Download MOLECULAR GENETICS You Are Here*

MOLECULAR GENETICS
You Are Here*
molecular basis of inheritance
Genes ---> Enzymes ---> Metabolism (phenotype)
Central Dogma of Molecular Biology*
DNA -transcription--> RNA -translation--> Protein
Student CD Activity - 13.2 Events Protein Synthesis: INFORMATION
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
T thymidine, G guanine
--> polynucle
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 polynucleotid
3 nucleotides = 1 codon (word) = 1 amino acid
- 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
≈
500,000,000 possible codons (words or amino acids)
- average page your textbook = approx 850 words
thus, human genome is equal to 590,000 pages or 470 copies of bio text boo
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, &
have roughly the same number of nucleotides in their genomes -- about 3 billion
bp.
Experimental Proof of DNA as Genetic Material...
1. Transformation Experiments of Fred Griffith... (1920's)
Streptococcus pneumoniae - pathogenic S strain & benign R
transforming 'principle'* (converting R to S cells) is the genetic ele
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 experimen
VIRAL REPLICATION* [ pic 1 phage infection & pic-2* & 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/ou
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 &
literature references
Replication of DNA...
&
Quicktime movie of DNA structure
myDNAi timeline*
James D. Watson Resigns
(Arthur Kornberg - 1959 Nobel - died 10/26/
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 Replication
DNA polymerase: enzyme that copies DNA...
prokaryotic
Pol I-IV eukaryotic 
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...
proofreads* & bidirectional synthesis*... & EM pic*
Replication forks - leading & lagging strands - Campbell figure*
Pol III (pic)
[quickti
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 DN
DNA ligase repairs Okazaki fragments (seals lagging strand 3' open
student CD Activity - 12.1 - Overview of DNA Synthesis
Structure of DNA polymerase III*
copies both strands simultaneously, as DNA is Threaded Through a Repliso
a "replication machine", which may be stationary by anchoring in nuclear m
Continuous & Discontinuous replication occur simultaneously in both st
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 replic
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' h
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 syn
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)
Transcription - RNA polymerase
Student CD Activity 13.1 - Transcription
RNA*polymerase - in bacteria Sigma factor* binds promoter & initiates* co
[pnpase]
Student CD Activity 15.1 - DNA Regulatory R
transcription factors* are needed to recognize specific DNA sequence [
binds to promoter DNA region [ activators & transcription factors*] *
makes a complimentary copy* of one of the two DNA strands [sense st
Quicktime movie of transcription*
myDNAi Roger Kornberg's movie of transcription (20
Nobel)*
Kinds of RNA [table*]
tRNA -
structure*
small, 80n, anticodon sequence, single strand with 2ndary
function = picks up aa & transports it to ribosome
rRNA -
3 individual pieces of RNA - make up the organelle = RIBOSOM
primary transcript is processed into the 3 pieces of
rRNA pieces (picture*) & recall structure of ribosome
hnRNA -
heterogeneous nuclear RNA : large Primary Transcript RNA
function - is the precursor of mRNA in eukaryotes
mRNA -
intermediate sizes - 100n to 400n ( split genes*)
function - codes for amino acid sequence
primary transcript
were not sam
processing (cutting) of introns & exons*
Splicesome splicing of eucaryotic genes*
[glossary] (Sumanas, Inc. advanced an
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*
there 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) - scRNA is a component of the protein-RNA c
that recognizes the signal sequence of polypeptides targeted to the ER small nucleolar RNA (snoRNA)
- aids in processing of pre-rRNA transcripts f
ribosome subunit formation in the nucleolu
micro RNA's (micro-RNA) - also called antisense RNA & interfereing RNA; c7short (20-24 nucleotide) RNAs that bind to mRNA inhibiting it.
