1. Gene Expression and DNA Technology
Set 3b
Test on 7/28

2. Gene Expression
What causes DNA (genes) to be transcribed (mRNA) and then translated (amino acids/ polypeptides/proteins)?
What makes one gene expressed, and another not?
Would really want to make ALL your proteins?

3. Operons Operons turn genes on and off in prokaryotes.
A simple switch, based on the cell environment.
Ex.) lac operon turns on the lactase, which breaks down lactose when lactose is present.

4. Parts of an Operon
Regulatory gene (DNA) – where repressor protein is coded for
Not part of the operon, technically.
Promoter (DNA) – where RNA polymerase binds
Operator (DNA) – where the repressor protein binds
Structural gene/loci – the functional gene that is controlled.

5. Repressors Repressors stop RNA polymerase from coding the operon.
Can be on “active” in the presence of a chemical, or off in it’s presence.
Ex) lac repressor is active when there is no lactose.
Inactive when there is lactose.
Ex) trp repressor is active when there is tryptophan.
Inactive when there is no tryptophan.

6. Differentiation
Different cells become different because they make different proteins.
Different types of cells make different proteins because different combinations of genes are active in each type
Muscle cell
Pancreas cells
Blood cells
Fiure 11.2

7. Cell division in culture
Differentiated cells can retain all of their genetic potential
Especially in plants.
Most differentiated cells retain a complete set of genes
Your brain cells have all the DNA for your white blood cells, and visa versa
Root of carrot plant
Root cells cultured in nutrient medium
Cell division in culture
Plantlet
Adult Plant
Single cell
Figure 11.3

8. DNA packing in eukaryotic chromosomes helps regulate gene expression
A chromosome contains DNA wound around clusters of histone proteins
This beaded fiber is further wound and folded
DNA packing blocks gene expression by preventing access of transcription proteins to the DNA
Presumably…
DNA double helix (2-nm diameter)
Histones
Linker
“Beads on a string”
Nucleosome (10-nm diameter)
Tight helical fiber (30-nm diameter)
Supercoil
(300-nm diameter)
Metaphase chromosome
700 nm
TEM

9. Epigenetic Inheritance
Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence
A new field of genetics… Allows traits to be passed differently than just in the DNA.
This can be accomplished by acetylation or methylation of histones
Basically, changing the proteins that the DNA coils around, which can be passed in the gametes on to the later generation.

10. Histone acetylation seems to loosen chromatin structure and thereby enhance transcription
Addition of methyl groups to certain bases in DNA is associated with reduced transcription in some species
Epigenetics NOVA movie
Figure 19.4 b
(b) Acetylation of histone tails promotes loose chromatin structure that permits transcription
Unacetylated histones
Acetylated histones

11. Two cell populations in adult
In female mammals, one X chromosome is inactive in each somatic (body) cell
Called a “Barr Body”
Early embryo
X chromosomes
Allele for orange fur
Allele for black fur
Cell division and random X chromosome inactivation
Two cell populations in adult
Active X
Inactive X
Orange fur
Black fur
Figure 11.5

12. Complex assemblies of proteins control eukaryotic transcription
A variety of regulatory proteins interact with DNA and with each other to turn the transcription of genes on or off
Enhancers
Promoter
Gene
DNA
Activator proteins
Other proteins
Transcription factors
RNA polymerase
Bending of DNA
Transcription
Figure 11.6
More than just a single repressor protein.
Multiple activators, many proteins involved.
Changes the shape of DNA, often from “far” away.

13. Eukaryotic RNA may be spliced in more than one way
After transcription, alternative splicing may generate two or more types of mRNA from the same transcript
DNA
RNA
transcript
mRNA
Exons or
RNA splicing
Figure 11.7

14. Regulation at the translation stage and later
Breakdown of mRNA
The lifetime of an mRNA molecule helps determine how much protein is made
Initiation of Translation
Among the many molecules involved in translation are a great many proteins that control the start of polypeptide synthesis
Protein Activation
After translation is complete polypeptides may require alteration to become functional
Protein Breakdown
Some of the proteins that trigger metabolic changes in cells are broken down within a few minutes or hours

15. Blockage of translation
RNA interference by single-stranded RNAi can lead to degradation of an mRNA or block its translation
RNAi NOVA movie
Recent data shows that RNAi can also work as a decoy for promoters, binding transcription factors so that they don’t initiate transcription.
The micro-
RNA (miRNA)
precursor folds
back on itself,
held together
by hydrogen
bonds. 1 2
An enzyme
called Dicer moves
along the double-
stranded RNA,
cutting it into
shorter segments.
One strand of
each short double-
stranded RNA is
degraded; the other
strand (miRNA) then
associates with a
complex of proteins. 3
The bound
miRNA can base-pair
with any target
mRNA that contains
the complementary
sequence. 4
The miRNA-protein
complex prevents gene
expression either by
degrading the target
mRNA or by blocking
its translation. 5 5
Chromatin changes
Transcription
RNA processing
mRNA
degradation
Translation
Protein processing
and degradation
Degradation of mRNA OR
Blockage of translation
Target mRNA
miRNA
Protein
complex
Dicer
Hydrogen
bond
Figure 19.9

