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FCH 532 Lecture 6

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FCH 532 Lecture 6 Chapter 5 Figure 5-51 A degenerate oligonucleotide probe. Figure 5-52 Colony (in situ) hybridization. Figure 5-53 Chromosome walking. – PowerPoint PPT presentation

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Title: FCH 532 Lecture 6


1
FCH 532 Lecture 6
  • Chapter 5

2
Figure 5-51 A degenerate oligonucleotide probe.
Page 111
3
Figure 5-52 Colony (in situ) hybridization.
Page 113
4
Figure 5-53 Chromosome walking.
Page 113
5
Figure 5-56 Construction of a recombinant DNA
molecule by directional cloning.
Page 117
6
Figure 5-54 The polymerase chain reaction (PCR).
  • Thought up by Kerry Mullis in 1985.
  • Amplify DNA up to 10 kb.
  • Heat denatured DNA is incubated with
  • DNA polymerase
  • dNTPs
  • Two oligonucleotide primers
  • Heat stable poymerases used
  • Taq
  • Pfu

Page 114
7
PCR
  • Amplified DNA can be used for RFLP analysis,
    Southern blotting, and sequencing.
  • Can be used for rapid detection of diseases nad
    mutations.
  • Can be used to identify DNA from hair, sperm,
    blood by amplification of short tandem repeats
    (STRs)-segments of repeating DNA sequence (2 -7
    bp) such as (CA)n and (ATGC)n
  • STRs are genetically variable and can be used as
    markers for individuality. The number of tandem
    repeats of STR are unique to an individual.
  • STRs are amplified from unique sequence outside
    the tandem repeats.
  • RNA can be amplified by PCR first reverse
    transcribing it to DNA (cDNA) through reverse
    transcriptase.

8
Figure 5-57 Site-directed mutagenesis.
Allows for the customization of a
protein. Oligonucleotide containing a short gene
segment with the desired altered base sequence
corresponding to the new amino acid sequence is
used as a primer in the reaction. In this case
used DNA polymerase I. Can also use PCR to
amplify a gene of interest and insert a mutation
in the primer.
Page 118
9
Production of proteins
  • Cloned structural genes can be inserted into an
    expression vector to produce recombinant protein.
  • Relaxed control plasmid with an efficient
    promoter can produce up to 30 of the total
    cellular protein as the inserted structural gene.
  • Inclusion bodies-large amounts of insoluble and
    denatured protein. The protein must be extracted
    and renatured by dissolving in a chaotrope like
    urea or guanididium chloride and slowly
    renaturing the protein.

10
Figure 5-55 Electron micrograph of an inclusion
body of the protein prochymosin in an E. coli
cell.
Page 116
11
Production of proteins
  • Can engineer a signal sequence to target the
    protein to the periplasmic space of the bacteria
    so it folds properly.
  • Toxic proteins can be placed under an inducible
    promoter (lac) promoter in a plasmid that also
    has the gene for the lac repressor protein.
  • Binding of the lac repressor will prevent the
    expression from the lac promoter.
  • After cells have grown to high density, an
    inducer (isopropylthiogalactoside-IPTG, a
    synthetic nonmetabolizable analog of allolactose)
    is added to release the lac repressor protein.

12
Reporter genes can be used to monitor
transcription
  • Rate at which a gene is expressed dependent on
    upstream control sequences.
  • Replace the gene you want to monitor with a
    reporter gene.
  • Reporter genes encode proteins that can be easily
    detected by some assay. lacZ can be assayed
    with x-gal and the production of blue color.
  • Another reporter is the green fluorescent protein
    (GFP) which produces a bioluminiscent protein
    when irradiated with UV or 400nm light.

13
Replace geneX with reporter gene in the correct
reading frame
In the presence of X-gal, expression will produce
the blue color.
14
Figure 5-58 Use of green fluorescent protein
(GFP) as a reporter gene.
Page 119
15
Transgenic organisms
  • Organisms expressing a foreign gene are
    considered transgenic.
  • Foreign gene referred to as transgene.
  • For the change to be permanent, transgene must be
    stably integrated into germ cell.
  • Established in mice by microinjection of DNA into
    a pronucleus of a fertilized ovum.
  • Can also be accomplished in an embryonic stem
    cell.

16
Figure 5-59 Microinjection of DNA into the
pronucleus of a fertilized mouse ovum.
Page 119
17
Nucleic acid sequencing
  • Development of DNA sequencing techniques has
    spurred the huge amount of DNA sequence data (gt35
    billion nucleotides in 2003 and growing!)
  • Complete genomes determined for over 110
    prokaryotes and over 11 eukaryotes.

