MLS 047: Molecular Biology and Diagnostics

Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

Lesson Title: Plasmids, Transcription Regulation and Materials:

Epigenetics
Lesson Objectives:
At the end of this lesson, I can:
1. Describe plasmids and its general types.
2. Discuss the factors responsible for the regulation of
RNA synthesis.
3. Discuss the types of gene regulation.
Student Activity Sheets
References:
Buckingham, L. and Flaws, M. (2013).
Molecular Diagnostics: Fundamentals,
Methods, & Clinical Applications (2nd ed.).
Philadelphia: F.A. Davis Company
McPherson and Pincus. (2012). HENRY’s
Clinical Diagnosis and Management by
Laboratory Methods (22nd ed.). Singapore:
Elsevier Pte. Ltd.
Rifai, Nader et al. (2018). Principles and
Applications of Molecular Diagnostics.
Elsevier Inc., Netherlands
Weaver, R.F. (2012). Molecular Biology (5th
ed.). McGraw-Hill Companies, Inc., New
York

Productivity Tip:
“Keep going! Start your day with a smile and grit. Properly arrange your study materials on your table and
always stick to your working hours. Strive harder to achieve your goals today!

A. LESSON PREVIEW/REVIEW
Introduction (2 mins)

Welcome to our new lesson today about plasmids, regulation of transcription and epigenetics! Though
microorganisms such as bacteria and fungi have their own chromosomes, they may also possess an
extrachromosomal DNAs which are called plasmids. They are very significant in various biomedical researches
since they would introduce specific traits such as antibiotic resistance to the microorganism via recombinant
DNA technology. A systematic regulation of transcription is essential to produce a viable and functional protein
for certain cellular responses. Further, modification of gene expression can be demonstrated by epigenetic
regulations. Analysis of these modifications is a substantial target for diagnostic, prognostic and therapeutic
applications.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

Activity 1: What I Know Chart, PART 1 (3 mins)

Instruction: Please write under the WHAT I KNOW column the things that you already knew about the
questions for today’s topic. Please ONLY answer the “What I Know” column and wait until you finish answering all
the activities before you answer the “What I Learned” column.

What I Know Questions: What I Learned (Activity 4)

1. What is the importance of a

plasmid?

2. What is regulation of
transcription?

3. What is histone modification in

epigenetics?
B. MAIN LESSON

Activity 2: Content Notes (13 mins)

I. Plasmids
- are extrachromosomal, small, circular, double stranded DNA molecules.
- replicate independently of the chromosome.
- also carry a limited amount of genetic information.
- are packaged by supercoiling.

Figure 4.1 Plasmids. Plasmids are small extrachromosomal DNA duplexes that can carry genetic information.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

- are not essential for growth but may confer a selective advantage such as antibacterial resistance (up to
7 different resistance genes on one plasmid). Plasmids with antibiotic-resistance genes are called
resistance transfer factors (RTF) or R factors while those carrying resistance to bacteriocins, toxin
proteins produced by bacteria, are called colicinogenic factors.
- are transferred from cell to cell by conjugation. Efficient vehicle for transferring genes from one cell to
another, thus producing new recombinant organisms.

A. General Types of Plasmids

1. Large plasmids
- are usually conjugative.
- occur in small numbers such as one or two copies per chromosome.
2. Small plasmids
- are non-conjugative but can also carry antibiotic-resistance genes.
- they can also be mobilized by a large conjugative plasmid.
- may occur in the cell as 20 copiers per chromosome.
II. Transcription Regulation
- Regulation of transcription especially in initiation process is crucial in the production of a functional
proteins, since a huge amount of information in gene expression is coming from this stage of
transcription. There should be an assembly of proteins such as RNA polymerase complex and general or
specific transcription factors at the gene’s transcription initiation site.
- Prokaryotic regulatory sequences are found near the gene, however both proximal and distal regulatory
elements are found in the eukaryotic genes.
- Enhancers and silencers are examples of distal regulatory elements that respectively stimulate or
dampen expression of distant genes.

Figure 4.2 Proximal and distal regulatory

elements. Proximal regulatory elements in
prokaryotes are closed to the structural genes
while in eukaryotes, distal elements are located
far from the genes they control and proximal
elements can be located in or around the genes
they control. Promoters, rich in A-T sequences,
are important for the initiation of mRNA
transcription.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

A. Two Types of Factors Responsible for RNA Synthesis Regulation

1. Cis factors
- DNA sequences that mark places on the DNA involved in the initiation and control of
RNA synthesis.
2. Trans factors
- Proteins that bind to the cis sequences and direct the assembly of transcription
complexes at the proper gene.
- Also bind to enhancers and promoters and selectively stimulate or inhibit mRNA
transcription.

