Outline of Molecular Biology 2005

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Outline of Molecular Biology 2005 Major
references Genes VIII, 2004 (G) Molecular
Biology (WCB/McGraw-Hill, 3rd Edition, 2005) by
R. Weaver (M) Topics ___________________________
__________________________________ 1.
Introduction (i) Definitions of Molecular
biology, (ii) A brief history From transmission
genetics to molecular genetics to molecular
biology today. (iii) Central dogma, colinearity
of genes and proteins, and genetic code, (iv)
Definition of gene, (v) Approaches to study the
function of genes (classical genetic approach and
reverse genetic approach). M Ch.1,3 G
Ch.1 2. DNA structure and the molecular nature
of genes (i) DNA structure, (ii) physical
chemistry of nucleic acids, (iii) Cot analysis,
genome complexity, and repetitive sequences, (iv)
topology of DNA, (v) packaging of DNA, (vi)
chromosomes and nucleosomes, (vii) organization
of genes on chromosomes. M Ch. 2 G Ch.
2,3,4,19,20 3. Methods in molecular biology
(please avoid those already covered in
Biochemistry). M Ch. 4,5 4. Transcription in
prokaryotes (i) transcription apparatus, (ii)
processes of initiation, elongation and
termination, (iii) operons and regulation. M
Ch.6,7,8 G Ch.9-12 5. Transcription in
eukaryotes (i) eukaryotic RNA polymerase and
their promoters, (ii) trasncriptional factors,
activators and silencers, (iii) regulation. M
Ch.10-13 G Ch. 21-23 6. Post-transcriptional
events (i) splicing, (ii) capping and
polyadenylation, (iii) other events. M
Ch.14,15,16 G Ch.24,25 7. Translation (i)
mRNA, tRNA and ribosomes, (ii) initiation,
elongation, and termination. M Ch. 17,18,19 G
Ch. 5,6,7 8. Protein targeting and
post-translational events. G Ch. 8,27 9. DNA
metabolism (i) DNA replication, (ii) DNA
recombination (homologus recombination,
site-specific recombination, and transposition),
(iii) DNA repair. M Ch. 20-23 G Ch.13-18 10.
Immune diversity. G Ch.26 11. Cell cycle and
growth regulation. G Ch. 29 12. Signal
transduction, gradients and cascades. G Ch. 28,
31 13. Oncogenes and cancer. G Ch. 30 14.
Genomics M Ch.24)
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Molecular BiologyIntroduction

• Definition(s) the study of gene structure and
  function at the molecular level.
• Molecular biology grew out of the disciplines of
  genetics and biochemistry.
• A brief history from transmission genetics to
  molecular genetics to molecular biology.

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(mRNA is discovered in 1961)
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Transmission genetics

• Mendels laws of inheritance (i) genes
  (particulate factor) can exist in different
  alleles, (ii) principle of dominance, (iii)
  principle of segregation, (iv) random assortment.
• The chromosome theory of inheritance (i) if
  chromosomes carry the genes, their number should
  be reduced by half in gametes-and they are, (ii)
  genes are arranged in linear fashion on
  chromosomes, (iii) genes on the same chromosome
  tends to be inherited together, i.e., they are
  linked.
• Genetic recombination and mapping (i) genes on
  the same chromosome may not show perfect genetic
  linkage, (ii) observation of crossing over
  between homologous chromosomes during meiosis,
  (iii) recombination between two homologus
  chromosomes can produce nonparental
  combinations, the farther apart two genes are on
  a chromosome the more likely such recombination
  between them will be. A recombination frequency
  of 1 correspond to a map distance of one
  centimorgan (named after Morgan).
• Physical evidence for recombination a direct
  relationship between a region of chromosome and a
  gene is established.

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Molecular genetics/Molecular Biology

• DNA is the genetic material.
• One gene-one enzyme hypothesis.
• DNA structure is deduced in 1953.
• DNA is replicated semiconservatively and the
  discovery of DNA polymerase.
• How genes function?
• Central dogma paradigm.

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Genetic material can be provided by DNA or RNA

• Central dogma Genes are perpetuated as sequences
  of nucleic acid, but function by being expressed
  in the form of proteins. Replication is
  responsible for the inheritance of genetic
  information. Transcription and translation are
  responsible for its conversion from one form to
  another. Flow of genetic information is usually
  unidirectional, i.e., DNA to RNA to protein.
• Cells use DNA as genetic material, while some
  viruses use RNA.

