Molecular Basis for Relationship between Genotype and Phenotype

1. Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription replication RNA translation amino acid sequence protein function phenotype organism

2. Refer to Figure 7-24 from Introduction to Genetic Analysis, Griffiths etal., 2012.

3. Initiation at Origin of Replication Prokaryotes: Fixed origin DnaA proteins DnaB (helicase) Eukaryotes: Multiple origins ORC protein complex Cdc6 and Cdt1 MCM complex (helicase)

4. Overview of DNA Synthesis DNA polymerases synthesize new strands in 5’ to 3’ direction. Primase makes RNA primer. Lagging strand DNA consists of Okazaki fragments. In E. coli, pol I fills in gaps in the lagging strand and removes RNA primer. Fragments are joined by DNA ligase.

6. The Problem of Replicating Chromosome Ends telomere 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ A B A B A B RNA primer removal and DNA ligation + + DNA Replication 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Last section of lagging strand cannot be primed. Result is a shorter chromosome after each round of replication.

7. The Problem of Replicating Chromosome Ends Here is another angle. Note again that the last section of lagging strand cannot be primed. A shorter chromosome is produced after each round of replication. It is theorized that as cells age (generations), telomeres shorten, eventually leading to their death. Is there a mechanism to maintain chromsome length?

8. Telomere Lengthening Telomerase (a reverse transcriptase) adds repeats to telomeric DNA. It carries an RNA molecule that serves as template for DNA synthesis.

9. Telomere Lengthening

10. The Telomeric Cap Structure Telomeric end is protected by a “cap”. It consists of TRF1 and TRF2 (that bind to telomeric repeats) and proteins such as WRN that bind to TRF1 and TRF2.

12. Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription RNA translation amino acid sequence protein function phenotype organism

13. Making Recombinant DNA: Donor DNA Genomic DNA: DNA obtained from chromosomes of an organism Complementary DNA (cDNA): double-stranded DNA version of mRNA obtained by reverse transcription Chemically Synthesized DNA: DNA sequence obtained by automated chemical reactions

14. Cutting DNA: Restriction Endonucleases

15. Formation of a recombinant DNA molecule Circular ds DNA is cut with one restriction enzyme. Both restriction fragments are linear and have sticky ends (in this case). Linear ds DNA is cut with the same restriction enzyme. By complementary base pairing, the sticky ends can hybridize. The result is a recombinant DNA molecule.

16. Inserting a gene into a recombinant DNA plasmid Vector is a cloning vehicle. Both vector and donor DNA are cut with the same restriction enzyme. Restriction fragments are mixed; sticky ends hybridize. Recombinant vector is the result. DNA ligase seals gaps by forming phophodiester linkages.

17. How amplification works Recombinant vectors are introduced into bacterial host cells. Replication and cell division produce many copies of the recombinant vector. Clones of donor DNA fragments result.