Figure 4.3 Cis factor and Trans factor. Cis elements or

factors (top) are DNA sequences recognized by transcription
factors while the transcription factors or trans factors are the
DNA-binding proteins that would bind to the cis sequences.

A. Operon
- A series of structural genes transcribed together on one mRNA and subsequently separated
into individual proteins.
- It brings about coordinate expression of proteins required at the same time among
microorganisms with small genomes.
- Modes of regulation in prokaryotes include induction as found in the lac operon, repression
as found in the arg operon (negative control), and activation as in mal operon (positive
control).
Ex. Lactose Operon
- contains three structural genes: LacZ, LacY, and LacA, which are all required for lactose
metabolism. The regulator or repressor gene in lac operon codes for the repressor
protein trans factor that binds to the operator.
- β -galactosidase is a LacZ gene product that hydrolyzes lactose into glucose and
galactose.
- Lactose permease is the LacY gene product that transports lactose in the cell.
- Thiogalactosidase transacetylase is the LacA gene product that transacetylates
galactosides.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

- LacI gene encodes a protein which acts as a repressor in the gene expression of the
operon.
Note: In the absence of lactose, the repressor binds to the operator sequence, upstream
of the lacZ gene, and prevents operon transcription. On the other hand, if lactose is
present, it binds to the repressor and causes conformational change of the protein thus
lowered its affinity to the operator sequence.

III. Epigenetics
- This focuses on the regulation of gene expression by nonheritable and heritable modifications, and not
dependent on DNA sequence.
- This commonly involves in chromatin change which subsequently facilitates or blocks gene
transcription.
- Epigenetic changes are recognized to be significant in tissue-specific gene transcription, and alterations
may be factors in cancers, aging, and stress response.
A. Three Major Areas of Epigenetic Modifications
1. Histone Modification
- a mechanism that controls chromatin conformation. Chromatin is a nuclear DNA that is
compacted onto nucleosomes (DNA wrapped around with eight histone proteins).
- Chromatin proteins may be acetylated, deacytylated or methylated.
- affects the activity of chromatin-associated protein and transcription factors that increase
or decrease gene expression.
- Specific histones are reversibly and posttranslationally modified at their N-terminal tails
and globular regions to change the chromatin from a euchromatin state to a
heterochromatin state.
1.1 Euchromatin has more acetylated histones and less methylated histones thereby
making it transcriptionally active
1.2 Heterochromatin is transcriptionally silent and made up of more condensed
nucleosome fibers.

2. DNA Methylation
- an epigenetic change that is important in X chromosome inactivation, gene imprinting
(gamete-specific silencing of genes) such as Prader-Willi and Angelman Syndrome, and
cancer.
- Methylation of cytosine to form 5-methylctosine is the most common methylation event.
- occurs in cytosine-guanine (CpG)-rich sequence s in the DNA, specifically found in the first
exons, promoter regions and also in the 3’ end of genes.
- decreases the affinity of transcription factors to a DNA promoter and enhances the
binding of methylation-specific transcription factors.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

- Methylation of cytosine residues in the promoter regions of tumor suppressor genes is a

mechanism of inactivation of these genes in cancer.
- Imprinting maintains the balanced expression of genes in growth and embryonic
development by selective methylation of homologous genes.

Figure 4.4 Epigenetics. DNA

methylation of CpG island regions
indicated by Me in and around gene
promoters is associated with loss of
gene expression and silencing of the
gene. When CpG islands are
unmethylated, shown by absence of
Me, gene expression is unaffected.
Modifications of the tails of histone
proteins such as methylation,
acetylation, and phosphorylation,
shown as Me, Ac, and P, respectively,
can increase gene expression.
3. Noncoding RNAs
- Short noncoding RNAs or small RNAs consist of microRNAs, small interfering RNAs
and piwi interacting RNAs. These small RNAs could function as posttranscriptional
regulators of gene expression.
- Specific miRNAs were found abundantly in certain tumors while siRNAs are
recognized as a new RNA-based immune system against viruses.
a. MicroRNAs or miRNAs- regulate gene expression by binding to a specific
sequence of the mRNA and inhibiting its protein translation at the
ribosome.
b. Small interfering RNAs or siRNAs- inhibit translation by initiating
degradation of the targeted mRNA or short term silencing of protein-
coding genes.
c. Piwi interacting RNAs or piRNAs- function in the repression of
transposons and are important in the development of gametes in many
multicellular eukaryotic species.

Activity 3: Skill-building Activities (18 mins + 2 mins checking)

Instruction: Illustrate the general structure and two states of the lac operon.

