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Control of Gene Expression

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									         BIOTECHNOLOGY UNIT:
• “The Immortal Life of Henrietta Lacks”
• Dr. Wayne Grody- Guest Speaker
• “GATTACA”
• Ch18&19 Pro&Eukaryotic control of gene expression
• Ch11 Cell Signaling Cell Communication
• Ch20&21 Biotechnology…manipulating 
  microorganisms to do our bidding!!!! Tools/techniques
• LAB 6 MOLECULAR BIOLOGY
    – Bacterial transformation
    – Restriction Enzymes/DNA Digests
    – Gel Electrophoresis (DNA fingerprinting)
• BIOTECH RESEARCH PAPER & SYMPOSIUM
Control of Gene Expression

         Chapter 18
                   VOCABULARY
 Hetero-       Eu-          Methylation   Transcrition   Histones
 chromatin     chromatin                  factors        nucleosome
                                                         s
 Operon/       Gene         Inducible/    ATP            Jacob & 
 Operator/     experssion   Repressible   ADP            Manod
 Promoter                                 AMP   cAMP
 Galactosida
 se
               Glucose
                      VOCABULARY
                            Galactose     Lactose        Transcription 
                                                         factor
 Negative      Positive     Inducer/      CRP            Regulatory 
 regulation    regulation   Corepressor                  Gene
                   QUESTIONS TO ANSWER

Who?      What? Where? When               Which     Why? How?
                       ?                  ?
                   Q & A
• Q:What is gene expression?
• A: Activating a gene to produce a protein.
• Q: What is an operon?
• A: The genes for creating an enzyme and the
  genes that regulate their transcription.
• Q: What makes up an operon?
• Answer:
  1) Promoter region
  2) Operator region
  3) Structural genes
          OPERON
Promoter, Operator, Structural 
           Genes 
   Control of gene expression
• The expression of genetic material controls cell 
  products, and these products determine the 
  metabolism and nature of the cell. 
• Gene expression is regulated by both 
  environmental signals and developmental 
  cascades or stages. 
• Cell signaling mechanisms can also modulate 
  and control gene expression. 
• Thus, structure and function in biology involve 
  two interacting aspects: the presence of 
  necessary genetic information and the correct 
  and timely expression of this information.
Gene expression 
 can be under:
a) Negative
   regulation: When
   the operon is
   turned off by
   chemicals.
repressible or
   inducible
          or
b) Positive
   regulation: When
   gene expression is
   stimulated by
        2 Types of Negative 
      Regulation Part 1: The trp
               operon
        (repressible operon)
•   What does repressible mean?
•   This operon is located in E. coli bacteria.
•   The purpose of the operon is to create the
    enzymes that synthesize the amino acid
    tryptophan.
•   When the operon is “ON:
1) RNA polymerase binds to the promoter region.
2) RNA polymerase crosses over the operator region because 
      the repressor is inactive and therefore does not bind to the o
3) The structural genes are transcribed and tryptophan is synthesiz
OPERON “ON”
    Switching the trp operon “off”
•  Done by a repressor: a protein that binds to
   the operator region.
• It blocks the attachment of RNA polymerase to
   the promoter region.
1) A gene called a regulatory gene (located away 
   from the operon) produces the trp operon
   repressor.
2) The repressor protein is allosteric having an
   active and inactive state/shape.
3) At first the repressor protein is in its inactive form.
4) When the amino acid tryptophan binds to the 
  repressor the repressor becomes its active form and
  binds to the operator region… blocking
  transcription.
5) Since tryptophan assists in turning the operon off it
  is called a co-repressor.
6) When the levels of tryptophan drop the repressor
  loses its tryptophan, changes shape, and the
  repressor is released from the operator , initiating
TURNING THE OPERON 
        OFF 
         regulatory gene              operator




