lecture 3 -the diversity of genomes and the tree of life
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PowerPoint for BI 520-01 Spring 2013TRANSCRIPT
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Chapter 1
© Garland Science 2008
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Cells Can Be Powered by a Variety of Free Energy Sources
Phototrophic – organisms that obtain their energy from harvesting the energy from sunlight (e.g., many types of bacteria, plants and algae).
• We and virtually all living things that we ordinarily see around us depend on this major input of free energy.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Cells Can Be Powered by a Variety of Free Energy Sources
Organotrophic – organisms that obtain their energy from feeding on other living things (e.g., animals, fungi, gut microflora).
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Some Cells Fix Nitrogen and Carbon Dioxide for OthersLithotrophic - 'lithos' (rock) and 'troph' (consumer) - an organism that uses inorganic substrates to obtain reducing equivalents for use in biosynthesis (e.g., carbon dioxide fixation) or energy conservation via aerobic or anaerobic respiration.
Diazotrophs are bacteria that fix atmospheric nitrogen gas into a more usable form such as ammonia.
Methanogens are archaea that produce methane as a metabolic byproduct in anoxic conditions.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
The Greatest Biochemical Diversity Exists Among Prokaryotic Cells
Epulopiscium fishelsoni (0.6
mm)
Thiomargarita namibiensis (0.75 mm)
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
In extending their capacity to live in biochemically diverse habitats, eukaryotes went down the path of symbiosis, rather than reinventing
the “metabolic wheel”.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
In fact, the origin of eukaryotes is thought to arise from the merging of symbiotic
prokaryotic cells!
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
The Tree of Life Has Three Primary Branches:Bacteria, Archaea, and Eukaryotes
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
• rRNA is the most conserved (least variable) gene in all cells.
• For this reason, genes that encode the rRNA (rDNA) are sequenced to identify an organism's taxonomic group, calculate related groups, and estimate rates of species divergence.
• Many thousands of rRNA sequences are known and stored in specialized databases such as RDP-II and the European SSU database.
Ribosomal Genes
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Prokaryotes have 70S ribosomes, each consisting of a small (30S) and a large (50S) subunit. Their large subunit is composed of a 5S rRNA subunit (consisting of 120 nucleotides), a 23S rRNA subunit (2900 nucleotides) and 34 proteins. The 30S subunit has a 1540 nucleotide RNA subunit (16S rRNA) bound to 21 proteins.
Eukaryotes have 80S ribosomes, each consisting of a small (40S) and large (60S) subunit. Their large subunit is composed of a 5S RNA (120 nucleotides), a 28S RNA (4700 nucleotides), a 5.8S subunit (160 nucleotides) and ~49 proteins. The 40S subunit has a 1900 nucleotide (18S rRNA) RNA and ~33 proteins.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Both prokaryotic and eukaryotic ribosomes can be broken down into two subunits
Type Size Large subunit Small subunit
prokaryotic 70S 50S (5S, 23S) 30S (16S)
eukaryotic 80S 60S (5S, 5.8S, 28S) 40S (18S)
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
The ribosomes found in mitochondria of eukaryotes also consist of large and small subunits bound together with proteins into one 70S particle.
These organelles are believed to be descendants of bacteria and as such their ribosomes are similar to those of bacteria (e.g., they have a 12S and 16S rRNA subunit)
Roughly 200 genes are universal to life
Model Organisms
Chapter 1
Model organisms are those with a wealth of biological data that make them attractive to study as examples for other species – including humans – that are more difficult to study directly.
• Often chosen because they are easy to manipulate experimentally. This usually will include characteristics such as short life-cycle, techniques for genetic manipulation (inbred strains, stem cell lines, and methods of transformation) and ease of care. They also consider size, accessibility, conservation of mechanisms, and potential economic benefit.
• Sometimes, the genome arrangement facilitates the sequencing of the model organism's genome, for example, by being very small or concise (e.g. yeast, Arabidopsis, or pufferfish).
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Genetic models have short generation times, such as the fruit fly (D. melanogaster) and nematode worm (C. elegans) and thus, we can study genes, etc., easier in them.
