The traditional monoclonal antibody (mAb) production process usually starts with generation of mAb-producing cells (i.e. hybridomas) by fusing myeloma cells with desired antibody-producing splenocytes (e.g. B cells). These B cells are typically sourced from animals, usually mice. After cell fusion, large numbers of clones are screened and selected on the basis of antigen specificity and immunoglobulin class. Once candidate hybridoma cell lines are identified, each "hit" is confirmed, validated, and characterized using a variety of downstream functional assays. Upon completion, the clones are scaled up where additional downstream bioprocesses occur.

1. DNA: The Genetic Material
Chapter 14

2. 2
The Genetic Material
Frederick Griffith, 1928
studied Streptococcus pneumoniae, a
pathogenic bacterium causing pneumonia
there are 2 strains of Streptococcus:
- S strain is virulent
- R strain is nonvirulent
Griffith infected mice with these strains
hoping to understand the difference
between the strains

3. 3
The Genetic Material
Griffith’s results:
- live S strain cells killed the mice
- live R strain cells did not kill the mice
- heat-killed S strain cells did not kill the
mice
- heat-killed S strain + live R strain cells
killed the mice

4. 4

5. 5
The Genetic Material
Griffith’s conclusion:
- information specifying virulence passed
from the dead S strain cells into the live R
strain cells
- Griffith called the transfer of this information
transformation

6. 6
The Genetic Material
Avery, MacLeod, & McCarty, 1944
repeated Griffith’s experiment using purified
cell extracts and discovered:
- removal of all protein from the
transforming material did not destroy its
ability to transform R strain cells
- DNA-digesting enzymes destroyed all
transforming ability
- the transforming material is DNA

7. 7
The Genetic Material
Hershey & Chase, 1952
- investigated bacteriophages: viruses that
infect bacteria
- the bacteriophage was composed of only
DNA and protein
- they wanted to determine which of these
molecules is the genetic material that is
injected into the bacteria

8. 8
The Genetic Material
- Bacteriophage DNA was labeled with
radioactive phosphorus (32P)
- Bacteriophage protein was labeled with
radioactive sulfur (35S)
- radioactive molecules were tracked
- only the bacteriophage DNA (as indicated
by the 32P) entered the bacteria and was
used to produce more bacteriophage
- conclusion: DNA is the genetic material

9. 9

10. 10
DNA Structure
DNA is a nucleic acid.
The building blocks of DNA are
nucleotides, each composed of:
–a 5-carbon sugar called deoxyribose
–a phosphate group (PO4)
–a nitrogenous base
• adenine, thymine, cytosine, guanine

11. 11

12. 12
DNA Structure
The nucleotide structure consists of
–the nitrogenous base attached to the 1’
carbon of deoxyribose
–the phosphate group attached to the 5’
carbon of deoxyribose
–a free hydroxyl group (-OH) at the 3’
carbon of deoxyribose

13. 13

14. 14
DNA Structure
Nucleotides are connected to each other to
form a long chain
phosphodiester bond: bond between
adjacent nucleotides
–formed between the phosphate group of
one nucleotide and the 3’ –OH of the
next nucleotide
The chain of nucleotides has a 5’ to 3’
orientation.

15. 15

16. 16
DNA Structure
Determining the 3-dimmensional structure of
DNA involved the work of a few scientists:
–Erwin Chargaff determined that
• amount of adenine = amount of thymine
• amount of cytosine = amount of guanine
This is known as Chargaff’s Rules

17. 17
DNA Structure
Rosalind Franklin and Maurice Wilkins
–Franklin performed X-ray diffraction
studies to identify the 3-D structure
–discovered that DNA is helical
–discovered that the molecule has a
diameter of 2nm and makes a complete
turn of the helix every 3.4 nm

18. 18
DNA Structure
James Watson and Francis Crick, 1953
–deduced the structure of DNA using
evidence from Chargaff, Franklin, and
others
–proposed a double helix structure

19. 19
DNA Structure
The double helix consists of:
–2 sugar-phosphate backbones
–nitrogenous bases toward the interior of
the molecule
–bases form hydrogen bonds with
complementary bases on the opposite
sugar-phosphate backbone

20. 20

21. 21
DNA Structure
The two strands of nucleotides are
antiparallel to each other
–one is oriented 5’ to 3’, the other 3’ to 5’
The two strands wrap around each other to
create the helical shape of the molecule.

