MOLECULAR GENETICS                             You Are Here*
                            molecular basis of inheritance 
        Genes   --->   Enzymes   --->   Metabolism  (phenotype) 
                     Central Dogma of Molecular Biology*
       DNA  -transcription-->  RNA  -translation-->  Protein
                                                     Concept Activity -17.1  Overview of Protein Synthesis - INFORMATION FLOW 

   
       What is a GENE
= ?    DNA is the genetic material...
                                         
[ but what about, retroviruses, as HIV & TMV, contain RNA ]
              - a discrete piece of deoxyribonucleic acid
              - linear polymer of repeating nucleotide monomers
                              nucleotides* -->   A   adenine,      C   cytosine
                                                             thymidine,     guanine     --> polynucleotide*

                                                     
Technology with a Twist - Understanding Genetics

 

 


 




 

 
  
INFORMATION PROCESSING    & the    CENTRAL DOGMA
 

     - the letters of the genetic alphabet... are the nucleotides A, T, G, & C
of DNA
             - the unit of information is CODON   =   genetic 'word'
                                  a triplet sequence of nucleotides   'CAT'  in a polynucleotide
                                  3 nucleotides  =  1 codon (word)  =  1 amino acid in a polypeptide
             - the definition of (codon) word = amino acid

   

     - Size of Human Genome:  3,000,000,000 base pairs  or 1.5b in single strand of DNA genes
                                          
    500,000,000 possible codons (words or amino acids)
             - average page your textbook  =  approx 850 words
                 thus, human genome is equal to 588,000 pages   or  470 copies of bio text book
                  reading at 3 bases/sec it would take you about 47.6 years  @  8h/d - 7d/w

                                       
WOW... extreme nanotechnology
 
   
  
Mice & humans (indeed, most or all mammals including dogs, cats, rabbits, monkeys, & apes)
         have roughly the same number of nucleotides in their genomes -- about 3 billion bp.
         It is estimated that 99.9% of the 3billion n's of human genome is same person to person.

                                            

 

 

 

 

 
 
Experimental Proof of DNA as Genetic Material...

      1.  Transformation Experiments of Fred Griffith... (1920's)   
          
Streptococcus pneumoniaepathogenic S strain  &  benign R 
                        transforming 'principle'* (converting R to S cells)  is the genetic element
   

      2.  Oswald Avery,  Colin MacLeod,  &   Maclyn McCarty...
(1940's)
                        suggest the transforming substance* is DNA molecules
, but...
  

       3.  Alfred  Hershey   &   Martha  Chase's*  
1952   bacteriophage experiments*...
               VIRAL REPLICATION*   
[ phage infection &  & lytic/lysogenic*
                        a genetically controlled biological activity
(viral reproduction)
                        they did novel experiment...
1st real use radioisotopes in biology*
               CONCLUSION - DNA is genetic material because  
                  (32P) nucleic acid not (35S) protein guides* viral replication
                                                        Sumanas, Inc. animation - Life cycle of HIV virus
 

 

 

 

 

 


 

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

  Nobel prize
*JD Watson, Francis Crick, Maurice Wilkins,  
but [ Erwin Chargaff & Rosy Franklin ]...
  Race for the Double Helix "Life Story" - a BBC dramatization of the discovery of DNA.

  

  used two approaches to decipher structure:
          1. model building - figure*    (are the bases in/out; are the sugar-P's in/out?)
          2. x-ray diffraction* pattern* favor a DNA helix of constant diameter*  

  

   we know now: DNA is a double stranded, helical, 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,   dimensions*,   models of DNA structure*
                     john kyrk's animation of DNA  &   Quicktime movie of DNA structure
 
            literature references            &   myDNAi timeline*      

 

 

 

 

 

Replication of DNA...      (Arthur Kornberg -  1959 Nobel  -  died 10/26/07)    
             copying of DNA into DNA is structurally obvious???    [figure*
 
