|What is somatic cell hybridization?||
The physical mapping of genes on a chromosome and NOT the location on the chromosome.
E.g. HeXA gene is on chromosome 15
|What does somatic cell hybridization accomplish?||
It creates a hybrid from rodent cells and human fibroblast or lymphocytes in the presence of the Sendai virus.
It allows a researcher to isolate hybrid clones containing different human chromosomes that have in one way or another helped restore function to a mutant rodent gene.
|What is the HAT medium?||
A selection medium for hybrid cell lines; contains hypoxanthine; aminopterin; thymidine. Only cell lines expressing both hypoxanthine phosphoribosyl transferase (HPRT+) and thymidine kinase (TK+) can survive in this medium. Aminopterin inhibits de novo synthesis of nucleosides, while HPRT and TK supply them from hypoxanthine and thymidine.
What are the two pathways for DNA replication?
During which circumstance(s) is one or the other chosen?
Main DNA pathway and Salvage pathway.
In the presence of the HAT medium, the Salvage pathway is chosen and becomes dependent on the HPRT or TK in the medium.
|What was the tetrad analysis used for?||
It proved that the four strand stage of recombination is the correct one.
|The tetrad analysis used which organism?||
|A T, PD, and NPD tetrad have howm any parentals and recombination progeny?||
T: 2P and 2R
|Which crossover(s) give us a PD, T, and NPD tetrad?||
PD: no crossover & dco (2 strand)
T: sco, dco (3 strand)
NPD: dco (4 strand)
|What is the recombination frequency formula for tetrad analysis?||
RF = (1/2*T + NPD / Total tetrads) * 100%
|What are the three types of bacterial markers?||
2. Amino acids
3. Sugars (Carbon Source)
|What does it mean if a bacterium is lac-? his-?||
lac-: cannot utilize lactose at all even if lactose is provided in the environment (true for all sugars)
his-: cannot produce histamine because that pathway is mutated but it can work if the histamine is provided in the medium.
|What is the formula for calculating phage recombinant frequency?||
RF = (2 * # of λ plaques / total plaques) * 100%
|What is the lowest RF%?||
It is 0.01% (1 nucleotide apart) in the rII region of phages.
|Most phage mutations are of what type?||
Point mutations - single nucleotide changes.
|What is the size of virus, bacteria, and eukaryotic genetic material?||
Virus: 100,000 - 200,000 bp
Bacteria: 4,600,000 bp
Eukaryote: 3,000,000,000 bp
|What do viral genes produce?||
Capsid and tail fibers
|What is the chromosome organization of viruses?||
Circular or linear
RNA or DNA
SS or DS
|Viruses have space restrictions what do they do package their genetic material?||
Overlapping reading frames
|The smallest and largest viruses have how many genes?||
3 and 300 respectively
|What is the bacterial chromosome organization?||
Mostly circular with little linear chromosomes.
Bacterial chromosomes mostly contain little __________ DNA.
Most of it is made up of _______.
|These genes can move around between bacteria and viruses in very low frequency.||
|E. coli DNA is naturally _______________ supercoiled.||
|What is negative supercoiled and positive supercoiled DNA?||
Negative: Same DNA length, 1 less 360 degree turn
Positive: Same DNA length, 1 more 360 degree turn
|How many histones make up a nucleosome? What is this complex called? What are the individual histones called?||
2A, 2B, 3, 4
|You can synthesize with these four parts.||
1. 4 dNTPs
2. DNA fragment
3. DNA polymerase
4. Mg++ ions
|What is the proofreading activity of DNA polymerase?||
3' -> 5' exonuclease activity
|What is semidiscontinuous DNA replication?||
The leading strand is continuous ly replicated without stopping.
The lagging strand is composed of Okazaki fragments which will need to be ligated together everything time the replication fork moves ahead.
|True or False. DNA polymerase creates a phosphodiester bond from the 3' 0H of last nucleotide to the 5' phosphate of the next nucleotide.||
|Explain the model of initiation as it pertains to DNA replication.||
1. Gyrase relaxes supercoiled DNA
2. Initiator proteins wrap around ORI to form a complex
3. DNA Helicase binds to initiator proteins and denatures DNA using ATP
4. Primase binds to helicase forming a complex called primosome
5. Primase synthesize a short RNA primter for DNA polymerase action to begin
|These proteins stabilized the unwound single strand of DNA and prevent it from annealing.||
S.S. DNA binding proteins
|What are the 3 checkpoints in Interphase and Mitosis?||
|Explain what the function of 6 DNA polymerases in eukaryotes.||
DNA Repair: alpha, delta, epsilon
DNA Replication: beta, xi
Mitochondrial Replication: gamma
|What is a replicon?||
A chromosomal region whose replication is controlled by a single adjacent DNA replication initiation site.
