MOLECULAR GENETICS...    DNA is molecular basis of inheritance          DNA toons...
         DNA molecules are transcribed as "software" and blindly copied by polymerase enzymes, as the "hardware"
         for the next generation. If the copying of the software by the hardware is a process that allows for errors,
         then the replication of the software by the hardware is a process that is evolvable.

.....
                  Genes
   --->   Enzymes   --->   Metabolism  (phenotype) 
                                                                                Overview of the Central Dogma (3 min)*
                                       DNA    --transcription-->    RNA    --translation-->    Protein
                                               Central Dogma of  Molecular  Biology*

 DNA animated the Era of OMIC's of modern Molecular Biology - ["OME" has the sense of the whole]
 Genomics - study of the genomes of organisms (sequencing, mapping, & gene interactions)
 Proteomics - study of the entire complement of proteins in a system or organism
 Transcriptomics -
all messenger RNA (mRNA) or "transcripts," made in one cell or a population of cells 
 Metabolomics -
complete map of all small-molecule metabolites in the human body
 Structural Genomics - determination of 10, 20, & 3D structure of all proteins encoded by genome
 BioInformatics - application of information technology to field of molecular genetics- BIG DATA.
 Pharmacogenomics
genetic basis for heritable & inter-individual variation in drug responses       

             



 
 
 
 
 
 
 
 
 
 

 
 
 
 

 What is a GENE = ?   How do we define what a gene is??? 
 

    
         - we seen that it is a basic unit of information in all living organisms
                                      responsible for a morphological trait (
free ear lobes)...
             - and DNA is the likely chemical for this genetic material, thus a gene is...
  
          - a segment of DNA specifies a morphological trait (round/wrinkled)
             - it's a discrete piece of deoxyribonucleic acid
                                      that  directs the synthesis of a specific protein
            
- it's a linear polymer of repeating nucleotide monomers

                                   nucleotides    -->   A   adenine,      C   cytosine
                                                                     thymidine,      guanine
  

                                      yields    -->  a 
polynucleotide strand* 

      What about, retroviruses, as HIV, Covid-19, & TMV, which contain RNA
                which molecularly replicate, transcribe, and translate RNA.

                   ...
maybe our definition of a gene may need to be broader.
gene
                    


                

             

 

 

 


 

   
          DNA     and      INFORMATION PROCESSING  
 

        - the letters of the genetic alphabet... are just the 4 nucleotides A, T, G, & C
of DNA.
        - the unit of genetic information... or the genetic 'word'  is called a  CODON, which is  
                     a triplet sequence of nucleotides, such as  
p5'-CAT-3'p   in DNA makes a codon
                     &  these 3 nucleotides  =  1 codon (word) & code for an  amino acid  in a polypeptide.
        - thus the definition of codon (a genetic word)   is an 
amino acid   5'-AUG-3' = 'MET'
        
                (DNA)
  p5'-CAT-3'p  -->  (mRNA)   5'-AUG-3'  -->  (amino acid)   -->  N-methionine-C*
        

        - Informational size of a Human Genome:

              
 3,088,286,401 base pairs  [A:T & G:C]  or   6.18 billion nucleotides in a human cell.
              
1,029,428,800 possible codons (amino acids "words") in one human DNA strand.
                  average page your textbook    approx 850 words
                  thus, human genome is equal to 1,211,093 pages   or   841 copies of bio book (1440 pg)
                  reading at
3 bases/sec it would take you about 32.64 years  @  a 24/7 rate

                       but, only about 80% or all DNA has any metabolic function =
986,874 pages,
                              and only 1.5% codes for proteins = 12,111 pages or 8.4 bio books.

  
                WOW... nanotechnology animation*    &    how DNA is packaged*
         

      
Of MICE and MEN... nearly every human gene is also found in mice, and
                   98.8% of Chimp & Human DNA is the same. A 1.2% difference is 35 million bp.
                   Many mammals including dogs, cats, rabbits, monkeys, & apes have roughly
                   the same number of nucleotides in their genomes -- about 3 billion bp.

 
                 Estimates suggest that Humans share 99.9% of their DNA person to person.

           

 
 
   
       
 
 
 

 

 

 

 

 

 
Experimental Proof that DNA is the Genetic Material...

    1.  Bacterial Transformation Experiments of Fred Griffith for a pneimina vaccine (1928)...
            he observed process whereby the bacterium distinctly changed its form & function...
            he studied Streptococcus pneumoniae   -   pathogenic S strain  &  benign R*
                and showed the transformation of R to S cells, via a genetic transfer process,
            he called it a 'Transforming Principle'*, converting one strain to a different strain.
   

    2.  Oswald Avery,  Colin MacLeod,  &   Maclyn McCarty... 
  (pic - 1940's Rockerfeller Institute, NY)
            experimantally suggest the
transforming substance* is DNA molecules
, but... 
 
   3.  Alfred Hershey & Martha Chase's (1952 Cold Spring Harbor, L.I.) virus replications experiment...
             
Using a genetically controlled biological activity, bacteriophage experiments*,
             their inquiry based experiments established DNA as the genetic basis of heredity.
            
An explanation of Hershey & Chase's experiment  =   anim bacteriophage exp*    
            
the use of radioisotopes in biology* (32P) DNA not (35S) protein guides viral replication

                         Conclusion   - 
DNA is accepted as the Genetic Material...                                  
 

 

 

                        

                         

 


 

 

 


 

Structure of DNA...
Discovery of Double Helix*         Watson's book
  

  1962 Nobel prize JD Watson, Francis Crick, Maurice Wilkins
                              [??? what role  did Erwin Chargaff
& Rosy Franklin play???] 
   
