Ch 6 DNA and Biotechnology Flashcards
DNA and RNA
Polymers that together create molecules integral to life
DNA is recorded from 5’-3’
Nucleosides
Five-carbon sugar (pentose) bonded to a nitrogenous base formed by covalently linking the base to C-1’ of the sugar
Comprise nucleotides
Nucleotides
Formed when one or more phosphate groups are attached to C-5’ of a nucleoside
Often named according to number of phosphates present.
High energy because of energy of repulsion between negative charges on phosphate groups
Building blocks of DNA
Ribose
Five-carbon sugar (pentose) in a ring with O between C-1 and C-4, another C and OH off of C-4 and Ohs
Deoxyribose
“Deoxygenates” C-2 on pentose to just H.
Base Adenine
Nucleoside: Adenosine (deoxyadenosine)
Nucleotides: AMP (dAMP), ADP (dADP), and ATP (dATP)
Guanine Base
Nucleoside: Guanosine (deoxyguanosine)
Nucleotides: GMP (dGMP), GDP (dGDP), GTP (dGTP)
Cystosine
Nucleoside: Cytidine (deoxycytidine)
Nucleotides: CMP (dCMP), CDP (dCDP), CTP (dCTP)
Uracil
Nucleoside: Uridine (deoxyuridine), UMP (dUMP), UDP (dUDP), UTP (dUTP)
Thymine
Nucleoside: (deoxythymidine) no thymidine because it appears almost exclusively in DNA
Nucleotides: (dTMP), (dTDP), (dTTP)
Purines
Aromatic, Contain two rings in their structure
The two found in nucleic acids are adenine (A) and guanine (G) both found in both RNA and DNA
Pyrimidines
Aromatic, Contain only one ring in their structure
The three are cytosine (C), Thymine (T), and uracil (U)
Aromatic
Unusually stable ring system that adheres to these:
- Compound is cyclic
- Compound is planar
- Compound is conjugated (has alternating single and multiple bonds, or lone pairs, creating at least one unhybridized p-orbital for each atom in the ring)
- The compound has 4n+2 pi electrons (Huckel’s rule)
Watson-Crick Model
Key features: the two strands of DNA are antiparallel or oriented in opposite directions
- the sugar-phosphate backbone is on the outside of the helix with the nitrogenous bases on the inside
- specific base pairing rules (complementary base pairing) A to T (or U) and G to C (C-G is three hydrogen bonds making it stronger
- %A=%T and %C=%G total purines are = to total pyrimidines Chargaff’s rules
B-DNA
Right handed helix makes a turn every 3.4 nm and contains about 10 bases within that span
Most DNA
Z-DNA
Zigzag appearance
Left handed helix turn every 4.6 nm and contains about 12 bases within each turn
Denaturing of DNA
Disruption of hydrogen bonds
No covalent bonds between nucleotides in the backbone of the DNA break
Heat, alkaline pH, and chemicals like formaldehyde and urea cause this
Renealing DNA
If denaturing condition is slowly removed two strands can become paired again.
Probe DNA
DNA with a known sequence
Histones
Small basic proteins around which the DNA that make up a chromosome are wound, creating a chromatin
There are two copies of H2A, H2B, H3, and H4 that form a histone core and about 200 base pairs of DNA are wrapped around the protein complex
Nucleosome
Formed from the 200 base pairs of DNA that are wrapped around a histone core in chromosome
Nucleoproteins
Proteins that associate with DNA
Histones fall into this category
Heterochromatin
Small percentage of chromatin that remains compacted during interphase of cell cycle
Transcriptionally silent
Euchromatin
Dispersed chromatin during interphase and has genetically active DNA
Telomere
Repeating unit at the end of replication (TTAGGG). Some is lost each replication but can be replaced by the enzyme telomerase. However this accounts for limited replication
Telomeres have high GC-content that creates strong strand attractions at the end of chromosomes to prevent unraveling
Centromeres
Region of DNA found in center of chromosomes
Composed of heterochromatin with repeating sequence and high GC content allowing two sister chromatids to remain connected until microtubules separate during anaphase
Replisome
Also called replication complex - in DNA replication is a set of specialized proteins that assist the DNA polymerases
Origins of replication
Point at which dna unwinds to begin replication
Replication forks
Produced as DNA replication proceeds in both directions from the origins of replication
Helicase
The enzyme responsible for unwinding DNA generating two single-stranded template strands ahead of the polymerase
Single-stranded DNA binding proteins
Required to hold the separated strands of DNA apart during process of replication
They bind to unraveled strand preventing both reassociation of the dna strands and the degradation of dna by nucleases
DNA Topoisomerases
Introduce negative supercoils to relieve torsional stress of winding
Work ahead of helicase by nicking one or both strands allowing relaxation of torsional pressure and then resealing the cuts.
Parental strands
Serve as template for daughter strands
Semi conservative
DNA replication is considered to be this because the parent strand is conserved in the two daughter strands
DNA Polymerase
Responsible for reading the DNA template or parent strand and synthesizing the new daughter strand
Can read template strand in a 3’ to 5’ prime direction while synthesizing complementary strand in the 5’ to 3’ direction
Leading strand
Strand that is copied in a continuous fashion in the same direction as the advancing replication fork
Lagging strand
Strand that is copied in a direction opposite the direction of the replication fork. DNA polymerase cannot synthesize directly because it only synthesizes in 3’ to 5’ direction.
