UNIT 2 - pages.stolaf.edu · Okazaki fragment 2 DNA pol III makes Okazaki fragment 2. 5 ...

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UNIT 2 A. Mendel and the Gene Idea (Ch11) B.  Chromosomal Basis of Inheritance (Ch12) C. Molecular Basis of Inheritance (Ch13) D. Gene Expression from Gene to Protein (Ch 14) Where are we going?

CH13More discoveries concerning the heritable material!What is it?What might its structure be?Replication and how it worksListen to an interview of WatsonWe will try to keep our eye on big picture as we look over details of replication!


Figure 13.21b

Loops

30-nm fiber

300-nm fiber

Replicated chromosome (1,400 nm)

Scaffold

Chromatid (700 nm)

Reminder of structure!


Figure 13.21a

Histone tail Histones

H1

DNA double helix (2 nm in diameter)

Nucleosome (10 nm in diameter)

Histones are a protein


Figure 13.21

Histone tail Histones H1

DNA double helix (2 nm in diameter)

Nucleosome (10 nm in diameter)

Loops

30-nm fiber

300-nm fiber

Replicated chromosome (1,400 nm)

Scaffold

Chromatid (700 nm)


Remember Morgan sex linked traits..He confirmed that genes are on chromosomes

But chromosomes are made up of protein AND DNA…which one is doing the important job of harboring information?

We will focus on these 3 classic experiments highlighted in this chapter……•  Griffith (Fred)•  Hershey and Chase (Alfred and Martha)•  Meselson and Stahl (Matt and Frank)


Early 1900s……everyone was focusing on proteins as hereditary material

DNA was big but monotonous seemed to have no specific cellular function

Proteins were enzymes! And had structural jobs in cells!

Proteins were also known to be polymers of numerous amino acids.

This 20 amino acid "alphabet” of proteins could be arranged into more unique information-carrying structures than the four-letter alphabet of DNA


http://en.wikipedia.org/wiki/File:Fred_Griffith_and_%22Bobby%22_1936.jpg

Griffith (Trying to develop a vaccine)Turned to a bacterial pathogen…Streptococcus pneumoniae

Is it gram positive or negative?


This species (Streptococcus pneumoniae) is one of the species that can cause meningitis (along with Neisseria meningitidis). What is meningitis?Did you get your meningitis vaccine?

Note you can also get viral meningitis but usually not as dangerous-no vaccine.

Streptococcus pneumoniae is also a cause of pneumonia along with a bunch of other organisms…

What is pneumonia?


https://blogs.scientificamerican.com/guest-blog/why-is-meningitis-still-causing-deaths-on-u-s-college-campuses/


So what did he do??

2 strains what do we mean by strains?

One virulent and not so virulent (What do we mean by virulent?)


Figure 13.2

Living S cells

Mouse healthy Results

Experiment

Mouse healthy Mouse dies

Living S cells

Living R cells

Heat-killed S cells

Mixture of heat-killed S cells and living R cells

Mouse dies

Work by Avery identified the transforming substance as DNA (but others not convinced)


Transformation-did not really understand mechanism

•  Can we do this-pick up DNA from our environment?

•  Why does this ability freak us out….what do we feed chickens pigs and cows to make them grow fast?

•  Where do we put their manure waste??

•  Where can this manure waste go?


Hershey and Chase (1952) (Alfred and Martha)Their work also pointed to DNA rather than proteins…

Bacteriophages what are they???? (worked with one called T2)

E. coli bacterial cell-who is E. coli?

At this time they knew that viruses could infect bacterial cells with “heritable material” and …...that material that is put into bacterial cells hijacks or reprograms the cell to makes new viruses.

Is it the coat (protein) or is it the stuff inside (DNA)?

Which is actually entering the cell and reprogramming it?


Some phages grown in media for a couple hrs with radioactive Sulphur…which should be incorporated into some coat proteins (Methionine, Cysteine)

Other phages grown in media for a couple hrs with radioactive Phosphorus….which should be incorporated into DNA


These phages were allowed to infect bacteria.

Knocked off phage from cell and then centrifuged to concentrate cells. Where is Phosphorus (DNA)? Where is Sulfur (Protein Coat)??

Batch with P (DNA) labeled, found in cells.

Batch with S (Protein Coat) labeled, found in liquid

Was the protein coat passed on?

Thus, the Hershey–Chase experiment helped confirm that DNA, not protein, is the genetic material.


Figure 13.4a

Labeled phages infect cells.

