Final Exam Flashcards
What did Mendel study by looking at peas compared to yeasts
Diploid organisms, whereas yeast were haploid
What are mendel’s two laws?
Equal transmission: same number of genes are transmitted
Independent assortment: segregation of one gene is independent of the segregation of the other gene
What are alleles?
The different forms of one genes a diploid organism can have
When looking at inheritance of genes, we are looking at what type of cell?
We are looking at reproductive cells (gametes)
Each gamete of has ____ chance to have either member of a gene pair
Equal
When do gametes unite at random?
During fertilization
What is the pattern of inheritance when looking at one gene with two alleles
3:1 or 1:1
What is a pedigree analysis?
When looking at a family tree and the pattern of inheritance
What progeny do we see when crossing two homozygote, one dominant, one recessive?
All heterozygous
What progeny do we see when crossing two hétérozygote?
3:1 phenotype
1:2:1 genotype
1/4 homo dominant, 1/2 hétérozygote, 1/4 homo recessive
What progeny do you get with crossing hétérozygote with homo recessive?
1:1 progeny
This is called a test cross?
What does a test cross tell you?
If you progeny has half one phenotype and half the phenotype of homo recessive, then the cross with done with a heterozygote
If they all give the other phenotype, you know the cross was done with a homozygote dominant individual
how do you find the probability of a progeny with 2 genes that are unlinked ?
You multiply the probability of each gene genotype
What are the expected ratio for crossing two heterozygote genes with two heterozygote genes?
9:3:3:1
What is the progeny for one homo and one hetero?
1:1:1:1
How do you count the number of possible combination of gametes?
Multiply the # of gametes of one individual of different genes, with the # of gametes of other individual of different genes
What is the difference between eukaryotic and prokaryotic chromosomes?
Prokaryotic have circular chromosomes, eukaryotic have linear
What is a karyotype?
The complete set of chromosomes of a organism. Gives structure and number of chromosomes
Humans have how many pairs of chromosomes?
23 - 22 autosomal and 1 pair that is sex linked
What are the different types of centromeres?
Telocentric
Afrocentric
Meta centric
What results from mitosis?
Two daughter cells that have the same diploid genetic material
What results from meiosis?
2 cell divisions that results in 4 haploid gametes (reproductive cells that will fuse)
What occurs in the following stages of mitosis?
Prophase: chromosome condense, nuclear membrane breaks
Métaphase: nuclear spindle becomes prominent, chromosomes move at equatorial plane
Anaphase: pairs of sister chromatids separate
Telophase: nuclear membrane reforms around each daughter cell, by end, cytoplasm has been divided
In meiosis or mitosis can chromosomes cross over?
In meiosis, because of the way they align
What occurs in the following phases of meiosis?
Prophase I: chromosomes condense
Métaphase I: nuclear membrane disappears, take position at equatorial plane.
Anaphase I: chromosomes start moving to poles
Telophase I: no nuclear membrane forms, proceeds to prophase II
Prophase II: -
Métaphase II: chromosomes arrange themselves on equatorial plane, chromatids partly dissociates
Anaphase II: centromeres split, chromatids are pulled to opposite poles
Telophase II: nuclei reforms around chromosomes at pole
In what phase of meiosis do the chromosomes become fully condensed?
Prophase i
At what phase of meiosis do the chromosomes align at the equatorial plane?
Métaphase I and Métaphase II
At what phase of meiosis do the centromeres divide vs. Do not divide?
Metaphase I vs. Metaphase II
At what phase of meiosis do the chromosomes start moving to poles?
In Anaphase I Anaphase II (chromatids get pulled apart)
At what phase does the nuclei disappear and reforms?
Métaphase disappears
Telophase reappears
What are some differences between mitosis and meiosis?
Meiosis:
- results in 4 haploid cells
- One synthesis, two divisions
- crossing over can occurs at I
- centromeres do not divide in Anaphase I, but yes in Anaphase II
Mitosis:
- one cell division per synthesis
- results in 2 diploid daughter cells
- no crossing over
- centromeres divide at anaphase
Name the characteristics of a model organism?
- fast reproductive cycle
- give rise to many offsprings
- small size
- small genome
- inexpensive- important biological property
What patterns of inheritance do we see with sex-linked genes?
They do not follow Mendelian rations
Disproportionate frequencies along male and females
Mothers tend to pass to sons, fathers to daughters
If a gene is sex-linked and the mother is heterozygous, what percentage of sons will have the trait? If she is homozygous or trait?
Half of the sons
All of the sons
If the father has an x dominant disease, what percentage of daughters will have it?
All daugthers will have it and show phenotype if the disease is dominant
In drosophila, if specie has XO, is it male or female? XXY
What is it based on?
Male
Female
Based on the proportion of XX to autosomal set of chromosomes
What kind of chromosomes do organelles (mitochondria and chloroplasts) have, how inherited are they?
They have circular chromosomes and is inherited from the maternal gametes. Do not follow mendel’s laws
What are gene maps used for? What can it be used for?
To calculate the distance between genes on the chromosomes by measuring the recombination.
Can be used to:
- isolate and clone genes
- clearly distinguish diff genes that affect the same trait
- Link traits to markers
What happens to inheritance if two genes are linked?
They will not abort independently in the gametes
When will two link genes assort independently?
If there are recombination
how do crossovers occur?
By double stranded break
More likely when genes are further apart - frequency is a measure of the distance between genes
How do you get the % recombinant?
You test cross the heterozygous F1 progeny with homozygous recessive
How do you measure % recombinant?
