1KEY CONCEPTS from LECTURE 1:

1. Prok. vs. Euk. cells2. The players - DNA, RNA, protein3. The processes - replication, transcription,

translation4. DNA is the genetic material5. Chemical composition and structure of DNA6. Denaturation of DNA7. Supercoiling and relaxing of circular DNA

10.6: Organelle DNA (p. 438-444)1. Mito. and chloroplast

DNA evolved from bacteria2. Circular DNA3. Encode structural RNAs and some prot.4. Cytoplasmic inheritance5. Sim. of mito. ribosomes to bacterial6. Slight variations in the genetic code7. Mutations in mtDNA

Fig 8-6

Endosymbiont Hypothesis of Mitochondriaand Chloroplast Origins

Mitochondrion ChloroplastFig 8-3

Multiple mtDNAs/ Mitochondrion

Fig 10-35

Ethidium bromide - DNA DiOC6 - mito

2Cytoplasmic Inheritance

Fig 10-36

1. Petite yeastmutants containdeletions in mtDNA

2. Mate WT to Petite

3. What is the phenotype of daughter cells?

Cytoplasmic Inheritance

Fig 10-36

1. Daughters from mating divide

2. Petite phenotypereappears!

Maternal (mostly) Cytoplasmic InheritanceDiseasesLebers Hereditary Optic Neuropathy - progressive vision loss

BUT highly variable and ONLY passed from mother

Single ntd. change in mtDNA = change in mRNA coding forelectron transport protein

Embryo receives maternal mito (but Paternal Inheritance ofMito. DNA NE J of Med. 2002)

Heteroplasmy - mixture of WT and mutant mtDNA

Maternal mtDNA Inheritance

Identification

Reunite families via mtDNA evidence

Dr. Mary-Claire King - identified a variable segment of mtDNA that could be used as a marker to link mother to child

3Mt DNA encodes: rRNAstRNAssome proteins (intron-less mRNAs)

(Some nuclear encoded proteins, RNAs imported into mito)

Fig 10-37

Differences in mt and nuclear encoded gene expression

Translational machinery, antibiotic sensitivity

The genetic code (i.e. the UGA STOP codonis read as Trp in mito of mammals toyeast, but not in mito of plants)

10.1 Molecular Definition of a Gene (p.405-408

1. Gene definition2. Introns3. Simple Euk.

transcription units4. Complex Euk.

transcription units5. Cell type specific gene expression

3.4 Billion Base Pairs of DNA

Fig. 10-1. Structure of genes and chromosomes

4What is a gene?A gene is the entire nucleic acid sequencethat is necessary for the synthesis of a functional gene product.

This includes: coding regiontranscriptional control regions

(enhancers)splice sitespolyA sites

What about sequences that code for rRNA, tRNA, etc?

2 Types of Transcription Units in Eukaryotes

Fig. 10-2a

1- 1 gene, 1 mRNA, 1 protein

a-d are examples of mutations that could alter gene expression - HOW?

1 2 3

2-

Fig. 10-2b

1 gene, multiple mRNAs and proteins

1. Alternative splicing

2. Alternative PolyA sites

3. Alternative promoters

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

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 vertebrates3. Biological complexity = genome size 4. 3 classes of genomic DNA5. Solitary vs. duplicated genes6. Gene families7. Genes in tandem arrays8. Simple-sequence DNA repeats9. DNA fingerprinting/ Southern Blotting

5Lots of Nonfunctional DNAGenes in 80kb region of DNA:

Why do unicellular organisms typically have more gene richregions compared to the gene-poor desert regions foundin vertebrates?

Fig. 10-3

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Solitary and Duplicated Genes

SOLITARY GENES: found only once in the genome

DUPLICATED GENES: multiple genes of close butnot identical sequences

- usually within 5-50kb of one another- gene families encode homologous proteins

that constitute a protein family

The -globin gene family:

Gene Duplication from Unequal Crossing Over

Fig. 10-4

L1 - homologous non-coding DNA

6Pseudogenes can Arise from Gene Duplication

Pseudo because they contain mutations that terminate translation or block mRNA processing

Sequence drift

Fig. 10-3a

Tandemly Repeated Genes

Encode identical or nearly identical gene products

Head to tail copies

Examples: rRNAstRNAshistone coding genes

Simple-sequence DNA - repetitious DNA

Fig. 4-33Fig. 10-5 - FISH of a Simple Sequence DNA site

Satellite DNA: 14-500 bp repeats in tandem, 20-100kb (at centromeresand telomeres)

Microsatellites: 1-13bp repeatsin tandem, up to 150bpbackward slippage during DNA rep.expanded microsatellites can causedisease

7DNA Fingerprinting using Simple Sequence Repeat (SSR) DNA

Minisatellites: 15-100bp repeatsin tandem, 1-5kb regions

Sequences of SSRs are highly conserved BUT the number of repeats vary.

Unequal crossing over causes differences in SSR number

Fig. 10-6

Detecting SSRs - DNA Fingerprinting 1. Digest genomic DNA

2. Separate bands byelectrophoresis

3. Probe with different minisatellite sequences

What is this technique?

What other technique couldbe used to detect SSRdifferences?

Stats - match to 1 specific probe1/100 or 10-2; test 5 probes (10-2)510-10, 1/10 billion chance of match!

Fig. 10-7

+

_

Southern Hybridization

1. Digest DNA withrestriction enzyme

2. Separate 3.Transfer 4. Fix 5. Hybridize 6. Detect

Fig. 9-26

Fig. 9-10

8DNA Fingerprint Analysis Example

Suspects A (lane 2) and B (lane 4) are accused of raping a female victim (lane 7 and samples fromvictim lanes 3, 6)

What can you conclude from this data?

10.3 Mobile DNA (p414-424)1. Mobile DNA elements1. DNA transposons; retrotransposons2. Short direct repeats3. enzymes encoded by mobile elements4. abundance5. LTR retrotransposons6. LINES, SINES7. movement of LINES and SINES9. Alu elements10. processed pseudogenes11. evolution

Mobile DNA elements = transposable elements

Barbara McClintock (~1950) - Genes canmove on and between chromosomes.

Developed the transposon theory by studying corn kernel coloration.

Nobel prize for Medicine, 1983

2 Types of Transposition

Fig. 10-8

cut & paste copy & paste

9Bacterial DNA transposons -IS (insertion sequences)

transposaseFig. 10-9

Target-site direct repeats -hallmarks of all mobile elements

Transposition of a Bacterial IS

3 Functions of Transposase:1. Blunt cut at specific

sites in donor DNA2. Staggered cut in

target DNA3. Ligation of donor

to target

Fig. 10-10

Fig. 10-11

Eukaryotic Retrotransposons1. LTR (long terminal repeat) containing2. Non-LTR containing

Reverse transcriptaseIntegrase

Fig. 10-12

Retrotransposons Require an RNA Intermediate

10

Reverse Transcriptase (RT) makes DNA from RNAIntegrase inserts the DNA into the Genome

Fig. 10-13

Evidence that a Transposon goes through an RNA intermediate

Fig. 10-14

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

Fig. 10-15

ORF1 - RNA binding proteinORF2 - RT, DNA endonuclease

11

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 regionsthat control gene expression

Complete sequencing and characterization of 21,243 full length human cDNAs Ota et al., 2004, Nat. Gen.

Sequenced genome # predicted genesH. sapiens 32,000

D. melanogaster 13,338

C. elegans 18,266

S. cerevisiae 6,000

5,481 unique cDNAs with no protein coding potential!