Regulatory gene codes for the repressor which may bind to the operator
Types of Negative Regulation 
Part 2: The lac operon
(inducible operon)
• What does inducible mean?
• This operon is also located in E. coli bacteria.
• It was first discovered by: Francois Jacob and 
  Jacques Monod (1961) called the “Jacob and
  Monod model”
• The purpose of the operon is to produce the enzyme 
  B-galactosidase that splits (via hydrolysis) lactose
  into glucose and galactose.
• This operon is normally off because the repressor
  protein is formed in its active shape, thus binds to the
  operator region blocking RNA polymerase.
• If the RNA polymerase is being blocked 
  how does transcription ever occur???
 Done by an inducer: a molecule 
 that binds to and inactivates the 
                   repressor.
1) The molecule allolactose (an isomer of lactose) binds to
     the repressor, inducing an allosteric change.
2)   The repressor is released from the operator region.
3)   The RNA polymerase can move along the template
     strand catalyzing the synthesis of mRNA.
4)   When the lactose levels decrease the repressor binds to
     the operator region and transcription is shut down.
Figure 18.21a  The lac operon: regulated synthesis of inducible enzymes
Figure 18.21b  The lac operon: regulated synthesis of inducible enzymes
     Compare and contrast:
• The enzymes produced by the trp 
  operon are called repressible enzymes
  and are involved in anabolic pathways.
• The enzymes produced by the lac 
  operon are called inducible enzymes
  and are involved in catabolic pathways.
      Types of Positive
         Regulation: 
A closer look at the lac operon

• In order for the lac operon to 
  produce enzymes in large 
  quantities, a second factor must 
  exist… 
• a low concentration of glucose.
       How does E. coli sense the low levels of
      glucose and how is this relayed to the lac
                      operon?




By the molecule cyclic AMP or cAMP.
cAMP is present in large quantities when the glucose levels are
    low.
1) The cAMP binds to an allosteric protein called cAMP
    receptor protein or CRP
2) The activated CRP binds to a site within the lac promoter
    adjacent to the TATA box.
3) The attachment of CRP makes it easier for RNA
    polymerase to bind to the promoter region.
Figure 18.22a  Positive control: cAMP receptor protein
        Figure 18.22b  Positive control: cAMP receptor protein




CRP is known as an activator protein because it
activates transcription. 

If the levels of glucose increase the levels of
cAMP decrease and the CRP is released from its
binding site.
Figure 18-22x  cAMP
Figure 18.20b  The trp operon: regulated synthesis of repressible enzymes (Layer 2)
Figure 18.21b  The lac operon: regulated synthesis of inducible enzymes
Figure 18.22a  Positive control: cAMP receptor protein
Figure 18.22b  Positive control: cAMP receptor protein
 The Organization and Control 
   of Eukaryotic Genomes
          Chapter 19
    How is eukaryotic gene expression
     different from prokaryotic gene
                expression?
1. Importance of cell specialization in multicelluar Euk’s.
2. Greater size of genome of Euk’s and chromatin 
   structure:
-single, circular, chromosome in PROKARYOTES
-double, linear, protein enhanced in EUKARYOTES* 
   Histones/Nucleosomes = DNA is coiled around 
   bundles of 8 or 9 histone proteins to form DNA-histone 
   complexes called nucleosomes. 
       1. Euchromatin = regions where DNA is loosely 
   bound                 to nucleosomes and is actively 
   transcribed.
       2. Heterochromatin = regions where 
   nucleosomes are                   more tightly 
Figure 19.1  Levels of chromatin packing
DNA Packing is the first level
of control of eukaryotic gene
         expression
Figure 19.0  Chromatin in a developing salamander ovum
     Only _3_% of 
  eukaryotic DNA is 
translated into protein 
products, compared to 
  almost _100_% of 
   prokaryotic DNA. 