    Dramatizations of DNA's discovery
: Race for the Double Helix (1987) & Photograph 51 (2015)
    and maybe one of the most Egregious Ripoff in the History of Science [by Howard Markel Oct 2021]
  
    Watson & Crick, as theoretical biologists, used two approaches to decipher structure:
          1. model building - figure
*    (are the bases in/out
*?)
          2. x-ray diffraction* and photo 51 favor a DNA helix of constant
diameter*

   
 
     we now know : DNA is a double stranded*,   the helixof polynucleotide chains,
                           made of 4 nucleotides - A, T, G, C   (purine & pyrimidines)
                           in 2 polynucleotide strands   (polymer chains)
                           head-tail polarity [5'-----3'] - strands run
antiparallel 
                           held together via weak H-Bonds & complimentary pairing

                Chargaff's rule....    A:T**        G:C
                                              A  +  G  /   T  +  C  =  1.0       

      Fig's:  
sugar-P-backbone & base pairing*, (question*?),  dimensions*,
                       Summary animation of DNA Structure*view   -    structure DNA & RNA**view@home
           myDNAi timeline   &    literature references   --> [Nature171, 737-738(25 April 1953



 
 
 
 
 
 
 
 
 
          view@home
 

 

 

   
 
REPLICATION OF DNA... copying of DNA into DNA seems structurally obvious*???   figure  
 
1.   Patterns of Replication* - 3 possibilities:  
ConservativeSemi-conservative,  &  Dispersive 
                                            
what was needed was a procedure to tell difference between parental & new DNA
      Matt Meselson & Frank Stahl 1958 - devised an experiment to establish semi-conservative
           replication of DNA -     Explanations of the Meslson-Stahl Experiment*view  
           the experimental design* to separate parental  15N-DNA from 14N-new DNA- (OLD vs. NEW DNA)?
           the beauty of their experiment is one can predict what results ought to be before doing it...      
           the results of the DNA centrifugation experiment
  -->  actual results*  another fig-2
    
DNA Replication is a  semi-conservative process.                                 view@home-1

2.  DNA POLYMERASE: [EC 2.7.7.7] copies DNA...    prokaryotic Pol I-IV    eukaryotic α,β,γ, & δ 
         model polymerase is prokaryotic Pol III (pic)   req: 4-deoxy-NTP's  &  a 3'-ssDNA template strand
                    DNA polymerase reads template strand and
adds a complimentary nucleotide
*view                       
 
                   reads 3' to 5' and synthesizes in 5' to 3' direction  -->  templated polymerization*...             
                    DNA polymerase III proofreads*  &   the error rate of base copying
* 
                   origin of replication & bidirectional synthesis of DNA*  &  
EM pic
*
        the two DNA strands are antiparallel (run opposite directions) -->
--> Overview animation*3.5min      
                     both strands are copied at same time
*     leading*  &  lagging strand synthesis.

   

 


                         
 

 

 

 

 

 

 

 

 

 
the Model for studying the mechanisms of DNA Replication
is bacterial DNA polymerase III...
  

    all of the Enzymes* involved form a Replication Complex called a Replisome:
    the bacterial enzymes include*:    
            helicase - untwists DNA 
            topoisomerase [DNA gyrase] - removes supercoils.....  [animation]
            single strand binding proteins - stabilize replication fork,
            Primase - makes RNA primer terminus on leading strand & pieces on lagging strand
            POL III - synthesizes new DNA strands 
            DNA polymerase I - an exonuclease removes RNA primer, 1 base at a time, & adds DNA bases
            DNA ligase repairs Okazaki fragments (seals lagging strand 3' open holes)
  
    
    Structure of DNA polymerase III Replisome
*
                copies both strands simultaneously, as DNA is Threaded Through a Replisome...
                a "replication machine", which may be stationary by anchoring in nuclear matrix
                Continuous & Discontinuous replication occur simultaneously in both strands

                           Rates of DNA synthesis:  myDNAi movie of - replication*view    
   
                                                                                                

 

 

     

 

     

 
 
 

            
 
                    

 

   
 
 
 
  

 

 

 

 

 

 
 
GENE Expression...   DNA  -->  RNA  --> PROTEIN

   
the Central Dogma of Molecular Biology describes flow of genetic information
*
  

               DNA sequence ------->  RNA sequence ----->  amino acid sequence
                       CAT                              AUG                              MET
    
a triplet sequence in DNA -->  codon in mRNA ----->   amino acid in protein
  

               Information : triplet sequence in DNA is the genetic word [a
codon]  
  

                   Transcription - copying of DNA sequences into RNA
      
             Translation   - copying of RNA sequences into protein
 

   
One Gene One Enzyme* - 1941 hypothesis that each gene directly
                                               makes on enzyme
 
(Beadle & Tatum 1958 Nobel)
    Compare Events:

         Procaryotes*  vs.  Eucaryotes*  =   Separation of labor 
         Differences DNA vs. RNA
(
bases & sugars)  and its single stranded

   
      

 

 

 

 

 

 

 

     

 

 
 

 Transcription is via  RNA polymerase...                                                                                        
        RNA polymerasetranscribes DNA into an RNA product*.  
             - in bacteria Sigma factor* binds promoter &  initiates copying of DNA*
             - makes a
complimentary copy
* of just   ONE*  of the two DNA strands 
           
- activators & transcription factors* are needed to recognize specific DNA sequences
           - Transcription factors* via protein motifs bind to promoter DNA region
                                                                                                 Transcription: the movie*view@home     
            
     Cells make many KINDS of RNA  [table*]
                                            
          
tRNA -    small,  80n,  anticodon sequence, single strand with 2ndary structure*  
                                 function = picks up aa & transports it to ribosome  [
Mahlon Hoagland]  
  
          
rRNA -    3 individual pieces of RNA - make up the organelle = RIBOSOME
                                 a primary transcript of rRNA is processed into
 the 3 pieces of
                                     
recall structure of ribosome
  &  rRNApieces*    
                                

 

 

 

 

     

 

 

 

 

 
  Primary Transcript   functions as the precursor of mRNA molecules in eukaryotes.  (fig*)  
                                    is a large
 1:1 RNA copy of  a DNA segment that gets cut up to make mRNA.                    

