Kul.

9
Model (Micro) Organism

Dr. Rika Indri Astuti

Mk. Pengantar Bioteknologi
Departemen Biologi, FMIPA, IPB
2019/2020
Memahami  fenomena  fisiologi/gene0ka  pada  level  molekuler  suatu  
organisme  (manusia)  è  kompleks/sulit  

Reduksi    

focused on a few central chose to use the simplest

molecular mechanisms organisms (bacteria and
(replication, transcription, bacteriophages) in which to
protein synthesis and the study these phenomena.
control of gene activity)    
The biological question determines the model organism
•  bacterium Escherichia coli,
•  yeast Saccharomyces cerevisiae,
•  worm Caenorhabditis elegans and
•  fruit fly Drosophila melanogaster
•  rodent, Mus musculus
•  plant, Arabidopsis thaliana
•  fish, Zebra fish , Danio rerio
•  frog, Xenopus laevis
Penggunaan Lalat Buah untuk Mengamati Fenomena
Gen Terpaut Kromosom Sex

Morgan Hunt (Columbia University,

1933), Embriologist

•  The first solid evidence associating

a specific gene with a specific
chromosome

•  Convincing evidence that

chromosomes are the location of
Medel`s heritable factors (gene)

•  He discovered that the gene for

eye color in fruit flies is a sex
linked trait carried on the X
Why fruit flies : chromosome
•  Quick breeding (2 weeks)
•  Have only 4 pairs of chromosomes (3 auotosomes, 1 pair of sex crhomosome)
•  Produce many offspring (hundreds)
 SAKSIKAN VIDEO BERIKUT (Video 1. Zhong Zhong dan Hua Hua)

https://www.youtube.com/watch?v=gFJkPWTl2PA

Kloning primata pertama tersebut diperuntukkan

untuk pengujian penyakit degeneratif yang lebih
akurat 
(menggunakan dua organisme primata yang secara genetik sama
persis (hasil klon)).
Iden0cal  monkeys  as  organism  model    
-­‐  Gene0c  disorder  
-­‐  Test  drug  

h@ps://www.nature.com/ar0cles/
d41586-­‐018-­‐01027-­‐z  
 Tiap  Organisme  Model  memiliki  Aplikasi  Riset  
yang  Berbeda  

h@ps://www.nature.com/scitable/topicpage/the-­‐use-­‐of-­‐animal-­‐models-­‐in-­‐studying-­‐855  
Untuk  mengetahui  kelebihan  masing-­‐masing  organisme  model,  kunjungi  situs  berikut    
 h@ps://www.yourgenome.org/facts/what-­‐are-­‐model-­‐organisms  
 
Fokus  Materi  ini:  Mikrob  sebagai  
Organisme  Model  
 Model  (micro)  Organisms  
a)  Mengurangi permasalahan etika pemakaian hewan sebagai
hewan percobaan.
b)  Dapat menjadi landasan pengembangan metode analisis baru,
terutama untuk standarisasi suatu analisis.
c)  Representasi dari organisme tingkat tinggi untuk berbagai
fenomena biologis

Technical advantages
•  Relatively easy to grow and maintain in a restricted space
•  Relatively easy to provide necessary nutrients for growth
•  Relatively short generation time (birth è reproduction è birth)
•  Relatively well understood growth and development as well as its
genetic aspects
•  Closely resemble others organisms or systems
 Model  microorganisms  

Bacteria
•  Escherichia coli

Yeast
•  Saccharomyces cerevisiae
•  Schizosaccharomyces pombe

Amoeba
•  Dictyostelium discoideum
motility, chemotaxis, phagocytosis, endocytic vesicle
traffic, cell adhesion, pattern formation, caspase-
independent cell death, and, more recently, autophagy
and social evolution

Dictyostelium discoideum    
Escherichia  coli  

•  Many strains are avirulent

•  Small and rapid reproduction
•  Easy to culture
•  Genome is single chromosome –haploid ,
small genome, 4.5 million bp/4000 genes,
completely sequenced
•  Laboratory procedures are well established
•  Relatively easy to manipulate, physiologically
and genetically
•  Gene deletion library is available (Keio
library)

Favorable in the study of genetics, biochemistry

and physiology :
E. coli K-12
 Keio  Collec>on  
•  a set of single-gene deletion mutants of Escherichia coli K-12
•  Developed as a collaboration between the Institute for Advanced Biosciences,
Keio University (Japan), Nara Institute of Science and Technology (Japan), and
Purdue University
•  Used for studying function of particular gene(s)

