1. EUKARYOTIC GENE EXPRESSION

2. DNA PACKING Histones Nucleosomes (1st) 30nm fibers (2nd)
Looped domains (3rd)
Heterochromatin –
not transcribed,
metaphase chromatid
Euchromatin – open,
actively transcribed
Chromosome location specific

3. GENOME ORGANIZATION - DNA LEVEL
Repetitive DNA (tandem repeats)
10-15% in mammals
short sequences (10) repeated in series, 100,000’s times
Fragile X: 100’s instead of 30 triplet repeats
Huntington’s: CAG repeats
# of repeats correlates with severity and age of onset.
Regular, mini & micro satellites
Interspersed Repetitive DNA
Scattered, 25-40%, similar but not identical
Alu elements: family of sequences 5% primate genome, about 300 bp, many transcribed; most transposons

4. Telomeres &
centromeres
Useful in
fingerprinting

5. Chromosome puff
MULTIGENE FAMILY:
Collection of similar
or identical genes
Salamander RNA
3 kinds of rRNA
after processing,
S-sedimentation
Rates due to differences in density

6. NON-IDENTICAL MULTIGENE FAMILY
2 related families that code for globins
4 subunits,
2α & 2β
Nonfunctional,
very similar to
functional genes

7. Gene Amplification: the temporary increase in the number of copies of a gene
rRNA in amphibians
rRNA in developing ovum, large # of ribosomes  burst of protein synthesis after fertilization
Extra copies cannot replicate & are broken down
Selective Gene Loss: occurs in certain insects, whole chromosomes or parts of chromosomes may be lost early in development.

8. REARRANGEMENTS
Transposons: can prevent normal functioning, may ↑ or ↓production, or be activated, 10% of human genome
Retrotransposons: use an RNA intermediate
Immunoglobulin Genes: code for antibodies, genes become rearranged as immune cells differentiate

9. Retrotransposon Movement: like retrovirus reproduction, can populate the genome in huge numbers

10. DNA Rearrangement maturation of an immunoglobulin gene
The joining of V, J & C regions of DNA in random combinations  enormous
variety of antibody-producing lymphocytes
Hundreds of V regions
Several Junction regions
1-2 Constant regions

11. CONTROL OF GENE EXPRESSION
Cellular differentiation – divergence in form & function as cells become specialized during development
3-5% expressed at any given time (liver vs skin)
DNA Methylation – genes not expressed, methyl group (CH3) attached
Barr body – heavy methylation, inactive X, heterochromatin
Genomic Imprinting – turning off alleles
Histone Acetylation – acetyl groups on histone a.a. causing shape change, grip DNA less tightly, easier access to genes for transcription

12. OPPORTUNITIES FOR CONTROL
OF GENE EXPRESSION
DNA packing, methylation, acetylation
Transcription (most important)
RNA Processing
Transport to cytoplasm
Degradation of mRNA
Translation
Cleavage, Chemical modification, Transport to cellular destination
Degradation of protein

13. TRANSCRIPTIONAL CONTROLS
Transcription factors
Equivalent to repressors & activators
Bind to specific sites (TATA box)
May be near promoter
Optimum binding – 50 ish factors
“read” DNA without unzipping to find appropriate gene (zinc fingers & leucine zippers)
Promoter, Enhancer, Suppressor sequences

14. Transcription initiation – controlled by transcription factors (proteins) that interact w/ DNA & each other.
Typical Eukaryotic Gene – promoter, terminator, distal & proximal control elements (key to high levels of transcription)
Promoter regions bind to RNA polymerases I,II,III (tRNA’s)

15. Model for enhancer action
Activator proteins bind to enhancer sequences
DNA bends, activators closer to promoter
Protein-binding domains attach to transcription factors, form active transcription initiation complex on promoter

16. DNA Binding Domain: 3-D part of a transcription factor that binds to DNA
Found in many regulatory proteins
α helix β sheet
held by zinc atom
2 α helices w/ spaced leucines coil

17. POST-TRANSCRIPTIONAL CONTROLS
mRNA processing – mG cap? poly A tail?
Alternative splicing – same primary transcript, different introns & exons
mRNA degradation – prokaryotic mRNA’s last a few minutes; eukaryotic- hours, can be days even weeks (hemoglobin)
Translation inhibited by masked mRNA prior to fertilization (activated in embryo)
Protein processing & degradation