                            
                
   mRNA -   has sizes can range from 100n to 4000n - and is made from the primary transcript
                                    and is made by cutting the primary transcript into small segments.
   
   
                Split gene experiments* revealed the primary transcript & mRNA are not the same size.
                                    the initial DNA transcript is composed of Exons and Introns
*.

Processing (cutting) of introns & exons*               
Splicesome splicing of eucaryotic genes*      &     Spliceosome animation        
         
Introns requires specific sequences to be recognized by splicesomes to be cut out.
       
Role of introns - may be equivalent to blank spaces in sentence structure?
 

  final structure of mRNA*  
              
caps & tails...            
      
  
 
             
    
  role of 5' CAP and Poly-A Tails*          &      nuclear export of mature mRNA*
  summary eukaryotic RNA processing&     mRNA processing: tails & caps*view@home    

Alternative splicing* of exons allows a single gene  to code many proteins via mRNAs
                                 a thought question on splicing? *
           
          
      


  

 

 

 

 

 

 

 

 

 

 


 
Other Classes of RNA Molecules include: 
  
  
snRNP's  small nuclear RNA - plays a structural and catalytic role in spliceosome*
            
called  "snurps"...  there are 5 making up a spliceosome [U1, U2, U4, U5, & U6],
             all
urideine rich - they participate in several RNA-RNA and RNA-protein interactions

   SRP (Signal Recognition Particle): contains a small cytoplasmic 7s-scRNA
             component  as part of the receptor complex, which recognizes the signal sequence
           
 of polypeptides targeted to the ER -
figure
*  
 
  
snoRNA (small nucleolar RNA) -  are noncoding RNAs located in the nucleolus, which
              are involved in rRNA modifications by chemically modifying them via methylation
              and pseudouridylations to generate mature RNA molecules...
 

  
Micro RNAs (& siRNA):   function in gene silencing & gene regulation.  
   are single-stranded RNA molecules of 20-24 nucleotides in length, that are not translated
   into protein (non-coding RNA) and form short
stem-loop structures
that are partially
  
complimentary to mRNAs & that can block translation
* by downregulating gene expressions.
  
   
     FDA approves 1st RNAi drug treatment (august 2018) for
ONPATTRO a therapeutic that
        treats polyneuropathy in hereditary mutation in amyloidogenic transthyretin amyloidosis
        which results in protein misfolds that form aggregates and deposits amyloid plaque
        in multiple tissue and organs in the body.

                
                                                           
  
 

 

 

 

 

 


 

 

 

 

 

 




 

TRANSLATION - Protein Synthesis...    basics of translation animation*
       process of making a protein in a specific amino acid sequence 
               from a
unique mRNA sequence...  
[ Model*  process* ]
                                           
polypeptides are built on the ribosome on a polysome.

SEQUENCE DETAILS
...
  
        [the Discovery of tRNA by Paul Zamecnik  - JBC article 1958]
   
      1st   add an amino acid to tRNA  -- >  tRNA
* -->  aa-tRNA    - ACTIVATION       + animation*     

      2nd  assemble players [
ribosome
, mRNA, aa-tRNA]             - INITIATION         + animation*         
      3rd   adding new aa's via peptidyl transferase                
      - ELONGATION*   + animation *         
      4th   stopping the process                                            
      - TERMINATION    + animation*   
                                                                            

           Review the processes:   RNA Processing summary & translation summary fig*
                                                           
initiation, elongation, &
termination in animations
                                                                           Real-time animation of translation*view@home

     
                            

 

 

 
 
 
 
 
 
 

 

 

 

 

 GENETIC CODE...  is the genetic alphabet spelling the genetic information of proteins.

      ..is the sequence of nucleotides in DNA,
but because mRNA
        sequences specify sequence of amino acids in a protein        
      the code is routinely shown as a  mRNA CODE
the code is in mRNA

       Coding ratio how many nucleotides are needed to specify 1 amino acid  ?
               
1n = 4 singlets,     2n = 16 doublets,     3n = 64 triplet codons      a Triplet Code
*
        
   
       S. Ochoa (NYU)  (1959 Nobel) - discovered polynucleotide phosphorylase (it's an exonuclease)
              
Np-Np-Np-Np ---- Np-Np-Np + Np but, can be reveresed to make SYNTHETIC mRNA                
       Marshall Nirenberg
(1968 Nobel) - made synthetic mRNA's using in vitro systems
                5'-UUU-3' = phe         U + C -->  UUU,  UUC,  UCC,  CCC
                                                                    UCU,  CUC,  CCU,  CUU
= mixes gave codons

    the Genetic CODE* - has 64 possible mRNA triplet codons:     new view of genetic code*
          61 code for an amino acid,    
1 is an
initiator (start) codon (AUG = nf-MET), and  
            3 are molecular 'periods' or stop codons
(UGA, UAA, & UAG); code is
universal,
           redundant, but non-ambiguous, and exhibits "
wobble
*".
   "the sequencing of DNA of dozens of species, from viruses to humans, documents that
             we're all connected to the commonality of the genetic code in evolution
"  jcv (2007
)

 

 

 

 

 

 

 

 

 
   
 
GENETIC CHANGE... a change in DNA nucleotide sequence (= change in mRNA)
              - done in 2 significant natural ways... via  mutation & recombination   
[glossary]
  
                                                                                                
    1. MUTATION -  a permanent change in a cell's and progeny's DNA*that results in
                               a different codon = different amino acid sequence in a protein

         Point mutation*  - a single to few nucleotides change...
             - rate: E. coli = 1/1,000 per generation  and  humans = 150-200 per generation.
                             ex: - deletions, insertions,
frame-shift mutations     [
CAT]    = proof of a triplet codon

                                   - trinucleotide repeats - CAG repeats in Huntington's disease... 
 
