Subject: Microbiology Second Semester A.Y.

2013-2014
Topic: 1.04 – Basic Concepts 2: Microbial Genetics
Lecturer: Dr. Eric Constantine G. Valera
Date: 30 June 2014

OUTLINE KEEP THIS IN MIND

I. Introduction V. Mechanism of Recombination Recall the distinction between eukaryotes and prokaryotes. One key
A. Definition of Terms VI. Mechanism of Gene Transfer distinction is that prokaryotes, unlike eukaryotes, do not have
B. Organization of genes A. Conjugation
membrane bound organelles such as a nucleus and mitochondria. All
II. Prokaryotic Chromosomes B. Transduction
A. Replicons C. Transformation
their contents are simply found inside the cell membrane, instead of
B. Pathogenicity island D. Restrictions on Gene Transfer being segregated into distinct compartments. Generally, bacteria are
C. Plasmids VII.Bacterial Mutations prokaryotic. This would imply that they do not have a distinct
D. Transposons A. Spontaneous Mutation nucleus. Bacteria and Archaea are classified as Prokaryotes.
E. Bacteriophages B. Reversion
III. Bacterial Gene Expression C. Suppression B. Organization of Genes
A. Replication VIII. Genetic Engineering
B. Transcription A. Cloning
C. Translation B. Steps in cloning Table 1. Three basic differences between DNA and RNA:
D. Summary of Gene Expression IX. DNA Characterization Parameters DNA RNA
IV. Gene Regulation Mechanisms A. Restriction Mapping Single-stranded
A. Activation B. Sequencing (However, RNA can, by
B. Attenuation C. Hybridization Number of Double-stranded
complementary base pairing,
C. Repression strands coiled in a helix
form intra-strand double
helixes, as seen in tRNA.)
OBJECTIVES Deoxyribose Ribose
At the end of the lecture, the student should be able to: Pentose- (no hydroxyl group (With hydroxyl groups that
1. Summarize basic genetic concepts on DNA and RNA phosphate attached to the make RNA less stable and
2. Explain concepts on bacterial gene recombination, transfer and backbone pentose ring in more prone to hydrolysis)
mutation the 2’ position)
3. Discuss concepts of bacterial genetic engineering Complementary base
4. Describe tools involved in DNA characterization Complementary
to adenine (A) is uracil (U),
base to adenine
Base pairs which is
References: (A) is thymine (T)
an unmethylated form of
 Jawetz, Melnick & Adelberg’s Medical Microbiology, 26th Edition thymine
 Dr. Valera’s powerpoint, audio recording and video links Cytosine (C) Cytosine (C)
Base pairs Guanine (G) Guanine (G)
Legend: present Adenine (A) Adenine (A)
 Links inside the double-lined boxes are the videos from Dr. Thymine (T) Uracil (U)
Valera. Some links are different from those provided, but the
videos are the same (we just used shorter URLs). Please take II. PROKARYOTIC CHROMOSOMES
time to watch!
 Bold – important terms REVIEW
 Italicized – important concepts / as stated by lecturer / from Mitosis: Type of cell division which results in two identical
Jawetz / from the videos daughter cells each having the same number and kind of
chromosomes as the parent cell. It is typical of ordinary
I. INTRODUCTION tissue growth.
A. Definition of Terms
 Genetics – study of genes, heredity, and variation in living Meiosis: Type of cell division which results in four daughter
organisms cells each with half the number of chromosomes of the
 Chromosome – threadlike structure of proteins and compacted parent cell, as in the production of gametes and plant
nucleic acids which carry genetic information in the form of genes spores. Note that mitosis has copies of both pairs of parent
o Histones – proteins that package and organize DNA into chromosomes.
structural units called nucleosomes. They make the DNA more
compact, allowing it to fit into the nucleus of the cell. Note Binary Fission: Method of asexual reproduction. It is the
however that these are usually for eukaryotes. In prokaryotes, most common form of reproduction in prokaryotes and
the analogue would be Bacterial DNA binding protein, which occurs in some single-celled eukaryotes. After replicating
are histone-like proteins (not discussed by sir). its genetic material, the cell divides into two identical
 Genome – total genetic content contained in a haploid set of daughter cells.
chromosomes in eukaryotes, in a single chromosome in bacteria,
or in the DNA or RNA of viruses.
o please note that the word “genome” refers to the whole set of
genes of an organism regardless of being haploid or diploid.
 Genes – distinct sequence of nucleotides forming part of a
chromosome that codes for a cellular function, protein, or process.

