Gene (遺伝子)

Synthetic biology (合成生物学)

nuclein extracted from red blood cell, yeast, yolk, salmon soft roe, etc

Structure

N-base___ nucleoside
thymine___thymidine
adenine___adenosine
guanine___guanosine
uracil_____uridine
cytosine__ cytodine

cf. nucleoside: [base]–[sugar] → triphosphate deoxynucleoside, distributed in nuclei

A-dR: deoxyadenosine_A-R: adenosine___dATP
G-dR: deoxyguanidine_G-R: guanosine___dGTP
C-dR: deoxycytidine___C-R: cytidine_____dCTP: phospholipid
__________________________________ synthesis
T-dR: deoxythymidine__×______________dGTP: protein synthesis
×__________________U-R: uridine

DNA (= deoxyribose): H bounded at C₂' ↔ RNA (= ribose): OH bounded at C₂'

________________Ribose_____________Deoxyribose
sugar conformation
2'-exo 3'-end_______________2'-endo 3'-exo

                    relative   base  lg (μm)       MW
                    volume     pair

DNA virus    SV40             4,000      1.6       3.1·106
             λ phage        53,000     16        31·106
    bacteria E. coli      3,400,000 12,000     2,320·106
RNA          m-RNA     5%    random               -4·105
             t-RNA    15%     70-80                2.5·104
             r-RNA    80%       120                3.6·104
                              1,700                0.55·106
                              3,700                1.2·106

G, A

C, U, T

Abnormal base (異常塩基)

7-Methyl guanosine (7mG)___Inosine (I)

N⁶-ΔIsopentenyl 2 methylthioadenosine (2ms6iA)

Extraction of DNA from DNA-protein complex

1. cell disruption
2. celll fractination – nuclei, organelle, chromatin
3. protein-denature (DNA, protein – disordinate)
   1. pronase: use SDS to remove possibility that RNase is working
   2. detergent: denature protein (Ex. sarkosyl SDS)
   3. salt concentration = 1-2 M NaCl
   4. phenol, chloroform

0.1M Tris-HCl (pH 7.5-8.0), 0.1-0.2 M NaCl, 1-5 mM EDTA, homogenized with 1-2% SDS

relaxed circlar DNA____super coil
_______
Twisting returns when one part is cut (constraint: distortion occuring in the DNA chain due to forcibly approaching)

DNA polymerase (DNA合成酵素)

dXTP = uptake into primer DNA

60': purified = Kornberg enzymes (= DNA polymerase I, poly I)

MW 10900, single-strand peptide, one SS-bond
→ function = mending enzyme for DNA

response to both 5'-end and 3'-end
fragments dichotomize by papain has activity → all of them form 5'-mononucleotide

5' → 3': transfer the next nucleotide to 3'-OH, indicator free 3'-OH
→ these are mending enzymes

1. template (鋳型) – synthesized DNA is complementary to the template
2. primer (出発点)

____________Exonuclease III______DNA polymerase I
______________________partly single______repair
cf. exonuclease endonuclease
__cut from outside______________cut from inside

discovered defective mutant of DNA polymerase I(A) from 3478 strains → DNA polymerase I is not essential for DNA synthesis

DNA polymerase II

DNA polymerase III: RNA as primer, DNA as template, 5' → 3'

discovered reverse transcriptase = RNA-dependent DNA polymerase

dna genge: engaged in gene replication
dna B, dna C = primer RNA
dna E (pol C) = DNA polymerase III
dna G = primer
dna Z = poly III &gamm;a subunit
pol A, pol B
lig (ligase)

DNA polymerase

DNA polymerase I

DNA polymerase III* |α 140| Poly III (dna E, pol C)
                    |ε 25 |
                    |θ 10 |
                    |J 83
                    |γ 32   dna Z      |factor II
                    |δ 32   factor III |
                    |β 40   factor I – copol III*

Three DNA polymerases, α, β and γ, in eukaryotes

high ion intensity > 25 mM
N-ethylmaleimide (NEM) – restraint the activation by attaching SH (sensitive)

utilizing oligo12-18, poly(rA)
SH-group, nonsensitive to NEM (→ distinguished from α)
stimulate (activity↑) under high ion intensity
not related to cell cycle? – chromatin bound polymerase

favor oligo
NEM sensitive
like-mitochondrial DNA

Plant DNA polymerase

mashing up pea seedlings &larr ; collecting supernatant after centrifugation → ammonium sulfate concentration gradient → extracting animal-like α, β DNA plymerases

DNA synthesis by Vicia fava
Measuring the activity of ribonucleoside-5'-diphosphate reductase → DNA polymerase governs the supply of substrates

DNA repair (DNA修復)

neck____Enzyme (= ligase joining enzyme)
-------------__→__-------------
------ ------______-------------