Add up the ones that recombined and divide by total progeny
What is the 3-point cross?
Used to map the distance between 3 genes and their order
What are the most frequent and less frequent in a 3 point cross?
Parental types are most frequent, double cross over are the least frequent
How do you find distance between two genes?
Calculate their % recombination (add up and divide)
What is interference and what is the formula?
Probability of double crossover is the product of the independent event
1- (observed DBC/Expected DBC)
What is the maximum distance that can be mapped between two genes ad why?
50cm, means it happens 100% of the time - gametes get 50.
More than that the genes are too far apart that they will assort independently.
What is the chi square test for and what is the formula?
Used to decide whether data fits a certain ration (ie: these genes are linked or not linked) Sum of (o-E)2/E
Should probability of this occurrence be lower than 5%, accept null hypothesis, should it be higher, deviation is probably due to chance, therefore reject null hypothesis
What are characteristics of bacteria?
Cells do not have nucleus or organelles Have circular chromosomes Typically haploid Have genes and phenotypes Have cell division, not meiosis We mostly look at e-coli
Why are bacteria good to study?
Large population
Rate mutations can be selected
Fast regeneration time
Because haploid, genotype=phenotype
What is the formula for find the original concentration of colonies
Colonies/total serial dilution
What are the 3 major classes of genes for bacterial genetics?
Résistance to bacteria
Requirement of a nutrient to grow
Ability to grow on one particular compound as sole carbon
What is a phototroph ?
Can grow on minimal medium
What is an autotrophs?
Requires a particular nutrient in order to grow
What is bacterial matin?
Under the control of the fertility factor (plasmid)
Describe bacterial matin conjugation
The fertility factor (plasmid) is a freely replicating circular piece of DNA (1/50 of bacterial DNA length)
F factor can be transferred to F- recipient.
If f factor integrate into the bacterial chromosome, becomes HFr (high frequency recombination)
HFr, can transfer copy of chrosomes to recipient F-. You have exogenote and endogenote.
In what sequence does the HFr incorporate into recipient bacteria?
Begin at the section closest to HFr, HFr is last to enter the cell
how is the HFr integrated?
By double recombination
What is the F-
Bacterial cell that can receive DNA (F factor) but not donate DNA
What is F+
bacteria that has the fertility factor as a plasmid, it can donate it readily
Only small portion of F+ inter grate it in chromosomes to become HFr
What is Hfr?
Bacteria like cell that has the fertility factor integrated in the chrosome. Can transfer to F- by starting at place adjacent to Hfr
What is F’?
Have episode that contains part of the bacterial chromosome
What is conjugation?
Transfer of DNA from one F+ to F- bacteria
What is a virus?
Small particles made up of DNA/RNA and proteins
Dépend on cellular machine of host
Called bacteriophages
What is transduction?
Phage infects cell with its DNA, which then gets replicated and degrades. Phage retakes its DNA and goes to infect another bacterial cell which can then recombine in the chromosomal DNA
Sometimes bacterial chrosome gets picked up and get reintegrated in new bacterial cell
What is transformation?
DNA can be introduced directly into cell in lab, heat shock and electroporation. They are transferred in segments
What are the different methods of gene mapping?
- timed mating (timing of passage becomes a measure of distance between genes)
- mapping by recombination frequency
- mapping by transformation
- mapping by phage transduction (measuring rate of co-transduction max 2 minutes)
What are two important generalized concepts?
Genes generally encode proteins
Proteins are generally enzymes
What are enzyme composed of?
Proteins
What catalysés a substrate?
An enzyme
What was the beagle and Tatum hypothesis?
Biosynthetic pathway where mutations occured in genes encoding the pathway
Name the different types of inheritance pattern (all of them)
- one gene inheritance with dominance
- 2 gene inheritance with dominance
- incomplete dominance
- co-dominance
- recessive lethal alleles
- 3 or more alleles of a single gene
- multiple genes affecting trait
- epistasis
- duplicated genes
What happens when an enzyme has reduced amount?
Can become a dominant mutation because:
it can cause it be non functional:
- if part of a complex
- if an activity was released from normal constraint
OR
It could mean partial functionality (incomplete dominance)
What inheritance should we expect for incomplete dominance?
1:2:1, where heterozygous is a mix between both
What pattern do we expect fo co-dominance?
1:2:1 (same as regular Mendelian ratios)
What inheritance doo we expect for recessive lethal allele?
2:1
They never breed true, the homozygous recessive individual will always die, therefore you will never have a partent that is true
Inheritance of Multiple alleles?
Typical 1:2:1 or 1:1
Multiple genes affecting the same trait?
Should expect the 2 gene inheritance pattern 9:3:3:1
Inheritance of duplicated genes?
1:15
Epistasis, what is it and inheritance?
When alleles of one gene, masks the effects of alleles of another gene
9:4:3
What is a complementation test?
To test whether two mutations are in the same gene or different gene.
Should the progeny be normal, both parent complemented and mutations are on different genes (biosynthethic pathway is an example)
What is a protein?
Long structure of polypeptide chains, composed of Amino acids
There are 20 AA
Each protein has a unique AA sequence
What s a merodiploid?
A partial diploid organism, where a chromosomes fragment was introduced by conjugation, transformation or transduction
What are the four basic nucleotides and what are they a base of?
Adenine
Guanine
Cytosine
Thymine
They are nitrogen bases
What are the different components of DNA?
Phosphate base attached to deoxyribose sugar, attached to nitrogen base, which attaches to to another nitrogen base with hydrogen bonds
Who discovered the A=T and G=C rule?
