Ch. 16 – Molecular Basis of Inheritance (DNA) - PowerPoint Presentation

Slide1

Ch. 16 – Molecular Basis of Inheritance (DNA)

1

Slide2

Griffith -

Transformation

Since Morgan showed that genes are found on chromosomes,

the new issue was whether the genetic material was in the proteins of the chromosomes or the DNA

. Most thought the

PROTEINS

because there are so many more possibilities and at that time, they didn’t know a lot about DNA.

Griffith did an experiment where he used

streptococcus pneumoniae (which is a bacteria that causes pneumonia in mammals) and injected it into mice. He used harmless strains, and the mice were fine. He used harmful strains, and the mice died. Then he used heat to kill the harmful bacteria and injected that into the mice. The mice were fine. Lastly, he mixed the dead harmful bacteria with live harmless bacteria and injected the mixture into the mice. The mice died.

He concluded that there must be something from the dead bacteria that gets incorporated into the live bacteria to convert it from harmless into harmful…thus killing the mice. He called this process transformation.

Fredrick Griffith

2

Slide3

Transformation

= the process when a cell takes up DNA from the environment and assimilates it into its own genome. It can then start to express some of these “newly acquired” genes on its own.

Transformation was first observed by Fredrick Griffith with his mice.

Transformation

3

Slide4

Avery – Transforming Agent = DNA

Oswald Avery wanted to know what this “transforming agent” that Griffith found was. He focused on DNA, RNA and proteins. He purified each of the chemicals from the harmful bacteria and used each one, individually, to try to transform the harmless bacteria into the virulent strain. The only molecule that successfully transformed the bacteria was DNA. Thus, he found that DNA was the transforming agent .

Even though Avery and his team (McCarty and MacLeod) proved that DNA was the transforming agent, people were still skeptical that it was DNA that carried the genetic material. Because they really didn’t know about the structure of DNA, they didn’t understand how it could possibly carry all that information.

4

Slide5

Alfred Hershey and Martha Chase did more work in determining that DNA was the part of the chromosome that carried the genetic information. They worked with the T2 phage (a

phage

or

bacteriophage

is a virus that infects the bacteria E. coli) and exposed it to radioactive sulfur (which was incorporated into the protein coat) and radioactive phosphorus (which was incorporated into the DNA).

They let the different phages infect different batches of non-radioactive E. coli cells. Soon after the infection, the cells were whirled in a blender and then centrifuged. In the centrifuge, the cells formed a pellet at the bottom and the rest of the liquid formed a supernatant. They then checked for radioactivity. Here were their results:

35

S (proteins) – radioactivity in supernatant 32P (DNA) – radioactivity in pellet (cells)SO…THE PART THAT GOT PUT INTO THE BACTERIA CELLS WAS THE DNA (not protein)!!!

Hershey and Chase – DNA is genetic material5

Slide6

Bacteriophage

A

bacteriophage

is a virus that infects bacteria. A common one is T2, and this infects

E. coli

. Phages are used a lot in scientific experiments (we will see more of this in the next few chapters).

6

Slide7

Hershey and Chase Experiment - Specifics

7

Slide8

Chargaff – Base Pairing Rules

Erwin Chargaff did work with DNA from many different types of organisms. He found that “In the DNA of any one species, the amounts of the four nitrogenous bases are not all equal but are present in a characteristic ratio.”

He found that in humans,

A = 30.9% C = 19.8%

T = 29.4% G = 19.9%

These are now known as Chargaff’s Rules.

This information helped Watson and Crick come up with the structure of DNA

8

Slide9

Franklin & Wilkins – X-ray crystallography

The race to find the correct structure of DNA was in full swing in the 1950’s.

Linus

Pauling came up with one idea that had 3 chains all linked together. This was incorrect. Jim Watson and Francis Crick visited Cambridge where they saw an X-ray picture of DNA done by Rosalind Franklin (this was unpublished work). They knew she had concluded that the sugar-phosphate backbones were on the outside of the double helix. This was enough information to determine that the shape was a helix and Jim Watson was able to calculate important information from this image about DNA.

X-ray crystallography is a technique where a picture is made as X-rays are deflected off of purified DNA samples. The patterns can be mathematically analyzed to figure out important dimensions of the molecules.

Rosalind Franklin

Maurice Wilkins

9

Slide10

Watson and Crick – DNA Structure

While Jim Watson is still living, Francis Crick died in 2004.