                                                                              
         single nucleotide base substitutions* :
                     non-sense = change to no amino acid [STOP codon]  
UCA --> UAA      ser to non                                                                                                  
                     mis-sense = different amino acid                                   UCA --> UUA      ser to
leu 

                         Sickle Cell Anemia*  - a mis-sense mutation...
                                                                                       - 
one base pair change = multiple effects in SCA-pleiotropy*
                     other point mutation examples:
                              1) in a blood disease, thalassemia, a form of anemia (change at intron site)
                              2) mutant AMPD1 which can lead to muscle cramps & early fatigue 

                                   
adenosine-monophosphate-deaminase-1 =  is a C to a T change

    
 
                 EFFECT =   no effect,   detrimental (lethal),   +/- functionality,   beneficial

                                                                        

  
                                                

 

 

 

 

 

 

 

 

 


   
2. 
Recombinant DNA...  DNA made artificially by combining DNA's from different cells
       using laboratory techniques that
often insert NEW (foreign) DNA into recipient cells.
  
   Natural Recomination processes:
        
   1. 
Fertilization* - sperm inserted into recipient egg cell --> zygote   [n + n =
2n]
  

   2. exchange of homologous chromatids via Crossing over* = new gene combo's
  
   3. Transformation*  - absorption of 'foreign' DNA by recipient cells changes cell

 

   4. BACTERIAL CONJUGATION*   - involves DNA plasmids*  (
F+ or R = resistance)
                 conjugation may result in genetic recombination in bacteria  [Hfr*]   
  

   5. Virus
*replication has 2 phases =  lysis vs. lysogeny*   &    [figure]
          
TRANSDUCTION* is transfer of foreign DNA into a cell via a
viral vector

                 general transduction - pieces of bacterial DNA are
                               packaged w viral DNA during viral replication & lead to recombination 
                 restricted transduction - a temperate phage goes lytic
                               carrying adjacent bacterial DNA into virus particle & recombination

                             bacteriophages shuttle bacterial genes between ecosystems

   
                                                          

 

 

 

 

 

 

 


 

 

 
6.
Biotechnology  -   Genetic Engineering   -   Designer Genes 

     RECOMBINANT DNA TECHNOLOGY...  involves laboratory-made DNA molecules
        and a collection of experimental techniques, which allow for the isolation, copying,
        and insertion of new DNA sequences into host-recipient cells by a number of
        unique laboratory protocols &
methodologies.
  

     Restriction Endonucleases
glossary       Cut DNA at unique sequences*    
       
... Group of bacterial enzyme that cut DNA with 2 incisions through sugar-P's making small
               pieces about 15-20,000 np's each; 
cuts are unequal or blunt
* @ unique DNA sequences.
        ... To date some 3,000 restriction endonucleases are known that recognize 250 cut sites
               and some 800 restriction endonuclease are available commercially.
                                                                 
-  Werner Arber, Daniel Nathans, & Hamilton O. Smith 1978 Nobel
          
Eco-R1*-figure*          @ many are palindromes...    ["never odd or even"  cartoon
cartoon                                               
            
5' G.AATTC 3'          5' G . . . . .      +      AATTC 3'
          3' CTTAA.G 5'          3' CTTAA               . . . .  G 5'

         
                        DNA's cut this way have STICKY (complimentary) ENDS & can be 
spliced* 
                        with other DNA molecules to make Recombinant DNA
* producing
                       
new genes combos and manufacture proteins for medical use.

            Anecdote:  Origins of Recombinant DNA Technology*

 









 

 

 

 

 
           
Some Typical Procedures in Biotechnology
(Genetic Engineering)...   

                    involving restriction enzymes   &    A:T   G:C   pairings
            

A.  Techniques involved in Cloning a Gene...


      1.  via a plasmid
*  [ human shotgun plasmid cloning             &   animation*]    

      2.  Libraries
g...       [ whole genome library*                             &  animation of DNA fragment library*]

        
3. 
cDNA genes*   [ synthetic genes made from mRNA          &   cDNA library* ]

      4. 
Probes
g...          [ a DNA probe of interest* + synthetic DNA & Hybridization & DNA Probes*
                          
       (how to find a gene w/a probe  +  cDNAg  +   reverse transcriptaseg)
   

      5. 
Polymerase Chain Raction CR anim* is a method to amplify small pieces of DNA
               Taq pol  &    PCR image         Covid PCR test*     &     a PCR long animview@home    
               Bio-Rad 1000 Thermal Cycler    &   the PCR Song        &   a cartoon   &  "We are the World" 
   
 


 

 







 

 

 
B.  Visualization of DNA...   to locate a gene (or its activity) -->  Restriction Maps.  
                               and other practical applications in Forensics & Gene Expression.    
    

        1.  Restriction Maps
g... the banding pattern seen in gel electropherograms by
                DNA treated with restriction enzymes and then electrophoresed =  "a fingerprint"

                    
electrophoresis - Video*    &   DNA electrophoresis*   &    DNA-electropherogram    
               

   
    2.  DNAg fingerprint*...    CSI Miami - how to make one*    a murder case*   a rape case*
                                                 
                                              Glowing gloves & CSI   &   DNA fingerprints in Health & Society
       
       
3.  Single Gene Expression during development via RT-PCR-cDNA methodologies
                       and Differential Gene Activity --->  
fig 20.11
*
   
 

       4.  DNA  
Micro-chip arrays*   -->   chip animation  
                          monitor gene expression in thousands of genes & changes DNA microarrays
           
               contain 1,000s of short synthetic ssDNA gene sequences that are are fabricated onto
                         
a glass slide by high speed robotics akin to Intel chip making.

