Cellular and Molecular Biology

CELLULAR AND MOLECULAR BIOLOGY FINAL EXAMINATION

DENNIS NABOR MUÑOZ, RN, RM, LPT

Graduate Student, Master of Science in Biology
Ateneo de Davao University

Submitted To
Maria Cristina S. Delas Llagas, Ph.D
Faculty/Chairman of Biological Science Department
Ateneo de Davao University

1. Discuss in detail how DNA is Synthesized in prokaryotes. Use diagrams if needed.

Replication is an enzymatic process in which synthesis of a daughter or progeny duplex

DNA molecule, identical to the parental duplex DNA occurs. Rate of replication in E.Coli
(prokaryotic cell) is 1500 nucleotides per second. To complete replication of whole E.Coli
genome it takes 40 minutes. Rate of replication in eukaryotes is about 10 - 100 nucleotides per
second. To complete replication of simple eukaryotic genome 6 - 8 hours required. In
prokaryotic circular DNA only one replication fork is present but in eukaryotic DNA several
replication forks are present. Space between two-replication forks in eukaryotes is about 20kbps
apart (Alberts, 2017).

THE REPLICATION OF CIRCULAR DNA IN E. COLI (Prokaryotic duplex DNA

replication) (q REPLICATION) (Alberts, 2017).

The synthesis or replication of DNA molecule can be divided into three stages

I. Initiation (Formation of Replisome)

II. Elongation (Initiation of synthesis and elongation)
III. Termination

I) Initiation

The replication begins at a specific initiation point called OriC point or replicon.
(Replicon: It is a unit of the genome in which DNA is replicated; it contains an origin for
initiation of replication) It is the point of DNA open up and form open complex leading to the
formation of prepriming complex to initiate replication process (Quiñones-Valles, Espíndola-
Serna & Martiń ez-Antonio, 2011).

Figure 1. Arrangement of Sequence in the E. coli Replication origin (OriC). Consensus

sequences (p. 104) for key repeated elements are shown. N represents any of the four nucleotides.
The horizontal arrows indicate the orientations of the nucleotide sequences (left-to-right arrow
denotes a sequence in the top strand; right-to-left, in the bottom strand). FIS and IHF are binding
sites for proteins described in the text. R sites are bound by DnaA. I sites are additional DnaA-
binding sites (with different sequences), bound by DnaA only when the protein is complexed with
ATP. Excerpt from Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2010). Lehninger principles
of biochemistry. 6th Edition. New York: Worth Publishers. p. 959
 Cellular and Molecular Biology

Initiation The E. coli replication origin, called oriC, consists of 245 base pairs, many of
which are highly conserved among bacteria The key sequences for this discussion are two series
of short repeats; three repeats of a 13 base pair sequence and four repeats of a 9 base pair
sequence (Lehninger., et al., 2017).

The OriC site is situated at 74" minute near the ilv gene. The OriC site consists of 245
basepairs, of which three of 13 base pair sequences are highly conserved in many bacteria and
forms the consensus sequences (GATCTNTTNTTTT). Close to OriC site, there are four of 9
base pair sequences each (TTATCCACA) (Lehninger., et al., 2017)

The sequence of reactions in the initiation process is as follows:

a. Dna A protein recognizes and binds up to four 9bp repeats in OriC to form a complex of
negatively supercoiled OriC DNA wrapped around a central core of Dna A protein
monomers. This process requires the presence of the histone like HU or 1 HC proteins to
facility DNA bending.
 Cellular and Molecular Biology

Figure 2. Initiation Process. The sequence of reactions in the initiation process is explained
from a to f. Excerpt from Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2010). Lehninger
principles of biochemistry. 6th Edition. New York: Worth Publishers. p. 989

b. Dna A protein subunits then successively melt three tandemly repeated 13bp segments in the
presence of ATP at >=22*C (open complex).
c. The Dna A protein then guides a Dna B - Dna C complex into the melted region to form a so
called prepriming complex. The Dna C is subsequently released. Dna B further unwinds
open complex to form prepriming complex.
d. DNA gyrase, single stranded binding protein (SSB), Rep protein and Helicase - II are bound
to prepriming complex and now complex is called as priming complex.
e. In the presence of gyrase and SSB, helicases further unwinds the DNA in both directions so as
to permit entry of primase and RNA polymerase. Then RNA polymerase forms primer for
leading strand synthesis while primase in the form of primosome synthesis primer for lagging
strand synthesis.
f. To the above complex, DNA polymerase - III will bind and forms replisome.
 Cellular and Molecular Biology

REPLISOME: It is the multiprotein structure that assembles at the bacterial replicating fork to
undertake synthesis of DNA. It contains DNA polymerase and other enzymes.

Figure 3. E. coli replisome. Schematic representation of the E. coli replisome depicting

coordinated DNA synthesis. Three DnaG primase monomers are shown interacting with the DnaB
helicase, adding an RNA primer (green) onto the SSB-coated lagging strand. (Nathan, Samir,
Slobodan, Karin, Patrick, Nicholas, & Antoine, 2008).

II) ELONGATION:

Now the stage is set for the initiation of synthesis and the elongation to proceed. But this
occurs in two mechanistically different pathways in the 5'-->3' template strand and 3'--
>5' template strand (Kaguni, 2011)

a) Initiation of synthesis and Elongation on the 5'-->3' template (synthesis of leading

strand) (If replication fork moves in 3'-->5' direction)

The DNA daughter strand that is synthesized continuously on 5'-->3' template is called
leading strand. DNA pol-III synthesizes DNA by adding 5'-P of deoxynucleotide to 3'-OH group
of the already presenting fragment. Thus chain grows in 5'-->3' direction. The reaction catalyzed
by DNA pol-III is very fast. The enzyme is much more active than DNA pol - I and can add 9000
nucleotides per minute at 37*C. The RNA primer that was initially added by RNA polymerase is
degraded by RNase.

b) Initiation of synthesis and Elongation on 3'-->5' template when fork moves in 3'-->5'
direction (Synthesis of lagging strand)
 Cellular and Molecular Biology

The daughter DNA strand which is synthesized in discontinuous complex fashion on the
3'-->5' template is called lagging strand. It occurs in the following steps:

Figure 5. The Okazaki Fragment. These are short, newly synthesized DNA fragments that are
formed on the lagging template strand during DNA replication. They are complementary to the
lagging template strand, together forming short double-stranded DNA sections (Wolański,
Donczew, Zawilak-Pawlik, & Zakrzewska-Czerwińska, 2014).

i) Synthesis of Okazaki fragment:

To the RNA primer synthesized by primosome, 1000-2000 nucleotides are added by DNA
pol-III to synthesis Okazaki fragments.

ii) Excision of RNA primer:

When the Okazaki fragment synthesis was completed up to RNA primer, then RNA
primer was removed by DNA pol - I using its 5'-->3' exonuclease activity.
 Cellular and Molecular Biology

Figure 6. Synthesis of Okazaki fragments. (a) At intervals, primase synthesizes an RNA primer
for a new Okazaki fragment. Note that if we consider the two template strands as lying side by
side, lagging strand synthesis formally proceeds in the opposite direction from fork movement. (b)
Each primer is extended by DNA polymerase III. (c) DNA synthesis continues until the fragment
extends as far as the primer of the previously added Okazaki fragment. A new primer is
synthesized near the replication fork to begin the process again. (Lehninger., et al., 2017).

iii) Filling the gap (Nick translation)

The gap created by the removal of primer, is filled up by DNA pol - I using the 3'-OH of
nearby Okazaki fragment by its polymerizing activity.

iv) Joining of Okazaki fragment: (Nick sealing)

Finally, the nick existing between the fragments are sealed by DNA ligase which catalyze
the formation of phosphodiester bond between a 3'-OH at the end of one strand and a 5' -
phosphate at the other end of another fragment. The enzyme requires NAD for during this
reaction (Helmstetter, 1968)
 Cellular and Molecular Biology

Figure 7. The Joining of Okazaki Fragments.

