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FCH 532 Lecture 2
Webpage:
http://www.esf.edu/chemistry/nomura/fch
532/
Genetics review
Chapter 1
Nucleic acids overview
Chapter 5
Bacterial genetics
• Advantages can be grown quickly (20 min doubling times).
• Bacteria usually haploid-phenotype indicates genotype
(usually).
• Bacterial genetics started in the 1940s for procedures to
isolate mutants.
• Mutants can be detected and selected for by their ability
or inability to grow under certain conditions.
• Example: wild-type E. coli can grow on medium with glucose
as the sole carbon source. However mutants unable to
synthesize leucine require its presence in the growth medium.
• Mutants that are resistant to an antibiotic can grow whereas
wild-type cells cannot.
• Some mutants have proteins that are temperature sensitive.
• Use replica plating to screen for colonies with mutant
attributes.
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Figure 1-30 Replica plating.
Viruses
• Viruses are infectious particles consisting of a nucleic acid
molecule enclosed by a capsid (protective coat made of
protein).
• A virus specifically adsorbs to a susceptible cell and injects its
nucleic acid.
• The viral chromosome takes over the cell to produce new
viruses.
• At the end the viral infection causes the lysis of the cell to
release the viruses.
• Not living organisms since in the absence of their host, they
are biologically inert.
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Figure 1-31 The life cycle of
a virus.
Viral complementation and
recombination
• Bacteriophages-bacterial viruses aka phages
• Form plaques (clear spots) on a “lawn” of bacteria.
• A mutant phage is detected by its ability to produce
progeny under “permissive conditions” and
inability to produce progeny under “restrictive
conditions”
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Figure 1-32 Screening for
viral mutants.
Viral complementation and
recombination
• If two different mutant varieties of phage are used to
infect a bacterium, it could yield progeny under
conditions under which neither of the mutants alone
could reproduce through complementation.
• Each mutant phage supplies a function that the
other mutant cannot.
• Each mutation is part of a different
complementation group.
• Also undergo recombination but differently than
eukaryotes.
Viral complementation and
recombination
• Bacteriophages reproduce so rapidly that you can detect
recombination with a frequency of 1 in 108.
• Benzer carried out studies of the rII region of
bacteriophage T4 chromosome.
• 4000 bp, approx. 2% of the T4 chromosome.
• 2 complementation groups rIIA and rIIB (rapid lysis
genes).
• In a permissive host (E. coli B) a mutation that
inactivates the product of either gene causes the
formation of plaques that are larger than wild-type.
• In a restrictive host (E. coli K12()), only the wild-type
phage can cause lysis.
• However, if K12() is infected with 2 different rII mutants,
recombination can take place within the same gene.
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Figure 1-33 Viral recombination.
Viral complementation and
recombination
• Benzer also demonstrated that complementation
between 2 mutants in the same complementation group
yields progeny in the restrictive host when the 2 mutation
are in the cis configuration (on the same chromosome)
but do not if in the trans configuration (on physically
different chromosomes)
• This is due to the fact that when both mutations
physically occur in the same gene, the other gene will be
intact.
• Cistron the functional genetic unit of this type of cistrans test.
• Determined the the unit of recombination is about the
size of a single base pair!
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Figure 1-34 The cis-trans
test.
Nucleotides and nucleic acids
1.
2.
3.
4.
5.
Form monomeric units of nucleic acids-storage and
expression of genetic information.
Nucleoside triphosphates (i.e., ATP, GTP) are compounds
that store energy from energy releasing pathways (glycolysis,
electron transport) and are use to supply energy for energyrequiring reactions in the cell.
Most metabolic pathways are regulated in part by levels of
nucleotides such as ATP and ADP.
Nucleotide derivatives like nicotinamide adenine dinucleotide
(NADH), flavin adenine nucleotide (FAD) and coenzyme A
are required for many reactions.
Have catalytic activities in in enzymelike nucleic acids
(ribozymes).
10.1 RNA and DNA Chemical Structures
• Nucleic acids are sequence variable, linear
polymers of monomeric units called nucleotides.
• A nucleotide is composed of three chemical
parts:
1. An aromatic cyclic compound containing
C and N atoms: “nitrogenous bases”
2. A five-carbon carbohydrate (sugar): an
aldopentose.
3. 1,2, or 3 charged phosphate groups.
General structure of a nucleotide showing the
three fundamental units:
1. Purine or pyrimidine nitrogenous base.
2. An aldopentose (D-ribose or 2’-deoxy-D-ribose).
3. 1-3 charged phosphate groups.
Figure 10.3 The aldopentoses in RNA and DNA
a) b-D-Ribose
b) b-D-2-Deoxyribose
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Figure 5-1
Chemical structures of (a) ribonucleotides
and (b) deoxyribonucleotides.
Phosphate group can be bonded to the C5’ or C3’ of the pentose.