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 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

Wow, that’s AWESOME!

Note: Please check your answers against the Key to Corrections found at the end of this SAS. Write your score on
your paper.

Activity 4: What I Know Chart, PART 2 (2 mins)

Instruction: Please go back to Activity 1 “What I Know Chart” and finish answering the third column “What I
Learned. Do this in 2 minutes. Are there any changes in your answer to the given questions? If there are, then
congratulations for you have learned something new today.

Activity 5: Check for Understanding (5 mins)

Instruction: Write your best answer before each item.

________________1. It is a noncoding RNA that inhibits protein translation and abundant in certain tumor-specific
genes.
________________2. This commonly involves genetic control by factors other than an individual’s DNA sequence.
________________3. It is a gene which encodes protein that represses the gene expression of lac operon.
________________4. This affects the activity of chromatin-associated protein and transcription factors which
increases or decreases gene expression.

This document is the property of PHINMA EDUCATION

 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

______________5. A type of plasmid that is non-conjugative but can carry antibiotic-resistance genes.
______________6. These are proteins that might displace histones from nucleosomes directly and bind to the cis
sequences for the regulation of gene expression.
______________7. These are small, extrachromosomal DNA molecules that is distinct from a cell’s chromosomal
DNA and with genetic advantages such as carrying antibiotic-resistance genes.
______________8. A type of chromatin that contains DNA segments which are transcriptionally active and has
more acetylated histones than methylated ones.
______________9. This is a cluster of genes that shares the same promoter and transcribed together to give a
single mRNA molecule.
_____________10. These are distal regulatory DNA sequence that can strongly stimulate expression of distant
genes.

B. LESSON WRAP-UP

Activity 6: Thinking about Learning (5 mins)

A. Work Tracker

Wow! You are almost done with this session!

Let’s track your progress. Please shade the session number you just completed.

Period 1 Period 2 Period 3

1 2 3 4 5 P1 Exam 6 7 8 9 P2 Exam 10 11 12 13 P3 Exam

Congratulations!

B. Think about your Learning

Can we talk? Please tell me something about today’s lesson. Write your answer on the opposite box.

1. Tell me at least 3 things you’ve learned from

today’s lesson.
2. What are those 2 things in the lesson you found
interesting?
3. Ask me 1 thing that made you curious about the
lesson but we were not able to discuss.

This document is the property of PHINMA EDUCATION

 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

FAQs
1. What are the similarities and differences between siRNAs and miRNAs?
Both small interfering RNAs (siRNAs) and microRNAs (miRNAs) are noncoding RNA molecules and proteomic
tools substantially used for the regulation of gene expression. These both types of RNA molecules undergo
similar methods of gene silencing effects at the post-transcriptional level through a process called RNA
interference (RNAi), nevertheless their cellular mechanisms of action and clinical applications are different from
each other.
Further, the siRNAs which were processed by Dicer by transcription from dsRNA and having 21-23 nucleotides
with two nucleotide overhangs at the 3’ end would activate the RNA-induced silencing complex (RISC), and
then eventually the guide strand of each siRNA would bind to single target mRNA with full complementarity
resulting to specific gene silencing through endonucleolytic cleavage. On the other hand, the miRNAs were
generated from pre-miRNA and processed by Dicer, and subsequently, the guide strand of each miRNA
through partial complementarity binding would target the 3’ untranslated region of target mRNA.

2. Discuss the nucleosome structure of chromatin. What happens to the nucleosomes during remodeling?
The eukaryotic genome is organized by nucleosome which is then apparently the continuous basic structural
unit of chromatin- a DNA-protein complex that forms chromosomes. The nucleosome structure of chromatin
makes the DNA to be tightly coiled in eukaryotic nucleus, but chromatin remodeling would modify histones in
nucleosomes with the aid of energy from ATP hydrolysis to expose the regions of DNA to transcription factors
and RNA polymerases. The constitutive effects of these processes would control gene expression such as
specifically turning on and off the genes. Nevertheless, there still have been researches nowadays aim to
thoroughly investigate the complex mechanisms of nucleosomes especially on how they work systematically in
contribution to the variable patterns of eukaryotic gene expression.

KEY TO CORRECTIONS

Activity 3.

General Structure of the Lac Operon

Two States of Lac Operon

This document is the property of PHINMA EDUCATION

 MLS 047: Molecular Biology and Diagnostics
Student Activity Sheet Module #4

Name: _________________________________________________________________ Class number: _______

Section: ____________ Schedule: ________________________________________ Date: ________________

This document is the property of PHINMA EDUCATION