18. ALTERNATIVE mRNA SPLICING

19. PROTEIN DEGRADATION

20. EUKARYOTIC vs PROKARYOTIC
Genes spread out
1 promoter = 1 gene
Many introns
Processing
2 copies of DNA (2n)
Paired, rod shaped chrom.
3 polymerases
Nucleus, separation of transcription/translation
Operons
1 promoter=multiple genes
Lack introns
No processing
1 copy of DNA (n)
Single circular chrom.
1 polymerase
No nucleus, simultaneous transcription/translation

21. CANCER
Mutations in genes that regulate growth & division, chemical carcinogens, physical mutagens (X-rays, viruses)
Oncogenes – cancer causing
Proto-oncogenes – normal gene  oncogene
Tumor-suppressor genes – prevent uncontrolled division
ras gene – proto-oncogene
p53 gene – tumor suppressor gene

22. Proto-oncogenes  Oncogenes

23. ras gene
G protein
Relays growth signal
Result – stimulation of cell cycle

24. p53 – transcription factor, activates p21, product blocks CDK’s

26. MODEL: DEVELOPMENT OF COLORECTAL CANCER

27. DNA TECHNOLOGY

28. TERMINOLOGY
Recombinant DNA – DNA in which genes from two different sources are combined in vitro into the same molecule.
Genetic engineering – direct manipulation of genes for practical purposes.
Biotechnology – manipulation of organisms or their components to make useful products.
Gene cloning – method for preparing well defined, gene sized pieces of DNA in multiple identical copies.

29. BACTERIAL PLASMIDS FOR CLONING

30. RESTRICTION ENZYMES Cut up foreign DNA. Endonucleases RESTRICTION SITE
Recognition sequence
Usually symmetrical
RESTRICTION FRAGMENT
Piece of DNA cut by specific enzyme
STICKY END
Single strand end of restriction fragment

31. Cloning a human gene in a bacterial plasmid

34. USING A NUCLEIC ACID PROBE TO IDENTIFY A CLONED GENE
Cloned gene of interest on a plasmid, probe is short length of radioactive single stranded DNA complimentary to part of gene.
2) Result – single stranded DNA stuck to filter paper
3) Probe DNA hybridizes with complimentary DNA on filter
4) Filter laid on photographic film, radioactive areas expose film (autoradiography)

35. MAKING COMPLEMENTARY DNA (cDNA) FOR A EUKARYOTIC GENE
Expression vector - Cloning vector w/ prokaryotic promoter upstream of restriction site where eukaryotic gene can be inserted.
cDNA – made in vitro using mRNA template & reverse transcriptase (DNA w/o introns)

36. TERMINOLOGY Yeast artificial chromosomes (YACs) –
vectors that combine the essentials of a eukaryotic chromosome ( origin for replication, centromere, 2 telomeres) w/ foreign DNA.
Electroporation – application of electric pulse to solution containing cells creating temporary hole in membrane allowing DNA to enter.
Genomic library – complete set of thousands of recombinant plasmid clones, each carrying copies of a particular segment from the initial genome.
cDNA library – library containing a collection of genes (represents only part of a cell’s genome – only the genes that were transcribed in the starting cells)

37. The same 3 foreign genome fragments in a phage library
Shown - 3 of the thousands of “books” in the library. Each is a bacterial clone containing one particular variety of foreign genome fragment in its recombinant plasmid
The same 3 foreign genome
fragments in a phage library

38. PCR Polymerase chain reaction
Technique to quickly amplify (copy many times) a piece of DNA w/o using cells
Start w/ double stranded DNA (“target”), add to polymerase, nucleotide supply & primers
5 minutes per cycle

39. DNA ANALYSIS & GENOMICS
Genomics – the study of whole sets of genes and their interactions
Gel electrophoresis – separates macromolecules (nucleic acids or
proteins) based on size, electrical charge and other physical
properties.
Southern blotting – hybridization technique that enables
researchers to determine the presence of certain nucleotide
Sequences in a sample of DNA.
Restriction fragment length polymorphisms RFLPs - differences
in DNA sequence on homologous chromosomes that can result in
different restriction fragment patterns. Scattered abundantly
throughout genomes