                         
By passing cDNA probes of a cell's mRNA over slide with ssDNA of all a cell's genes,
                          the fluorescently tagged cDNA probes are so easy to see in slide's wells.

           normal vs. cancer cells
*   &   making DNA microchips  &  DNA chips - normal vs. cancer cells*view  

          

 


 

 

                                      
                                           

 

         

 

 

 

 

 

 



 

Some Other Practical Applications of DNA Biotechnology
-
What's been Done in Medicine...
 
       diseases often involve changes in gene expression and can be visulaized in lab.
 

    

     a. 
RFLP -
 Restriction Fragment Length Polymorphism - markers may be inherited with disease
                       
what is RFLP
*  this polymorphism is the basis of a genetic test for
                        the Sickle Cell trait -->
DdeI cuts* 
Genetic Info Nondiscrimination Act  
                      
     b.  Gene Therapy... idea is to replace defective genes via
microinjection*
                                     allowing recombination to replace defective gene with newly injected gene.
                                           
Vectors*   ...using viral vectors to fix a defective gene
          Trials:   1. Sept. 1990 a patient with
ADA Deficiency -Ashanti DeSilva  video then - now
                       2. in 2000 - SCIDs [severe combined immunodeficiency - a single gene enzyme defect]. 
                               C
linical trials in 20 patients triggered 5  cases of leukemia & 1 death: trials stopped.
                               retroviral vector inserted the repair gene into bone marrow cells genes incorrectly.
                    

   
 
   c. 
CRISPR-Cas9 short animation* CRISPR structure*  CRISPR-Cuts*  CRISPR Potential      
                   
          2020 Nobel   &   Doudna explains CRISPR   &  Treatment of a Patient (SSA) 
                                           anecdote:   Story of Discovery of CRISPR system.

    
     d. 
Pictorial Chronology of the Gene...   &   Genetic Milestones from Peas to now... 

     e.  What is involved in Genetic Engineering today
*:

          back to Bb         next lecture  'Genomics'
           
                     .                    .    
                                                                                            

 

   

 

 

 

 

 

 

 

 





 
 
 
 
 
 
 
 
 
 
 
 
 
 
 




  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 




SKIP EVERYTHING BELOW THIS POINTS....

      
  How do cells Control Gene Expression?

       How do we know a gene has been active (turned on) within cells????
                           one looks for a gene's product, i.e., an RNA or a protein

                           an increase in enzyme activity implies gene action?   
                           no enzyme activity suggests no gene action. 


              but, what about pre-existing inactive enzymes converting into --> active forms 
                                    ZYMOGENS        - proelastase    ----->   elastase
                                                                - trypsinogen   ----->  
trypsin

 
             thus, we have 2 possibilities: 
                                   1)  pre-existing inactive enzyme --> active
                                   2)  de novo (new)  enzyme synthesis by gene action.

      

 

 

 

 

 

 
 
How do cell control gene expression...        by Turning On/Off Genes* ...  
   

    a model:   in Prokaryotes:     LACTOSE OPERON - Jacob & Monod (1965 Nobel Prize)
                    in bacterial DNA, a cluster of contiguous genes, called an operon,
                    are transcribed from one promoter making a single long mRNA

  

                     
glucose     E. coli  (grown on)   lactose
                  NO beta-galactosidase                      beta-galacotsidase  (gene expression)
                                                                                lac  --->  glu + gal
 
  
   OPERON = series of mapable-linked genes that can be induced (on) to make several enzymes.
                  
p    Rg      crp      p  O    Sg1        Sg2        Sg3   
       p                    promoter region   -  binds RNA polymerase    [TATAAT box for RNA polymerase]    figure
       Rg (I gene)    regulator gene      -  LacI gene makes repressor protein -  no lactose is present
*
       O                   operator site        -  DNA regions that binds repressor protein      
figure
    
       S                   structural genes   -  make enzyme proteins         but what if regulator binds lactose
*
                                                          
                           animation - Lac Operon*  the Lac Operon is Inducible     

 
med
                        appl.            Tryptophan Operon is off when TRP is present & is this a Repressible Operon
* 
                          

                              

 

 

 

 

 

 

 

 
 
Control of Gene Expression...     in   EUKARYOTES
                                                                                                       

    Gene Action of the Exome (expressed genes)  has significantly greater complexity
      

              there are
no operons present that map as genes linked together ,
                                                  i.e., no polycistronic mRNAs.
                          but regulator proteins, inducers & repressors are present;

    
              and there's much
more DNA, - but  it's inside a compartment  (nucleus)
              have
many more promoters   - sites where RNA polymerase binds
                 
     enhancer sequence      - sites where enhancers/transcription factors bind
                 
     transcription factors      - proteins that help transcription
              and the individual
genes are not contiguous with each other, thus no operons


             
Levels for eukaryotic controls*   - transcriptional,   translational,   post-translational

              
there are multiple places for control* - of whether a gene makes a protein or not..

                    

         Summation -  Control Gene Expression* & Differential Gene Activity animation*

      back                                                                                                       

    

                                                                                            

 

 

 

 

















 

 

  
 
Some examples of how Eukaryotic gene expression controls work: 
  

        
1.    Differential Gene Activity...   is the selective expression of genes as crystallin gene*   
                     i.e., different cell types express different genes [liver vs.
lens cell]
       
              role of activators in selective gene expression (Differential Gene Activity*) 
               
                                                 ex: Growth factors & Steroid Hormones (figure*)
  

        2.   Epigenetic activation/repression of genome...  heritable changes in phenotype
                      without alteration of DNA sequence, rather by chemical modification to
                     
DNA or nucleosome histones (methylation, acetylation, phosphorylations).
                      examples:  Barr bodies (inactivation of one X chromosome & imprinting).
              Epigenetics
modifications* can carry over to all a cell's progeny in an organism,
                                    a cell's memory of gene action can be inherited by progeny cells.