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Important approaches used in microbial and/or
molecular genetics

• Gene mutations use mutagens to produce
  mutations in any gene of interest. (mutation is
  defined as any change in the nucleotide sequence
  of DNA).
• Genetic analysis by recombination studies (eg.,
  genetic mapping of genes).

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Gene-protein relations

• One gene-one enzyme hypothesis (Beadle and
  Tatum).
• Gene mutations and altered proteins (Ingrams
  work on hemoglobin A).
• Colinearity of gene and protein (Yanofsky) the
  linear sequence of nucleotides in a gene
  determines the linear sequence of amino acids in
  a protein.
• One gene-one protein, to one gene-one
  polypeptide.

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How genes work?

• Proteins are known to be made in ribosomes. So
  there must be a messenger to carry information
  from DNA to ribosomes.
• rRNA as the messenger?
• The discovery of mRNA.
• Transcription the making of RNA from DNA by RNA
  polymerase.
• The discovery of tRNAs (4S RNA), the adaptor
  molecule.
• Deduction of genetic code (i) synthetic mRNA,
  (ii) binding of aminoacyl-tRNA to trinucleotides.
• Translation the making of polypeptide from RNA.

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Use C13 and N15 to label old ribosome
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The nature of multiple alleles

• Mutation in a given gene produces a new allele in
  that gene. Some alleles may produce a
  gain-of-function phenotype, while others may
  produce a loss-of-function phenotype.
• The relationship between two alleles may be
  dominant, recessive, or co-dominant.
• Examples of the ABO blood group.

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Gene, DNA and Chromosomes

• DNA is a chain-like molecule composed of subunits
  called deoxyribonucleotides. Most organisms use
  DNA as genetic material.
• What is gene?
• Genes are are segments of DNA that code for
  polypeptides and RNAs.
• What is chromosome?
• Chromosome is a complex of DNA, RNA and proteins.
  Each chromosome consists of one DNA molecule.

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Classical genetic approach to study the function
of a gene

• Use mutagens (or transposons) to produce
  mutations in a gene of interest, i.e., by
  screening mutants with the desired phenotypes.
• Locate the gene by genetic approaches or newer
  molecular approaches.
• Clone the gene of interest, and study the
  structure of the gene (eg., by sequencing).
• Express the protein product and study its
  biochemical function.

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Reverse genetic approach to study the function of
gene

• Identify the protein of interest (function
  unknown).
• Determine the partial amino acids sequence of the
  protein.
• Deduce the potential nucleotide sequences
  encoding this region of protein.
• Use the deduced nucleotide sequence to design
  probe for searching the gene of interest (eg., in
  a cDNA library).
• Isolate the gene of interest and study the
  structure of gene by DNA sequencing. The function
  of gene may be studied by (i) expressing the
  protein product, and study the function of the
  expressed protein biochemically (ii) other
  approaches such as gene knock-out.

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Objectives of Ist lecture

• To know the major discoveries in the history of
  genetics that are important in the development of
  todays Molecular Biology.
• Students are required to know the definition and
  precise meaning of the following terms frequently
  used in Genetics gene, mutation, allele, locus,
  chromosome, homozygous, heterozygous, linkage,
  crossing-over, genotype and phenotype.
• How to study the structure and function of gene?
  Two examples were provided in this lecture. Are
  there other approaches?

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DNA structures

• Three major families of DNA.
• A-from DNA in low humidity.
• B-form DNA in high humidity, in aqueous
  solution.
• Z-form DNA with specific sequences under special
  conditions.
• Forces stabilizing the duplex.
• Hydrogen bonds (3-6 kcal/mol)
• Base stacking (vertical base-base hydrophobic
  interactions, 3.8-14.6 kcal/mol).

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Models of DNA structure
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Bonds affecting the structure of DNA
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Computer graphic models of A-, B-, and Z-DNA.
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Comparison of A, B, and Z forms of DNA
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Unusual DNA structures

• Z-DNA (repeating units of dinucleotides Pu-Py).
• Hairpins and cruciforms (palindrome or inverted
  sequences).
• H-DNA (polypurine or polypyrimidine tract that
  also contains a mirror repeat, Pu-Py structure)
• G-quartet (G-rich DNA, eg., telomeric DNA)

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H-DNA
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Physical chemistry of DNA

• Denaturation-renaturation The two strand of DNA
  may be separated (denaturation). Two
  complementary single-stranded DNA may unite to
  form duplex (renaturation).
• Hyperchromic shift the absorbance of DNA
  increases about 30-40 upon denaturation.
• Density of DNA depends on GC contents. (satellite
  DNA, separation of CCC and linear DNA).