III) TERMINATION:

Figure 8. E. coli Replication Termination. Termination occurs when the two replicating forks
meet each other on the opposite side of circular E.Coli DNA. Termination sites like A, B, C, D, E
and F are found to present in DNA. Of these sites, Ter A terminates the counter clockwise moving
fork while ter C terminates the clockwise moving forks. The other sites are backup
sites. Termination at these sites are possible because, at these sites tus protein (Termination
utilizing substance) will bound to Dna B protein and inhibits its helicase activity. And Dna B
protein released and termination result (Jameson, and Wilkinson, 2017).

The arrest of DNA replication in Escherichia coli is triggered by the encounter of a

replisome with a Tus protein-Ter DNA complex. A replication fork can pass through a Tus-
 Cellular and Molecular Biology

Ter complex when traveling in one direction but not the other, and the chromosomal Ter sites are
oriented so replication forks can enter, but not exit, the terminus region. The Tus-Ter complex
acts by blocking the action of the replicative DnaB helicase, but details of the mechanism are
uncertain. One proposed mechanism involves a specific interaction between Tus-Ter and the
helicase that prevents further DNA unwinding, while another is that the Tus-Ter complex itself is
sufficient to block the helicase in a polar manner, without the need for specific protein-protein
interactions. Therefore, it is possible to explain polar fork arrest by a mechanism involving only
the Tus-Ter interaction, there are also strong indications of a role for specific Tus-DnaB
interactions.

After the complete synthesis, two duplex DNA are found to be catenated (knotted). This
catenation removed by the action of topoisomerase. Finally, from single parental duplex DNA,
two progeny duplex DNA synthesized.

REGULATION OF PROKARYOTIC REPLICATION:

Especially initiation of replication is regulated. Dna A protein when available in high

concentration then ratio of DNA to cell mass is quiet high but at low Dna A concentration, the
ratio found to be low. This shows that Dna A protein regulates the initiation of replication.

The sequence most commonly methylated in E.Coli is GATC including in three of 13mer
sequence. Thus, the observation that E.Coli defective in the GATC methylation enzyme are very
inefficiently replicated, suggests that the DNA replication trigger also responds to the level of
OriC methylation.

Reference:

1. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York:
Garland Science; 2002. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21054/
2. Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2010). Lehninger principles of biochemistry.
6th Edition. New York: Worth Publishers.
3. César Quiñones-Valles, Laura Espiń dola-Serna and Agustino Martiń ez-Antonio (2011).
Mechanisms and Controls of DNA Replication in Bacteria, Fundamental Aspects of DNA
Replication, Dr. Jelena Kusic-Tisma (Ed.), InTech, DOI: 10.5772/19313. Available from:
https://mts.intechopen.com/books/fundamental-aspects-of-dna-replication/mechanisms-and-
controls-of-dna-replication-in-bacteria
4. Nathan A., T., Samir M., H., Slobodan, J., Karin V., L., Patrick M., S., Nicholas E., D., &
Antoine M., van O. (2008). Single-Molecule Studies of Fork Dynamics of Escherichia
coli DNA Replication. Nature Structural & Molecular Biology, 15(2), 170–176.
http://doi.org/10.1038/nsmb.1381
5. Kaguni, J. M. (2011). Replication initiation at the Escherichia coli chromosomal
origin. Current Opinion in Chemical Biology, 15(5), 606–613.
http://doi.org/10.1016/j.cbpa.2011.07.016
6. Wolański, M., Donczew, R., Zawilak-Pawlik, A., & Zakrzewska-Czerwińska, J. (2014). oriC-
encoded instructions for the initiation of bacterial chromosome replication. Frontiers in
Microbiology, 5, 735. http://doi.org/10.3389/fmicb.2014.00735
7. Jameson, K. H., & Wilkinson, A. J. (2017). Control of Initiation of DNA Replication
in Bacillus subtilis and Escherichia coli. Genes, 8(1), 22.
http://doi.org/10.3390/genes8010022
8. Helmstetter, C. E. (1968). Origin and Sequence of Chromosome Replication in Escherichia
coli B/r. Journal of Bacteriology, 95(5), 1634–1641.
9. Hill, N. S., Kadoya, R., Chattoraj, D. K., & Levin, P. A. (2012). Cell Size and the Initiation of
DNA Replication in Bacteria. PLoS Genetics, 8(3), e1002549.
http://doi.org/10.1371/journal.pgen.1002549
10. Windgassen, T. A., Wessel, S. R., Bhattacharyya, B., & Keck, J. L. (2018). Mechanisms of
bacterial DNA replication restart. Nucleic Acids Research, 46(2), 504–519.
http://doi.org/10.1093/nar/gkx1203
 Cellular and Molecular Biology

11. Bussiere DE, Bastia D (March 1999). "Termination of DNA replication of bacterial and
plasmid chromosomes". Molecular Microbiology. 31 (6): 1611–8. doi:10.1046/j.1365-
2958.1999.01287.x. PMID 10209736.
12. Neylon, C., Kralicek, A. V., Hill, T. M., & Dixon, N. E. (2005). Replication Termination
in Escherichia coli: Structure and Antihelicase Activity of the Tus-
Ter Complex. Microbiology and Molecular Biology Reviews, 69(3), 501–526.
http://doi.org/10.1128/MMBR.69.3.501-526.2005
13. Cooper, S.; Helmstetter, C. E. (1968-02-14). "Chromosome replication and the division cycle
of Escherichia coli B/r". Journal of Molecular Biology. 31 (3): 519–540. doi:10.1016/0022-
2836(68)90425-7. ISSN 0022-2836. PMID 4866337
 Cellular and Molecular Biology

2. Discuss how DNA is amplified using a Thermal Cycler. Discuss the steps involved and relate
this with how DNA is synthesized in our cells.

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a
laboratory apparatus most commonly used to amplification of a short defined segments of
DNA in vitro via the PCR or polymerase chain reaction (Smith, 2009)..

PCR involves three processing steps: denaturation, annealing and then extension by DNA
polymerase (Smith, 2009):

1. Step 1, the double-stranded DNA is heated (Denaturation 95–98◦C) and separates into two
complementary single strands.
2. Step 2 (60◦C), the synthetic oligonucleotide primers (chemically synthesised short-chain
nucleotides) – short sequences of nucleotides (usually about 20 nucleotide base pairs long) –
are added and bind to the single strands in places where the strand’s DNA complements their
own. Annealing (40-60◦C)
3. Step 3 (37◦C), the primers are extended by DNA polymerase in the presence of all four
deoxynucleoside triphosphates, resulting in the synthesis of new DNA strands that are
complementary to the template strands. (70◦ – 74◦)

The completion of the three steps comprises a cycle and the real power of PCR is that, with
25–30 cycles, this experimental synthesis leads to massive amplification of DNA which can
then
be used for analytical purposes. A major recent advance has been the development of
automated thermal cyclers (PCR machines), which allow the entire PCR to be performed
automatically in several hours (Smith, 2009)..