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 1 of 11
Edited By: David Whang
Characteristics of the prokaryotic genome:
 Most prokaryotic genes are carried on the bacterial chromosome Table 2. Examples of Metabolic Activities determined by Plasmids
 Bacterial genes are haploid (with only few exceptions)
 Most prokaryotic genomes (90%) consist of single circular DNA
molecule containing from 580 kbp to more than 5220 kbp of DNA
 Few bacteria have genomes consisting of two circular DNA
molecules
 Many bacteria contain additional genes on plasmids that range in
size from several to 100 kbp
o Plasmids – small DNA molecule physically separate from, and
can replicate independently of chromosomal DNA. They
usually contain genes that confer antibiotic resistance or
virulence factors. They are most commonly double stranded.

Figure 1: A bacterial cell showing the distinction of a bacterial DNA
(red) and a plasmid (blue).
A. Replicons
 Covalently closed DNA circles (such as the entire bacterial
chromosome and plasmids), which contain genetic information
necessary for their own replication
B. Pathogenicity Island
 Specific genes for pathogenic determinants that are often
clustered together in the DNA
 Genomic island that converts a "harmless" bacterium to a
pathogen when they are integrated into the bacteria’s genome.
 Distinct region of DNA which is present in pathogenic bacteria BUT
absent in nonpathogenic strains of the same species
 Reason why some bacterial species and subspecies are efficient at
causing disease in higher organisms compared to others from the
same genus.
Characteristics of pathogenicity islands:
 These gene segments can be quite large (up to 200 kbp) and
encode a collection of virulence genes
 Have a different G+C content from the rest of the genome
 Closely linked on the chromosome to tRNA genes
 Flanked by direct repeats
 Contain diverse genes important for pathogenesis, including,
antibiotic resistance, adhesins, invasins, and exotoxins, as well as
genes that can be involved in genetic mobilization
D. Transposons
 Segments of DNA that include genes that can migrate from one
locus to another
 Usually enters the cell by being carried on a plasmid
 Can create insertion mutations
 Do not carry genetic information required to couple their own
replication to cell division
 Propagation depends on their physical integration with a bacterial
replicon
 Insert in random pattern but favour regions encoding tRNAs
 Insertion elements or Insertion sequence (IS) elements:
o Short transposons (0.75-2 kbp long)
o Produce majority of insertion mutations
o Carry only the genes for enzymes needed to promote their own
transposition to another genetic locus
o Cannot replicate on their own
 Movements of transposons:
o From plasmid to host/bacterial genome
o From one site to another site on the host genome (transposons
replicate, leaving a copy in the original position)
o From the host genome to a plasmid
Transposons: Shifting Segments of the Genome
http://highered.mheducation.com/sites/9834092339/student_view
0/chapter24/transposons__shifting_segments_of_the_genome.ht
ml

C. Plasmids
 In contrast to “housekeeping genes” (genes essential for bacterial  Two methods of transposition:
growth), plasmids carry genes with independent evolutionary o Cut-and-Paste Mechanism – no copy of the transposon is left
origins and which are associated with specialized functions: in the original position
o Mediate own transfer from one organism to another o Copy-and-Paste Mechanism – a copy of the transposon is
o Genetic acquisition retained in the original position
o DNA rearrangement
o Confer antibiotic resistance and virulence factors
 A consequence of such functions has been observed in the
swift spread among bacterial populations of plasmid-borne
resistance to antibiotics after their liberal use in hospitals

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 2 of 11
Edited by: David Whang
 Figure 3. Two methods of transposition

