FCH 532 Lecture 2

Webpage: http://www.esf.edu/chemistry/nomura/fch532/

Genetics reviewChapter 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.

Figure 1-30 Replica plating.

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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.

Figure 1-31 The life cycle of a virus.

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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”

Figure 1-32 Screening for viral mutants.

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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.

Figure 1-33 Viral recombination.

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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 cis-trans test.• Determined the the unit of recombination is about the size of a single

base pair!

Figure 1-34 The cis-trans test.

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Nucleotides and nucleic acids

1. Form monomeric units of nucleic acids-storage and expression of genetic information.

2. 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 energy-requiring reactions in the cell.

3. Most metabolic pathways are regulated in part by levels of nucleotides such as ATP and ADP.

4. Nucleotide derivatives like nicotinamide adenine dinucleotide (NADH), flavin adenine nucleotide (FAD) and coenzyme A are required for many reactions.

5. Have catalytic activities in in enzymelike nucleic acids (ribozymes).

• 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.

10.1 RNA and DNA Chemical Structures

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) D-Ribose b) D-2-Deoxyribose

Figure 5-1 Chemical structures of (a) ribonucleotides and (b) deoxyribonucleotides.

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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 N-glycosidic 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)

nucleoside-OH(5') HO P

O

O-

O- nucleoside P

O

O-

O-

O H2O

• Linkage of nitrogenous base and aldopentose forms nucleoside.

• Nucleotide is formed when phosphoryl group is linked to carbohydrate hydroxyl group

Figure 10.6 Structures for three types of nucleotides

AMPADPATP

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.