During PCR, oligonucleotide primer molecules are bound at low temperature to templates
of heat-denatured DNA and extended on their 3' end using a thermostable DNA polymerase.
The DNA denaturation, primer annealing, and extension is repeated several times under
program control to accumulate many identical copies of the DNA sequence between the
primers. A microcomputer system controls the flow of 96 degrees Celsius and 37 degrees
Celsius water through a 24-well sample holder so that the temperature in the samples in the
holder varies as required for DNA denaturation, primer annealing, and DNA
polymerization. (Weier and Gray, 1988)

A typical amplification reaction includes target DNA, a thermostable DNA polymerase,

two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and
magnesium. Once assembled, the reaction is placed in a thermal cycler, an instrument that
subjects the reaction to a series of different temperatures for set amounts of time. This series
of temperature and time adjustments is referred to as one cycle of amplification. Each PCR
cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten
cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a
factor of more than a million in a matter of hours (Promega, 2018).
 Cellular and Molecular Biology

Figure 1. Schematic diagram of the PCR process. Amplifying DNA by the polymerase
chain reaction. Start with a DNA duplex (top) and heat it to separate its two strands. Then
add short, single-stranded DNA primers (purple and yellow) complementary to sequences on
either side of the region (X, 250 bp) to be amplified. The primers hybridize to the appropriate
sites on the separated DNA strands; now a special heat-stable DNA polymerase uses these
primers to start synthesis of complementary DNA strands. The arrows represent newly made
DNA in which replication has stopped at the tip of the arrowhead. At the end of cycle 1, two
DNA duplexes are present, including the region to be amplified, whereas we started with only
one. The 59→39 polarities of all DNA strands and primers are indicated. The same principles
apply in every cycle thereafter (Promega, 2018; Smith, 2009).

Figure 2. Temperature profile of a PCR cycle is controlled by the Thermal Cycler

Program. Each cycle of PCR includes steps for template denaturation, primer annealing
and primer extension (Smith, 2009).
 Cellular and Molecular Biology

The initial step denatures the target DNA by heating it to 94°C or higher for 15 seconds to
2 minutes. In the denaturation process, the two intertwined strands of DNA separate from one
another, producing the necessary single-stranded DNA template for replication by the
thermostable DNA polymerase. In the next step of a cycle, the temperature is reduced to
approximately 40–60°C. At this temperature, the oligonucleotide primers can form stable
associations (anneal) with the denatured target DNA and serve as primers for the DNA
polymerase. This step lasts approximately 15–60 seconds. Finally, the synthesis of new DNA
begins as the reaction temperature is raised to the optimum for the DNA polymerase. For most
thermostable DNA polymerases, this temperature is in the range of 70–74°C. The extension
step lasts approximately 1–2 minutes. The next cycle begins with a return to 94°C for
denaturation (Promega, 2018).
Each step of the cycle should be optimized for each template and primer pair combination. If
the temperature during the annealing and extension steps are similar, these two steps can be
combined into a single step in which both primer annealing and extension take place. After
20–40 cycles, the amplified product may be analyzed for size, quantity, sequence, etc., or used
in further experimental procedures (Promega, 2018).

EUKARYOTIC DNA REPLICATION

The mechanism of eukaryotic DNA replication is similar to that of prokaryotic DNA

replication. However, eukaryotic DNA replication requires special consideration due to
differences in DNA sizes, unique linear DNA end structures called telomeres, and distinctive
DNA packaging that involves complexes with histones. Unlike prokaryotes, most eukaryotes are
multicellular organisms, except for the unicellular eukaryotes such as yeast, flagellates, and
ciliates. Therefore, DNA replication in eukaryotes is a highly regulated process and usually
requires extracellular signals to coordinate the specialized cell divisions in different tissues of
multicellular organisms. External signals are delivered to cells during the G1 phase of the cell
cycle and activate the synthesis of cyclins. Cyclins form complexes with cyclin-dependent kinases
(CDK), which, in turn, stimulate the synthesis of S phase proteins such as DNA polymerases and
thymidylate synthase. These complexes prepare cells for DNA replication during the S phase.

Initiation of DNA replication in eukaryotes begins with the binding of the origin
recognition complex (ORC) to origins of replication during the G1 phase of the cell cycle. The
ORC complex then serves as a platform for forming much more complicated pre-replicative
complexes (pre-RCs). Formation of pre-RCs involves the assembly of cell division cycle 6p
(Cdc6p) protein, DNA replication factor Cdt1p, mini-chromosome maintenance complex (Mcm
2p-7p), and other proteins. Pre-RCs formed during the G1 phase are converted to the initiation
complex during cell cycle transition from G1 to S by the action of two kinases: cyclin-dependent
kinase (CDK) and Dbf4-dependent kinase (DDK). Formation of an initiation complex, which
includes helicase activity, unwinds the DNA double helix at the origin site . The DNA polymerase
α-primase complex synthesizes the first primer. It initiates DNA replication on the leading strand
and Okazaki fragments on the lagging strand. In addition to the polymerase α-primase, two DNA
polymerases, δ and ε, are required for DNA replication. Polymerase δ is the major polymerase in
leading-strand synthesis; polymerases δ and ε are the major polymerases in lagging-strand
synthesis. This is similar to the DNA polymerase I and III in the lagging-strand synthesis of
prokaryotes. In eukaryotes, Okazaki fragments generated during lagging-strand synthesis are
shorter than those in E. coli (up to 200 bases in eukaryotes versus up to 2000 bases long in E.
coli). Also, eukaryotic DNA replication is initiated by forming many replication forks at multiple
origins to complete DNA replication in the time available during the S phase of a cell cycle.

Two key structural features of eukaryotic DNA that are different from prokaryotic DNA
are the presence of histone complexes and telomere structures. Histones are responsible for the
structural organization of DNA in eukaryotic chromosomes. The positive charge of histones, due
to the presence of numerous lysine and arginine residues, is a major feature of the molecules,
 Cellular and Molecular Biology

enabling them to bind the negatively charged phosphate backbones of DNA. Pairs of four
different histones (H2A, H2B, H3, and H4) combine to form an eight-protein bead around which
DNA is wound. This bead-like structure is called a nucleosome A nucleosome has a diameter of
10 nm and contains approximately 200 base pairs. Each nucleosome is linked to an adjacent one
by a short segment of DNA (linker) and another histone (H1). The DNA in a nucleosome is
further condensed by the formation of thicker structures called chromatin fibers, and ultimately
DNA must be condensed to fit into the metaphase chromosome that is observed at mitosis.
Despite the dense packing of DNA in chromosomes, it must be accessible to regulatory proteins
during replication and gene expression. At a higher level of organization, chromosomes are
divided into regions called euchromatin and heterochromatin. Transcription of genes seems to
be confined to euchromatin regions, while DNA in heterochromatin regions is genetically
inactive.