  

        3.   
Molecular turnover - 
animation*  -->  life mRNA's*  &  longevity of some proteins*   
  

        4.   Processing of RNA transcript   (figure*)
                        cut/spliced in nucleus and capped for transport
                           
     intron - pieces cut out (non gene-proteins)   
                           
     exons - pieces transported to cytoplasm
                                            
alternative splicing  =   figure 18.11*  

      5.  Summation - McGraw-Hill animation on control of gene expression*AdobeView@Home 
                                                                

 

 

 

 

 

  
 
   Eukaryotic gene expression controls...    examples from cancer: 
  

     Cancer often results from gene changes affecting cell cycle control proteins
            cancer genes, such as adenomatous polyposis coli, which cause 15% of colorectal cancers,
            are  tumor suppressor genes, a
type of  Oncogenesg
... (an oncogene is a cell cycle gene
               that has mutated & is now expressed at high levels turning a normal cell into a tumor cell)
.  

    

     Examples of human cancer Proto-oncogens & Oncogenes: a presumptive
* & a cancer gene...
         
Ras a proto-oncogene (a cell division activator) involved in some 30% of human cancers:
                   is a
G-protein that promotes other cell division proteins... = over-expression = tumors.
                   a Ras gene mutation --> hyperactive Ras protein --> cell division  
fig 18.24
*

          p53 a tumor suppressor geneg is involved in  50% human cancers:          fig 18.25* 
                             p53 is a transcription factor that promotes the synthesis
                             of cell cycle inhibiting proteins...           

                    
             thus a p53 mutation --> leads to excess cell division (cancer tumor).

          BRAC1 & BRAC2 are tumor suppressor genes involved in 50% of human breast cancers
                      that when mutated prevents repair of DNA damage & unregulated cell division.

                                                                                                                      



 

 

 

 

 

 Genes and Evolution:      Modern synthetic Darwinian evolution predicts that Natural Selection
  
                                 over time can lead to permanent changes in the the DNA that is inheritable.

   

   but recent studies have evoked a new science called EPIGENETICS...
                       study of changes in gene activity that do not involve alteration to the genetic code,
                       but changes that are still
are passed to at least one successive generation.
       
      epigenetics changes the DNA as a biological response to an environmental stressor,
                      
changes that can be inherited through many generations via epigenetic marks,
   but if you
remove the stressor epigenetic marks fade & the DNA code will revert to normal
              somewhat
Lamarckian (1744-1829) animals acquire traits with their life span (giraffe).   
  

       
ex: 1. drug geldanamycin produces outgrowth of Drosophila eyes that can last for 13 generations
                        no change is DNA sequences and generations 2-13 were not exposed to drug.
             2.
166 fathers who smoked before age 11 had sons who had a significantly higher BMI
                        than control kids of 14,024 fathers.
             3. a
diet rich in B-vitamins (folic acid & B12 - CH3 donors) fed to pregnant agouti mice
                        normal pups, but those w/o B-vitamins produces pus with yellow coats & diabetes.

 
           
analogy: the genome is the hardware and epigenetics is the software:
                         one can load windows on a Mac; you'll have the same chip in the Mac (same genome)
                         but the software will produce a different outcome - a different cell type.      end.
       Home |About |News |Syllabus |Lecture-outlines |Links |FAQ |Sitemap |Contact      

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
 

MST II
restriction cuts of normal sickle beta-gene      
                       
( pink is DNA sequence  &  blue = 4 gel fragments)

           _________|__________________|CCTNAGG GAA _____________|____________
                              
                  a                  b                      c                       d
   In 1978, Yuet Wai Kan and Andrees Dozy of the University of California-San Francisco showed that the restriction enzyme Mst II, which cuts normal b globin DNA at a particular site, but will not recognize and therefore will not cut DNA that contains the sickle cell mutation. Mst II recognized the sequence CCTNAGG (where N = any nucleotide). Sickle cell disease is due to a single point mutation in the beta globin gene on chromosme 11 that changes CCTGAGG to CCTGTGG.

MST II restriction cuts of recessive sickle beta-gene       (blue = 3 gel fragments)

           _________|___________________CCTGTGG ________________|____________
                              
                  a                                x                                     d

  

 

 

 

 


 
Sickle Cell disease occurs when the DNA sequence for glutamic acid is converted to valine. This results from a change in the nucleotide T to A. This change eliminates a site recognized by the restriction enzyme DdeI.

     Restriction enzyme:   DdeI* (recognition sequence:  5'-C^TNAG-3')
    Southern blotting probe:   fragment of -globin coding sequence
      Pattern result: normal cell =   3 fragments (1 large, a 201bp piece, and a 175bp piece
                              sickle cell  =   2 fragments (1 large, and a 376bp piece)

      fig 20.9*    Thus the number of RFLP piece can indicate presence of defective alleles.

  

 

 

 

 

 

 

 

 

 

  
reading frame is 1 codon = CAT    point mutations at hot spots - [fig]
       mutational hotspots in bacteria - sequences more susceptible to change than others

        C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T    (MET)
                     ↑ ↑

1st point insertion or deletion (where chemical mutagen analogs act during replication)

        C-A-T-X-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A     = mutant bacteria    

2nd insertion or deletion

        C-A-T-X-Y-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C     = mutant bacteria

3rd insertion or deletion

        C-A-T-X-Y-Z-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T-C-A-T  = normal cells  
 
   back                hot spots where mutations occur with greater frequency.
                             provides evidence in support of a triplet codon.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



The index case for AMPD deficiency was a 18 year old female with calf pain that revealed a mis-sense mutation at nucleotide 143 in codon 48 of exon 3 where C changed to T resulting in a change of proline to leucine. This mutant allele is found in 12% of Caucasians and 19% of African Americans and results in the AMPD deficiency in muscle biopsies and results in exercise-induced metabolic myopathy in humans.

ref: A life Decoded by J. Craig Venter, Viking Press, 2007, C2, pg 28

  back 

 

 

 

 

 

 

 

 

 

 