Okazaki fragment
Since DNA polymerase can only synthesize in 3’ to 5’ direction these fragments are developed for synthesis of the lagging strand
Primase
This is before the replication begins, primase synthesizes a short primer of RNA (roughly 10 nucleotides) in the 5’ to 3’ direction for the DNA to hook onto as it splits
Constantly laid down for lagging strand while leading strand requires only one
DNA polymerase III
Prokaryotes only
Synthesizes daughter strand of DNA in the 5’ to 3’ manner
DNA polymerases alpha, delta and epsilon
Eukaryotes only
Begins synthesizing daughter strands in the 5’ to 3’ manner
Removal of RNA after synthesis in prokaryotes
DNA polymerase I
Removal of RNA after DNA replication in eukaryotes
Carried out by RNase H
Adds nucleotides where RNA primer had been, in prokaryotic dna replication
DNA polymerase
Adds dna nucleotides where RNA primer had been in dna replication of eukaryotes
DNA polymerase delta
DNA ligase
After DNA replication this seals the ends of DNA molecules together creating one continuous strand
Five classic DNA polymerases in eukaryotes
Alpha, beta, gamma, delta, and epsilon
Alpha, delta and epsilon work together to synthesize both the leading and lagging strands; delta also fills in gaps left behind when RNA primers are removed
Gamma replicates mitochondrial dna
Beta and epsilon- dna repair
Delta and epsilon assisted by PCNA protein which assembles a trimer to form sliding clamp
Sliding clamp
Clamp that helps strengthen interaction bt DNA polymerases delta and epsilon and the template strand
Oncogenes
Mutated genes that cause cancer
Before mutation they are referred to as proto-oncogenes
Antioncogenes
Tumor suppressor genes (p53 and Rb)
Encode proteins that inhibit the cell cycle or participate in dna repair processes
Proofreading
In part of the polymerase - detects incorrectly paired bases during replication
Distinguishes the template strand from daughter strand because template is more methylated
Occurs during S cell cycle
Mismatch repair
G2 phase is cell cycle
Enzymes coded by genes MSH2 and MLH1 which detect and remove errors introduced in replication that were missed during the S phase of the cell cycle.
Nucleotide excision repair
Eliminates thymine dimers introduced from UV light
A cut and patch process
Specific proteins scan the DNA molecule molecule and recognize the lesion
An excision endonuclease makes nicks on both sides of thymine dimer and removes it
Occurs during G1, G2 cell cycle
Base excision repair
Detects small non-helix distorting mutations
First affected base is recognized and removed by glycosylase enzyme, leaving behind an apurinic/apyrimidine (AP) site also called an abasic site
AP site is then recognized by an AP endonuclease that removes damaged sequence and lets DNA polymerase and DNA ligase repair the gap
Occurs during G1, G2 cell cycle
Recombinant DNA
Allows dna fragment from any source to be multiplied by either gene cloning or polymerase chain reaction (PCR) which provides a means of analyzing and altering genes and proteins
DNA cloning
Technique that can produce large amounts of a desired sequence
DNA to be cloned is often present in small amounts and is part of a heterogeneous mixture of other DNA
The goal is a large quantity of one type
Vector or recombinant vector
In DNA cloning a piece of nucleic acid into which the dna is litigated by the investigator
Restriction enzymes (restriction endonucleases)
Enzymes that recognize specific DNA sequences
The 5’ to 3’ orientation of both strands is identical
Isolated from bacteria
Allows fragments to be inserted directly into the vector sometimes.
DNA libraries
Large collections of known DNA sequences
Genomic libraries
DNA library that contains large fragments of DNA and includes both coding (exon) and noncoding (intron) regions of genome
cDNA (complementary DNA) library
DNA library constructed by reverse transcribing processes mRNA
Lacks noncoding regions and only includes the genes that are expressed in the tissue from which the mRNA was isolated
Sometimes called expression libraries
Hybridization
Joining is complementary base pair sequences
Can be DNA-DNA or DNA-RNA recognition
Vital part of polymerase chain reaction and Southern blotting
Polymerase chain reaction (PCR)
Automated process that can produce millions of copies of a DNA sequence without amplifying the DNA in bacteria
Knowing sequences that flank the desired region of DNA allows for amplification of the sequence between
Primers
PCR requires primers complementary to the DNA that flanks the region of interest, nucleotides (dATP, dTTP, dCTP, dGTP) and DNA polymerase
Also needs heat
Must use DNA polymerase from Thermus aquaticus - bacteria because ours cannot survive at high heat.
Preferred gel for DNA electrophoresis
Agarose gel
Southern blot
Used to detect presence and quantity of various DNA strands in a sample.
DNA is cut by restriction enzymes and then separated by gel electrophoresis
DNA fragments are then carefully transferred to a membrane, retaining separation
Split strands bound to radioisotopes and indicates presence of desired sequence
Dideoxyribonucleotide
A modified base that is added in lower concentrations in DNA sequencing
Once added the polymerase can no longer add to the chain
Include ddATP, ddCTP, ddGTP, ddTTP)
Gene therapy
Potential cures for individuals with inherited diseases
Transferring a normal gene into the affected tissues
Transgenic mice
Altered at their germ line by introducing a cloned gene into fertilized ova or into embryonic stem cells
Cloned gene is called a transgene
Knockout mice
Mice in which a gene has been intentionally deleted.
Chimera
Blastocyst that has been given transgene but also has the original cells that lacks the transgene and has patches of cells
