the OMICs Era of modern Molecular Biology -
Genomics - study of the genomes of
organisms (sequencing, mapping, & gene interactions)
Proteomics - study of the entire
complement of proteins in a system or organism
Transcriptomics - all messenger RNA (mRNA) or
"transcripts," made in one cell or a population of cells
Metabolomics- complete map of all small-molecule
metabolites in the human body (human
metabolome) Structural
Genomics - determination of 10,
20,
& 3D structure of all proteins in a given organism
Informatics - application of
information technology to field of molecular biology
Pharmacogenomics- identifies genetic
basis for heritable & interindividual variation in response to drugs
What is a GENE = ?
a basic unit of heredity in a living
organism
DNA is the genetic material...
- a
segment of DNA that specifies a morphological trait
- a discrete piece of deoxyribonucleic acid
-
linear polymer of repeating nucleotide monomers
nucleotides* --> A
adenine, C cytosine
T thymidine, G guanine
- the letters of the genetic alphabet...
are the nucleotides
A, T,
G,
& C
of DNA
- the unit of information is
CODON = genetic 'word'
a triplet sequence of nucleotides
'CAT'
in a polynucleotide
3 nucleotides = 1 codon (word) = 1 amino
acid in a polypeptide
- the
definition of (codon) word =
amino acid
- Size of Human Genome:
≈ 3,000,000,000
base pairs or 1.5b in single strand
of DNA genes ≈500,000,000
possible
codons (words or
amino acids)
- average page your textbook = approx
850 words
thus, human genome is equal to
588,000 pages or
490 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. It is
estimated that 99.9%
of the 3billion n's
of human genome is same person to person. chimp
& human DNA differ by only 15 million basesWhat makes us
Human?
used two approaches to decipher structure:
1.
model building -
figure*
(are the bases in/out; are the sugar-P's in/out?)
2.
x-ray diffraction*pattern*
favor a DNA helix of constant
diameter*
Model of
Replication we'll look at 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 (RNA polymerase)
POL III - synthesizes new DNA strands
DNA polymerase I - removes RNA primer 1 base
at a time, adds DNA bases
DNA ligase repairs Okazaki fragments (seals
lagging strand 3' open holes)
Concept
Activity - DNA Replication Review* Structure of DNA polymerase III*
copies
both strands simultaneously, as DNA is Threaded
Through a
Replisome
a "replication
machine", which may be stationary by anchoring in nuclear matrix
Continuous & Discontinuous replication occur simultaneously
in both strands
EVENTS:
1. DNA pol III binds
at the origin of replication site in the template
strand
2. DNA is unwound by
replisome complex using
helicase & topoisomerase
3. all polymerases require a preexisting DNA
strand (PRIMER) to start replication,
thus
Primase adds a single short primer to
the LEADING strand
and adds many primers to the
LAGGING strand
4. DNA pol III is a
dimer adding new nucleotides to both strands
primers
direction of reading is 3' ---> 5' on template
direction of synthesis of new strand is 5" ---> 3'
rate of synthesis is substantial 400 nucleotide/sec
5. DNA pol I removes
primer at 5' end replacing with DNA bases, leaves 3' hole
6. DNA ligase seals 3'
holes of Okazaki fragments on lagging strand -
cartoon
the sequence of
events
in detail*
and
DNA
Repair*
GENE Expression
the Central Dogma of Molecular Biology depicts flow of genetic information
Transcription - copying of
DNA sequences into RNA
Translation - copying of
RNA sequences into protein
DNA sequence -------> RNA sequence ----->
amino acid sequence
TAC
AUG
MET
a
triplet sequence in DNA --> codon in mRNA ----> amino acid in protein
Information : triplet sequence in DNA is the genetic word [a codon]
small nuclear RNA (snRNP's) - plays a structural and catalytic role in
spliceosome*
there are 5 "snurps" making a
spliceosome [U1, U2, U4, U5, & U6];
they and participate in several RNA-RNA and RNA-protein interactions
SRP (signal recognition particle):
7s-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 RNAs [miRNA]
single-stranded RNA molecules of 21-23 nucleotides in length, that
are not translated into
protein (non-coding RNA) and form short stem-loop
structure*that are partially complimentary
to mRNAs and that can down-regulate or
possibly activate gene expression.
some 400 miRNAs
are known in the human genome & function as
small interfering RNAs (siRNA)
by hybridizing to mRNA, forming a
dsRNA duplex
and
blocking translation*.
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 -->
1) BARR
Body* & 2)CCD*& 3)heart
disease
The ability of transfected synthetic
small interfering siRNAs to suppress
the expression of
specific transcripts has proved a useful
technique to probe gene function in
mammalian cells.
2006 Nobel Prize goes to A.Fire & C.Mello in 2008 UCSF
researchers R. Place & L. Li (& others) showed
(PNAS 105:1608-13)
some microRNAs
may Activate genes via acting at promoter
sites. There are
3 possible models
of activation.
the non-protein coding RNA transcripts likely
regulate much of the genome.