Using information from Chargaff, Franklin, and Wilkins, Jim Watson and Francis Crick discovered the

structure of DNA

. They found that the bases match up on the inside of the chain, and the phosphates and sugars are on the outside of the chain. They also figured out that the strands are

antiparallel

, with the subunits running in the opposite direction. Along with Wilkins, they won the

Nobel Prize

for their work.With their idea, they also proposed the semi-conservative model of DNA Replication.

10The nitrogenous bases are paired in specific combinations:

adenine with thymine and guanine with cytosine. Only a

pyrimidine-purine pair

produces the diameter indicated by the X-ray data.

Based on details of their structure,

adenine forms two hydrogen bonds only with thymine

, and

guanine forms three hydrogen bonds only with cytosine

(

Chargaff’s Rules

.) The

base-pairing rules dictate the combinations of nitrogenous

bases that form the “rungs” of

DNA

. The

linear sequence of the four bases can be varied in countless ways, and each gene has a unique order of nitrogenous bases

.

Slide11

Meselson & Stahl – DNA Replication

Watson and Crick proposed a method by which DNA replicates called the semi-conservative model.

Meselson

and Stahl came up with an experiment to support their hypothesis.

The 3 models of DNA replication are:

Conservative

– one whole piece of DNA has both of the strands from the parent DNA and the other one has two totally new strands

Dispersive – random pieces from the parent DNA show up in the new DNA in both strands Semiconservative – Each new DNA molecule has one strand from the parent and one strand that’s new11

Slide12

Meselson & Stahl Experiment

They cultured E. coli for a few generations using a medium with “heavy nitrogen” (

15

N). This way, the “parent” DNA would have this heavier type of nitrogen. They then put the bacteria into a medium with lighter nitrogen (

14

N). This way, any NEW DNA they made would have the

14

N in it, rather than 15N. They could then test the densities of the DNA by extracting it from the cells and centrifuging it. These are their results. Proved the

Semiconservative model of DNA replication 12

Slide13

Models of DNA Replication

Watson and Crick hypothesized that the

semiconservative

model was how DNA replicated.

Meselson

and Stahl confirmed this when they did their experiments using the heavy isotope of nitrogen.

13

Slide14

Structure of DNA

The structure of DNA is a double helix. It is two chains held together in the middle by hydrogen bonds between nitrogenous bases. The backbone of the molecule is made up of phosphates and sugars. The bond between the phosphates and sugars is called the

phosphodiester

linkage

.

A

nucleotide

is the basic unit of DNA. It is composed of a sugar, phosphate, and nitrogen base.

Between A-T, there are 2 hydrogen bonds; Between C-G, there are 3 hydrogen bonds holding the bases together. 14

Slide15

Double Helix

15

Slide16

DNA Replication in PROKARYOTES

The replication of a DNA molecule in

PROKARYOTES

begins at

a special site

called

the origin of replication.

Enzymes separate the strands, forming a replication “bubble.”Replication proceeds in both directions until the entire molecule is copied.16

Slide17

DNA Replication in Eukaryotes

The Beginning

There is a special site where replication starts. This sequence of DNA is recognized by proteins that come in and separate the 2 strands.

Replication proceeds

both ways

. This is called

the origin of replication

; or the replication bubble and forms a replication fork.

In eukaryotes, there a lots of origins, but in prokaryotes, there is only one origin. 17

Slide18

Unwinding DNA

18

Several kinds of proteins participate in the unwinding of parental strands of DNA.

Helicases

untwist the double helix and separate the template DNA strands at the replication fork.

Single-strand binding proteins

bind to unpaired DNA strands, stabilizing them. Unwound sections of parental DNA strands are now available to serve as templates for the synthesis of new complementary DNA strands.

Slide19

Primers

T

he

enzymes that synthesize DNA

cannot

initiate

synthesis of a polynucleotide. They can only add nucleotides to the end of an existing chain that is base-paired with the template strand.The initial nucleotide chain is a short

stretch of RNA called a primer, synthesized by the enzyme primase. Primase starts a complementary RNA chain from a single RNA nucleotide, adding RNA nucleotides one at a time, using the parental DNA strand as a template. The RNA primers are eventually replaced with DNA nucleotides by DNA polymerase

. 19

Slide20

DNA Replication – Elongating the Strand

20

Enzymes called

DNA polymerases

catalyze the synthesis of new DNA by

adding nucleotides to a preexisting chain

. (NOTE: there are many DNA polymerases in eukaryotes)

The two strands of DNA in the double helix are antiparallel.

Slide21

Antiparallel Strands of DNA

The two strands of DNA run antiparallel – think 2 pencils going in the opposite directions.

