Lecture BIOD 9: Microbial Genetics

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Lecture BIOD 9 Microbial Genetics
- Genetics is the study of what genes are, how
they carry information, how their information is
expressed, and how they are replicated and passed
to subsequent generations or other organisms.
- DNA is the genetic material for all living
organisms. In bacteria, there is a single
circular (closed) chromosome which is a polymer
of DNA (nucleic acid).
- The DNA in a chromosome exists as one long
double helix associated with various proteins
that regulate genetic activity.
- The DNA in a cell is replicated before the cell
divides, so each daughter cell receives the same
genetic information.
- Gene is a segment of DNA, a sequence of
nucleotides, that codes for a functional product,
usually a protein.
- When a gene is expressed, DNA is transcribed to
produce an mRNA mRNA is then translated into
proteins.
- Clone is a population of cells that are
genetically identical.
- Genome all the genes present in a cell or
virus.
- Genotype the specific set of genes an organism
possesses.
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- Phenotype the collection of characteristics of
an organism that an investigator can observe.

• Nucleic Acids Structure

- DNA made from subunits called nucleotides.

• - Each nucleotide contains
• Purine (Adenine or A, Guanine or G) or
  Pyrimidine (Cytosine or C, Thymine or T) bases.
• Deoxyribose sugar.
• 1, 2, or 3 phosphate groups.

- RNA nucleotides containing ribose sugar.
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- Nucleotides are named according to of
phosphates e.g., dATP deoxy adenosine
triphosphate, whereas dAMP deoxy adenosine
monophosphate.
-Nucleotides in RNA don't have deoxyribose, don't
have prefix "d" names like ATP, ADP, AMP refer
to RNA nucleotides.
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- Purine Pyrimidine bases are attached to
deoxyribose sugar, free to rotate. In DNA, form
specific base pairs A with T (2 H-bonds), G with
C (3 H-bonds).
- Two chains of DNA face in opposite directions,
called antiparallel (defined by which way 3' and
5' sides of sugar molecule are facing).
- In a linear DNA molecule, one strand has free
3'-end, other (complementary) strand has 5'-end.
5'-CAGCTAGAGTCATCG-3' 3'-GTCGATCTCAGTAGC-5'

• DNA Replication

- During DNA replication, the two strands of the
double helix separate at the replication fork,
and each strand is used as a template by DNA
polymerases to synthesize two new strands of DNA
according to the rules of nitrogenous base
pairing.
- The result of DNA replication is two new
strands of DNA, each having a base sequence
complementary to one of the original strands.
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- DNA is synthesized in one chemical direction
called 5' to 3' (5' is phosphate end 3' is
hydroxyl end of deoxyribose).
- Because each double-stranded DNA molecule
contains one original and one new strand, the
replication process is called semiconservative.
- DNA polymerase proofreads new molecules of DNA
and removes mismatched bases before continuing
DNA synthesis.
- Each daughter bacterium receives a chromosome
identical to the parent's.

• Transcription

- During transcription, the enzyme RNA polymerase
synthesizes a strand of RNA from one strand of
double-stranded DNA, which serves as a template.
- RNA is synthesized from nucleotides containing
the bases A, C, G, and U, which pair with the
bases of the DNA sense strand.
- The starting point for transcription, where RNA
polymerase binds to DNA, is the promoter site
the region of DNA that is the endpoint of
transcription is the terminator site RNA is
synthesized in the 5' gt 3' direction.
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(No Transcript)
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• Translation

- Translation is the process in which the
information in the nucleotide base sequence of
mRNA is used to dictate the amino acid sequence
of a protein.
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- The mRNA associates with ribosomes, which
consist of rRNA and protein.
- Three-base segments of mRNA that specify amino
acids are called codons.
- The genetic code refers to the relationship
among the nucleotide base sequence of DNA, the
corresponding codons of mRNA, and the amino acids
for which the codons code.
- The genetic code is degenerate that is, most
amino acids are coded for by more than one codon.


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- Of the 64 codons, 61 are sense codons (which
code for amino acids), and 3 are nonsense codons
(stop codons) which do not code for amino acids
and are stop signals for translation.
- The start codon, AUG, normally codes for
methionine (formylmethionine at the beginning or
a protein).
- Specific amino acids are attached to molecules
of tRNA. Another portion of the tRNA has a base
triplet called an anticodon.
- The base pairing of codon and anticodon at the
ribosome results in specific amino acids being
brought to the site of protein synthesis.
- The ribosome moves along the mRNA strand as
amino acids are joined to form a growing
polypeptide mRNA is read in the 5' gt 3'
direction.
- Translation ends when the ribosome reaches a
stop codon on the mRNA.
- In prokaryotes, translation can begin before
transcription is complete.
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• Regulation of Gene Expression in Bacteria

- Gene is a linear sequence of nucleotides that
is within the genomic nucleic acid molecule, and
that has a fixed start point and end point
- It has controlling elements (e.g., promoters)
that regulate expression of a gene.
- It encodes a polypeptide, a tRNA, or an rRNA.
- With some exceptions, genes are not overlapping.
- The segment that encodes a single polypeptide
is also called a cistron.
- Procaryotic coding information is normally
continuous although some bacterial genes are
interrupted.
- Most Eucaryotic genes have coding sequences
(exons) that are interrupted by noncoding
sequences (introns).
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- Several structural genes (for protein)
controlled by a single promoter.
- mRNA is polycistronic has multiple start
stop signals, codes for multiple polypeptides.
- Some genes are expressed constitutively always
translated when cells synthesizing protein.
(Examples ribosomal genes, genes for replication
transcription, genes for central metabolism
enzymes, etc.)
- Other genes are regulated (controlled) by
various Genetic Regulatory Mechanisms proteins
are synthesized only as they are needed.
- The control of Genetic regulatory mechanisms,
such as Repression and Induction, is aimed at
mRNA synthesis.
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• Repression

- Gene is regulated by negative control in
absence of specific repressor, gene is
transcribed just like constitutive gene.
- In order to regulate, must add specific block.
Must say "no" otherwise gene is not
down-regulated.
- An example of a repressible genetic system is
the synthesis of the amino acid tryptophan in E.
coli.