Batch 1: Radioactive sulfur (35S) in phage protein Experiment

Agitation frees outside phage parts from cells.

Centrifuged cells form a pellet.

Radioactivity (phage protein) found in liquid

Radioactive protein

Centrifuge

Pellet

1 2 3

4


Figure 13.4b

Batch 2: Radioactive phosphorus (32P) in phage DNA

Radioactivity (phage DNA) found in pellet

Radioactive DNA

Centrifuge Pellet

Labeled phages infect cells.

Agitation frees outside phage parts from cells.

Centrifuged cells form a pellet.

1 2 3

4

Experiment


Watson-Crick Model predicted….

Each of two daughter molecules would have one parental strand and one newly made!

© 2014 Pearson Education, Inc. http://www.dnaftb.org/20/gallery.html

The annual "degree granting" party of Max Delbrück's phage group held in the house shared by Meselson and Stahl at Caltech. (L-R) Harry Rubin, Max Delbrück, Rene Cohen, Matt Meselson, Frank Stahl


Meselson and Stahl-clever experiment…What did they do?? STABLE ISOTOPES!

•  Grew up some E. coli in a medium with heavy N (or 15N)-incorporated into DNA bases (A,C,T,G).

•  Transferred to a light N (or 14N) medium and let E. coli replicate there.

•  Then start taking subsamples of this mix over time.

•  Can separate by weight using centrifuge…get bands or layers

Asked…what N (heavy or light) is going to be in this population of E. coli at different times?


Figure 13.11a

DNA sample centrifuged after first replication

DNA sample centrifuged after second replication

Bacteria cultured in medium with 15N (heavy isotope)

Bacteria transferred to medium with 14N (lighter isotope)

Less dense

More dense

Experiment

Results

1

3 4

2


Figure 13.10

(a) Conservative model

(b) Semiconservative model

(c) Dispersive model

Parent cell First

replication Second

replication

Dark blue=heavy 15N

Move from heavy into light N media-what will happen when you centrifuge?


Figure 13.10

(a) Conservative model

(b) Semiconservative model

(c) Dispersive model

Parent cell First

replication Second

replication

Dark blue=heavy 15N

Second Replication….How many bands?


Figure 13.1

Watson and Crickhttp://www.ted.com/talks/james_watson_on_how_he_discovered_dna.html


Watson interview!

What did you think was interesting?What was unusual?Surprising?Insights into how science works?


First some info about structure of DNA!


Figure 13.7

(c) Space-filling model

(a) Key features of DNA structure

(b) Partial chemical structure

3ʹ end

5ʹ end

3ʹ end

5ʹ end

Hydrogen bond

T A

C G

C G

3.4 nm

T A

T A

C G

C G

T

A

1 nm

0.34 nm

T A T

A

C G

C G

C

G

C G

T A

T A

C G C

G C G


Figure 13.7a

(a) Key features of DNA structure

3.4 nm

T A

C G

C

G

T

A

1 nm

0.34 nm

T A T

A

C G

C G

C

G

C

G

T A

T A

C G C

G C G


Figure 13.7b

3ʹ end

5ʹ end

3ʹ end

5ʹ end

Hydrogen bond

T A

C G

C G

T A

Phosphate group

12

What are the blue pentagons?

•  A nucleotide is a phosphate molecule + a sugar molecule + a nitrogen rich base

Nitrogen rich bases A, T, G, C

A and G have double rings -two double rings would not fit so A bonds with T and G with C.


Figure 13.5

Sugar– phosphate backbone

DNA nucleotide

Nitrogenous bases

3ʹ end

5ʹ end

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)


Figure 13.14

Pyro- phosphate

New strand

Phosphate

Nucleotide

5ʹ 3ʹ Template strand

Sugar Base

5ʹ

3ʹ

5ʹ

3ʹ

5ʹ 3ʹ

DNA poly-

merase T

A T

C G

A

C G

C

P

P i P

i 2

A T

C G

A

C G

C


Figure 13.9-3

(a) Parental molecule

(b) Separation of parental strands into templates

(c) Formation of new strands complementary to template strands

through addition of nucleotides

T A

C G

C G

T A

T A T A

T A

C G

C G

T A

T A

C G

C G

T A

T A

T A

C G

C G

T A

T A


DNA replicationWhere does it take place? When does it take place?How does it work??


