Download MOLECULAR GENETICS You Are Here* Genes --

MOLECULAR GENETICS
You Are Here*
Genes ---> Enzymes ---> Metabolism
Central Dogma of Molecular Biology*
DNA -transcription--> RNA -translation--> Protein
Student CD Activity - 13.2 Events 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
T thymidine, G guanine --> polynucleotide*
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INFORMATION PROCESSING
& the
CENTRAL DOGMA
- the letters of the genetic alphabet... are the nucleotides A, T, G, & C
- unit of information is CODON = genetic 'word'
a triplet sequence of nucleotides CAT in a polynucleotide
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 genes
≈ 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 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.
Experimental Proof of DNA as Genetic Material...
1. Transformation* Experiments of Fred Griffith... (1920's)
Streptococcus pneumoniae - pathogenic S strain & benign R
transforming 'principle' is 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* [ 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
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Structure of DNA .....
Discovery of Double Helix...
Nobel prize* - JD Watson, Francis Crick, Maurice Wilkins,
Watson's book
but [ Erwin Chargaff & Rosy Franklin ]....
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 constant diameter* of DNA 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
eplication of DNA...
(Arthur Kornberg - 1959 Nobel)
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  & 
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*
Pol III (pic)
[quicktime movie]
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Replication forks - leading & lagging strands
- Campbell figure*
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)
student CD Activity - 12.1 - Overview of DNA Synthesis
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 1,000 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*
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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* copying*
[pnpase]
Student CD Activity 15.1 - DNA Regulatory Regions
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*
Kinds of RNA [table*]
tRNA -
structure*
small, 80n, anticodon sequence, single strand with 2ndary
function = picks up aa & transports it to ribosome
5
rRNA -
3 pieces of RNA - make up the organelle = ribosome
rDNA processing of primary transcript into
rRNA pieces* (pic) and
structure of ribosome*
hnRNA -
heterogeneous nuclear RNA : large Primary Transcript RNA
function - is the precursor of mRNA in eukaryotes
mRNA -
intermediate size - 100n to 400n ( split genes*)
function - codes for amino acid sequence
structure of mRNA* role of 5' CAP and Poly-A Tails*
[glossary]
processing of introns & exons*
splicing of eucaryotic genes*
[glossary]
(Sumanas, Inc. advanced animation)
summary of eukaryotic RNA processing*
Other classes of RNA:
small nuclear RNA (snRNP's) - plays a structural and catalytic role in spliceosome*
SRP (signal recognition particle) - 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.
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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 - figure*
[BARR Body*]
What are Introns? and What is the Role of Intron DNA?
Percentage of non-coding DNA during evolution*
fall 2005 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.
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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?
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...
1. add an amino acid to tRNA -- > aa-tRNA
- ACTIVATION*
[glossary]
2. assemble players [ribosome*, mRNA, aa-tRNA] - INITIATION*
3. adding new aa's - peptidyl transferase
4. stopping the process
- ELONGATION*
- TERMINATION*
Student CD Activity - 13.2 Events in Protein Synthesis
Review the processes - initiation, elongation, & termination
Quicktime movie of translation
Review figures & parts [ components, locations, AA-site, & advanced animation ]
Summary fig*
Nobel Committee static animations of Central Dogma
GENETIC CODE...
...is the sequence of nucleotides in DNA, but 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
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1n = 4 triplets, 2n= 16 triplets, 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
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 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.
cell
Recombination (Recombinant DNA) newly combined DNA's that
[glossary]*
can change genotype by insertion of NEW (foreign) DNA molecules into recipient
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1. fertilization* [n + n = 2n] sperm into egg cell --> zygote
2. exchange of homologous chromatids via crossing over*
3. transformation* - absorption of 'foreign' DNA by recipient cells
4. BACTERIAL CONJUGATION* - involves DNA plasmidsg* (F+ & R = resistance)
conjugation is 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 & methodologies
Restriction Endonucleases* - diplotomic cuts at unique [glossary]*
Eco-R1 figure*
DNA sequences, mostly palindromes... [Never odd or even]
▼
5' GAATTC 3'
3' CTTAAG 5'
5' G . . . . .
