Molecular Genetics

What are chromosomes made of?

I. Introduction

• A. Current definition of a gene• 1. a unit factor controlling the inheritance

of some external trait• 2. a spot on a chromosome which

influences some trait• 3. a poppit bead on a string of poppet

beads in the laboratory• 4. going to flesh out in more detail what a

gene is made of and what it does in our next unit

• B. Two questions are going to be answered in this part of the course

• 1. What is a gene made of?• 2. What do genes do? How do genes

control the activity of a cell?

II. Two traits are necessary to be a good candidate for the stuff genes are made of

• A. a genetic molecule must be able to replicate with very few mistakes

• B. a good genetic candidate must be able to direct the activities of the cell’s cytoplasm from the confines of the nucleus

III. Protein vs. DNA

• A. Basic structure of the two

• 1. protein• 2. DNA• B. Which is likely

candidate?

B. Series of historical experiments that determined the chemical nature of chromosomes

1. Friedrich Miescher (1869)– a. 1869-not too long ago– b. exposed cells to pepsin-a

proteolytic enzyme– c. the proteins in the cell’s

cytoplasm were liquefied but the contents of the nucleus were left relatively intact

– d. it was believed at this time that the genes or controlling factors of inheritance were housed in the cell’s nucleus

2. Feulgen• a. developed a stain specific

for nucleic acids• b. what region of the cell took

up the stain?• c. all the somatic cells of an

individual stained with the same intensity-what does that tell us about developmental processes?

• d. the germ cells of an organism stain with half the intensity as the somatic cells

3. Griffiths Work

a.Work involved two different strains of pneumococcus

b.Smooth-virulent strain

c. Rough-benign strain

d. protocol

e. results

• Dead S bacteria transformed the living R bacteria into a virulent strain

• Later determined that the fraction of the dead S bacteria that was the transforming agent was DNA

4. Herschey and Chase

a. structure of a bacteriophage

b. how do viruses work?

c. phage infection

d. protocol

IV. Molecular Structure of DNA

• A. Monomers of nucleic acids are nucleotides which consist of three basic parts

• 1. pentose sugar• 2. phosphate and nitrogen

base • 3. notice that there are two

classes of nitrogen containing bases

• a. purines• b. pyrimidines

Four examples of DNA nucleotides

B. Polymers

• 1. Polymers of nucleotides are produced by dehydration synthesis of nucleotides in a 5’ to 3’ direction

• 2. this represents a single strand of DNA

• 3. If another nucleotide were to join this existing strand-to where would it attach? Which end?

C. Watson and Crick built their model of DNA on the work of others

Early sixties

Done in England

Physically built model seen to fit data of others

Pieced together information

Which one is which?

1. Chargaff’s rule

• a. a number of species were investigated

• b. the percentage of different bases found in the DNA of each species was investigated

• c. found that the amount of adenine was about the same as the thymine

• d. the amount of guanine was about the same as the amount of cytosine

• e. at the time, did not know what to make of the discovery

• f. Chargaff data • h. analysis

2. Rosalind Franklin and X-ray diffraction study

2. Rosalind Franklin

• a. Watson came across knowledge of the work at a tea party

• b. had not been exposed to the information before

• c. like a light bulb going off in his head• d. recurring periodicities seen• -.34 nm• -3.4 nm• -2.0nm

3. Work of Linus Pauling

• a. worked on helical nature of proteins

• b. saw parallel in nucleic acid structure

• c. denied travel visa to attend a DNA conference that was being held in England

• d. linked to the communist movement in USA because he had opposed nuclear bomb used against Japan

Diary of Pauling

• Diary shows complementary base pairing before proposed by Watson and Crick

I believe that Linus Pauling won the Nobel Peace prize later in life

V. DNA double helix

• A. Complementary base pairing as suggested by work of Chargaff and Franklin

B. Double helix

C. Properties

• 1. antiparallel• 2. complimentary base pairing• 3. periodicities seen• 4. covalent vs. hydrogen bonding