DO  NOT  study ANY OF THE material below

         
  Z-DNA

 Gene expressions in   pharmacogenomics    &   toxicogenomics  via microarrays

  1 cM = about 1 Mb

  TRANSPOSONS - pieces of DNA prone to moving & creating repeat sequences
                 LINE - long interspersed nuclear element holds promoter & 2 genes: RT &
integrase

    an anomaly - RNA Recoding*

 

          Simple Tandem Repeats (short- 5n to 6n)  or trinucleotide (3n) repeats can undergo an increase in copy
                     number by a process of dynamic mutation; # of tandem repeats is unique to a genetic indiv.
                     Variation in the length of these repeats is polymorphic.     figure*
                                   individual A has ACA repeated 65 times @ loci 121, 118, and 129
                                   individual B has a different repeat pattern at these loci
                     STR'sa can cause genetic diseases as well:

                                      CCG trinucleotide occur in fragile sites on human chromosomes (folate-sensitive group).
                                            fragile X (FRAXA) is responsible for familial mental retardation.
                                            another FRAXE is responsible for a rarer mild form of mental retardation.
                                            mutations of AGC repeats give rise to a number of neurological disorders.

 
 3.  Forensics - DNA fingerprinting is the vogue judicial modus operandi
                    
 a murder case* &  a rape case*  + DNA prints in Health & Society & DNA Forensic Science
                     DNA fingerprinting usually looks a 5 RFLP markers and blood is tested via
                     Southern Blotting (20.10) using probes for these alleles
  

 4.  Environmental Clean-up...
            bacteria can extract heavy metals (Cu, Pb, Ni) from the environment
            & convert them into non-toxic compounds
                          genetically modified bacteria may be the "miner's" of the future
 

   
 

 


 5.  Franken Food...  genetically modified (GM) animals & agricultural crops
        Transgenics - organisms with inserted foreign DNA in their genomes
             
 Animals
*  -  GFP novelties*  +  Dolly
                                -  animal cloning companies   --->   mammalian cloning success?
                               
-  "pharm" animals (20.18*)  --->   transgenic animal movie
                                     
sheep carry human blood protein gene that inhibits enzymes in cystic fibrosis;
                                          
    artificially insemination, microinjection of human gene, fertilized ova are put
                                              into a surrogate sheep:
                                              chimeras mated to produce homozygote- Milk tested for active protein.

              
Plants      - genetically modified crop plants - fig 20.19*
 
                               -  to get Ti plasmids in = a DNA gun*   Purdue University Gene Gun movie
                        
                          -  Frankenfood  &  Edible Vaccines
                                                  
-  National Plant Genome Initiative Plan    update  future   

 6.  Synthetic Biology...   artificially manufactured biological systems
                                - virus models
*                            (synthetic Biology)

                                            An overview of biotechnology  
                                                 History of Biotechnology    
                                                 Human Genome Project & Biotech Companies
   
                                      HHMI  funded  DNA  Interactive  tutorial

 
 

 

 

 

What are Introns? and What is the Role of Intron DNA?
don't really know, but Percentage of non-coding DNA during evolution*  goes up.

 

   summer & fall 2006: skip this material
 INTRONS - DNA Junk or sophisticated Genetic Control Elements?
    
Current dogma of Molecular Biology
           DNA --> RNA --> Proteins,   (proteins supposedly regulate gene expression)     figure
*
  
in 1977 Phillip Sharp & Richard Roberts discovered DNA contains introns
                intervening DNA segments that do NOT code for proteins
                a primary RNA transcript is processed by splicing to assemble protein coding exons

  
   Presence of Introns:  Absent in prokaryotes: they have few non-coding DNA sequences
                as eukaryotic complexity grows so does non-coding DNA    [figure]
                
makes up greater than 95% of the DNA
                
less than 1.5% of human genome encodes proteins, but all of DNA is transcribed
                40% of human genome is Transposons & repeat genetic elements.

  
   Evolutionary Origins?  may have been self-splicing mobile genetic elements
                                    that inserted themselves into host genomes 
                           Advent of Spliceosomes: catalytic RNA/protein complexes
                                    that snip RNAs out of mRNAs,
                            would encourage introns to proliferate, mutate, evolve

 

   fall 2005 skip ALL OF THIS MATERIAL
  
Role of Introns?  Not Junk, but rather Genetic Control Elements          [figure*]
           
Micro RNAs - derived from introns? - occur in plants, animals, & fungi
                         a) help control timing of developmental processes as cell proliferation,
                                    apoptosis, and stem cell maintenance
                         b) help tag chromatin with methyl and acetyl groups
                         c) may help in alternative splicing mechanisms              

   COMPLEXITY:    to build a complex structure one must have bricks & mortar,
                      as well as an architectural plan.
           
DNA, therefore should contain both - the materials and the plan:
                      a) component molecules - proteins, carbs, lipids, and nucleic acids:
                                     all known living organism use the same bricks and mortar
                      b) the difference between Man & Monkey is the architectural plan

            Where is the Architectural Information?   we've always assumed in the regulatory proteins
                      Maybe it's in the non-coding mirco-RNAs (intronic elements)

            Thus the greater proportion of the genome of complex organisms, the introns, isn't junk,
                      but rather, it is functional RNA that regulates time dependent complexity?

 
                           A Gene Sweep betting pool winner with closest bet (25,947) was Lee Rowen.