present in MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, & plants (ar
seems to help regulate gene expression by controlling the timing of developmental events via mRN
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. pictu
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
4. stopping the process
- ELONGATION*
- TERMINATION*
Student CD Activity - 13.2 Events in Protein Syn
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 co
...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 mR
Np-Np-Np-Np <---->
Np-Np-Np + Np
Marshall Nirenberg (1968 Nobel) - synthetic mRNA's
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
- 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]
Student CD Activity - 11.2 - Triplet Coding
- 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
[g
can change genotype via insertion of NEW (foreign) DNA molecules into reci
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
4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R = resist
conjugation may be a primitive sex-like reproduction in bacteria [Hfr
5. VIRAL TRANSDUCTION - 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 & metho
Restriction Endonucleases-[glossary]*... diplotomic cuts* at unique DNA seque
Eco-R1-figure*
mostly palindromes... [Never odd or e
▼
5' GAATTC 3'
3' CTTAAG 5'
5' G . . . . .
3' CTTAA
+
AATTC 3'
. . . . G 5'
campbell 7/e movie*
▲
DNA's cut this way have sticky (complimentary) ends & can be reannea
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 analysi
making copies of gene DNA
1. via a plasmid* [ A.E. fig & human shotgun plasmid cloning & My DNAi movie]
2. Librariesg...
3. Probesg...
[ library figure* & Sumanas animation - DNA fingerprint library ]
[ cDNAg
cDNA figure*
4. Polymerase Chain Reactiong
&
&
&
reverse transcriptaseg
cDNA library* ]
figure 20.7*
&
animation* +
Sumanas, Inc. ani
PCR reaction protocol &
Xeroxing DNA
& Ta
polymerase
B. Detection of a Gene... Locating a gene (or its activity) - Restriction Maps.
1. Restriction mapsg...
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
3. DNA Probe Hybridizationg... to detect specific DNA with a probe
4. Comparing Restriction Fragments...
fig 20.5*
to a probe
Southerng Blotting fig*
Sumanas, Inc. animation - DNA electrophoresis & blot
one can detect specific gene sequence in samples by binding to labeled probes
5. DNA micro-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 maki
cDNA (mRNA's) are fluorescently tagged so easy to see in slide's wells.
[microchips arrays made simultaneously by phopshoramidite method of Caruthe
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 gene
in the human genome and identified another 2,188 DNA segments that are
predicted to be protein-coding genes
=
21,787 genes
mtDNA & Y-chromosome DNA aid in search for our human ancestry
Practical Applications of DNA Technology - Some examples of What's been Do
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 infectio
b. RFLP - Restriction Fragment Length Analysis - markers often inherited with
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
c. Gene Therapy... idea is to replace defective genes via microinjection of
requires VECTORS - fig 20.16* (patient: ADA Deficiency & Ashanti DeSilva u
SCID (severe combined immunodeficiency - a single gene enzyme defec
clinical trials in 2000 resulted in 2 of 9 cured, but they developed leuk
a retroviral vector inserted a repair gene in bone marrow cells
near genes involved in blood cell division, thus leukemia. trials sto
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 RN
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 f
ZYMOGENS
- pepsinogen -----> pepsin
- trypsinogen -----> trypsin
thus, we
have 2 possibilities:
1) pre-existing inactive enzyme --> active
2) de novo (new) enzyme synthesis (gene act
Mechanism of Gene Action (turning on/off genes)
in PROCARYOTES.
model: LACTOSE OPERON - Jacob & Monod
E. coli
[glossary]*
(grown on)
glucose
lactose
NO beta-galactosidase
beta-galacotsidase
lac ---> glu + gal
OPERON* = series of mapable-linked genes controlling synthesis of protein
p
Rg (i gene)
p
O
S
Rg
crp
p
O Sg1 Sg2 Sg3
regulator - makes repressor protein
promoter - binds RNA polymerase
operator - binds repressor protein
structural - make enzyme proteins
Sumanas, Inc. animation - Lac Operon*
Control of Gene Expression
-
what if regulator binds
figure
figure
figure*
Catabolic Repression*
in EUKARYOTES
Mechanism of Gene Action (turning on/off genes) is more complex
much more DNA
& it's inside a compartment (nucleus)
and, there are no operons present
have many more promoters - sites where RNA polymerase binds
enhancer sequence - sites where enhancers/transcription factors bind
transcription factors - proteins that help transcription
but, individual genes are not contiguous, thus no operons
3 levels for eukaryotic controls* - transcriptional, translational, post-translatio
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 ce
1.
role of activators in selective gene expression (Differential Gene Activity*
ex: Steroid Hormones (figure*)
2. Molecular turnover - ½ 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 C17.11*
and some examples*
ex. cont.
