BACTERIAL GENETIC

I. Organization of Genetic Information-

General Concept

• A. Deoxyribonucleid Acid (DNA)

- Containing chains of nucleotide monomers
- Each nucletide contains a sugar, a base and
phosphate group. The sugar is 2’
deoxyribose which has five carbons named
1’, 2’ etc.
- There are four types of base : adenine and
guanine have two carbon–nitrogen rings and
are purines, thymine and cytosin have a
single ring and are pyrimidine. The bases are
attached to the 1’ carbon of deoxyribose. A
sugar plus a base is formed a nucleotide. A
nucleotide has phoshate attached to the 5’
carbon of the sugar.
- The polinucleotide has a free 5’ phosphate
at one end (5’) and a free 3’ OH (3’) at the
other end. It can be read 5’-3’ or 3’-5’.
- Polynucleotides are extremely long and
encodes the genetic information.
- Double stranded composed of
complementary base pairs (A-T or G-C )
joined by hydrogen bonds.
B. Ribonucleotid Acid (RNA)
• Single stranded
• Transcribes and translate DNA bound genetic
instruction for protein synthesis
• Substitutes uracil for the thymine base used by
DNA : the complementary base pairs for RNA
are A-U or G-C.
• Is found in three types :
1. Messenger RNA (mRNA)
- mRNA acts as template for protein synthesis.
- RNA transribed by RNA polymerase
2. Ribosomal RNA (rRNA)

• Is a structural component of ribosomes

• Acts as a substrate for protein syndromes
• Ribosomes have a large and small subunit with
characteristic sizes, described in terms of
sedimentation (S) values. Prokaryotic ribosome
are 70S with 50S and 30S subunits. They
contains three rRNAs ( 23S, 16S and 5S).
• Eucaryote ribosome are 80S with 60S and 40S
subunits and contain four rRNAs (28S,18S,5.8S
and 5S)
 3. Transfer RNA (tRNA)

• Transfer RNAs are small molecules that

bring amino acids together for protein
synthesis in an order specified by a s of
different tRNAs each of which binds a
specific amino acid. Each tRNA also binds
a specific codon in mRNA allowing it to
place its amino acid in the correct
position.
II. Comparison of Prokaryotic and Eukaryotic
Genomes
A. Eukaryotic Genome
1. Structure
a. Eukaryote are diploid with two
homologous copies of each chromosome
b. Virtually all genetic information is
contained in two or more linear
chromosome located within a membrane
bound nucleus
 1. Structure (continued)

c. Eukaryotic genome contain introns (DNA

sequences not translated into gene products)
and exons (DNA sequences translated into
gene products). The introns must be
removed from RNA molecules by process
called splicing which leaves the exons and
the coding continous.
 1. Structure (continued)

d. Certain eukaryotic organelles (mitochondria,

chloroplast) contain a self replicating, circular,
double stranded DNA molecule (plasmid)
relating to their intracellular function.
B. Prokaryotic genome

1. Structure
a. Most prokaryote are haploid (single
chromosome)
b. Genes of essential for bacterial growth
are carried on a single, supercoilled circular
DNA molecule called circular bacterial
chromosome encoding generally several
thousand genes. They are not enclosed in a
membrane- circumscribed nucleus. Some
protein may help to package the DNA is
called histon
c. Many bacteria contain additional, specialized
genes or smaller extrachromosomal plasmid. The
plasmids replicate independently. Some exist in
transmissible and non transmissible forms and
may be integrated into bacterial chromosome.
Many different plasmid exist. These include
Resistance (R) , fertility (F), col plasmid,
degradative plasmid and virulence plasmid.
Bacterial may contains of several types of
plasmid.
d. Specialized information may also be carried on
transposons, moveable genetic elements that
cannot self-replicated. Transposons contain
insertion sequences and can transfer their
information by inserting themselves into other
loci in the same or other genetic elements ( e.g.
plasmids, chromosomes, viral DNA )
III. DNA REPLICATION :