If bound to C5’ it is a 5’-nucleotide, if bound to C3’, it is a 3’nucleotide.
If there is no phosphate group it is a nucleoside.
A nucleoside consists of a purine or pyrimidine base linked
to a carbohydrate (ribose or deoxyribose) by an Nglycosidic bond.
Two numbering systems (primed ‘ and unprimed) are necessary to
distinguish the two rings.
Nucleotides and nucleic acids
•
•
•
•
•
•
In naturally occurring nucleotides and nucleosides, the bond
linking the nitrogenous base to the pentose C1’ atom
(glycosidic bond) extends from the same side of the ribose
ring as the C4’- C5’ bond.
Nucleotide are moderately strong acids.
Nitrogenous bases are planar, aromatic, heterocyclic
molecules.
Derivatives of purines or pyrimidines.
Pyrimidines are 6-membered rings similar in structure to
benzenes and have N at at the 1 and 3 positions.
Purines are heterocyclic compounds consisting of a
pyrimidine attached to an imidazole group.
Figure 10.2 The major and some minor heterocyclic bases
in RNA and DNA. All are derived from purine or pyrimidine.
Figure 10.2 The major and some minor heterocyclic bases
in RNA and DNA. All are derived from purine or pyrimidine.
Purines and Pyrimidines with Physiological Activity
•
•
•
•
5-fluorouracil (Adrucil) - used in cancer treatment
Fluorine atom is very electronegative.
Analog of thymine, inhibitor of DNA synthesis.
Inhibits enzyme thymidylate synthase.
Figure 10.2 The major and some minor heterocyclic bases
in RNA and DNA. All are derived from purine or pyrimidine.
Figure 10.2 The major and some minor heterocyclic bases
in RNA and DNA. All are derived from purine or pyrimidine.
RNA, DNA contains quantities of methylated bases.
Methyl groups added by enzymes after incorporation into
nucleic acids
Purines and Pyrimidines with Physiological Activity
•
•
•
Trimethylated derivative of purine ring.
Found in plants Coffea arabica, Camellia thea, Cola
acuminata and Cola nitida.
Inhibits enzyme phosphodiesterase involved in cell
signaling (cAMP).
Purines and Pyrimidines with Physiological Activity
•
•
•
•
Antivirial agent with trade name of Zovirax.
Used to treat herpes viral infections.
Inhibits enzyme DNA polymerase of herpes simplex.
Activated in cell via phosphorylation of side chain hydroxyl group
by kinase enzyme.
Purines and Pyrimidines with Physiological Activity
•
•
•
•
6-mercaptopurine (Purinthol)
Blocks synthesis of nucleic acids.
Effective in treatment of leukemia.
Affects rapidly growing cancer cells.
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Formation of nucleotides (phosphorylation)
•
•
Linkage of nitrogenous base and aldopentose
forms nucleoside.
Nucleotide is formed when phosphoryl group is
linked to carbohydrate hydroxyl group
O
O
nucleoside-OH(5') + HO P OO
-
nucleoside
O
P OO-
+
H2O
Figure 10.6 Structures for three types of nucleotides
AMP
ADP
ATP
Nucleotides and nucleic acids
•
•
•
•
In naturally occurring nucleotides and nucleosides, the bond
linking the nitrogenous base to the pentose C1’ atom
(glycosidic bond) extends from the same side of the ribose
ring as the C4’- C5’ bond.
Nucleotide are moderately strong acids.
Nitrogenous bases are planar, aromatic, heterocyclic
molecules.
Derivatives of purine or pyrimidine.
Phosphodiester bonds linking mononucleotides into nucleic acids.
• The phosphodiester
bonds are between
the 3’ carbon and the
5’ carbon of the
second nucleotide.
• This gives direction to
the nucleic acids!!!
• One end has a free 5’
OH
• The other end has a
free 3’ OH
• The 3’,5’ phosphodiester
bonds are highlighted
with green
Structure of AZT and DDI drugs used to treat AIDS
3’-azidodeoxythymidine
2’,3’-dideoxyinosine
DNA base composition follows
Chargaff’s Rules
•
•
•
•
•
•
DNA has equal numbers of adenine and thymine
residues (A = T)
DNA also has equal numbers of guanine and cytosine
residues (G = C)
Structural basis for Chargaff’s rules lie in the hydrogen
bonds between the bases. G always hydrogen bonds
with C and A always forms base pairs with T.
Base composition of a specific organism is characteristic
of that organism (independent of tissue type).
DNA composition varies among different organisms. It
ranges from 25% to 75% G + C in different species of
bacteria.
RNA, when forming duplexes, also follows Chargaff’s
rules
Figure 1-16 Double-stranded
DNA.
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•Each DNA base is hydrogen
bonded to a base on the
opposite strand forming a base
pair.
•A bonds with T and G bonds
with C forming complementary
strands.