Upgrade to remove ads
Only $2.99/month

Biochem Chapter 6: DNA and Biotech

Terms in this set (78)

Where is DNA found in cell?
Chromosomes in nucleus (some in mito and chloro)
NucleoSide
Sugar and nitrogenous base (covalent link at C-1 of sugar)
NucleoTide
1+ phosphate groups attached to C5 of nucleoside

High energy molecules (repulsion of negative charges on phosphates)
Breaking phosphate bonds is endo/exothermic
Exothermic! (exception to the general rule)
Sugar-phosphate backbone
read 5' to 3'

Phosphate group links 3' of one sugar to 5' of the next
3' end of DNA has
free OH group on C3 of sugar
Purines
Adenine and guanine (2 rings)
Pyrimidines
Cytosine, uracil, thymine (1 ring)
Characteristics of aromatic compounds
Cyclic, planar, conjugated, Huckel's rule
Huckel's rule
Aromatic cmpd characteristic: 4n+2 pi electrons
Watson-Crick model of DNA structure
double-helical
antiparallel strands
bases inside, sugar-phosphate backbone outside
complementary base-pairing: AT/CG
H-bonds b/w AT and CG
AT: 2 h-bonds
GC: 3 h-bonds
Chargaff's rule:
A = T and C = G
Total purines = total pyrimidines
B-DNA
Right handed helix; 10 bases per turn; major and minor grooves for protein binding
Z-DNA
Left handed; 12 bases per turn; high GC content or high salt concentration - unstable
Denaturing of DNA
Disrupt H-bonding by heat, alkaline pH, formaldehyde, urea
Reannealing
Slowly remove denaturing condition
Chromosomes
46 in humans; compacted DNA units
Histones
DNA is wound around these basic proteins -- forms chromatin
Nucelosome
Histone-DNA complex
Histone proteins
H2A, H2B, H3, H4
H1 seals off DNA as it enters/leaves nucleosome
Nucleproteins
Proteins that associate with DNA
Heterochromatin
Chromatin that remains compact during interphase - transcriptionally silent (often highly repetitive DNA)
Euchromatin
Genetically active DNA; dispersed chromatin
Telomere
Repeating units at end of DNA; some is lost in each round of replication; high GC content seals off ends of chromosomes and prevent unraveling
Telomerase
Enzyme that replaces telomeres - more expressed in rapidly dividing cells
Centromeres
DNA region in center of chromosomes - heterochromatin (high GC) - allows sister chromatids to remain connected until separation during anaphase
Replisome
Set of specialized proteins that assist DNA polymerases
Origins of replication
Points where DNA unwinds to begin replication
Replication forks
DNA replication goes in both direction
Bacterial chromosome
Closed, ds circular DNA with one origin of replication
Eukaryotic replication
Multiple origins of replication; replication forks meet at centromere
Helicase
Enzyme that unwinds DNA
ss DNA binding proteins
Bind unraveled strands to prevent reassociation and degradation by nucleases
Supercoiling
DNA wraps in on itself is helical structure is unwind
DNA gyrase (toposiomerase II)
Nicks and reseals DNA to relieve supercoiling
Semiconservative replication
Each new double strand has one parental and one daughter strand
DNA polymerases
read template and synthesize daughter - read 3 to 5 and synthesize 5 to 3
Leading strand
Advances into replication fork
Lagging strand
Synthesized out of fork (parental is 5 --> 3); synthesized in small strands = Okazaki fragments
Primase
Lays down short RNA primer
Main polymerases in euk and prok
DNA pol alpha and delta (euk)
DNA pol III (prok)
Proteins that remove RNA primers and add DNA
Prok: DNA pol I
Euk: RNase H and DNA pol delta
Ligase
Seals DNA ends together
Eukaryotic DNA pols
alpha and delta: synthesize leading and lagging (delta replaces RNA primers)
gamma: replicates mito DNA
beta and epsilon: DNA repair
delta and epsilon: along with PCNA protein assemble into sliding clamp
Chromosome ends
5' end becomes shorter each cycle - telomeres
Cancer cells
Excessive proliferation; no longer subject to controls on cell cycle
Metastasis
Migration of cancer cells by local invasion thru blood or lymph
Oncogenes
Mutated genes that cause cancer - encode cell cycle-related proteins
Proto-oncongenes
oncogenes before mutation - abnormal alleles more active than normal proteins (mutations tend to be dominant)
Tumor suppressor genes
p53 or retinblastoma - proteins that inhibit cell cycle or repair DNA - "anitoncogenes" - mutation of these result in loss of tumor suppression - need to inactivate both alleles for loss of function
Proofreading
Detecting incorrect pairs (h-bonding unstable) and replace with correct one - detect daughter strand b/c it has less methylation
Lagging/leading - higher prob of mutation?
Lagging - ligase lacks proofreading ability, constantly stopping and starting
Mismatch repair
During G2 phase;
MSH2 and MLH1 genes encode enzymes that detect and remove errors (MutS and MutL in prok)
Nucleotide excision repair (G1/G2)
Removing TT dimers made by UV exposure