Delesi  gen:  ORF  gen  tertentu    

didelesi/dihilangkan  melalui  
reaksi  homologi  rekombinasi  
(hal  14)  
 Gene  Dele0on  Library  
Satu  sumur  ((A)  mengandung  koloni  bakteri  
yang  satu  gen  terdelesi  (mis.  Gen  A)  

Satu  sumur  (B)  mengandung  koloni  bakteri  

yang  satu  gen  terdelesi  (mis.  Gen  B),  dst  

Tiap  sumur  mengandung  koloni  khamir  

yang  delesi  gen  nya  berbeda-­‐beda    

Jika  bakteri  memiliki  (misal)  10.000  gen  ,maka  ada  10.  000  koloni  mutan  delesi  yang  
berbeda  beda.    
 Pelajari  :  
h@ps://www.ncbi.nlm.nih.gov/pmc/ar0cles/PMC1681482/  
 Keio  Collec>on  
•  Keio collection of mutant strains was designed to create in-frame deletions
upon excision of a kanamycin resistance cassette.  
•  Each deleted gene is replaced with a kanamycin resistance cassette that can be
excised by FLP recognition target (FRT) recombination
 Keio  Collec>on  :  Applica>on  
•  Determine the E. coli genes important for cell morphology

doi:  10.1128/mBio.01977-­‐16.7  March  2017  mBio  vol.  8  no.  2  e01977-­‐16  

 E. coli dapat digunakan untuk mempelajari
kejadian penyakit kanker pada manusia
Kanker  diketahui  dapat  disebabkan  oleh  kegagalan  memperbaiki  DNA  akibat  
mutasi  (failure  in  DNA  repair)  

•  DNA mismatch repair (MMR) is a highly conserved biological pathway that

plays a key role in maintaining genomic stability

•  The specificity of MMR is primarily for base-base mismatches and insertion/

deletion mispairs generated during DNA replication and recombination

•  Escherichia coli MutS and MutL and their eukaryotic homologs, MutSα and
MutLα (MSH2 and MLH1), respectively, are key players in MMR-associated
genome maintenance

•  Defects in MMR are associated with genome-wide instability, predisposition to

certain types of cancer including hereditary non-polyposis colorectal cancer,
resistance to certain chemotherapeutic agents, and abnormalities in
meiosis and sterility in mammalian systems

Li,  Cell  Research  (2008)  18:85-­‐98.  

 DNA  Repair  Systems  

Comprehensive  study  in  DNA  

repair  systems  are  mostly  
conducted  in  E.  coli  
Mekanisme DNA repair pada E. coli homolog (mirip) dengan pada
Manusia
When  there  is  mistmatch  :  
 
 
The  MutS  and  MutL  form  
complexes  (homodimer  in  E.  coli,  
heterodimer  in  eukaryote)  
 
The  downstream  protein  
requirements  for  eukaryo0c  
mismatch  repair  also  remain  to  be  
established  (as  indicated  by  
ques0on  marks),  and  these  may  
differ  for  the  repair  of  different  
heteroduplexes.  
 
 SSB,  single-­‐strand  DNA-­‐binding  
protein;  RPA,  replica<on  protein  A,  
a  eukaryo<c  single-­‐strand  DNA-­‐
binding  protein.  
 Penelitian melaporkan: Kegagalan pada berbagai mekanisme DNA
repair menjadi penyebab terjadinya berbagai tipe tumor/kanker

The  choice  of  repair  

mechanism  is  largely  
defined  by  the  type  of  
lesion,  but  factors  such  
as  the  stage  of  the  cell  
cycle  also  have  a  role  

Key  proteins  involved  in  

each  DNA  damage  
response  mechanism,  
the  tumour  types  
usually  characterized  by  
DNA  repair  defects  

BER,  base  excision  repair;  NER,  nucleo0de  excision  repair;  NHEJ,  non-­‐homologous  end-­‐joining.    
Christopher  J.  Lord  &  Alan  Ashworth,  Nature  481,  287–294  (19  January  2012)  
 Yeast    
(Saccharomyces  cerevisiae  and  Schizosaccharomyces  
pombe)  
Yeast is one of the most widely used eukaryotic
model organisms.
It has been used as a model to study
aging,
regulation of gene expression ,
signal transduction
cell cycle
metabolism
apoptosis
neurodegenerative disorders
and many other biological processes.

For example, up to 30% of genes implicated in human disease may have

orthologs in the yeast proteome
 Linking  Yeast  to  Human  
Gene Deletion library :
•  Saccharomyces cerevisiae
(Winzeler et al. 1999 and Giaever et al.
2002).