During DNA replication, the histone complexes of nucleosomes are separated; the leading
strand retains the old histones. The lagging strand remains free of histone complexes while new
histones are made and assembled. Since histones have greater affinity for double-stranded DNA,
newly synthesized histone octamers are quickly added as the lagging strand is polymerized.
Since DNA in eukaryotic chromosomes is a linear molecule, problems arise when replication
comes to the ends of the DNA. Synthesis of the lagging strand at each end of the DNA requires a
primer so that replication can proceed in a 5′ to 3′ direction. This becomes impossible at the ends
of the DNA, and the portion of RNA primer at the 5′ end of both leading and lagging strands is
lost each time a chromosome is replicated. Thus, at each mitosis of a somatic cell, the DNA in its
chromosomes becomes shorter and shorter. To prevent the loss of essential genetic information
during replication, the ends of DNA in chromosomes contain special structures called telomeres.
Human telomeres are repeated end sequences of (TTAGGG)nand have typical sizes of 15–20 kb
at birth. At each round of DNA replication, the telomere sequences of eukaryotic chromosomes
are shortened. This is the case for normal somatic cells, and the number of DNA replications/cell
divisions is linked to the timing of cell death. However, germline and cancer cells contain
enzymes called telomerases to extend the 5′ end of lagging strands The extension of telomere
sequences by telomerases in these cells contributes to their immortality. Human telomerase is a
reverse transcriptase that contains a short stretch of RNA sequence, AUCCCAAUC. This short
stretch of RNA serves as a template for telomere extension and plays a major role in leading
strand extension; when DNA replication is completed, telomerase binds to the 3′ end of the
leading strand. This establishes base pairing with the short stretch of RNA sequence the
telomerase carries and adds a 6-nucleotide sequence (GGTTAG) to the 3′ end of the strand
(Figure 22.9). After leading-strand extension on the 3′ end by the telomerase is completed, DNA
polymerase α completes the extension of the 5′ end of lagging strand and DNA ligase seals the
nick on the lagging strand left by DNA polymerase α. Since up to 90% of tumors contain
telomerases, which confer their immortality, telomerase inhibitors are being tested as a cancer
therapy.

Reference
1. Weier HU, Gray JW.( 1988 ). A programmable system to perform the polymerase chain
reaction. DNA. Jul-Aug;7(6):441-7. PubMed PMID: 3203600.
2. Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2010). Lehninger principles of
biochemistry. 6th Edition. New York: Worth Publishers.
3. Smith, J. (2009). Biotechnology. Cambridge: Cambridge University Press.
doi:10.1017/CBO9780511802751
4. Promega Corporation. (2018, April 10). PCR Amplification, Protocols and background
information about PCR and RT-PCR.Retrieved from
https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-
applications-guide/pcr-amplification/
5. Leman, A. R., & Noguchi, E. (2013). The Replication Fork: Understanding the Eukaryotic
Replication Machinery and the Challenges to Genome Duplication. Genes, 4(1), 1–32.
http://doi.org/10.3390/genes4010001
 Cellular and Molecular Biology

6. Meselson M., Stahl F.W. The replication of DNA in Escherichia coli. Proc. Natl. Acad.
Sci. USA. 1958;44:671–682. doi: 10.1073/pnas.44.7.671. [PMC free article] [Cross Ref]
7. Bessman M.J., Lehman I.R., Simms E.S., Kornberg A. Enzymatic synthesis of
deoxyribonucleic acid. II. General properties of the reaction. J. Biol. Chem.
1958;233:171–177.
8. Lehman I.R., Bessman M.J., Simms E.S., Kornberg A. Enzymatic synthesis of
deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme
from Escherichia coli. J. Biol. Chem. 1958;233:163–170.
9. Okazaki R., Okazaki T., Sakabe K., Sugimoto K., Sugino A. Mechanism of DNA chain
growth. I. Possible discontinuity and unusual secondary structure of newly synthesized
chains. Proc. Natl. Acad. Sci. USA. 1968;59:598–605. doi: 10.1073/pnas.59.2.598. [PMC
free article] [Cross Ref]
10. Wold M.S., Kelly T. Purification and characterization of replication protein A, a cellular
protein required for in vitro replication of simian virus 40 DNA. Proc. Natl. Acad. Sci.
USA. 1988;85:2523–2527. doi: 10.1073/pnas.85.8.2523. [PMC free article] [Cross Ref]
11. Alani E., Thresher R., Griffith J.D., Kolodner R.D. Characterization of DNA-binding and
strand-exchange stimulation properties of y-RPA, a yeast single-strand-DNA-binding
protein. J. Mol. Biol. 1992;227:54–71. doi: 10.1016/0022-2836(92)90681-9. [Cross Ref]
12. Siegal G., Turchi J.J., Myers T.W., Bambara R.A. A 5' to 3' exonuclease functionally
interacts with calf DNA polymerase ε Proc. Natl. Acad. Sci. USA. 1992;89:9377–9381.
[PMC free article]
13. Goulian M., Richards S.H., Heard C.J., Bigsby B.M. Discontinuous DNA synthesis by
purified mammalian proteins. J. Biol. Chem. 1990;265:18461–18471.
14. Waga S., Bauer G., Stillman B. Reconstitution of complete SV40 DNA replication with
purified replication factors. J. Biol. Chem. 1994;269:10923–10934.
15. Budd M., Campbell J.L. Temperature-sensitive mutations in the yeast DNA polymerase I
gene. Proc. Natl. Acad. Sci. USA. 1987;84:2838–2842. doi: 10.1073/pnas.84.9.2838.
[PMC free article] [Cross Ref]

3. From a journal: What is DNA fingerprinting? Its applications? How is it done in the
laboratory?
 Cellular and Molecular Biology

DNA fingerprinting, also known as DNA typing or DNA profiling, has a variety of
applications, ranging from aiding in criminal investigations to wildlife management. DNA
Fingerprinting is the method in which variable elements are isolated and identified within the base
pair sequence of DNA. A sample of cells such as skin, hair and blood cells are first obtained and
then DNA is extracted from the cells and purified. Essentially, DNA profiling is an identification
process based on genetic information. Every creature, excluding identical (zygotic) twins,
triplets, and so on is genetically distinct (Alexander, Fang, Rozowsky, Snyder, & Gerstein, 2010)
DNA Fingerprinting can be used to match a sample to a known source, compare two samples
to see how similar they are to determine relatedness, check the compatibility of biological
transplants, aid in plant breeding, identify skeletal remains, and help breeding programs in zoos
and animal reserves. Only a very small sample is needed to analyze DNA, for example,
commonly DNA is collected from an individual by obtaining a blood sample or a skin cell sample
using a cheek swab (Kirby, 1993).

Applications of DNA Fingerprinting

DNA Fingerprinting has become one of the most useful applications in molecular
biology and biological research. “Since the first forensic use of DNA fingerprinting in 1987, the
technology to analyze an individual's genetic profile has become more sensitive, easier and
cheaper to use and more widely available” (Collins, 2002). Forensic investigation, genetic
counseling, genetic therapy, disease detection, selective plant breeding, captive breeding
programs, paternity testing, transplant compatibility, identification of remains, and anthropology
were all able to progress due to the advances in DNA typing (Mishra, Sathyan, & Shukla, 2015).

Criminal Investigations

Criminal investigations have taken on a much more accurate approach since DNA
fingerprinting was developed. Before DNA could be analyzed, the only way to differentiate
biological evidence was by blood type. But blood type was not a very discriminatory piece of
evidence and therefore could easily lead an investigation in the wrong direction. “In criminal
investigations, DNA from samples of hair, bodily fluids or skin at a crime scene are compared
with those obtained from suspected perpetrators (Mishra, Sathyan, & Shukla, 2015).