• Induction

- In the presence of certain chemicals
(inducers), cells synthesize more enzymes. This
process is called induction.
- An example of an inducible genetic system is
the production of b -galactosidase by E. coli in
the presence of lactose, so lactose can be
metabolized.

• The Operon Model of Gene Expression

- Operon is set of genes transcribed as a
polycistronic message and controlled by a
regulatory protein.
- In the operon model (e.g. lac operon) for an
inducible system,a regulatory gene codes for
repressor protein.
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- In the absence of lactose (inducer)
- In the presence of lactose (inducer)
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• An Inducible Operon (Lac Operon) in the Absence
  of an Inducer (Lactose)

- The regulator gene codes for an active
repressor protein.
-The repressor protein then binds to the operator
region of the operon.
- With the active repressor protein bound to the
operator region, RNA polymerase (the enzyme
responsible for the transcription of genes) is
unable to bind to the promoter region of the
operon.
- If RNA polymerase does not bind to the promoter
region, the three enzyme genes (Z, Y, and A) are
not transcribed into mRNA.
-Without the transcription of the three enzyme
genes, the three enzymes needed for the
utilization of the sugar lactose by the bacterium
are not synthesized.
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• An Inducible Operon (Lac Operon) in the Presence
  of an Inducer (Lactose)

- The regulator gene codes for an active
repressor protein.
- Lactose, the inducer molecule binds to the
active repressor protein.
- The binding of the inducer alters the shape of
the allosteric repressor causing it to become
inactivated.
- The inactivated repressor protein is then
unable to bind to the operator region.
- Since the inactive repressor protein is unable
to bind to the operator region, RNA polymerase
(the enzyme responsible for the transcription of
genes) is now able to bind to the promoter region
of the operon.
- RNA polymerase is now able to transcribe the
three enzyme genes (Z, Y, and A) into mRNA.
- With the transcription of these genes, the
three enzymes needed for the bacterium to utilize
the sugar lactose are now synthesized.
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• Genetic Transfer and Recombination

- Genetic recombination is the transfer of DNA
from one organism to another. The transferred
donor DNA may then be integrated into the
recipient's nucleoid by various mechanisms.
- Natural mechanisms of genetic recombination in
bacteria include transformation, transduction
conjungation.

• Plasmids

- A plasmid is an "extra-chromosomal" piece of
bacterial DNA.
- Plasmids are stably maintained within bacterial
cells, replicating fast enough that they are
passed on to bacterial progeny as the bacteria
divide.
- Like bacterial chromosomes, plasmids are
circular, double-stranded DNA.
-The major difference between chromosomes and
plasmids is that they are much smaller than
chromosomes, and they tend to carry genes that
are not essential except in certain environments.
- There are several types of plasmids, including
conjugative plasmids, dissimilation plasmids,
plasmids carrying genes for toxins or
bacteriocins, and resistance factors.
- R PLASMID A plasmid having genes coding for
multiple antibiotic resistance and often a sex
pilus.
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- F PLASMID A plasmid coding only for a sex
pilus.

• Transformation

- A process during which DNA fragment from a
dead, degraded bacterium enters a competent
recipient bacterium.
- The DNA taken up is exchanged for a piece of
DNA of the recipient.
- This process was first demonstrated in
Streptococcus pneumoniae, and occurs naturally
among a few genera of bacteria.
- A bacterial cell that is capable of being
transformed (i.e., of taking up DNA directly from
the environment) is said to be competent.
- Bacteria that are not naturally competent
(e.g., E. coli) often can be manipulated in the
laboratory in such a way that they become able to
pick up environmental DNA.
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• Conjugation

- This process requires contact between living
cells ('bacterial sex').
- One type of genetic donor cell is an F
recipient cells are F-. F cells contain plasmids
called F factors these plasmids are transferred
to the F- cells during conjugation.
- Donating bacteria is described as being male,
and the recipient then becomes an F male and can
make a sex pilus. Conjugation serves to convert
the recipient bacteria also to a male.
- When the plasmid becomes incorporated into the
chromosome, the cell is called an Hfr
(high-frequency recombinant).
- At the end of Hfr conjugation there was
transfer of some donor chromosomal DNA, but
usually not a complete F plasmid, the recipient
bacterium usually remains F-.
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• Transduction

- Transduction is the transfer of fragments of
DNA from one bacterium to another bacterium by a
bacteriophage.
- Bacteriophages are viruses that infect bacteria.
- Generalized Transduction by Lytic Bacteriophage
is done following these steps
1. A lytic bacteriophage adsorbs to a susceptible
bacterium.
2. The bacteriophage genome enters the bacterium.
The genome directs the bacterium's metabolic
machinery to manufacture bacteriophage components
and enzymes.
3. Occasionally during maturation, a
bacteriophage head or capsid assembles around a
fragment of donor bacterium's nucleoid or around
a plasmid instead of a phage genome by mistake.
4. The bacteriophages are released.
5. The bacteriophage carrying the donor
bacterium's DNA adsorbs to a recipient bacterium.
6. The bacteriophage inserts the donor
bacterium's DNA it is carrying into the recipient
bacterium. DNA is exchanged for some of the
recipient's DNA