Eukaryotic gene expression controls:
5. cancer often results from gene changes affecting cell cycle control.
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
a Ras mutation --> hyperactive Ras protein --> cell division fig 19.12
p53 (tumor suppressor geneg = 50% human cancers)
fig 19.12b
p53 is a transcription factor that promotes the synthesis of cell cyc
inhibiting proteins
[DNA damage --> active p53 --> p51 gene --> protein binds
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 hum
Organization of the Genome* the structural organization of genome in eukaryotes influence its exp
Size of Human genome:
3million+ base pairs, or some 500,000 pages of journal Nature.
reading a 5 bases/sec it would take you about 60 year @ 8h/d 7d/w
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 segments, regulator genes, operators ?
BEST ≈ "a GENE is a region of DNA that CODES for an RNA"
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
restriction enzyme Mst II, which cuts normal b globin DNA at a particular site, but will not recognize an
therefore will not cut DNA that contains the sickle cell mutation. Mst II recognized the sequence CC
(where N = any nucleotide). Sickle cell disease is due to a single point mutation in the beta globin gene
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.
results from a change in the nucleotide T to A. This change eliminates a site recognized by
restriction enzyme DdeI.
Restriction enzyme: DdeI (recognition sequence: 5'-C^TNAG-3')
Southern blotting probe: fragment of ß-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 (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-T1st 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 = norma
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 increa
number by a process of dynamic mutation; # of tandem repeats is unique to a genet
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 gr
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
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 futur
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
fibrosis;
Plants
artificially insemination, microinjection of human gene, fertilized ova are put
into a surrogate sheep:
chimeras mated to produce homozygote- Milk tested for active protein.
- 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 prote
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?
MOLECULAR GENETICS
You Are Here*
molecular basis of inheritance
Genes ---> Enzymes ---> Metabolism (phenotype)
Central Dogma of Molecular Biology*
DNA -transcription--> RNA -translation--> Protein
Student CD Activity - 13.2 Events Protein Synthesis: INFORMATION
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
T thymidine, G guanine
--> polynucle
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 polynucleotid
3 nucleotides = 1 codon (word) = 1 amino acid
- 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
≈
500,000,000 possible codons (words or amino acids)
- average page your textbook = approx 850 words
thus, human genome is equal to 590,000 pages or 470 copies of bio text boo
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, &
have roughly the same number of nucleotides in their genomes -- about 3 billion
bp.
Experimental Proof of DNA as Genetic Material...
1. Transformation Experiments of Fred Griffith... (1920's)
Streptococcus pneumoniae - pathogenic S strain & benign R
transforming 'principle'* (converting R to S cells) is the genetic ele
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 experimen
VIRAL REPLICATION* [ pic 1 phage infection & pic-2* & 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/ou
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 &
literature references
Replication of DNA...
&
Quicktime movie of DNA structure
myDNAi timeline*
James D. Watson Resigns
(Arthur Kornberg - 1959 Nobel - died 10/26/
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 Replication
DNA polymerase: enzyme that copies DNA...
prokaryotic
Pol I-IV eukaryotic 
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...