In Zoology

In Zoology, DNA fingerprinting determine the genetic identity of individuals and measure
genetic variation in natural populations, allowing true genetic relationships among individuals to
be determined, rather than them being inferred from field observations (Mishra, Sathyan, &
Shukla, 2015).

Parentage testing

DNA fingerprinting is an advantageous technique in cases, such as, of establishing the

paternity of disputed offspring or cases of baby swapping. Tis method replaced ABO blood
antigen systems which cannot establish paternity but can conclusively exclude an alleged father
from being a candidate (Mishra, Sathyan, & Shukla, 2015).

Botany

DNA fingerprinting is an essential tool for genotype identification in both wild plant and
cultivated species. DNA profiling is used for protection of biodiversity, identifying markers for
traits, identification of gene diversity and variation (Mishra, Sathyan, & Shukla, 2015).

Molecular Archaeology
 Cellular and Molecular Biology

Another application of DNA Fingerprinting is Molecular Archaeology. This branch of

archaeology uses old samples of DNA or mtDNA to discover information about past civilizations.
Human and animal bloodlines can be followed; migration and traveling patterns can be
determined, and even cultural practices of civilizations can be determined by using DNA analysis.
DNA material can be obtained from a variety of old sources such as “biological remains, skeletal
remains, body tissues, hair, teeth, and in some cases fossils” (Christianson, 2000).

Parental Testing

“Excluding criminal investigation, the principal use of DNA fingerprinting is parentage

testing” (Collins, 2002). Paternity or Maternity Identifications Tests have become readily
available since the development of DNA Fingerprinting. These tests are performed when the
mother or father of a child is unknown or is being disputed. Most often it is the paternity of the
child that is unknown, but with new developments in fertility procedures there are more cases
where the maternity of a child needs to be determined (Mishra, Sathyan, & Shukla, 2015).

Procedure to create a DNA fingerprinting.

The steps involve others techniques used in Molecular Biology, such as polymerase chain
reaction (PCR) and electrophoresis among others. The following are the steps to generate a DNA
fingerprinting.

1. The DNA is extracted from the nuclei of any cell in the body.
2. The DNA molecules are broken with the help of enzyme restriction endonuclease (called
chemical knife) that cuts them into fragments. The fragments of DNA also contain the
VNTRs.
3. The fragments are separated according to size by gel electrophoresis in agarose gel.
4. The separated fragments of single-stranded DNA are transferred onto a nylon membrane.
Radioactive DNA probes having repeated base sequences complementary to possible VNTRs
are poured over the nylon membrane. Some of them will bind to the of single-stranded
VNTRs. The method of hybridization of DNA with probes is called Southern Blotting.
5. The nylon membrane is washed to remove extra probes.
6. An X-ray film is exposed to the nylon membrane to mark the places where the radioactive
DNA probes have bound to the DNA fragments. These places are marked as dark bands when
X-ray film is developed. This is known as autoradiography.
7. The dark bands on X-ray film represent the DNA fingerprints (DNA profiles)

These steps are shown better in figure 1.

Figure 1. Steps in the DNA Fingerprinting (Garcia and Miño, 2012).

CONCLUSION
 Cellular and Molecular Biology

DNA fingerprinting technique has become an important tool for scientific research, because it
allows identifying patterns in the known coding region of genetic material that makes every
individual unique, for that reason, areas as forensic investigations and parentage testing have found
an instrument to convict criminals, identify victims, and solved parentage disputes.

Since Alec Jeffreys developed DNA fingerprinting technique in 1984, the technique has gone
through for many adjustments, from southern blot to PCR methods, from mini satellites to micro
satellites and new markers have been developed according to the needs of research fields for
example in Anthropological genetics, botany, and zoology.

DNA fingerprinting has allowed molecular biology to be developed to lengths we could

not have imagined previously. As this technique is developed further into new applications, we
will only discover more about the human genome and how we may be able to harness that
knowledge to aide humankind. In the years to come, advances in gene therapy and disease
detection are sure to be one of the most researched areas involving DNA analysis and will
hopefully yield breakthrough information. We cannot predict what this research may lead to, or
how many mysteries may be solved, but we can tell from its growth in the past twenty years that
DNA fingerprinting applications in the years to come will offer similarly exciting wisdom.

Reference
1. Alexander, R., Fang, G., Rozowsky, J., Snyder, M., & Gerstein, M. (2010). DNA Finger
printing. Nature Reviews Genetics. Retrieved from
http://www.nature.com/nrg/journal/v11/n8/fg_tab/nrg2814_T2.html
2. Kirby, L. (1993). DNA fingerprinting: an introduction. United Kingdom: Palgrave Macmillan.
3. Jobling, M. (2013). Curiosity in the genes: the DNA fingerprinting story. Investigative
Genetics.
4. Matheson, S. (2016). DNA phenotyping: snapshot of a criminal. Cambridge.
5. Sethi, S., Hazari, P., Inderjeet, & Khare, R. (2016). DNA fingerprinting technology: and
exhaustive review. India: Biochemiae acta.
6. Roewer, L. (2013). DNA fingerprinting in forensics: past, present, future. Investigative
genetics, 4(1), 22.9
7. Mishra, A., Sathyan, S., & Shukla, S. K. (2015). Application of DNA Fingerprinting in an
Alleged Case of Paternity. Biochemistry and Analytical Biochemistry, 4(2), 1.
8. Crawford, M. H., & Beaty, K. G. (2013). DNA fingerprinting in anthropological genetics:
past, present, future. Investigative genetics, 4(1)
9. Garcia D and Miño K. (2017). DNA fingerprinting. Bionatura. 2 ( 4).

4. Differentiate Protein synthesis in prokaryotes and eukaryotes.
Summary of difference between Prokaryotic and Eukaryotic Protein Synthesis (Harvey,
2011)
Sl. Prokaryotes
No.
1 Transcription and translation are
continuous process and occurs
simultaneously in the cytoplasm
2 The primary transcript is processed after
5’ end of mRNA is immediately
available for translation
3 Transcription initiation machinery is
simple since DNA is not associated with
any histone proteins
4 Only one type of RNA polymerase
enzyme, which synthesize all types of
RNA in the cell (mRNA, rRNA, tRNA)
5
Ribosome 70S type
6
70S Ribosome composed of 50S larger
subunit and 30S smaller subunit
7
Larger subunit of ribosome with two
rRNA molecules 5S and 23S rRNA
8
Smaller subunit of ribosome (30S) with
16S rRNA
9
Smaller subunit of ribosome with 21
proteins
10
Larger subunit of ribosome with 36
proteins
11
Ribosome mass 2700 kd
12
Endoplasmic reticulum absent and
hence protein synthesizing ribosome
freely distributed in the cytoplasm
13
mRNA is usually polycystronic
Cellular and Molecular Biology
Eukaryotes
Transcription and translation are separate
process, transcription occurs in the nucleus
whereas translation occurs in the cytoplasm
transcription and then it is transported to the
cytoplasm, then only the cytoplasmic ribosomes
can
initiate translation
Transcription machinery is very complex
since the genetic material is associated with
protein
Three types of RNA polymerase in the cell.
RNA Polymerase I for rRNA synthesis. RNA
polymerase II for mRNA synthesis. RNA
polymerase III for tRNA and 5S rRNA
synthesis
Ribosome 80S type
80S Ribosome composed of 60S larger
subunit and 40S smaller subunit
Larger subunit of ribosome with three
rRNA molecules: 5S, 5.8S and 28S
rRNA
Smaller subunit of ribosome (40S) with 18S
rRNA
Smaller subunit of ribosome with ~ 33 proteins
Larger subunit of ribosome with ~ 49 proteins
Ribosome mass 4200 kd
Endoplasmic reticulum present, protein
synthesizing ribosome usually attached to the ER
mRNA is always monocystronic
 Cellular and Molecular Biology

