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What is (a) gene? Central Dogma
• 學習目標 :
• To know the history of searching heredity unit and the definition of gene.
• To understand how genes are regulated in various organisms (from genotype to phenotype).
What is (a) gene? Central Dogma
• 1. Discovery of DNA as a genetic material.
• 2. Classic genetics, modern genetics and molecular genetics.
• 3. Different levels of gene regulation (transcriptional, translational and post-translational levels).
• 4. New frontier of small RNA regulation.
決定生物的藍圖• 龍生龍• 鳳生鳳• 老鼠生出來的…… ..
• 遺傳的觀念
生命怎麼變的如此複雜 ?
生物演化的三步曲:變異、遺傳與天擇
variation, inheritance and natural selection!
Heredity unit = Gene
Genetics and Evolution
Gregor Mendel (1823-1884)
孟德爾豌豆雜交實驗
Mendel cannot reproduce his results on peas in hawkweed!
Mendel 豌豆實驗結果分析
Important terms in Mendelian genetics.• Character: color of peas.
• Trait: yellow or white.
• Gene: unit of heredity.
• Allele: version of a gene produces a specific trait.
• Homozygous: having two copies of the same alleles for a given gene.
• Heterozygous: having two different alleles for a given gene.
Insight of genetics
• Partial Dominance: dilute concentration.• Codominance: human ABO blood group (AB)• Overdominance: sickle-cell anemia• Dominant: gain-of-function.• Recessive: loss-of-function.• Negative dominant mutation. • Genetic suppression: intra vs intergenic
suppression!
Sex linkage• Thomas Hunt Morgan in The Fly Room!
(Columbia University 1910)• Fruit Flies (Drosophila melanogaster)
http://nobelprize.org/nobel_prizes/medicine/articles/lewis/index.html
© 2007 Paul Billiet ODWS
Hypothesis
A cross between the F1 flies should give us: 3 red eye : 1 white eye
F2 Phenotypes Red eye White eye
Numbers 3470
82%
782
18%
So far so good
© 2007 Paul Billiet ODWS
An interesting observation
F2 Phenotypes Red-eyed males
Red-eyed
females
White-eyed males
White-eyed femal
es
Numbers 1011 2459 782 0
24% 58% 18% 0%
© 2007 Paul Billiet ODWS
Chromosome linkage
Physical map
Generation of fly mutants by x-ray
radiation
Genetics• classic genetics 古典遺傳學• modern genetics 近代遺傳學
• molecular genetics 分子遺傳學
• 尋找遺傳物質及基本單位
The Search for the Genetic Material: Scientific Inquiry
• When T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material
• The key factor in determining the genetic material was choosing appropriate experimental organisms
• The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them
© 2011 Pearson Education, Inc.
Milestones in DNA History
• 1869 Johann Friedrich Miescher identifies a weakly acidic substance of unknown function in the nuclei of human white blood cells. This substance will later be called deoxyribonucleic acid, or DNA.
• 1912 Physicist Sir William Henry Bragg, and his son, Sir William Lawrence Bragg, discover that they can deduce the atomic structure of crystals from their X-ray diffraction patterns. This scientiFic tool will be key in helping Watson and Crick determine DNA's structure.
• 1924 Microscope studies using stains for DNA and protein show that both substances are present in chromosomes.
• 1928 Franklin Griffith, a British medical officer, discovers that genetic information can be transferred from heat-killed bacteria cells to live ones. This phenomenon, called transformation, provides the first evidence that the genetic material is a heat-stable chemical.
肺炎雙球菌的實驗
• 1944 Oswald Avery, and his colleagues Maclyn McCarty and Colin MacLeod, identify Griffith's transforming agent as DNA. However, their discovery is greeted with skepticism, in part because many scientists still believe that DNA is too simple a molecule to be the genetic material.
• 1949 Erwin Chargaff, a biochemist, reports that DNA composition is speciesspecific; that is, that the amount of DNA and its nitrogenous bases varies from one species to another. In addition, Chargaff finds that the amount of adenine equals the amount of thymine, and the amount of guanine equals the amount of cytosine in DNA from every species.
DNA是攜帶遺傳資訊的分子! How ?
奧斯卡.阿佛來 (Oscar Avery) 1943
Figure 16.4-3
Bacterial cell
Phage
Batch 1:Radioactivesulfur(35S)
Radioactiveprotein
DNA
Batch 2:Radioactivephosphorus(32P)
RadioactiveDNA
Emptyproteinshell
PhageDNA
Centrifuge
Centrifuge
Radioactivity(phage protein)in liquid
Pellet (bacterialcells and contents)
PelletRadioactivity(phage DNA)in pellet
EXPERIMENT
• 1953 James Watson and Francis Crick discover the molecular structure of DNA.
