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Dr.Alia Shoeib
Antibiotics and Plasmids
Plant &Microbiology Dept.
Micro.623
Definition
Molecular biology is the study of biology at a molecular level. Molecular
biology chiefly concerns itself with understanding the interactions between
the various systems of a cell, including the interrelationship of DNA, RNA
and protein synthesis and learning how these interactions are regulated.
Molecular genetics is the field of biology which studies the structure and
function of genes at a molecular level. Molecular genetics employs the
methods of genetics and molecular biology.
MOLECULAR GENETICS
Genes ---> Enzymes ---> Metabolism
Central Dogma of Molecular Biology*
DNA -transcription--> RNA -translation--> Protein
What is a GENE = ?
DNA is the genetic material...
- a discrete piece of deoxyribonucleic acid
- linear polymer of repeating nucleotide monomers
nucleotides* --> A adenine, C cytosine
T thymidine, G guanine -->
polynucleotide*
INFORMATION PROCESSING & the CENTRAL DOGMA
- the letters of the genetic alphabet... are the nucleotides A, T,
G, & C
Current dogma of Molecular Biology
DNA --> RNA --> Proteins, (proteins supposedly
regulate gene expression) figure*
- unit of information is CODON = genetic 'word'
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a triplet sequence of nucleotides CAT in a
polynucleotide
3 nucleotides = 1 codon (word) = 1 amino
acid
- the definition of (codon) word = amino acid
Size of Human Genome: ≈ 3,000,000,000 base pairs or 1.5b in
single strand genes
≈ 500,000,000 possible codons (words
or amino acids)
1. Transformation* Experiments of Fred Griffith... (1920's)
Streptococcus pneumoniae - pathogenic S strain & benign R
transforming
'principle'
is
genetic
element
2. Oswald Avery, Colin MacLeod, & Maclyn McCarty... (1940's)
suggest the transforming substance* is DNA molecules, but...
Gene expression is the multi-step process by which a gene's information is
converted into the structures and functions of a cell, following the central
dogma of molecular biology.
Recombinant DNA is DNA that has been created artificially. DNA from
two or more sources is incorporated into a single recombinant molecule.
A bacterial artificial chromosome (BAC) is a DNA construct, based on a
fertility plasmid, used for transforming and cloning in bacteria, usually E.
coli. Its usual insert size is 150 kbp, with a range from 100 to 300 kbp.
BACs are often used to sequence the genetic code of organisms in genome
projects, for example the Human Genome Project. A short piece of the
organism's DNA is amplified as an insert in BACs, and then sequenced.
Finally, the sequenced parts are rearranged in silico, resulting in the genomic
sequence of the organism.
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To be useful, the recombinant molecule must be replicated many times to
provide material for analysis, sequencing, etc. Producing many identical
copies of the same recombinant molecule is called cloning.
Cloning can be done in vitro, by a process called the polymerase chain
reaction (PCR). Here, however, we shall examine how cloning is done in
vivo.
Cloning in vivo can be done in



unicellular prokaryotes like E. coli
unicellular eukaryotes like yeast and
in mammalian cells grown in tissue culture.
In every case, the recombinant DNA must be taken up by the cell in a form
in which it can be replicated and expressed. This is achieved by
incorporating the DNA in a vector. an example of cloning using E. coli as
the host and a plasmid as the vector.
vector
Plasmids are sometimes called "vectors", because they can take DNA from one organism
to the next. Not all vectors are plasmids, however. We commonly use engineered viruses,
for example bacteriophage lambda, which can carry large pieces of foreign DNA.
Plasmids
In addition to the nucleoid, many bacteria often contain small
nonchromosomal DNA molecules called plasmids. Plasmids usually contain
between 5 and 100 genes. Plasmids are not essential for normal bacterial
growth and bacteria may lose or gain them without harm
Transposons (transposable elements or "jumping genes") are small pieces of
DNA that encode enzymes that transpose the transposon, that is, move it
from one DNA location to another. Transposons may be found as part of a
bacterium's nucleoid (conjugative transposons) or in plasmids and are
usually between one and twelve genes long. A transposon contains a number
of genes, coding for antibiotic resistance or other traits, flanked at both ends
by insertion sequences coding for an enzyme called transpoase. Transpoase
is the enzyme that catalyzes the cutting and resealing of the DNA during
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transposition. Thus, such transposons are able to cut themselves out of a
bacterial nucleoid or a plasmid and insert themselves into another nucleoid or
plasmid and contribute in the transmission of antibiotic resistance among a
population of bacteria.
TRANSPOSONS - pieces of DNA prone to moving & creating repeat
sequences
LINE - long interspersed nuclear element holds promoter & 2
genes: RT & integrase
Plasmids can also acquire a number of different antibiotic resistance genes by
means of integrons. Integrons are transposons that can carry multiple gene
clusters called gene cassettes that move as a unit from one piece of DNA
to another. An enzyme called integrase enables these gene cassettes to
integrate and accumulate within the integron. In this way, a number of
different antibiotic resistance genes can be transferred as a unit from one
bacterium to another.
Biotechnology
Restriction Enzymes :
definition: enzymes that cut DNA in specific places
function: inactivate foreign DNA which can derange metabolism
breaks only palindrom sequences, i.e. those exhibiting two-fold symmetry
examples
Important in DNA research, i.e. sequencing, hybridization
Companies purify and market restriction enzymes
Modification Enzymes
protect destruction of native DNA by native restriction enzymes
mechanism
A nucleotide can be a substrate for a particular restriction enzyme or
modification enzyme but not both
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Gene Amplification: polymerase chain reaction (PCR)
to make multiple copies of a specific gene
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Dr.Alia Shoeib
Plant &Microbiology Dept.
Antibiotics and Plasmids
Micro.623
GENE Expression
the Central Dogma of Molecular Biology depicts flow of genetic information
Transcription - copying of DNA sequence into RNA
Translation - copying of RNA sequence into protein
DNA sequence -------> RNA sequence -----> amino acid sequence
TAC
AUG
MET
triplet sequence in DNA --> codon in mRNA ----> amino acid in protein
Information : triplet sequence in DNA is the genetic word [codon]
GENETIC CODE...
...is the sequence of nucleotides in DNA, but shown as a mRNA code*
...specifies sequence of amino acids to be linked into the protein
GENETIC CHANGE - a change in DNA nucleotide sequence
- 2 significant ways mutation & recombination
1. MUTATION - a change in an organism's DNA*that results in
a different codon = different amino acid sequence
Point mutation - a single to few nucleotides change
- deletions, insertions, frame-shift mutations* [CAT]
2. Recombination (Recombinant DNA) newly combined DNA's
that
[glossary]*
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can change genotype by insertion of NEW (foreign) DNA molecules
into recipient cell
3. transformation* - absorption of 'foreign' DNA by recipient cells
4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R
= resistance)
conjugation is primitive sex-like reproduction in
bacteria [Hfr*]
5. VIRAL TRANSDUCTION - via a viral vector ( lysogeny &
TRANSDUCTION* )
general transduction - pieces of bacterial DNA are
packaged w viral DNA during viral replication
restricted transduction - a temperate phage goes lytic
carrying adjacent bacterial DNA into virus particle
6. DESIGNER GENES - man-made recombinant DNA molecules
Designer Genes - Genetic Engineering - Biotechnology
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