1

Bi 1 “Drugs and the Brain”

Lecture 15

Thursday, April 27, 2006

The Human Genome

For today’s lecture,

It’s appropriate inspect the memorials

to Norman Davidson

on the walls of this lecture room.

2

from Lecture 15:

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

receptor

tsqiG protein

enzymechannel effector

The Bi1 intellectual journey

3

Where does the Human Genome Sequencing Project stand today?

Essentially finished!

http://www.genome.gov/11006929

3.2 billion base pairs (nearly 10 orders of magnitude)

Major effort from information technology (60% of the professionals are software experts)

. . . but annotating the human genome has just begun

What is a Genome?

The genome is the information set containing the totality of DNA sequence that

specifies a species (on average)

or an individual member of a species.

4

Lander et al

5

Importance of DNA structure

A double molecule

Information content in base-pairing

Chemistry of base-pairing

Organization of the genome

Lectures 15, 17, 18:Relationship between sequence and function

6

DNA structure

Requires Swiss-PDB viewer on your computer

http://www.its.caltech.edu/~lester/Bi-1/DNA.pdb

Compute and view H-bondsRender in solid 3-D

7

Two types of base pairs in DNA: C-G pairs are more stable(Watson-Crick base pairing)

A-T base pair2 hydrogen bonds

C-G base pair3 hydrogen bonds

dA

dT

dC

dG base

ribose (sugar)

phosphate

8

Norman Davidson wrote,

“Some time around 1958 or 1959 I was thinking about switching to biology-related

research . . . I learned that ion channels were selective for either sodium ions or

for potassium ions. This fascinated me because I knew from my undergraduate

analytical chemistry course how difficult this separation was. . . I told Bernard Katz

about my interest in doing something chemical about ion channels. He advised me

to forget about it because . . . it would be impossible to isolate a sufficient quantity

to do anything chemical.”

26

no stimulus; spontaneous “miniature”postsynaptic potentials

repeated stimuli to presynaptic neuron

5 mV

50 - 1000 channels (differs among types of synapse).

This is the contents of a single vesicle.

Electrophysiological analysis of quantal synaptic transmission

(slide 3)

Analysis of Quantal Synaptic Transmission

00.10.20.30.40.50.60.70.80.9

1

1 2 3 4 5 6Amplitude of Postsynaptic Response (mV)

Fra

ctio

n o

f O

bse

rvat

ion

s

Stimulated

Spontaneous

0 1 2 3 4 5

from Lecture 9

Therefore, Davidson

began by studying the

chemistry of DNA

9

1. The hydrogen bonds that form double-stranded DNA are easily disrupted by heating.

2. Some dyes fluoresce when they bind to double-stranded DNA.

Physical Chemistry of DNA Hybridization:

Studied at Caltech in ‘60’s and ‘70’sby Norman Davidson

10

from Lecture 15:

kinase

phosphorylatedprotein

cAMPCa2+

intracellularmessenger

receptor

tsqiG protein

enzymechannel effector

The Bi1 intellectual journey

Beginning in 1980, Norman Davidson

used his skills at molecular biology to

find the genes for several of these

molecules. His intellectual journey.

11

GC content is quite nonrandom

Lander et al

Expectations from random variation:

Coefficient of variation = 100/10,000 = 1%

100000,10

12

Humans have 22 pairs of chromosomes, plus the X and Y.Males are XY; females are XX.

A. Each chromosome is “painted” with a unique combination of fluorescent dyes

B. Photoshop: we have moved the chromosomes to form pairs

© Garland; Little Alberts Fig 5-12

13

Humans have 22 pairs of chromosomes, plus the X and Y.Males are XY; females are XX.

A. Each chromosome is “painted” with a unique combination of fluorescent dyes

B. We have arranged the chromosomes to form pairs.

© Garland; Little Alberts Fig 5-12

14

“To find who’s the tallest,

we start with the smallest . . .

We start with the smallest. Then what do we do?

We line them all up. Back to back. Two by two.

Taller and taller. And, when we are through,

we finally will find one who’s taller than who.

But you have be smart and keep watching their feet.

Because sometimes they stand on their tiptoes and cheat.“

Dr. Seuss explains fluorescence microscopy of chromosomes.

“Happy Birthday to You”, 1959.

15Little Alberts Fig 10-16

Genes can be localized crudely by

hybridizing a fluorescent nucleotide probe to chromosomes

2 m

6 distinct genes are probed in this image

Seuss 1959

16

An older staining method

reveals dark bands in the

chromosomes.

(termed p12, q21, etc)

The genome sequence

reveals that these bands

are AT-rich.

#21, 45 Mb

#1, 279 Mbshort arm,p

long arm,q

1 mLittle Alberts 5-13© Garland

17

What happens in this room? We make enough DNA to sequence.

