Fundamental Molecular Biology

1
Fundamental Molecular Biology

• BL 424 Ch 4 Molecular Biology
• Student Learning Outcomes
• Explain essential principles of molecular
  biology
• expression of genetic information DNA ? RNA ?
  protein.
• 2. Explain basic tools of recombinant DNA
• gene cloning, DNA sequencing, PCR.
• 3. Describe tools to detect specific nucleic
  acids and proteins
• Southern, Northern, Western, hybridization
• 4. Describe how tools of recombinant DNA permit
  detailed analysis of gene function in prokaryotes
  and eukaryotes, including construction of
  transgenic organisms

2
The structure of DNA

• DNA is genetic material (Figs. 4.5, 4.6)
• double-helical structure (antiparallel chains),
• complementary bases A-T, C-G
• semi-conservative replication
• 5 ? 3 direction of synthesis
• leading, lagging strands

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Fig 4.2 Chromosomes at meiosis and fertilization

• Eukaryotes most cells of plants, animals are
  diploid
• 2 copies of each chromosome.
• Meiosis segregates chromosomes ? haploid gametes
• Fertilization restores diploid progeny.

• Haploid prokaryotes duplicate DNA
• divide by fission

Fig. 4.2
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Heredity, Genes, and DNA

• Classic Mendelian transmission genetics
• Gene determines polypeptide or structural RNA
• Alleles alternate versions of genes, encode
  traits
• One copy (allele) specifying each trait is
  inherited from each parent.
• Genotype genetic makeup of an individual.
• Phenotype resulting physical appearance.

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Fig 4.1 Inheritance of dominant and recessive
genes

• Ex Parental strains with identical alleles of
  gene specifying yellow (Y) or green (y) seeds,
  are crossed YY x yy.
• Progeny (F1 generation) are hybrids yellow
  seeds
• yellow is termed dominant, green recessive.
• Genotype of F1 generation is Yy.
• Phenotype is yellow.
• Phenotype of F2 shows
• Recessive and dominant
• 2 alleles per individual
• 1 allele per gamete

Fig. 4.1
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Fig 4.3 Gene segregation and linkage

• Dihybrid crosses
• Genes on different chromosomes segregate
  independently
• Genes on same chromosome mostly stay together -
  linked

Fig. 4.3
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Fig 4.8 Colinearity of genes and proteins

• Colinearity of genes and proteins
• revealed by positions of mutations
• 5-end of gene is NH2-end of protein
• 3 end of gene is COOH- end of protein

Fig. 4.8 mutations of TrpA gene of E. coli
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Central dogma

• Central dogma of molecular biology
• Genetic information DNA ? RNA ? Protein
• RNA polymerase synthesizes RNA from
• DNA templates (transcription)
• complementary base pairing T-A, A-U C-G
• Proteins are synthesized on ribosomes from
• mRNA templates (translation)
• Ribosomal RNA (rRNA) sites of
• protein synthesis on ribosomes
• Transfer RNAs (tRNAs) adaptor
• molecules that align charged
• amino acids on mRNA template
• Triplet code 3 nucleotides specify
• 1 amino acid degenerate code

Fig. 4.9
Fig. 4.10
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Fig 4.13 Reverse transcription and retrovirus
replication

• Retroviruses, group of RNA tumor viruses
  replicate via synthesis of a DNA intermediate,
• Forms DNA provirus that integrates in host (Ex.
  HIV)
• RT carried by virus critical for forming DNA copy

• Reverse transcriptase
• (RT) can make DNA copies of any RNA molecule
• (cDNA from mRNA)
• Clone copy of mRNAs of eukaryotic cells to study

Fig. 4.13
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Recombinant DNA

• Recombinant DNA technology (gene cloning)
• Permits isolation, sequence, analysis and
  manipulation of individual genes from any cell.
• Enables detailed molecular studies of structure
  and function of genes and genomes
• Revolutionized understanding of cell biology
• Series of tools
• Restriction enzymes, ligase
• Plasmids, other vectors
• Gel electrophoresis
• Transformation of bacteria,
• Introduction of DNA into other cell types

