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Sunday, 25 November 2007

genetic engineering (5)

RA synthesis
5'->3' direction
no primer
DNA dependent RNA polymerase (RNA polymerase)
ATP, GTP, CTP, UTP
anticoding strand, template strand


promotor -RNA polymerase (alpha, beta, delta)binding site - 5' ustream to transcription initiation site (+1)
termination -Rho protein, inverted repeat
prokaryote - -35 and - 10 consensus sequence- Pribnow box
Eukaryote -75 CAAT box -5'GGNCAATCT3', -25 TATA box,Hogness box - 5' TATAAA3'
higher organisms
RNA pol 1 polA-rRNA except 5 SrRNA, super repeat promotor
RNA pol2 polB-mRNA- normal promotor, AAUAAA-polyadenylation signal (20-30 b before 3')
RNA pol3 polC-tRNA, 5srRNA, scRNA, internal control region (ICR)

Posttranscriptional modification
tRNA- methylation, oxygen-->sulfur
rRNA-methylation, shortening
mRNA
-7 methyl guanosine capping
-poly Adenylation tail
-splicing -exclude intron-snRNPs, some self splicing

alpha interferon - no intron
beta globin -2 intron
factor VIII-25 intron
human thyroglobulin- > 40 intron

genetic engineering (4)

DNA replication
semiconservative
1.helix-destabilizing protein-helicase
2.single strand DNA binding protein (SBP, DBP)
3. DNA gyrase or topoisomerase-replication fork
4.primase-synthesize small RNA primer
5.DNA polymerase
E coli- pol3 leading strand 5'-->3', lagging strand small fragment (Ogazaki fragment), pol I 5'->3' exonuclease)
polI (C-terminal -polymerase, 3'-5' exonuclease -Klenov fragment, short N-terminal, 5'-3' exonuclease) -proofreading
Mammals-alpha-nuclear-polymerase, beta-nuclear-repair, delta -nuclear similar alpha, gamma-mitochondria-mitochondrial DNAreplication
6. DNA ligase
bidirectional
E coli- 1 replicon
Yeast-500 replicon
Drosophila-3500 replicon
Toad-15000 replicon
Mice 25000 replicon
plant -35000 replicon

Model
1. Theta model-circular DNA-bacteria, mitochondria
2. Sigma model-rolling circle model: phage lambda circular dsDNA-nick, unwheel, produce linear DNA-concatamer, catenated DNA, phage M13 circular ssDNA --> circular dsDNA--> linear ssDNA-->circular ssDNA
3. Y-shaped model-phage T7 linear dsDNA, higher organisms- chromosome

telomerase - terminal deoxynucleotidyltransferase - repeat CCCTAA---> inverted to complementary
found in egg and sperm only
so shorten of chromosome each replication 50-60 times --.senescence

Genetic engineering (3)

Structure and type of RNA
RNA - 5X DNA

transfer RNA (tRNA)80-100 type
5S ribosomal RNA 1-2 type
5.8 Sribosomal RNA 1 type
16 S ribosomal RNA 1 type
23 S ribosomal RNA 1 type
18 S ribosomal RNA 1 type
28 S ribosomal RNA 1 type
messenger RNA (mRNA) many thousands type
heterogeneous nuclear RNA (hnRNA) many thousands type
small cytoplasmic RNA (scRNA)
small nuclear RNA (snRNA)

Size, organization, complexity of Genomes
Size and organization of genomes
1000 bp to 10000 mbp
dsDNA except virus: ssDNA, dsDNA, ssRNA, dsRNA, linear or circular, single or multiple
bacteria-circular dsDNA+protein
eukaryote-linear dsDNA (multile)+histone, non histone protein

left hand super helix 1.8 rounds, 145 bpDNA(2nm)+2H2A, 2H2B, 2H3, 2H4- nucleosome core particle(10nm)--> solenoid(30nm)-->filament(300nm)-->supercoiled(700nm)-->chromosome (metaphase 1400nm)
60 bp linker DNA+H1

heterochromatin-inactive-centromere, telomere
active chromatin-10%

packing ratio=DNA length/Final length average 1000-2000

Complexity of genome
unique sequence, repetitive sequence
slow component 1 copies-45% of genome
intermediate component 350 copies-30% of genome
fast component 500000 copies-25% of genome

satellite, minisatellite, microsatellite

Important of DNA
DNA replication
DNA transcription
Complexity of genome

Genetic engineering (2)

Structure and function of Deoxyribonucleic Acid (DNA)
Structure and behavior of DNA and RNA (Ribonucleic acid)
Chemical composition
Nucleotide=Base+Sugar+Phosphate group
Nucleotide=(Purine: adenine(A), guanine(G) or Pyrimidine: Cytosine(C), Thymidine (T-DNA only), uracil (U-RNA only)+ Deoxyribose (DNA) or Ribose(RNA)
5' --> 3 '
Structure of DNA
B-DNA
double helix
antiparallel
base -hydrogen bone- base
A=T, G-triple bond-C in horizontal plane
sugar back bone
phosphate outside
3.4 nm height for each round 10 base
diameter 2 nm
A-DNA
relative humidity 75%, Sodium, Potassium, or Cesium
base diagonal 20 degree, each base differ 0.26 nm
11-12 bp each round
diameter 2.3 nm
antiparallel
RNA, or DNA-RNA hybrid
Z-DNA
poly dGC,polydAC
each base differ 0.37 nm
12 bp per each round
diameter 1.8 nm
Zigzag-like
stabilized with replace C in poly dGC with 5-methyl C
Denature (melting)and renature(association, annealing)
melting temperature (Tm) -temperature that have equal ssDNA and dsDNA
pHm-pH that have equal ssDNA and dsDNA
urea and formamide-decrease Tm
monovalent or divalent cation concentration-increase Tm
G+C/A+T increase Tm. pHm
GC increase 1%, Tm increase 0.4 degree
renaturation between different species of nucleic acid-hybridization: solution, filter
Other qualities of DNA
1. UV absorption: absorbance (A) or optical density (OD) peak at 260
OD of 1 mg/ml of DNA, RNAA, oligonucleotide = 20, 25, 30
OD increase with temperature (ssDNA-> increase OD)
2. Acid-base:stable at pH 4-5, depurination at pH <3, base donot react with DNA, but destroy phosphodiester bond of RNA (react at OH)
3.sedimentation
velocity sedimentation: generate gradient column of sucrose or potassium citrate-add DNA mixture-centrifuge-highMW down, low MW up
equilibrium sedimentation:CS-->spin down centrifuge-->diffusion until equilibrium-add Mixture buoncy of DNA=Cs, protein-top, RNA-bottom

Genetic engineering (1)

Introduction
Basic genetics to genetic engineering
1866 Mendel - Principle of Inheritance
1900 de Vries, Correns, van Tschermak - rediscover Mendel's rule
1905 Bateson - word "Genetics"
1901 Johannsen - word "Genes"
1928 Avery, Macleod, McCarthy- DNA is genetic material
1953 Watson, Crick - double helix DNA
Beginning of genetic engineering techniques
1970 Nathans, Smith - restriction enzymes
1970 reverse transcriptase
1976 antibody diversities
1977 split genes: introns and exons
1977 Sequencing techniques
1982 Supermice: Growth Hormone Gene Transfer
1983 Ribozymes
1985 Polymerase chain reaction (PCR), DNA fingerprint
1990 CFTR gene for cystic fibrosis
1993 IT15 gene for Huntington disease
1997 Telomerase gene
2003 First draft of Human Genome