Chargaff
What did Griffith’s study show? And who else helped in confirming and how?
Showed the existence of transformation between bacteria with the virus in a mouse
Mcleod and Mcarthy showed that it was DNA
Hershey and chase confirmed that it was DNA with their isotopes
What is the Experiment of Hershey and chase?
Use isotopes of phosphorous and Sulfur (DNA only has phosphorous, not sulfur)
32P and 35S, which was found in phage and bacterial cell (only 32P entered the bacteria cell
What did Watson and crick propose?
Worked with an X-ray to propose the semi-conservative model of replication
Who proved the semi-conservative model and how?
Meselson and stahl
Used heavier isotope of Nitrogen
15N
First generation showed a mix of both (middle density)
Second generation showed 2 middle bands and 2 light 14N bands
What is DNA composed of?
C,N,O,P,H
What are DNA strands held by?
Hydrogen bonds
Which nucleotide pairing is held by 3H bonds instead of 2?
G-C
Which part of the DNA is held by covalent bonds?
Deoxyribose (sugar) and phosphodiester
What is the directionality of DNA?
5’ to 3’
How can two strands of DNA be separated?
By heat and enzymes
What is DNA replication? And when does it occur in the life cycle of a cell?
It occurs in the S phase
One DNA double helix gives rise to two double helix strands. Each strand is replicated
What is needed for replication?
- DNA template
- Deoxyribonucleotide triphosphate (acts as substrate to add nucleotide)
- Multiple enzymes:
≥topoisomerase
≥Helicase
≥Primase
≥DNA pol - Primer (DNA or RNA)
Which enzyme is required for unwinding of the double strand?
Topoisomerase
Which enzyme breaks the hydrogen bonds
Helicase
What does RNA primate do?
Builds the RNA primer
What does DNA polymerase do?
- It is the enzyme that synthesizes the new DNA
- Must use a DNA strand as a template
- Moves in the 3’ to 5’ direction but adding nucleotide in the 5’ to 3’ direction of the new strand
- Pol III is the principle DNA replication enzyme
In what direction does the new strand elongate?
What direction does the DNA pol move?
Elongate in the 5’ to 3’ direction
DNA pol moves in the 3’ to 5’ direction
What is exonuclease activity and what does it provide? Which enzyme allows that?
- Cleaving one nucleotide at a time and provide proofreading and editing
- Pol I and III
What are the two different strands formed during DNA replication and how do they differ?
Leading strands:
- Pol III moves in the 3’ to 5’ towards the fork
- pol III synthesizes in a continuous manner
- only need one primer
Lagging strands:
- pol III moves int eh 3’ to 5’ away from the fork
- synthesizes in short fragments using RNA primers ( made by Primase)
- create okosaki fragment
- pol I removes primer
- Ligage rejoins the new segment
- need multiple primers
What is an Okazaki fragment?
Fragment of newly synthesized DNA with the Primer
What enzyme removes the primer?
DNA pol I
What enzyme connects the selected strands?
DNA ligase
What direction does the DNA Pol I move?
Always in the 3’ to 5’
What direction do the new DNA synthesize?
Always int he 5’ to 3’
how does DNA pol III work at the fork?
Uses primer to synthesize new DNA
Will detach and go the the fork once again
Give the details of DNA replication (full steps)
- DNA double helix is opened by topoisomerase
- Helicase enzyme breaks the hydrogen bonds
For lagging strand: - Primase synthesizes RNA primer (for the lagging strand)
- primer is the starting point for DNA pol III
- Okazaki fragment is created
- Pol I removes primer
- Ligase joins the new segments
- DNA pol III goes back towards the fork where it starts again with primer
For leading strand:
- DNA pol III synthesizes new strand from primer
- moves towards the fork
What is the role of DNA pol i?
Removes the primer and fills gap by synthesis of short fragments
What is the role of DNA pol II
DNA repair, replacement of new DNA synthesized that went wrong
Which enzyme is responsible for joints segments?
DNA ligase
Which enzyme is responsible for synthesis on most of the DNA
DNA pol I
Which enzyme is responsible for unwinding the double helix stand?
Topoisomerase
Which enzyme is responsible for DNA repair and short segment synthesis?
DNA pol II
What it’s he starting point of DNA pol III
RNA primer, which was synthesized by Primase
How does replication occur in e.coli compared to eukaryote?
E.coli:
-single origin of replication and moves in both direction
Eukaryotes:
- multiple orginin of replication
- moves in both direction
What occurs in eukaryotic replication after the primer is removed?
There is a terminal gap that cannot be filled by DNA pol
what fills in the terminal gap in eukaryotic cells? And how?
Telomerase
- it is a reverse transcriptase which has its own internal RNA primers
What are the two enzymes that fills gap, but in different places?
Ligase - to fill within the DNA strand
Telomerase - to fill at the end, the terminal gap
how does telomerase work?
- Has internal RNA that is complementary to 3’ overhang
- add complementary nucleotide, moves along to add again
- then Primase and DNA pol can synthesize the missing parts of strands
What is the AZT drug?
Treatment for HIV used to inhibit reverse transcriptase where DNA pol cannot attach the next nucleotide because it has an N instead of an O
how do you replicate in a test tube?
- Heat (will meld DNA to single strand)
- add primer, DNA pol and nucleotides
- DNA will synthesize in 5’ to 3’
What is Gene transcription?
Synthesis of RNA (ribonucleic acid) from DNA template
What is translation?
MRNA is translated into a protein product
What is the purpose of mRNA?
