General Biology/Genetics/DNA, The Genetic Material

DNA DNA stands for Deoxyribose Nucleic Acid. That is, a nucleic acid with two sugars. DNA is the hereditary material of cells and is considered the blueprint of life. DNA is found in all kingdoms of life. Even most viruses have DNA. A molecule of DNA is chemically stable (it does not have a 2-prime alcohol group.) When someone says DNA, they may be referring to one's genetic material on multiple levels: They may be speaking about a single deoxyribose nucleic acid molecule, a section of a double helix, a section of a chromosome, or one's entire hereditary composition. antiparallel Double helix Semiconservative replication Sequence of nucleotides encodes functional RNA or polypeptide Historical perspective Mitosis and meiosis Regular distribution of chromosomes suggested that they contain hereditary information Bridges/Morgan, using Drosophila melanogaster showed that genes are on chromosomes (1910s) Hammerling: nucleus contains hereditary information (1930s) Griffith: transformation of bacteria (1928) Avery, MacLeod, McCarty: transforming substance is DNA (1944) Hershey, Chase: DNA is hereditary material of viruses (1952) Rosalind Franklin Watson and Crick: structure of DNA (1953) Hershey-Chase Experiment Two batches of isotopically labeled bacteriophage T2 particles were prepared. One was labeled with 32P in the phosphate groups of the DNA, the other with 35S in the sulfur-containing amino acids of the protein coats (capsids). (Note that DNA contains no sulfur and viral protein contains no phosphorus.) The two batches of labeled phage were then allowed to infect separate suspensions of unlabeled bacteria. Each suspension of phage-infected cells was agitated in a blender to shear the viral capsids from the bacteria. The bacteria and empty viral coats (called “ghosts”) were then separated by centrifugation. The cells infected with the 32P-labeled phage were found to contain 32P, indicating that the labeled viral DNA had entered the cells; the viral ghosts contained no radioactivity. The cells infected with 35S-labeled phage were found to have no radioactivity after blender treatment, but the viral ghosts contained 35S. Progeny virus particles (not shown) were produced in both batches of bacteria some time after the viral coats were removed, indicating that the genetic message for their replication had been introduced by viral DNA, not by viral protein. DNA/RNA components Miescher: discovered DNA, 1869 Structure of DNA DNA is in a double helix structure made up of nucleotides. The "backbone" of the double helix is composed of phosphates connected to a five-carbon sugar called deoxyribose. The "rungs" are composed of organic compounds, purines and pyrimidines. Purines contain Adenine(A) and Guanine(G) and have two rings in their structures. Pyrimidines contain Cytosine(C) and Thymine (T) and have one ring in their structures. Chemical structure of DNA Polynucleotide Phosphodiester bonds between nucleotides 5’-pGpTpCpGpTpApApTp-OH 3’ Chargaff’s rules, in DNA: equimolar amounts A = T G = C 3D structure of DNA James Watson and Francis Crick (1953) Nucleotide Keto and amino forms of bases Chargaff’s rules X-ray crystallographic data (Rosalind Franklin) Franklin X-ray diffraction of DNA crystals revealed regular pattern explained by antiparallel double helix DNA model Double helix of polynucleotides antiparallel 3’-5’ phosphodiester bonds Base pairs held by hydrogen bonds AT GC There are about 10 base pairs per turn of helix model has predictive power mode of DNA replication encoding of genetic information DNA replication Conservative model One double helix of both old strands One double helix of two new strands Dispersed Each strand mixture of old new Semiconservative Meselson-Stahl experiment confirmed its viability over the previous two grew E. coli bacterium in a culture containing 15N (a heavy isotope of nitrogen) bacterium assimilated the 15N into their DNA a similar process was then done using 14N, a lighter isotope following centrifugation, the densities were observed to be that of combined in the middle, and 14N on top, thereby confirming the semiconservative model DNA replication Semiconservative New nucleotides added to 3’ –OH Replication fork Replication complex DNA polymerase Associated enzymes/proteins Energy from phosphate bonds of triphosphate nucleotide substrates (dNTP) DNA polymerases Prokaryotes, E. coli 3 DNA polymerases III is main enzyme for DNA replication ~1000 nt/sec Eukaryotes 6 DNA polymerases Add nucleotide to 3’ –OH end All require primer, i.e., free 3’ –OH DNA replication complex Helicase "unzips" the DNA double helix Primase: synthesize RNA primer Single-strand binding proteins DNA gyrase (topoisomerase) DNA polymerase III DNA polymerase I (remove primer, fill gaps) DNA replication 5’ → 3’ replication Nucleotide addition at 3’ –OH No exceptions New strands are oriented in opposite direction due to 5’ → 3’ constraint Leading strand: continuous replication Lagging strand: discontinuous replication contains multiple Okazaki fragments Joined by DNA ligase DNA replication fork primer required by all DNA polymerases Replication units Replicon A region of DNA that is replicated from a single origin. What is gene? Garrod “inborn errors of metabolism” Alkaptonuria: enzyme deficiency Beadle and Tatum One gene one enzyme Genetic and biochemical analysis in Neurospora Today: gene is sequence of nucleotides encoding functional RNA molecule or the amino acid sequence of a polypeptide References This text is based on notes very generously donated by Paul Doerder, Ph.D., of the Cleveland State University.