E. Bacteriophages
 Viruses associated with prokaryotes. They are viruses that infect
and replicate within bacteria Figure 5. Lytic cycle versus Lysogenic Cycle. Note that in the lytic cycle
 Constitute the largest of all viral groups (left), the viruses replicate first within the cell it initially infected and
 Nucleic acid molecules: then lyses the cell. On the other hand, the lysogenic cycle (right), the
o Surrounded by a protein coat viral DNA (green) is incorporated onto the host DNA and replication
o dsDNA (most often), ssDNA, dsRNA and ssRNA of the host cell produces daughter cells with the viral DNA. It can
o Sometimes contain unusual bases such as then switch to the lytic cycle as seen.
hydroxymethylcytosine
 Exhibit a wide variety of morphologies III. BACTERIAL GENE EXPRESSION
 Usually contain specialized syringe-like structures (tails) that A. Replication
bind to receptors on the cell surface and inject the phage nucleic
acid into a host cell
Bacterial DNA Synthesis
 When the phage is loaded with nucleic acid, it takes on a different
form than when the nucleic acid is absent https://highered.mcgraw-
hill.com/sites/dl/free/0072835125/126997/animation17.html

http://highered.mcgraw-
hill.com/sites/0072552980/student_view0/chapter9/animations.ht
ml#

 Semi-conservative & bi-directional starting at ori (origin) locus (at

replication fork) and ending at ter (termination) locus
 Origin – contains an AT-rich region adjacent to sequences that are
recognized by the DNA-binding protein, DNAa.
Figure 4. Bacteriophage structure

 Types of propagation:
 Lytic cycle
 Phages produce many copies of themselves inside host cell
 After much replication, the cell lyses (hence called lytic
cycle) and releases all the viruses that replicated within
 The newly produced viruses can now infect other cells and
repeat the process Figure 6. DNAa and AT-rich region at the Origin
 Lysogenic cycle
 DNA material of bacteriophages incorporate with the  The binding of the DnaA protein to DNA boxes initiates a process
bacterial (host) chromosome, forming a prophage that leads to an opening of the AT-rich region
 Replication of the host chromosome also replicates the  The replication bubble in the DNA molecule is opened further by
DNA of the virus helicase, and the replication forks at each end of the bubble are
 No damage to host cells but imposes higher risk of harming expanded
the immune system unknowingly. After a certain number of  Single-strand binding proteins (SSBPs) then attach to each single
replications, it can change to the lytic cycle stranded DNA molecule to stabilize the open bubble and

