Lecture 3 -the diversity of genomes and the tree of life
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Dolly the sheep was the first mammal to be cloned from an adult somatic cell, demonstrating that differentiated cells could be reprogrammed to pluripotency. Prior to Dolly, all cloning had involved embryonic cells or cells that had not yet differentiated. Dolly showed that the cell specialization process could be reversed, proving the possibility of therapeutic cloning and stem cell research using somatic cell nuclear transfer techniques. Her successful cloning from an adult cell was a major scientific breakthrough.
Lecture 3 -the diversity of genomes and the tree of life
- 1. Chapter 1 ©
- 2. 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.
- 3. 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).
- 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 that atmospheric nitrogen gas into a more produce methane as a metabolic usable form such as ammonia. byproduct in anoxic conditions.
- 5. The Greatest Biochemical Diversity Exists Among Prokaryotic Cells Thiomargarita namibiensis (0.75 mm) Epulopiscium fishelsoni (0.6 mm)
- 6. In extending their capacity to live in biochemically diverse habitats, eukaryotes went down the path of symbiosis, rather than reinventing the “metabolic wheel”.
- 9. In fact, the origin of eukaryotes is thought to arise from the merging of symbiotic prokaryotic cells!
- 11. The Tree of Life Has Three Primary Branches: Bacteria, Archaea, and Eukaryotes
- 12. Ribosomal Genes •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.
- 13. 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.
- 14. 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)
- 15. 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)
- 17. 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).
- 18. 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.
- 19. Organism Genome Sequenced Homologous Recombination Biochemistry 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
- 20. Escherichia coli Our model prokaryote
- 21. 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.
- 22. Saccharomyces cerevisiae The model organism that’s been in our hearts for as long as we can remember!
- 23. 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.
- 25. 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
- 27. Because a zebrafish embryo is completely transparent… …it is widely used to study morphological development of vertebrates.
- 28. The mouse is one of the major model organisms for medicine
- 29. 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.
- 30. Chapter 1 ©
- 31. 2006 Craig C. Mello and Andrew Fire's received a noble prize for RNAi
- 32. The Nobel Prize in Physiology or Medicine, 2007 Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their discoveries of "principles for introducing specific gene modifications 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
- 33. Go 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
- 34. 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.
- 37. 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
- 38. Why? 1. Study gene function and regulation 2. 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).
- 39. term used to refer to an organism that contains genes from other organisms
- 40. Transgenic Transgenic Transgenic Bacteria Plants animals Produce Stronger plants More clotting factors More production insulin production HGH Pest resistance
- 41. The organism of choice for mammalian genetic engineers. - small - hardy - short life cycle - genetics possible - many useful strains and tools
- 43. Vector with a transgene tk1 & tk2 - Herpes Simplex Virus thymidine kinase genes (make cells susceptible to gancyclovir) Neo - neomycin resistance gene Homologous regions - homologous to the chromosomal target Transgene - foreign gene
- 44. Example of what happens with N-H recombination Nonhomologous 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-->
- 45. What happens with HR Homologous 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.
- 46. 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.
- 47. How to make a transgenic mouse. Transfe ction With DNA Embryonic Stem ce lls Blasto cyst (ES ce lls) (mouse) Grow in culture . Select for those that carry the transgene. Inject into a blastocyst
- 48. 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.
- 49. (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.
- 51. member of a population of genetically identical organisms produced from a single cell
- 52. “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.
Editor's Notes
- #33: First KO mice using their ES cell technology were produced in 1989 – now its like an industry