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115 Cards in this Set

  • Front
  • Back
Semi-conservative replication
DNA synthesis uses each strand of the parent to make a new strand of DNA.
Number of hydrogen bonds in C:G base pairs
Number of hydrogen bonds in A:T base pairs
What is required for DNA synthesis in vivo?
1. Template
2. Primer with 3'OH
3. Building blocks of 5' dNTPs
4. Energy in phosphate bonds
5. DNA polymerase
What additionally is required for DNA synthesis in vitro?
1. Mg
2. Buffer
3. Taq DNA polymerase
What is the proofreading mechanism found in DNA polymerase?
3'-5' exonuclease activity
Taq polymerase
1. Heat stable
2. Non-proof reading
3. Used in in vitro PCR reactions
Reverse Transcriptase
Makes DNA from an RNA template and inserts it into genome.
Reverse transcriptase found in human stem cells and cancer cells. it prevents telomere shortening using its own RNA template to extend DNA.
Topoisomerase I
Relieves strain due to supercoiling by cleaving one strand of the DNA and releasing pressure.
Topoisomerase II
Relieves strain due to supercoiling by cleaving both strands of the DNA and releasing pressure.
Topoisomerase II found in bacteria that is an isoenzyme so can be targeted specifically by flouroquinone that will not affect the human.
Actively transcribed genes
Inactive genes
B form of DNA
1. Biological form
2. Right handed helix
3. 10 bp/turn
A form of DNA
1. Right handed
2. 11 bp/turn
3. ssDNA
Z form of DNA
1. Left handed
2. 12 bp/turn
3. Active chromatin
Cleave within the DNA strand.
Cleave the ends of the DNA strand
Importance of methylation in DNA synthesis
Proofreading ability. The parental strand is methylated and the new strand is not methylated until it has been checked for errors.
Repair mechanism with double strand DNA breaks
1. Quick and dirty ligation of broken strands
2. In important genetic or modulary regions use the other chromosome as template.
P53 role in repair
Prevents cell replication before DNA repair has occurred.
1. Defense mechanism in lower eukaryotes and plants.
2. Around 25 base pairs long
3. Have 5’ phosphates and 3’ hydroxyl groups and 2 to 3 nucleotide overhands on the 3’ end
4. All that helps with binding to RISC
5. siRNA is unwound by RISC activity and antisense strand is left to bind to target mRNA
1. Family of RNase III enzymes
2. Recognize and process dsRNA into siRNA
3. Each dicer enzyme has an amino terminal helicase domain, 2 RNase III catalytic domains, dsRNA binding domain and a PAZ domain
3. Dicer is thought to act as a dimer of 2 enzymes – why?
We know that siRNAs are around 25 bp long
dsRNA cleavage is performed by the tandem RNase III catalytic domains - a single dicer enzyme will cleave dsRNA into 12-13 bp fragments
Put them together in a dimer, the 2 internal catalytic domains are made non-functional and the remaining catalytic domains are spaced far enough from each other to generate 25 bp fragments
1. RISC – RNA-induced silencing complex
2. Components of RISC are still unknown… but what’s is known so far:
2 RNA binding proteins – bind the siRNA
RNA/DNA Helicase – unwinding of the siRNA
Translation Initiation Factor – mutations of this component affect the initiation step
RNA-Dependent RNA Pol – may be a part of the RISC complex – may not – but it plays a role in the triggering and amplification of the silencing effect
Transmembrane Protein – seems to be the protein involved in the systemic spread of the RNAi… found in plants and so far in animals it’s only found in c.elegans
Plasmid cloning vectors
1. Plasmid contains origin of replication, antibiotic resistance gene, and polylinker region
2. new DNA with sticky ends is annealed to plasmid
3. Amplification occurs with antibiotic (the plasmid should be resistant) as the selection marker
4. You can prepare a genomic library in a plasmid vector
Lambda phage cloning
Similar to plasmid cloning but with large amounts of DNA
Yeast artificial chromosomes
Have the advantage of being eukaryotic and can hold large amounts of DNA>
Genomic libraries
Have all the DNA in the body including:
1. Introns
2. Promoters
3. Non-transcribed spacer DNA
cDNA libraries
Have only expressed post-modified genes in mRNA form.
Complimentary endonucleases
DNA is inserted into a cloning vector after cleavage with these that create a direction.
2 methods to produce DNA probes
1. Separate DNA strands and add radioactive dNTPs
2. Use polynucleotide kinase to transfer radioactive phosphate to 5' end of DNA
Chemical Method (Maxam-Gilbert) of DNA sequencing
1. 4 test tubes used
2. Chemical will attach to designated nucleotide and break the strand
3. Results in a bunch of different sized strands that are sequenced
4. DNA footprinting also uses chemicals to determine where specific proteins bind.
Enzymatic method (Sanger's dedeoxy) of DNA sequencing
1. Make 4 different vials and add a specific dideoxy "chain terminating" nucleotide into each vial
2. Results in stoppage in synthesis when these are encorporated
3. Anti-viral drugs use the same dideoxy method
Automated Sequencing of DNA
Uses fluorescent tags on dideoxy nucleotides that can be read by a machine as a certain color.