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 3 of 11
Edited by: David Whang
 subsequently topoisomerases bind to relieve the supercoiling of
the double strand DNA created by the unwinding of the strands
 DNA primase – synthesize short RNA primers
 Single primers are synthesized in the origin to make the two
leading strands
 DNA Polymerase III – extends each primer by synthesizing a
daughter strand of the DNA in 5’-3’ direction as it moves towards
the replication fork
Remember!
DNA polymerase III reads the template strand in a 3’-5’ direction and
ADDS NEW NUCLEOTIDES in a 5’-3’ direction. In the exam, please
take note of this especially during the exams.
Figure 7: A replication fork.
(A) Notice that the LEADING STRAND (top left) being synthesized
continuously. The template strand (blue) is READ in a 3’-5’ direction,
and the new DNA nucleotides (red) are synthesized in a 5’-3’
direction TOWARDS the replication fork.
(B) Notice first that the LAGGING STRAND’S (bottom left) template
strand (blue) is in a 5’-3’ direction, meaning that the complementary
nucleotides have to be added in a 3’-5’ direction. Remember that
DNA Pol III can ONLY READ in a 3’-5’ direction, and ADD in a 5’-3’
direction. To remedy this, RNA primers first add SHORT DNA
SEQUENCES (red, notice there are two in the figure) IN A 5’-3’
fashion, AWAY from the replication fork (towards the left side of THIS
PAGE). Since this is now in the 5’-3’ direction, DNA pol III can simply
continue adding to the free 3’ end AWAY from the replication fork. It
will meet with the previous Okazaki fragment, (notice there are two
short Okazaki fragments) and hence the synthesis in the lagging
strand is said to be DISCONTINUOUS.
 Each lagging strand is made away from the replication fork
 DNA polymerase I – extends the primers by synthesizing a short
segment of the daughter strand called an Okazaki fragment (5’-3’
direction) until it reaches the previous RNA primase from the
previously made Okazaki fragment
B. Transcription
Bacterial mRNA Synthesis
https://highered.mcgraw-
hill.com/sites/dl/free/0072835125/126997/animation21.html
 Process by which the information in a strand of DNA is copied into
a new molecule of messenger RNA (mRNA)
Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in
Edited by: David Whang
 Operon – a group of adjacent genes controlled by a set of proteins
interacting with specific sequences in DNA molecule; leads to
initiation of transcription or termination of transcript
 Transcription – initiated just beyond the promoter region;
terminated at the termination site
o Promoter region – a short sequence on the DNA that
PROMOTES binding of proteins required for initiation. This is
NOT where initiation starts
Figure 7. Promoter and Termination Sites of the DNA
 The sequence of the promoter at positions 35 and 10 nucleotides
upstream from the transcription start site are critical to initiating
transcription.
 Core RNA polymerase and sigma factor = form a holoenzyme
 This holoenzyme binds to the promoter region of the DNA
molecule forming a closed complex between RNA polymerase and
DNA molecule
 RNA polymerase forms a single polyribonucleotide strand, the
mRNA, using DNA as a template
 The mRNA has a nucleotide sequence complementary to a
template strand in the DNA double helix if read in the 3′–5′
direction. Thus, an mRNA is oriented in a 5′–3′ direction
C. Translation
Bacterial Protein Synthesis
http://highered.mheducation.com/sites/0072552980/student_view
0/chapter9/animations.html#
(I highly encourage you to watch this video) 
 Process by which cellular ribosomes create proteins
 Amino acids are enzymatically activated and transferred to specific
adapter molecules of RNA, called transfer RNA (tRNA). Each
adapter molecule has a triplet of bases (anticodon) complementary
to a triplet of bases on mRNA, and at one end its specific amino
acid. The triplet of bases on mRNA is called the codon specific for
that amino acid
 mRNA and tRNA come together on the surface of the
ribosome. As each tRNA finds its complementary nucleotide triplet
on mRNA, the amino acid that it carries is put into peptide linkage
with the amino acid of the preceding tRNA molecule
 The enzyme peptidyltransferase (which is actually the 23S rRNA)
catalyzes the formation of the peptide bond. The ribosome moves
along the mRNA, the polypeptide growing sequentially until the
entire mRNA molecule has been translated into a corresponding
sequence of amino acids
Page 4 of 11
D. Summary
Bacterial Gene Expression
https://highered.mcgraw-
hill.com/sites/dl/free/0072835125/126997/animation1.html
 The sequence of the nucleotide bases in DNA carried genetic
information in units that are called genes
 Structural genes encode the information for specific proteins
 To create a protein, a gene must first be transcribed into a
sequence of nucleotide bases in an mRNA molecule
 The mRNA sequence is translated into an amino acid sequence of
protein
 The sequence of amino acids in a protein molecule determines the
shape and characteristics of a protein. Thus, each structural gene
specifies a specific protein that carries out a specific function based
on its chemical characteristics and molecular shape
IV. GENE REGULATION MECHANISMS
A. Activation
Combination of Switches: The lac Operon
http://www.mheducation.ca/school/applets/abbio/ch18/lacoperon
_combination_o.swf
1. The lac operon
REMEMBER
An operon is a group of genes that is under the control
of a single operator site.
 Genes associated with fermentation of lactose (lac) in E. coli
 Has a Coding region, which contains three structural genes
coding for three proteins necessary for the use of lactose as a
food source
 lacZ gene – encodes β-galactosidase, the enzyme that
hydrolyzes lactose to galactose and glucose
 lacY gene – encodes permease
 lacA gene – encodes transacetylase
 These proteins are ONLY NEEDED when lactose is available and
the normal food source, glucose, is in short supply
Figure 8. The lac operon
 Regulatory region has two swiches:
o CAP binding site
 CAP means cAMP-binding protein (Jawetz et al., 2013) or
catabolite activator protein (McGraw-Hill video)
Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in
Edited by: David Whang
 In front of the promoter for the lac operon
 ON only when glucose levels are low
o Operator site
 Next to the promoter for lac operon
 Is turned ON only when lactose is present
o Both must be ON to allow expression of the lac operon genes
 When glucose level is LOW:
o High level of cAMP builds up in the cell
o cAMP binds to CAP
o CAP-cAMP complex binds to CAP-binding site
o DNA bends, allowing RNA polymerase to bind at the
promoter
o Promoter activated  lac genes expressed
Figure 9. Promoter is activated when glucose level is low
 When glucose level is HIGH:
o cAMP levels are low
o cAMP does not bind to CAP
o Unactivated CAP is unable to bind to the DNA
o RNA polymerase does not associate to the lac promoter
o Promoter inactivated  lac genes not expressed
2. The lac repressor
REMEMBER
A repressor is a regulatory protein that can bind to the
operator site and prevent transcription.
 When glucose is present but lactose is ABSENT:
o Repressor binds to the operator
o Repressor covers part of the promoter
o RNA polymerase is prevented from binding to the promoter
and making mRNA
o Promoter inactivated  lac genes not expressed
Figure 10. When lactose is absent, repressor is active while promoter
is inactive
Page 5 of 11
  When glucose is present and lactose is also PRESENT:
o Lactose isomer binds to repressor and inactivates it
o Repressor is prevented from binding to the operator site
o RNA polymerase can associate to the promoter
o Promoter activated  lac genes expressed
o Transcription of lac genes are said to be “induced” by lactose