22. 22

23. 23
DNA Replication
Matthew Meselson & Franklin Stahl, 1958
investigated the process of DNA replication
considered 3 possible mechanisms:
–conservative model
–semiconservative model
–dispersive model

24. 24

25. 25
DNA Replication
Bacterial cells were grown in a heavy
isotope of nitrogen, 15N
all the DNA incorporated 15N
cells were switched to media containing
lighter 14N
DNA was extracted from the cells at various
time intervals

26. 26
DNA Replication
The DNA from different time points was
analyzed for ratio of 15N to 14N it contained
After 1 round of DNA replication, the DNA
consisted of a 14N-15N hybrid molecule
After 2 rounds of replication, the DNA
contained 2 types of molecules:
–half the DNA was 14N-15N hybrid
–half the DNA was composed of 14N

27. 27

28. 28
DNA Replication
Meselson and Stahl concluded that the
mechanism of DNA replication is the
semiconservative model.
Each strand of DNA acts as a template for
the synthesis of a new strand.

29. 29
DNA Replication
DNA replication includes:
–initiation – replication begins at an
origin of replication
–elongation – new strands of DNA are
synthesized by DNA polymerase
–termination – replication is terminated
differently in prokaryotes and eukaryotes

30. 30
Prokaryotic DNA Replication
The chromosome of a prokaryote is a
circular molecule of DNA.
Replication begins at one origin of
replication and proceeds in both
directions around the chromosome.

31. 31

32. 32
Prokaryotic DNA Replication
The double helix is unwound by the enzyme
helicase
DNA polymerase III (pol III) is the main
polymerase responsible for the majority of
DNA synthesis
DNA polymerase III adds nucleotides to the
3’ end of the daughter strand of DNA

33. 33

34. 34
Prokaryotic DNA Replication
DNA replication is semidiscontinuous.
–pol III can only add nucleotides to the 3’
end of the newly synthesized strand
–DNA strands are antiparallel to each other
leading strand is synthesized continuously (in
the same direction as the replication fork)
lagging strand is synthesized discontinuously
creating Okazaki fragments

35. 35

36. 36
Prokaryotic DNA Replication
The enzymes for DNA replication are
contained within the replisome.
The replisome consists of
–the primosome - composed of primase
and helicase
–2 DNA polymerase III molecules
The replication fork moves in 1 direction,
synthesizing both strands simultaneously.

37. 37

38. 38
Eukaryotic DNA Replication
The larger size and complex packaging of
eukaryotic chromosomes means they
must be replicated from multiple origins of
replication.
The enzymes of eukaryotic DNA replication
are more complex than those of
prokaryotic cells.

39. 39
Eukaryotic DNA Replication
Synthesizing the ends of the chromosomes
is difficult because of the lack of a primer.
With each round of DNA replication, the
linear eukaryotic chromosome becomes
shorter.

40. 40

41. 41
Eukaryotic DNA Replication
telomeres – repeated DNA sequence on the
ends of eukaryotic chromosomes
–produced by telomerase
telomerase contains an RNA region that is
used as a template so a DNA primer can
be produced

42. 42

43. 43
DNA Repair
- DNA-damaging agents
- repair mechanisms
- specific vs. nonspecific mechanisms

44. 44
DNA Repair
Mistakes during DNA replication can lead to
changes in the DNA sequence and DNA
damage.
DNA can also be damaged by chemical or
physical agents called mutagens.
Repair mechanisms may be used to correct
these problems.

45. 45
DNA Repair
DNA repair mechanisms can be:
–specific – targeting a particular type of
DNA damage
• photorepair of thymine dimers
–non-specific – able to repair many
different kinds of DNA damage
• excision repair to correct damaged
or mismatched nitrogenous bases

46. 46

47. 47