A genetic unit of replication including a length of DNA and its site for initiation of replication.
|Mitochondrial genome contains how many protein, rRNA, and tRNA genes?||
Protein: 13 genes
rRNA: 2 genes
tRNA: 23 genes
|What is the C-paradox?||
Higher, more evolved organisms have less DNA than their less complex neighbors (fish, reptiles, and amphibians).
DNA complexity != Amount of DNA
|How many base pairs make up1 3/4 turns of the nucleosome?||
|The chromatin fiber of packed nucoleosome follow this model of organization.||
|The domain loops are attached to __________ by way of the ______.||
|What is the function of the domain loops of the extended section of the chromosome?||
The loops have structural and/or regulatory roles.
|This complex protects chromosome ends from nuclease breakdown?||
|What is telomerase?||
It is a reverse transcriptase that extends the 3' of the strand and polymerizes the 5' end by laying down RNA primer.
What is the breakdown of coding and non-coding DNA?
What makes up the coding portion?
3 and 97 respectively
The coding portion is made of protein producing genes including histone and hemoglobin genes but NOT rRNA genes.
|The hemoglobin genes make up what two families?||
alpha and beta
|True or False. rRNA genes bring their own RNA polymerase I.||
|What is satellite DNA?||
It is clustered DNA found in many organisms; the clustered DNA is mostly made up of introns.
In humans, this portion of DNA is satellite.
What is the function of this DNA?
Alphoid DNA (171 bp, lots or repeats)
Found in the region of centriomeric DNA -- hypothesized to bind sister chromatids together before S phase completes.
|What is mini satellite DNA?||
Mini satellite (VNTRs)
6 to 64 nucleotides long
1 to 20 Kb in size
Use probe hybridization to identify individuals uniquely by fingerprinting.
|What is micro satellite DNA?||
Micro satellite (STRs)
1 to 5 nucleotides long; mostly 2 to 3
Use PCR to establish genomic profile
|What are the primary markers of genetic mapping?||
Micro satellites and now SNPs (single nucleotide morphosim).
|What are two types of interspersed sequences?||
LINEs and SINEs
|SINEs and LINEs make up how much of the genome?||
|Which family is an example of the SINEs? Explain the family.||
280 bp and > 1 million copies
Made up of processed pseudo-genes -- no regulatory genes, no introns, and no promoters.
Located in gene rich regions
|Which family is an example of the LINEs? Explain the family.||
6.1 Kb and 512,000 copies
Carry reverse transcriptase, retransposition occurs.
Located in gene sparse regions
|What are pseudogenes?||
They are genes that have had too many mutations in them causing them to not work any longer.
|What is the Holliday Model of Recombination and Double Break Model?||
These models demonstrated that the proposed physical intermediates existed in the form of the Greek Xi (chi) letter.
Heteroduplex is formed when the strands switch pairing relationships and bond to the other strand.
|Besides minimal medium Neurosporra Crassa needs one other nutrient, what is it?||
|One gene creates one ________________||
|Antibody and TCR genes come together to form one polypeptide, what is this process called?||
Alternative RNA splicing
|PKU- affects which process and allows what to increase in concentration?||
It affects phenylalanine hydroxylase from transforming phenylalanine into tyrosine.
Phenylpyruvic acid increases in concentration and leads to mental retardation.
|What changed in the 6th nucleotide of the beta globin of human hemoglobin to cause it to become SCA?||
A missense mutation occurred with valine being substituted for glutamic acid.
|What is colinearity? How was it proved?||
A series of nucleotides will code for amino acids in the correct, sequential order.
Yanofsky proved this theory with the TrpA gene in E. Coli
|Transformation of E. Coli occurs through ____________ or _______________||
Chemical shock or electrophoration
What are the types of restriction enzymes?
How do they function?
Types I and II
I: Cuts DNA from the origin
II: Cuts DNA within specific sequences/recognition sites; e.g. EcoR1
|How many nucleotides long do restriction enzymes of Type II recognize?||
4, 6, or 8
1/(4^4) = 1/256
1/(4^6) = 1/4096
1/(4^8) = 1/65536
|Can blunt ends be annealed together by large amounts of ligase?||
|Which vector is used for recombination? What features does it have?||
- Small size but holds large insert
- Contains ORI
- Contains antibiotic resistance gene
- Contains polylinker
|What is special about pUC and the polylinker?||
The polylinker is inserted in between the gene for beta-galactosidase but it still works.
|When XGal is added to a colony that can utilize beta-galactosidase, what is observed?||
alpha complementation is observed along with blue colonies
|What is alpha complementation?||
Where two separate portion of the peptide fragments are combined to yield a functional b-galactosidase protein.
|True or False. When the polylinker portion of the pUC vector is filled with insert, it destroys the beta-galactosidase activity.||
|What is the formula for number of clones needed to insert vectors into?||
N = ln (1 - P) / ln (1 - F)
|What are vectors can be used for cloning?||
|What is cDNA?||
Duplex DNA with exons and no introns.
No introns = no promotor, no regulatory genes