Rapid Pace of Disease Gene Discovery

Susceptibility genesTotal disease genes identified- 1,112 (as of yr 2000)

• 1940’s- T.H. Morgan’s group showed that genes are located on chromosomes

• But…. Are _________ or DNA the genetic material??

• Most believed it to be protein

• However, this was not consistent with experiments with microorganisms, like bacteria and viruses.

1. The search for genetic material lead to DNA

History

DNA Structure and Function Chapter 10

• 1928- Frederick _______ - He studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals.

• One strain, the R strain, was harmless.

• The other strain, the S strain, was ___________.

• Experiment- Griffith mixed _____________ S strain with live R strain bacteria and injected this into a mouse.

• Result- Mouse died, but the S strain was recovered from the mouse’s blood. S R Killed S R + Killed S

LivesDies Lives Dies

Injection

• Griffith called this phenomenon, ______________-a change in genotype and phenotype due to the assimilation of a foreign substance (now known to be DNA) by a cell.

Live S found

Fig. 16.1

• What was the transforming substance in Grifith’s experiments??

• Finally in 1944, Oswald_______, Maclyn McCarty and Colin MacLeod announced that the transforming substance was _____ .

• Still, many biologists were skeptical.

• In part, this reflected a belief that the genes of bacteria could not be similar in composition and function to those of more complex organisms.

• In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the phage T2

• Phage T2 is a ______ that infects bacteria

• Viruses consist of a DNA (sometimes ____ ) enclosed by a protective coat of protein.• To replicate, a virus infects a host cell and takes over the cell’s metabolic _________________ .

What is a virus??

• The T2 phage, consisting almost entirely of DNA and protein, attacks Escherichia coli (E. coli), a common _______________ bacteria of mammals.

• This phage can quickly turn an E. coli cell into a ___________ factory that releases phages when the cell ruptures.

Fig. 16.2a

• The Hershey and Chase Experiment-Designed to determine the source of genetic material in the phage

• Procedure: Two parallel experiments#1Grow T2 phage in the presence of _____________sulfur,

marking the ___________ but not DNA

#2 Grow T2 phage in the presence of _____________ phosphorus , marking the _______ but not proteins

•Allow each batch to infect separate E. coli cultures.

•Shortly after infection, use blender to “shake loose” any remaining phage components

•Recall that sulfur is a component of some_____________.

•Recall that __________is a component of DNA

Fig. 5.29

ATP

• Now centrifuge – bacteria pellet and phage do not.

• Tested the pellet and ____________ of the separate experiments for the presence of radioactivity.

Hershey-Chase experiment (cont.)virus particle labeled with 35S

virus particle labeled with 32P

bacterial cell (cutaway view)

label outside cell

label inside cell

• Results of Hershey-Chase experiment-

• If used radioactive sulfur (proteins labeled)- most of the radioactivity was in the______________, not in the pellet.

• If used radioactive phosphorous(DNA labeled)- most of the ______________ was in the pellet with the bacteria.

•Conclusion- injected _______ of the phage provides the _________ information for new viruses.

Hershey-Chase experiment (cont.)

• Circumstantial evidence that DNA is the genetic material in eukaryotes

1. Cells double the amount of DNA in a cell prior to ________________ and then distribute the DNA equally to each daughter

2. Diploid sets of chromosomes have twice as much DNA as the __________ sets in gametes of the same organism.

• 1947- Erwin Chargaff had developed a series of rules based on a survey of DNA composition in organisms.

• Known that DNA was a polymer of nucleotides consisting of a nitrogenous base, deoxyribose, and a phosphate group.

• Known that there were four bases -adenine (A), thymine (T), guanine (G), or cytosine (C).

• Chargaff noted that the four bases are found in characteristic, but not necessarily equal, ratios

• He also found a peculiar regularity in the ratios of nucleotide bases which are known as Chargaff’s rules.

Chargaff’s rules-

• 1. The number of adenines was approximately equal to the number of thymines (%T = %A).

• 2. The number of guanines was approximately equal to the number of cytosines (%G = %C).