14
mRNA can acts as the template for mRNA can act as the template for a single
the synthesis of many polypeptide
polypeptides
15
Only one type of translation Two types of translation initiation
initiation mechanism (cap mechanisms. (1) Cap depended and (2) Cap
independent) independent
16
May have many start sites and SD
Always have only one start site which is
sequences (Shine‐Dalgarno sequence)
located towards the 5’ region of mRNA
all along the mRNA
17
SD sequence present 8 nucleotide
SD sequence is absent in mRNA
upstream of start codon. SD sequence act
as the ribosome binding site
18
Kozak sequence present in the mRNA which is
Kozak sequence absent in mRMA
located few nucleotide upstream of start site.
Kozak sequence assists initiation process of
translation
19
Initiation codon is usually AUG, Initiation codon is AUG. occasionally GUG or
occasionally GUG or UUG. CUG.
20
Smaller subunit of ribosome (30S)
Smaller subunit of ribosome (40S) recognize
recognize the SD sequence in the
the 5’ cap of mRNA during initiation
mRNA during translation initiation
21
First tRNA is special type namely Met‐ First tRNA is Met‐tRNA
tRNAf
22
First amino acid in the protein No fomylation of methionine, the first amino acid,
synthesis (methionine) will be will occurs
formylated
23
Seven types of initiation factors are required for
Only three types of initiation factors are
translation, they are eIF1, eIF2, eIF3, eIF4,
required for translation, they are IF1, IF2,
eIF5A, eIF5B, eIF6
IF3
24
Elongation factor are EF – Tu and EF ‐ Elongation factors are eEF1 and eEF2
Ts
25
Speed of translation: ~20 amino Speed of translation ~1 amino acid/second
acids/second
26
Termination is facilitated by three Termination is facilitated by only one release
release factors RF1, RF2, RF3 factor eRF1
27 Only the formyl group from the first
amino acid (methionine) is removed Usually the un‐formylated first methionine as
from the polypeptide after protein such is removed from the polypeptide after
synthesis protein synthesis
28
Life span of mRNA is short, few seconds Life span of mRNA long, few hours to a
to few minutes day or sometimes more

29
IF3 prevents the association of
ribosomal subunits in the absence of
initiation complex
30
Post translational modifications of
proteins takes place in the cytoplasm
Figure 1. Initiation of translation. The separate components are depicted at the left of the figure.
(Klug, & Cummings,2003)
Cellular and Molecular Biology
eIF3 prevents the association of ribosomal
subunits in the absence of initiation complex
Post translational modifications usually takes
place in the endoplasmic reticulum or Golgi
bodies or in the cytoplasm
 Cellular and Molecular Biology

Figure 2. Elongation of the growing polypeptide chain during translation (Klug, &
Cummings,2003)
.
 Cellular and Molecular Biology

Figure 3. Termination of the process of translation (Klug, & Cummings,2003)

Transcription in Eukaryotes Differs from Prokaryotic Transcription in Several Ways

Much of our knowledge of transcription has been derived from studies of prokaryotes. Most of
the general aspects of the mechanics of these processes are similar in eukaryotes, but there are
several notable differences (Klug, & Cummings,2003):
1. Transcription in eukaryotes occurs within the nucleus under the direction of three separate
forms of RNA polymerase. Unlike the prokaryotic process, in eukaryotes the RNA transcript
is not free to associate with ribosomes prior to the completion of transcription. For the mRNA
to be translated, it must move out of the nucleus into the cytoplasm.

2. Initiation of transcription of eukaryotic genes requires the compact chromatin fiber,

characterized by nucleosome coiling, to be uncoiled and the DNA to be made accessible to
RNA polymerase and other regulatory proteins. This transition, referred to as chromatin
remodeling, reflects the dynamics involved in the conformational change that occurs as the
DNA helix is opened.

3. Initiation and regulation of transcription entail a more extensive interaction between cis-
acting DNA sequences and trans-acting protein factors involved in stimulating and initiating
transcription. Eukaryotic RNA polymerases, for example, rely on transcription factors (TFs)
to scan and bind to DNA. In addition to promoters, other control units, called enhancers and
silencers, may be located in the 5’ regulatory region upstream from the initiation point, but
they have also been found within the gene or even in the 3’ downstream region, beyond the
coding sequence.

4. Alteration of the primary RNA transcript to produce mature eukaryotic mRNA involves many
complex stages referred to generally as “processing.” An initial processing step involves the
addition of a 5’ cap and a 3’ tail to most transcripts destined to become mRNAs. The initial
 Cellular and Molecular Biology

(or primary) transcripts are most often much larger than those that are eventually translated
into protein. Sometimes called pre-mRNAs, these primary transcripts are found only in the
nucleus and referred to collectively as heterogeneous nuclear RNA (hnRNA) Alberts,
Johnson, Lewis, Raff, Roberts, Walter, 2002).

Figure 1. Comparison of Ribosomal unit between Prokaryotic and Eukaryotic

Reference
1. Klug, W. S., & Cummings, M. R. (2003). Concepts of genetics. Upper Saddle River, N.J:
Prentice Hall.
2. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002).
Molecular biology of the cell. New York: Garland Science.
3. Harvey, Richard A. (2011). Lippincott's illustrated reviews: Biochemistry. Philadelphia
:Wolters Kluwer Health,
 Cellular and Molecular Biology

5. Cite an article that Discusses posttranslational modifications and its implications.

Definition:

Post-translational modifications (PTM) are modifications that occur on a protein, catalyzed by

enzymes, after its translation by ribosomes is complete. Post-translational modification generally
refers to the addition of a functional group covalently to a protein as in phosphorylation and
neddylation, but also refers to proteolytic processing and folding processes necessary for a protein
to mature functionally (Nature, 2018). PTMs play an important part in modifying the end product
of expression, contribute to biological processes and diseased conditions, playing a key role in
many cellular processes such as cellular differentiation, protein degradation, signaling and
regulatory processes, regulation of gene expression, and protein-protein interactions (Proteintech,
2018).

Title of the Study

Current strategies and findings in clinically relevant post-translational modification-specific

proteomics
 Cellular and Molecular Biology

Figure 1 Frequently reported post-translational protein modifications.