Southern Blot
1. Electrophoresis to separate different DNA fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids
Northern Blot
1. Electrophoresis to separate different RNA fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids
Western Blot
1. Electrophoresis to separate different protein fragment lengths
2. Heat denatured
3. Radioactive probe attached to filter for hybridization
4. Sample is exposed to film to identify hybrids
Restriction fragment length polymorphism
Detects mutations in gene sequences by knowing the digestion patterns of certain endonucleases. You cleave the DNA and identify the digested pieces
1. Use southern blotting
2. Prenatal screening for sickle cell and phenylketonuria
Polymerase Chain Reaction
Used to make copies of genetic information
1. Heat to separate strands
2. Add primers to hybridize and add dNTPs
3. Heat and use Taq polymerase to transcribe
4. Repeat many cycles
Variable number of tandem repeats
Unique sequences outside of the coding regions of DNA that are amplified for identification purposes.
Transcription methodology
1. Synthesis of RNA from DNA templates
2. Uses RNA polymerase
3. Helicase and topo I for accuracy
4. Promoters, enhancers, repressors, and insulators for regulation
DNA sequence required for initiating transcription; always orientation dependent.
Sequences that enhance rate of transcription. Generally position and orientation independent and can be large distances away.
Work on promoters and decrease the activation of a gene. Often a long way away from the gene.
Sequences on either side of a gene that insulate from the influence of adjacent genes.
Coupled Transcription/Translation
Occurs in prokaryotes because of the lack of nucleus.
Uncoupled Transcription/Translation
Occurs in eukaryotes because the nucleus is enclosed by membrane.
One strand of Prokaryotic mRNA produces multiple proteins.
One strand of eukaryotic mRNA produces one protein.
Effect of methylation on transcription
Methylation on promoters leads to inactivation of genes.
Effect of acetylation on transcription
Acetylation on histones leads to activation of genes.
Eukaryotic mRNA processing
1. 5' guanylmethyl cap
2. 3' polyadenine tail
3. Removal of introns and alternate and trans splicing
Alternative splicing
Ligates together different combinations of exons from the same gene.
Ligates together different combinations of exons from different genes.
snRNP spliceosome
Made of 7 subunits of snRNA and proteins that comes together to cleave out introns and splice together exons.
Required things for translation
Synthesis of proteins from mRNA template:
1. mRNA
2. Ribosomes
3. tRNAs
4. Amino acids
5. Protein factors
6. Energy (ATP, GTP)
Two functions of tRNA
1. Bind correct amino acid at 3' end through aminoacylsynthases
2. Bind triplet codon on mRNA using anti-condon segment
Wobble hypothesis
The third position in anti-codon pairs loosely leading to imperfect pairing and the necessity for less separate tRNAs. Increases rate of protein synthesis.
Positioning of ribosome on mRNA in prokaryotes
The 16S subunit binds 3' end of mRNA at a specific sequence and then reads and starts translation.
Positioning of ribosome on mRNA in eukaryotes
Ribosome starts at the 5' cap and scans for the start codon (AUG)
1. Initiation factors aid small ribosomal subunit with initiator tRNA bound (AUG) to find start codon.
2. Large subunit binds and the next tRNA
1. Each new amino acid gets transferred to growing chain.
2. Acceptor site - entry site for tRNA
3. Peptide site - place of peptide bond synthesis
4. Exit site - place of tRNA removal
When it reaches a stop codon a tRNA like release factor comes and binds to ribosome releasing protein.
Multiple ribosomes translating a single mRNA at once on RER surface or in cytosol.
Proteins are folded and undergo post-translational modifications
Silent mutation
The switched nucleotide codes for the same amino acid so the result protein is the same.
Missense mutation
The switched nucleotide codes for a different amino acid so the result protein is different.
Nonsense mutation
The switched nucleotide codes for a stop codon so the protein is truncated or nonfunctional.
Addition/deletion mutations
Addition or deletion of a single nucleotide resulting in a frame shift.
Frame-shift mutation
Changes the entire reading frame resulting in either a completely different protein or a shortened or nonexistant protein.
Untranslated regions
At the 5' and 3' end of mRNA confer stability and places for regulatory proteins to bind.
Method of transferrin and aconitase activity.
1. Iron binding to transferrin removes inhibition
2. Iron binding to aconitase induces degradation
3. In low iron environment - inhibits TR translation and stabilizes aconitase.
A protein responsible for storage of iron in a cell.
A surface receptor that brings iron into the cell.
Recombinant proteins
Use a plasmid to clone a desired gene into a bacteria, grow up bacteria, and purify protein for use as medication.