18
Nucleic acid sequencing
  • Development of DNA sequencing techniques has
    spurred the huge amount of DNA sequence data (gt35
    billion nucleotides in 2003 and growing!)
  • Complete genomes determined for over 110
    prokaryotes and over 11 eukaryotes.

19
Table 7-3a Some Sequenced Genomes
Page 177
20
Table 7-3b Some Sequenced Genomes.
Page 177
21
Nucleic acid sequencing
  • Chain terminator method (aka dideoxy sequencing)-
    used to sequence long stretches of DNA.
  • Utilizes DNA polymerase to synthesize single
    stranded DNA.
  • Assembles the four deoxynucleoside triphosphates
    (dNTPs) into a complementary sequence.
  • Initiates from a primer sequence.
  • Sequence is terminated after the incorporation of
    23-dideoxynucleoside triphosphate (ddNTP)

P
Base
P
P
OCH2
O
H
H
H
H
H
H
22
Figure 7-14 Flow diagram of the chain-terminator
(dideoxy) method of DNA sequencing.
Page 178
23
Figure 7-15 Autoradiograph of a sequencing gel.
A G C T A G C T
Page 179
24
  • Chain-terminator method has been automated.
  • Instead of radioactivity, use fluoresence-labeling
    techniques.
  • 2 types used
  • Four reaction/one gel systems - primers used in
    each of the four chain extension reactions are
    5-linked to a differently fluorescing dye.
    Loaded into a single lane of a gel. As each
    exits, the fluorescence is detected.
  • One reaction/one gel system - Each of the four
    ddNTPs used to terminate the chain extension is
    linked to a different fluroescing dye. The
    extension is carried out in a single vessel and
    the mixture is loaded into a single lane.
  • Advanced systems use capillaries instead of slab
    gels.

Page 179
25
Genome sequencing
  • In order to sequence entire genomes, segments
    need to be assembled into contigs (contiguous
    blocks) to establish the correct order of the
    sequence.
  • Chromosome walking may be one way to do so, but
    is prohibitively expensive.
  • Two methods have been used recently
  • 1. Conventional genome sequencing-low resolution
    maps made by identifying landmarks in 250 kb
    inserts in YACs.
  • Landmarks are 200-300 bp segments, aka sequence
    tagged sites(STSs)-2 clones with the same STS
    overlap.
  • STS-containing inserts are sheared randomly into
    40kB segments and cloned into cosmid
    vectors-used to create high resolution maps.
  • The cosmid inserts are fragmented to smaller
    sizes and sequenced.
  • Cosmid inserts are assembled by using the STS
    sequence overlaps and cosmid walking.
  • Cannot be used effectively with sequences
    containing high amounts of repetitive sequence.
    (Use expressed sequence tags (ESTs)).

26
Genome sequencing
  • 2. Shotgun strategy-
  • genome library is randomly fragmented
  • large amount of cloned fragments are sequenced.
  • Genome is assembled by identifying overlaps
    between pairs of fragments.
  • The probability that a base is not sequenced is
    e-c,
  • c is the redundancy of coverage, c LN/G,
  • where L is the average length of the cloned
    inserts in base pairs,
  • N is the number of inserts sequenced,
  • and G is the length of the genome in base pairs.
  • The aggregate length of the gaps between contigs
    is G e-c and the average gap size is G/N.
  • Bacterial genomes-shotgun strategy is
    straightforward. Gaps are filled in by
    synthesizing PCR primers and finishing a genome.
  • Eukaryotic genomes-larger size so it must be
    carried out in stages using BACs and then
    identifying 500 bp sequences from each to yield
    sequence tagged connnectors (STCs or BAC ends)
  • This allows assembly via the overlapping of STCs.

27
Figure 7-17 Genome sequencing strategies.
Page 180
28
Human genome
  • 2.2 billion nucleotide sequence 90 complete
    because of highly repetitive sequence.
  • About half of the human genome consists of
    various repeating sequences.
  • Only 28 of the genome is transcribed to RNA
  • Only 1.1 to 1.4 of the genome (5 of the
    transcribed RNA) encodes protein.
  • Only 30,000 protein encoding genes (open reading
    frames or ORFs) identified. Predicted 50,000 -
    140,000 ORFs.
  • Only a small fraction of human protein families
    are unique to vertebrates most occur in other
    life forms.
  • Two randomly selected human genomes differ, on
    average, by only 1 nucleotide per 1250 that is,
    any 2 people are likely to be gt99.9 identical.

