Lecture 9 DNA Structure & Replication What is a Gene? Mendel s work left a key question unanswered: What is a gene? The work of Sutton and Morgan established that genes reside on chromosomes But chromosomes contain proteins and DNA So which one is the hereditary material Several experiments ultimately revealed the nature of the genetic material The Griffith Experiment In 1928, Frederick Griffith discovered transformation while working on Streptococcus pneumoniae The bacterium exists in two strains S Forms smooth colonies in a culture dish Cells produce a polysaccharide coat and can cause disease R Forms rough colonies in a culture dish Cells do not produce a polysaccharide coat and are therefore harmless 1
How Griffith Discovered Transformation Thus, the dead S bacteria somehow transformed the live R bacteria into live S bacteria The Avery and Hershey-Chase Experiments Two key experiments that demonstrated conclusively that DNA, and not protein, is the hereditary material swald Avery and his coworkers Colin MacLeod and Maclyn McCarty published their results in 1944 Alfred Hershey and Martha Chase published their results in 1952 The Avery Experiments Avery and his colleagues prepared the same mixture of dead S and live R bacteria as Griffith did They then subjected it to various experiments All of the experiments revealed that the properties of the transforming principle resembled those of DNA 1. Same chemistry and physical properties as DNA 2. Not affected by lipid and protein extraction 3. Not destroyed by protein- or RNA-digesting enzymes 4. Destroyed by DNA-digesting enzymes 2
The Hershey-Chase Experiment Viruses that infect bacteria have a simple structure DNA core surrounded by a protein coat Hershey and Chase used two different radioactive isotopes to label the protein and DNA Incubation of the labeled viruses with host bacteria revealed that only the DNA entered the cell Therefore, DNA is the genetic material Thus, viral DNA directs the production of new viruses Discovering the Structure of DNA Chemically, DNA is made up of nucleotides Each nucleotide has a central sugar, a phosphate group and an organic base The bases are of two main types: urines Large bases: Adenine (A) Guanine (G) 5-C sugar Nitrogenous base yrimidines Small bases: Cytosine (C) Thymine (T) More Key bservations About DNA Erwin Chargaff made key DNA observations that became known as Chargaff s rule urines = yrimidines A = T C = G Rosalind Franklin s X-ray diffraction experiments revealed that DNA had the shape of a coiled spring or helix Rosalind Franklin (1920-1958) 3
The Structure of DNA Revealed In 1953, James Watson and Francis Crick deduced that DNA was a double helix They came to their conclusion using Tinker toy models and the research of Chargaff and Franklin James Watson (1928- ) Francis Crick (1916-2004) The DNA Double Helix The two DNA strands are held together by weak hydrogen bonds between complementary base pairs A and T C and G If the sequence on one strand is ATACGCAT TATGCGTA The other s sequence must be Dimensions suggested by X-ray diffraction The two possible base pairs Each chain is a complementary mirror image of the other So either can be used as template to reconstruct the other 3 possible methods for DNA replication Daughter DNAs contain one old and one new strand riginal DNA molecule is preserved ld and new DNA are dispersed in daughter molecules 4
Evidence for Semi-Conservative Replication These three mechanisms were tested in 1958 by Matthew Meselson and Franklin Stahl verview of How DNA Copies Itself DNA helices begin unwinding from the nucleosomes Helicase untwists the double helix and exposes complementary strands The enzyme primase puts down a short piece of RNA termed the primer DNA polymerase reads along each naked single strand adding the complementary nucleotide The site of replication is the replication bubble Each nucleotide strand serves as a template for building a new complementary strand LAY DNA Replication verview How nucleotides are added in DNA replication Thus, DNA replication is semiconservative Template strand H 3 C Sugarphosphate backbone T A C G A A 5 New strand 5 G A T 3 H T DNA polymerase H Template strand New strand H 3 5 C G T A A T C G A T yrophosphate A 3 H 5 5
Summary of How DNA Copies Itself The process of DNA replication can be summarized as such The enzyme helicase first unwinds the double helix The enzyme primase puts down a short piece of RNA termed the primer DNA polymerase reads along each naked single strand adding the complementary nucleotide DNA polymerase can only build a strand of DNA in one direction The leading strand is made continuously from one primer The lagging strand is assembled in segments created from many primers DNA Replication Since DNA polymerase only works in one direction: A continuous leading strand is synthesized A discontinuous lagging strand is synthesized DNA ligase splices together the short segments of the discontinuous strand Two new telomeres are also synthesized This process is called semiconservative replication DNA Replication (cont.) RNA primers are removed and replaced with DNA Ligase joins the ends of newly-synthesized DNA Mechanisms exist for DNA proofreading and repair LAY DNA Replication 6
Transcription The path of genetic information is often called the central dogma DNA RNA rotein A cell uses three kinds of RNA to make proteins Messenger RNA (mrna) Transfer RNA (trna) Ribosomal RNA (rrna) Transcription Gene expression is the use of information in DNA to direct the production of proteins It occurs in two stages Transcription The transcriber is RNA polymerase It binds to one DNA strand at a site called the promoter It then moves along the DNA pairing complementary nucleotides It disengages at a stop signal 7
Working with DNA Key techniques used by today s genetic engineers include CR amplification Used to increase the amounts of DNA cdna formation Used to build genes from their mrna DNA fingerprinting Used to identify particular individuals 8