage and exchange
What happened in 1931 concerning crossing over?
H. Crighton & B. McClintock (corn) & C. Stern: direct evidence that genetic recombination depends on reciprocal exchange of chromosomes
- physical markers used identify specific chromosomes
- genetic markers used as points of reference for recombination
What did H. Sturtevant propose?
Recombination frequencies (RF) can be used as a measure physical distance between two linked genes
1 % RF = 1 map unit (m.u.) = 1 centiMorgan (cM)
What is important about recombination frequency percentage?
Never exceeds 50%
- RF Unlinked genes = 50% because of independent assortment
- RF linked genes cannot exceed 50% know dealing with linked
– no crossovers = parental types
– single & double crossovers –> 1:1 parental:recombinant types on average
Parentals 50-100%
Recombinants 0-50%
What is important about mapping genes?
To determine correct gene order
- Left-right orientation is arbitrary
- best: summing many small intervening distances
(start with homozygous parent because then no crossing over)
What are limitations of two point crosses?
- Difficult to determine gene order if two genes are close together
- Actual distances between genes do not always add up
- Pairwise crosses are time and labor consuming
What is the benefit of three point crosses? What are the three things to consider in analysis?
Faster & more accurate mapping
- double cross between genes: should see gene from parent gene from other parent gene from first parent
Analysis:
- most common: parental configuration
- least common: double crossovers
- double crossover shows which gene in middle
What is the use of the Chi square test? What can it do?
It pinpoints the probability that ratios are evidence of linkage
- “goodness of fit” between observed and expected values
- can reject the null but not prove a hypothesis
(Cut off 0.05) > 0.05 no difference, no linkage, < 0.05 difference, linkage
How is the Null Hypothesis used in the Chi square test?
- Null hypothesis: observed values are no different from expected values
- linkage studies: null hypothesis = no linkage
- then expect 1:1:1:1 in F2 progeny
What information is needed for the chi-square test? Then what?
breeding experiment:
- total # progeny
- # progeny classes
- # offspring observed in each class
–> calculate number of offspring expected in each class if no linkage
What is the equation for the chi-square test?
x^2 = (# observed - # expected)^2/# expected
- Consider: degrees of freedom (df) = N-1
How is p value determined for chi-square test?
Chi-square value and df
- probability that deviation from expected numbers had occurred by chance
How do we know the genetic function of DNA? And where does this lead us? What is it dependent on?
By knowing the molecular structure
Leads to an increased understanding of the biochemical process
Depends on: proteins to read the information as DNA itself is chemically inert
What are phosphodiester bonds?
Covalent bonds between adjacent nucleotides (in the backbone) that are fairly strong between 3’ (growing end) and 5’ (toward Phosphate)
What was Griffith’s contribution to DNA?
- Bacterial transformation experiments showed DNA as the substance of genes
- Streptococcus pneumoniae: S - virulent, R - nonvirulent,
- S dead: mouse alive, S dead & R alive: mouse dead
(1928)
What were Avery, MacLeod, and McCarty’s contribution to DNA?
Identified DNA as the transforming principle
- Protease, RNase, DNase, ultracentrifugation (fats),
- Only DNase meant no transformation of S to R cells (no DNA)
What were R. Franklin and M. Wilkins contribution to DNA?
XR diffraction
- helical structure w/ 20 A diameter
- Space between repeating units: 3.4 A
- Complete turn every 34 A (10 nucleotides)
What was E. Chargaff’s contribution to DNA? What is the reason for this?
A:T ratio is 1:1 and G:C ratio is 1:1
A:T has two H bonds
G:C has three H bonds
What are the two groups of nitrogenous bases?
Purines:
- Adenine
- Guanine
Pyrimidines:
- Cytosine
- Uracil
- Thymine
How do the DNA strands relate to one another? What are the kinds of spirals/backbones?
- Antiparallel
- B-form DNA: right-handed spiral & smooth backbone (MOST DNA)
- Z-form DNA: left-handed & irregular backbone
What are the ways DNA are read?
- most of the time from unwound DNA
- some information from double-stranded DNA (DNA-binding proteins regulate gene expression)
What are the three chemical differences between RNA and DNA?