Methods to make recombinant proteins
1. Plasmid
2. Expression of recombinant genes in mammary glands so proteins are released in milk.
Recombinant proteins used in medicine
1. Tissue plasminogen activator
2. Blood clotting factors
3. Interleukins
4. Enzymes for research
5. Safer vaccines
6. Insulin
Transgenic Mice
Can be used to make disease models because of similarities between human and mouse genes
1. Gene replacement
2. Gene knock-out
3. Gene addition

Grow mouse stem cells in culture, alter DNA and then introduce them into early embryo, breeding these to get purebreds.
Severe Combined Immune Deficiency
Results from adenosine deaminase deficiency which causes an inability to make quickly dividing cells like B and T lymphocytes.
Adenosine deaminase
Needed for the break down of adenosine from food and for the turnover of nucleotides in the cell.
Treatment for SCID
Gene therapy:
1. Take stem cells from patient
2. Add the ADA gene using a viral vector
3. Introduce patient's cells back into system
Long tandem repeat segments
Used by viruses as location of placement into the genome.
Cloning sequence when using a viral vector
LTR-Enhancer-promoter-gene cDNA-poly(A) signal-LTR
Attacks a fusion protein made only in cancer cells of chronic myeloid leukemia. Prevents these from becoming cancerous until blood turns over and there is no longer cancer.
Fusion of chromsomes 23 and 9 result in this mutant gene that causes chronic myeloid leukemia.
Methods of Oncogene Discovery
Random genes from a cDNA library introduced into cells. Those that show overgrowth phenotype are oncogenes. Led to the discovery of Ras oncogene.
Dicer in humans
We have dicer and new research is looking at using it to chop up viral or cancer mRNA in human disease.
Enzyme that separates DNA strands.
Enzyme that synthesizes RNA primers.
Single stranded DNA binding proteins
Keeps DNA protected and exposed for copying.
DNA polymerase
Multi-subunit enzyme that adds one nucleotide at a time to the 3'-OH of primer RNA or elongating DNA chain.
250 bp of DNA covered in histones.
Normal base to abnormal base mutations and repair
Mutation: Caused by covelent bond formation of carcinogens or thymine dimerization which distort DNA helix.

Repair: Detected by DNA repair enzymes due to deformation of helix. Endonucleases cleave on either side of the damaged DNA and exonucleases cleave further along these nicks. DNA polymerase repairs and DNA ligase fuses the backbone.
Enzyme that cleaves DNA or RNA in the middle.
Enzyme that cleaves DNA or RNA from the end.
Normal base to normal base mutations and repair
Mutation: An incorrect amino acid is incorporated into a DNA strand.

Repair: Methylation occurs on the parent strand before DNA synthesis. The newly created strand has no methylation so the cell knows which one has the mutation on it when the base pairs do not match up. Mismatches are removed and repaired by DNA polymerase and ligase.
Two ways to repair double stranded breaks in DNA
1. Nonhomologous end joining in which the ends are simply ligated back together
2. Homologous end joining - copying process involved where the complete sequence is restored by copying from the second chromosome.
Tumor suppressor protein that recognizes damaged DNA and leads to cell cycle arrest so that cells do not replicate damaged DNA.
Self versus foreign macromolecules in humans
Our immune system functions at the protein level, recognizing our own proteins from foreign proteins.
Self defense for bacteria
Restriction enzymes that degrade foreign DNA. They protect their own DNA by methylation at sites of action of restriction enzymes.
Self defense for lower eukaryotes and plants
siRNA which degrades foreign RNA potentially from viruses.
Digest cellular DNA and plasmid DNA with a restriction enzyme, anneal, join with DNA ligase. Plasmid introduced into bacteria, copied, and purified.
Anti-viral drugs
Modified nucleotides called nucleosides are used. They incorporate into growing DNA chains and prevent further chain elongation. AZT.
Intervening sequences between exons. Few genes in lower eukaryotes. Most genes in higher eukaryotes.
Altering codons after transcription. Infrequent in humans; common in lower eukaryotes.
Cap binding proteins
Bind to the guanylmethyl cap and allow it to interact with the polyA tail to facilitate circularization of mRNA.
PolyA binding proteins
Bind to the poly A tail to stabilize mRNA and form a circle with cap binding proteins.
Soluble protein translation
Soluble proteins are translated on free-polyribosomes.
Secreted protein translation
Translated on the endoplasmic reticulum.
3' untranslated regions
Determine stability (halflife) of the mRNA.
5' untranslated regions
Regulates its own translation by binding molecules that can prevent translation machinery from being able to move.
Degrades RNA. Several ribonucleases that work by removing polyA tail and removing cap structure allowing RNA to be degraded by exonucleases from 5' and 3' ends.
Macrocytic anemia
Results from deficiency in folic acid or vitamin B12. Causes problems with DNA synthesis which means while the cell is trying to replicate its DNA it is growing large.
Microcytic anemia
Results from deficiency in iron which leads to problems with hemoglobin synthesis resulting in tiny cells.