ChaFRADIC: Charge-based fractional diagonal chromatography; COFRADIC; Combined

fractional diagonal chromatography; PTM: Post-translational modification; SCX: Strong cation
exchange chromatography; TAILS: Terminal amine isotopic labeling of substrates.
 Cellular and Molecular Biology

Figure 2. Frequency of human PTMs. Summary of human PTMs which, according to UniProt
and PhosphoSitePlus, have been detected (A) frequently, (B) less frequently and (C) rarely. For
UniProt, the percentage of entries with experimental evidence is given (ECO:0000269). (D) The
high number of known PTMs is in stark contrast to the limited knowledge about their
involvement in disease.PTM: Post-translational modification.
 Cellular and Molecular Biology

Figure 3. Protein copy number distribution in HeLa cells [221] and copy numbers of some
prominent cancer biomarkers. (*indicates a membrane protein). Dashed lines give the limits of
detection when analyzing a certain number of cells, assuming a full quantitative recovery and a
limit of detection of 100 amol. If only 600 cells are available, approximately 20% of the proteome
will be covered.

Summary

Implications in the study of PTM

 LC-mass spectrometry (MS)–based post-translational modification (PTM) research enables

the quantification of hundreds to thousands of PTMs in a single experiment. However, the
link between aberrant PTM patterns and disease or during drug treatment is still poorly
understood and is one of the main goals of LC-MS–based PTM research.
 Typical workflows for analyzing PTMs are conducted on the peptide level, which allows
specific enrichment of modified peptides from the bulk of non-modified peptides, prior to LC-
MS analysis. Thus, the low abundance of PTMs can be overcome in the light of the dynamic
range of mammalian cells.
 State-of-the-art workflows enable quantitative analysis from less than 100 µg of cell lysate.
Whereas in cell culture-based experiments sufficient sample is readily available, clinical
applications with patient samples require highest sensitivity and robustness.
 Cellular and Molecular Biology

 In clinical proteomics, label-free quantification, super-SILAC and chemical labels can be

employed for large-scale quantitative discovery. Modified peptides that may serve as
biomarkers can be validated with larger cohorts using targeted MS methods such as multiple
reaction monitoring (MRM) or parallel reaction monitoring (PRM). These can also be
designed for diagnostic purposes.
 Aberrant protein phosphorylation has been connected to a wide variety of diseases.
Nowadays, various sensitive phosphopeptide enrichment methods such as Ti4+-IMAC, TiO2-
MOAC and electrostatic repulsion-hydrophilic interaction liquid chromatography (ERLIC)
are established. Particularly, ERLIC is a simple and sensitive method for enrichment and
simultaneous fractionation of both singly and multiphosphorylated peptides.
 Glycosylation is an extremely heterogeneous group of PTMs that is characterized by the
attachment of complex carbohydrate structures to proteins. Various cancer biomarkers are
glycoproteins and several studies indicate that both glycosylation levels and glycan structures
are potential biomarkers. Although dedicated enrichment methods are available for
glycopeptides, site-specific analysis of glycosylation structure is challenging. Therefore, most
studies focus on monitoring glycosylation sites rather than resolving glycan structures.
 Proteolytic processing is a ubiquitous, non-reversible PTM. Generated ‘neo’-N-termini can be
enriched using methods such as combined fractional diagonal chromatography (COFRADIC),
terminal amine isotopic labeling of substrates (TAILS) and charge-based fractional diagonal
chromatography (ChaFRADIC). These enable the identification of protease substrates as well
as their distinct cleavage sites. Charge-based fractional diagonal chromatography has been
demonstrated to be a sensitive and straightforward method that might be applicable for
clinical proteomics. The analysis of ‘neo’-C-termini, however, is still more challenging.
 The role of PTM crosstalk is still not well understood. The current limitations in performing
real large-scale PTM crosstalk analyses render the development of adequate clinical
biomarker assays that target peptides with different modifications extremely challenging and
rather unlikely for the near future.

Reference
1.
2. Pagel, O., Loroch, S., Sickmann, A., & Zahedi, R. P. (2015). Current strategies and
findings in clinically relevant post-translational modification-specific proteomics.
Expert Review of Proteomics, 12(3), 235–253.
http://doi.org/10.1586/14789450.2015.1042867
 Cellular and Molecular Biology

6. Cite an article explaining how a gene is regulated?

Mechanism in Gene Regulation

Gene regulation refers to the mechanisms that act to induce or repress the expression of a
gene. These include structural and chemical changes to the genetic material, binding of proteins to
specific DNA elements to regulate transcription, or mechanisms that modulate translation of
mRNA (Nature, 2018).

Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are
repressed or turned off. The process of turning genes on and off is known as gene regulation.
Gene regulation is an important part of normal development. Genes are turned on and off in
different patterns during development to make a brain cell look and act different from a liver cell
or a muscle cell, for example. Gene regulation also allows cells to react quickly to changes in
their environments. Although we know that the regulation of genes is critical for life, this
complex process is not yet fully understood.

Gene regulation can occur at any point during gene expression, but most commonly
occurs at the level of transcription (when the information in a gene’s DNA is transferred to
mRNA). Signals from the environment or from other cells activate proteins called transcription
factors. These proteins bind to regulatory regions of a gene and increase or decrease the level of
transcription. By controlling the level of transcription, this process can determine the amount of
protein product that is made by a gene at any given time (Henetic Home Reference, 2018.

Genes can't control an organism on their own; rather, they must interact with and respond
to the organism's environment. Some genes are constitutive, or always "on," regardless of
environmental conditions. Such genes are among the most important elements of a cell's genome,
and they control the ability of DNA to replicate, express itself, and repair itself. These genes also
control protein synthesis and much of an organism's central metabolism. In contrast, regulated
genes are needed only occasionally — but how do these genes get turned "on" and "off"? What
specific molecules control when they are expressed?
It turns out that the regulation of such genes differs between prokaryotes and eukaryotes.
For prokaryotes, most regulatory proteins are negative and therefore turn genes off. Here, the cells
rely on protein–small molecule binding, in which a ligand or small molecule signals the state of
the cell and whether gene expression is needed. The repressor or activator protein binds near its
regulatory target: the gene. Some regulatory proteins must have a ligand attached to them to be
able to bind, whereas others are unable to bind when attached to a ligand. In prokaryotes, most
regulatory proteins are specific to one gene, although there are a few proteins that act more
widely. For instance, some repressors bind near the start of mRNA production for an entire
operon, or cluster of coregulated genes. Furthermore, some repressors have a fine-tuning system
known as attenuation, which uses mRNA structure to stop both transcription and translation
depending on the concentration of an operon's end-product enzymes. (In eukaryotes, there is no
exact equivalent of attenuation, because transcription occurs in the nucleus and translation occurs
in the cytoplasm, making this sort of coordinated effect impossible.) Yet another layer of
prokaryotic regulation affects the structure of RNA polymerase, which turns on large groups of
genes. Here, the sigma factor of RNA polymerase changes several times to produce heat- and
desiccation-resistant spores. Here, the articles on prokaryotic regulation delve into each of these
topics, leading to primary literature in many cases.
For eukaryotes, cell-cell differences are determined by expression of different sets of
genes. For instance, an undifferentiated fertilized egg looks and acts quite different from a skin
cell, a neuron, or a muscle cell because of differences in the genes each cell expresses. A cancer
cell acts different from a normal cell for the same reason: It expresses different genes. (Using
microarray analysis, scientists can use such differences to assist in diagnosis and selection of
 Cellular and Molecular Biology