- Sugar (deoxyribose/ribose)
- Nitrogenous Base: thymine & uracil
- Strand: DNA - Double, RNA - single
What are the three models of DNA replication?
1) Semiconservative: each strand is a template for a new strand by insertion of complementary base pair producing two identical daughter double helices (Watson-Crick)
2) Conservative: parental double helix remains intact & daughter helices newly synthesized
3) Dispersive: both strands of daughter helices contain original and newly synthesized DNA
What are the three requirements for DNA polymerase action?
1) Four dinucleotide triphosphates (for incorporation into chain and energy)
2) Single-stranded template (unwound by other proteins)
3) Primer with exposed 3’ hydroxyl
How are strands read verses synthesized?
Read 3’-5’
Synthesized 5’-3’
What are the four aspects of the mechanisms of DNA replication in initiation?
1) initiator protein: binds to origin of replication (replication will occur in two directions)
2) Helicase: unwinds the helix (regions are worked on in replication bubble- proceeds in two directions)
3) Single-strand binding proteins: keep the DNA helix open (get in way of bases so do not snap back together)
4) Primase: synthesizes RNA primer
What is the nature of the binding primers?
They are complementary and antiparallel to each template strand
What are the six aspects of the mechanism of DNA replication: Elongation?
Elongation: the correct nucleotide sequence is copied from template strand to newly synthesized strand of DNA
1) DNA polymerase III: catalyzes phosphodiester bond formation between adjacent nucleotide (polymerization)
2) Leading strand: continuous synthesis
3) Lagging strand: discontinuous synthesis
4) Okazaki fragment: short DNA fragments on lagging strand
5) DNA polymerase I: replaces RNA primer with DNA sequence
6) DNA ligase: covalently joins successive Okazaki fragments (phosphodiester bonds)
What is important about a circular bacterial chromosome and DNA replication?
Since replication proceeds in two directions from a single Origin of replication
- Unwinding creates supercoiled DNA
What is the function of DNA topoisomerase?
relax supercoils by cutting sugar phosphate backbone bonds strands of DNA
- unwound broken strands sealed by ligase
(synthesis continues bidirectionally until replication forks meet)
What are the recombinations at the DNA level?
(new combinations of alleles created by two types of events in meiosis)
- Independent assortment:
- Crossing over:
What is independent assortment?
each pair homologous chromosomes segregated freely from the other (new allele combinations of unliked genes)
What is crossing over?
two homologous chromosomes exchange portions of DNA
(new allele combinations for genes, ensures proper chromosome segregation during meiosis)
What are mutations in DNA replication and what are the two categories and the types?
Changes in replicated DNA base sequence
- Forward mutation: wild-type allele to different allele
- Reverse mutation: mutant allele back to wild-type
Types: substitution, deletion, insertion
What is substitution and what are the two types?
Mutation by replacement of a base by another base
- Transition: purine by purine or pyrimidine by pyrimidine
- Transversion: purine by pyrimidine (& vice versa)
What is deletion?
Mutation by block of one or more base pairs lost from DNA
What is insertion?
Mutation by block of one or more base pairs being added to DNA
What are the two mutations rates?
1) Parents to children
- rate: 1 x 10^-8
- each child 60 mutations
- most don’t influence phenotypes
2) In sperm
- rate: 2-4 x 10^-8
- Because continual mitosis
- Older fathers means more mutations
What did Luria-Delbruck do in 1943?
Fluctuation test
Purpose: origin of bacterial resistance to phage infection
Procedure: Infect wild-type bacteria with phage: use velvet to replicate bacterial colonies
Result: Majority of cells die but some grow and divide
Questions:
- Cells undergo biochemical alteration?
- Cells have heritable mutations for resistance?
- Cell mutations by chance or response to phage?
Conclusion: by chance
What are the four natural processes that damage DNA?
- Depurination: mutation 75% of time, 1000/hr in every cell, Guanine no longer function as Guanine
- Deamination of Cytosine: changed to U lead to A-T after replication
- X-Rays: break phosphate backbone (deletion)
- UV light: thymine dimers (adjacent thymines form abnormal covalent bonds)
What are trinucleotide repeats?