It carries the information of a gene (from DNA) ito the cytoplasm for translation
What is the difference between RNA and DNA?
RNA
- has a sugar phosphate backbone composed of ribose (OH), not deoxyribose (H)
- single stranded
- has uracil instead of Thymine (methyl group differentiation)
What are the different RNA classes? What do they do?
- ribosomal RNA: part of translation complex
- transfer RNA: brings AA to ribosome
- messenger RNA: protein encoding transcriptase of Gene. Strand that gets translated into a protein.
in what direction is the RNA transcribed?
5’ to 3’
Are genes on the same stands transcribed in the same direction?
Yes
The RNA strand is synthesized from which strand and is complementary or identical to which strand
Complementary to template
But identical to coding strand
What are under rna that were recently discovered?
Small nuclear RNA (snRNA) - involved in splicing
Small nucleolar RNA (snoRNA) - involved in processing
Micro RNA- involved in mRNA degradation and gene reg
Piti interacting (piRNAs) - role in suppression of viruses
In prokaryotes, what enzyme is used to transcribe the DNA stand?
RNA polymerase ( a multimeric complex)
Contains:
- beta, alpha + sigma factor = holoenzyme.
What does the sigma factor do?
Provide specificity in choosing the promoter (where initiation of transcription begins)
What are different promoters in prokaryotes for transcription?
TATA box (-10) Pro now box (-35)
How is termination of transcription controlled in prokaryotes?
- mostly controlled by rho factor
Or - hair pin in RNA transcript
Prokaryotic transcripts tend to be ___, meaning genes are cotranscribed into a single RNA
Polycystronic
What do you call genes that are polycistronic?
An opérons?
What is an operon?
A single transcript that can encode multiple different proteins (ie: lac operon)
What are difference between eukaryotic and prokaryotic transcription of rna?
Prokaryotes:
- RNA pol (one type), multimeric complex with sigma factor
- TATA (-10) ad pro now box )-35)
- termination controlled by rho factor or hairpin loop
- polycistronic
- no 5’ cap
- if there is a poly A tail, message for degradation
Eukaryotes:
- use diff enzymes depending on type of proteins
- promoters are TATAAT (-30) and CAAT (-50)
- termination is capped with 7-methyl guanosine, there if a poly A tail that signals cleavage
- are regulons and not operons, meaning one rna encodes one protein
- have splicing before it is exported to cytosol ask for translation
Name the different enzymes used for protein production(in transcription) in prokaryotes
RNA pol I ≥ for rRNA
RNA pol II ≥ copies of genes
RNA pol III ≥ tRNA
What are the promoters sued in eukaryotic transcription?
TATAAT (-30)
CAT (-50)
What is a polydenylation signal and what does it do?
The addition of a poly A tail
Indicates the need for cleaving about 20bp after the poly A tail
Where is the 7-methylguanosine added?
Ont he 5’ end of the transcribed RNA
Where does the splicing occur? What is it and what enzyme does it?
- Splicing occurs after the transcription
- occurs in the nucleus before it is exported to cytoplasm
- removes introns
- spliceosome does it
What is translation?
When mRNA is translated into synthesis of polypeptide chains
What is a polypeptide chain?
Many Amino acids
Amino acids are triplets of nucleotides called codon
Several codons represent the same AA
What is the start and stop signals?
AUG - start
UAG and UAA and UGA - stop
What are long open reading frames?
Long sequence of nucleotide without interruptions
- usually 80 AA or longer
In prok a gene is approx: 950 base pairs, one gene = several proteins
In euk : 1300 base pairs, 1 gene = several proteins
What are the different structures of a mature mRNA
- A 5’ UTR
- AUG start codon and a stop codon
- an ORF
- 3’ UTR followed by a poly A tail
what are the three phases of translation in prokaryotes?
Initiation
Elongation
Termination
What is the shine-Dalgarmo sequence?
Sequence that is 8 base pairs from start codon that helps recruit the ribosomes
In what direction does the ribosome move on the mRNA stand?
From 5’ to 3’
In what direction do the tRNA moves?
From A site to P site to E site, towards 5 ‘ end
What is the role of tRNA in translation?
TRNA is charged and has anticodon complementary to mRNA. It brings AA to A site, moves to P site and after moves to E site and gets released.
How is tRNA charged? What energy is used?
Amino Acyl tRNA synthetase adds an AA to tRNA, in orde for it to be brought to complex.
ATP is used
what is the wobble hypothesis?
That the 3rd base nucleotide does not always follow Watson-crick rules. It can pair with another nucleotide
In prokaryotes, translation occurs ____ mRNA is fully transcribed
Before
Describe the initiation steps of translation in prokaryotes
1- binding of IF3 to 30S - becomes initiation factor
2- complex binds to mRNA
3- IF2 binds to n-formyl methionine tRNA - which brings it to P site
4- GTP hydrolysis brings dissociation of IF3 and IF2, allows 50S to associate with 30S
5- charged tRNA can now be brought to A- site
What kind of reaction allows dissociation of Initiation factors in initiation phase, what does it allow?
GTP hydrolysis
Allows 50S to associate with complex
Where does IF2 and N-formal methionine tRNA first binds?
The P site
What does the IF3 and 30S complex do?
Allows complex to bind to mRNA and prevents 50s to associate, until ready
What does the 30s subunit consist of?
50 s?
16s rna and 21 proteins
23s , 5s rna and 31 proteins
Describe the elongation steps of translation. What is it?
Addition of charged tRNA in the A site
1- Elongation factor -Tu (EF-Tu) binds to EF-ts which is a GTP exchange factor
2- EF-Tu gets charged with GTP and releases EF-Ts.