40. GEL ELECTROPHORESIS Negatively charged DNA migrates toward
positive electrode.
Longer fragments travel more slowly
DNA samples arranged in bands along a “lane” according to size.
Shorter fragments travel farthest
3 DNA samples placed in wells.
Electrodes attached & voltage applied

41. Using restriction fragment patterns to distinguish DNA from different alleles
2 homologous segments w/ different alleles
Electrophoresis separates the fragments; allele 1has 3 fragments, allele 2 has 2
Addition of binding dye, fragments fluoresce pink; shown: 6 samples cut w/ a restriction enzyme

42. RESTRICTION FRAGMENT ANALYSIS BY SOUTHERN BLOTTING
DNA denatured &
transferred to paper
Radioactivity exposes film, image forms – bands w/ DNA base-pairs w/ probe
Probe complimentary to gene of interest

43. MAPPING GENOMES AT THE DNA LEVEL
Human Genome Project – effort to map the entire human nucleotide sequence for each chromosome.
Genetic (Linkage) Mapping – construction of a linkage map using various genetic markers.
Physical Mapping: Ordering DNA Fragments
chromosome walking: make fragments that overlap, then use probes of the ends to find the overlaps
Bacterial artificial chromosome (BAC): artificial version of a bacterial chromosome that can carry inserts of 100,000 – 500,000 base pairs
DNA Sequencing – determining the nucleotide sequence of a DNA segment or an entire genome. 3 sequencing methods
Alternative Approaches to Whole-Genome Sequencing

44. Chromosome walking Prepare probe to match 3’ end
Cut starting DNA w/ 2 restriction
enzymes & clone fragments
3) Use probe 1 to screen library II
for DNA fragments that overlap the
known gene
4) Isolate DNA from tagged clone,
prepare probe 2 to match 3’ end of
that segment.
5) Use probe 2 to screen library I for
an overlapping fragment farther along
6) Repeat 4&5 with new probes &
Alternating libraries to “walk” down DNA
7) Result – DNA map w/ series of
known markers (sequences) in a
Known order & separated by known
distances

45. SANGER METHOD 4 portions each incubated w/ primer DNA polymerase
4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP
Different one of the 4 nucleotides in modified dideoxy (dd) form

46. SANGER METHOD SANGER METHOD
4 portions each incubated w/
primer
DNA polymerase
4 deoxyribonucleoside triphosphates: dATP, dGTP, dCTP, dTTP
Different one of the 4 nucleotides in modified dideoxy (dd) form
Synthesis of new strands begins
at primer & continues until a
dideoxyribonucleotide is
incorporated, which prevents
further synthesis.
SANGER
METHOD
SANGER
METHOD
Eventually, a set of
labeled strands
of various lengths
is generated.

47. SANGER METHOD New strands separated by electrophoresis
Sequence can be read from bands on autoradiograph and original template sequence deduced.
Longest fragment ends with a ddG, so G must be the last base in the sequence

48. ALTNERATIVE STRATEGIES FOR SEQUENCING AN ENTIRE GENOME
- The arrangement of DNA fragments in order depends on their having overlapping regions
Cut DNA of chromosome into small fragments
Clone fragments in plasmid or phage vectors
Sequence fragments
Assemble overall sequence

50. DNA microarray assay for gene expression
Researcher simultaneously test all the genes expressed in particular tissue for hybridization with an array of short DNA sequences representing thousands of genes.
Fluorescence intensity indicates relative amount of mRNA in tissue.

51. Practical Applications of DNA Technology
Diagnosis of Diseases – PCR & labeled probes
Human Gene Therapy – replacing defective genes
Pharmaceutical Products – vectors, vaccines
Forensics - DNA fingerprinting, simple tandem repeats
Environmental Uses – mining, sewage treatment, detoxifying microbes (oil spills etc.)
Agricultural Uses – transgenic organisms, “pharm” animals, Ti plasmid, herbicide resistant crops
Genetically Modified Organisms – safety & ethical questions

52. Using RFLP markers to detect presence of disease causing alleles

53. Gene Therapy

54. DNA Fingerprinting

55. Using the Ti plasmid as a vector for genetic engineering