• Human DNA is 30.9% adenine, 29.4% thymine, 19.9% guanine and 19.8% cytosine.

Nucleotide Bases

phosphate group

deoxyribose

ADENINE (A)

THYMINE (T)

CYTOSINE (C)

GUANINE (G)

• By the beginnings of the 1950’s, the race was on to move from the structure of a single DNA strand to the three-dimensional structure of DNA.

• Among the scientists working on the problem were Linus ______, in California, and Maurice Wilkins and Rosalind ________, in London.

Watson and Crick discovered the double helix by building models to conform to X-ray data

Fig. 16.3

• The ________group of one nucleotide is attached to the

_____ of the next nucleotide

in line.

• The result is a “__________ ” of alternating phosphates and sugars, from which the bases project.

The basic structure of DNA was known by 1952

Bases

T

A

C

G

Fig. 16.5

The _______________ chains of each strand are like the side ropes of a rope ladder.

The double helix- a _________ homology

The ladder forms a twist every ____ bases

Pairs of nitrogen bases, one from each strand, form rungs.

• In addition, Watson and Crick determined that chemical side groups off the nitrogen bases would form hydrogen bonds, connecting the two strands.

• Adenine could form two ________ bonds only with thymine

• Guanine could form ______hydrogen bonds only with cytosine.

• This finding _________Chargaff’s rules.

Fig. 16.6

• The linear sequence of the four bases can be varied in countless ways.

• Each gene has a unique order of nitrogen bases.

• In April 1953, Watson and Crick published a succinct, one-page paper in Nature reporting their double helix model of DNA.

The Nobel Prize in Chemistry was awarded in later years

• In a second paper, Watson and Crick published their _____________for how DNA replicates.

Base pairing is at the heart of DNA replication

Base pairing enables existing DNA strands to serve as templates for new complimentary strands

Fig. 16.7

Double-stranded

DNA

1.Separate strands

2.Add complementary bases

3.Connect bases

When a cell copies a DNA molecule, each strand serves as a _________

for ordering nucleotides into a new ________________ strand.

DNA Replication

newnew old old

• Each parent strand

remains ________

• Every DNA

molecule is half

“____” and half

“_____”

• Bacterium E. coli (5 million base pairs- )- _______________

• Human cells- (6 billion base pairs)- ___________

DNA replication requires a large number of enzymes and other proteins

A. DNA replication is fast and efficient

Time to copy the entire genome and divide into daughter cells:

• Less than one error per __________nucleotides.

Accuracy

• More than a dozen enzymes and other proteins required

Machinery

B. Where does replication start? –

Bacteria- one site in DNA recognized by specific proteins

Answer- The ______________________

• Eukaryotes- ______________ of origin sites per chromosome. • At each origin site, the DNA strands separate forming a

replication “______ ” with ______________ at each end.

• The replication bubbles _______ as the DNA is replicated and eventually ______ .

Fig. 16.10

Origin of replication (cont.) 15.3

15.215.1

14

13.3

13.11211

11.111.2

12

13.113.213.3

14

15

21

2223.1

23.3

31.131.231.3

3233.133.334

35.1

• DNA polymerases add the ____________ (nitrogen base, deoxyribose, and a triphosphate tail).

• Rate of elongation

• ____nucleotides/second in bacteria

• ___per second in human cells. The raw nucleotides are nucleoside triphosphates.

Fig. 16.11

Two phosphates are removed upon addition of nucleotide

One

phosphate connects

nucleotides

Fig. 16.16

• At the replication fork

• the leading stand is copied ______________ from a single primer

• the lagging strand is copied in ____________ using many primers.

Summary of DNA replication

• Nucleotide pairing errors occur at a rate of one error per __________ base pairs.

• Yet the final error rate is only one per billion nucleotides.

Enzymes proofread DNA during its replication and repair damage in existing DNA

a. ______________ proofreads each new nucleotide against the template nucleotide as soon as it is added. If incorrect, it corrects it.

How do we account for this discrepency??

Answer- ______________

b. Each cell continually monitors and repairs its genetic material, with over ______ repair enzymes identified in humans.