• 1962 Francis Crick, James Watson, and Maurice Wilkins receive the Nobel Prize for determining the molecular structure of DNA.
•
Watson and Crick 華生與克立克
Rosalind Franklin 和她的 DNA X- 光繞射圖
• 1961 Sidney Brenner and Francis Crick establish that groups of three nucleotide bases, or codons, are used to specify individual amino acids.
• 1966 The genetic code is deciphered when biochemical analysis reveals which codons determine which amino acids.
Figure 17.5 Second mRNA base
Fir
st m
RN
A b
ase
(5 e
nd
of
cod
on
)
Th
ird
mR
NA
bas
e (3
en
d o
f co
do
n)
UUU
UUC
UUA
CUU
CUC
CUA
CUG
Phe
Leu
Leu
Ile
UCU
UCC
UCA
UCG
Ser
CCU
CCC
CCA
CCG
UAU
UACTyr
Pro
Thr
UAA Stop
UAG Stop
UGA Stop
UGU
UGCCys
UGG Trp
GC
U
U
C
A
U
U
C
C
CA
U
A
A
A
G
G
His
Gln
Asn
Lys
Asp
CAU CGU
CAC
CAA
CAG
CGC
CGA
CGG
G
AUU
AUC
AUA
ACU
ACC
ACA
AAU
AAC
AAA
AGU
AGC
AGA
Arg
Ser
Arg
Gly
ACGAUG AAG AGG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
GAU
GAC
GAA
GAG
Val Ala
GGU
GGC
GGA
GGGGlu
Gly
G
U
C
A
Met orstart
UUG
G
• 1970 Hamilton Smith, at Johns Hopkins Medical School, isolates the first restriction enzyme, an enzyme that cuts DNA at a very specific nucleotide sequence. Over the next few years, several more restriction enzymes will be isolated.
• 1972 Stanley Cohen and Herbert Boyer combine their efforts to create recombinant DNA. This technology will be the beginning of the biotechnology industry.
•
• 1976 Herbert Boyer cofounds Genentech, the first firm founded in the United States to apply recombinant DNA technology
• 1978 Somatostatin, which regulates human growth hormones, is the first human protein made using recombinant technology.
尋找基因的實驗CONCLUSION From the growth patterns of the mutants, Beadle and Tatum deduced that each mutant was unable
to carry out one step in the pathway for synthesizing arginine, presumably because it lacked the necessary enzyme. Because each of their mutants was mutated in a single gene, they concluded that each mutated gene must normally dictate the production of one enzyme. Their results supported the one gene–one enzyme hypothesis and also confirmed the arginine pathway. (Notice that a mutant can grow only if supplied with a compound made after the defective step.)
Class IMutants(mutationin gene A)
Class IIMutants(mutationin gene B)
Class IIIMutants(mutationin gene C)Wild type
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor
Ornithine Ornithine Ornithine Ornithine
Citrulline Citrulline Citrulline Citrulline
Arginine Arginine Arginine Arginine
EnzymeA
EnzymeB
EnzymeC
A A A
B B B
C C C
Figure 15.15
Down syndrome
Figure 16.23
5 m
Central dogma of molecular biology : a process of decoding
Genetic code in DNA: A, T, G, C
Genetic code in RNA: A, U, G, C
20 amino acids in protein
The Nobel Prize in Physiology or Medicine 1975 was awarded jointly
to David Baltimore, Renato Dulbecco and Howard Martin Temin "for their discoveries concerning the interaction between tumour viruses and the genetic material of the cell".
The Nobel Prize in Physiology or Medicine 1975
David Baltimore Renato Dulbecco Howard Martin Temin
Ribozymeribozyme (ribonucleic acid enzyme) is an RNA molecule that is
capable of catalyzing specific biochemical reactions, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. Also termed catalytic RNA, ribozymes function within the ribosome (as part of the large subunit ribosomal RNA) to link amino acids during protein synthesis, and in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.
Evolution of enzymes that catalyze nucleic acids (RNA and DNA)
RNA dependent RNA polymerase
RNA to RNA
RNA dependent DNA polymerase (RT)
RNA to DNA (cDNA)
DNA dependent DNA polymerase
DNA to DNA
DNA dependent RNA polymerase
DNA to RNA (mENA, tRNA, rRNA)
細胞
電腦
硬體
2D 資訊在磁碟複製、長久、穩定
軟體硬體
DNA複製、長久、穩定
RNA暫時、不穩定
蛋白質執行工作或通訊
RAM暫時、不穩定
銀幕或其他機器執行工作或通訊
細胞像電腦?電腦像細胞?