- repeats of three nucleotide
- Unstable trinucleotide repeats cause 20 human disease genes: Fragile X syndrome, Huntington disease
- Children of carriers may have expanded number of repeats
25 ok, 50 not good, 200 bad
What are the two DNA repair mechanisms?
Accurate repair systems:
Error-prone repair systems
What is proofreading?
What DNA polymerase does to recognize and excise mismatches in DNA (rare incorrect information of bases by DNA polymerase)
What does DNA glycosylases do? What comes after?
Fixes damaged DNA post replication (usually chemical change in base ie deamination of C)
- endonuclease cleans up surrounding bases
- DNA polymerase patches the hole
- DNA ligase reattaches backbone
How does is DNA damage from UV light repaired?
- UvrA - UvrV complex: scans for double helix distortions
- UvrB - UvrC complex: nicks damaged DNA
- DNA polymerase fills the gap
What directionality do polypeptides have?
N terminus (amino acid side) and C terminus (carboxyl side)
What causes sickles cell disease?
Gly for Val at 6th amino acid affecting 3D structure of hemoglobin B chain
- Abnormal protein aggregates causes sickle shape of RBC
How is sickle-cell anemia pleiotropic?
(Gene functioning in several pathways)
A) Rapid destruction of sickle cells –> anemia –> fatigue, heart damage, overactivity of bone marrow
B) Clumping of cells, interference with circulation –> local failures in blood supply –> Damage to heart, kidney, muscle/joints, brain, lung, GI
C) Accumulation of RBC in spleen –> enlargement & damage to spleen
What are the two steps of gene expression?
- Transcription: RNA polymerase transcribes DNA to produce an RNA transcript
- Translation: Ribosomes translate the mRNA sequence to synthesize a polypeptide (follows the genetic code)
What are the names of the strands involved in gene expression?
- Template strand: of DNA is complementary to mRNA
- RNA-like strand: of DNA has the same polarity and sequence of mRNA
What is the DNA, mRNA, and polypeptide directionality?
DNA read 3’ to 5’
mRNA is transcribed 5’ to 3’
polypeptide: N to C terminus
What are the seven aspects of the genetic code?
1) It has triplet codons
2) Codons are non-overlapping
3) Three nonsense codons don’t encode an amino acid; UAA, UAG, and UGA
4) It is unambiguous
5) Reading frame is established from fixed starting point - AUG (translation initiation)
6) mRNAs and polypeptides have corresponding polarities
7) Mutations: frameshift, missense, and nonsense
What are the aspects of the accurate repair system for DNA?
- Base alterations reversal (excision repair)
- Bases/nucleotides damage homology-dependent repair
- Replication error corrections
- Double-strand break repair
What are the aspects of the error-prone repair systems?
- SOS systems
- Microhomology-mediated end-joining (MMEJ)
What is in vitro? Why does this work?
Translational systems from one organisms can use mRNA from another organism to generate protein
- works because most cells same basic genetic code
- perfect correspondence between codons & amino acids
Exceptions: ciliates, mitochondria, some prokaryotes
What is the transcription process in prokaryotes?
- Catalyzed: RNA polymerase which is directed by…
- Promoters: DNA sequence signal start transcription
- Function: adds nucleosides 5’-3’ forming phosphodiester bonds using ribonucleotide triphosphates (A/C/G/UTP), energy from hydrolysis of NTP bonds
- Terminators: sequence signal stop transcription
What is the initiation step of transcription in prokaryotes?
RNA polymerase binds to promoter sequence
- Sigma factor binds to RNA polymerase making holoenzyme
- Region of DNA unwound to form promoter complex
- Phosphodiester bonds formed between first two nucleotides
What is the elongation step of transcription in prokaryotes?
- Sigma factor separates from RNA polymerase leaving core enzyme
- Core RNA polymerase loses affinity for promoter moves 3’-5’ on template strand
- nucleotides added to 3’ of mRNA
What is the termination phase of transcription in prokaryotes?
Signal end
- Extrinsic kind: require rho factor
- Intrinsic kind: no additional factors required
- Usually form hairpin loops (intramolecular H-bonding)
What is the difference between prokaryotic and eukaryotic transcription?
Eukaryotes often have enhancers
- may be thousands of base pairs away from promoter
- Required for efficient transcription
What is the difference between the product of transcription for eukaryotic or prokaryotic organisms?
