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

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.

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

Ribonucleotides : Building Blocks for RNA Refer to Figure 8-2 from Introduction to Genetic Analysis, Griffiths et al., 2015.

Complementarity and Asymmetry in RNA Synthesis Only one strand of DNA is used as template for RNA synthesis. RNA bases are complementary to bases of template DNA. Template strand is antiparallel to RNA transcript.

RNA Polymerase RNA polymerase in E. coli consists of 4 different subunits (see model below). s recognizes the promoter. Holoenzyme is needed for correct initiation of transcription. RNA polymerase adds ribonucleotides in 5’ to 3’ direction. A single type of RNA polymerase transcribes RNA in prokaryotes.

Promoter Sequences in E. coli Refer to Figure 8-7 from Introduction to Genetic Analysis, Griffiths et al., 2015. Promoters signal transcription in prokaryotes.

Transcription Initiation in Prokaryotes Refer to Figure 8-8 from Introduction to Genetic Analysis, Griffiths et al., 2015. s subunit positions RNA polymerase for correct initiation. Upon initiation of transcription, s subunit dissociates.

Elongation NTP + (NMP)n (NMP)n+1 + PPi RNA polymerase adds ribonucleotides in 5’ to 3’ direction. RNA polymerase catalyzes the following reaction: DNA Mg++ RNA polymerase NTP + (NMP)n (NMP)n+1 + PPi

Termination Termination of transcription occurs beyond the coding sequence of a gene. This region is 3’ untranslated region (3’ UTR), which is recognized by RNA polymerase.

RNA polymerase recognizes signals for chain termination. (1) Intrinsic: Termination site on template DNA consists of GC-rich sequences followed by A’s. Intra-molecular hydrogen bonding causes formation of hairpin loop. (2) rho factor (hexameric protein) dependent: These termination signals do not produce hairpin loops. rho binds to RNA at rut site. rho pulls RNA away from RNA polymerase. rut site In E. coli, this structure signals release of RNA polymerase, thus terminating transcription.