1. Cellular Anatomy typical cell: 1. nucleus
2. cell membrane = plasma membrane
3. cytoplasm
-cytosol
-cytoskeleton
4. cytoplasmic organelles
-membranous
-non-membranous

2. The Typical Cell
TEM of a typical eukaryotic
cell

3. The Nucleus: Information Central
nucleus contains most of the cell’s genes and is usually the most conspicuous organelle
bound by a double, phospholipid membrane = nuclear membrane
each membrane is a phospholipid bilayer
the nuclear envelope connects to the endoplasmic reticulum (protein synthesis)
Nucleus
Rough ER
Nucleolus
Chromatin
Nuclear envelope:
Inner membrane
Outer membrane
Nuclear pore
Ribosome
Pore complex
Close-up of nuclear envelope
nuclear membrane - contains nuclear pores = channels of over 100 proteins
allows entry and exit of materials e.g. mRNA
Pore complexes (TEM)
0.25 m

4. Nucleus is comprised of the following
1. Nucleoplasm
2. Nucleolus
3. Chromatin

5. A. Nucleoplasm:
specialized fluid of the nucleus
suspends the DNA in its form as chromatin
contains a network of filaments = nuclear matrix or nuclear lamina
matrix gives the nucleus its shape
matrix helps organize the chromatin
also helps in DNA replication & transcription
1 m
Nuclear lamina (TEM)

6. B. Nucleolus: “little nucleus”
dense body DNA, RNA and protein with no defining
membrane
site of ribosome production and rRNA synthesis (via transcription)
site for the assembly of rRNA with the two protein subunits of the ribosome
two ribosome subunits migrate out through nuclear pores – for assembly in the cytoplasm
nucleolus can be very prominent in cells that produce high amounts of proteins (e.g. neurons)

7. loosely coiled fibers of DNA wrapped around proteins = histones
C. Chromatin:
loosely coiled fibers of DNA wrapped around proteins = histones
found only in eukaryotic cells
Function of Chromatin:
to package DNA into a smaller volume to fit in the cell
to strengthen the DNA to allow mitosis
to prevent DNA damage
to control gene expression and DNA replication

8. described as a “beads on a string” model
C. Chromatin:
described as a “beads on a string” model
histone
- the histone DNA complex is
called a nucleosome
DNA in between nucleosomes
is called “linker DNA” – 50bp
the entire complex is known as
the 10nm fiber or euchromatin

9. Chromatin Organization
-10nm fiber = euchromatin
10nm fiber is packed further into a 30nm fiber = heterochromatin
compacted further by forming “loops”
eventually condenses into a chromosome
Nucleosome
Euchromatin
Heterochromatin
DNA
Further looping and condensing
Chromosome

10. chromatin arrangement is critical to DNA function
organization of chromatin depends on the cell cycle – i.e. the stage of cell replication
during interphase of the cell cycle - two forms of chromatin: euchromatin and heterochromatin (more condensed form of chromatin)
with cell replication - heterochromatin condenses even more -> chromosomes
condensing of chromatin through its various forms – regulated by specific proteins
e.g. protein called condensin
chromatin arrangement is critical to DNA function
more condensed the less access the molecular “machinery” of gene expression has
human karyotype

11. Histones complex of small, basic proteins
core of 4 proteins: H2A, H2B, H3, H4
2 complexes = 8 total proteins per histone
DNA wraps around this core = 10nm fiber (euchromatin)
plus 2 linker histones H1, H5
linkers interact with the DNA and pack it into a thicker 30nm fiber (heterochromatin)
many histone amino acids are positively charged
so DNA-histone interaction is just an attraction between negatively charged DNA and positively charged histones
not dependent upon DNA sequence
proteins of the histone make ionic bonds to the acidic sugar-PO4 backbone of the DNA helix
bonds between histone and DNA can be modified by enzymes
e.g. methylation, phosphorylation
this modification can change the interaction between the DNA and the histone
can make the DNA either more or less accessible to the replication machinery
modifications can either make replication easier or harder

12. The Search for the Genetic Material
early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists
when T. H. Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material
key factor in determining the genetic material was choosing appropriate experimental organisms
role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them

13. DNA as the source of genetic material
1928
Frederick Griffith – working on a vaccine for pneumonia (Streptococcus pneumonia)
worked with 2 strains – one harmless, one pathnogenic
killed the pathenogenic bacteria and mixed them with cultures of living, harmless bacteria
some of the harmless bacteria became pathenogenic
their progeny remained pathenogenic
DNA had to have been transferred from path. to harmless when he mixed the bacteria
called this transfer = transformation (assimilation of external by a cell)
Living S cells (control)
Living R cells (control)
Heat-killed S cells (control)
Mixture of heat-killed S cells and living R cells
Mouse dies
Mouse healthy
Living S cells
EXPERIMENT
RESULTS