   Patterns of Replication* =
 conservative,  semi-conservative,  &  dispersive 
             Matt Meselson & Frank Stahl  1958 - experimental design*
                    can we separate 
15N-DNA   from   14N-DNA  - (OLD DNA from NEW DNA)?
                    
sedimentation of DNA's  (sucrose gradients  -->  CsCl gradients*  &  picture*)
             we can predict results...  figure*
 &   overview &  all possible results 
                                                  Sumanas, Inc. animation - Meselson-Stahl DNA Replications*   

  DNA polymerase: enzyme that copies DNA...  prokaryotic Pol I-IV  eukaryotic a & d 
    
 Pol III (pic)    req: 4-deoxy-NTP's ssDNA template piece       
                    reads template and adds a complimentary nucleotide
*                 
 
                   reads 3' to 5' and synthesizes in 5' to 3' direction...               [quicktime movie]
                           proofreads*  &    bidirectional synthesis*...  &   EM pic*  
     Replication forks - leading*  &  lagging*  strands - Campbell figure* 
 
      
   Arthur Kornberg - 1st to synthesize DNA in test tube, died 26 Oct 2007


                         
 

 

 

 

 
  

Model of Replication
is bacterial with DNA polymerase III...
  

          several enzymes* form a Replication Complex (Replisome) & include:    
                    helicase - untwists DNA 
                    topoisomerase [DNA gyrase] - removes supercoils,
                    single strand binding proteins - stabilize replication fork,
                    Primase - makes RNA primer
                    POL III - synthesizes new DNA strands 
                    DNA polymerase I - removes RNA primer 1 base at a time, adds DNA bases
                    DNA ligase repairs Okazaki fragments (seals lagging strand 3' open holes)  
                                                                                         Concept Activity - DNA Replication Review
   

  Structure of DNA polymerase III
*
          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
   
 
   

  

 


  
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 adds many 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
                            the sequence of  
events in detail*     and       DNA Repair*
  

      Rates of DNA synthesis:        
myDNAi movie of replication*
                                 
native polymerase: 400 bases/sec with 1 error per 109 bases
                                 
artificial: phophoramidite method (Marvin Caruthers, U.Colorado); ssDNA synthesis
                          on polystyrene bead @ 1 base/300 sec with error rate of 1/100b      

 

 

 

 

 

 
 
GENE Expression 
     
the Central Dogma of Molecular Biology depicts flow of genetic information
        
          Transcription - copying of DNA sequence into RNA
      
            Translation     - copying of RNA sequence into protein
  

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

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

  
   

 Compare Events:
         Procaryotes*  vs.  Eucaryotes*  =   Separation of labor 
         Differences DNA vs. RNA (
bases & sugars)  and its single stranded
        
Flow of Gene Information (FIG*) - One Gene - One enzyme (Beadle & Tatum)
   

                                                              
18.3 - Overview: Control of Gene Expression                                           

 

 

 

 

 

 Transcription - RNA polymerase           Concept Activity 17.2 - Transcription                                                                          
        RNA*polymerase  - in bacteria Sigma factor* binds promoter &  initiates*  copying*
                      
[pnpase]                                                   
             transcription factors* are needed to recognize specific DNA sequence [motif*] 
             binds to
promoter DNA region [ activators & transcription factors*]   *     
             makes a complimentary copy
* of one  of the two DNA strands  [sense strand
                
Quicktime movie of transcription*     myDNAi Roger Kornberg's movie of transcription (2006 Nobel)*
                                                       
   
     Kinds of RNA  [table*]                                            
          
tRNA -    small,  80n,  anticodon sequence, single strand with 2ndary structure*  
                                 function = picks up aa & transports it to ribosome 
  
          
rRNA -    3 individual pieces of RNA - make up the organelle = RIBOSOME
                                 primary transcript is processed into
 the 3 pieces of
                                          
rRNA pieces (picture*)   &    recall structure of ribosome 
                              

 

 

 

 