       Sizewise…
     several million 
    nucleotide pairs
           vs.
   2 x 10 8 pairs per 
      chromosome
(that’s x 46 in humans!)
             Figure 19.x1a  Chromatin




HETEROCHROMATIN                EUCHROMATIN
•      Repetitive DNA = noncoding segments not 
    transcribed within a gene
           » CENTROMERE- center
           » TELOMERE- ends (telomerase)
           » PSEUDOGENES = not transcribed, almost
             identical to a coding gene. May represent 
             evolutionary precursor—mutated over the years. 
•       TRANSPOSONS or “jumping genes”= can move 
    to a new location on the same chromosome or to a 
    different chromosome. Discovered by Barbara
    McKlintock (maise)
           Have the effect of a mutation… can change the 
              expression of a gene
           1. Turn on or off its expression
           2. Have no effect at all
        Enduring understanding 3.B:
 Expression of genetic information involves
    cellular and molecular mechanisms.


• Essential knowledge 3.B.1:
• Gene regulation results in differential
  gene expression, leading to cell
  specialization.
        Enduring understanding 3.B:
 Expression of genetic information involves
    cellular and molecular mechanisms.


• Essential knowledge 3.B.1:
• Gene regulation results in differential
  gene expression, leading to cell
  specialization.
    Both DNA regulatory sequences,
      regulatory genes, and small
    regulatory RNAs are involved in
           gene expression.
• Regulatory sequences are stretches of DNA 
  that interact with regulatory proteins to control 
  transcription. (ex. promoter, terminator, 
  enhancer)
• A regulatory gene is a sequence of DNA 
  encoding a regulatory protein (repressor & 
  activator) or RNA (miRNA & siRNA) blocks 
  translation on a transcribed mRNA by binding 
  to it.
  – Micro RNA
  – Small Interfering RNA
– RNA interference (RNAi) is a biological
  process in which RNA molecules inhibit
  gene expression, typically by causing the
  destruction of specific mRNA molecules.
– Two types of small ribonucleic acid (RNA) 
  molecules – microRNA (miRNA) and small 
  interfering RNA (siRNA) – are central to RNA 
  interference. RNAs are the direct products of 
  genes, and these small RNAs can bind to 
  other specific messenger RNA (mRNA) 
  molecules and either increase or decrease 
  their activity, for example by preventing an 
  mRNA from producing a protein. RNA 
  interference has an important role in 
  defending cells against parasitic nucleotide 
  sequences – viruses and transposons – but 
  also in directing development as well as 
  gene expression in general.
   In eukaryotes, gene expression is
     complex and control involves
regulatory genes, regulatory elements
  and transcription factors that act in
                   concert.
• Transcription factors bind to specific DNA 
  sequences and/or other regulatory 
  proteins.
• Some of these transcription factors are 
  activators (increase expression), while 
  others are repressors (decrease 
  expression).
• The combination of transcription factors 
  binding to the regulatory regions at any 
  one time determines how much, if any, of 
       Controlling Eukaryotic Gene
                Expression
• Under positive control
• Transcription will not take 
  place without the
  assembly of the
  transcription complex
• Transcription Factors  
  are regulatory proteins
  that bond to the
  enhancer region, the        • Once the transcription factor
  promoter (TATA box)
                              have assembled around the 
  and to each other.
                              promoter, they are called a
                              transcription complex.
       Areas that regulate eukaryotic
               transcription:
a) The Enhancer Region: causes the chromosome to 