14. DNA as the source of genetic material
transformation now defined as – change in genotype and phenotype due to the assimilation of external DNA by a cell
1944 -Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that the transforming substance was DNA
14 year project!!
possible transforming agents: DNA, RNA or protein
broke open heat-killed bacteria and extracted their contents
treated each sample with chemicals that inactivated either DNA, RNA or protein
tested the modified samples for their ability to transform live, non-pathenogenic bacteria
only DNA worked in transforming harmless bacteria into pathogenic bacteria
lost this function once they were inactivated with chemicals
many biologists remained skeptical, mainly because little was known about DNA

15. Viral DNA Can Program Cells
more evidence for DNA as the genetic material - studies of viruses that infect bacteria
called bacteriophages (or phages)
Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2
phage that normally infects E. coli strains
composed of DNA and protein
they designed an experiment showing that only the DNA enters an E. coli cell during infection

16. Viral DNA Can Program Cells
labelled phages with radioactive sulfur to label proteins (batch #1) or phosphorus to label DNA (batch #2)
mixed the labelled phages with bacteria to cause infection
separated the bacteria from the phages and looked for what radioactive signal was in the isolated bacteria
confirmed and measured the presence of radioactive phosphorus in the transformed bacteria
Proteins labelled
DNA labelled

17. Chargaff’s Rules
DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group
Erwin Chargaff reported that DNA composition varies from one species to the next
BUT he also noticed two other things
these 2 findings became known as Chargaff’s rules
1. WHILE the base composition of DNA varies between species
2. the number of A and T bases are equal and the number of G and C bases are equal in any species

18. Watson & Crick 1950s – three groups working on the structure of DNA
1. Linus Pauling – Cal Tech
2. Maurice Wilkins and Rosalind Franklin – King’s College, London, UK
3. American James Watson and Englishman Francis Crick – Cavendish Laboratory, Cambridge, UK
1953- Watson and Crick constructed a model for DNA – two complementary strands that run anti-parallel to one another in a double helix

19. Watson & Crick
based their work on Rosalind Franklin’s X-ray crystallography findings
X-rays bent as they passed through the strands of purified DNA
photo 51 – suggested a double helix, with the bases facing inward, making a full turn every 3.4nm, 10 base pairs every full turn
1962 – Nobel prize – Watson, Crick and Wilkins (Franklin dies in 1958)
(a) Rosalind Franklin
(b)
Franklin’s X-ray diffraction photograph of DNA

20. Nucleic acids nucleotide: two types: DNA, RNA C,H,O,N,P
building blocks = nucleotides
nucleotide:
5 carbon sugar (pentose)
phosphate group (negative charge) located at the 5’ carbon
organic base located at the 1’carbon
sugar and the base is known as a nucleoside
bases: 5 types: adenine (A)
cytosine (C)
guanine (G)
thymine (T)
uracil (U)
Sugar-phosphate backbone
5 end
5C
3C
3 end
(a) Polynucleotide, or nucleic acid
(b) Nucleotide
Phosphate group
Sugar (pentose)
Nucleoside
Nitrogenous base
1C

21. Naming nucleotides
nucleotide is conventionally named “based” on its base
adenine, guanine etc…
but the true name is a combination of the nucleoside and the number of phosphate groups
nucleoside= combination of a base and a sugar
e.g. adenine + sugar = adenosine
so “adenine” is really called
e.g. RNA - adenosine monophosphate (AMP)
e.g. DNA - deoxy-adenosine monophosphate (dAMP)

22. (c) Nucleoside components Pyrimidines
Nitrogenous bases
Cytosine (C)
Thymine (T, in DNA)
Uracil (U, in RNA)
Adenine (A)
Guanine (G)
Sugars
Deoxyribose (in DNA)
Ribose (in RNA)
(c) Nucleoside components
Pyrimidines
Purines
There are two families of nitrogenous bases
1. Pyrimidines (cytosine, thymine, and uracil) have a single six-membered ring
2. Purines (adenine and guanine) have a six-membered ring fused to a five-membered ring
Figure 5.26 Components of nucleic acids.

