KEY CONCEPTS from LECTURE 1:

1. Prok. vs. Euk. cells

2. The players - DNA, RNA, protein
3. The processes - replication, transcription,
translation
4. DNA is the genetic material
5. Chemical composition and structure of DNA
6. Denaturation of DNA
7. Supercoiling and relaxing of circular DNA

Endosymbiont Hypothesis of Mitochondria

and Chloroplast Origins

Mitochondrion

10.6: Organelle DNA (p. 438-444)

1. Mito. and chloroplast
DNA evolved from bacteria
2. Circular DNA
3. Encode structural RNAs and some prot.
4. Cytoplasmic inheritance
5. Sim. of mito. ribosomes to bacterial
6. Slight variations in the genetic code
7. Mutations in mtDNA
Fig 8-6

Multiple mtDNAs/ Mitochondrion

Fig 8-3

Chloroplast

Fig 10-35

Ethidium bromide - DNA

DiOC6 - mito

Cytoplasmic Inheritance

Cytoplasmic Inheritance

1. Petite yeast
mutants contain
deletions in mtDNA

1. Daughters from
mating divide
2. Petite phenotype
reappears!

2. Mate WT to Petite
3. What is the phenotype
of daughter cells?

Fig 10-36
Fig 10-36

Maternal (mostly) Cytoplasmic Inheritance

Diseases

Maternal mtDNA Inheritance

Lebers Hereditary Optic Neuropathy - progressive vision loss

Identification

BUT highly variable and ONLY passed from mother

Reunite families via mtDNA evidence

Single ntd. change in mtDNA = change in mRNA coding for

electron transport protein

Dr. Mary-Claire King - identified a

variable segment of mtDNA that
could be used as a marker to link
mother to child

Embryo receives maternal mito (but Paternal Inheritance of

Mito. DNA NE J of Med. 2002)
Heteroplasmy - mixture of WT and mutant mtDNA

Mt DNA encodes: rRNAs

tRNAs
some proteins (intron-less mRNAs)

Differences in mt and nuclear

encoded gene expression
Translational machinery, antibiotic sensitivity
The genetic code (i.e. the UGA STOP codon
is read as Trp in mito of mammals to
yeast, but not in mito of plants)

Fig 10-37

(Some nuclear encoded proteins, RNAs imported into mito)

10.1 Molecular Definition of a Gene

(p.405-408

Fig. 10-1. Structure of genes and chromosomes

1. Gene definition
2. Introns
3. Simple Euk.
transcription units
4. Complex Euk.
transcription units

3.4 Billion Base Pairs of DNA

5. Cell type specific gene expression

What is a gene?

2 Types of Transcription Units in Eukaryotes

A gene is the entire nucleic acid sequence

that is necessary for the synthesis of a
functional gene product.
This includes:

coding region
transcriptional control regions
(enhancers)
splice sites
polyA sites

What about sequences that code for

rRNA, tRNA, etc?

2-

1 gene, multiple mRNAs and proteins

1. Alternative splicing

2. Alternative PolyA sites

3. Alternative promoters

Fig. 10-2b

1-

1 gene, 1 mRNA, 1 protein

3
Fig. 10-2a

a-d are examples of mutations that could

alter gene expression - HOW?

10.2: Chromosomal Organization of

Genes and Noncoding DNA (p. 408-414)
1. Dense gene arrays in Prok. and simple Euk.
2. Abundant nonfunctional DNA in vertebrates
3. Biological complexity = genome size
4. 3 classes of genomic DNA
5. Solitary vs. duplicated genes
6. Gene families
7. Genes in tandem arrays
8. Simple-sequence DNA repeats
9. DNA fingerprinting/ Southern Blotting

How could the example mutations a-e affect gene expression?

Lots of Nonfunctional DNA

Fig. 10-3

Why do unicellular organisms typically have more gene rich

regions compared to the gene-poor desert regions found
in vertebrates?