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Fig 4.14 EcoRI digestion and gel electrophoresis
of ? DNA

• Restriction endonucleases (RE)
• Enzymes cleave DNA at specific sequences
• Ex. EcoRI cleaves 5-GAATTC-3
• About 100 different enzymes
• for specific recognition
• Fragments separated by
• gel electrophoresis
• Smaller molecules move
• more rapidly
• Stain DNA to visualize

Fig. 4.14
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Fig 4.16 Generation of a recombinant DNA molecule
Recombinant DNA gene cloning DNA fragment
inserted into DNA molecule (a vector such as a
plasmid) capable of independent replication in
host cell. Recombinant plasmids introduced into
E. coli (transformation) Select plasmid
(antibiotic resistance) Plasmid replicates
with bacteria get millions of copies in culture
Fig. 4.16
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Fig 4.17 Joining of DNA molecules

• RE often cleave staggered sites, leaving
  overhanging single-stranded regions (5-PO4
  3-OH)
• DNA ligase seals ends (5-PO4 3-OH)

Fig. 4.17
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Fig 4.18 cDNA cloning

• Cloned inserts can be
• genomic DNA or cDNA
• mRNA is copied using
• reverse transcriptase (RT)
• Specific primer is often
• poly(dT) for eukaryotes
• (binds poly(A) on mRNA)
• Add linker sequences for
• easier cloning.

Fig. 4.18
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Fig 4.19 Cloning in plasmid vectors

• Review molecular cloning

Fig. 4.19
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Fig 4.21 Expression of cloned genes in bacteria

• Bacterial expression vectors
• contain regulatable promoters
• Inserted genes are expressed
• at high levels
• Expression in eukaryotic cells
• may be needed if
• posttranslational modifications
• (phosphorylation, sugars)
• are required
• (also needs eukaryotic promoters).
• Consider cloning

Fig. 4.21
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DNA sequencing

• DNA sequencing gives order of bases
• understand genes, genomes, structure, function

Dideoxy method uses premature termination of DNA
synthesis. DNA synthesis is initiated with
synthetic primer. Dideoxynucleotides included
with normal nucleotides each ddNTP labeled
different fluorescent dye ddNTPs stop DNA
synthesis because no 3? OH group for addition
of next dNTP.
Fig. 4.20 ddNTP
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Fig 4.20 DNA sequencing (Part 2)
Dideoxynucleotides stop DNA synthesis because no
3? OH Get series of fragments, partial copies of
target, terminated. Fragments separated by gel
electrophoresis laser beam excites fluorescent
dyes, and records color at each position.
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Detection of Nucleic Acids and Proteins

• 3.Detection of specific nucleic acids, proteins
• Polymerase chain reaction (PCR) amplifies DNA
• Nucleic acid hybridization detects nucleic acids
• Southern DNA on gel
• Northern RNA on gel
• Microarrays - all the mRNAs

• Antibodies detect proteins
• Western proteins on gel
• Immunofluorescence
• Immunoprecipitation

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Detection of Nucleic Acids and Proteins

• Polymerase chain reaction (PCR) amplifies DNA
• Repeated replication of segment of DNA specific
  primers
• Rounds of denature at 95oC,
• anneal to primer (55oC)
• synthesis of DNA (68oC)
• Heat-stable DNA polymerase
• from bacteria of hot springs
• (Thermus aquaticus (Taq)

Fig. 4.23 PCR
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Fig 4.24 Detection of DNA by nucleic acid
hybridization

• Nucleic acid hybridization
• uses complementary
• base pairing to
• detect specific
• nucleic acid sequences
• DNA or RNA probes

Fig. 4.24
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Fig 4.25 Southern blotting