18

Two ways to amplify a DNA sequence 1. Plasmid cloning in bacteria (0.500-10 kb):

Little Alberts Fig. 10-22© Garland

“small, circular double-stranded DNA molecules that are separate from the larger bacterial chromosome”

recombine (“splice”), with base pairing

19

The “Bacterial Artificial Chromosome” (BAC) ~120 kb

The Goldilocks plasmid: not too small, not too large

Mel Simon

5

A single BACin a fluorescencemicroscope

20

> 12 nt

100 - 10,000 nucleotide pairs

Two ways to amplify a DNA sequence 2. The polymerase chain reaction (PCR)

DNA polymerase requires a region of

double-stranded DNA

Little Alberts Fig. 10-27© Garland

21

PCR amplifies DNA exponentially

DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

fragment of DNA

to be detected

heat to separate DNA

strands

heat to separate DNA

strands

heat to separate DNA

strands

Little Alberts Fig. 10-27-2© Garland

22

DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

DNAsynthesis

cool tobind primers

fragment of DNA

to be detected

heat to separate DNA

strands

heat to separate DNA

strands

heat to separate DNA

strands

PCR amplification uses 15 to 40 cycles (3 - 5 min each) in a sealed tube

DNA polymerase (enzyme)

plusdATPdGTPdCTPdTTPDNA templateprimers

23

In fact, PCR uses dozens of sealed tubes simultaneously in a heated and cooled metal block

24

Thermostable DNA polymerase is obtained fromThermococcus litoralis ,an archaebacteria first isolated from deep submarine vents.

This organism can grow at 98o C.

Confirming that PCR can detect single molecules:

In experiments on individual sperm, only 50% of the sperm had signals for a gene on the Y chromosome; but all were positive for genes on autosomes.

25

Lee Hood ‘60

Fluorescence applied to DNA sequencing

practical limit:500 bp for each distinct DNA molecule

The peaks broaden as nucleotides are added with statistical fluctuations around an average rate. This limits the length of each run.

26

The new genome vision:

“New technologies that can sequence the entire genome of any person for less than $1,000.”

http://www.genome.gov/11006929

27

Proceedings of the National Academy of Sciences, 2003

Ido Braslavsky, Benedict Hebert, Emil Kartalov ‘96, Stephen R. QuakeDept of Applied Physics, Caltech

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

28

Total internal reflection fluorescence microscopy will enable single-molecule sequencing

Dichroic mirrors

Braslavsky et al, 2003

Experiments use single-molecule fluorescence, FRET, and photobleaching

10 m

Condensing Lens

29

Restriction enzymes cut DNA to manageable lengths

Uniqueness / fragment lengths:4-base hitter: 1 in 44 = 256 6-base hitter: 1 in 46 = 40968-base hitter: 1 in 48 = 65,536

a “6-base hitter”

Part of Little Alberts Fig. 10-4© Garland

Most restriction enzymes have 2 identical subunits

30

Lander et al, Figure 2

50-200 MBon each

chromosome

~ 100 kB

~ 1 kB

31

Cumulative pace of Disease Gene Discovery (1981-2003)

Number of disease

genes identified

1600

1200

800

400

0‘83 ‘85 ‘87 ‘89 ‘91 ‘93 ‘95 ‘97 ‘99 ‘01‘81 ‘03

32

New orthologs and paralogs of common drug targets identified by searching the draft human genone sequence (Lander et al, Table 27)

To be discussed in Lecture 25

33

Exemplar Genomes fully or partially sequenced

E. coli 4.6 ~ 4,300

Yeast 12.5 ~ 6,300

Mustard weed 120 ~ 25,000

Worm 97 ~ 22,000

Drosophila 120 ~ 15,000

Mouse 2,700 ~23,000

Human 3,300 ~23,000

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Genome

Rice 466 ~48,000

Mosquito 278 ~15,000

Organism genome size number of genes(common) (Mb)

See also Little Alberts 1-40

34

Humans have only about as many genes as worm,

and 50% more than fly.

However, human genes differ in two ways from those in worm or fly.

1. Human genes are spread out over much larger regions of genomic DNA

2. Human genes are used to construct more alternative transcripts.

Result: humans have ~ 5 times as many protein products as worms or

flies (Lecture 17).

35

More than 3 million SNPs in the human genome have been identified.

This collection should allow researchers to conduct genome-wide

linkage mapping of the genes in the human population.

Basically: hunt for a gene for a phenotype (such as a disease) by

asking which people have both the phenotype and one version of the

polymorphism. This means that the phenotype is near the gene that

contains the polymorphism.

Single-nucleotide polymorphisms (SNPs)

36

Genomics and genetics in Bi 1

“Drugs and the Brain”

15. The human genome

17. DNA to mRNA

18. From mRNA to protein

20. Genetic diversity and genetic animals

20. An Exemplar Simple Genetic Disease: Cystic Fibrosis, Cholera, and Osmosis

21. Two other exemplar simple genetic diseases: Long-QT syndrome and some Epilepsies

22. Schizophrenia and the complex genetics of psychiatric diseases 25. Evolution 1: Inferences from Molecular Biology

27. Evolution 2: The eye as an example

37

Bi 1

“Drugs and the Brain”

End of Lecture 15

For today’s lecture,

It’s appropriate inspect the memorials

to Norman Davidson

on the walls of this lecture room.