   loop and make contact with the Promoter regions. 
• Located thousands of nucleotides away from the
   promoter. 
• Activator proteins bind to the enhancer regions and 
   then to the transcription complex after the DNA loops.
• When the activator proteins (special transcription 
   factors) bind to the transcription complex,  RNA
   polymerase is positioned over the promoter region
   and the rate of transcription increases.
b) The Silencer Region: a repressor
   region.
• Located close to the enhancer region.
• Repressor Proteins bind to the 
   silencer sites prevent the activator
   proteins from binding to the enhancer
   region.
Turning On A Gene
Turning On A Eukaryotic 
         Gene
Figure 19.8  A eukaryotic gene and its transcript
ALTERNATIVE RNA SPLICING
Control of Translation
Protein Processing
cancer
•   What is cancer? 
•   Unregulated cell growth and division.
•   What causes cancer? 
•   Damage to the genes regulating the cell 
    division cycle. 
       • Usually by carcinogens (cancer causing 
         agents).
• Tumor = a cluster of cancerous cells
• Metastases = When cells leave the tumor, 
  spread, grow new tumors. 
• Sarcomas = Tumors of the cells in connective 
  tissue, muscle or bone.
• Carcinoma = Tumors of cells in epithelial tissue 
  like skin. 
• The three deadliest human cancers:
      • Lung …smoking
      • Colorectal …diet
      • Breast…causes is still unknown, however 
        some forms are inherited as the genes 
        BRCA1 and BRCA 2.
• Genes that cause cancer are called 
  oncogenes.
• The normal versions of these genes are called 
  proto-oncogenes and they code for proteins 
  that stimulate normal cell growth and division.
• How do proto-oncogenes become 
  oncogenes?
     • Translocation, or movement of fragments of 
       chromosomes(break off and attach somewhere else) 
       that result in being around an active promoter.
     • Amplification, or increasing the number of copies of the 
       gene in the cell. 
• Two genes that are significant: ras  gene and 
  the p53 gene. 
• The ras gene creates a protein that influences 
  the cell cycle. 
• The mutated ras protein is hyperactive, 
  leading to excessive cell division. 
• The p53 gene becomes active when DNA is 
  damaged and creates a tumor suppressing 
  protein. 
• Mutating the p53 gene can lead to the 
  formation of tumors. 
• Chemicals in cigarette smoke induce p53
• 15% of the cancers worldwide are associated 
  with viral infections. Certain viruses can insert 
  oncogenes others may insert DNA into proto-
  oncogenes, turning them into oncogenes. 
Figure 19.13  Genetic changes that can turn proto-ocogenes into oncogenes
Figure 19.14  Signaling pathways that regulate cell growth (Layer 1)
Figure 19.14  Signaling pathways that regulate cell growth (Layer 2)
Figure 19.14  Signaling pathways that regulate cell growth (Layer 3)
Figure 19.15  A multi-step model for the development of colorectal cancer
• The basic structure of viruses includes 
  a protein capsid that surrounds and 
  protects the genetic information 
  (genome) that can be either DNA or 
  RNA. 
• Viruses have a mechanism of 
  replication that is dependent on the host 
  metabolic machinery to produce 
  necessary viral components and viral 
• Some classes of viruses use RNA 
  without a DNA intermediate; however, 
  retroviruses, such as HIV, use a DNA 
  intermediate for replication of their 
  genetic material. 
• Some viruses introduce variation into 
  the host genetic material. 
  – When the host is bacterial, it is referred to 
    as lysogenesis; 
  – whereas in eukaryotic cells, this is referred 
    to as transformation. 
• Since viruses use the host metabolic 
  pathways, they experience the same potential 
  as the host for genetic variation that results 
  from DNA metabolism.
        SUMMARY
Viral & Bacterial Control of 
     Gene Expression