- This is the process by which a cell

copies its DNA. Replication is necessary
so that the genetic information present
in cells can be passed on the daughter
cells following cell division. The DNA is
copied by enzymes called DNA
polymerases. These act on single
stranded DNA synthesizing a new strand
complementary to the original strand.
- Replication fork : During DNA replication
the double helix of a cell’s entire DNA is
progressively unwound producing segments
of single stranded DNA which can be copied
by DNA polymerases. Unwinding of the
double helix begins at a distinct position
called the replication origin. The region
where the helix unwinds and new DNA is
synthesized is called the replication fork.
 At the replication fork a number of distinct
events occur :

- Separation the double helix. This achieved

by the action of a helicase enzyme.
Following separation of the strand, single
strand binding (SSB) protein attaches to to
the DNA and prevents the double helix
form reforming.
- Synthesis of leading and lagging strands.
Synthesis of DNA by DNA polymerases occurs
only in the 5’3’ direction. As the two strands of
the double helix run in the opposite directions
(one strand run 5’3’ and the other 3’5’ )
slightly different mechanism are required to
replicate each. One strand called leading strand,
is copied in the some direction as the unwinding
helix and so can be synthesized continuously.
The lagging strand is synthesized in the opposite
direction and must be copied discontinuously. The
lagging strand is synthesized as a series of
segments known okazaki fragment.
IV. GENE EXPRESSION

The biological information in a DNA

molecule is contained in its base sequence.
Gene expression is the process by which this
information is made available to the cell.
The use information is described by the
central dogma which states that information
is tranferred from DNA to RNA to protein.
During gene expression, DNA molecule copy their
information by directing the synthesis of an RNA
molecule of complementary sequence. This
process is known as transcription.
The RNA then directs the synthesis of a
polypeptide whose amino acid sequence is
determined by the base sequence of the RNA.
The process is known as translation.
- Transcription : The first stage of gene expression
and it involves the synthesis of RNA from DNA
template by RNA polymerase. RNA polymerase
has sigma factor. The RNA is synthesized from
the template DNA strand and has the some
sequence as the non template (sense) strand.
Initiation begins at the gene promotor.
RNA polymerase recognizes the -10 box and –35
box sequence elements, and the sigma factor
binds to –35 box. The double helix then
dissociates at the –10 box to open the promoter
and the RNA synthesis begins.
Termination pallindromic sequences can form
stem-loop structures in RNA and act as signals
for transcription termination.
- Translation

Transfer RNA (tRNA) deliver amino acids to

the ribosome for protein synthesis in an
order specified by the messenger RNA
(mRNA) sequence. Translation occurs three
stages (initiation, elongation and
termination)
- Initiation : Translation begins with binding
of the small ribosomal subunit to the mRNA
at the Shine Dalgarno sequence. The sub
unit migrates down stream to the AUG
initiation codon (initiator tRNA methionine 
tRNAmet )
- Elongation : Following initiation the large
ribosomal subunit binds the initiation
complex forming the A and P site. The P site
occupied by tRNAmet. A second charge tRNA
enter the A site and peptidyltransferase
forms a peptide bond between two amino
acids.
Following peptide bond formation the
ribosome translocated to the next codon the
dipeptide bound to the second tRNA moves
to the P site expelling the initiator tRNA. A
third charge tRNA enters the A site and the
elongation cycle repeats.
- Termination : Translation ends when a
termination codon enters the A site. There
are no tRNA able to binds to the termination
codons (recognize as stop codons UAA and
UAG). Instead protein knows as release
factor enter the A site and cause the
completed polypeptide to release.
V. GENE TRANSFER BETWEEN ORGANISM

- Most bacteria are haploid and lack meiosis.

However they can undergo recombination.
An artificial process for achieving this is
transformation, where DNA from one
bacterial strain is mixed with another. DNA
is taken up only by competent cells which
is ready to take the transformation process.
- May also occurs as the crossing over of
homologous chromosomes or by non
homologous means (e.g. movement of
plasmids or transposons, insertion a viral
genes).
- can results in the acquisition of new
characteristic (e.g. antigens, toxins,
antibiotic resistance )
- Occurs via three mechanisms : Conjugation,
transduction, and transformation.
A. CONJUGATION
- Is a one way transfer of genetic material
(usually plasmid) from donor to recipient by
means of physical contact.
- Typically involves three types of plamids :
(In many cases plasmid can moved from one
bacterium to another and even between
bacteria of different species. The transfer
process is directed by genes carried on plasmid.
The ability of plasmid to moved between
bacteria to study transfer of gene from the
bacteria chromosome)
- 1 F* Cell :The first plasmid to be shown to
move between bacteria was F plasmid (the
bacteria contains F plasmid F*  fertility
factor as a males).
F* cells mediating the creation of sex pilus
necessary for conjugal transfer of the F plasmid
to the recipient.
F* can integrate into choromosomal DNA
creating high frequency recombination (Hfr)
donors from which choromosomal DNA is readily
transferred.
 - 2 R factors : Contain genes conferring drug
resistance. Frequently, the resistance genes are
carried on transposons.
Express resistance phenotype through natural
selection.
.
- 3 F’ and R’ : Combination fertility or resistance
plasmids in which limited regions of chromosomal
DNA can be replicated and transferred by
conjugation independently of the chromosome.
 B. TRANSDUCTION