Figure 13. When tryptophan levels are low, repressor is inactive while
promoter is active

 When tryptophan levels are HIGH:

o Two tryptophan molecules bind to the repressor
o Orientation of the helix-turn-helix motifs in the repressor is
altered, causing it to be activated
Figure 11. When lactose is present, repressor is inactive while o Activated repressor binds to the operator located within the
promoter is active trp promoter (recognition helices in the repressor fit to
B. Attenuation adjacent major grooves of the DNA)
o Promoter inactivated  trp genes not expressed

Attenuation
http://highered.mheducation.com/sites/dl/free/0072835125/1269
97/animation28.html

The Trp operon

 Five genes (trpA, trpB, trpC, trpD, trpE) that are transcribed
together as a unit
 Encodes enzymes needed for the biosynthesis of the amino acid
tryptophan
 Regulated both by repressor and by attenuation/weakening
 Attenuation in the trp operon can prevent expression of
tryptophan biosynthesis genes when levels of tryptophan are
high, essentially “turning off” the trp operon when it isn’t needed
Figure 12. The Trp Operon
C. Repression
The Tryptophan Repressor
http://highered.mheducation.com/sites/dl/free/0072437316/1200
80/bio26.swf
The Trp repressor
 Is a helix-turn-helix regulatory protein
 When tryptophan levels are LOW:
o Repressor is inactive
o RNA polymerase binds to promoter site
o Promoter activated  trp genes expressed
Figure 14. When tryptophan levels are high, repressor is active while
promoter is inactive
V. MECHANISM OF RECOMBINATION
 Donor DNA that does not carry information necessary for its own
replication must recombine with recipient DNA to become
established in a recipient strain
 Exchange of small pieces of genome (a few genes at a time)
o Homologous
 Consequence of close similarity in the sequences of donor
and recepient DNA
 Almost always involves exchange between genes that share
common ancestry
 Requires a set of Rec gene (reciprocal transfer)
o Non-homologous
 Result of enzyme-catalyzed recombination between two
dissimilar DNA sequences
 Depends on enzymes encoded by the integrated DNA and is
most clearly exemplified by insertion of DNA into a
recipient to form a copy of donor transposon
 Gene conversion (non-reciprocal transfer) in the acquisition
of genetic diversity

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 6 of 11
Edited by: David Whang
 VI. MECHANISM OF GENE TRANSFER
A. Conjugation
 A process of genetic transfer between bacterial cells that requires
direct contact between the cells. Many, but not all, species of
bacteria can conjugate

 Requires donor cell to recipient cell contact to transfer only one

strand of DNA
 Recipient cell completes structure of dsDNA by synthesizing strand
that complements the strand acquired from donor cells
 Plasmids are most frequently transferred by conjugation