16. Page 217, right column Very important image. Figure 11.9 NUCLEUS
Chromosome
Gene
RNA transcript
mRNA in nucleus
mRNA in cytoplasm
Polypeptide
Active protein
Breakdown of protein
Cleavage / modification /
activation
Translation
Breakdown of mRNA
CYTOPLASM
Flow through nuclear envelope
Splicing
Addition of cap and tail
Transcription
DNA unpacking
Other changes to DNA
Exon
Intron
Cap
Tail
Broken- down mRNA
Broken- down protein
Figure 11.9
Page 217, right column
Very important image.

17. Viruses
Viruses inject their DNA code into the cells of the infected cells.
“DNA viruses” use DNA to put into the cells
Retroviruses use RNA, which is converted to DNA, which is then put into the cells
Then the virus uses the cell’s processes to make new virus proteins and DNA/RNA to create new virus particles

18. Cloning and DNA technology

19. To Clone or Not to Clone?
A clone is an individual created by asexual reproduction and thus is genetically identical to a single parent

20. ANIMAL CLONING Nuclear transplantation can be used to clone animals
Nuclear transplantation can be used to clone animals
Remove nucleus from egg cell
Add somatic cell
from adult donor
Grow in culture to produce an
early embryo (blastocyst)
Implant blastocyst in surrogate mother
Remove embryonic stem cells from blastocyst and grow in culture
Induce stem cells to form specialized cells (therapeutic cloning)
Clone of donor is born
(reproductive cloning)
Donor cell
Nucleus from donor cell
Figure 11.10

21. Therapeutic cloning can produce stem cells with great medical potential
Like embryonic stem cells, adult stem cells can perpetuate themselves in culture and give rise to differentiated cells
Adult stem cells in bone
marrow
Cultured embryonic stem cells
Different culture conditions
Heart muscle cells
Different types of differentiated cells
Nerve cells
Blood cells
Figure 11.12

22. Unlike embryonic stem cells adult stem cells normally give rise to only a very limited range of cell types

23. Stem Cells Initial cells that can become many different types of cells
Important for development, because all cells start as one
There are genes, such as the Hox gene which control segmentation
Which then controls development

24. Signal transduction pathways convert molecular messages to cell responses
This is an important part of development.
How it gets “going”, tells which cell to do what…
Signaling cell
Signal molecule
Receptor protein
Plasma membrane
Target cell
Relay proteins
Transcription factor (activated)
Transcription
Nucleus
DNA
mRNA
New protein
Translation 1 2 3 4 5 6
Figure 11.14

25. THE GENETIC BASIS OF CANCER
Cancer cells, which divide uncontrollably result from mutations in genes whose protein products affect the cell cycle

26. Proto-Oncogenes
A mutation can change a proto-oncogene (a normal gene that promotes cell division) into an oncogene, which causes cells to divide excessively
Proto-oncogene DNA
Mutation within the gene
Multiple copies of the gene
Gene moved to new DNA locus, under new controls
Oncogene
New promoter
Hyperactive growth- stimulating protein in normal amount
Normal growth- stimulating protein in excess
Figure 11.16A

27. Tumor-Suppressor Genes
Mutations that inactivate tumor suppressor genes have similar effects as oncogenes
Tumor-suppressor gene
Mutated tumor-suppressor gene
Normal growth- inhibiting protein
Cell division under control
Defective, nonfunctioning protein
Cell division not under control
Figure 11.16B

28. Oncogene proteins and faulty tumor-suppressor proteins can interfere with normal signal transduction pathways
Growth-inhibiting factor
Receptor
Nonfunctional transcription factor (product of faulty p53 tumor-suppressor gene)
Relay proteins
Transcription factor (activated)
Transcription
Translation
Protein that inhibits cell division
cannot trigger transcription
Protein absent (cell division not inhibited)
Normal product
of p53 gene
Figure 11.17B
Growth factor
Target cell
Receptor
Hyperactive relay protein (product of ras oncogene) issues signals on its own
Normal product of ras gene
Relay proteins
Transcription factor (activated)
DNA
Nucleus
Transcription
Translation
Protein that stimulates cell division
Figure 11.17A

29. Cancers result from a series of genetic changes in a cell lineage
Colon cancer develops in a stepwise fashion
Colon wall 1
Increased cell division
Oncogene activated 2
Growth of polyp
Tumor-suppressor gene inactivated 3
Growth of malignant tumor (carcinoma)
Second tumor- suppressor gene inactivated
Cellular changes:
DNA changes:
Figure 11.18A

30. Accumulation of mutations can also lead to cancer
UV radiaton
Smoking/Tobacco use
Chromosomes
mutation 1 2 3 4
mutations
Normal cell
Malignant cell
Figure 11.18B