• Southern blotting detects specific genes (DNA).
• DNA digested with RE,
• Fragments separated by gel electrophoresis.
• DNA fragments transferred to membrane (blotted).
• Filter incubated with labeled nucleic acid probe
• Northern blotting
• detects RNA
• separate RNA on gel,
• transfer, hybridize
• with specific probe
• Sizes, amount mRNA
• Different tissues

Fig. 4.25
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Fig 4.26 Screening a recombinant library by
hybridization

• Recombinant DNA libraries collections of clones
  containing all genomic or mRNA sequences of
  particular cell type. (vector can be plasmid,
  virus)
• Ex. Clone random fragments in vector, test for
  specific gene

Fig. 4.26
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Fig 4.27 DNA microarrays

• Hybridization to DNA microarrays allows 1000s of
  genes analyzed simultaneously.
• DNA microarray on glass slide has
  oligonucleotides or fragments of cDNAs printed by
  robotic system in tiny spots
• Compare expression
• in two cell types
• (cancer vs. normal)
• Isolate mRNA
• Use RT then PCR
• with different dyes
• Ex. Cancer red,
• Normal green
• If equal, yellow color

Fig. 4.27
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Fig 4.28 Fluorescence in situ hybridization

• In situ hybridization detects homologous DNA or
  RNA sequences in chromosomes or intact cells.
• Hybridization of fluorescent probes to specific
  cells or
• subcellular structures
• seen by microscope

Different probe for each human chromosome
Fig. 4.28
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Detection of Nucleic Acids and Proteins

• Antibodies detect specific proteins
• Antibodies - proteins from immune cells (B
  lymphocytes) - react to foreign molecules
  (antigens).
• Different antibodies recognize unique antigens
• Antibodies can detect proteins in intact cells.
• Cells stained with antibodies
• labeled with fluorescent dyes,
• or tags visible by electron microscopy.

Fig. 4.31 Human Cells in culture actin (blue),
tubulin (yellow), nuclear stain (red)
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Fig 4.29 Western blotting
Immunoblotting (Western blotting). Proteins
separated by size on SDS-polyacrylamide gel
electrophoresis (SDS-PAGE). SDS detergent binds,
denatures proteins, gives charge Small
proteins faster Transfer to membrane
Antibodies bind to specific proteins
Fig. 4.29
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Fig 4.30 Immunoprecipitation
Immunoprecipitation Purifies specific
proteins. Cells (radioactive proteins) incubated
with antibodies Antigen-antibody complexes are
isolated and electrophoresed. Co-immunoprecipitati
on asks which proteins are bound together in
complexes Antibody purifies one, ask which other
proteins
Fig. 4.30
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Gene Function in Eukaryotes

• Analysis of gene function
• Revealed by altered phenotypes of mutant
  organisms.
• Study function of cloned gene by reintroducing
  it into eukaryotic cells
• Can use specific mutations in genes, deletions of
  genes,
• or add specific genes (can have conditional (ts)
  mutants)
• Use embryonic stem cells in culture, then
  transfer to whole animals or plants
•  Transgenic organisms have altered genomic DNA
• Genetically modified organisms (GMO)

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Fig 4.32 Cloning of yeast genes
Model eukaryote yeast Transform yeast with
plasmids carrying selectable genes (prototrophic,
LEU) Yeast vectors are shuttle vectors that
reproduce in E. coli

• Yeast
• grow as haploid or diploid
• easily grown in culture, reproduce rapidly (90
  min),
• small genome.
• Mutants available for every gene
• ts mutants for essential genes

Fig. 4.32
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Fig 4.33 Introduction of DNA into animal cells
Cloned DNA can be introduced into plant and
animal cells (gene transfer, transfection).
In most cells, DNA is transcribed for several
days transient expression. In 1 or less of
cells, DNA integrates into genome and is stably
transferred to progeny cells (can select)
Fig. 4.33
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Fig 4.34 Retroviral vectors
Animal viruses, especially retroviruses, are
vectors to introduce cloned DNAs into cells.
Fig. 4.34
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Fig 4.35 Production of transgenic mice
Transgenic mice model system Cloned genes in
germ line of multicellular organisms Microinject
cloned DNA into pronucleus of fertilized egg
Check offspring for gene (fur color, check by
Southern blot). Easier to add a new gene
can be inserted anywhere
Fig. 4.35
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Fig 4.36 Introduction of genes into mice via
embryonic stem cells