T₄ phase gene 30 = ligase → However, the mutant (ts B50) does not synthesize normal ligase under high temperature

Trigger of DNA synthesis (DNA合生開始)

Semi-conservative DNA replication (半保存的DNA複製)

Table. Structure of polynucleotide double helices

λ-DNA: eye shape = double strand
______________start at gene 0 of which length is 3 μm

NA, cytokinine c–basic + 2M ammonium acetate
sub-mutant → genetic code
UAA, UGA, UAG: nonsense code
TS mutant → used for DNA synthesis

30, 32, 41, 42, 43, 45, 46, 47, 56, 62
30: DNA ligase (→ bound with DNA)
32: DNA binding protein
43: DNA polymerase

Replication (複製)

nucleolus-DNA, r-RNA genes → replications [special example]
amplification (増幅): specfice gens increasing reversively at a life cycle in higher organisms; R-RNA genes increasing from somatic cells

1st: Caris type (θ) → 2nd: Rolling type

a___________b_____________c

starting the replication to the inverse direction just after the replication of a repeating this process

Sites for DNA replication (DNA複製の場所)

Tradescantia - prochromosome

chromatin fiber ≈ 230Å

at metaphase on Chinese hamster, etc. → chromatin bounded with nuclear membreane

Haplopappus gracilis, n = 2
Condensed chromatin → synthesis occurring at the late S stage
Diffused chromain → synthesis occurring at the early S stage

Alkaptonuria, cystinuria (シスチン尿症), pentosuria (ペントース尿症), achromia or albinism (色素欠乏症)

One gene-one enzyme theory

• mRNA (messenger RNA)
• rRNA (ribosomal RNA)
• tRNA (transfer RNA)

16S ribosomal RNA (16S rRNA)

16S ribosomal databases

Reversed genetics (改変遺伝学)

isolation of gene → assay of gene structure → restriction map (制限酵素地図)
            +1                                                   +n
-----↓-----↓===↓===↓===↓===↓======↓===↓------↓------↓------
           5' ←----------------------------------------→ 3' gene

designed alteration of the gene

fraction test of the mutant (mutated) genes (J Genetics 57: 581-598, 1982)

→ investigate the mechanisms of transcription =
I. in vitro (cell free) system
II. cultured cells: injection, Ca phosphate gel
III. fertilized egg

I. identification of signal DNA sequence

II. types of cells

III. fertilized egg

→ test of regulated expression → identification of signal DNA sequences

Finding out the component responsible for the regulated function

(Suzuki & Brown 1972)

Isolation of sil fibroin mRNA

G/G content = high
G: (30 + α mol%) + (9.3 + α) ≈ 40 + α
C: (9.3 + α) + (2 + α) + (1) + (1) ≈ 12 + α
G + C = 57–60% ⇒ such mRNA should be present

60% (w/w):_____(Gly-Ala-Gly-Ala-Gly-[Ser-Gly-(Ala-Gly)₂]₈-
_________________Ser-Gly-Ala-Ala-Tyr)_n = 58 A.A.
40%:______5.8% Gly-Ala-Gly-Ala-Gly-Ala-Gly-Try
_______________------------------------- alliance
__________5.6%
__________4.5%
⇒ consider codon
G_↑G_↑X-UCZ or AG_↑U/C-_↑G_↑CY-G_↑G_↑X-G_↑CY-G_↑G_↑X

in Rnase T1 (cut 3'-side of G)

Residue     G  XG  CYG  XUCZG  Total
Nucleotide  3    4      6          5         18
*: if X or Y is G, the bond is cut → X and Y are not G

Advantages of silk fibroin = 16000 nucleotides, 4.4% of total DNA

Detection of fibroin genes

Ex. fibroin mRNA 90%: 1 copy/haploid → 90
___contaminant 10%: 100 copy/haploid → 1000

not matched with the fact

total genome G + C = 39%
fibroin mRNA (gene) ~60%

DNA in rear-part silk gland cell ........ 1-3
DNA in middle-part silk gland cell .... 1-3
DNA in the whole body .................... 1-3
⇒ number of copies in the whole body is equivalent

fibroin gene = 0.002 × 2 (%) of total genome
[Conclusion] (Gage & Manning 1976): 1 copy/haploid
Cf. analbumin gene (Sullivan et al. 1973), globin gene (Harrison et al. 1974)

4 kinds out of ca. 50-70% kinds
amplification: can not be observed in general genes
Cf. Prokaryotes – Sarmonella phase variation (transition)

Structural analysis of the genes

           ├──→ 
       ──┼───────── early gene
    5' ──┼───────── late gene (functional)
           │←──

Mature mRNA

========== "intron" sequence

all sequences are transcribed at first, and then unnecessary portions are discarded

complementary DNA obtained from the mRNA of mouse globrin (Tilghman et al. 1977)