(亞瑟.孔伯)Arthur Kornberg(亞瑟.孔伯)Arthur Kornberg
• 1956 找到複製 DNA 的酵素
• 1959 Nobel prize winner• His son, Roger Kornberg
received Nobel Prize in 2006 for his study of structure basis of gene transcription in eucaryotes.
Characteristic of DNA synthesis - I
• Primers absolutely necessary– Usually short stretches of RNA or RNA-DNA– Some virus use proteins primers.
Characteristic of DNA synthesis - II
• 5’ to 3’ directionality– Leading strand vs. lagging strand– End problems for linear DNA molecules
when replication starts internally
Okazaki fragments: discontinue synthesis!
Figure 16.20a
Ends of parentalDNA strands
Leading strand
Lagging strand
Last fragment Next-to-last fragment
Lagging strand RNA primer
Parental strand Removal of primers andreplacement with DNAwhere a 3 end is available
3
3
3
5
5
5
Proofreading and Repairing DNA• DNA polymerases proofread newly made DNA,
replacing any incorrect nucleotides• In mismatch repair of DNA, repair enzymes correct
errors in base pairing• DNA can be damaged by exposure to harmful chemical
or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes
• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA
© 2011 Pearson Education, Inc.
Figure 16.21
1 m
DNA (genetic code)
To make a unique protein with a specific amino acid sequence
through transcription and translation
Gene expression
(Expression of information)
mRNA
How many polymerase?
• DNA dependent DNA polymerase– For DNA replication and repair.– 5 known Prokaryotic DNA polymerases.– at least 15 Eukaryotic DNA polymerase
• DNA dependent RNA polymerase– For gene transcription.
• RNA dependent RNA polymerase– For RNA virus genome replication
• RNA dependent DNA polymerase– Reverse transcriptase of retrovirus– Telemerase to make telemere structure
In 1977, when viral mRNA was hybridized with its DNA, some loops were observed.
Figure 17.12-3 RNA transcript (pre-mRNA)5
Exon 1
Protein
snRNA
snRNPs
Intron Exon 2
Other proteins
Spliceosome
5
Spliceosomecomponents
Cut-outintronmRNA
5Exon 1 Exon 2
Figure 17.11
5 Exon Intron Exon
5CapPre-mRNACodonnumbers
130 31104
mRNA 5Cap
5
Intron Exon
3 UTR
Introns cut out andexons spliced together
3
105 146
Poly-A tail
Codingsegment
Poly-A tail
UTR1146
How many genes do we have ?
DNA (genetic code)
whatwherewhen
how much
Regulation of gene expression at different level!
Transcription factors turn genes on and off.
Transcription factors are proteins that bind to a specific base sequence in DNA.
… AGCCTACCAAAAAAGGTTCCACG……TCGGATGGTTTTTTCCAAGGTGC…
Nontemplatestrand of DNA
RNA nucleotides
RNApolymerase
Templatestrand of DNA
3
35
5
5
3
Newly madeRNA
Direction of transcription
A
A A A
AA
A
T
TT
T
TTT G
GG
C
C C
CC
G
C CC A AA
U
U
U
end
Figure 17.9
Promoters, enhancers, silencers etc.
Figure 17.26TRANSCRIPTION
DNA
RNApolymerase
ExonRNAtranscript
RNAPROCESSING
NUCLEUS
Intron
RNA transcript(pre-mRNA)
Poly-A
Poly-A
Aminoacyl-tRNA synthetase
AMINO ACIDACTIVATION
Aminoacid
tRNA
5 C
ap
Poly-A
3
GrowingpolypeptidemRNA
Aminoacyl(charged)tRNA
Anticodon
Ribosomalsubunits
A
AE
TRANSLATION
5 Cap
CYTOPLASM
P
E
Codon
Ribosome
5
3
Figure 16.22a
DNA double helix(2 nm in diameter)
DNA, the double helix
Nucleosome(10 nm in diameter)
Histones
Histones
Histonetail
H1
Nucleosomes, or “beads ona string” (10-nm fiber)
Figure 16.22b
30-nm fiber
30-nm fiber
Loops Scaffold
300-nm fiber
Chromatid(700 nm)
Replicatedchromosome(1,400 nm)
Looped domains(300-nm fiber) Metaphase
chromosome
DNA
Nucleosome
Epigenetic chromatin regulation
A. Modification at the DNA level
1. cytosine methylation
B. Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitilation
5. Different types of histones
The five nucleotides that make up the DNA
Maintenance of methylation
Brandeis, M., Ariel, M. & Cedar, H. (1993) Bioessays 15, 709-713.