3' CTTAA
+
AATTC 3'
. . . . G 5'
▲
DNA's cut this way have sticky ends & can be recombined (reannealed)
or spliced* w other DNA molecules to produce new genes combos
Procedures of Biotechnology?
A. Technology involved in Cloning a Gene...
[Genome Biology Research]
[animation* & the tools of genetic analysis]
making copies of gene DNA
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1. via a plasmid* [ A.E. fig & human shotgun plasmid cloning ]
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* +
PCR reaction protocol &
Sumanas, Inc. animation*
Xeroxing DNA & Taq
polymerase
B. Detection of a Gene... Locating a gene (or its activity)
Restriction Fragments & Maps.
1. Restriction mapsg...
via gel electrophoresis* & DNA-electropherogram*
2. DNA fingerprintg...
How to make one*
3. DNA Probe Hybridizationg... to detect specific DNA with a probe
fig 20.5*
4. Comparing Restriction Fragments... to a probe
Southerng Blotting* fig*
Sumanas, Inc. animation - DNA electrophoresis & blotting
one can detect specific gene sequence in samples by binding to labeled probes
5. DNA* micro-arrays - to 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
cDNA (mRNA's) are flourescently tagged so easy to see in wells.
Sumanas animation - DNA chip technology*
5. Gene Sequencing* - Human Genome Project
dideoxy method (Sanger)
strategy - shotgun approach* developed by Celera Genomics
random fragments are sequenced and then ordered
relative to each other via overlap & supercomputing
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Student CD Activity - 16.1 - Sequencing Strategies
Surprising Size Estimates of Human Genome
&
figure*
NHGRI researchers*< A> have confirmed the existence of 19,599 protein-coding genes
< A>in the human genome and identified another 2,188 DNA segments that are
< A> predicted to be protein-coding genes
= < A> <
A>21,787 genes
mtDNA & Y-chromosome DNA aid in search for our human ancestry
Practical Applications of DNA Technology - What's been Done...
1. Medical... disease often involves changes in gene expression
a. disease/infection diagnosis:
PCR & labeled probes from pathogens can help identify microbe types...
isolate HIV RNA --RT--> cDNA --PCR--> probe can ID AIDS infection
b. RLFP - Restriction Length Fragment Analysis - markers often inherited with disease
what is RFLP* genetic testing & polymorphism ---> RFLP markers to disease*
MST II cuts Sickle Cell* & DdeI cuts Sickle Cell*
paternity testing via DNA fingerprinting
c. Gene Therapy... idea is to replace defective genes using VECTORS*
and microinjection of DNA* & fig 20.16* & 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 lukemia:
the retroviral vector inserted the repair gene near bone marrow cell
genes involved in blood cell division, thus lukemia. trials stopped.
2. Pharmaceutical Products... manufactured drugs
Recombinant bacteria* = Humulin
&
protropin (an ethical dilemma)*
Student CD Activity - 17.1 - Producing Human Growth Hormone
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
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*
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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.
4. Environmental Clean-up...
bacteria can extract heavy metals (Cu, Pb, Ni) from the environment
& convert them into not 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 --->
- "pharm" animals (20.18*) - sheep carry human blood protein gene
that inhibits enzymes in cystic fibrosis patients;
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*
- 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
Control of Gene Expression
How do we know a gene has been active (turned on)
we look for gene's product, i.e., protein or RNA
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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 circumstances:
1) pre-existing inactive enzyme --> active
2) de novo (new) enzyme synthesis (gene action)
Mechanism of Gene Action (turning on/off genes)
model: LACTOSE OPERON - Jacob & Monod
E. coli
(grown on)
glucose
in PROCARYOTES...