 CHECK FOR UNDERSTANDING
  Both positive and negative control
       mechanisms regulate gene
       expression in bacteria and
                      viruses.
1. The expression of specific genes can be turned on by 
     the presence of an _________. 
2.   The expression of specific genes can be inhibited by 
     the presence of a _________. 
3.   Inducers and repressors are small molecules that 
     interact with ____________ and/or regulatory 
     sequences.
4.   Regulatory proteins inhibit gene expression by 
     binding to _________and blocking transcription 
     (negative control).
5.   Regulatory proteins stimulate gene expression by 
     binding to DNA and stimulating transcription 
     (___________) or binding to _________ to inactivate 
     repressor function.
6.   Certain genes are continuously expressed; that is, 
    Both positive and negative control
         mechanisms regulate gene
         expression in bacteria and
•                      viruses.
    The expression of specific genes can be turned on by 
    the presence of an inducer. 
•   The expression of specific genes can be inhibited by 
    the presence of a repressor. 
•   Inducers and repressors are small molecules that 
    interact with regulatory proteins and/or regulatory 
    sequences.
•   Regulatory proteins inhibit gene expression by binding 
    to DNA and blocking transcription (negative control).
•   Regulatory proteins stimulate gene expression by 
    binding to DNA and stimulating transcription (positive
    control) or binding to repressors to inactivate 
    repressor function.
•   Certain genes are continuously expressed; that is, 
    they are always turned “on,” e.g., the ribosomal
Gene regulation accounts for some of
 the phenotypic differences between
    organisms with similar genes.
QUESTIONS:
1. describe the connection between the regulation of gene
   expression and observed differences between different
   kinds of organisms.
2. describe the connection between the regulation of gene
   expression and observed differences between individuals
   in a population.
3. explain how the regulation of gene expression is
   essential for the processes and structures that support
   efficient cell function.
4. use representations to describe how gene regulation
   influences cell products and function.
Gene regulation accounts for some of
 the phenotypic differences between
    organisms with similar genes.
QUESTIONS:
1. describe the connection between the regulation of gene expression and observed
   differences between different kinds of organisms. Structure and function in biology 
   result from the presence of genetic information and the correct expression of this 
   information. 
2. describe the connection between the regulation of gene expression and observed
   differences between individuals in a population. The expression of the genetic material 
   controls cell products, and these products determine the metabolism and nature of the cell. 
   Most cells within an organism contain the same set of genetic instructions, but the 
   differential expression of specific genes determines the specialization of cells. 
3. explain how the regulation of gene expression is essential for the processes and
   structures that support efficient cell function. Some genes are continually expressed, 
   while the expression of most is regulated; regulation allows more efficient energy utilization, 
   resulting in increased metabolic fitness. 
4. use representations to describe how gene regulation influences cell products and
   function. Gene expression is controlled by environmental signals and developmental 
   cascades that involve both regulatory and structural genes. A variety of different gene 
   regulatory systems are found in nature. Two of the best studied are the inducible and the 
   repressible regulatory systems (i.e., operons) in bacteria, and several regulatory pathways 
   that are conserved across phyla use a combination of positive and negative regulatory 
   motifs. In eukaryotes, gene regulation and expression are more complex and involve many 
   factors, including a suite of regulatory molecules.
DNA TECHNOLOGY AND 
     GENOMICS
      Chapter 20
 Ch 20 & 21   VOCABULARY
 put a + by the terms you know and – by the ones you don’t.
GMO    Clone         Vaccine        biotechnology   Genetic
                                                    engineering
GFP    ligation      plasmid        endonuclease    technology
PCR    insulin       Gene           Transgenic      Gel 
                     expression                     electrophoresis
DNA    Gene          Restriction    Somatic cell     DNA Fingerprint
       therapy       enzyme         Nuclear transfer
HGH    Dolly         vector         Biomedical      agriculture
RFLP   interleukin   ethical        interferon      transformation