 

  hnRNA -  heterogeneous nuclear RNA : large Primary Transcript RNA of genes
                               function - it is the precursor molecule of mRNA in eukaryotes 
                            
                
  mRNA -   intermediate sizes - 100n to 400n    ( split genes*)    primary transcript & mRNA
                              function - codes for amino acid sequence               
were not same size?
 processing (cutting) of introns & exons*
   Splicesome splicing of eucaryotic genes*  [glossary]  (Sumanas, Inc. advanced animation)
  
  
 
   structure of mRNA*caps & tails    
 
   role of 5' CAP and Poly-A Tails*  [glossary]          luciferase
 summary of eukaryotic RNA processing*
 
      


  
 

 

 

 

    Other classes of RNA:
  

   small nuclear RNA  (snRNP's)
 - plays a structural and catalytic role in spliceosome*
                    t
here are 5 snRNP's making a spliceosome [U1, U2, U4, U5, & U6];
                    they and participate in several RNA-RNA and RNA-protein interactions

   SRP (signal recognition particle) - srpRNA is a component of the protein-RNA complex
            that recognizes the signal sequence of polypeptides targeted to the ER -
figure
*

   small nucleolar RNA (snoRNA)    - aids in processing of pre-rRNA transcripts for
                                                            ribosome subunit formation in the nucleolus

   micro RNA's (micro-RNA) - also called antisense RNA & interfereing RNA c7-fig 19.9
                     short (20-24 nucleotide) RNAs that bind to mRNA inhibiting it.    
figure
     
  present in MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, &  plants (arabidopsis);
       seems to help regulate gene expression by controlling the timing of developmental events via mRNA action
       also inhibits translation of target mRNAs.
ex:        siRNA   -->   [BARR Body*]
  
 
                   
  
           
                                                           
  
 

 

 

 

 

 

 

TRANSLATION - Making a Protein   
       process of making a protein in a specific amino acid sequence 
                                                          from a unique mRNA sequence...  
[ E.M. picture* ]
      
polypeptides are built on the ribosome (pic) on a polysome [ animation*]
  
   Sequence of 4 Steps in Translation...                                    [glossary]                            
 
             1. add an amino acid to tRNA  -- >   aa-tRNA           
- ACTIVATION*
                                                                                                                                                                 

             2. assemble players [ribosome
*, mRNA, aa-tRNA] - INITIATION*
  

             3. adding new aa's via peptidyl transferase            
- ELONGATION*
         

             4. stopping the process                                            
 - TERMINATION*
                                                                           
Concept CD Activity - 17.4 Events in Translation
 
                     Review the processes - initiation, elongation, & termination
                                     myDNAi real-time movie of translation*     &      Quicktime movie of translation       
                      Review figures & parts:    Summary fig*
                                           [
components locations,  AA-site, advanced animation ]
                                           [
Nobel Committee static animations of Central Dogma ]
     
                                            

 

 

 

 
 GENETIC CODE...   
    

       ...is the sequence of nucleotides in DNA, but routinely shown as a  mRNA  code*
       ...specifies sequence of amino acids to be linked into the protein 
  
          coding ratio* - # of n's...    how many nucleotides specify 1 aa
                    
1n = 4 singlets,  2n= 16 doublets,  3n = 64 triplets
                                                                                     Student CD Activity - 11.2 - Triplet Coding
  

         
S. Ochoa  (1959 Nobel) - polynucleotide phosphorylase  can make SYNTHETIC mRNA
                                                         Np-Np-Np-Np 
<---->    Np-Np-Np   +   Np

  
          Marshall Nirenberg
(1968 Nobel) - synthetic mRNA's used in an in vitro system 
                5'-UUU-3' = phe         U + C -->  UUU,  UUC,  UCC,  CCC
                                                                     UCU,  CUC,  CCU,  CUU
  
         the Genetic CODE*  -   64 triplet codons [61 = aa   &   3 stop codons]
                      universal (
but some anomalies),  1 initiator codon (AUG), 
                      redundant but non-ambiguous, and exhibits "wobble
*".
 