TRANSLATION - Making a Protein
process of making a protein in a
specific amino
acid sequence
from a
unique mRNA sequence... [
fig*
&
E.M. picture*]
polypeptides are built on the
ribosome on a
polysome
[
animation*]
...is the sequence of nucleotides in
DNA, but routinely shown as a
mRNAcode*
...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
Marshall Nirenberg
(1968 Nobel)
-
synthetic mRNA's used in an in vitro
system
5'-UUU-3' =
phe U
+ C --> UUU, UUC, UCC, CCC
UCU, CUC, CCU, CUU the Genetic CODE*-
64 possible triplet codons:
61
= an amino acid, 1
initiator (start) codon (AUG
= nf-MET),
&
& 3 are 'molecular periods' or stop codons
(UGA, UAA, & UAG); code is universal
(but some
anomalies),
redundant,
but non-ambiguous, and exhibits "wobble*".
"the sequencing of DNA of dozens of species, from viruses
to humans documents that
we're all
connected to the commonality of the genetic code in evolution"
jcv (2007)
GENETIC CHANGE
- a change in DNA nucleotide sequence
(= change in mRNA)
- 2
significant ways mutation &
recombination
[glossary]µ
1.
MUTATION -
a permanent change in an organism's DNA*that results in
a different
codon = different amino acid sequence Point mutation* -
a single to few nucleotides change...
- deletions, insertions, frame-shift mutations* [CAT]
-
single nucleotide base substitutions* :
non-sense = change to no amino acid (a
STOP codon)
UCA --> UAA ser to
non
mis-sense = different amino acid
UCA --> UUA ser to leu Sickle Cell Anemia* - a mis-sense mutation...
(SCA-pleiotropy)
another point mutation blood disease -
thalassemia adenosine-monophosphate
deaminase 1 - a C to a Tchange produces
mutant AMPD1 which can lead to cramps & early fatigue - Effects = no effect, detrimental (lethal), +/- functionality, beneficial
2.
Recombination
(Recombinant DNA) newly
combined DNA's that
[glossary]*
can change genotype via
insertion of NEW
(foreign) DNA
molecules into recipient cell
1.
fertilization* - sperm inserted into
recipient egg cell* --> zygote [n +
n = 2n]
2. exchange of homologous chromatids via
crossing over* = new gene combo's
3.
transformation* -
absorption of 'foreign' DNA by recipient cells
changes cell
4.
BACTERIAL CONJUGATION* -
involves
DNA
plasmids* (F+
or
R
= resistance) conjugation
may be a
primitive sex-like reproduction in bacteria [Hfr*]
5.
VIRAL TRANSDUCTION - insertion via a
viral vector(lysogeny*&TRANSDUCTION*)
general transduction - pieces of bacterial DNA are
packaged w viral DNA during viral replication
restricted transduction - a temperate phage goes lytic
carrying adjacent bacterial DNA into virus particle
6. DESIGNER GENES
-
man-made recombinant DNA molecules
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
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*cartoon
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*
4.
Measuring single gene expression via
RT-PCR methodologies -
fig 20.13* 5. DNAmicro
chipsarrays
- 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. Sumanas animation - DNA chip technology*&myDNAi
DNA microarrays*
Frederick
Sanger (Cambridge U.) in May 1975 produced 1st complete sequence of viral
genome
of phage ΦΧ=174 (5,375 np's); followed
by human mitochondrion with 17K np's.
today: strategy -
shotgun approach*
&
ESTs
developed by Celera Genomics
random cDNA fragments (from a library
500-800 nuc long) are sequenced & then ordered
relative to each other via overlap & supercomputing;
these Expressed Sequence Tags
(ESTs) represent portions of expressed genes.
methodology -
dideoxy
procedure*
& sequence reading* (development
by
Fred Sanger)
uses terminator nucleotides for they randomly stop action of polymerase
when they are incorporated into growing chain marking its end.
Lee Hood of CIT used fluorescent tags instead of
Sanger's isotopes, later formed
company (Applied Biosysytems) that made automated sequencing machine; JCV got
1st.
Using methods for cloning DNA fragments on the order of 100 kb in
length,
automated DNA
sequencing techniques,
and computer algorithms to piece together the stored sequence data
researchers have determined the
entire genome sequence of a human and many key
experimental organisms (including
viruses, bacteria, archaea, yeast, C. elegans, Drosophila, mouse,
and man). This vast amount of
data is stored and organized in two primary data banks: the GenBank at
the NIH in Bethesda, MD and EMBL Sequence Database at the European Molecular
Biology
Laboratory in Heidelberg, Germany and are available via the internet.
Practical
Applications of DNA Technology -
Some examples of What's been Done...