GENERATE YOUR OWN QUESTIONS:
1pt question words: Who? What? Where? When? 
2pt question words: Which? How? 
3pt question words: Why? 
Genetic Engineering is the application of 
   molecular genetics for practical purposes.
Uses:
1) Identify genes for specific traits
2) Transfer genes for a specific trait from one 
   organism to another.
Tools for manipulating genes:
1) Restriction enzymes (endonucleases)
2) Cloning vector (bacterial plasmid)
Transgenic/Recombinant organisms contain 
   DNA that was not part of their original 
                genome.


                    Green fluorescent 
                    protein (GFP) is 
                    responsible for the 
                    green bioluminescence 
                    of the jellyfish Aequorea
                    victoria.

                    This is a GM mouse!
    5. The genetic composition of cells can be
altered by incorporation of exogenous DNA into
   the cells. As a basis for understanding this
                     concept:
a.Students know the general structures and functions 
  of DNA, RNA, and protein.
b. Students know how to apply base-pairing rules to 
  explain precise copying of DNA during 
  semiconservative replication and transcription of 
  information from DNA into mRNA. 
    5. The genetic composition of cells can be
altered by incorporation of exogenous DNA into
   the cells. As a basis for understanding this
                     concept:
c.  Students know how genetic engineering (biotechnology) is 
   used to produce novel biomedical and agricultural products. 
d.* Students know how basic DNA technology (restriction 
   digestion by endonucleases, gel electrophoresis, ligation, 
   and transformation) is used to construct recombinant DNA 
   molecules. 
e.* Students know how exogenous DNA can be inserted into 
   bacterial cells to alter their genetic makeup and support 
   expression of new protein products.
Recombinant organisms contain 
 DNA that was not part of their 
      original genome.
                  The green 
                  fluorescent protein 
                  (GFP) created by 
                  these transgenic 
                  mice is responsible 
                  for the green 
                  bioluminescence of 
                  the jellyfish
                  Aequorea victoria.
              This GMO is a GM mouse!
    Practical Uses of DNA
        Technology:
• Gene Therapy
• Pharmaceuticals- HGH, Interferons, 
  Interleukins etc.
• Vaccines- solution that contains a 
  harmless version of a virus or bacterium 
  to stimulate an immune response & 
  formation of “memory” cells.
• Increased Agricultural Yields- ex. crops 
  that don’t need fertilizer.
           Ethical Issues
• Describe two potential safety and 
  environmental problems that could 
  result from genetic engineering.
Golden rice contains beta-carotene, which our bodies use to make vitamin A… 
normal rice does not. Vitamin A deficiency results in blindness & lowered immunity. 
Figure 20.x2  Injecting DNA



                               Somatic
                               Cell
                               Nuclear
                               Transfer




                              What does that mean
“Pharm” animals create other species proteins secreted in their milk, ex. spider’s silk.
Figure 20.16  One type of gene therapy procedure
  We owe the field of Genetic 
Engineering to the bacterial cell.
    1) Plasmids: small circular 
          pieces of DNA.

Plasmids often contain 
      genes for antibiotic
      resistance, thus 
protection from fungi.
Ex. Penicillin
        Ampicillin, 
      Amoxycillin
2) Conjugation:
        • Bacteria exchanging 
          plasmids.
        • Pili = cytoplasmic 
          extensions used to 
          contact other 
          bacteria.
        • Plasmids are 
          exchanged through 
          the pili.
     3) Restriction endonucleases
• Molecular “scissors” 
   that cut DNA at specific 
   sequences.
• Provide protection for 
   bacteria against viruses.
* More details to follow.
 
4)Transformation:
        •  bacteria can incorporate new 
           DNA into their genome from 
           the external environment.
        We will do this in the next lab
        • Give E. Coli a plasmid that 
           contains:
        1) Gene for Ampicillin 
           resistance protein
        2) Gene for B galactosidase 
           enzyme to digest xgal sugar 
           to make blue protein.
  How restriction 
endonucleases work
         • The enzyme will 
           recognize a specific 
           sequence of DNA & 
           cut it at that specific 
           place in a specific 
           way.
• For example E. coli bacteria have a restriction 
  endonuclease called EcoRI.
• It will recognize the site:      5’-- GAATTC--3’
                                    3’--CTTAAG--5’
• Many recognition sites are palindrome sequences- read
  the same forwards/backwards- see 2 strands.
• It will cut the DNA between:    G    AATTC
                                     CTTAA    G
• The results are “sticky ends” or short, single strands of 
  bases.
• If two complementary sticky ends pair up, they can be 
  joined by DNA ligase.
Restriction Enzymes
RECOMBINANT PLASMIDS
          • Stanley Cohen and 
            Herbert Boyer created 
            the first recombinant 
            plasmids in 1973.
          • In one of the first 
            recombinant 
            experiments with animal 
            DNA, Cohen and Boyer 
            spliced an amphibian 
            gene into a bacterial 
            plasmid.
1972
UCSF & Stanford scientists  met at a conference in 
Hawaii on bacterial plasmids. Over lunch of hot pastrami
sandwiches they decided to pool their resources. Within four
the joint labs succeeded in cloning predetermined
segments of DNA. This paved the way for a new, huge, 
International industry that has created products such as:
Human growth hormome(HGH), human insulin, and a heart 
medication to remove blood clots.
  How to create a 
recombinant plasmid
1) Treat plasmid and 
    amphibian DNA with the 
    same restriction 
    endonuclease (they used 
    EcoRI)
2) This creates the same 
    sticky ends on plasmid 
    and amphibian DNA.
3) Place both together with 
    DNA ligase to join the 
    amphibian gene with the 
    plasmid.
  The recombinant 
  plasmid is inserted 
  (transformed) into 
  bacterial cells and the 
  bacteria made 
  amphibian mRNA.