proofreads* & bidirectional synthesis*... & EM pic*
Replication forks - leading & lagging strands - Campbell figure*
Pol III (pic)
[quickti
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 DN
DNA ligase repairs Okazaki fragments (seals lagging strand 3' open
student CD Activity - 12.1 - Overview of DNA Synthesis
Structure of DNA polymerase III*
copies both strands simultaneously, as DNA is Threaded Through a Repliso
a "replication machine", which may be stationary by anchoring in nuclear m
Continuous & Discontinuous replication occur simultaneously in both st
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 replic
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' h
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 syn
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)
Transcription - RNA polymerase
Student CD Activity 13.1 - Transcription
RNA*polymerase - in bacteria Sigma factor* binds promoter & initiates* co
[pnpase]
Student CD Activity 15.1 - DNA Regulatory R
transcription factors* are needed to recognize specific DNA sequence [
binds to promoter DNA region [ activators & transcription factors*] *
makes a complimentary copy* of one of the two DNA strands [sense st
Quicktime movie of transcription*
myDNAi Roger Kornberg's movie of transcription (20
Nobel)*
Kinds of RNA [table*]
tRNA -
structure*
small, 80n, anticodon sequence, single strand with 2ndary
function = picks up aa & transports it to ribosome
rRNA -
3 individual pieces of RNA - make up the organelle = RIBOSOM
primary transcript is processed into the 3 pieces of
rRNA pieces (picture*) & recall structure of ribosome
hnRNA -
heterogeneous nuclear RNA : large Primary Transcript RNA
function - is the precursor of mRNA in eukaryotes
mRNA -
intermediate sizes - 100n to 400n ( split genes*)
function - codes for amino acid sequence
primary transcript
were not sam
processing (cutting) of introns & exons*
Splicesome splicing of eucaryotic genes*
[glossary] (Sumanas, Inc. advanced an
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*
there 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) - scRNA is a component of the protein-RNA c
that recognizes the signal sequence of polypeptides targeted to the ER small nucleolar RNA (snoRNA)
- aids in processing of pre-rRNA transcripts f
ribosome subunit formation in the nucleolu
micro RNA's (micro-RNA) - also called antisense RNA & interfereing RNA; c7short (20-24 nucleotide) RNAs that bind to mRNA inhibiting it.
present in MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, & plants (ar
seems to help regulate gene expression by controlling the timing of developmental events via mRN
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. pictu
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
4. stopping the process
- ELONGATION*
- TERMINATION*
Student CD Activity - 13.2 Events in Protein Syn
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 co
...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 mR
Np-Np-Np-Np <---->
Np-Np-Np + Np
Marshall Nirenberg (1968 Nobel) - synthetic mRNA's
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
- 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]
Student CD Activity - 11.2 - Triplet Coding
- 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
[g
can change genotype via insertion of NEW (foreign) DNA molecules into reci
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
4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R = resist
conjugation may be a primitive sex-like reproduction in bacteria [Hfr
5. VIRAL TRANSDUCTION - 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 & metho
Restriction Endonucleases-[glossary]*... diplotomic cuts* at unique DNA seque
Eco-R1-figure*
mostly palindromes... [Never odd or e
▼
5' GAATTC 3'
3' CTTAAG 5'
5' G . . . . .
3' CTTAA
+
AATTC 3'
. . . . G 5'
campbell 7/e movie*
▲
DNA's cut this way have sticky (complimentary) ends & can be reannea
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 analysi
making copies of gene DNA
1. via a plasmid* [ A.E. fig & human shotgun plasmid cloning & My DNAi movie]
2. Librariesg...
3. Probesg...
[ library figure* & Sumanas animation - DNA fingerprint library ]
[ cDNAg
cDNA figure*
4. Polymerase Chain Reactiong
&
&
&
reverse transcriptaseg
cDNA library* ]
figure 20.7*
&
animation* +
Sumanas, Inc. ani
PCR reaction protocol &
Xeroxing DNA
& Ta
polymerase
B. Detection of a Gene... Locating a gene (or its activity) - Restriction Maps.
1. Restriction mapsg...
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
3. DNA Probe Hybridizationg... to detect specific DNA with a probe
4. Comparing Restriction Fragments...
fig 20.5*
to a probe
Southerng Blotting fig*
Sumanas, Inc. animation - DNA electrophoresis & blot
one can detect specific gene sequence in samples by binding to labeled probes
5. DNA micro-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 maki
cDNA (mRNA's) are fluorescently tagged so easy to see in slide's wells.