            5'                         3'
    ────┼────────────┼───
    ────┼────────────┼───
            └──↑─────────┘
            1 kb  1kb  intron   15 kb   ⇒ mRNA = 16 kb

the lengths of fibloin fibers are different between the strains - the lengths of DNA are proportionally changed

── exon, ━━ itron

⇒ 1 globrin gene(Tsujimoto & Suzuki 1979ab)
Figure (a) shows that mRNA is artificially cut at the point (↑)

⇒ (ProNAS 74: 4406, 1980)

========================⇒ mRNA
____↓ reverse transcription
========================⇒
←------------------------------------------
____↓_________↑
⇐========================
------------------------------------------→

Southern blotting

⇒ two fragments obtained by using enzyme A

hybridization with cDNA

friend cells 8 l
↓ DM 80
globin ↑

R-loop

β⁺ thalassemia (地中海性貧血症) – produced globrin somehow
______ex. 1____int. 1__ex. 2__int. 2__ex. 3
---------▓▓▓▓▓▓░░░░▓▓▓▓░░░░▓▓▓▓▓---------×---
_______________→|__|←

×: mutation having the genes in the outside

mutation [normal = G, β⁺ = A] at 21 bp
slow transcription rate at beginning ← splicing can not be made to mature mRNA

ex. 1______int. 1_____ex. 2
▓▓▓▓▓▓▓░░░░░░░▓▓▓▓
__________┃_┃___┃┗━━━━━┓
__________┃_┃____C*CCT*T*A*G*
__________┃_┗━━━━┓
___________C*TAT*T*GG* - normal
___________C*TAT*T*A*G* - β⁺

(Kole & Weissman 1982)

in vivo splicing

_________________Nomal splicing__Abnormal splicing
1 Ca/Ph gel β⁺gene_____100%____________0%
2 Ca/Ph gel β⁺gene______10%___________90%

Hela cell whole extract
succeed in transcription of most cellular components extracted by 0.42 M (NH₄)₂SO₄ are transcribed in vitro
protein = 1-10 mg ⇔ protein obtained by in vitro splicing = 20 mg/ml (high concentration)
240 μm Hela extract
10 μCi ³²P-UTP, CTP, GTP, ATP
100-200 μg/ml DNA (human β globin DNA)
400 μl reaction mixture

Trancation assay

Synthesizing precursor in vitro

        ↓ phenol extraction   ↓ no phenol
        ↓ new + extract       ↓ extract
        ↓ incubation          ↓ incubation
        ↓                     ↓
        RNA degraded            Stable

probably proteins connected to the synthesized RNA - (possibly) due to splicing
--------|====|========|================|------------
    5'  +1   +66    ⇓      1037       ex. 2                   fibroin
                       970 bp = intron
probably transcription starts from +1

Cell → Total cellular RNA: *³²P
mRNA → (reverse transcriptase, RT) copy DNA → cloning (hybridized with natural DNA) ※ shotgun method

SI mapping method: method to determine initiation point

actual signal for starting transcription (ex. AGT) = promoter

Hybrids arrest = ex. clone for few numbers of mRNA

Use of in vitro transcription system (生体外翻訳系利用)

Type I gene = rRNA: rDNA = class I gene
Type II gene = general mRNA: genes for mRNAs = class II gene
Type III gene = tRNA, 5S RNA: tDNA, 5S DNA = class III gene

firstly, transcription of type III gene was analyzed

Manley et al. (1980): Hela cell extract
- Promoter on fibroin genes
trancation assay (Tsujimoto et al. 1981) pBR322______________+1_______+514:

  ------------===============▆▆▆▆▆▆▆▆▆▆▆
	                     =========▶
  ----------------===========▆▆▆▆▆▆▆▆▆▆▆
  ---------------------======▆▆▆▆▆▆▆▆▆▆▆
  -------------------------==▆▆▆▆▆▆▆▆▆▆▆

_________↑__↑____↑__ ↑
_________becoming inactive how long the genes are cut
__
*: Class II genes = TATA box (majority): important for transcription

these can be separated by an electrophoresis because the lengths (or masses) of mRNA are different

signal can be activated when the region of -29~+6 is present

⇒ analyze this sequence - single base is solely important (Hirose, Takeuchi & Suzuki 1982)

Used the method proposed by Manley et al. = 0.4 M (NH₄)₂SO₄
trancation assay: synthesized by ³²P-UTP, CTP, GTP and ATP


expression level of transcription is two to three times hihger when -860 is contained in silk gland (or serictery) than when not
Used the extraction of Hela cell and stink bug


____A and B are the same

slow-growing mutant, called poky

poky (mother, ♀) → poky (all children)
poky (father, ♂) → normal (all children)