 

Visualizing Restriction Fragments...    to a probe of a gene (cDNA)
               
Southern Blotting
g   fig*       Sumanas animation - DNA electrophoresis & blotting*view@home
                    one can detect specific gene sequence by binding to labeled probes to DNA fragments

       d.  'Human Microbiome Project'... sequencing human microbes to correlate with human health  

 

---> RFLP markers to disease* 

& Newer VECTORS -    [ animation of translation*view@home]

                                [ Virus infection - phage pics - Replication  role of tubulins ] 

                      DNA replication animation quiz*view@home  
         

   Review of the Events:
          1.  DNA pol III binds at the origin of replication site in the template strand
          2.  DNA is unwound by replisome complex using helicase & topoisomerase
          3.  all polymerases require a preexisting DNA strand (PRIMER) to start replication,
                  thus Primase adds a single short primer to the LEADING strand
                  and also adds primers to the LAGGING strand
          4. DNA pol III is a dimer adding new nucleotides to both strands primers
                  direction of reading is 3' ---> 5' on template
                  direction of synthesis of new strand is 5" ---> 3'
                  rate of synthesis is substantial 400 nucleotide/sec
          5. DNA pol I removes primer at 5' end replacing with DNA bases, leaves 3' hole
          6. DNA ligase seals 3' holes of Okazaki fragments on lagging strand -
cartoon
    
        
Review of the sequence of  
events in detail- lagging strand   and 


and       BioFlix anim. of Replication*view@home                       
         DNA Repair mechanisms - 2015 Nobel Prize in Chemistry*
  

 
         Linear DNA replication, Telomeres, and Aging

          Sumanas advanced animation


            - de novo gene origins         

                              current events [Ebola poster]                        


                             DNA @ 60 Poster & sequencing costs  


   rmaceuticals...   Recombinant bacteria*Humulin pen  &  protropin (an ethical dilemma)

                                   How the Maize genome was sequenced by NSF's Plant Genome Inititive

Forensic Analyses     

  e.  Genetically Modified Organisms   [1st by FDA =  animationAtlantic Salmon*picture)
 
    
   
2.  control of the Human genome...   How is gene expression regulated???

 



 

  SKIP this one page 
  
Micro RNAs (& siRNA):  function in gene silencing & gene regulation  
   are single-stranded RNA molecules of 20-24 nucleotides in length, that are not translated
   into protein (non-coding RNA) and form short
stem-loop structures
that are partially
  
complimentary to mRNAs and that can down-regulate or possibly activate gene expression.
 


   some 400 miRNAs are known in the human genome & function as small interfering RNAs
   (siRNA) or miRISC's by hybridizing to mRNA, forming a dsRNA duplex  & block translation
*

         found in MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, &  plants (arabidopsis);
        ex:    
miRNA -->    1) BARR Body*     &     2)  CCD     &      3) heart disease
                                             2) TMK-ebola siRNA Drug
   FDA approved Sep 22, 2014

     The ability of transfected synthetic small interfering miRNAs to suppress the expression of
      specific transcripts is a useful technique to probe gene function in mammalian cells.

      Such non-protein coding RNA transcripts likely regulate much of the genome via - miRNA's.

        new roles* for siRNA, miRNA,  and Crispr-gene editing...

 


      

C.  Sequencing Genes   &   the  Human Genome Project   moon-like project est: $3 bil & 15 yr
     Frederick Sanger (Cambridge U.) in May 1975 produced 1st complete sequence of viral genome
                           of phage
ΦΧ-174 (5,375 np's) -  followed by human mitochondrion with 17K np's.

 1st:    methodology - dideoxy*method* & reading* & procedures   =   sequencing labs*  
                     used terminator nucleotides for they randomly stop action of polymerase
                     when they are incorporated into growing chain marking its end.  
  dideoxy video
 next:  strategy - shotgun approach* shotgun video
                     random DNA fragments (from a library 500-800n long) are sequenced using automated 
                     sequencing machines & then ordered relative to each other via overlap & supercomputing.

                                     
next generation Sequencing    &     Genome Sequencing Standards
 
timeline:   in 2003 (13thyr) 1st "complete" human genome sequence was published ($3 billion).
 costs:       in 2007 Craig Venter's Genome (dideoxy method)  =  $7mil ($10 per 1,000 bases).
                 James Watson's genome (April 2008) =  $1mil  (20 per 1,000 bases)

                 CoFactor Genomics was able to do it next for about $22,900  (4 per 1,000 bases)
                 EBay bidder (in 2009) paid $68,000 for sequencing his genome.
                
NHGRI cost estimates - per million bases & per genome bases* & genome* & fetal genome
 $1,000 genomesNovogene's $850 Sequencing  Your Ancestry DNA   +   23and me   
                 Sequence Your Genome   &   medical whole genome analysis    future of medicine 


     Arthur Kornberg (1918-2007) - 1st to synthesize DNA in test tube won;  

fork 1959 Nobel 


      
  (polynucleotide phosphorylase)

                  additional animations:  BioFlix anim. of Transcription, Translation & Protein Traffic*
                                                   Summary of the Molecular Biology of the Gene*view@home-7 min
                                                                                
Virtual Cell animation of translation*view@home 
              and:   
 Life Cycle of a mRNA  &   Nobel Committee on the Central Dogma   &   Virtual Cell animation of Translation

                             additional animations:  BioFlix anim. of Transcription, Translation & Protein Traffic*
                                                   Summary of the Molecular Biology of the Gene*view@home-7 min
                                                                                
Virtual Cell animation of translation*view@home 
              and:   
 Life Cycle of a mRNA  &   Nobel Committee on the Central Dogma   &   Virtual Cell animation of Translation

          CRISPR a gene editing technique* - 

   
               What's been done   medical roles for RNAs...   

        Luekemia immunotherapy... inserts genes into T-cells for cancer receptors (fig*)  


                                                   MST II also cuts Sickle Cell)               

             copying of DNA into DNA seems structurally obvious*???

                
               How is the transcription of genes regulatedlater?

                       DNA Learning Center video  Eukaryotic Initiation Complex formation is complex
                             
Roger Kornberg's real-time animation of transcription (2006 Nobel)*view@home  x    

                 
E.M. picture*

        &    fig 20.13    

       How is the transcription of genes regulated   .

        
  Long CRISPR-description    HTR

                   
    animation of their experimental design*