– Importance of accessibility of information– Where is information stored in the molecule

• 5. The information in the DNA molecule is carried in the base pair sequence of the molecule

• 6. Notice also that because of complimentary base pairing, the two sides of the molecule are mirror images of each other

• 7. each strand contains the information needed to produce the second strand

• 8. Watson and Crick proposed an early model of replication in their first paper describing the structure of DNA

VI. Replication• A. Size of the problem• 1. approximately 40,000 human genes in a nucleus• 2. the length of the DNA in one human cell is

approximately 3 meters• 3. (length of 1 bp)(number of bp per cell)(number of cells in the

body) = (0.34 x 10-9 m)(6 x 109)(1013) = 2 x 1013 meters• 4. That is the equivalent distance from the earth to the sun and

back.

B. Semi-conservative model as proposed

C. There were three possible modes of replication

1. conservative mode-the parent molecule would remain intact while the newly synthesized molecule is totally brand new

2. semi-conservative model-each new molecule of DNA is half new and the other half comes as a template from the parent

3. dispersive model-the two strands of the old molecule would break up and be scattered in the composition of the two new molecules

D. Work of Meselsohn and Stahl

1. Verified the semiconservative nature of DNA replication

2. Used heavy nitrogen technology introduced by Kornberg along with in vitro DNA replication

3. Protocol

E. Replication-the real story

• 1. Mentioned above the sheer size of the DNA molecules in the nucleus

• a. the amount of information held in the nucleus is enough to fill 600 volumes of 500 pages each

• b. the enzymes copying DNA add 100 nucleotides together per second in mammals

• c. 1000 nucleotides together per second in bacterial cells

• d. huge amount of information to be processed

2. Very accurate process-not a duplication event

• a. one in every billion base pairs there is a mistake

• b. in the typing of the 600 volumes above, that would translate to about 4 mistakes

3. Replication origins

• replilcation can start at the beginning of the molecule as well as internally at special recognition sites

4. Helicases unwind double helix

• a. double helix must be unwound in order for the templates to be accessed by free nucleotides

• b. single stranded binding proteins also keep the separated strands separated

5. DNA polynucleotide strands cannot start from nothing

• a. the first nucleotide must connect to something else

• b. RNA primer piece must be laid down firstc. notice that with many replication forks, this piece of primer must be laid down every time a new section of DNA is manu.

6. Nucleotides add to a growing chain in the 5’ to 3’ direction

a. new bases cannot add onto the 5’ end of the polynucleotide chain

b. DNA is an antiparallel molecule

c. at the replication fork this causes confusion

d. DNA polymerase can only operate in one direction

6. cont.

e. terms to know: leading strand, lagging strand, Okazaki fragments, ligase

f. the Okazaki fragments are 1000-2000 fragments long in bacteria

g. the Okazaki fragments are 100-300 fragments long in mammals

h. the Okazaki fragments are joined by an enzyme called ligase

7. Summary Slide

8. The final problem

• a. Chromosomes shorten with repeated divisions

• b. The position where the primer RNA was found on the leading strand leaves a gap at the end with each replication

• c. The daughter molecules get shorter and shorter

• d. Important information can be eroded

• e. telomeres are basically like buffers• f. able to be eroded away without getting into the coding sequence

of the chromosomes• g. repeating nucleotide base sequence TTAGGG• h. there may be over 100 to 1000 of these repeats

8. The final problem cont.

• i. telomerase not present in all cells

• j. lack of enzyme and shortening of chromosomes may be a built in clock for life of cell

• k. telomerase is present in germ lines

• l. also present in tumors which exhibit uncontrolled mitotic divisions

VII.

VII. What is a chromosome anyways?

2

• A. Recall that each nucleus possesses about 3 meters of DNA

• B. It appears as chromatin in a nondividing cell

• C. In a mitotic cell the chromatin must be organized to accomplish an efficient division process

D. Terms to remember

• 1. Histone proteins• 2. Nucleosomes• 3. Scaffolding proteins