Imprinting is maintained by DNA methylation
Genomic imprinting
Some genes are expressed only from the maternal genome and some only from the
paternal genome
It is estimated that about 40 genes are imprinted and they can be found on
several different chromosomes
For example - igf2, h19, igf2r and genes involved in the Angelman and Prader Willi syndromes
Histone modification
EXPANDING THE GENE CONCEPT EXPANDING THE GENE CONCEPT BEYOND THE PROTEIN ENCODING BEYOND THE PROTEIN ENCODING
SEQUENCESES of DNASEQUENCESES of DNA::
TRANSCRIPTION OF SOME GENES PRODUCES NONCODING RNAs
Non-Coding RNA: Formerly known as “JUNK” A Key to Eukaryotic Complexity?
Types of RNATypes of RNA
CODINGCODINGIn translationIn translation (mRNA) (mRNA)
NON-CODINGNON-CODINGIn translation (tRNAs and rRNAs)In translation (tRNAs and rRNAs)
IIn RNA processingn RNA processingRegulatory RNAs: lincRNA, Riboswitch Regulatory RNAs: lincRNA, Riboswitch
and microRNAand microRNA
A decade of riboswitch Cell January 17, 2013 page 17
What are lincRNAs?What are lincRNAs?LLarge arge iintergenic ntergenic nnononccoding RNAs (lincRNAs) oding RNAs (lincRNAs) are emerging as key regulators of diverse are emerging as key regulators of diverse cellular processes. cellular processes.
Determining the function of individual Determining the function of individual lincRNAs remains a challenge. lincRNAs remains a challenge.
Recent advances in RNA sequencing (RNA-seq) Recent advances in RNA sequencing (RNA-seq) and computational methods allow for an and computational methods allow for an unprecedented analysis of such transcripts.unprecedented analysis of such transcripts.
Cell July 3, 2013. Page 26
Cell nucleus is a highly organized structure just like a Rome city!
24 MAY 2013 VOL 340 SCIENCE page 910
Long Noncoding RNAs May Alter Chromosome’s 3D Structure
What are miRNAs?
• Small non-coding double stranded RNAs• Approximately 19-22 nt long• Repress activity of complementary mRNAs• Regulate 30% of mammalian gene products• 1 miRNA = hundreds of mRNAs• Many are conserved between vertebrates and
invertebrates
Genomic OrganizationGenomic Organization
miRNA processing
Microprocessor Complex
Differences in miRNA Mode of Action
Extended thinking
•miRNA and Cancer
CORRELATION OF MIR EXPRESSION WITH PROGRESSION AND PROGNOSIS OF GASTRIC CANCER*
CORRELATION OF MIR EXPRESSION WITH PROGRESSION AND PROGNOSIS OF GASTRIC CANCER*
PATIENTS: 181 patients from 2 cohorts (Japan)
CLASSIFICATION: Stages I-IV Diffuse vs. Intestinal type
ANALYSIS: • Custom miR microarray chip (Ohio State Univ.)
• miR expression in 160 paired samples (tumor vs. non-tumor)• Correlations of miR expression vs. stage, type and prognosis (survival)
* Lancet Oncol. 11,136, 2010
MiRs AS PROGNOSTIC FACTORS: GASTRIC CANCER SURVIVAL*
MiRs AS PROGNOSTIC FACTORS: GASTRIC CANCER SURVIVAL*
0
1
2
3
4
5
StagesI-II
StagesIII-IV
3.2
high low high low low high01
234
567
89
10
Let-7g
miR-199
miR-495
HAZ
ARD
RAT
IO(d
isea
se fr
ee s
urvi
val)
HAZ
ARD
RAT
IO(d
isea
se fr
ee s
urvi
val)
Intestinal-Type Gastric Cancer
I-II III-IV I-II III-IV I-II III-IV
ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER BREAST CANCER (Mol Cancer Therap. 11, 108-118,
2012).
ANTIMETASTATIC ACTIVITY OF AHR AGONISTS IN ER BREAST CANCER (Mol Cancer Therap. 11, 108-118,
2012).
Normal cells Preneoplastic cells
Cancer cells(Invasive carcinoma)
Metastasis
SOX4
SOX4 and other miR-335 regulated proteins
miR-335
miR-335
SOX4
SOX4 and other miR-335 regulated metastatic
mRNAs
arntAhRLigand activated Ahr
RNA world• Carry information (DNA)• Catalyze chemical reaction (protein enzyme)• Nutrient sensor to control gene expression
(protein receptor)• Broadly control gene expression through
mRNA stability, translational efficiency etc. (protein activator or repressor)
• Global control nuclear and chromosome structure.(Histone code)