[glossary]*
lactose
NO beta-galactosidase
beta-galacotsidase
lac -----> glu + gal
OPERON = series of mapable-linked genes* controlling synthesis of protein
p
Rg
promoter operator regulator structural -
crp
O Sg1 Sg2 Sg3
p
binds RNA polymerase
binds repressor protein
makes repressor protein
make enzyme proteins
Sumanas, Inc. animation - Lac Operon*
Control of Gene Expression
-
figure*
figure*
what if regulator binds lactose*
Catabolic Repression*
in EUKARYOTES
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Mechanism of Gene Action (turning on/off genes) is more complex
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
3 levels for controls* - transcriptional, translational, post-translational
multiple places for control* - of whether a gene make a protein or not
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*)
2. Molecular turnover - ½ life mRNA's* &
longevity of some proteins*
3. Steroid Hormones (figure*)
4. Processing of RNA transcript (figure*)
cut/spliced in nucleus and capped for transport
intron - pieces cut out (non gene-proteins)
exons - pieces transported to cytoplasm
alternative splicing = figure 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 Oncogenes*
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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 19.12a*
p53 (tumor suppressor geneg = 50% human cancers)
fig 19.12b*
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
Organization of Human Genome (esp. non-protein coding genes)
highly condensed DNA (heterochromatin) is often not expressed and
chemical modification of histones in genomic DNA often regulates DNA's action
ACYLATION (-COCH3) of histones (lys) unfolds chromatin
c7-fig 19.4*
METHYLATION of nucleotides leads (often to C) favors condensation
and leads to inactive DNA... once CH3 passed generationally.
differences*
Twins show majors chromatin
Non-Coding Genes and Genome Structure:
in prokaryotes: DNA codes for proteins, tRNA, or rRNA (avg. gene = 1,000np)
non-coding DNA is mostly promoters; generally no introns
in eukaryotes: most DNA is non-coding (mostly introns)
avg. gene is 27,000np
c7 fig 19.14*
non-protein coding DNA genome makeup:
largest portion of human genome is Transposable Genetic Elements
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2 types: 1. transposons (cut & paste) - c7-fig 19.16a*
a genetic element that moves by means of a DNA intermediate
2. retro-transposon (RNA intermediate) - c7-fig 19.16b*
move via a transcript of the retrotransposon DNA
in humans one group of transposable genes is the Alu elements
≈ 10% of human genome (1.4 million copies - some 500,000 Alu elements
each Alu element is about 300 n long with a poly-A tail, non-coding
copy themselves & reinsert into genome randomly (like a genomic parasite)
1 new insert per every 100-200 births
5% of human alternately splice genes hold Alu pieces
origins of Alu ? --> piece jumped into intron --mutation--> created new 5' or 3' splice site
resulting in exon that was alterantely splice into mRNA
Alu pieces are transcribed into RNA whose function is unknown
next largest portion of Human Genome is Repetitive DNA...
15% of human genome is Repetitive DNA.
⅓ is large piece repeats (10K-300K np) often copied from 1 chromosome location
3% is single DNA short sequence repeats of --GTTAC-repeats sequences can be from 5n (short) yup to 500n
most are located at teleomers and centromeres
next largest portion of Human Genome is Intronic DNA...
1.5% of Human genome is exonic DNA (i.e., some 25,000 genes)
½ of exonic DNA is "traditional" single gene unique sequence DNA
24.0% is intronic multigene family DNA...
ex: identical repeats- rDNA genes - tandem repeats of DNA coding for rRNA
advantage: can make millions of ribosomes as needed for protein synthesis
mass production; primary transcript is processed c7 fig 19.17a*
ex: non-identical repeats- globin genes -->  & β polypeptides of Hb
chromosome 16 holds  gene family
c7 fig 19.17b* different forms of globin genes
chromosome 11 holds β gene family
are expressed at different developmental stages.
includes psuedogenes (non-functional nucleotides sequences)
possible origins of gene families --> c7 fig 19.19* mutation, transposition, duplication
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EXON Shuffling... can lead to significant genome rearrangement
(evolutionarily
significant)
exons are mixed and matched due to meiotic division errors
occurs via unequal crossing over at non-sister transposable element sites c7 fig 19.18*
recall that proteins often have a modular architecture
made up of discrete structural & functional regions called domains
different exons code for the different domains of a protein - c7 fig 17.12*
ex: TPA (tissue plasmogen activator) an extracellular protein that prevents blood clotting
protein has 4 domains - 3 each coded by different exon & 1 duplicate
each exon is also found in other proteins
origin may have been by exon shuffling - c7 fig 19.20*
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, must also 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:
promoters, enhancers, regulator genes, operators,
also segments that code for rRNA, tRNA, & snRNP's
BEST = "A GENE is a region of DNA that CODES for an RNA"
end.