23. DNA is double stranded: The DNA double helix
DNA strand is known as a polynucleotide chain
2 sugars of adjacent nucleotides are joined by a phosphodiester bond
the phosphodiester bond can only form between a 3’OH “down” to a 5’PO4 group
so that the DNA chain grows in a 5’ to 3’ direction

24. -the DNA strand must “grow” in a specific direction because of the nature of the phosphodiester bond
-this direction is called 5’ to 3’ because one end is a 5’phosphate group, the other end is a 3’ OH group
-the complementary strand also grows this way = two anti-parallel strands of DNA (opposite directions of growth)

25. Why 5’ to 3’?? the DNA chain grows in a 5’ to 3’ direction
why can’t you form a phosphodiester bond from 3’ to 5’??
the first thing that happens is the bases pair up with each other
THEN the phosphodiester bond forms
the incoming nucleotide is in its triphosphate form

26. Why 5’ to 3’??
a magnesium ion is associated with the outer two phosphate groups
this weakens the phosphate bonds between these groups
and allows for a successful nucleophilic attack by the 3’OH group in the nucleotide that is already bound to the DNA chain
results in the loss of the outer 2 phosphate groups (due to a shift in electrons) and the formation of a 3’-5’ phosphodiester bond
Nucleophilic attack
Mg2+

27. single DNA chain is 2.2 to 2.6nm (22 to 26 Angstroms) wide – each nucleotide is 3.3A long
DNA exists as a double helix – backbone made of alternating sugars and phosphates from the nucleotide
bases are inside the helix – away from the aqueous environment of the cell
helix makes 1 full turn every 3.4nm
~10bp every turn
so one base is 0.34nm wide
helix is a right hand twist
clockwise twist

28. 2 DNA strands are held together by Hydrogen bonds between the bases
not very strong, so DNA helix is easily separated by high heat, salt, mechanical forces etc…
this is known as denaturation
Watson and Crick found that the helix was uniform in its diameter
proposed that the base pairing had to be consistent throughout the helix
proposed = DNA held together by complementary base pairs
the base pairing rule is purine-pyrimidine
purine-purine pairing would produce a bulge in the helix
pyrimidine-pyrimidine pairing would produce a “dent”
A to T – two H bonds
C to G - three H bonds
C-G pairings are stronger – DNA is harder to separate in those regions (“GC rich”)

29. helix also exhibits additional levels of coiling = supercoiling
DNA can either supercoil in the direction of the helix (positive) or in the opposite direction (negative)
degree of supercoiling can determine the strength of the interaction between the two DNA chains

30. Naming the DNA strands DNA is a double helix of complementary sequence
to simplify things we call one strand “sense” the other “anti-sense”
sense strand – has same sequence as the mRNA strand that will be translated into a protein
anti-sense strand – template used to create this mRNA
when written out – the sense strand is the top strand – with the 5’PO4 on the left and the 3’OH on the right

31. -double stranded DNA helix is
unwound into individual strands
-enzyme = DNA polymerase attaches
to each strand
-polymerase moves along the DNA
strand and attaches the complementary
base
e.g. C is read – attaches a G

32. Models of DNA replication
conservative: means that the replicated DNA helices are entirely parental or daughter
semi-conservative: means that the replicated DNA helices are 50% parental DNA and 50% daughter DNA
dispersive: means that the replicated DNA helices are a mixture of parental and daughter DNA

33. Replication is Semi-conservative
labelled bacterial DNA first with a heavy 15N isotope to label the parental strands
replication - added a labelled a lighter 14N isotope that would label the replicated DNA daughter strands
continued to label bacteria with the lighter isotope for another round of replication
isolated the DNA & based on their “weight” they could tell which DNA strands had the 15N label or the 14N label
found that after the 1st replication – hybrids of both 15/14N strands
after the second replication – mixture of hybrid DNA and light 14N DNA helices
only way to explain this = Semi-conservative replication

34. DNA replication – the players
DNA polymerase III = reads the DNA strand and lays down a complementary base to create a complementary “daughter” strand of new DNA
Helicase/dnaB= enzyme that “melts” or unzips the double-helix of the parental DNA
single stranded binding proteins/SSBs – hold the unwound DNA helix “open” – allows for the action of the replication machinery
RNA polymerase (Primase/dnaG) = creates a small RNA “oligo” primer which binds onto the DNA template and allows the DNA polymerase III to attach to the DNA template
DNA ligase = joins up the fragments of DNA made during replication

35. DNA polymerase
polymerase = enzyme that catalyzes the addition of bonds between two nucleotides of either DNA or RNA
catalyzes the bond between bases
also catalyzes the phosphodiester bond within the backbone
in bacteria – three polymerases that act in replication
replication polymerase = DNA polymerase III
DNA polymerase I – removes RNA nucleotides found in the primer(exonuclease activity) and fills in the gaps
also corrects mistakes made upon replication = “proof-reading”
DNA polymerase II – proof reading
in eukaryotic cells – 11 polymerases !!
same idea as bacterial replication

36. Problems with Replication
first problem: DNA is a helix
DNA polymerases are unable to melt duplex DNA in order to separate the two strands that are to be copied
solution: binding of a “helicase” enzyme unwinds the two strands
second problem: unwinding the DNA produces supercoiling in the regions ahead
solution: action of topoisomerases to “nick” the DNA helix, unwind the supercoils and connect the strand back together

