Lecture 3 -the diversity of genomes and the tree of life


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Spring 2013

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  • First KO mice using their ES cell technology were produced in 1989 – now its like an industry
  • Lecture 3 -the diversity of genomes and the tree of life

    1. 1. Chapter 1 ©
    2. 2. Cells Can Be Powered by a Variety of Free Energy SourcesPhototrophic – organisms that obtain their energy from harvesting the energyfrom 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.
    3. 3. Cells Can Be Powered by a Variety of Free Energy SourcesOrganotrophic – organisms that obtain their energy from feeding on other livingthings (e.g., animals, fungi, gut microflora).
    4. 4. Some Cells Fix Nitrogen and Carbon Dioxide for Others Lithotrophic - 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 Methanogens are archaea thatatmospheric nitrogen gas into a more produce methane as a metabolicusable form such as ammonia. byproduct in anoxic conditions.
    5. 5. The Greatest Biochemical Diversity Exists Among Prokaryotic CellsThiomargarita namibiensis (0.75 mm) Epulopiscium fishelsoni (0.6 mm)
    6. 6. In extending their capacity to live in biochemically diversehabitats, eukaryotes went down the path of symbiosis, rather than reinventing the “metabolic wheel”.
    7. 7. In fact, the origin of eukaryotes is thought to arise from the merging of symbiotic prokaryotic cells!
    8. 8. The Tree of Life Has Three Primary Branches: Bacteria, Archaea, and Eukaryotes
    9. 9. Ribosomal Genes•rRNA is the most conserved (least variable) gene in all cells.•For this reason, genes that encode the rRNA (rDNA) aresequenced to identify an organisms taxonomicgroup, calculate related groups, and estimate rates of speciesdivergence.•Many thousands of rRNA sequences are known and storedin specialized databases such as RDP-II and the EuropeanSSU database.
    10. 10. 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 120nucleotides), a 23S rRNA subunit (2900 nucleotides) and 34 proteins. The 30S subunithas 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 40Ssubunit has a 1900 nucleotide (18S rRNA) RNA and ~33 proteins.
    11. 11. 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)
    12. 12. The ribosomes found in mitochondria of eukaryotes also consistof large and small subunits bound together with proteins intoone 70S particle.These organelles are believed to be descendants of bacteriaand as such their ribosomes are similar to those of bacteria(e.g., they have a 12S and 16S rRNA subunit)
    13. 13. Chapter 1Model organisms are those with a wealth of biological data that make themattractive to study as examples for other species – including humans – that aremore difficult to study directly.•Often chosen because they are easy to manipulate experimentally. This usuallywill include characteristics such as short life-cycle, techniques for geneticmanipulation (inbred strains, stem cell lines, and methods of transformation) andease of care. They also consider size, accessibility, conservation ofmechanisms, and potential economic benefit.•Sometimes, the genome arrangement facilitates the sequencing of the modelorganisms genome, for example, by being very small or concise (e.g.yeast, Arabidopsis, or pufferfish).
    14. 14. Genetic models have short generation times, such as the fruit fly (D.melanogaster) and nematode worm (C. elegans) and thus, we canstudy genes, etc., easier in them.Experimental models (S. cerevisiae and E.coli) are easily manipulatedand observable traits have been documented.Genomic models, with a pivotal position in the evolutionary tree.Historically, model organisms include a handful of species withextensive genomic research data, such as the NIH model organisms.Yet, as comparative molecular biology has become morecommon, some researchers have sought model organisms from awider assortment of lineages on the tree of life.
    15. 15. Organism Genome Sequenced Homologous Recombination BiochemistryProkaryoteEscherichia coli Yes Yes ExcellentEukaryote, unicellularDictyostelium discoideum Yes Yes ExcellentSaccharomyces cerevisiae Yes Yes GoodSchizosaccharomyces pombe Yes Yes GoodChlamydomonas reinhardtii Yes No GoodTetrahymena thermophila Yes Yes GoodEukaryote, multicellularCaenorhabditis elegans Yes Difficult Not so goodDrosophila melanogaster Yes Difficult GoodArabidopsis thaliana Yes No PoorVertebrateDanio rerio Yes Difficult? GoodMus musculus Yes Yes GoodHomo sapiens Yes Yes Good
    16. 16. Escherichia coli Our model prokaryote
    17. 17. E. coli is by far the most frequently used model organism because of its smallsize, short generation time, ease of culture, and amenity to genetic manipulation.Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratoryenvironment, and, unlike wild type strains, have lost their ability to thrive in theintestine. They have also been “removed” of their toxins.Genetic information is easily transferred to E. coli, and thus we use it to amplifyDNA and express proteins (insulin). But it lacks post-translational modifications andhas codon issues, etc., most of which can be overcome.