Experimental models (S. cerevisiae and E.coli) are easily manipulated and observable traits have been documented.
Genomic models, with a pivotal position in the evolutionary tree.
Historically, model organisms include a handful of species with extensive genomic research data, such as the NIH model organisms. Yet, as comparative molecular biology has become more common, some researchers have sought model organisms from a wider assortment of lineages on the tree of life.
NIH Model organismsOrganism Genome
SequencedHomologous
RecombinationBiochemis
try
Prokaryote
Escherichia coli Yes Yes Excellent
Eukaryote, unicellular
Dictyostelium discoideum Yes Yes Excellent
Saccharomyces cerevisiae Yes Yes Good
Schizosaccharomyces pombe
Yes Yes Good
Chlamydomonas reinhardtii
Yes No Good
Tetrahymena thermophila Yes Yes Good
Eukaryote, multicellular
Caenorhabditis elegans Yes Difficult Not so good
Drosophila melanogaster Yes Difficult Good
Arabidopsis thaliana Yes No Poor
Vertebrate
Danio rerio Yes Difficult? Good
Mus musculus Yes Yes Good
Homo sapiens Yes Yes Good
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Escherichia coli
Our model prokaryote
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
E. coli is by far the most frequently used model organism because of its small size, short generation time, ease of culture, and amenity to genetic manipulation.
Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratory environment, and, unlike wild type strains, have lost their ability to thrive in the intestine. They have also been “removed” of their toxins.
Genetic information is easily transferred to E. coli, and thus we use it to amplify DNA and express proteins (insulin). But it lacks post-translational modifications and has codon issues, etc., most of which can be overcome.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Saccharomyces cerevisiae
The model organism that’s been in our hearts for as long as we can remember!
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Civilization owes yeast for its use since ancient times in baking and brewing.
It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology.
Many proteins important in human biology were first discovered by studying their homologs in yeast.
• cell cycle proteins, signaling proteins, and protein-processing enzymes.
As a eukaryote, S. cerevisiae shares the complex internal cell structure of plants and animals without the high percentage of non-coding DNA that can confound research in higher eukaryotes.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Drosophila melanogaster
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
One of the most commonly used model organisms in biology, including studies in genetics, physiology and life history evolution.• They are small and easily raised. • Morphology is easy to identify.• Has a short generation time (~10 days at RT) • Has a high fecundity• Males and females are readily distinguished
(virgins)• It has only four pairs of chromosomes.• Genetic transformation techniques have been
available since 1987.• About 75% of known human disease genes have
a recognizable match in the genetic code of fruit flies
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Zebrafish (Zebra danio)
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Because a zebrafish embryo is completely transparent…
…it is widely used to study morphological development of vertebrates.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
The mouse is one of the major model organisms for medicine
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
Mice are by far the most genetically altered laboratory mammal.
They are the primary model organism for most human diseases, including cancer, because almost every human gene has a mouse homolog.
Knock-out mice make this even more important.
THE DIVERSITY OF GENOMES AND THE TREE OF LIFE
• Genes Can Be Transferred Between Organisms, Both in the Laboratory and in Nature
• Sex Results in Horizontal Exchanges of Genetic Information Within a Species
Chapter 1
© Garland Science 2008
2006Craig C. Mello and Andrew Fire's received a noble prize for RNAi
The Nobel Prize in Physiology or Medicine, 2007
Mario R. Capecchi, Martin J. Evans and Oliver Smithiesfor their discoveries of "principles for introducing specific gene modifications in mice by the use of embryonic stem cells"
M. CapecchiUniv. of Utah
Sir M. EvansCardiff Univ., UK
O. SmithiesUNC Chapel Hill
Cell TransformationGo to this website to perform your gel
electrophoresis
http://learn.genetics.utah.edu/content/labs/gel/
Once you understand the process, use your DNA detective skills to help solve a mystery.
http://www.pbs.org/wgbh/nova/sheppard/analyze.html
Or google NOVA DNA Fingerprint NOVA Online | Killer's Trail | Create a DNA Fingerprint
Plasmid - circular DNA molecule found in bacteria
genetic marker - gene that makes it possible to distinguish organisms that carry a plasmid with foreign DNA from those that don’t
Recombinant DNA – DNA that has been created artificially. DNA from two or more sources is incorporated into a single recombinant molecule.