29
Human genome
  • 2.2 billion nucleotide sequence 90 complete
    because of highly repetitive sequence.
  • About half of the human genome consists of
    various repeating sequences.
  • Only 28 of the genome is transcribed to RNA
  • Only 1.1 to 1.4 of the genome (5 of the
    transcribed RNA) encodes protein.
  • Only 30,000 protein encoding genes (open reading
    frames or ORFs) identified. Predicted 50,000 -
    140,000 ORFs.
  • Only a small fraction of human protein families
    are unique to vertebrates most occur in other
    life forms.
  • Two randomly selected human genomes differ, on
    average, by only 1 nucleotide per 1250 that is,
    any 2 people are likely to be gt99.9 identical.

30
Chemical evolution
  • Evolutionary aspects of amino acid sequences.
  • Change stem from random mutational events that
    alter a proteins primary structure.
  • Mutational change must offer a selective
    advantage or at least, not decrease fitness.
  • Most mutations are deleterious and often lethal
    so they are not reproduced.
  • Sometimes mutations occur that increase fitness
    of the host in its natural environment.
  • Example Sickle-cell anemia.

31
Figure 7-18a Scanning electron microscope of
human erythrocytes. (a) Normal human erythrocytes
revealing their biconcave disklike shape.
Page 183
32
Figure 7-18b Scanning electron microscope of
human erythrocytes. (b) Sickled erythrocytes from
an individual with sickle-cell anemia.
Page 183
33
Figure 7-20 A map indicating the regions of the
world where malaria caused by P. falciparum was
prevalent before 1930.
Page 184
34
Chemical evolution
  • Pauling and co-workers showed that normal human
    hemoglobin (HbA) is more electronegative than
    sickle-cell hemoglobin (HbS).
  • Sickle-cell anemia is inherited according to the
    laws of Mendelian genetics.
  • Homozygous for HbS is almost all HbS,
    phenotypesickle cell anemia.
  • Heterozygous for HbS is 40 HBs,
    phenotypesickle cell trait.
  • Homozygous for HbA, normal human hemoglobin.

35
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36
Mutations in a- or b-globin genes can cause
disease state
  • Sickle cell anemia E6 to V6
  • Causes V6 to bind to hydrophobic pocket in
    deoxy-Hb
  • Polymerizes to form long filaments
  • Cause sickling of cells
  • Sickle cell trait offers advantage against
    malaria
  • Cells sickle under low oxygen conditions and if
    infected with Plasmodium falciparum.
  • Causes the preferential removal of infected
    erythrocytes from circulation.

37
Variations in homologous proteins
  • Similar proteins from related species likely
    derived from the same ancestor.
  • A protein that is well adapted to its function
    will continue to evolve.
  • Neutral drift-mutational changes in a protein
    that dont affect its function over time.
  • Homologous proteins-evolutionarily related
    proteins.
  • Comparison of the primary structures of
    homologous structures can be used to identify
    which residues are essential to its function,
    lesser significance, and little function.
  • Invariant residue-the same side chain at a
    particular position in the amino acid sequence of
    related proteins.
  • If an invariant residue is observed between
    related proteins, it is likely necessary to some
    essential function of the protein.
  • Other amino acids may have less stringent side
    chain requirements-where amino acids may be
    conservatively substituted-(be substituted with
    an amino acid with similar properties).
  • If many amino acids tolerated at a specific
    position - hypervariable.

38
Cytochrome c
  • Cytochrome c is nearly universal eukaryotic
    protein necessary for electron transport.
  • Vertebrates 103-104 residues up to 8 more aas in
    other phyla.
  • Similarities are observed in an alignment.
  • 38 of 105 residues are invariant and the others
    are conservatively substituted.
  • 8 positions are hypervariable.
  • His 18 and Met 80 form bonds with the redox Fe of
    the heme group.

39
Table 7-4a Amino Acid Sequences of Cytochromes c
from 38 species.
Page 184
40
Page 185
41
Cytochrome c
  • Evolutionary differences between two homologous
    proteins are determined by counting the amino
    acid differences between them.
  • Order of differences parallels taxonomy and can
    be put into a table.
  • This data can be used to construct a phylogenetic
    tree-a tree that indicates ancestral
    relationships among organisms and their proteins.

42
Page 186
43
Figure 7-21 Phylogenic tree of cytochrome c.
  • Each branch point indicates a possible common
    ancestor to everything above it.
  • Relative evolutionary distances between
    neighboring branch points are expressed as the
    number of amino acid differences per 100 residues
    of the protein (percentage of accepted point
    mutations or PAM units).

Page 187
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