Bacterial Conjugation
https://highered.mcgraw-
hill.com/sites/dl/free/0072835125/126997/animation6.html

 F factor (fertility factor)

o Small DNA circle
o Required for conjugation Figure 15. Bacterial Conjugation and Recombination
+
o F : strains of bacteria containing the F factor; donor; produces
Pilus to connect with another recipient cell B. Transduction
-
o F : strains of bacteria without the F factor

From video: Bacterial Transduction

 To begin conjugation, the F factor is cut at the origin of transfer by
a protein assembly called Relaxosome, which associates with the
strand to be transferred (T-DNA strand)
 Accessory proteins of the relaxosome are released but a portion of
relaxosome called Relaxase remains attached to T-DNA. This T-
DNA/relaxase complex is recognized by a coupling factor and
+
transferred to the exporter (which is a complex in the F cell that is
contiguous with the pilus)
 The exporter pumps the T-DNA/relaxase complex into the recipient
cell. Once the entire T-DNA molecule is transferred to the recipient
cell, relaxase joins the ends to make a circular DNA molecule. As it
replicated to become double stranded
 In donor cell, the F factor cell was also replicated to become
double stranded. This actually occurred as the T-DNA was being
transferred to the recipient cell
 In the end, both cells wind up with a complete double stranded
copy of the F factor. Their connection through the pilus is released;
+
each is now F cells that can go to conjugate with other cells
Other Bacterial Conjugation Videos
http://highered.mcgraw-
hill.com/sites/0072552980/student_view0/chapter9/animations.ht
ml#
https://highered.mcgraw-
hill.com/sites/dl/free/0072835125/126997/animation7.html
http://highered.mcgraw-
hill.com/sites/0072552980/student_view0/chapter9/animations.ht
ml#
 Donor DNA is carried in a phage coat and is transferred into the
recipient by the mechanism used in phage infection
 Is a phage-mediated genetic recombination in bacteria
 Even a lytic phage population may contain some particles in which
the phage cot surrounds DNA derived from the bacterium rather
than from the phage
 Temperate phages preferred vehicles for gene transfer because
infection of recipient bacteria under conditions that favor lysogeny
minimizes cell lysis and thus favors survival of recombinant strains
 A recipient bacterium carrying an appropriate prophage may form
a repressor that renders the cell immune to lytic infection
 The size of DNA in transducing particles is usually no more than
several percent of bacterial chromosome, therefore a co-
transduction (transfers more than one gene at a time), is limited to
linked bacterial genes

http://highered.mcgraw-
hill.com/sites/0072552980/student_view0/chapter9/animations.ht
ml#

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 7 of 11
Edited by: David Whang
 Figure 17. Bacterial Transformation

RECIPIENT CELL
(integration with
DONOR CELL recipient gene)
(transfers small gene
fragment) RECIPIENT CELL
(establishment as
independent replicon)

Figure 16. Bacterial Transduction

Figure 17. Bacterial diversity results from genetic exchange
C. Transformation
D. Restrictions on Gene Transfer
1. Restriction Endonucleases
Bacterial Transformation  Cleavage of donor DNA before becoming a part of the
recombinant replicon
http://highered.mcgraw-
hill.com/sites/0072556781/student_view0/chapter13/animation_q
uiz_1.html Restriction Endonucleases
http://highered.mcgraw-
 Direct uptake of “naked” donor DNA by the recipient cell depends hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/
on their competence for transformation 0072437316/120078/bio37.swf::Restriction+Endonucleases
 Types:
o Forced Transformation
 Induced in the laboratory where after treatment with high
salt and temperature shock, many bacteria are rendered
competent for the uptake of extracellular plasmid
o Natural Transformation
 An active process demanding specific proteins produced by
the recipient cell
 Uptake sequences are species-specific, thus restricting
genetic exchange to a single species. The DNA that is not
incorporated can be degraded and used as source of
nutrient to support microbial growth
Figure 18. The EcoRI restriction endonuclease
 Natural Competence
o Unusual among bacteria and some of these strains are Table 3. Types & Activities of Restriction Enzymes
transformable only in the presence of competence factors Type Activity
produced only at a specific point in growth cycle
o Found in several genera and include Bacillus subtilis, Cleaves DNA at random sites far from its
I
Harmophilus influenza, Neisseria gonnohoeae, N. meningitides recognition sequence
and Streptococcus pneumonia Cleaves DNA at defined positions close or
II
within its recognition sequence
Cleaves outside its recognition sequence with
IIG both Rease and Mtase enzymatic activities in
the same protein
IIP Cleaves symmetric targets and cleavage sites