[microchips arrays made simultaneously by phopshoramidite method of Caruthe
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 gene
in the human genome and identified another 2,188 DNA segments that are
predicted to be protein-coding genes
=
21,787 genes
mtDNA & Y-chromosome DNA aid in search for our human ancestry
Practical Applications of DNA Technology - Some examples of What's been Do
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 infectio
b. RFLP - Restriction Fragment Length Analysis - markers often inherited with
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
c. Gene Therapy... idea is to replace defective genes via microinjection of
requires VECTORS - fig 20.16* (patient: ADA Deficiency & Ashanti DeSilva u
SCID (severe combined immunodeficiency - a single gene enzyme defec
clinical trials in 2000 resulted in 2 of 9 cured, but they developed leuk
a retroviral vector inserted a repair gene in bone marrow cells
near genes involved in blood cell division, thus leukemia. trials sto
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 RN
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 f
ZYMOGENS
- pepsinogen -----> pepsin
- trypsinogen -----> trypsin
thus, we
have 2 possibilities:
1) pre-existing inactive enzyme --> active
2) de novo (new) enzyme synthesis (gene act
Mechanism of Gene Action (turning on/off genes)
in PROCARYOTES.
model: LACTOSE OPERON - Jacob & Monod
E. coli
[glossary]*
(grown on)
glucose
lactose
NO beta-galactosidase
beta-galacotsidase
lac ---> glu + gal
OPERON* = series of mapable-linked genes controlling synthesis of protein
p
Rg (i gene)
p
O
S
Rg
crp
p
O Sg1 Sg2 Sg3
regulator - makes repressor protein
promoter - binds RNA polymerase
operator - binds repressor protein
structural - make enzyme proteins
Sumanas, Inc. animation - Lac Operon*
Control of Gene Expression
-
what if regulator binds
figure
figure
figure*
Catabolic Repression*
in EUKARYOTES
Mechanism of Gene Action (turning on/off genes) is more complex
much more DNA
& it's inside a compartment (nucleus)
and, there are no operons present
have many more promoters - sites where RNA polymerase binds
enhancer sequence - sites where enhancers/transcription factors bind
transcription factors - proteins that help transcription
but, individual genes are not contiguous, thus no operons
3 levels for eukaryotic controls* - transcriptional, translational, post-translatio
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 ce
1.
role of activators in selective gene expression (Differential Gene Activity*
ex: Steroid Hormones (figure*)
2. Molecular turnover - ½ 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 C17.11*
and some examples*
ex. cont.
Eukaryotic gene expression controls:
5. cancer often results from gene changes affecting cell cycle control.
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
a Ras mutation --> hyperactive Ras protein --> cell division fig 19.12
p53 (tumor suppressor geneg = 50% human cancers)
fig 19.12b
p53 is a transcription factor that promotes the synthesis of cell cyc
inhibiting proteins
[DNA damage --> active p53 --> p51 gene --> protein binds
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 hum
Organization of the Genome* the structural organization of genome in eukaryotes influence its exp
Size of Human genome:
3million+ base pairs, or some 500,000 pages of journal Nature.
reading a 5 bases/sec it would take you about 60 year @ 8h/d 7d/w
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 segments, regulator genes, operators ?
BEST ≈ "a GENE is a region of DNA that CODES for an RNA"
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
restriction enzyme Mst II, which cuts normal b globin DNA at a particular site, but will not recognize an
therefore will not cut DNA that contains the sickle cell mutation. Mst II recognized the sequence CC
(where N = any nucleotide). Sickle cell disease is due to a single point mutation in the beta globin gene
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.
results from a change in the nucleotide T to A. This change eliminates a site recognized by
restriction enzyme DdeI.
Restriction enzyme: DdeI (recognition sequence: 5'-C^TNAG-3')
Southern blotting probe: fragment of ß-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 (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-T1st 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 = norma
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 increa
number by a process of dynamic mutation; # of tandem repeats is unique to a genet
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 gr
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
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 futur
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
fibrosis;
Plants
artificially insemination, microinjection of human gene, fertilized ova are put
into a surrogate sheep:
chimeras mated to produce homozygote- Milk tested for active protein.
- 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 prote
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?