But not the protein… we’ll 
  see why in a minute.

You can clone the gene 
  using the bacterial cells
When scientists create plasmids 
one problem they encounter is 
that prokaryotes cannot modify 
    mRNA by removing the 
 eukaryotic intron sequences. 

Introns often prevent translation.
      To overcome this problem, a DNA gene 
     is created using the modified mRNA as a 
                     template.
1.   Modified mRNA is isolated from the cytoplasm.
2.   An enzyme called reverse transcriptase creates a strand of 
     DNA from the mRNA template.
3.   The newly synthesized strand of DNA can act as a template 
     for the complementary strand.
4.   This type of DNA, synthesized without the intron sequence is 
     called complementary DNA or cDNA.
5.   The cDNA can be successfully inserted into plasmids, 
     transcribed and translated by bacteria.
Figure 20.5  Making complementary DNA (cDNA) for a eukaryotic gene
Cloning A Gene
Screening for the Recombinant Plasmid
     (How to find the few bacteria
 transformed w/ recombinant plasmid
           from the rest!!!!)
• A recombinant plasmid should contain:
• 1) A gene for antibiotic resistance.
• 2) A functional gene containing a restriction 
  site.
   – Ex. THE GFP gene

• Why?
You can do this two ways:
1. They are grown on a media containing an 
   antibiotic.
Only the bacteria that took up the plasmid will
   survive.
2. Knock out a gene- this let’s you know if you 
   successfully spliced your “gene of interest”  into 
   the plasmid in the first place.
If a gene on the plasmid contains a restriction site, 
   then that gene will be rendered useless when 
   the foreign gene of interest is inserted.

Ex. The organisms that don’t “Glow” have the 
  recombinant plasmid and do… ex. make HGH.
For Example:
• The "Z gene" on a plasmid produces an enzyme 
  that metabolizes the sugar X-Gal.
• When the gene is functioning X-Gal is broken 
  down into a blue product. 
• If the restriction site is within the "Z gene" and the 
  gene of interest is inserted there, the bacteria that 
  have this plasmid cannot metabolize X-gal.
• When they are cultured on a petri dish, using an X
  -gal media, these bacteria will appear white.
• Other bacteria that have a plasmid without the 
  gene of interest will appear blue.
Other techniques:

•   X  Antibody staining
•   X    Radioactive DNA probe
• Restriction Digest of DNA/Gel 
  Electrophoresis
DNA FINGERPRINT/ RFLP analysis
Restriction Digest of DNA
Restriction Fragments separated by Gel Electrophoresis
                                DNA FINGERPRINT 
              Cut DNA at specific sequences w/ restriction enzymes.
Each different sample is cut at different locations- makes different sized fragments
    Loaded onto gel. Electric current runs through. Pulls - DNA to + charge.
          Small fragments move fastest, Large fragments left at the top.
                          Unique banding pattern forms
         Making a DNA Fingerprint
• A DNA sample is extracted 
  from nucleated cells.
• The DNA is amplified using 
  P.C.R.
• The DNA is cut into fragments 
  by restriction enzymes.
• The stained fragments are 
  placed into a gel, and are 
  moved by an electrical current.
• Comparison is made between 
  DNA samples.