31. Researchers can insert desired genes into plasmids, creating recombinant DNA and insert those plasmids into bacteria
Bacterium
Bacterial
chromosome
Plasmid 1
isolated 3
Gene inserted
into plasmid 2
DNA
Cell containing gene
of interest
Gene of
interest
Recombinant DNA
(plasmid) 4
Plasmid put into
bacterial cell
Recombinant
bacterium 5
Cell multiplies with
gene of interest
Copies of protein
Copies of gene
Clone of cells
Gene for pest
resistance
inserted into
plants
Gene used to alter bacteria
for cleaning up toxic waste
Protein used to dissolve blood clots in heart attack therapy
Protein used to make snow form at higher temperature
Figure 12.1

32. Restriction enzymes cut DNA. DNA ligase puts it back together.
Creating recombinant DNA using restriction enzymes and DNA ligase
Restriction enzyme recognition sequence
G A A T T C
C T T A A G
DNA 1 2 3 4
C T T A A
A AT TC G
Addition of a DNA fragment from another source
Two (or more) fragments stick together by base-pairing
G A AT T C
C T TA A G 5
DNA ligase
pastes the strand
Restriction enzyme cuts the DNA into fragments
Recombinant DNA molecule
Sticky end
Restriction enzymes cut DNA.
DNA ligase puts it back together.
Figure 12.2

33. Cloning a gene in a bacterial plasmid
E.coli 1
Isolate DNA from two sources
Human cell 2
Cut both DNAs with the same restriction enzyme
Plasmid
DNA
Gene V
Sticky ends 3
Mix the DNAs; they join by base-pairing 4
Add DNA ligase to bond the DNA covalently
Recombinant DNA plasmid
Gene V 5
Put plasmid into bacterium by transformation
Recombinant bacterium 6
Clone the bacterium
Figure 12.3
Bacterial clone carrying many copies of the human gene

34. Recombinant cells and organisms can mass-produce gene products
Table 12.6

35. Therapeutic hormones
In 1982, humulin, human insulin produced by bacteria
Became the first recombinant drug approved by the Food and Drug Administration
Figure 12.7A

36. Gene therapy (or the alteration of an afflicted individual’s genes) may someday help treat a variety of diseases
Cloned gene
(normal allele) 1
Insert normal gene
into virus 2
Infect bone marrow
cell with virus 3
Viral DNA inserts
into chromosome 4
Inject cells
into patient
Bone
marrow
Bone marrow
cell from patient
Viral nucleic
acid
Retrovirus
Figure 12.13

37. Gel electrophoresis sorts DNA molecules by size+ –
Power
source
Gel
Mixture of DNA
molecules of
different sizes
Longer
molecules
Shorter
Completed gel
Figure 12.10

38. How Restriction Fragments Reflect DNA Sequence
Restriction fragment length polymorphisms (RFLPs) reflect differences in the sequences of DNA samples
Crime scene
Suspect w x y z
Cut
DNA from chromosomes C G A T
Figure 12.11A

39. After digestion by restriction enzymes the fragments are run through a gel– +
Longer
fragments
Shorter x w y z 1 2
Figure 12.11B

40. Detecting a harmful allele using restriction fragment analysis1 2 3 4 5
Restriction fragment
preparation
Gel electrophoresis
Blotting
Radioactive probe
Detection of radioactivity
(autoradiography) I II
III
Restriction
fragments
Filter paper
Probe
Radioactive, single-
stranded DNA (probe)
Film
Figure 12.11C

41. DNA profiling can help solve crimes
Figure 12.12B
Defendant’s
blood
Blood from
defendant’s clothes
Victim’s
Figure 12.12A

42. The polymerase chain reaction (PCR) method is used to amplify DNA sequences. It can be used to clone a small sample of DNA quickly, producing enough copies for analysis 1 2 4 8
Initial
DNA
segment
Number of DNA molecules
Figure 12.14

43. gene within plant chromosome
Recombinant DNA technology can be used to produce new genetic varieties of plants and animals, genetically modified (GM) organisms
Agrobacterium tumefaciens
DNA containing
gene for desired trait
Plant cell Ti
plasmid 1
Insertion of gene
into plasmid using
restriction enzyme
and DNA ligase 2
Introduction
into plant
cells in
culture 3
Regeneration
of plant
Recombinant
Ti plasmid
T DNA carrying new
gene within plant chromosome
T DNA
Plant with new trait
Restriction
site
Figure 12.18A

44. Sowing A Gene Revolution, Scientific American, (September 2007), 297, 104-111

45. Sowing A Gene Revolution, Scientific American, (September 2007), 297, 104-111

46. Transgenic organisms are those that have had genes from other organisms inserted into their genomes
Chimera: Organism that has cells or genes from another individual of the same or a different species.
Figure 12.18B

47. Examples of Transgenics
Cows, sheep or goats that produce proteins in their milk e.g. tPA (tissue plasminogen activator)
Mice to study what a gene does or as a disease model.
Cows that produce milk with less cholesterol or lactose
Influenza resistant pigs