 

 

 

 

 

 

 

 


 
GENETIC CHANGE - a change in DNA nucleotide sequence (= change in mRNA)
                             - 2 significant ways  mutation & recombination       
[glossary]
                                                                                                
    1. MUTATION -  a permanent change in an organism's DNA*that results in
                                 a different codon = different amino acid sequence
         Point mutation*  - a single to few nucleotides change...
             - deletions, insertions, frame-shift mutations*     [CAT]
 
                                                                               
             - single nucleotide base substitutions* :
                     non-sense = change to no amino acid (a STOP codon)
                                                     UCA --> UAA      ser to non
                     mis-sense = different amino acid 
                                                     UCA --> UUA      ser to leu 
                             Sickle Cell Anemia*  - a mis-sense mutation... (
SCA-pleiotropy)
                                          another point mutation blood disease - thalassemia
    - Effects = no effect, detrimental (lethal), +/- functionality, beneficial   

 

 

 

 


2. 
Recombination (Recombinant DNA) newly combined DNA's that            [glossary]*
      can change genotype via insertion of NEW (foreign) DNA molecules into recipient cell
     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
g*  (F+ & R = resistance)
                 conjugation may be a primitive sex-like reproduction in bacteria  [Hfr*]   
     5. VIRAL TRANSDUCTION - insertion via a viral vector
 (lysogeny* & TRANSDUCTION*)
                 general transduction - pieces of bacterial DNA are 
                                             packaged w viral DNA during viral replication
                 restricted transduction - a temperate phage goes lytic
                                            carrying adjacent bacterial DNA into virus particle
     6. DESIGNER GENES    -   man-made recombinant DNA molecules
 

 

 

 

 

 

Designer Genes - Genetic Engineering - Biotechnology

   RECOMBINANT DNA TECHNOLOGY...
         a collection of experimental techniques, which allow for 
                  isolation, copying, & insertion of new DNA sequences into 
                  host-recipient cells by A NUMBER OF  laboratory protocols & methodologies

     Restriction Endonucleases-
[glossary]*... diplotomic cuts (unequal) at unique DNA sequences
           
Eco-R1-figure*                                  @ mostly palindromes...    [never odd or even]
                  

          
5' GAATTC 3'          5' G . . . . .      +      AATTC 3'
        3' CTTAAG 5'          3' CTTAA               . . . .  G 5'

        
                                                                                 campbell 7/e movie*
               DNA's cut this way have STICKY (complimentary) ENDS & can be reannealed
               or spliced*  w other DNA molecules to produce new genes combos

    and sealed via DNA ligase.                      myDNAi movie of restriction enzyme action*
 

 

 

 

 

 

 
 Procedures of Biotechnology?   
[Genome Biology Research]
            

   A.  Technology involved in Cloning a Gene...    [animation*  &  the tools of genetic analysis]
                       
making copies of gene DNA

            1.  via a plasmid
*  [ A.E. fig  &  human shotgun plasmid cloning  &  My DNAi movie*]   

            2.  Libraries
g...    [ library figure*  &  BAC's*   &  Sumanas animation - DNA fingerprint library ]

            3.  Probes
g...        [ cDNAg   &   reverse transcriptaseg   &   DNA Probe Hybridizationg...
                                  cDNA figure*  &    cDNA library*  &    a probe for a gene of interest*
                                  finding a gene with a probe among a library
*]
   

            4. 
Polymerase Chain Reactiong     &     figure 20.7*   &   animation* +  Sumanas, Inc. animation*   
                                            the PCR song                                               PCR reaction protocol   &    Xeroxing DNA    &   Taq polymerase


 


 

 

 

 

 

 
   B.  Detection of a Gene...   Locating a gene (or its activity) -  Restriction Maps.
  

        1.  Restriction maps
g...     via gel electrophoresis*   &   DNA-electropherogram*
                          
   
    2.  DNA fingerprintg...     CSI Miami - how to make one*
                      
a murder case* &  a rape case*  + DNA prints in Health & Society & DNA Forensic Science   
  

       
3.  Comparing Restriction Fragments...    to a probe
               
Southern
g  Blotting  fig*            Sumanas, Inc. animation - DNA electrophoresis & blotting*
                    one can detect specific gene sequence in samples by binding to labeled probes
  
       4.  Measuring single gene expression via RT-PCR methodologies - fig 20.13
*
   
       5.  DNA
micro chips* arrays - monitor gene expression in thousands of genes & changes
                   
by passing cDNA of the cell's mRNA over slide with ssDNA of all cell's genes;
                   
DNA microchips are fabricated by high speed robotics akin to Intel chip making
                   
cDNA (mRNA's) are fluorescently tagged so easy to see in slide's wells.
                  