Paternity Testing: Who’s the daddy?     A         or           B
     Making a DNA Fingerprint… cont.
• Smaller fragments migrate the 
  farthest and the result is a 
  column of dark DNA bands 
  that extend across the gel.
• The amount of DNA between
  restriction sites varies from
  individual to individual of the
  same species. The differences
  are called restriction
  fragment length
  polymorphisms or RFLP’s.  
  RFLP’s result in unique
  restriction fragment patterns
  on a gel.
Using the circle provided, construct a labled diagram of the restriction
     map of the plasmid. Explain how you developed your map.
b) Describe how: recombinant DNA technology 
could be used to insert a gene of interest into a 
  bacterium. Recombinant bacteria could be 
 identified. Expression of the gene of interest 
               could be ensured.
b) Describe how: recombinant DNA technology could be used to insert a gene 
    of interest into a bacterium. Recombinant bacteria could be identified. 
             Expression of the gene of interest could be ensured.
c) Discuss how a specific genetically modified organism might 
 provide a benefit for humans and at the same time,  pose a 
             threat to a population or ecosystem.
c) Discuss how a specific genetically modified organism might 
 provide a benefit for humans and at the same time,  pose a 
             threat to a population or ecosystem.
 Polymerase Chain Reaction 
a way to make millions of copies of DNA!!!

What you need:
     1. DNA sample
     2. Free nucleotides
        – A heat resistant DNA polymerase 
        – Example: Taq polymerase
     3. Primers: short segments(20-30bases)
        of DNA complementary to the ends of
        the DNA being copied.
     What to do:
1)    Denature the original strand 
      of DNA with heat.
2)    Cool the mixture, allowing 
      the primers to bind (anneal) 
      to the DNA.
3)    The DNA polymerase binds 
      free nucleotides to the 
      primer using the original 
      DNA strand as a template. 
      This creates two copies of 
      the DNA sample.
4)    Repeat.
                   Gel Electrophoresis
•   Technique used to separate 
    restriction fragments.
•   DNA fragments of different lengths 
    are separated as they diffuse 
    through a gelatinous material under 
    the influence of an electric field.
•   Since DNA is negatively charged 
    (phosphate groups), it moves toward 
    the positive electrode.
•   Shorter fragments move 
    further/faster than longer ones so a 
    pattern is made.
Figure 20.15  RFLP markers close to a gene
Figure 20.x1a  Laboratory worker reviewing DNA band pattern
Figure 20.x1b  DNA study in CDC laboratory
               APPLICATIONS 
                       of 
              Gel Electrophoresis
1. Compare DNA fragments of closely related species 
   to determine evolutionary relationships.

2. CSI. Compare restriction fragments between 
      individuals of the same species- murder, rape.

    Fragments differ in length because of polymorphisms, slight 
    differences in DNA sequences. These fragments are called 
    restriction fragment length polymorphisms, or RFLP’s.
Figure 20.6  Genomic libraries
Figure 20.13  Alternative strategies for sequencing an entire genome
Table 20.1  Genome Sizes and Numbers of Genes
Figure 20.14a  DNA microarray assay for gene expression
Figure 20.14b  DNA microarray assay for gene expression
Figure 20.19  Using the Ti plasmid as a vector for genetic engineering in plants
   DNA, and in some cases RNA, is the
      primary source of heritable
              information.
CHECKING FOR UNDERSTANDING:
• Genetic engineering techniques can manipulate 
  the heritable information of DNA and, in special 
  cases, RNA. 
  Q: What are three genetic engineering techniques?
• Illustrative examples of products of genetic
  engineering include: 
  Q: What are three examples of products of Genetic 
   Engineering?
DNA, and in some cases RNA, is the
   primary source of heritable
           information.
• Genetic engineering techniques can manipulate 
  the heritable information of DNA and, in special 
  cases, RNA. 
  – ex.Electrophoresis , Plasmid-based transformation , 
    Restriction enzyme analysis of DNA , Polymerase 
    Chain Reaction (PCR)
• Illustrative examples of products of genetic
  engineering include: 
  – Genetically modified foods, Transgenic animals, Cloned 
    animals, Pharmaceuticals, such as human insulin or 
    factor X

								
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