    18. 18. Saccharomyces cerevisiaeThe model organism that’s been in our hearts for as long as we can remember!
    19. 19. Civilization owes yeast for its use since ancient times inbaking and brewing.It is one of the most intensively studied eukaryotic modelorganisms in molecular and cell biology.Many proteins important in human biology were firstdiscovered by studying their homologs in yeast. •cell cycle proteins, signaling proteins, and protein- processing enzymes.As a eukaryote, S. cerevisiae shares the complex internalcell structure of plants and animals without the highpercentage of non-coding DNA that can confound researchin higher eukaryotes.
    20. 20. Drosophila melanogaster
    21. 21. One of the most commonly used model organisms inbiology, including studies in genetics, physiology and lifehistory 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 availablesince 1987.•About 75% of known human disease genes have arecognizable match in the genetic code of fruit flies
    22. 22. Zebrafish (Zebra danio)
    23. 23. Because a zebrafish embryo is completely transparent… …it is widely used to study morphological development of vertebrates.
    24. 24. The mouse is one of the major model organisms for medicine
    25. 25. Mice are by far the most geneticallyaltered laboratory mammal.They are the primary model organismfor most human diseases, includingcancer, because almost every humangene has a mouse homolog.Knock-out mice make this even moreimportant.
    26. 26. Chapter 1 ©
    27. 27. 2006Craig C. Mello andAndrew Fires receiveda noble prize for RNAi
    28. 28. The Nobel Prize in Physiology or Medicine, 2007Mario R. Capecchi, Martin J. Evans and Oliver Smithiesfor their discoveries of "principles for introducing specific genemodifications in mice by the use of embryonic stem cells" M. Capecchi Sir M. Evans O. Smithies Univ. of Utah Cardiff Univ., UK UNC Chapel Hill
    29. 29.  Go to this website to perform your gel electrophoresishttp://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 | Killers Trail | Create a DNA Fingerprint
    30. 30.  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.
    31. 31. Transforming Bacteria
    32. 32. Transforming Plants
    33. 33. 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
    34. 34.  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).
    35. 35.  term used to refer to an organism that contains genes from other organisms
    36. 36. Transgenic Transgenic TransgenicBacteria Plants animalsProduce Stronger plants More clotting factors More production insulin production HGH Pest resistance
    37. 37. The organism of choice for mammaliangenetic engineers. - small - hardy - short life cycle - genetics possible - many useful strains and tools
    38. 38. Vector with a transgenetk1 & tk2 - Herpes Simplex Virus thymidine kinase genes (make cells susceptible to gancyclovir)Neo - neomycin resistance geneHomologous regions - homologous to the chromosomal targetTransgene - foreign gene
    39. 39. Example of what happens with N-H recombinationNonhomologous r e combination homolo gous homolo gous tk 1 se quence neo tr ansge ne se quence tk 2 chr omo some homolo gous homolo gous tk 1 se quence tr ansge ne se quence tk 2 neo chr omo some Transformed cells are neo-resistant, but gancyclovir sensitive. homol-->
    40. 40. What happens with HRHomologous re combinants homolo gous homolo gous tk 1 se quence neo tr ansge ne se quence tk 2 chr omo some homolo gous homolo gous se quence neo tr ansge ne se quence chr omo some 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.
    41. 41. To knock-out a gene: 1.1. Insert neo gene into the target KO gene.2. Transform KO plasmid into embryonic stem cells.3. Perform double- selection to get cells with the homologous KO 2,3. integration (neo & gangcyclovir resistant).4. Inject cells with the knocked-out gene into a blastocyst.
    42. 42. How to make a transgenic mouse. Transfe ction With DNA Embryonic Stem ce llsBlasto cyst (ES ce lls) (mouse) Grow in culture . Select for those that carry the transgene. Inject into a blastocyst
    43. 43. Inject into a blastocyst Implant int o pseudo pregn an t mou se Chimeric mouse Ident if y of f spring w hich carry t he t ransgene in t heir germline.
    44. 44. (a) If the recipient stem cells are from a brown mouse, and thetransgenic cells are injected into a black (female) mouse, chimeras areeasily identified by their Brown/Black phenotype.(b) To get a completely transgenic KO mouse (where all cells have KOgene), mate the chimera with a black mouse. Some of the progeny willbe brown (its dominant), because some of the germ line cells will befrom the KO cells. ½ the brown mice will have the transgeneKO, because the paternal germ-line cell was probably heterozygous.(c) To get a homozygous KO mouse (both chromosomes have the KOtransgene), cross two brown transgenic heterozygotes. ~1/4 will behomozygous at the transgene locus.
    45. 45. Not necessarily 3:1
    46. 46.  member of a population of genetically identical organisms produced from a single cell
    47. 47.  “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.