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CELL BIO II Experiment #4:
CELL BIO II Experiment #4:
*'''Introduction'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
*'''Introduction'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
  Statement of objectives or aims of the experiment in the student’s own words.
<pre>Statement of objectives or aims of the experiment in the student’s own words.
   (not to be copied from the Lab Manual)
   (not to be copied from the Lab Manual)</pre>
*'''MATERIALS AND METHODS'''<span style="font-weight:bold;color:OrangeRed;"> 0 points</span> ''':'''
*'''MATERIALS AND METHODS'''<span style="font-weight:bold;color:OrangeRed;"> 0 points</span> ''':'''
  This should be a brief synopsis and must include any changes or deviations  
<pre>This should be a brief synopsis and must include any changes or deviations from the procedures  
  from the procedures outlined in the Lab Manual. Specify which organisms were  
outlined in the Lab Manual. Specify which organisms were used to create the phylogram.</pre>
  used to create the phylogram.
*'''RESULTS'''<span style="font-weight:bold;color:OrangeRed;"> 4 points</span> ''':'''
*'''RESULTS'''<span style="font-weight:bold;color:OrangeRed;"> 4 points</span> ''':'''
  A print out of the phylogram will suffice.
<pre>A print out of the phylogram will suffice.</pre>
*'''DISCUSSION'''<span style="font-weight:bold;color:OrangeRed;"> 4 points</span> ''':'''
*'''DISCUSSION'''<span style="font-weight:bold;color:OrangeRed;"> 4 points</span> ''':'''
  Responses to discussion questions.
<pre>Responses to discussion questions.</pre>
*'''SUMMARY |CONCLUSION'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
*'''SUMMARY |CONCLUSION'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
  Two sentence summary of your findings.
<pre>Two sentence summary of your findings.</pre>
*'''REFERENCES'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
*'''REFERENCES'''<span style="font-weight:bold;color:OrangeRed;"> 1 point</span> ''':'''
  Credit is given for pertinent references obtained from sources other than the Lab Manual.
<pre>Credit is given for pertinent references obtained from sources other than the Lab Manual.
   This point is in addition to the 10 for the lab report..
   This point is in addition to the 10 for the lab report..</pre>


===INTRODUCTION===
===INTRODUCTION===
Line 51: Line 50:
| Evolution can be defined as descent with modification.  In other words, changes in the nucleotide sequence of an organsim’s genomic DNA is inherited by the next generation.  According to this, all organisms are related through descent from an ancestor that lived in the distant past.  Since that time, about 4 billion years ago, life has undergone an extensive process of change as new kinds of organisms arose from other kinds existing in the past.<br /> The evolutionary history of a group is called a phylogeny, and can be represented by a phylogram (Figure 1).  A major goal of evolutionary analysis is to understand this history.  We do not have direct knowledge of the path of evolution, as by definition, extinct organisms no longer exist.  Therefore, phylogeny must be inferred indirectly.  Originally, evolutionary analysis was based upon the organisms’ morphology and metabolism.  This is the basis for the Linnaean classification scheme (the “Five Kingdoms” scheme).  However, this method can lead to mistaken relationships.  Different species living in the same environment may have similar morphologies in order to deal with specific environmental factors.  Thus these similarities have nothing to do with how related the organisms are, but are a direct result of shared surroundings.  However, with the advent of genomics, organisms can be grouped based upon their sequence relatedness.  Since evolution is a process of inherited nucleotide change, analyzing DNA sequence differences allows for the reconstruction of a better phylogenetic history.<br/>
| Evolution can be defined as descent with modification.  In other words, changes in the nucleotide sequence of an organsim’s genomic DNA is inherited by the next generation.  According to this, all organisms are related through descent from an ancestor that lived in the distant past.  Since that time, about 4 billion years ago, life has undergone an extensive process of change as new kinds of organisms arose from other kinds existing in the past.<br /> The evolutionary history of a group is called a phylogeny, and can be represented by a phylogram (Figure 1).  A major goal of evolutionary analysis is to understand this history.  We do not have direct knowledge of the path of evolution, as by definition, extinct organisms no longer exist.  Therefore, phylogeny must be inferred indirectly.  Originally, evolutionary analysis was based upon the organisms’ morphology and metabolism.  This is the basis for the Linnaean classification scheme (the “Five Kingdoms” scheme).  However, this method can lead to mistaken relationships.  Different species living in the same environment may have similar morphologies in order to deal with specific environmental factors.  Thus these similarities have nothing to do with how related the organisms are, but are a direct result of shared surroundings.  However, with the advent of genomics, organisms can be grouped based upon their sequence relatedness.  Since evolution is a process of inherited nucleotide change, analyzing DNA sequence differences allows for the reconstruction of a better phylogenetic history.<br/>
|-
|-
|[[File:TreeLife.png|thumb|center|alt=The Tree of Life.|Tree of life based on 16S ribosomal RNA (image credit: NR Pace, Science 1997)]]
|[[File:TreeLife.PNG|center|alt=The Tree of Life.|Tree of life based on 16S ribosomal RNA (image credit: NR Pace, Science 1997)]]
|-style="background-color:powderblue;"
|-style="background-color:powderblue;"
|Of course, when comparing DNA sequences, the question of which genes to use arises.  The most widely used genes are those coding for the 16S rRNA gene in prokaryotes and the 18S rRNA gene in eukaryotes.  These genes code for small subunit ribosomal RNA and are used for evolutionary analysis because they 1) are found in all organisms, 2) are functionally conserved, 3) vary only slightly between organisms (their nucleotide sequence changed slowly throughout evolution), and 4) have adequate length.  In this lab, you will be performing evolutionary analysis by constructing a phylogram of 15 microbes spanning bacteria, archaea and eukarya.  You will find and download rRNA sequences, align them and use that alignment to create a phylogram.
|Of course, when comparing DNA sequences, the question of which genes to use arises.  The most widely used genes are those coding for the 16S rRNA gene in prokaryotes and the 18S rRNA gene in eukaryotes.  These genes code for small subunit ribosomal RNA and are used for evolutionary analysis because they 1) are found in all organisms, 2) are functionally conserved, 3) vary only slightly between organisms (their nucleotide sequence changed slowly throughout evolution), and 4) have adequate length.  In this lab, you will be performing evolutionary analysis by constructing a phylogram of 15 microbes spanning bacteria, archaea and eukarya.  You will find and download rRNA sequences, align them and use that alignment to create a phylogram.
Line 305: Line 304:
|-
|-
|
|
[[ File:Phylo.PNG|center|thumb|Phylogram with internal nodes (a, b, c, d) and tips (1, 2, 3, 4, 5).  Nodes at the tips are species that exist today, and internal nodes are extinct ancestors.]]
[[ File:Phylo.PNG|center|Phylogram with internal nodes (a, b, c, d) and tips (1, 2, 3, 4, 5).  Nodes at the tips are species that exist today, and internal nodes are extinct ancestors.]]
|-style="background-color:powderblue;"
|-style="background-color:powderblue;"
|A rooted tree shows the unique path from an ancestor (internal node) to each strain.  Trees are rooted by inclusion of an outgroup in the analysis.  An outgroup is an organism that is less closely related to the other organisms under study than the organisms are to each other.
|A rooted tree shows the unique path from an ancestor (internal node) to each strain.  Trees are rooted by inclusion of an outgroup in the analysis.  An outgroup is an organism that is less closely related to the other organisms under study than the organisms are to each other.

