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

Refer to Figure 7-26 from Introduction to Genetic Analysis, Griffiths et al., 2015.

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.

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?

Telomere Lengthening Telomerase (a reverse transcriptase) adds repeats to telomeric DNA. It carries an RNA molecule that serves as template for DNA synthesis. Refer to Figure 7-27 from Introduction to Genetic Analysis, Griffiths et al., 2015.

Telomere Lengthening Refer to Figure 7-27 from Introduction to Genetic Analysis, Griffiths et al., 2015.

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**. * Werner Syndrome protein ** TTAGGG Repeat Binding Factor Refer to Figure 7-28 from Introduction to Genetic Analysis, Griffiths et al., 2015.

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

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

Cutting DNA: Restriction Endonucleases

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. Refer to Figure 10-2 from Introduction to Genetic Analysis, Griffiths et al., 2015.

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. Refer to Figure 10-5 from Introduction to Genetic Analysis, Griffiths et al., 2015.

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. Refer to Figure 10-8 from Introduction to Genetic Analysis, Griffiths et al., 2015.

Choice of Cloning Vectors: Criteria Small Size: Convenience of manipulation Capability of Prolific Replication: Ease of amplification of donor DNA fragment Convenient Restriction Sites: Single location for insertion of donor DNA Ease of Identification: Quick recovery of recombinant DNA

Examples of Cloning Vectors Bacterial Plasmids: * Circular double-stranded DNA * Replicates independently of chromosomal DNA * Selectable markers for transformation Bacteriophages: * Phage l - clone DNA up to 15 kb

Vectors for Larger DNA Inserts Fosmids: Hybrid between l phage DNA and plasmid DNA - can carry inserts 35-kb to 45-kb PAC: P1 Artificial Chromosome (derivative of bacteriophage P1) - can carry inserts 80-kb to 100-kb BAC: Bacterial Artificial Chromosome (derivative of F plasmid) - can carry inserts 150-kb to 300 kb YAC: Yeast Artificial Chromosome - can carry inserts larger than 300-kb

Modes of delivering recombinant DNA into bacterial cells Refer to Figure 10-11 from Introduction to Genetic Analysis, Griffiths et al., 2015. (a) Plasmid DNA is introduced into host cell by transformation. (b) Fosmids are introduced in phage heads by transduction. Once inside, they replicate as large plasmids. (c) Phage vectors are introduced by infection.