PowerPoint for BI 520-01 Spring 2013

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.

24. Drosophila melanogaster

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

26. Zebrafish (Zebra danio)

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.

35. Transforming Bacteria

36. Transforming Plants

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.

50. Not necessarily 3:1

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.