             [microchips arrays made simultaneously by phopshoramidite method of Caruthers]
 
        Sumanas animation - DNA chip technology*      &            myDNAi DNA microarrays*

                                                                                                                            
 

 

 

 

 

        5.  Gene Sequencing - Human Genome Project  

                     strategy - shotgun approach
* developed by Celera Genomics
 
                                             random fragments are sequenced and then ordered
                                              relative to each other via overlap & supercomputing

                                   
Student CD Activity - 16.1 - Sequencing Strategies
                     methodology  dideoxy procedure* (development by Fred Sanger)
   
 
          Surprising Size Estimates of Human Genome    &    figure*
                        
NHGRI researchers* have confirmed the existence of 19,599 protein-coding genes
                     in the human genome and identified another 2,188 DNA segments that are
                     predicted to be protein-coding genes    = 
 
21,787 genes

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

                              mtDNA & Y-chromosome DNA aid in search for our human ancestry

     descriptions of DNA sequencing, microarrays, Northern/Southern Blotting, & protein analysis*

 

 

 

 

 

 

 

 Practical Applications of DNA Technology - Some examples of What's been Done...
      

 1.  Medical... disease often involves changes in gene expression
           a.  disease/infection diagnosis:

                        PCR & labeled DNA probes from pathogens can help identify microbe types...
                             isolate HIV RNA  --RT--> cDNA --PCR--> probe     can ID... AIDS infection

           b. 
RFLP -  Restriction Fragment Length  Analysis - markers often inherited with disease
                        what is RFLP
*   genetic testing & polymorphism ---> RFLP markers to disease 
                    DdeI cuts
Sickle gene*    (also MST II cuts Sickle Cell)
                      
      fragment analysis (DNA fingerprinting) also used for paternity testing  + GINA   
           c.  Gene Therapy... idea is to replace defective genes via
 microinjection of DNA* 
                    
requires
VECTORS - fig 20.22 (patient: ADA Deficiency & Ashanti DeSilva update)
                                    SCID [severe combined immunodeficiency - a single gene enzyme defect],
                                    clinical trials in 2000 resulted in 2 of 9 cured, but they developed leukemia:
                                         a retroviral vector inserted a repair gene in bone marrow cells
                                         near genes involved in blood cell division, thus leukemia.  trials stopped.

   
 2.  Pharmaceutical Products...   manufactured drugs 
                  Recombinant bacteria*  =  Humulin       &         protropin (an ethical dilemma)
*    
 
                                                                                                 Student CD Activity - 17.1 - Producing Human Growth Hormone
  
 

 

 

 

 

 

 





 

 

 

 

 

 

 

    
  Control of Gene Expression

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

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


              but, what about pre-existing inactive enzymes converting to --> active forms 
                                    ZYMOGENS        - pepsinogen    ----->   pepsin
                                                                 - trypsinogen   ----->   trypsin

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

 

 

 

 

 

 
 
Mechanism of Gene Action (turning on/off genes)  in  PROCARYOTES...
   

    model:  LACTOSE OPERON - Jacob & Monod                 
[glossary]*
  

                                                             E. coli  
(grown on)
                                
glucose                           lactose
            NO beta-galactosidase                                     beta-galacotsidase 
                                                                                                lac  --->  glu + gal 
  
   OPERON* = series of mapable-linked genes controlling synthesis of protein
                                    
p    Rg      crp      p  O   Sg1   Sg2   Sg3   
    p                    promoter  -  binds RNA polymerase        figure
    Rg (i gene)    regulator  -  makes repressor protein     figure
*
    O                   operator   -  binds repressor protein      figure
    
