1. 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

2. 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.

3. 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.

4. 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

5. 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

6. 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

7. Modes of delivering recombinant DNA into bacterial cells
Refer to Figure 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.

8. 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

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

11. 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.

12. 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.

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

14. 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.

15. 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 PPi

16. 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.

17. 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.