DNA

1.         Evidence for DNA as genetic material

            a.         Avery, et al. - Case Study p. 620N; Fig. 15.1C

            b.         Hershey and Chase - (Fig. 15.2C) used radioactive S to label the protein of a bacteriophage and allowed it to infect the host bacterium. After centrifugation, the radioactive protein was found in the supernatant. Showed that protein is not injected into host. Then, radioactive P was used to label the nucleic acid of the virus. After infection and centrifugation, the radiation was in the pellet with the bacterial cells. Showed that nucleic acid is injected into host bacterium during infection.

            c.         Chargaff - showed that in different species, DNA contained different amounts of the four nitrogen bases, suggesting that its composition was diverse. If the nucleotides were chosen randomly, species would be expected to have the same relative amounts of each nucleotide. Also showed that A = T and C = G

2.         Structure of DNA (Fig. 26.6N; Fig. 15.5C)

            a.         Wilkins and Franklin - used X-ray diffraction to attempt to find the structure of DNA

            b.         Watson and Crick - did little original work but interpreted the X-ray data to elucidate the structure of DNA

            c.         structure is 2 chains wound together in a spiral (double helix)

            d.         sides of the chains are made of alternating sugars and phosphates, like the sides of a ladder

            e.         the rungs of the ladder are made of the four nitrogen bases paired AT and CG (complementary base pairing) Why must A bond with T and C with G? Strands are held together by hydrogen bonding between bases.

            f.         note that the chains have direction - one end has a free phosphate (5'), the other a free hydroxyl (-OH) (3')

3.         Replication of DNA - process by which DNA doubles so that each daughter cell gets a copy during cell division. Important to be high fidelity.

            a.         3 models - Meselson and Stahl (Fig. 26.7N; Fig 15.8C) - Bacteria growing on a heavy isotope of N were allowed to grow for one generation (i.e., DNA replicated once) on a light isotope of N. The cells had DNA of one weight. This meant that it was constructed from half heavy (old) and half light (new) N. After a second generation (i.e., another replication) the cells had DNA of two distinct weights. Half was all light and half was a mixture of light and heavy. Showed that replication is semi-conservative.

            b.         Origin of Replication

                        i.         a specific sequences of nucleotides mark the origin (starting point)

                        ii.        humans have hundreds of origins from which replication proceeds on both strands in both directions

                        iii.       where the strands are separated to expose the bases is called the replication fork

                        iv.       helicase separates the strands. DNA polymerase then “reads” the exposed bases and adds new bases by complementary base pairing

            c.         Elongating a new strand

                        i.         after strands are separated, DNA polymerase reads the bases on the template strand and attaches complementary bases to form a new strand

                        ii.        DNA polymerase can only attach the 5' phosphate (P) of one nucleotide to the 3' hydroxyl (OH) of another nucleotide that is already part of a strand. The enzyme can only work by building a new strand in the 5' ➝ 3' direction

                        iii.       the new nucleotides (e.g., ATP) that are attached lose two of their three phosphates which provides the energy to form the bond

            d.         Problem of antiparallel strands

                        i.         the C5 phosphate of one nucleotide is attached to C3 of an adjacent nucleotide. Therefore, the strand has a free 3' OH at one end and a free 5' P at the other. The molecule is arranged with the strands going in opposite directions so the 3' end of one strand is aligned with the 5' end of the other

                        ii.        DNA polymerase adds nucleotides only to the 3' end but can only do this on one strand, the leading strand

                        iii.       the other strand has a 5' P at the end rather than a 3' OH as DNA polymerase needs. This strand, the lagging strand, must be made in short fragments (Okazaki fragments) going in the opposite direction from the leading strand. Another enzyme, DNA ligase, then fills in the gaps to join the fragments together

            e.         Error rate

                        i.         complementary base pairing allows 1/10,000 bp error rate

                        ii.        DNA polymerase checks for these errors by checking the width of the helix and reduces the rate to 1/10⁸. Why does DNA polymerase allow any errors at all?

                        iii.       constant exposure to chemicals, viruses, and radiation cause damage so that >50 known enzymes constantly check DNA for errors. These combined efforts reduce the error rate to 1/10⁹

DNA Technology

1.         DNA fingerprinting

            a.         DNA contains long sequences that are similar between many people but some sequences are unique to each individual - like a fingerprint

            b.         restriction enzymes are naturally occuring “scissor enzymes) in bacteria that cut DNA at specific sequences.

            c.         DNA is cut up into pieces by restriction enzymes, making strands of varying lengths

            d.         strands are separated by length using the technique of gel electrophoresis

            e.         each person has a unique DNA sequence, producing a unique collection of strands of various lengths, which results in a unique pattern of bands on the gel

2.         Recombinant DNA

            a.         bacterial plasmids

                        i.         a gene from one organism is spliced (inserted) into bacterial plasmid DNA

                        ii.        the gene is duplicated along with the bacterial DNA when the cell divides

                        iii.       the protein will be synthesized by the bacterial cell

                        iv.       often used as a means of cheaply, mass-producing protein (e.g., human growth hormone, insulin, etc.)

            b.         plant DNA

                        i.         bacterial DNA coding for an antibiotic is inserted in plant DNA

                        ii.        the plant is grown and produces the antibiotic

                        iii.       this confers resistance to various pests without having to spray chemicals on the plant

            c.         other organisms

                        i.         DNA for some protein (e.g., antibiotics) can be placed inside yeast cells

                        ii.        the yeast cells can be added to feed for some domestic animals and provide a dietary source of the protein

                        iii.       the animal then has a source of the protein without expensive supplements

            d.         research

                        i.         disease-causing genes can be inserted into animals to study the effects of the disease

            e.         gene therapy

                        i.         some diseases are caused by malfunctioning or damaged genes.

                        ii.        a correct copy can be inserted into cells to alleviate (or eliminate) the symptoms. e.g., cystic fibrosis, insulin-dependant diabetes