Figure 13.12

Replication fork

5ʹ

5ʹ 5ʹ 3ʹ

3ʹ

3ʹ

RNA primer

Origin of replication?Replication fork?3’ 5’? (skip)RNA primer (made by pink blob=primase). Other blobs (green=helicase=unzips)(blue-=topoisomerase)(gray=single stranded binding proteins)


Figure 13.15

Parental DNA

5ʹ 3ʹ

5ʹ

3ʹ

5ʹ

3ʹ

Continuous elongation in the 5ʹ to 3ʹ direction

5ʹ 3ʹ

5ʹ

3ʹ

DNA pol III

RNA primer

5ʹ 3ʹ

Origin of replication


Lagging strand

Lagging strand

Leading strand

Leading strand

Overview

Primer

DNA polymerase III (salmon blob)

Binds then ‘walks’ along, adding complementary nucleotide bases (A, C, G and T) to the strand of DNA (in the 5’ to 3’ direction-but you do not need to know this).It is making a leading strand!


Figure 13.16a

Origin of replication Lagging strand Lagging

strand

Overall directions of replication

Leading strand

Leading strand

Overview

Lets zoom in to #1 and look at the lagging strand.


Figure 13.16b-1

5ʹ 3ʹ

5ʹ

3ʹ RNA primer

Template strand

1


Figure 13.16b-2

5ʹ 3ʹ

5ʹ

3ʹ RNA primer.

RNA primer for fragment 1

Template strand

DNA pol III makes Okazaki fragment 1.

5ʹ 3ʹ

5ʹ

3ʹ

1

2

Once again DNA polymerase III binds and then ‘walks’ along adding new complementary nucleotide bases (A, C, G and T) to the strand of DNA.


Figure 13.16b-3

5ʹ 3ʹ

5ʹ

3ʹ RNA primer.

RNA primer for fragment 1

Template strand

Okazaki fragment 1

DNA pol III makes Okazaki fragment 1.

DNA pol III detaches.

5ʹ 3ʹ

5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ

1

2

3

What happened? Why did it stop? What is fragment called? Where is DNA pol III going to go next??


Figure 13.16c-1 RNA primer for fragment 2

Okazaki fragment 2 DNA pol III

makes Okazaki fragment 2.

5ʹ

3ʹ 5ʹ

3ʹ 4

Now you have all these bits what has to happen next? And who does that?

Can you leave the RNA primer pieces in there?


Figure 13.16c-2 RNA primer for fragment 2

Okazaki fragment 2 DNA pol III

makes Okazaki fragment 2.

DNA pol I replaces RNA with DNA.

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ 4

5

A different DNA polymerase comes in to replace them!(I vs III)


Figure 13.17

3ʹ 5ʹ


Lagging strand

Lagging strand

Overall directions of replication

Leading strand

Leading strand

Overview

5ʹ 3ʹ

5ʹ

3ʹ

Leading strand

Lagging strand

DNA ligase DNA pol I DNA pol III

Primase

DNA pol III Primer

5ʹ 3ʹ

5ʹ

3ʹ

Lagging strand template

Parental DNA

Helicase

Single-strand binding proteins

Leading strand template


Figure 13.14

Pyro- phosphate

New strand

Phosphate

Nucleotide

5ʹ 3ʹ Template strand

Sugar Base

5ʹ

3ʹ

5ʹ

3ʹ

5ʹ 3ʹ

DNA poly-

merase T

A T

C G

A

C G

C P i P

A T

C G

A

C G

C

Which DNA polymerase is this?


The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands.

Rank the primers (the red specks) in the order they were produced. If two primers were produced at the same time, overlap them.


The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands.

Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them.a and h then b and g then c and f and finally e and d


True of Leading strand, Lagging strand, or Both????

Daughter strand elongates away from replication fork

Multiple primers needed

Made in segments

Made continuously

Daughter strand elongates toward replication fork


True of Leading strand, Lagging strand, or Both????

Daughter strand elongates away from replication fork Lag

Multiple primers needed Lag

Made in segments Lag

Made continuouslyLead

Daughter strand elongates toward replication fork Lead


In an analysis of the nucleotide composition of DNA, which of the following will be found? (Imagine counting number of nucleotides of each type)

A = G and C = T

G + C = T + A

A = C

A + C = G + T


In an analysis of the nucleotide composition of DNA, which of the following will be found?

A = G and C = T

G + C = T + A

A = C

A + C = G + T


Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?

31%

42%

8%

16%

It cannot be determined from the information provided.

UNIT 2 - pages.stolaf.edu · Okazaki fragment 2 DNA pol III makes Okazaki fragment 2. 5 ...

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