5’ End

= Free Phosphate

3’ End

= Free Hydroxyl Group

21

Slide22

Replication

DNA polymerases can add nucleotides

only to the free 3



end

of a growing DNA strand.A new DNA strand can elongate only in the 5

3 direction.Along one template strand, DNA polymerase can synthesize a complementary strand continuously by elongating the new DNA in the mandatory 5

3 direction.22

Slide23

Leading Strand vs. Lagging Strand

The

leading strand

is a continuous strand and is elongating

TOWARDS the replication

fork

. It only needs ONE primer.

The

lagging strand is a continuous strand and is elongating AWAY from the replication fork. Small Okazaki fragments are needed…each one using a NEW primer. Since DNA Polymerase can only add to the 3’ end, only the strand with the 3’ end going towards the replication fork can add continuously. 23

Slide24

Okazaki fragments

Unlike the leading strand, which elongates continuously, the lagging stand is synthesized as a series of short segments called

Okazaki fragments

.

Although only one primer is required on the leading strand, each Okazaki fragment on the lagging strand must be primed separately.

Another

DNA polymerase, replaces the RNA nucleotides of the primers with DNA versions

.DNA ligase joins the sugar-phosphate backbones of all the Okazaki fragments into a continuous DNA strand.24

Slide25

Proteins used for Replication

DNA Polymerase

Important Enzymes

:

- DNA Polymerase

- Ligase

-

Primase - Helicase

- Single Strand Binding Proteins 25

Slide26

Proofreading in Replication

Sometimes the wrong nucleotide is placed in a spot, or sometimes the nucleotides are changed by UV light or other radioactive substances.

DNA polymerase

proofreads each new nucleotide against the template nucleotide as soon as it is added

If there is an error,

the part with the mistake is cut out using the enzyme

nuclease

. Then DNA polymerase fills in the gap with the correct nucleotides. Ligase then attaches the backbones to make one strand. This is called

nucleotide excision repair. (an example of mismatch repair)26

Slide27

Altered DNA nucleotides have evolutionary significance.

Mutations

can change the phenotype of an organism. If they occur in germ cells, which give rise to gametes, they can be passed on from generation to generation.

The

vast majority

of such changes are

harmful, but a very small percentage can be beneficial. Mutations are the source of the variation on which natural selection operates during evolution and are

ultimately responsible for the appearance of new species. The balance between accuracy of DNA replication or repair and a low mutation rate has allowed the evolution of the rich diversity of species we see on Earth today.27

Slide28

Shortening of DNA Strands

Because DNA Polymerase can only add to the 3’ end, the 5’ end can never be complete.

Therefore, after many, many replications, the DNA strands become shorter.

To prevent genes from eroding, chromosomes have

telomeres

.

28

Slide29

Telomeres – NON CODING!

Telomeres

are segments of

non-coding

DNA that are found in eukaryotic chromosomes at the

5’ end

. They usually consist of 1000’s of copies of a repetitive sequence.

They act as buffers to protect the organisms genes. Because they become shorter with every round of replication, telomeres are lengthened by the enzyme telomerase. This enzyme is found in embryonic cells, and in adults, in germ – line cells (not somatic cells). Normal shortening of telomeres may protect organisms from cancer by limiting the number of divisions that somatic cells can

undergo.29

Slide30

Prokaryotes

Most bacteria have a single, circular, double-stranded DNA molecule associated with a small amount of protein.

A bacterium has a dense region of DNA called the

nucleoid

.

30

Slide31

Structure of Chromatin

Levels of Chromatin Packing

:

1. Nucleosome

2. 30 nm chromatin fibers 3. Looped Domains

4. Chromosomes31In the cell, eukaryotic DNA

is packaged with protein to form chromatin.The packing of chromatin varies during the course of the cell cycle. Although interphase chromatin is generally much less condensed than the chromatin of mitotic chromosomes, it shows several of the same levels of higher-order packing.

Slide32

Heterochromatin

vs. Euchromatin

Heterochromatin

– very tightly coiled; therefore it is NOT transcribed

Euchromatin

– “true chromatin”; it is less compact and therefore the RNA polymerase can attach and it can get transcribed; more loosely packed32

Slide33

Energy Source for DNA Replication

The nucleotides that get added

by DNA Polymerase are

actually nucleoside triphosphates (NTP). These are nucleotides with 3 phosphates instead of 1. They are used as an energy source. The “extra” 2 phosphates break off to release energy (break bond = releases energy)

The 2 phosphate molecule is called pyrophosphate.

Pyrophosphate

NTP – nucleoside

triphosphate

33