3- this complex (EF-Tu- GTP) helps in bringing the charged Amynoacyl tRNA to the A site (facilitates the connection
4- once properly positioned - GTP hydrolysis, release of EF-Tu
5- ribosome catalyzes linkage of new AA to chain of AA
6- Ribosome moves along under control of EF-G bound to GTP (moves tRNA from P to A to E site) with GTP hydrolysis
What is the purpose of GTP, unlike ATP
Serves as directionality, not energy like ATP
When is GTP hydrolyzed in elongation and what does it do?
1- after positioning of aminoacyl-tRNA to A site
- releases GDP and EF-tu
2- also EF-G, which helps move tRNA from A to P to E site
What is the structure of a protein?
AA that re linked together by covalent polypeptide bonds
- added from amino end to carboxylase group
Describe steps of termination in translation
Terminate with a stop codon that appears at the A site
- tRA doe snot bind, instead RF1 (release factor) and RF3 binds and hydrolysis releases tRNA
What are mutations?
Permanent change in DNA sequence
If it changes the function of a protein, it has phenotypic consequences
Wha are the different kinds of mutations
- Point mutations
- missense mutation
- nonsense mutations
- synonymous mutations - Frameshift mutations
- Insertion or deletion of 3 nucleotides
- Large deletion or insertion
Describe point mutation and the different kinds?
One nucleotide is changed:
- Missense: changes condon to a different AA, can be very deleterious
- Nonsense: changes AA to a stop codon, produce trunkcated protein. Very deleterious
- Synonymous mutation: change in nucleotide that does not alter the sequence. No effect on protein
Describe frameshift mutations
Addition or loss of 1 or 2 nucleotide:
- changes all of the AA sequence
- often introduces a stop codon
- very deleterious
Describe insertion of deletion of 3 nucleotide
- if by 3 nucleotide not so bad
- could be correctio by a second mutation to correct the frame
- most common CFTR
Describe large deletions or insertions
- disrupts sequence and inactivates genes
What is an insertion mutations?
Transposable elements that can insert and cause mutations
how can point mutations be compensated?
By a suppressor mutation, which is a mutation int he anticodon of tRNA genes. This will prevent the termination of translation, and wont stop
Name the different factors associated with each phase of translation
Initiation:
IF2, IF3
GTP
Elongation:
EF-tu, EF-Ts, EF-g, Ribosome
Termination:
RF1, RF3
What is an operon and where do we find them?
mRNA that has multiple genes that encode different proteins
- found in prokaryotes
Name the difference operons that we looked at
Catabolic operon: -negative regulation: ≥Lac operon (break down lactose) - positive regulation: ≥ CAP (break down glucose)
Anabolic circuit:
≥trop operon (to make tryptophan)
≥attenuation (to activate the trip operon)
What is a regulon and name the regulon we looked at?
One gene on one mRNA encodes one protein
Catabolic
≥ Galactose regulon (Leloir pathway)
- to break down galactose
What is a merodiploid?
When a copy of the lac operon is carried on the F+ episode, (f’ factors) and make a temporary diploid
What are regulatory proteins?
Controls transcription depending on the bind of these regulatory proteins (activators or repressor)
When looking at operons, we are looking at what?
Genes that have not be transcribed yet
Looking at DNA strand that may or may not get transcribed
Describe the different site of lac operon (regulatory components)
I: synthesizes the repressor
P (promoter site) - where RNA pol binds
O (operator site) - where repressor binds
Z (B-galactose) - cleaves lactose
Y (permease) - facilitates lactose update in cells
A (transacetylase) - not essential
Describe the lac operon and how it works
Repressor which is synthesized by I gene, binds to Operator site.
RNA pol binds to promoter site
If Lactose is present, repressor unbinds from operator site and transcription of the 3 genes follow.
What kind of question with the lac operon?
If the gene B-galactose is being synthesized
What are the different types of mutations of lac operon. Are they Cis or trans acting, recessive or dominant
- Z-,Y-,A-: recessive, cis acting
- I-: synthesizes a repressor that cannot bind to operator site. Cis acting, recessive to I+. Operon i always on and encodes B-galatosidase
- Oc: operator site cannot bind repressor. Cis acting, dominant because you will see B-galactosidase.
- Is: is a super repressor. It will bind to O but will never be removed. Is trans acting and dominant. operon is always Off
- P-: promoter mutation, where RNA pol cannot bind, therefore can never make B-galactosidase. Is cis acting and recessive to p+, but epistasis to Oc and I-
what is the rule of thumb for mutations in lac operon?
P->Oc>Is>I-
What is an example of positive regulatory protein that explains why glucose is always used up before galactose?
CAP (catabolic et activator protein) regulator - when glucose levels are low, ATP is low and AMP is high
Describe the CAP regulation
- when there is low glucose, adenylate cyclase is activated
- transforms ATP to cAMP
- cAMP binds to CAP, which together, can bind to DNA and improve activity of promoter.
What happens when there is high glucose present? No lactose
Low cAMP, repressor binds and no lac genes are transcribed
what happens when there is high glucose but lactose
Repressor doesn’t bind because of lactose but cAMP/CAP didn’t improve promoter = little lac mRA
What happens when there is no glucose and lactose
No glucose, activation of adenylate cyclase, transforms ATP to cAMP, which binds to CAP, which improves activity of promoter
Lactose binds to repressor
LOTS of lac mRNA
What is the trp operon?
Is anabolic - tells when to make or not tryptophan
What does the trp gene encode?