(Protein end)
- Pro: primary transcript = mRNA
- Euk: primary transcript = processed to make mRNA: 5’ methylated cap, 3’ poly-A tail, introns removed by RNA splicing
What is a 5’ methylated cap?
A backward G added to the first nucleotide of a primary transcript
What is a 3’ poly A tail?
Adenosines added onto the 3’ end by Poly-A polymerase
What are Exons and Introns?
Exons: DNA sequence found in gene & mature mRNA (expressed)
Introns: DNA sequence found in gene but not mRNA (intervening regions)
What is RNA splicing? Creates, catalyst, alternatives
Two sequential cuts to remove an intron
- creates Lariat (loop)
- catalyst: spliceosome
- alternative splicing: produces different mRNAs from same primary transcript (ie intron in membrane-bound, exon in secreted Ig)
What are the three levels of tRNA structure?
Primary: nucleotide sequence
Secondary: (cloverleaf) b/c short complementary sequences
Tertiary: (L shape)b/c 3D folding
What connects tRNA and amino acids?
Aminoacyl-tRNA synthetase - recognizing specific amino acid and structural features of corresponding tRNA
What is the tRNA Wobble effect?
Some tRNAs recognize more than one codon
(wobble position: last position on the right, not as strong: more flexible)
What are the composition of Prokaryotic and Eukaryotic ribosomes?
- Pro: 70S, 50S & 30S (subunits)
- Euk: 80S, 60S & 40S (subunits)
Subunits are composed of RNA & Protein
What are the different functions of the ribosome subunits?
- Small subunit: binds to mRNA
- Large subunit: has peptidyl transferase activity
– Three distinct tRNA binding areas: Aminoacyl, Peptidyl, and Exit sites
What is the mechanism of translation?
- Initiation stage: start codon, AUG 5’ end (bacteria: initiator tRNA has formylated Met: fMet)
- Elongation stage: aa added to growing polypeptide, 5’-3’ direction along mRNA
- Termination stage: polypeptide synthesis stops at 3’ end (recognize nonsense codons, synthesis halted by release factors, release of ribosomes, polypeptides, and mRNA)
What is the initiation stage of translation in prokaryotes?
Ribosome binds to Shine-Dalgarno box (AGGAGG) and in AUG in 3 subsequential steps:
1) Small ribosomal subunit binds
2) fMet-tRNA positioned in P site
3) Large subunit binds
What is the initiation stage of translation in eukaryotes? (protein end)
Small ribosomal subunit binds to 5’ methylated cap then migrates to first AUG codon, initiator tRNA carries Met
What is the elongation phase of translation? (protein end)
- add amino acids to C-terminus of polypeptide
- Charged tRNAs into A site by elongation factors
What is the termination phase of translation? (protein end)
- stop codons do not have normal tRNAs anticodons
- Release factors bind to stop codons: release ribosomal subunit, mRNA, & polypeptide
What do polyribosomes do?
Several ribosomes translating same mRNA: simultaneous synthesis of many copies of polypeptide from single mRNA
(typical action)
What is enzymatic cleavage? What are the three types?
A form of post translational processing
1) N-terminal Met removal: fMet
2) Polyprotein processes (polyprotein cleaved into multiple smaller polypeptides) producing more protein by creating a long polypeptide (more efficient than starting and stopping) then cut into smaller polypeptides
3) Zymogen activation - inactive form of dangerous (digestive) protein has prosegment, cleaved to activate protein
Prokaryotic vs eukaryotic cells what is the big difference in replication because of the cell structure?
- Prokaryotes: do not have a nucleus so transcription and translation can happen simultaneously (leads to greater growth rate)
- Eukaryotes: Transcription in nucleus, translation in cytoplasm sequentially (takes time)
What are additional ways to have mutations? (not in coding sequence)
- Changes in enhancer region or promotor regions not part of DNA code (exons)
- splicing not only/all intron
What was the Human Genome Project? What has happened since? (Ch 9)
Completed in 2003 - an accurate sequence of the human genome
- now faster
- Thousands of species have been sequenced
What are the general ideas behind genome sequencing? (Ch 9)
- Fragmenting the genome (need some overlap)
- Cloning DNA fragments
- Sequencing DNA fragments
- Reconstructing the genome sequence from fragments
- Analyzing the information found in genomes
(info to grow, maintain, reproduce human)
(working on technology to read DNA strand in entirety)
Why were we able to fragment the DNA?