appropriate cancer treatment.) Interestingly, in eukaryotes, the default state of gene expression is
"off" rather than "on," as in prokaryotes. Why is this the case? The secret lies in chromatin, or the
complex of DNA and histone proteins found within the cellular nucleus. The histones are among
the most evolutionarily conserved proteins known; they are vital for the well-being of eukaryotes
and brook little change. When a specific gene is tightly bound with histone, that gene is "off." But
how, then, do eukaryotic genes manage to escape this silencing? This is where the histone code
comes into play. This code includes modifications of the histones' positively charged amino acids
to create some domains in which DNA is more open and others in which it is very tightly bound
up. DNA methylation is one mechanism that appears to be coordinated with histone
modifications, particularly those that lead to silencing of gene expression. Small noncoding RNAs
such as RNAi can also be involved in the regulatory processes that form "silent" chromatin. On
the other hand, when the tails of histone molecules are acetylated at specific locations, these
molecules have less interaction with DNA, thereby leaving it more open. The regulation of the
opening of such domains is a hot topic in research. For instance, researchers now know that
complexes of proteins called chromatin remodeling complexes use ATP to repackage DNA in
more open configurations. Scientists have also determined that it is possible for cells to maintain
the same histone code and DNA methylation patterns through many cell divisions. This
persistence without reliance on base pairing is called epigenetics, and there is abundant evidence
that epigenetic changes cause many human diseases.
For transcription to occur, the area around a prospective transcription zone needs to be
unwound. This is a complex process requiring the coordination of histone modifications,
transcription factor binding and other chromatin remodeling activities. Once the DNA is open,
specific DNA sequences are then accessible for specific proteins to bind. Many of these proteins
are activators, while others are repressors; in eukaryotes, all such proteins are often called
transcription factors (TFs). Each TF has a specific DNA binding domain that recognizes a 6-10
base-pair motif in the DNA, as well as an effector domain. In the test tube, scientists can find a
footprint of a TF if that protein binds to its matching motif in a piece of DNA. They can also see
whether TF binding slows the migration of DNA in gel electrophoresis.
For an activating TF, the effector domain recruits RNA polymerase II, the eukaryotic
mRNA-producing polymerase, to begin transcription of the corresponding gene. Some activating
TFs even turn on multiple genes at once. All TFs bind at the promoters just upstream of
eukaryotic genes, similar to bacterial regulatory proteins. However, they also bind at regions
called enhancers, which can be oriented forward or backwards and located upstream or
downstream or even in the introns of a gene, and still activate gene expression. Because many
genes are coregulated, studying gene expression across the whole genome via microarrays or
massively parallel sequencing allows investigators to see which groups of genes are coregulated
during differentiation, cancer, and other states and processes.
Most eukaryotes also make use of small noncoding RNAs to regulate gene expression. For
example, the enzyme Dicer finds double-stranded regions of RNA and cuts out short pieces that
can serve in a regulatory role. Argonaute is another enzyme that is important in regulation of
small noncoding RNA–dependent systems. Here we offfer an introductory article on these RNAs,
but more content is needed; please contact the editors if you are interested in contributing.
Imprinting is yet another process involved in eukaryotic gene regulation; this process
involves the silencing of one of the two alleles of a gene for a cell's entire life span. Imprinting
affects a minority of genes, but several important growth regulators are included. For some genes,
the maternal copy is always silenced, while for different genes, the paternal copy is always
silenced. The epigenetic marks placed on these genes during egg or sperm formation are faithfully
copied into each subsequent cell, thereby affecting these genes throughout the life of the
organism.
 Cellular and Molecular Biology

Still another mechanism that causes some genes to be silenced for an organism's entire
lifetime is X inactivation. In female mammals, for instance, one of the two copies of the X
chromosome is shut off and compacted greatly. This shutoff process requires transcription, the
participation of two noncoding RNAs (one of which coats the inactive X chromosome), and the
participation of a DNA-binding protein called CTCF. As the possible role of regulatory
noncoding RNAs in this process is investigated, more information regarding X inactivation will
no doubt be discovered.

Article Pertaining to Gene Regulation

Title: Selection to minimize noise in living systems and its implications for the evolution of gene
expression.
Result of the Study: Gene expression, like many biological processes, is subject to noise. This
noise has been measured on a global scale, but its general importance to the fitness of an
organism is unclear. Here, I show that noise in gene expression in yeast has evolved to prevent
harmful stochastic variation in the levels of genes that reduce fitness when their expression levels
change. Therefore, there has probably been widespread selection to minimize noise in gene
expression. Selection to minimize noise, because it results in gene expression that is stable to
stochastic variation in cellular components, may also constrain the ability of gene expression to
respond to non-stochastic variation. I present evidence that this has indeed been the case in yeast.
I therefore conclude that gene expression noise is an important biological trait, and one that
probably limits the evolvability of complex living systems
Article Reference:
 Lehner B. Selection to minimise noise in living systems and its implications for the evolution
of gene expression. Mol Syst Biol. 2008;4:170. doi:10.1038/msb.2008.11. Epub 2008 Mar 4.
PubMed PMID: 18319722; PubMed Central PMCID: PMC2290932.

Title: Directed evolution of promoters and tandem gene arrays for customizing RNA synthesis
rates and regulation.
Result of the Study:
Manipulating RNA synthesis rates is a primary method the cell uses to adjust its physiological
state. Therefore to design synthetic genetic networks and circuits, precise control of RNA
synthesis rates is of the utmost importance. Often, however, a native promoter does not exist that
has the precise characteristics required for a given application. Here, we describe two methods to
change the rates and regulation of RNA synthesis in cells to create RNA synthesis of a desired
specification. First, error-prone PCR is discussed for diversifying the properties of native
promoters, that is, changing the rate of synthesis in constitutive promoters and the induction
properties for an inducible promoter. Specifically, we describe techniques for generating
diversified promoter libraries of the constitutive promoters P(L)tetO-1 in Escherichia coli and
TEF1 in Saccharomyces cerevisiae as well as the inducible, oxygen-repressed promoter DAN1 in
S. cerevisiae. Beyond generating promoter libraries, we discuss techniques to quantify the
parameters of each new promoter. Promoter characteristics for each promoter in hand, the
designer can then pick and choose the promoters needed for the specific genetic circuit described
in silico. Second, Chemically Induced Chromosomal Evolution (CIChE) is presented as an
alternative method to finely adjust RNA synthesis rates in E. coli by variation of gene cassette
copy numbers in tandem gene arrays. Both techniques result in precisely defined RNA synthesis
and should be of great utility in synthetic biology.
Article Reference: Tyo KE, Nevoigt E, Stephanopoulos G. Directed evolution of promoters and
 Cellular and Molecular Biology

tandem gene arrays for customizing RNA synthesis rates and regulation. Methods Enzymol.
2011;497:135-55. doi: 10.1016/B978-0-12-385075-1.00006-8. PubMed PMID:21601085.

Title: Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic
hybrid promoters.

Result of the Study: A dynamic range of well-controlled constitutive and tunable promoters are
essential for metabolic engineering and synthetic biology applications in all host organisms. Here,
we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the
model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two
modular components-the enhancer element, consisting of tandem repeats or combinations of
upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility
of this approach with three main case studies. First, we establish a dynamic range of constitutive
promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast
promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to
impart synthetic regulation through a hybrid promoter approach by adding galactose activation
and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid
promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and
increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that
promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid
promoter approach can expand, enhance, and control promoter activity.
Article Reference:
Blazeck J, Garg R, Reed B, Alper HS. Controlling promoter strength and regulation in
Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng. 2012
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