Chapter 12

Scientists figured out “how genes work” years before they figured out “what genes are”

They didn’t know what they were, but they knew genes had to…

…be able to store information both for embryonic development and for changes throughout a lifetime

…be STABLE enough to be copied and passed to offspring during sexual intercourse

…be FRAGILE enough so that mutations are possible

In 1869, six years after Mendel’s first experiment, Swiss chemist Friedrich Miescher discovered a new chemical that contained phosphorus but not sulfur Neither lipids, proteins nor carbohydrates contained

phosphorus so he knew he discovered something new

Because it had acidic properties and was discovered inside of a nucleus they were called nucleic acids

Later, in the early 1900’s, it was discovered there were four types of nucleic acids, each with a separate nitrogenous base These were called nucleotides

In 1931, Frederick Griffith was working with mice and two strains of Streptococcus pneumoniae

One strain was “rough” in appearance and non-virulent

One strain was “smooth” in appearance and virulent

When injected with the rough (non-virulent) strain, mice lived

When injected with the smooth (virulent) strain, mice died.

Both as expected.

Also as expected, when he boiled the deadly smooth strand and injected the mice, the mice still lived

Finally, he injected the mice with BOILED smooth strands and ALIVE rough strands, neither of which are deadly.

The mice died.

Although the virulent strand had been killed, the non-virulent strand had absorbed the genetic material from the virulent strand

Even though the virulent cells were dead, the genetic material from the virulent were still present.

The living, non-virulent cells grave-robbed the genes from the dead, virulent cells and used those genes to become virulent.

Griffith’s experiment proved the concept of transformation, which means cells can take parts of other cells and use them for themselves

The “next experiment” for Griffith was to answer the following question: what was the molecule that the living cells grabbed from the dead cells?

Remember: We did not yet know about DNA, genes, chromosomes, etc

In the early 1900’s, good money said genetic information was made of proteins

We knew genes must be passed from cell to cell. Proteins can do this

Nucleic acids only have 4 different monomers (nucleotides). Proteins have 20 different monomers (amino acids.)

▪ Considering the trillions of genes that must exist, is it more likely they are built with 4 different monomers or 20?

We’re biased. We knew about proteins long before we knew about nucleic acids, and we don’t like change

▪ See: PS I and II

In 1952, Alfred Hershey and Martha Chase used a T2 bacteriophage to answer this question.

Using Griffith’s transformation procedure, they allowed bacteriophage viruses to absorb nucleotides and amino acids with radioactive phosphorus and sulfur Sulfur is found in proteins but not nucleic acids. Phosphorus is found in nucleic acids, but not proteins. The radiation emitted from these molecules would be visible

under special lights The bacteriophage viruses then infected numerous E.

coli cells by inserting their genetic material into the cell and having the cell build new viruses

Since genes are literally passed from parents to offspring, the radioactive markers on those genes would also be passed to the offspring of the viruses.

The specific color of radiation found in the offspring would show Hershey and Chase whether phosphorus or sulfur was passed to offspring Phosphorus color would indicate nucleic acids were passed

Sulfur color would indicate proteins were passed In the offspring, only phosphorus was detected. This

proved nucleic acids were passed from parents to offspring Therefore, genes were made of nucleic acids, not proteins.

Nucleic acids contains three sections

A Phosphate: phosphates connect nucleotides together

Ribose sugar: the sturdy, structural backbone

Nitrogenous base: the genetic code

There are two ribose sugars, one with an extra oxygen (ribose) and one missing an oxygen (deoxyribose)

The sugar determines whether it is DNA or RNA (deoxyribonucleic acid or ribonucleic acid).

For DNA, the four nucleotides are

Adenine (A)

Cytosine (C)

Guanine (G)

Thymine (T)

For RNA, Thymine is replaced with a fifth nucleotide

Uracil (U)

In the 1940’s Erwin Chargoff conducted experiments on DNA of various species.

He separated the individual nucleotides in an entire cell and weighed them.

His conclusions are now called “Chargoff’s rules.”

1. The amount of A, T, C, and G in DNA varies for each species.

2. In each species, for DNA, the amount of A = T and the amount of C = G.

Species A T G C

Bacillus Subtillus (Bacillus bacteria) 28.4 29.0 21.0 21.6

Escherichia coli (E. coli) 24.6 24.3 25.5 25.6

Neurospora crassa (Bread mold) 23.0 23.3 27.1 26.6

Zea mays (Corn) 25.6 25.3 24.5 24.6

Drosophila melanogaster (Fruit fly) 27.3 27.6 22.5 22.5

Homo Sapiens (Human) 31.0 31.5 19.1 18.4

In 1952, Rosalind Franklin used x-ray beams to take pictures of DNA.