The repressor
Describe the trp operon
- When there is tryptophan, the repressor will bind and prevent transcription
- when there is no tryptophan, the repressor will not bind and there will be transcription to make tryptophan
** presence of tryptophan activates the repressor
What is the difference between trp operon and lac operon
Presence of lactose, removes the repressor
Presence of tryptophan, activates the repressor
What is attenuation?
Fine-tune of the expression of trp operon
Works because of prokaryotes having transcription and translation coupled.
what happens in attenuation when there is plenty of tryp?
- Ribosome passes and reaches termination signal and falls off
- transcription terminators form 3-4
- RNA stops, RNA pol dissociates (not allowing the synthesis of the enzyme that makes more tryp)
what happens when there is not enough tryp?
- ribosome will stall
- 2-3 antiterminator will form
- allows RNA pol to continue along DNA, allowing production of the enzyme to make more TRP
What is the Galactose regulon?
- found i eukaryotes
- positive as something needs to “activate” promoters to allow RNA poly to access
what is the transcription factor in regulating galactose?
GAL 4 - binds as a dimer to Gal80 when there is no galactose.
what happens if galactose is present?
It bind to Gal 80, which detaches from Gal4, le loir pathway is on and allows for metabolism of galactose
How does gal4 act? What does Gal80 do?
Gal 80 is a repressor of gal4 not allowing for the transcription to occur.
Gal 4 binds as a dimer and activates the promoter by allowing RNA poly to access the promoter easier
How id a merodiploid form?
- From conjugation (or transformation, or transduction)
- f+ becomes Hfr
- Hfr introduces into f- and becomes an F-
Partial diploid for that locus
What is gene cloning?
- Refers to the insertion of the DNA of a gene into a vector (plasmid) that can replicate (insert into f- or as Hfr)
- can be transformed into bacteria where it will undergo many replications
- also called (genetic recombination technology)
What are the major needs for cloning?
1- introduction of DNA (by transformation)
2- molecules to propagate the fragment (vector /plasmid)
3- means to artificially recoding : restriction enzymes (cuts DNA) and ligage (sticks together)
what i the technique for transformation? What is it for?
To allow exogenous DNA to be taken up by e.coli
- salt wash
- heat shock
What are the two types of natural vectors?
1- f’ episome: plasmid where piece of DNA has recombined in f+ factor
2- lambda transduction phase : (propagated when carried bacterial DNA) - lysogeny
Name the different types of vectors:
- plasmid
- shuttle vectors
- lambda bacteriophage
- bacterial artificial chromosomes
- yeast artificial chromosomes
what is the major important i recombinant techniques?
Gene must integrate into the vector
What is an ideal vector?
- small
- carry markers (ie: antibiotic selectable markers)
- would propagate in e.coli)
- has restriction sites
What are two example to see if gene was transformed successfully?
- ampicillin resistance
- beta galactosidate
Allows to find bacteria that has new piece of DNA cloned
What is a polylinker?
Multiple clog in sites
- offers many choices in regards to which enzyme to use for closing
What are restriction sites?
Cut sites - DNA sequence that can be recognized by restriction enzymes
What are restriction endonucleases (restriction enzymes)? Who has them?
Enzymes that can cut DNA in a precise way.
Bacteria’s have these to prevent infection from foreign bacteria
What are the two types of restriction enzymes, how do they differ?
Type I: cuts elsewhere then the restriction site
Type II: work horse enzyme for recombination, recognizes a sequence and cuts at recognition site
who did the initial work on restriction enzymes and plasmids?
Boyer and cohen
Most plasmids have:
- origin of replication
- gene for antibiotic resistance (marker)
- multiple cloning sites (Polylinker)
- B-glactosidase
- lac promoter
What are the three types of restriction enzymes and how do they cut?
1- Eco RI - 5’ single stranded overhang
2- PST I - 3’ overhang
3 - Sma I - blunt ends
how do you calculate the expected frequency of a restriction site of DNA of cDNA?
1/4 to the n
N = # of bases in the recognition sequence
1/4 because chance of each nucleotide.
If given two nucleotides that can go there 1/2
How does electrophoresis work and what is it used for?
- Used to measure the size of DNA (size of the different fragments)
- you use gel and dye (ethics bromide) to stain DNA mol
- you have a ladder
- have undigested DNA
- then have it digested by different restriction enzymes, which give you different sizes.
Where do the smallest fragments travel in electrophoresis?
They travel the farthest down
what does size tell you in electrophoresis?
How far te cut sites are
If there are 3 fragments, it means you used ___ enzymes?
2
What is cDNA?
- MRNA that was converted to cDNA and clones (has no introns because made from mRNA)
- has 3’UTR
- can have 5’ UTR
- no introns
- no promoter
- done by reverse transcriptase
What is cDNA great for?
Deciphering the coding region of a gene
What is PCR, what does it stand for?
Polymerase Chain reaction
- allows direct amplification without the use of plasmid
- making lots of copies of a particular DNA fragment
what is the PCR cycle?
1 - denature DNA - 96 degrees
2- annealing of primers (primers stick to DNA) - 52 degrees
3- DNA synthesis from primer - 72 degrees
Do this again, many times
What enzyme is used in PCR?
TAQ, which cannot proofread - it extends the primers
What is the amplification of PCR?
After one cycle, # of DNA molecule has doubled - it is exponential
How can you tell if you have cloned the piece of DNA you want? Apart from electrophoresis?