• Embryonic stem (ES) cells for transgenic mice
• Cloned DNA put into ES cells in culture select
  drug-R
• Stably transformed cells introduced into mouse
  embryos
• Check gene is in germline, transfer to progeny
• Similar techniques to make other transgenic
  animals

Fig. 4.36
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Transgenic plants
Transgenic plants (genetically modified crops,
GMOs) have specific genes added or
deleted. Add DNA to cells in culture with DNA
gun, or use Ti plasmid with Agrobacterium (root
nodule symbiont). Many plants can regenerate
from callus tissue
Fig. 4.37
36
Many GFP transgenic animals and plants now exist
Widespread applications of GFP
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Fig 4.39 Gene inactivation by homologous
recombination

• Specific mutagenesis - homologous recombination
  of synthetic DNA to make particular mutations
• Powerful tool in studying function of eukaryotic
  genes
• Mutate one copy of gene to be cancer-causing
  oncogene
• More difficult to delete both copies (knockout)
• Easier to add a gene

Fig. 4.39 specific mutagenesis
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Fig 4.40 Production of mutant mice by homologous
recombination in ES cells

• Knockout mice
• Transgenic mice with both copies of a gene
  mutated
• Powerful tool
• May be lethal
• Techniques to have KO
• only in some tissues

Fig. 4.40
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Fig 4.41 Inhibition of gene expression by
antisense RNA or DNA

• Antisense nucleic acids
• Use RNA or single-stranded DNA complementary to
  mRNA of the gene of interest (antisense).
• Hybridize with mRNA and block translation into
  protein
• RNA interference (RNAi) (discovered in C.
  elegans)
• injection of double-stranded RNA inhibited
  expression of gene with complementary mRNA
  sequence
• Involves RISC complex binding mRNA, cleaving
  (Fig. 4.36)

Fig. 4.35 antisense
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Chapter 5

• BL 424 Chapter 5 Genomes brief
• Student learning outcomes
• Sequences of many genomes known
• Explain structure of eukaryotic chromosomes
  includes telomeres, centromeres
• Describe how eukaryotic DNA is linear, is
  compacted on nucleosomes (by histones)
• Explain that eukaryotic genes have introns, exons
• much of DNA is noncoding
• Splicing occurs on the primary transcript
• Alternative splicing provides additional proteins

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Fig 5.2 The structure of eukaryotic genes

• Gene coding sequences (exons) are separated by
  noncoding sequences (introns).
• Entire gene is transcribed to RNA introns
  removed by splicing only exons are included in
  mRNA.
• Average human gene 8 introns (gene 27 kb,
  coding 2.5 kb)

Fig. 5.2
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Alternative splicing

• Alternative splicing
• provides diversity of final proteins
• different tissues, different times of development

Fig. 5.3
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DNA is organized in nucleosomes in eukaryotes

• Eukaryotic DNA is linear, organized in
  nucleosomes
• Histones (basic small proteins) bind DNA

Fig. 5.11
44
Review

• Review questions
• 4.7. Starting with 2 sperm, how many copies of a
  specific gene sequence will be obtained after 10
  cycles of PCR? After 30 cycles?
• 4.12. Nucleic acids have net negative charge and
  are separated by electrophoresis on basis of
  size. Proteins have different charges, and so how
  are they separated by size in electrophoresis?
• 4.11. What is critical feature of cloning vector
  that permits isolation of stably transfected
  mammalian cells?
• 5.1. Many eukaryotic organisms have genomic sizes
  much larger than their complexity would seem to
  require explain the paradox.