The x-ray diffracted through a crystal and created an incredibly detailed image that showed the relative shape of DNA molecules.

Two of Franklin’s colleagues, James Watson and Francis Crick, used the image Franklin created to develop one of the most important discoveries of the 20th century: the DNA model

The model showed a double-helix structure; two strands of DNA attached to each other.

The strands matched nitrogenous base to nitrogenous base (A matched with T, C matched with G)

▪ Bases are connected using hydrogen bonds ▪ This proved Chargoff’s rules.

Each nucleotide was attached using the phosphates

Each strand faces the opposite direction

▪ This fit the size and shape of Franklin’s photograph

Adenine and Thymine are connected using two hydrogen bonds

Guanine and Cytosine are connected using three hydrogen bonds

This is how the two strands of DNA attach to each other

Pyramidines (Nitrogenous base is a single ring)

Thymine and Cytosine

Purines (Nitrogenous base is a double ring)

Adenine and Guanine

DNA Replication is the process of copying a DNA molecule

Watson and Crick’s model was so effective and accurate that immediately after publishing it they were easily able to develop their replication hypothesis

Each strand of DNA is used as a template for building a new strand

1. Unwinding of the DNA strand

The hydrogen bonds holding the strands together at the nitrogenous bases are “unzipped” by the enzyme helicase

After the strands are separated by helicase, an enzyme called RNA polymerase lays down an RNA primer on top of the DNA template

The RNA primer will act as a beacon to tell enzymes where to start copying DNA.

2 Base Pairing

New nucleotides are constantly being built in the ER and transported to the nucleus of cells

An enzyme called DNA polymerase attaches new nucleotides to the new strand of DNA at the RNA primer

Polymerase knows which nucleotide comes next because of the base pairing rules (A with T, C with G)

DNA polymerase always attaches new nucleotides to the 3’ carbon

Thus, DNA polymerase can only move in one direction, the 5’3’ direction

Because DNA strands face the opposite direction from each other, DNA polymerase works well on the strand moving in the 5’3’ direction

This is called the leading strand

For the other strand, polymerase attaches small sections of DNA called Okazaki fragments one section at a time.

This is called the lagging strand.

Replication was finally confirmed by Meselson and Stahl in 1958

Meselson and Stahl took a strand of DNA that contained 15N and allowed it to go through replication with free nucleotides that contained 14N nitrogen ions.

15N is heavier than 14N.

When placed in a centrifuge and allowed to sit for 2-3 days, the heavier DNA from the original strand (with 15N) will sink to the bottom and the lighter DNA that was replicated will float towards the top

After replicating DNA, if Watson and Crick’s model is correct then a centrifuge should show both strands of DNA At the bottom, two strands that contain the heavier, original

15N. Every other strand, built with the lighter 14N, would be floating above this strand.

If another model is correct, they should only see one or the other floating in the centrifuge

At the end of the experiment, both strands were visible in the centrifuge.

This proved semi conservative replication, which means each new copy of DNA has one recycled strand and one newly-formed strand.

Prokaryotic DNA is a single, circular structure called a plasmid.

Replication occurs in either one or both directions Bacteria takes approximately 40 minutes to copy

the entire genome at a rate of 106 base pairs/minute

Eukaryotic replication occurs at multiple origins and at a much slower rate

500-5000 bp/minute.

DNA polymerase not only attaches nucleotides together, but proofreads it’s work.

Mismatched nucleotides cause a kink in the strand which is easy to spot by the polymerase

The enzyme then excises (removes) the incorrect nucleotide and replaces it with the correct one.

Still, polymerase is so accurate that this is only necessary once per 100,000 base pairs.

After proofreading, the likelihood of a mutation is only one mistake every 1 billion base pairs

Other mutations occur due to mutagens, or environmental factors.

UV, radiation, organic chemicals such as tobacco smoke, pesticides, etc.

For these, the cell has DNA repair enzymes that go around and look for errors after replication is over.

Mutations may cause harm (cancer) or have no affect at all.

They also allow for the possibility for evolutionary changes