- DNA hybridization (norther or southern blot)
How do you do DNA hybridization, southern blot and northern blot
Southern:
After DNA digestion, electrophoresis and dénaturation (1 strand)
- you blot (transfer to membrane)
- hybridize with radioactive single stranded probe (will stick to complementary, even if not perfect)
Northern:
- RNA is used for electrophoresis instead of DNA
What is DNA hybridization? What doe sit show?
If two single stranded pieces of DNA tat are complementary willl spontaneously form a double helix, even if not perfect match.
- shows that the sequences are similar
What is autoradiographie used for?
Uses southern blot to help identify if 2 alleles are the same
Restriction fragment length polymorphism
- can tell you if two fragments are the same size as size of mutated allele
What is DNA sequencing?
Sequencing fragments of DNA using a primer (oligonucleotide to replicated DNA
What is the Sanger methods of DNA sequencing?
- PCR to amply the sample
- add regular deoxyribonucleotide to be able to extend strand
- add dideoxyribonucleotide - these are missing an oxygen and therefore terminate the sequence.
- add fluorescence to these ddNTP (A,G,C,T)
- they will terminate at different point and you will be able to sequence depending on colour
What does the CRISPR/Cas 9 do?
Improves specificity of genetic engineering?
What is cas 9?
Nucleases that is linked to RNA. RNA will direct it to the right sequence that is homologous, in order to cut it.
Therefore efficiently targets DNA of interest.
What percentage of cancers are sporadic?
75%
What are the genetics of cancer?
1- Genes can give stronger predisposition to specific cancers
2- other genetic factors can increase odds 2-3 folds
3- chemical compounds are clearly carcinogenic
4- env. and lifestyle can increase
What are oncogenes? What is a normal gene called?
Genes that if present, seem to put those cells at risk of cancer.
Pro-onco gene
What are differences between normal mammalian cells and tumor cells?
Normal:
- divides a limited # of times approx 50 x
- inhibition of growth with contact
Tumor cells:
- they were immortal
- no limit to # of division
- not inhibited by contact
- grow fo I
What gene was used to identify once genes?
NIH 3T3
What is the RAS oncogene?
- a small GTPase that switches from ON and OFF by hydrolysis of GTP
- have the ability to activate cancer
- RAS protein has 12th AA changed therefore could not hydrolyze GTP, therefore stayed ON
What are the dominant oncogenes? What do they do?
RAS
-found to link 2 signalling molecules:
≥receptor tyrosine kinase
≥ MAP kinase
MYC
- Encodes transcription factor
≥controls expression of large set of genes
SRC
- encodes a kinase
≥job is to add phosphate residue to various proteins
They are all key components of signalling pathways
What does receptor tyrosine kinase do? What is it also called?
Also called - epidermal growth factor receptor (HER)
Transmembrane protein that stimulates the proliferation of diff types of cells
What is a type of treatment for the epidermal growth factor?
Hereptin - monoclonal antibody against HER - which prevent dimerization and inhibits growth
What are two genetic diseases that we will be looking at?
- retinoblastoma
- breast cancer
What type of genetic disease is retinoblastoma?
2-hit model - 2 things have to go wrong in order to get the disease
Rob gene - 2 normal copies have to go wrong in order to be activates
- it is a recessive oncogene
- it is a tumor suppressor gene
why rb gene considered a tumor suppressor gene?
The normal gene is an inhibitor of transcritor facto E2F, which controls the start of DNA replication.
What occurs without the normal Rb gene that inhibits Transcriptor factor?
The E2F is too active and replicates inappropriately.
What percentage of breast cancer are hereditary directly from gene mutation inherited from parent?
5-10%
What are the two genes associated with breast cancer? What causes the disease?
BRCA1 - BRCA2
Loss of heterozygosity - where a mutation in somatic cell causes second copy and bring homozygosity
Breast cancer is ____ for inheritance and ____ at the cellular lever
Dominant
Recessive
What are recessive oncogenes?
Rb gene
BRCA1/2
P53
How could cancer be prevented?
- no smoking
- eat less fat, avoid over consumption
- exercise moderately
- drink moderately
- self breast check/ gynaecological/ testicular
- colonoscopy
- annual blood test for prostate
Diet to avoid cancer?
Carotenoids
Crucifierons vegetable (cabbage, broccoli etc
Fribers
Wha are the goal of anti cancer drugs?
- inhibit cell division: ≥ inhibiting DNA replication ≥disrupt separation during mitosis (disrupting spindle fibers, preventing cell division) - kill tumor cells ≥tiger apoptosis
What is apoptosis?
Organized cell suicide, when cells realize they have abnormalities
Name the different drugs that we are looking at?
- 5-flurouracil (5FU)
- Ara-C
- Vinca Alkaloids
- Gemcitabine (2’2’-diflurodeoxycytidine)
- monoclonal antibodies (2 types)
- bleomycin
- camptothecin
- methotrexate
- Iressa
- Gleevec
Describe 5-flurouracil
- used for colecteral caner
- analogue of Uracil/thymine
- inhibits Thymidylate synthase (precursor of DNA)
- inhibits DNA synthesis
- triggers programmed cell deat
Ara-C
CTP analogue OH in 2’ positio
-inhibits synthesis
Vince alkaloids types and what does it do?