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 8 of 11
Edited by: David Whang
 IIS Recognizes asymmetric sequences
Cleaves outside its recognition sequence and
III require 2 sequences in oposite orientations
within the same DNA
IV Cleaves modified DNA (example: methylated)

2. Plasmid incompatibility
 Incompatible plasmids result in one plasmid lost at a higher
than normal rate when the cell replicates

Figure 20. Mutation by Base Substitution

2. Insertion & Deletion (Frameshift)

Figure 19. Plasmid Incompatibility

VII. BACTERIAL MUTATION

A. Spontaneous Mutations
1. Base substitution (Point Mutation)
o One nucleotide is changed
o Consequence of mispairing between complementary bases
during replication
o Results in missense and nonsense mutations
 Missesnse – a different amino acid is produced
 Non-sense – the mutation produces a stop codon, abruptly
cutting the protein short
o Repair Mechanisms:
o Mismatch Repair Enzyme
 Mechanism that essentially proofreads a newly
synthesized strand to ensure that it is perfectly
complements its template
 Distinguish the newly synthesized strand from the
preexisting strand in the basis of methylation of
adenine in GATC sequences of preexisting strand.
o SOS response
 When DNA damage is too extensive
 Rescues cells in which DNA has been damaged
 A postreplication DNA repair system that allows DNA
replication to bypass lesions or error in DNA.
Mutation by Base Substitution
http://highered.mcgraw-
hill.com/sites/0072556781/student_view0/chapter11/animation_q
uiz_3.html
Figure 21. Insertion and Deletion Mutations
3. Rearrangement and Duplication
 Rearrangement is the result of deletions that remove large
portions of genes or even sets of genes
o Inversion is a rearrangement in which a DNA segment
undergoes breakage and is reversed end to end
o Transposition is the movement of a segment of DNA from
one place to another
 Duplication is frequently in tandem of comparable lengths of
DNA. This mutation is usually unstable and readily revert
B. Reversion
1. Phenotypic reversion
 Process of regaining an activity lost as a consequence of
mutation
2. Genotypic reversion
 Process of restoring the original DNA sequence
C. Suppression
1. Intragenic suppression
 Second mutation at a different site within the affected gene
restores the structure required for activity
2. Extragenic suppression
 Second mutation outside the affected gene restores the
structure required for activity

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 9 of 11
Edited by: David Whang
 VIII. GENETIC ENGINEERING
 Is the direct manipulation (DNA recombination) of an organism’s
genome to introduce desirable traits using biotechnology
 Site-directed mutagenesis

Figure 24. Sticky Ends

Each fragment has a single- stranded sequence of nucleotides on its
end that can hybridize with any piece of DNA that has also been cut
with the same restriction enzyme.

Step 3: Ligation
Generally, a plasmid contains 3 elements:
 Cloning/restriction site – where foreign DNA fragment can be
inserted
 Drug-resistance gene – destroys antibiotics to allow selective
Figure 22. Genetic Engineering growth of the host cell
 Replication origin – allows plasmid to replicate the host cell
“First, identify what you’re going to mix then isolate the gene that
you need, incorporate it with a plasmid, then place it in a bacteria and
transform that bacteria into whatever I want…” – Dr. Valera

A. Cloning
 Set of experimental methods used to assemble recombinant DNA
molecules and to direct their replication within host organism
 A method for isolating a particular sequence of DNA from a
complex mixture of different DNA sequences.
Figure 25. Cleaving of plasmid at restriction site
B. Steps in Cloning
Step 1: Isolate & Purify the desired gene The plasmid used for cloning has a single restriction site. When
Step 2: Fragment with a restriction enzyme. cleaved by the restriction enzyme generates the same cohesive ends
that are in the fragments of the DNA to be cloned.
Restriction enzyme (ex. EcoRl)
 Recognize specific regions of DNA for cleavage
 Are used to cleave the vector at the cloning site
 DNA sequences recognized are usually palindromes