Latest revision as of 20:38, 4 March 2013

EXPERIMENT # 4

BIOL 200 Cell Biology II LAB, Spring 2013

Hunter College of the City University of New York

Course information

Instructors: TBD

Class Hours: Room TBD HN; TBD

Office Hours: Room 830 HN; Thursdays 2-4pm or by appointment

Contact information:

  • Dr. Weigang Qiu: weigang@genectr.hunter.cuny.edu, 1-212-772-5296


Experiment #4

The Tree of Life and Molecular Identification of Microorganisms

Objective

To classify microorganisms and determine their relatedness using molecular sequences.

LAB REPORT GRADING GUIDE

CELL BIO II Experiment #4:

  • Introduction 1 point :
Statement of objectives or aims of the experiment in the student’s own words.
  (not to be copied from the Lab Manual)
  • MATERIALS AND METHODS 0 points :
This should be a brief synopsis and must include any changes or deviations from the procedures 
outlined in the Lab Manual. Specify which organisms were used to create the phylogram.
  • RESULTS 4 points :
A print out of the phylogram will suffice.
  • DISCUSSION 4 points :
Responses to discussion questions.
  • SUMMARY |CONCLUSION 1 point :
Two sentence summary of your findings.
  • REFERENCES 1 point :
Credit is given for pertinent references obtained from sources other than the Lab Manual.
  This point is in addition to the 10 for the lab report..

INTRODUCTION

MATERIALS

  • Required hardware: Computer

Table 1

Volume 1A (Gram-negative bacteria)

Escherichia coli

ACCESSION #174375

Helicobacter pylori

ACCESSION #402670

Salmonella typhi

ACCESSION #2826789

Serratia marcescens

ACCESSION #4582213

Treponema pallidum

ACCESSION #176249

Additional species: Agrobacterium tumefaciens, Boredetella pertussis, Thermus aquaticus, Yersinia pestis, Borrelia burgdorferi. (Note: To search for unlisted 16S sequences, type key words such as “yersinia AND 16S [gene]” in the NCBI GenBank search box.)

Volume 1B (Rikettsias and endosymbionts)

Baronella bacilliformis

ACCESSION #173825

Chlamydia trachomatis

ACCESSION #2576240

Rickettsia rickettsii

ACCESSION #538436

Additional species: Coxiella burnetii, Thermoplasma acidophilum

Volume 2A (Gram-positive bacteria)

Bacillus subtilis

ACCESSION #8980302

Dinococcus radiodurans

ACCESSION #145033

Staphylococcus aureus

ACCESSION #576603

Additional species: Bacillus anthracis, Clostridium botulinum, Lactobacillus acidophilus, Streptococcus pyogenes

Volume 2B (Mycobacteria and nocardia)

Mycobacterium haemophilum

ACCESSION #406086

Mycobacterium tuberculosis

ACCESSION #3929878

Additional species: Mycobacterium bovis, Nocardia orientalis

Volume 3A (Phototrophs, chemolithotrophs, sheathed bacteria, gliding bacteria)

Anabaena sp.

ACCESSION #39010

Cytophaga latercula

ACCESSION #37222646

Nitrobacter wiogradskyi

ACCESSION #402722

Additional species: Heliothrix oregonensis, Myxococcus fulvus, Thiobacillus ferrooxidans

Volume 3B (Archeobaceria)

''Methanococcus jannaschii

ACCESSION #175446

Thermotoga subterranean

ACCESSION #915213

Additional species: Desulfurococcus mucosus, Halobacterium salinarium, Pyrococcus woesei

Volume 4 (Actinomycetes)

Actinomyces bowdenii

ACCESSION #6456800

Actinomyces neuii

ACCESSION #433527

Actinomyces turicensis

ACCESSION #642970

Eukaryotic representative (used as outgroup for rooting the phylogenetic tree)

Saccharomyces cerevisiae

ACCESSION #172403

ANALYSIS

DISCUSSION

References

© Weigang Qiu, Hunter College, Last Update Jan 2013