Vincristine
Vinblastine
Vinorelbine
Disrupt the spindle fiber in mitosis - prevents dimerization of tubulin
Gemcitabine
- dCTP analogue incorporated into DNA
- inhibits chain elongation
- used for pancreatic cancer
Monoclonal antibodies
Made to target proteins found on surface of tumor cells
- rituximab (cd20 antigen target on B cells - leukaemia, lymphoma)
- trastuzumab (herceptin): target HER, which is overexpresed in 25-30% of breast cancer
Bleomycin
Causes single stranded/double stranded breaks
Camptothecin
Naturally occurring alkaloid
Inhibit topoisomerase
Methotrexate
Inhibit purine and thymidylate synthesis
Causes: misincorporation of dUTP into DNA
For leukaemia, lymphoma, breast etc
Iressa
Works again Tumors where HER 2 is amplified
Gleevec
Inhibits action of tyrosine kinase BCR1-Ab1 fusion oncogene
Why are there side effects?
Because they affect rapidly dividing cells, which are found
What is the frequency of genotypes in a population dependent upon?
The frequency of alleles that cause that trait
What does p and q stand for?
P = frequency of A
P = frequency of a
P+q=1
How do you find the frequency of a specific genotype?
P2 (homozygote dominant) +2pq (heterozygote) + q2 (homozygote recessive)
What are the conditions for following hardy Weinberg equilibrium?
Large population
Mating at random
No significant selection, migration, mutation
What would the chi square test be used for?
To check if population distribution is in hardy-W equilibrium
- must use # in population
- always use one degree of freedom
If given the frequency of a disease, how do you find the frequency of an allele?
50 = f(aa) =q2 square root gives you q P+q = 1
If asked the frequency of heterozygosity, what do you do?
Multiply, frequency of
What is an harem structure?
Single dominant male with many mates, not expected to ave H-W equilibrium
What is inbreeding and what does it cause?
Between close relatives (or in plants that self pollinate)
— it decreases heterozygosity, loose 50% (1/2) per heterozygote cross
Increases homozygozity
How is uniformity achieved?
Homozygote - true breeding
What is the inbreeding coefficient?
Probability of the offspring from consanguineous mating is homozygous by descent
What is the basic # of chromosomes in humans? # of chromosomes in gametes
X = 23
2X=46
N = # of chromosomes in gametes 2N = 46
What is polyploidy?
Have more than 2 copies of the genome
Tétraploïde = 2N = 4X = 28 x = 7
What is euploidy?
Changes in the number of whole chromosomes sets
What is aneuploidy?
Change in the # of individual chromosomes (of whole # of sets)
how can polyploidy be induced?
In plants with colchicine, which disrupts the spindle fibers and cells dont divide
- after one cycle, doubled chromosomes
- must have even # of chromosomes
What is autopolyploidy?
Doubling chromosomes with the same species
What is alloploidy?
Combined genome of 2 or more related species
What are the types of chromosomal changes?
1- duplications
2- deletions
3- inversions
4- translocations
What is duplications
Create imbalance in # of genes
Deletions?
Usually deleterious
Generates loss of genes
Inversion
1- paracentric inversion
More stable, as loss or addition, but induce recombination which produce invivable gametes and reduce surviving offspring
- Pericentric: includes centromeres
Lead to decrease map distance between genes because it redues the # of recombination in a populatio
Which chromosomal change causes recombinants to have a decreases distance?
Inversions
Translocations
Break and reciprocal fusion of non-homologous chromosomes
- switching piece of DNA from one chromosome to another
- reduce gamete viability and cause pseudolinkage
Caused by breakage and rejoining
What is Aneuploidy? What are the different types?
Arise from disjunction of chromosomes during meiosis
Nullsomis
- 2n-2 *missing two copies
Monosomics:
- 2n-1
- missing one X chromosomes
- Turner syndrome
Trisomies - 2n+1 - klinefelter syndro XXY (sterile men) Or - Down’s syndrome: chrosmomes 21,
What is genomics?
Study of genomes int heir entirety
How many genes do humans have appro?
20,000
What is genome structure?
Involves the analysis of gene sequence, gene #, order and physical nature of X
Functional genomics?
Studies the function of genes, gene expression, phenotype
Bioinformatics?
Use of computer analysis for structural or functional genomics
Proteomics?
Study of all proteins of an organism
how do we get a sequence?
Sanger gene sequencing method
how is gene sequencing done by Sanger, which were thre new approaches used?
Done my increments of 300-800 bp
Typical lens of a DNA sequence from one Sanger was about 500 bases long, but total 3 million base pairs, therefore requires 6 million runs
What are two distinc approaches used instead of Sanger?
1- human genome program: Broke genome into. Large fragments, clones as largement fragments in BAC and ordered into overlapping sets
2- Private consortium celera: whole genome shot gun approach. Broke genome into small fragments for sequencing and used powerful computer to fit pieces together
What is a contigus?
Contiguous sequences made by overlap of sequences in regions of identity.
What are today’s different technique to sequence a genome?
Illumina:
- millions of PCR amplified short fragments
- use fluorescent ATGC
- look under microscope
Ion torrent
- millions of PCR amplified short fragments on semi conductor
- detect hydrogen ions from each nucleotide incorporated
- chip computer
Pas bio
- traps DNA pol
- watches incorporation of not flucorecent in real time
- no amplification
- can be long pieces of DNA
What is Blastn comparison?
You identify gene coding region
1- compare cDNA sequence from same species
2- compare DNA sequence to known genes n related species
3- checki DNA for long ORFs
4- identify motifs of DNA sequence (promoters, intron-exon, terminators)
Which new technology uses hydrogen ion for genomic sequencing?
Ion torrent
Which technique does not amplify by PCR?
PacBio
Which technique uses fluorescence?
Illumina
What is different from illumina and Sanger?
Illumina séquences millions of fragments simultaneously per run, instead of one run with Sanger