→ mutation occurred on mitochondria

16569 base pairs - circular gene

☛ mitochondria

Mitochondrial gene (mt gene, ミトコンドリア遺伝子)

unstable: 16kbp (mammals). 20-100 kbp (protozoa, fungi), 60 kbp (tobacco), 2400 kbp (muskmelon)

☛ chloroplast

Chloroplast gene (葉緑体遺伝子, chl gene)

DNA extracted from chloroplats (later confirmed DNA in all plastids)

Chloroplast genome

Determined the sequence of chloroplast DNA (*: tRNA)
---███-----███████---░--▒-▒---███████---░░--▒▒--██--- DNA
Vals tRNA  16S mRNA  Ile* Aln*         23S        4.5S 5S  Asm

Plasmid (プラスミド)

Assay by using cultured cells (培養細胞利用検定法)

New castle desease virus = poly(I):poly(c) (Ohno & Taniguchi 1982)
Cells      +                      –             +
          Mouse              (Virus)    Human b    
FM3A  2187                 < 6            < 6
F1        729                   < 6           < 6
F2        729                   < 6       162 (6 × 27)
F3       2187 (729 × 3)  < 6         54 (6 × 9)
F4        729                  < 6         18 (6 × 3)
F5        729                  < 6             162

Chitin lysozyme gene¹1
Chitin lysozyme gene + thymidine kinase seequence

↓ (culture: various parts collected from chitin)
chichin oviduct cell (+++)
chichin macrophase (–)
chichin fibroblast (–)
rat fibroblast (–)

¹: + steroid hormone →

cells endocytose fluorescent substance to thymidine kinase
→ assay by the gene
→ the substances induced by hormone excite DNA

Δ-161~＋15: thimidine kinase gene

Assay system by using fertilized egg

tK
    █
    █      █
    █      █      █
    █      █      █
    █      █      █      █      ▄
-1700 -600 -340 -150   -90
  PMK

-1.7 kbp = remaining: 0K, -99 bp leaving: 0K

DNA marker (DNAマーカー)

Terminal Restriction Fragment Length Polymorphism (T-RFLP) Protocol

PCR amplify DNA using labeled primer(s)

Component = Working concentration
Unlabeled forward primer = 10 μM
Unlabeled reverse primer = 10 μM
HEX labeled forward primer = 10 μM
FAM labeled reverse primer = 10 μM
PerkinElmer PCR Buffer II = 10X
PerkinElmer MgCl₂ Solution = 25 mM
dNTP's = 2 mM (each)
BSA = 20 mg/mL
PerkinElmer AmpliTaq DNA Polymerase = 5 U/μL

                     Reaction
    Component        25 μL   50 μL   75 μL  100 μL
    Forward primer    0.50     1.00     1.50     2.00
    HEX forward pr.*  0.75     1.50     2.25     3.00
    Reverse primer    0.50     1.00     1.50     2.00
    FAM reverse pr.*  0.75     1.50     2.25     3.00
    Buffer            2.50     5.00     7.50    10.00
    MgCl2             3.75     7.50    11.25    15.00
    dNTP's            2.50     5.00     7.50    10.00
    BSA               0.05     0.10     0.15     0.20
    milliQ H2O       13.35    26.70    40.05    53.40
    Taq               0.10     0.20     0.30     0.40
    Template          1.50     3.00     4.50     6.00

* NOTE: Run separate PCR reactions for each labeled primer (i.e., run HEX labeled forward primer with unlabeled reverse primer; run FAM labeled reverse primer with unlabeled forward primer).

Thermal cycler (サーマルサイクラー)

- If positive, purify product
- If you have both HEX and FAM labeled product, purify separately and keep them separately until digestion (see below)

Digest the PCR product

  Component                    Working concentration 
  Labeled PCR product*         250-300 ng (each) per digest
  GibcoBRL REact buffer        10X
  (restriction enzyme specific)
  GibcoBRL restriction enzyme  10 U/μL
  milliQ H2O                   na

* NOTE: If you have both HEX and FAM labeled product, combine 250–300 ng of each labeled product in a single digest (i.e., 250 – 300 ng of HEX labeled product combined with 250–300 ng of FAM labeled product to give a total of 500–600 ng of labeled product in a 20 μL digest)

    Component            Volume (20 μL digest)
    Labeled PCR product  depends on PCR product
                         concentration 
    Buffer               2.00
    Restriction enzyme   1.50
    milliQ H2O           dilute digest to 20 μL
                         (PCR product + H2O = 16.5 μL
                         + buff. + re. enz. = 20 μL)

Digest purified PCR product with preferred restriction enzyme(s)

- If digesting with multiple restriction enzymes, do a separate digest for each restriction enzyme

Digested product is ready for T-RFLP analysis

Protocol provided by Brian Wade at the MSU Center for Microbial Ecology

including designing and constructing biological modules, biological systems, and biological machines

Mycoplasma mycoides JCVI-syn1.0