北京大学：《微生物学》课程PPT教学课件（英文版）Chapter 12 Microbial Evolution and Systematics

Chapter 12 Microbial Evolution and systematics Ribosomal rnas as evolutionary chronometers: The 16s rRNA or 18S rRNA Technique

Chapter 12 Microbial Evolution and Systematics Ribosomal RNAs as Evolutionary Chronometers: The 16S rRNA or 18S rRNA Technique

Ribosomal rnas as evolutionary chronometers D Reasons: Ancient molecules Functionally constant Universally distributed Moderately well conserved in sequence across broad phylogenetic distances

Ribosomal RNAs as Evolutionary Chronometers Reasons: • Ancient molecules • Functionally constant • Universally distributed • Moderately well conserved in sequence across broad phylogenetic distances

The 16s rRNA or 18S rRNA Technique 705 ribosome 30S subunit

The 16S rRNA or 18S rRNA Technique

The 16srRNA or 18S rRNA Technique 口 In Prokaryotes: SS rRNA is too small. contains limited info 23S rRNA iS too large, too difficult to manage 16srRNA has the right size for studies 口 In Eukaryotes 18S rRNA iS used for phylogenetic measurements

The 16S rRNA or 18S rRNA Technique In Prokaryotes: • 5S rRNA is too small, contains limited info • 23S rRNA is too large, too difficult to manage • 16S rRNA has the right size for studies In Eukaryotes: • 18S rRNA is used for phylogenetic measurements

The 16S rRNA or Organismic Isolate DNA 18S rRNA Techniqe ue 16 S RNA gene種 Heat to separate strands: add specific primers Primer extension with DNA polymerase Repeat above steps for many PCR cycles to yield multiple copies of 1 6S ribosomal RNA gene Run agarose gel and check for correct sized PCR product Purify PCR product Sequence by sanger method

The 16S rRNA or 18S rRNA Technique

The 16srRNA or 18S rRNA Technique Ribosomal Database Project[ROP]: http://www.cme.msu.edu/rop Compare your sequences with the database to find out the organisms you identify

The 16S rRNA or 18S rRNA Technique Ribosomal Database Project (RDP): • http://www.cme.msu.edu/RDP Compare your sequences with the database to find out the organisms you identify

圈 CUAGAGCUGAC Phylogenetic B CCUAGAGCUGGG Trees from DNA CCAAGACGUGGC GGUAGAUGUGCC Sequences Evolutionary distance Corrected evolutionary distance B 025 o Distance-Matrix 圈 Method for 圈 0.42 0.61 0.30 generating the trees 0.44 口 Evolutionar D 033 0.44 Distance (ED) Phylogenetic tree(computer-generated best fit to corrected evolutionary distances 口 Computer compare the sequence differences and build the 0.08 phylogenetic tree based on corrected D (c)

Phylogenetic Trees from DNA Sequences Distance-Matrix Method for generating the trees Evolutionary Distance (ED) Computer compare the sequence differences and build the phylogenetic tree based on corrected ED

Phylogenetic Trees from DNA Sequences Bacteria Archaea Eukarya Animals Gram-Crenarchaeota Plants y Korarcha Tri Thermodesulfobacteria Microsporic Diplomonads

Phylogenetic Trees from DNA Sequences

Signature Sequences: unique to certain group of organisms Applications: Phylogenetic Probes Table 18. 1 Signature sequences from 16S or 18S rRNA defining the three domains of living organisms Occurrence among Oligonucleotide sequencea pproximate position Archaea Bacteria Eukarya CACYYG 315 0 CYAAYUNYG 510 AAACUCAAA 910 100 AACUUAAAG 910 100 NUUAAUBCG 60 >95 0 YUYAABUG 000 100 CAACCYYCR 1110 >95 UUCCCG LCCCUG 05 100 CUCEUUG 0 UACAC 1400 0 99 100 100 Y=amy pyrmidine R any purine N any purine or pyrimidine Refer to Figure 18. 7 for numbering scheme of 16S, RNA Occurrence refers to percentage of organisms examnined in any domain

Signature Sequences: unique to certain group of organisms Applications: Phylogenetic Probes

Questions o Why is Ribosomal rNAs used as Evolutionary Chronometers o Why is 16s rRNA employed to study phylogenetics rather than the smaller 5s rrNR and the large 23S rRNA in Prokaryotic? o How to sequence 16s rRNA from inside the cells? o How to identify a prokaryotic or eu karyotic organism based on 16s or 18SrrNA? o How to build Phylogenetic Trees from DNA Sequences? o Why it is said the archaea closer to eu karya than bacteria is? o What is signature sequence and how can it be used?

Questions Why is Ribosomal RNAs used as Evolutionary Chronometers Why is 16S rRNA employed to study phylogenetics rather than the smaller 5S rRNR and the large 23S rRNA in Prokaryotics? How to sequence 16S rRNA from inside the cells? How to identify a prokaryotic or eukaryotic organism based on 16S or 18S rRNA? How to build Phylogenetic Trees from DNA Sequences? Why it is said the archaea closer to eukarya than bacteria is? What is signature sequence and how can it be used?