Download MICRO. 555 (555 Microbial Molecular Genetics) Dr.Afaf Ibrahim

MICRO. 555 (555 Microbial Molecular
Genetics)
Dr.Afaf Ibrahim Shehata
Botany and Microbiology Department
King Saud University
MOLECULAR GENETICS
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 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*
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
- 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 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
µ 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'* (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* [ 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
Replication of DNA...
(Arthur Kornberg - 1959 Nobel - died
10/26/07)
copying of DNA into DNA is structurally obvious??? [figure*]
Patterns of Replication* = conservative, semiconservative, & 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 IIV 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)
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 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
Transcription - RNA polymerase
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* 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
function - is the precursor of mRNA in eukaryotes
hnRNA - heterogeneous nuclear RNA : large Primary Transcript RNA
function - is the precursor 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*
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 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*]
Translation...
Sequence of 4 Steps in
[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
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
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
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... (SCApleiotropy)
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 plasmidsg* (F+ & R =
resistance)
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 & methodologies
Restriction Endonucleases-[glossary]*... diplotomic cuts* 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. Librariesg... [ library figure* & Sumanas animation - DNA fingerprint
library ]
3. Probesg...
[ cDNAg
& reverse transcriptaseg
cDNA figure* &
cDNA library* ]
4. Polymerase Chain Reactiong & figure
20.7* & animation* + Sumanas, Inc. animation*
PCR reaction
protocol & Xeroxing DNA & Taq 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 Science
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 - 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
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 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
c. Gene Therapy... idea is to replace defective genes via microinjection of
DNA*
requires VECTORS - fig 20.16* (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
glucose
NO beta-galactosidase
galacotsidase
(grown on)
lactose
beta-
OPERON* = series of mapable-linked genes controlling synthesis of
protein
p Rg crp p O Sg1 Sg2 Sg3
Rg (i gene) regulator - makes repressor protein what if regulator
binds lactose*
p
promoter - binds RNA polymerase
figure
O
operator - binds repressor protein
figure
S
structural - make enzyme proteins
figure*
Sumanas, Inc. animation - Lac Operon*
Repression
Catabolic
Control of Gene Expression - 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, posttranslational
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: 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 geneproteins)
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 19.12a
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 the Genome* the structural organization of genome
in eukaryotes influence its expression.
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 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
________________|____________
¥
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 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-AT1st 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-TC-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-AT-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-CA-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
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
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)
ð An overview of biotechnology
History of Biotechnology
Human Genome Project &
Biotech Companies
HHMI funded DNA Interactive tutori
al
.
What are Introns? and What is the Role of
Intron DNA?
don't really know, but Percentage of noncoding DNA during evolution* goes up.
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 noncoding 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
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
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?