- is phage – mediated transfer of host DNA

sequences

- can be performed by temperate phages or

virulent phage depending on how they
behave after infection of bacterium and
under special condition by lytic phages.
Occurs in two forms :
 In generalized transduction, the phage
randomly packages host DNA in a
bacteriophage coat and may transfer any
genes. The transducing particle contains
only host DNA.
 In specialized transduction, the lysogenic
phage favors the transfer of host DNA
segments near the site of prophage
integration. Specialized transducing phages
contain both viral and host genes.
 Lysogenic State
 Phage DNA is maintenance as a prophage
 Prophage is integrated to the chromosome of
bacterial ( does not always happen )
 Prophage exist as a plasmid like a molecule
in bacterial cell
C. TRANSFORMATION

- is uptake by a cell of a naked DNA

molecule or fragment from the medium
and the incorporation of this molecule
into the recipient chromosome.

- is used in recombinant DNA research and

commercially to introduce human genes
via vector into bacteria rapid and large
scale production of human gene
production.
VI. MUTATION

- Occur approximately once for any gene in

every 1 million cells.
- Is an induced or spontaneous heritable
alteration of the DNA sequence.
- Introduces variability into gene pool and
changes in phenotype.
- May be caused by various mutagens,
including ultraviolet light, acridine dyas
base, analogues and nitrous acid
A. Mutation types
1. Nucleotide subsitutions
- arise from mutagenic activity or
mispairing of complementary bases
during replication
- often do not significantly disrupt the
function of gene products
2. Frameshift mutations
- Result from insertion or deletion of
one or two base pairs, discrupting the
phase of the triplet – encoded DNA
message.
3. Deletions
- are usually large excisions of DNA,
dramatically altering the sequence of
coded proteins.
- may also result in frameshift mutations

4. Insertions
- Change genes and their products by
integration of new DNA via transposons.
B. Result of Mutation

1. Missense mutations
- Result in the substitution of one amino acid
for another.
- May be without phenotype effect (silent
mutation)
2. Nonsense mutations
- terminate protein synthesis and result in
truncated gene production
- Usually result in inactive protein products
C. Reversions

- Function lost to mutation may be regained

in two ways :
1. Genotype (true) reversion
- Is restoration at the site of DNA
alteration
2. Phenotypic (suppression) reversion
- is the restoration of an activity lost to
mutation, often by mutation at second
site (suppressor mutation)
VII. Application of In- vitro Genetics
1. Research
a. Plasmid vectors (cloned fragment)
- Origin of replication
- Selectable marker
- Cloning site
- Insertional inactivation
b. Bacteriophage vectors
ex. Yeast artificial chromosomes
c. Gene libraries
d. Recombinant clones : Hybridization
- Southern Blotting (Agarose gel electrophoresis)
- Western Blot
- Northern Blot
- Dot Hot Hybridization
- Selection hybridization
e. Making new genes
- DNA synthesis
- Protein engineering
- Mutagenesis
f. Cloning in animal and plants cells
g. Transgenic animals and plants cell
h. Live recombinant vaccines
e.g. Hepatitis B vaccine
2. Diagnosis and Epidemiology
 Gene probes for detection of pathogens
 Diagnostic use of PCR
 Genetic finger printing
 Human genetic diseases (gene disorders)
 Gene therapy
 RFLP analysis (Restriction Fragment Length
Polymorphism)
 Used to distinguish between closely related
species / between strains of the same species