Model Organisms & Tools of Cell Biology

1. Model Organisms & Tools of Cell Biology Lecture 3 Autumn 2007

2. Learning the Unknown • You are a car mechanic • Would you rather know a little bit about the working of every car every constructed… • … or everything about a representative one from each category? • Science is no different!

3. What is a Model Organism? • Many aspects of biology are similar in most or all organisms • It is much easier to study particular aspects in particular organisms - for instance, genetics is easier in small organisms that breed quickly, and very difficult in humans! • The most popular model organisms have strong advantages for experimental research • They become even more useful when other scientists have already worked on them, discovering techniques, genes and other useful information.

4. How many are there? • Many (about 80) • Mouse, rat, zebra fish, viruses, chicken, dog, hamster, slime mould, maize, tetrahymena, etc. • Many scientists have worked on all these over the years, and shared information extensively

5. Which are the main ones? • 1)E. coli (bacterium) • 2)Saccharomyces cerevisiae(yeast) • 3) Arabidopsis thaliana(weed) • 4) Drosophila melanogaster (fruit fly) • 5) Mus musculus (mouse) • 6) Homo sapiens (Man)

6. Arabidopsis thaliana (mustard plant) This is now the main model plant system for genetics. Its small genome, and the recent application of classical genetics has put it far ahead of other models of agricultural importance (tomato, tobacco, corn etc.) It's genome has been fully sequenced. 01_33_model plant.jpg

7. 01_34_Drosophila.jpg Drosophila sp. ‘Fruit Fly’ Usually the species Drosophila melanogaster - Easily raised in lab, rapid generations, mutations easily induced, many observable mutations. Many clues to development and genetics

8. 01_38_C.elegans.jpg • Caenorhabditis elegans, a nematode • (Usually called just C. elegans) • an excellent model for understanding the genetic control of development and physiology. • C. elegans was the first multicellular organism whose genome was completely sequenced • First to show fixed cell count in body • Gave important clues on programmed cell death

9. Saccharomyces cerevisiae, baker's yeast or budding yeast (used in brewing and baking) 01_32_model eucaryote.jpg Early studies on this enabled us to get a great grasp on the cell cycle

10. Humans • Regardless of how thoroughly we may understand other animal systems, sometimes there is no alternative but to study humans directly - i.e. breast cancers • The human animal is the most medically analyzed and documented of any species. • We have now completely sequenced our own genome too • Over the next decade or so we will understand more about our biology than ever before!

11. Where has knowledge of cells come from? • Can we see cells? • Yes and no, most are too small, but some we can see easily - the egg of a chicken is a large single cell • What are ‘The tools of Cell Biology’? • How big is the average egg? • Lets put it in perspective…

12. Size perspective 1X 10X -tissues 100X - cells 1000X - organelles 10,000X - organelles 100,000X - proteins 1,000,000X - atoms

13. Microscopes - aid us in seeing • First cells seen by Robert Hook, who made the first compound microscope • He looked at cork cells • The detail was lacking and limited by the quality of the instrument • Light microscopes are now much better

14. Light Microscopes • Many types exist • Light is either reflected from the specimen (Oblique illumination) or transmitted through the specimen (Bright field optical microscopy) • How small can we see? We can see all cells. • The lens are so good now that the limiting factor is now the wavelength of light itself. • The wavelength of the visible light used in optical microscopes is between 400 and 700 nanometers (nm). • The resolving powers of high-quality light microscopes are limited by the wavelength of imaging light to about 200 nanometers

15. Electron Microscopes • Sample placed in vacuum - thus dead • Four main types all using electron streams instead of light waves; • Transmission Electron Microscope (TEM) • Scanning Electron Microscope (SEM) • Reflection Electron Microscope (REM) • Scanning Transmission Electron Microscope (STEM) • Resolutions as low as 70pm have been obtained - single atoms • Read all about these on the course web site

16. Centrifugation - for analysis • Allows separation of • Cells • Organelles • DNA and other macromolecules • Uses gravitational and centrifugal forces to separate items based on density and size - one spins things at high speeds • Many types are used in cell biology…

17. Differential centrifugation • This is the most common method of fractionating cells • Fractionation is the separation of the different organelles within the cell • The speed determines which size and mass (density) of material is pelleted.

18. Isopycnic centrifugation • Isopycnic centrifugation or equilibrium centrifugation is a process used to isolate nucleic acids such as DNA. • Much high speeds and duration • Separates DNA based on base composition

19. Sucrose gradient centrifugation • Uses a decreasing concentration of sucrose in a tube • The particles travel through the gradient until they reach the point in the gradient at which their density matches that of the surrounding sucrose.

20. Other commonly used tools…

21. Mutation analysis - an important mechanism for discovery • Any organism which is different from the norm can be a candidate for biochemical analysis • Mutations are generally the cause of such differences • By analysis of these mutations one can gain insights or deduce the biochemical basis of important pathways…

22. Conditional Mutation Analysis - e.g. Temperature conditional The organism grows at one temperature (permissive) but fails to do so at a different one (restrictive). 01_35_Yeast mutation.jpg All the yeast colonies grow at one temperature (23C), but if one places the same cells at 35C some of the colonies fail to grow - what is wrong with the ones that fail to grow?

23. Gain-of-function mutations - e.g. biochemical function 01_36_S. pombe rescued.jpg

24. Sequence analysis has taught us that there are about 200-300 genes required for the most basic cell survival. Comparisons across species shows that many genes are common from simple bacteria to complex life forms, and that life uses similar processes 01_37_amino acid sequ.jpg

25. Genome sizes - a genome is the total haploid amount of DNA Amoeba proteus 290,000,000,000 (100 times the size of a human genome) Bufo bufo (cane toad) 6,900,000,000 Homo sapiens (Man) 3,000,000,000 (3 billion base pairs - 24 chromosomes - [22, X, Y]) Muntiacus muntjak vaginalis (Indian deer) 2,521,500,000 Boa constrictor (snake) 2,100,000,000 Rhinolophus ferrumequinum (bat) 1,929,400,000 Plasmodium falciparum (malaria parasite) 25,000,000 Human immunodeficiency virus type 1 (HIV) 19,750

26. There is no real correlation between the genome size and complexity 01_40_genome sizes.jpg

27. Genome size and number of genes Among the organisms whose genomes are sequenced, genome size does not correlate with the number of genes. Species Size of genome Number of genes Human 3.0 billion base pairs 25,000 ? Fruit fly (Drosophila melanogaster) 120 million base pairs 13,601 Baker's yeast (Saccharomyces cerevisiae) 12 million base pairs 6, 275 Worm (Caenorhabditis elegans) 97 million base pairs 19,000 E. coli 4.6 million base pairs 4,403 Arabidopsis (Arabidopsis thaliana) 125 million base pairs 25,000

28. Study… • Please read chapter 1 entirely, if you have not done so already • Sample questions for the exams will start appearing on the supporting web site this week! • Bye!