Vocab
Transforming Bacteria
Transforming Plants
Transforming Animal Cells
• Can be transformed similar to plants.
• Some eggs are large enough to physically inject new DNA by hand. Which can “Knock Out” a gene
Why?1. Study gene function and regulation2. Generate new organismic tools for other
fields of research.3. Cure genetic diseases.4. Improve agriculture and related raw
materials. 5. Generate new systems or sources for
bioengineered drugs (e.g., use plants instead of animals or bacteria).
Making Transgenic Plants and Animals
term used to refer to an organism that contains genes from other organisms
Transgenic Organisms
Transgenic OrganismsTransgenic Bacteria
Transgenic Plants
Transgenic animals
Produce clotting factors insulin HGH
Stronger plantsMore productionPest resistance
More production
The organism of choice for mammalian genetic engineers. - small - hardy - short life cycle - genetics possible - many useful strains and tools
Can occur by homologous (H) or non-homologous (N-H) recombination
Frequency of N-H >> H (by at least 5000-fold) in mammalian cells
If you want H integrants, which you need for knock-outs, you must have a selection scheme for those.
The Problem: DNA Integration
Vector with a transgene
tk1 & tk2 - Herpes Simplex Virus thymidine kinase genes (make cells susceptible to gancyclovir)
Neo - neomycin resistance geneHomologous regions - homologous to the chromosomal targetTransgene - foreign gene
Nonhomologous recombinationhomologoussequencetk1
homologoussequence neo tk2transgene
chromosome
homologoussequencetk1
homologoussequence neo tk2transgene
chromosome
homol-->
Example of what happens with N-H recombination
Transformed cells are neo-resistant, but gancyclovir sensitive.
Homologous recombinantshomologoussequencetk1
homologoussequence neo tk2transgene
chromosome
chromosome
homologoussequence
homologoussequence neo transgene
If DNA goes in by HR, transformed cells are both neo-resistant and gancyclovir-resistant!
Use double-selection to get only those cells with a homologous integration event.
What happens with HR
To knock-out a gene:
1. Insert neo gene into the target gene.
2. Transform KO plasmid into embryonic stem cells.
3. Perform double-selection to get cells with the homologous integration (neo & gangcyclovir resistant).
4. Inject cells with the knocked-out gene into a blastocyst.
1.
KO
KO 2,3.
Blastocyst
EmbryonicStemcells
(ES cells)
Transfection
Grow in culture.Select for those that carry the transgene.
Inject into a blastocyst
Implant intopseudopregnantmouse
Identify offspring whichcarry the transgene intheir germline.
(mouse)
With DNA
How to make a transgenic mouse.
Blastocyst
EmbryonicStemcells
(ES cells)
Transfection
Grow in culture.Select for those that carry the transgene.
Inject into a blastocyst
Implant intopseudopregnantmouse
Identify offspring whichcarry the transgene intheir germline.
Chimeric mouse
(a) If the recipient stem cells are from a brown mouse, and the transgenic cells are injected into a black (female) mouse, chimeras are easily identified by their Brown/Black phenotype.
(b) To get a completely transgenic KO mouse (where all cells have KO gene), mate the chimera with a black mouse. Some of the progeny will be brown (its dominant), because some of the germ line cells will be from the KO cells. ½ the brown mice will have the transgene KO, because the paternal germ-line cell was probably heterozygous.
(c) To get a homozygous KO mouse (both chromosomes have the KO transgene), cross two brown transgenic heterozygotes. ~1/4 will be homozygous at the transgene locus.
Not necessarily 3:1
Cloning member of a population of genetically
identical organisms produced from a single cell
“Dolly” “Dolly” was an important
break through not just because she was a mammal.
Frogs were cloned back in 1950’s
Why was dolly so special? Research and answer this
question for me.