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DNA content and C-value paradox
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Cot analysis of DNA reassociation
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Reassociation of DNA

• Renaturation of DNA depends on random collision
  of the complementary strands, and follows
  second-order kinetics. The rate of reassociation
  is dependent on temperature and is governed by
  the equation, dC/dt -kC2, where C is the
  concentration of DNA that is single-stranded at
  time t, and k is a reassociation rate constant.
• C/C0 1/1 x k C0 t
• When C/C0 ½, C0 t1/2 1/k
• C0 t1/2 is directly related to the amount of DNA
  in the genome (or called complexity)

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Examples of DNA reassociation

• Lets assume the genome sizes of bacteriophage P
  and bacteria B are 103 and 106 bp, respectively.
  And the DNA sequences are unique (i.e., no
  repetitive sequences).
• Imagine what would be the situation for Co of
  these two DNA? (say the average DNA fragment is
  103 bp and a total of 106 bp DNA is present in
  the reaction mixture).
• Phage DNA should contain 1000 copies of identical
  DNA fragments, but bacterial DNA should contain
  1000 copies of unique DNA fragments (none of them
  are the same).

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Cot curves of various polynucleotides
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The Cot1/2 is directly related to the complexity
(total length of different sequences) in the
genome.
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Hypothetical reassociation curve of a haploid DNA
content of 7 x 108 bp
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Cot curve of calf thymus DNA
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Eukaryotic genomes have several sequence
components

• Nonrepetitive DNA the complexity of the slow
  component corresponds with its physical size,
  i.e., unique sequences.
• Moderately repetitive DNA.component with a
  Cot1/2 of 10-2 and that of nonrepetitive DNA.
  Contains families of sequences that are not
  exactly the same, but are related. The complexity
  is made up of a variety of individual sequences,
  each much shorter, whose total length together
  comes to the putative complexity (eg.,6 x 105 bp
  in the Fig. shown above). Usually dispersed
  throughout the genome.
• Highly repetitive DNA component which
  reassociates before a Cot1/2 of 10-2. Usually
  forms discrete clusters.

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Most structural genes lie in nonrepetitive DNA
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Total gene number is known for several organisms
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How many genes are expressed?

• Hybridization of nonrepetitive DNA with an excess
  of RNA. The proportion of DNA that is bound at
  saturation identifies the complexity of the RNA
  population. Typically 1 of nonrepetitive DNA is
  used as template for mRNA.
• Kinetic analysis of reassociation between cDNA
  and mRNA (see next Figure).
• HDA (high density oligonucleotide arrays)
  analysis.

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The first component is ovalbumin mRNA. The
next component provides 15 of the reaction, with
a total complexity of 15 kb. This corresponds
to 7 to 8 mRNA species of average length of
2000 bp. The last component provides 35 of the
reaction with a total complexity of 26 Mb.This
corresponds to 13,000 mRNA species of average
length of 2000 bp.
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Organelle genomes are circular DNAs that code
for organelle proteins
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Satellite DNA
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Satellite DNA

• Satellite DNA are usually consisted of simple
  sequences that are tandemly repeated many times.
  
• Tandemly repeated sequences are especially liable
  to undergo misalignments during DNA metabolism,
  and thus the sizes of tandem clusters tend to be
  highly polymorphic, with wide variations between
  individuals. Useful in DNA fingerprinting.

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Satellite DNAs often lie in heterochromatin
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Objectives of 2nd lecture

• To know the general DNA structure, the three
  forms of DNA, and some unusual DNA structures.
• To know the bonds affecting DNA structure and
  forces that stabilize the DNAduplex.
• The basic physical chemistry of DNA and their
  applications in understanding the structure of
  genomic DNA.
• What is C-value paradox?
• DNA reassociation kinetics (Cot analysis), genome
  complexity, repetitive and non-repetitive DNA
  sequences, and satellite DNA.
• Most structural genes lie within non-repetitive
  sequences.
• The number of genes in different organisms and
  the number of genes expressed in a given cell
  type. How are these determined?