    S                    structural -  make enzyme proteins        what if regulator binds lactose
*
 
                          Sumanas, Inc. animation - Lac Operon*              Catabolic Repression*    
  

 

 

 

 

 

 

 

 
 
Control of Gene Expression  -    in   EUKARYOTES
                                                                                                       

    Mechanism of Gene Action (turning on/off genes) has greater complexity
      

                there are
no operons present
                and there's much more DNA   &   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
                but, the individual genes are not contiguous with each other, thus no operons


            
 3 levels for eukaryotic controls*   - transcriptional,   translational,   post-translational

          
multiple places for control* - of whether a gene make a protein or not
  

                                              McGraw-Hill higher Ed movie on control of gene expression

 

 

 

 

 

 

 
    
Some examples for Eukaryotic gene expression controls: 
  

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

        2.   Molecular turnover -  life mRNA's*   &    longevity of some proteins*
  

        3.   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*    and  some examples*.

 

 

 

 

 

 

 ex. cont.     Eukaryotic gene expression controls: 
  

 5.  cancer often results from gene changes affecting cell cycle control proteins:
                  cancer genes, such as adenomatous polyposis coli, which cause 15% of
                  colorectal cancers is a tumor suppressor gene, a type of  Oncogenesg
*
    

     2 kinds of human cancer genes:
          Ras (proto-oncogene) causes 30% human cancers:
                   is a G-protein that promotes other cell division proteins by over-expression-
                   a Ras mutation --> hyperactive Ras protein --> cell division   fig 18.21a
*

          p53 (tumor suppressor geneg = 50% human cancers)                    fig 18.21b*
                   p53 is a transcription factor that promotes the synthesis of cell cycle
                   inhibiting proteins           [
DNA damage --> active p53 --> p51 gene --> protein binds to
                                                                                                   cyclin dependent kinase stops cell division
]
                    
  thus a p53 mutation --> leads to excess cell division (cancer)                    
  

          - other cancer genes can lead to new gene actions resulting in cancer
                BRCA1 and BRCA2 (tumor suppressor genes) are involved in 50% of breast cancers in humans
                                                                                                                          
                                 


 
  

 

split genes DNA DNA2 chromatin

     Organization of the Genome   
                      the structural organization of genome in eukaryotes influences its expression.

           Size of Human genome:
                      3 billion+ base pairs, equaling some 500,000 pages of journal Nature.
                      reading at 5 bases/sec it would take you about 60 year @ 8h/d 7d/w
           yet, there are only about 21,787  protein coding genes.

       A definition of a GENE*.  



 

 

 

 

 

 Definition of a Gene
     Mendel's Particles... unit of heredity responsible for phenotype
     Morgan's Loci... he placed genes on a chromosome, i.e., 
                it's a cellular entity, that is part of chromosome & is mapable
     Watson & Crick... it's a sequence of specific nucleotides along the
                                           length of a double helical DNA molecule
            Molecular Definition...  
               length:  1 nucleotide = 0.34nm  thus tRNA = 81n x 0.34 = 27.5nm
               mass:    1 nucleotide = 340amu thus tRNA = 81n x 340 = 27,540amu

     Modern functional definition...  
          a DNA sequence coding for a specific polypeptide: but, also must include...
            
Split Genes...    presence of Introns & Exons :
                              eukaryotic genes contain non-coding segments (introns
                              and coding segments (
exons - that make proteins)

           
Others DNA pieces... any definition must also include:
                                    segments that code for rRNA, tRNA, & snRNP's
                                    also
promoters, enhancer segmentsregulator genes, operators?

     BEST
 ≈  "a segment of DNA corresponding to a single protein (or set of alternate
            protein variants) or a single catalytic or structural RNA molecule"   end.

 

 

 

 

 

 

 

 

 

 

 

 
 

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]

        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-

1st point insertion or deletion

        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    

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

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  
   back

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

DO  NOT  study the material below

 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 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?