•  S. pombe (Kim et al. 2010).

•  85% of nearly 5800 protein-coding of Saccharomyces genes are known

(higher than any other eukaryote)
•  Nearly 1000 yeast genes (i.e., ∼17%) are members of orthologous gene families
associated with human disease
•  For the majority of these genes their mammalian homolog is functional in yeast
and complements the yeast deletion mutant
 Yeast  dele>on  gene  collec>on  :  
Applica>on  
Koleksi  berbagai  khamir  mutan  delesi  (mis.  A,  B,  
C,  D)  lalu    masing-­‐masing  kultur  mutan  
diaplikasikan  senyawa  bioak0f  (potensial  
sebagai  obat).  
Gen  A        Gen  
  B           Gen  C          Gen  D          

Dianalisis  sel  mutan  mana  saja  yang  tetap  hidup  

dan  ma0  (amplifikasi  barcode/iden0fikasi  gen  
yang  terdelesi)  atau  analisis  ekspresi  gen  
dengan  microarray  

Sel  khamir  mutan  yang  ma0,  mengindikasikan  

bahwa  obat  X  bersifat  toksik  bagi  sel  yang  0dak  
memiliki  gen  C.    
 Study  in  Yeast    
Dr. Yoshinori Ohsumi has won the 2016 Nobel Prize in Physiology or Medicine for his
groundbreaking work on autophagy in yeast.

This is the process whereby

cells recycle their worn out
parts or where a cell eats
less essential bits of itself to
stay alive during times of
starvation
Physiological  importance  of  autophagy  
 Autophagy  in  yeast  
Macroautophagy entails the sequestration of
bulk cytoplasm or specific structures into
autophagomes

microautophagy (micropexophagy) è the

structures targeted to degradation are
recruited in proximity to the vacuole
membrane

micronucleophagy mediates the turnover

of part of the nuclear envelope and
content.

the  components  are  delivered  into  the  

interior  of  the  vacuole  are  degraded  by  
resident  hydrolases  

The  resul>ng  metabolites,  i.e.,  amino  acids,  sugars,  and  nucleo0des,  are  subsequently  transported  into  
the  cytoplasm  by  permeases  è  and  used  either  as  a  source  of  energy  or  as  building  blocks  for  the  
synthesis  of  new  macromolecules.  
 Yeast  for  comprehensive  autophagy  analysis  
Mechanism of autophagy is
conserved in yeast and
mammals

Autophagy is normal cellular

mechanism, induced by various
inducers and signals

- starvation
- environmental stress
- cytoplasmic materials
(protein  aggregates  and    
 organelles) Autophagy  is   Autophagy  is  
inhibited   induced  

Atg1 is important autophagy

modulator
(first discovery in yeast) Yongqiang  Chen,  Daniel  J.  Klionsky  
J  Cell  Sci  2011  124:  161-­‐170  
 Importance  of  Autophagy    
Important medical applications :
•  Chemotherapy resistance and
•  Amyloid-β aggregation in Alzheimer’s disease

•  a specialized form of autophagy that targets damaged mitochondria,

mitophagy, may not be working well in people with Parkinson’s disease
(protein PINK1).
•  Prolonging life span
 Yeast  as  Parkinson  Disease`s  model    
Different  pathways  and  their  dysfunc0ons  resul0ng  from  gene>c  modifica>ons  in  PD-­‐
related  genes  and  lead  to  an  increased  oxida>ve  stress.    
Yeast  models  of  PD  
 Yeast  models  of  PD  

The  PD-­‐associated  proteins  are  indicated  as  well  as  the  type  of  expression    
(heterologous  or  homologous).    
 Modeling  PD  in  yeast  
Quality  control  systems  and  aSyn  aggrega>on.  
aSyn can misfold and form oligomeric species that fibrillate and deposit into larger
aggregates, ultimately forming Lewy bodies. In healthy cells, the cellular quality
control systems are able to maintain proteostasis, avoiding this cascade of events.
Yeast  as  a  discovery  plaZorm  for  PD  
The discovery of small molecules, or natural products, which are able to
rescue aSyn toxicity and aggregation benefits from the combination of
approaches in yeast with those in other model systems.
 Studying  Life  Span  
Some conserved pathways and interventions of aging.
 Mekanisme  Penuaan  (longevity)  pada  khamir  
homolog  dengan  cacing,  lalat  buah,  mencit  dan  
manusia)  

Iwabu  et  al.,  2015  

 Saksikan  Video  1.    
Why  scien0st  need  model  organism?  
Thank  You  

h@p://www.yeastart.org/super-­‐yeast