37. Problems with Replication
another problem: the helix wants to re-form
solution: single-stranded binding proteins (SSBs) also required to prevent the DNA template from rewinding back up
still another problem: DNA polymerases cannot bind single stranded nucleic acids
unwinding the DNA gets rid of base pairing = Problem !
DNA polymerase III requires a “primer” to elongate off of
solution: enzyme called a “primase” makes a small piece of RNA that binds to the DNA strand and acts as a primer for the DNA polymerase – creates a temporary double stranded structure of RNA and DNA

38. Problems with the DNA polymerase
Biggest Problem: The two strands in the DNA duplex are opposite in chemical polarity
DNA polymerase can only form a phosphodiester bond between the 5’PO4 of an incoming new nucleotide and a 3’OH of a nucleotide already base paired to the DNA template
this means that new DNA strands can grow only in the 5 to 3 direction
this is okay when replicating the anti-sense strand because the complementary daughter strand that is being created will grow in the 5’ to 3’ direction
but there is a problem replicating the sense strand 3’ 5’
growing daughter strand
DNA
poly

39. DNA replication
replication starts at specific sequences of DNA = origins of replication
called oriC in bacteria
~240bp sequence – containing repetitive sequences – rich in As and Ts
also found in viruses
multiple origins are found in eukaryotic chromosomes
oriC is recognized by a protein complex that docks onto the oriC
this complex is comprised of at least five protein complexes –the helicase, the primase, DNA polymerase, clamp loading protein, infrastructure proteins that ‘tether’ the two DNA polymerases to each other

40. DNA replication
oriC is recognized by the helicase that unwinds the DNA
“melts” the DNA
forms a replication “bubble”
comprised of two replication forks
the forks are held open by SSBs
the helicase can actually act as a molecular “brake” that controls how fast replication happens
the helicase/primase/DNA polymerase moves along the parental DNA strand in the 3’ to 5’ direction

41. DNA replication: The Leading strand
REMEMBER – a complex of proteins binds at the oriC and moves along the parental DNA in the 3’ to 5’ direction
because of this one strand of parental DNA is able to be replicated continuously without any problems
new daughter strand grows in the 5’ to 3’ direction
the DNA strand that is made continuously = Leading Strand
the leading strand is uses the anti-sense strand of parental DNA as its template
the leading strand also grows in the same direction as the replication fork
Replication fork
direction

42. DNA replication: The Lagging strand
but since the second strand of DNA is in the opposite direction – it cannot be replicated continuously
this would require the daughter strand be made in the 3’ to 5’ direction
solution – sense strand is replicated in “chunks”
this daughter DNA strand being made is called the Lagging strand
‘chunks’ are called Okazaki fragments – 1000 to 2000 bps
(Reiji Okazaki)
as the helicase unwinds the parental duplex, primase creates multiple primers along the sense parent strand
DNA polymerase “clamps” onto the 3’ end of this DNA-RNA hybrid duplex and makes an Okazaki fragment

43. DNA polymerase III – 2 large complexes tethered together
core polymerase – three subunits:
alpha – active site for nucleotide addition – polymerase action
epsilon – exonuclease activity – part of the enzyme that removes incorrectly added nucleotides – “proof-reading”
can only back-up one nucleotide and correct it
so DNA polymerase III has to “catch” its mistakes as it makes them
sigma – proof-reading
beta – forms a beta-clamp – forms a donut-like clamp around the parental DNA
the gamma subunit: loads the b-clamp onto the DNA strand at the RNA primers and takes the clamp back off
now known as the Clamp-loading Protein

44. The big picture!!
leading and lagging strands are made at the same time
two core DNA polymerases III bind at each fork and replicate the DNA in the same direction!
how is this possible??
the parental/lagging strand is “looped” within the complex so that its orientation is the same as the parental/leading strand
IMPORTANT – the DNA polymerase III complex doesn’t move – the DNA template is “fed into” the complex and then fed back out
DNA polymerase III is anchored to the nuclear matrix
direction of polymerase movement (3’ to 5’)
DNA polymerase
SSBs
Helicase & Primase
(topoisomerase)

45. The big picture animation
for the “big picture”:
Leading
Daughter-
Antisense
parent
Sense parent
Beta clamp
Anti- Sense parent
alpha
helicase
primase
alpha
SSBs
RNA primer #2
RNA primer #1
Okazaki
Fragment #1
Beta clamp

46. Leading Daughter- Polymerase direction Antisense parent alpha helicase
RNA primer #3
helicase
alpha
primase
SSBs
RNA primer #2
Okazaki
Fragment #2
RNA primer #1
Okazaki
Fragment #1

47. Check out these animations!!