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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
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Sickle Cell disease occurs when the DNA sequence for glutamic acid is converted to valine. This
results from a change in the nucleotide A to T. This change eliminates a site recognized by the
restriction enzyme DdeI.
Restriction enzyme: DdeI (recognition sequence: 5'-GTNAG-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.
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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-T-
19
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
In the largest study of its kind, [80 sets of identical twins, ranging in age from 3 to 74
years] researchers have shown that 35% of twin pairs have significant differences in DNAmethylation and histone-modification profiles (Fraga et al, 2005).
M.F. Fraga et al, Epigenetic differences arise during the lifetime of monozygotic twins,
Proc. Natl. Acad. Sci. 102: 10604-9. July 26, 2005
back
Gene expressions in pharmacogenomics
& toxicogenomics via microarrays
Size of Human genome:
3million + bp, or some 500,000 pages of journal Nature
reading a 5 bases/sec it would take you about 60 year @ 8h/d 7d/w
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*
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Antibiotics
JAMA Patient Page:
Medical News
Learning Centers
Smart Parents' Health
Source
Bacteria are single-cell organisms that can cause a number of different
diseases. Viruses are extremely small infectious agents that also can cause
many diseases. Antibiotics work only against bacterial infections and are
completely ineffective against infections caused by viruses, such as the flu
or the common cold.
The September 12, 2001, issue of The Journal of the American Medical
Association includes an article about the overuse of antibiotics for sore
throat.
Development of Antibiotic Resistance
Some bacteria develop resistance to a previously effective antibiotic drug.
Resistance occurs when bacteria develop a method of growth that cannot be
interrupted by the antibiotic or when bacteria break down or inactivate the
antibiotic. Antibiotics no longer work against diseases caused by bacteria
that have developed resistance against them. Antibiotic resistance has
become one of the world’s most pressing public health problems.
Because overuse of antibiotics can cause bacteria to become resistant and
antibiotics cannot cure infections caused by viruses, antibiotics should be
prescribed only for bacterial infections.
Common Viral Illnesses
These illnesses are usually caused by viruses and generally should not be
treated with antibiotics:





Acute bronchitis — cough, fever
Common cold — stuffy nose, sore throat, sneezing, cough, headache
Influenza (flu) — fever and chills, body aches, headache, sore throat,
dry cough
Pharyngitis (sore throat) — except for strep throat, which is caused
by a type of bacteria
Viral gastroenteritis — most cases of vomiting and diarrhea are
caused by viruses
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Sources: Agency for Healthcare Research and Quality, Alliance for the
Prudent Use of Antibiotics, American Medical Association Complete Guide to
Your Children’s Health, American Medical Association Encyclopedia of
Medicine, American Pharmaceutical Association, Centers for Disease Control
and Prevention, Center for Drug Evaluation and Research, National Institute
of Allergy and Infectious Diseases.
For More Information
Alliance for the Prudent Use of Antibiotics (APUA)
(617) 636-0966
www.healthsci.tufts.edu/apua
Centers for Disease Control and Prevention
(800) 232-2522
www.cdc.gov/antibioticresistance
Agency for Healthcare Research and Quality
(800) 358-9295
www.ahrq.gov
Inform Yourself
To find this and previous JAMA Patient Pages, go to the Patient Page Index
on JAMA’s Web site at www.jama.com. A JAMA Patient Page on strep throat
was published on December 13, 2000.
Lise M. Stevens, M.A., Writer
Cassio Lynm, M.A., Illustrator
Richard M. Glass, M.D., Editor
The JAMA Patient Page is a public service of JAMA. The information and
recommendations appearing on this page are appropriate in most instances,
but they are not a substitute for medical diagnosis. For specific information
concerning your personal medical condition, JAMA suggests that you consult
your physician. This page may be reproduced noncommercially by
physicians and other health care professionals to share with patients. Any
other reproduction is subject to AMA approval. To purchase bulk reprints,
call (718) 946-7424.
© Copyright 2001 American Medical Association. All rights reserved.
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