Excision endonuclease nicks out damage
Polymerase fills in 5 --> 3 and ligase seals
Base excision repair (G1/2)
C deaminated to U
Base emoved by glycosylase, leaving abasic site
Recognized by AP endonuclease, and filled in with pol and ligase
Diff b/w oncogenes and tumor suppressor genes
Onco: cell cycle-promoting proteins (rapid cell cycling once mutated)

Tumor suppressor: code for repair or cell cycle inhibiting (unchecked cell cycle once mutated)

"Onco is stepping on the gas, mutated tumor suppressors are like losing brakes"
Difference b/w NER and BER
NER: correct lesions that distort the helix
BER: lesions small enough to not distort helix
Recombinant DNA tech
DNA fragment multiplied by cloning or PCR
DNA cloning
Produce large amts of desired seq
Recombination vector
DNA of interest ligated into vector (bacteria/virus) that can be transferred into host bacterium
Selecting recombinant bacteria
Include antibiotic resistance on vector - allows us to kill the bacteria that don't have it
Restriction enzymes
recognize and cut specific dsDNA sequences (palindromic) - some produce sticky ends - can paste genes into vectors
Formation of recombinant plasmid vector
Need ori, ampR and gene (w/ restriction enzyme site)
DNA libraries
Large collections of known sequences - fragments are randomly digested and cloned into vectors for further study
Genomic libraries
Large fragments of coding and noncoding
cDNA libraries
Made by reverse-transcription of processed mRNA - no noncoding regions and only includes genes expressed in the tissue from which we collected mRNA - also called expression libraries
cloning expressed genes by producing cDNA
Hybridization
Joining complementary base pair seq's
PCR
Produce copies of a DNA sequence

need complementary primer sequences and Taq polymerase
Gel electrophoresis
Separation by size and charge - agarose gel for DNA, since DNA is - charged, it will migrated to anode
Southern blot
Detecting presence and quantity of various DNA strands (separated by gel elec --> transferred t membrane --> probed with complementary DNA that is labelled for detection)
Sanger sequencing
Chain terminating ddNTPs; separate by gel electrophoresis, and can read off sequence
Gene therapy
Inherited disease with mutated or inactive genes - placing in a working copy: need efficient gene delivery vectors (modified viruses)
Transgenic mice
Altered at germ line by introducing cloned gene into embryonic stem cells
Knockout mice
Intentionally deleting a gene
Developing transgenic mice
Cloned gene may be microinjected into nucleus of newly fertilized ovum - implanted into surrogate mother
Safety and ethics of DNA cloning
Increased resistance in viruses and bacteria
Is it ethical to test for life-threatening diseases and terminate pregnancy?
YOU MIGHT ALSO LIKE...