Telomeres The leading strand of linear chromosomes can be replicated to the end The lagging strand addition of sequential RNA primers as polymerase moves toward the end of the chromosome – means an RNA primer is left “stranded” 1

2 Focusing on what happens at THIS chromosome END (because “upstream” there are Okazaki fragments generated From the fork moving in the opposite direction) This is NOT The chromosome end! (This is NOT a chromosome end)

(GGA -OH +PPP- T- OH )GGAT- OH (5’ exonuclease activity) Momentary detour to consider leading strand and its RNA primer… lagging leading

Telomeres This problem is resolved by repetitive sequences at the ends of chromosomes, called telomeres These repeats ensure that incomplete chromosome replication does not affect vital genes chromosomes WILL shorten with subsequent cell divisions, but they can shorten a lot before gene structure and expression is affected 4

Telomerase Telomeres are synthesized by the ribonucleoprotein telomerase which is active in germ cells Blackburn, Greider, and Szostak received the 2009 Nobel Prize for the discovery of telomeres and telomerase The RNA in telomerase is complementary to the telomere repeat sequence and acts as a template for addition of DNA – creating the long tandem repeats that are expendable for many cell divisions 5

6 = Reverse transcriptase (making DNA copy of RNA template) Telomere DNA up to 20kb long – so RNA primer of lagging strand can result in a “short” copy of the strand with no loss of important information

Telomeres, Aging, and Cancer Telomere length is important for chromosome stability, cell longevity, and reproductive success Telomerase is active in germ-line cells and some stem cells in eukaryotes Differentiated somatic cells and cells in culture have virtually no telomerase activity; such cells have limited life spans (30 to 50 cell divisions) Cancer cells are often somatic cells in which telomerase activity has been re-activated (so can divide indefinitely) 7

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 7.5 Molecular Genetic Analytical Methods Make Use of DNA Replication Processes Molecular biologists have used their understanding of DNA replication to develop new methods of molecular analysis Two widely used methods include polymerase chain reaction (PCR) and dideoxynucleotide DNA sequencing 8

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach The Polymerase Chain Reaction The polymerase chain reaction (PCR) is an automated version of DNA replication that produces millions of copies of a short target DNA segment You are carrying out PCRs in lab. You should understand why you need primers, dNTPs, and a heat-stable DNA polymerase for these reactions. Why do you not need helicase, SSB, primase, ligase, sliding clamp? Homework and lab work will test your understanding of PCR (knowledge will be tested on exams) 9

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Dideoxynucleotide DNA Sequencing The ultimate description of a DNA molecule is its precise sequence of bases The first DNA-sequencing protocols were developed by Maxam and Gilbert, and another by Sanger in 1977 The Sanger (dideoxynucleotide) method was most amenable to automation and is the method of choice today 10

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 11

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 12

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 13 Note: 5’end radioactively labelled primer What would happen if you added no dCTP and only added ddCTP?

5’ exonuclease: Recognizes free 5’ phosphate – then cuts adjacent phosphodiester bond Endonuclease: Recognizes base sequence – then cuts phosphodiester bond FREE 5’ phosphate FREE 3’ hydroxyl 3’ exonuclease: Recognizes free 3’ hydroxyl – then cuts adjacent phosphodiester bond Exonucleases vs Endonucleases

Hydrolysis of Phosphodiester bond

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 16

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Automated DNA Sequencing Automated DNA sequencers use a single reaction for each DNA sequence, in which all four ddNTPs are included Each ddNTP is labeled with a unique fluorescent marker The DNA is synthesized, and a mixture of fragments is produced and run on a DNA gel 17 What would happen if you ONLY added the four ddNTPs and added NO dNTPs?

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 18

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 19 Automated: All terminated chains are in one lane, but each chain has a different ddNTP so is one of four “colors”

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach “Next Generation” DNA Sequencing MANY different methods – but all involve a.small amounts of DNA b.PCR amplification c.Sequencing by synthesis; simultaneous sequencing of many targets Cut DNA into pieces and add “linkers” = defined DNA sequences at both ends of each piece. Physically separate each linker-DNA fragment from the next (e.g. bound to beads). Add primer (complement to linker) - PCR amplify to generate many identical molecules on the same bead. Add primer (complement to linker), polymerase and colored dNTPs – “identify” each dNTP as it is added (reversible stop to replication) Computer “reads” the sequence on thousands of beads (thousands of different DNA sequences) simultaneously 20

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Transcription… DNA into RNA (mRNA or pre-mRNA) Up to now, we have been describing DNA structure and the process of replicating DNA = semi-conservative DNA replication But why make and replicate DNA? It is the genetic material that dictates attributes of the organism = GENES leading to PHENOTYPES 21

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 1.3 Transcription and Translation Express Genes The central dogma of biology describes the flow of hereditary information; the original was proposed by Francis Crick 22

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Mechanisms of Gene Expression Shared by Eukarya, Bacteria, and Archaea (model systems for study) In transcription, one DNA strand is used to direct RNA synthesis – to give rise to mRNA Messenger RNA (mRNA) undergoes translation to produce proteins at nucleoprotein structures called ribosomes These processes deciphered in the 1960’s 23

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Types of RNA Several types of RNA are produced in a cell; messenger RNA (mRNA) is the only type that is translated Ribosomal RNA (rRNA) forms part of the ribosomes Transfer RNA (tRNA) carries amino acids to ribosomes, to be assembled into proteins 24

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Additional Features of an Updated Central Dogma Reverse transcription uses reverse transcriptase and an RNA template (from RNA-containing viruses) to produce complementary DNA Micro-RNAs are small RNA molecules with roles in regulation of gene expression in plants and animals 25

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 26 RNA Polymerase makes a complementary copy (the complement) of the Template strand, adding nucleotides to the mRNA from 5’ to 3’ CODING vs TEMPLATE DNA strands DNA and RNA Polymerases BOTH add nucleotides in 5’ to 3’ direction

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Identification of Messenger RNA In the late 1950s, researchers used the pulse-chase technique to follow the trail of newly synthesized RNA in eukaryotic cells how can you specifically “label” RNA (not DNA)? The pulse exposed cells to radioactive nucleotides followed by a chase of nonradioactive nucleotides The radiolabeled RNA molecules were then traced in the cells 27

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Experiments in Eukaryotes In eukaryotic cells that were pulsed with radioactive uracil, the radioactivity was initially concentrated in the nucleus Over a short period of time, the radioactivity moved to the cytoplasm This led researchers to conclude that RNA synthesized in the nucleus carried its information to the cytoplasm for translation into protein 28

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach RNA Synthesis RNA polymerase catalyzes the addition of each ribonucleotide to the 3 end of the nascent strand and forms the phosphodiester bonds between nucleotides Two phosphates are eliminated in the process, as in DNA synthesis 29

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach 30 Note: NTPs, not dNTPs…

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Bacterial Transcription will be covered in a later lecture (lectures 15 and/or 16) 31

Copyright © 2012 Pearson Education Inc. Genetics Analysis: An Integrated Approach Eukaryotes: similar 5 core subunits additional subunits