Solitary and Duplicated Genes

SOLITARY GENES: found only once in the genome

NONFUNCTIONAL FUNCTIONAL

Genes in 80kb region of DNA:

Gene Duplication from Unequal Crossing Over

L1 - homologous non-coding DNA

DUPLICATED GENES: multiple genes of close but

not identical sequences
- usually within 5-50kb of one another
- gene families encode homologous proteins
that constitute a protein family
The -globin gene family:
Fig. 10-4

Pseudogenes can Arise from Gene Duplication

Tandemly Repeated Genes

Encode identical or nearly identical gene products
Head to tail copies

Fig. 10-3a

Pseudo because they contain mutations that terminate

translation or block mRNA processing

Examples: rRNAs
tRNAs
histone coding genes

Sequence drift

Simple-sequence DNA - repetitious DNA

Satellite DNA: 14-500 bp repeats

in tandem, 20-100kb (at centromeres
and telomeres)
Microsatellites: 1-13bp repeats
in tandem, up to 150bp
backward slippage during DNA rep.
expanded microsatellites can cause
disease

Fig. 10-5 - FISH of a

Simple Sequence DNA site

Fig. 4-33

Unequal crossing over causes differences in SSR number

DNA Fingerprinting using

Simple Sequence Repeat (SSR) DNA
Minisatellites: 15-100bp repeats
in tandem, 1-5kb regions
Sequences of SSRs are highly conserved
BUT the number of repeats vary.

Fig. 10-6

Detecting SSRs - DNA Fingerprinting

1.

Southern Hybridization

Digest genomic DNA

2. Separate bands by
electrophoresis

1. Digest DNA with

restriction enzyme

3. Probe with different

minisatellite sequences
What is this technique?
Fig. 9-10

What other technique could

be used to detect SSR
differences?

Fig. 9-26

Stats - match to 1 specific probe

1/100 or 10-2; test 5 probes (10-2)5
10-10, 1/10 billion chance of match!

2. Separate

3.Transfer

4. Fix

5. Hybridize 6. Detect

Fig. 10-7

DNA Fingerprint Analysis Example

Suspects A (lane 2) and B (lane 4) are accused of

raping a female victim (lane 7 and samples from
victim lanes 3, 6)

10.3 Mobile DNA (p414-424)

1. Mobile DNA elements
1. DNA transposons; retrotransposons
2. Short direct repeats
3. enzymes encoded by mobile elements
4. abundance
5. LTR retrotransposons
6. LINES, SINES
7. movement of LINES and SINES
9. Alu elements
10. processed pseudogenes
11. evolution

What can you conclude from this data?

2 Types of Transposition

Mobile DNA elements = transposable elements

Barbara McClintock (~1950) - Genes can
move on and between chromosomes.
Developed the transposon theory by
studying corn kernel coloration.

cut & paste

copy & paste

Nobel prize for Medicine, 1983

Fig. 10-8

Transposition of a Bacterial IS

Bacterial DNA transposons IS (insertion sequences)

Fig. 10-9

3 Functions of Transposase:
1. Blunt cut at specific
sites in donor DNA
2. Staggered cut in
target DNA
3. Ligation of donor
to target

transposase
Target-site direct repeats hallmarks of all mobile elements

Fig. 10-10

Eukaryotic Retrotransposons
1. LTR (long terminal repeat) containing
2. Non-LTR containing

Retrotransposons Require an RNA Intermediate

Reverse transcriptase
Integrase
Fig. 10-11

Fig. 10-12

Reverse Transcriptase (RT) makes DNA from RNA

Integrase inserts the DNA into the Genome

Evidence that a Transposon goes

through an RNA intermediate

Fig. 10-14

Fig. 10-13

Non-LTR containing Retrotransposons

LINEs

- Long Interspersed Elements

- encode proteins that enable mobility
- ~6,000bp
- 21% of total human DNA (900,000!)

SINEs

- Short Interspersed Elements

- rely on LINE prot. for retrotransposition
- ~300bp
- ~13% of total human DNA (1,600,000!)

LINE Structure

ORF1 - RNA binding protein

ORF2 - RT, DNA endonuclease

Fig. 10-15

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Most SINEs contain Alu elements

-abundant throughout the genome
-homologous sequences support recombination
-exon shuffling

Mobile DNA elements and Evolution

1. Generation of gene families - duplications
- homologous sites for unequal crossing over

2. Creation of new genes - exon shuffling

3. Formation of complex regulatory regions

that control gene expression

Sequenced genome # predicted genes

H. sapiens

32,000

D. melanogaster

13,338

C. elegans

18,266

S. cerevisiae

6,000

Complete sequencing and characterization of 21,243

full length human cDNAs Ota et al., 2004, Nat. Gen.
5,481 unique cDNAs with no protein coding potential!

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