DNA, Chromosomes and Chromatin

Genetic information is stored in DNA, which synthesizes RNA, which leaves the nucleus for the cytoplasm where RNA gets degraded or goes on to synthesize proteins

• DNA generates more DNA through replication
• RNA can be used to generate DNA by reverse transcription
• DNA generates RNA through transcription

All of above processes must be regulated

Griffith (1928)

• Identified transforming principle. He worked with rough and smooth types of pneumonia bacteria. Smooth colonies have polysaccharide coating that makes them appear smooth
• injected rats with smooth bacteria -> they died
• injected rats with rough bacteria -> they lived
• heated smooth bacteria then injected into rats -> rats lived
• mixed heated smooth with rough and injected -> rats died

He deduced that smooth colonies break up when heated; when mixed with rough, the rough bacteria gains ability to infect and kill rats; observed that something was transferred from one cell to another to change the cell's behavior

Avery, et al. (1940's)

• Identified DNA as Griffith's transforming principle
• took extract (heated smooth bacteria) and treated it with DNAase (digests DNA) - then mixed with rough bacteria and injected into rats -> the rats lived
• in other side of experiment, treated extract with protease (digests proteins) -then mixed with rough bacteria and injected into rats -> rat died
• showed that DNA, not protein, has ability to transform cells

Hershey and Chase (1950)

Proved DNA was hereditary material of cell. They worked on bacterial virus (phage) comprised of protein coat around DNA. They wanted to find out which part of the phage (DNA or protein), produced new phages.

• labeled DNA with P32 (DNA would be radioactive, but proteins wouldn't) in one experiment and labeled proteins with S35 in another (proteins would be radioactive)
• allowed phages to infect cells, waited for phages to reproduce, then disrupted cells in a Waring Blender to produce a mixture in which the bacteria remained intact but the protein shells of the phage would be sheered off the bacteria and would be free in the solution
• mixture was then placed in centrifuge - in the first experiment (DNA labeled), the pellet containing whole bacteria was radioactive, which meant that the DNA was injected into the cell by the phage and that DNA could be passed on from first phages to their "offspring" phages. In the second experiment (proteins labeled), the bacterial pellet did not contain proteins labeled with S35, thus the proteins were not injected into the bacteria and could not be passed on to the "offspring" phages

DNA is built from nucleotides (adenine, thymine, cytosine, guanine). The basic structure consists of a sugar (deoxyribose) bonded with a nucleotide base (A,T,G,C) on one side and bonded to a phosphate on the other side. The bases protrude from sugar-phosphate backbone and form hydrogen bonds with the bases on the complementary strand (see figure 2.4 in Dealing with Genes)

Adenine and thymine always bond with each other, while guanine and cytosine always bond to each other. Because of the this relationship, one strand completely specifies the complementary strand and one strand is used to specify the other in replication

Directionality exists in DNA because the DNA strand has one 5' end and one 3' end. In a double stranded DNA the two strands run in opposite directions (they are antiparallel). The 3' end of one DNA strand lies at the same end of the double helix as the 5' end of the complementary strand.

The base pairs form a ladder and stack one on top of another, which serves to stabilize the structure.

The DNA structure is a right-handed double helix.

In all organisms but especially in eukaryotes, a double strand of DNA must be packed into small space. In eukaryotic chromatin, the DNA is packed with proteins. The first level of packing involves DNA interaction with histone proteins that form a sort of ball around which the DNA wraps itself; this structure is called the nucleosome.

Watson and Crick discovered the double helix structure and raised the question of how DNA was replicated. Two possibilities for DNA replication are:

• conservative replication keeps the original strands intact and produces an entirely new double stranded molecule
• semiconservative replication unwinds the original DNA into two single strands, each of which is used as a template to produce a complementary strand

Meselson and Stahl

grew cells in presence of heavy nitrogen isotope (N15 instead of N14) which created "heavy DNA"

after all DNA in the cell was heavy, the cells were placed in regular medium with N14 and were allowed to grow. They could then follow fate of heavy DNA when the cells were incubated in the N14 medium (this was called a pulse-chase experiment)

• if conservative replication was correct, one molecule of DNA would have heavy strands and the other (progeny) would have light DNA strands
• if semiconservative replication was correct, each molecule of progeny DNA would have one heavy strand and one light strand

Meselson and Stahl centrifuged the DNA to separate the molecules based on their densities.Their results supported semiconservative replication as the correct model.