Figure 26. Formation of recombinant plasmid

The sticky ends of the foreign and plasmid DNA molecules hybridize
and then are sealed into the phosphodiester linkages by the enzymes
DNA ligase, creating a recombinant plasmid.
Figure 23. Fragmentation by Restriction enzyme
Step 4: Transformation
Here, EcoRl cleaves the palindromic sequence GAATTC to produce
DNA fragments w/ sticky ends

Figure 27. Incorporation of DNA fragments into bacterial plasmids

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 10 of 11
Edited by: David Whang
 The host cells are added to the recombinant plasmids. In REVIEW QUESTIONS:
transformation, a few cells take up a recombinant, plasmid while
most other cells do not. 1. Mutations in bacteria can occur by which of the following
 The replication origin allows the plasmid to replicate by using the mechanisms?
host cell enzymes. (A) Base substitutions
 Plasmid replication is independent of host cell division, but (B) Deletions
plasmids are distributed to each daughter cell when the host cell (C) Insertions
divides. (D) Rearrangements
 As the plasmids replicate and the host cells multiply, the number of (E) All of the above
copies of the recombinant plasmid is greatly amplified.
 The multiple daughter cells form a colony/clone. 2. The form of genetic exchange in which donor DNA is introduced to
 Because all the host cells in a colony are derived from a single cell, the recipient by a bacterial virus is:
thay all contain copies of the same recombinant plasmid with its (A) Transformation
fragment of foreign DNA. (B) Conjugation
 A variety of assay methods can now be used on the bacterial (C) Transfection
colonies to determine which contains the particular DNA sequence (D) Transduction
we wish to isolate. (E) Horizontal transfer

IX. DNA CHARACTERIZATION 3. The form of genetic exchange in bacteria that is most susceptible
A. Restriction Mapping to the activity of deoxyribonuclease during the process of DNA
uptake is
 It is the process of obtaining structural information on a piece of
(A) Transformation
digested DNA by the use of restriction enzymes and then
(B) Conjugation
separating the resultant DNA fragments by gel electrophoresis
(C) Transfection
 A map to know where to find the gene
(D) Transduction
(E) All of the above
B. Sequencing
 It is the process of determining precise order of nucleotides within 4. Replication of which of the following requires physical integration
a DNA molecule with a bacterial replicon?
 Displays gene structure (A) Single-stranded DNA bacteriophage
 Deduce the amino acid sequence of gene products (B) Double-stranded DNA bacteriophage
 Analysis reveals regulatory regions that control gene expression & (C) Single-stranded RNA bacteriophage
genetic “hot spots” susceptible for mutation. (D) Plasmid
(E) Transposon
C. Hybridization
Hybridization Probes 5. The formation of a mating pair during the process of conjugation in
 Southern blot Escherichia coli requires:
o Method for detection of a specific DNA sequence in DNA (A) Lysis of the donor
samples (B) A sex pilus
o Hybridization of DNA to DNA (C) Transfer of both strands of DNA
 Northern blot (D) A restriction endonuclease
o technique to study gene expression by detection of RNA in a (E) Integration of a transposon
sample
o provide quantitative information about RNA synthesis
 Western blot (protein immunoblot)
o antibodies are used to detect cloned genes by binding to their
protein products.

Hydridization Techniques
 Restriction Fragment Length Polymorphism (RFLP)
o Demonstrated whenever the Southern blot pattern
(distribution of restriction sites) obtained with one individual is
substantially varied from the one obtained with another
individual
o Used to trace DNA from a small sample to its human donor.

 Polymerase Chain Reaction (PCR)

o Biochemical technology used to amplify a single or a few copies
of a piece of DNA across several orders of magnitude,
generating thousands to millions of copies of a particular DNA
sequence
Answers: E, D, A, E, B

Trans Group: Anne Tayo, Seline Tcheng, Liz Te, Krissy Tilla-in Page 11 of 11
Edited by: David Whang