Where does it come from and what is the purpose? (Ch 9)
Discovery of restriction enzymes “molecular scissors” that recognize specific sequence of bases
- cuts both strands of sugar-phosphate backbones
- generates restriction fragments (by digestion of DNA)
- hundreds now available
(straight or sticky ends)
- defense mechanism in bacteria against virus (chop up invading DNA)
What are the characteristics of recognition sites for restriction enzymes? (Ch 9)
Usually 4-8 bp
- often palindromic: base sequences of each strand are identical when read 5’ - 3’
- Each enzyme cuts at same place relative to its specific recognition sequence
What are the type of ends restriction enzymes produce? (Ch 9)
- Blunt ends: cuts are straight through both DNA strands at the line of symmetry
- Sticky ends: cuts are displaced equally on either side of the line of symmetry (5’ or 3’ overhangs)
- Will recombine after cut unless something gets in way
How do you calculate frequency of cutting? (Ch 9)
*
- Ave fragment length is 4^n
- n = number of bases in the recognition site
- 4-base recognition site occurs every 4^4 bp
– ave restriction fragment size = 256 bp
– 3 billion bp grnome/256 = 12 million fragments - 6-base recognition site occurs every 4^6 bp
– ave restriction fragment size is 4100 bp
– 3 billion bp genome/4100 = 700,000 fragments - 8-base recognition site occurs every 4^8
– ave restriction fragment size is 65,536 bp
– 3 billion bp genome/65,536 = 45,776 fragments
What is the purpose of gel electrophoresis? (Ch 9)
- separate DNA by size
- linear DNA: distance through gel depends on size
- determine size of fragments by comparison with markers of known size (ladder)
What is the purpose of molecular cloning? (Ch 9)
- allow for focusing on certain information (get around information overload)
- Definition: means to purify a specific DNA fragment away from all other fragments and make many identical copies of the fragment
What are the two steps of molecular cloning? (Ch 9)
- Insert DNA fragments into cloning vectors to make a recombinant DNA molecule
- Transport recombinant DNA into living cell to be copied
What are the main features of plasmid vectors (molecular cloning step 1)? (Ch 9)
- origin of replication (certain gene will be copied)
- known restriction site for cloning insert DNA
- selectable marker (ie antibiotic resistance) to recover plasmids
How are recombinant DNA molecules made with plasmid vectors? (Ch 9)
- Cut (digestion) plasmid and open up using restriction enzyme (results in complementary sticky ends
- insert gene of interest (plasmid may or may not take up gene)
- ligase seals phosphodiester backbones between vector and insert
What is step 2 of molecular cloning? (Ch 9)
Host cells take up and amplify recombinant DNA
- Transformation: the process by which a cell or organism takes up foreign DNA (via shock- daze w/o death)
- success ~ 0.1%
- Selection: only cells with plasmid grow in media with ampicillin
- cell multiplies with recombinant plasmid
What is the challenge of reading DNA without knowing where the gene is? (Ch 9)
- there are six potential reading frames due to stop codons and either strand can be template strand. LOOKING FOR ORF
- open reading-frame (ORF) = reading-frame uninterrupted by stop codons: indicates this is the reading frame to focus on
What is the arrangement of genes in the genome?
What is the exome and what is the percentage of the DNA is considered the exome? (Ch 9)
- 25,000 genes (humans)
- part corresponding to exons exome: DNA actually codes for proteins
- Most genome is non-coding
– Exome = ~ 2%
– Introns
– Centromeres, telomers, transposable elements
– Simple repeating sequences
What are the regions of the DNA that contain genes? (2 types) (Ch 9)
- Gene-rich regions: chromosomal regions that have many more genes than expected from average gene density over entire genome
- ie: class II region of major histocompatibility complex
- Gene deserts: Regions of >1 Mb that have no identifiable genes
- 3% human genome comprised of gene deserts
- Exist because hard to identify?
Biological significance of regions is unknown