1. Medical... disease often involves changes in gene expression a.
disease/infection diagnosis:
(HANAA) PCR & labeled DNA probes from
pathogens can help identify microbe types...
isolate HIV RNA --RT--> cDNA --PCR--> probe can
ID... viral infection
b. RFLP -
Restriction
Fragment Length Analysis
- markers often inherited with disease
what is RFLP*
genetic testing
& polymorphism --->
RFLP markers to disease DdeI cutsSickle
gene* (also
MST II cuts Sickle Cell) fragment
analysis (DNA fingerprinting) also used for
paternity testing +
GINA
c. Gene
Therapy... idea is to replace defective genes viamicroinjection of DNA* requiresVECTORS- fig 20.22*
(patient:ADA
Deficiency & AshantiDeSilvaupdate)
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.
d. 'human social genes'...
chromosome disorder map;
genes that predispose us to be "fat"?
e. 'Human
Microbiome Project'... sequencing human microbes to correlate with human
health 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...
Mechanism of Gene Action (turning on/off genes) has
greater
complexity
there are no
operons present
and there's much
more DNA & it's inside a compartment
(nucleus)
have
many
more promoters - sites where RNA polymerase binds
enhancer sequence - sites where enhancers/transcription factors bind
transcription factors - proteins that help transcription
but, the individual genes are not contiguous
with each other,
thus no operons
3. Processing of RNA transcript (figure*)
cut/spliced in nucleus and capped for transport
intron - pieces cut out (non gene-proteins)
exons - pieces transported to cytoplasm alternative splicing
= figure
18.11*
ex.
cont.
Eukaryotic gene expression controls:
5. cancer often results from gene changes
affecting cell cycle control proteins:
cancer genes, such as adenomatous polyposis coli,
which cause 15% of
colorectal cancers
is a tumor suppressor gene, a type of
Oncogenesg*
2 kinds of human cancer genes:
Ras
(proto-oncogene)
causes 30% human cancers:
is a
G-protein that promotes other cell division proteins
by over-expression-
a
Ras mutation --> hyperactive
Ras protein --> cell division
fig 18.21a*
p53
(tumor
suppressor geneg
= 50% human cancers)
fig 18.21b*
p53 is a transcription factor that promotes the synthesis of cell
cycle inhibiting proteins
[DNA damage --> active
p53 --> p51 gene --> protein binds to
cyclin dependent kinase stops cell division]
thus a p53 mutation --> leads to excess cell division (cancer)
- other cancer genes
can lead to new gene actions resulting in cancer
BRCA1 and BRCA2
(tumor suppressor genes) are involved in 50% of breast cancers in
humans
Organization of
the Genome the structural organization of genome
in eukaryotes influences its expression.
Size of Human genome:
3 billion+ base pairs,
equaling
some 500,000 pages of journal Nature.
reading at 5
bases/sec it would take you about 60 year @ 8h/d 7d/w
yet, there are only
about 21,787 protein coding genes.
Definition of a Gene:
Mendel's Particles... unit of heredity responsible for
phenotype
term Gene
was coined by Wilhelm Johanssen (1909) to describe
whatever it was that
parents
passed to offspring to develop same traits (a definition completely free of any hypothesis).
Morgan's Loci... he placed genes on a chromosome, i.e.,
it's a
cellular entity, that is part of chromosome &
ismapable.
Watson & Crick... sequence of specific
nucleotides along length of
double helical DNA 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
Functional definition...
DNA sequence coding for specific polypeptide:
but, also must include... Pseudogene... a mutated DNA segment no longer
making a protein [10-20K segments]
were once active, but evolution made them effectively dead 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 forrRNA,tRNA,snRNP's,
miRNA's &
also
promoters,
enhancer segments, regulator genes,
operators? Encode
Project- research effort to determine
functions of each piece of DNA
any Definition of a GENE:
maybe should trace an RNA transcript (exons) back to its DNA and that is
the GENE
smallest unit underlying an inherited trait: i.e., a collection of
exons.
≈ "a
segment of DNA corresponding to a single protein
(or set of alternate protein variants)
or a single catalytic or structural RNA molecule"
maybe we need a new term = dene...
any DNA sequence that promotes a role
in a cell.
coined by Evelyn Keller and David Harel
in journal PloS One 2(11): e123, Nov. 28, 2007
end.
MST II restriction cuts of normal sickle beta-gene
(
pink is DNA sequence & blue =
4 gel fragments)
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)
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.
TRANSPOSONS - pieces of DNA prone to
moving & creating repeat sequences LINE - long interspersed nuclear element holds promoter &
2 genes: RT & integrase
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.
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
INTRONS
- DNA Junk or
sophisticated Genetic Control Elements?
Current
dogma of Molecular Biology DNA --> RNA --> Proteins,
(proteins supposedly regulate gene expression)figure*
in 1977
Phillip
Sharp & Richard Roberts discovered DNA contains
introns
intervening DNA segments that do NOT code for proteins
a primary RNA transcript is processed by splicing to assemble protein coding
exons
Presence of Introns: Absent in
prokaryotes: they have few non-coding DNA sequences
as eukaryotic complexity grows so does non-coding DNA [figure]
makes up greater than
95% of the DNA
less than
1.5% of human genome encodes proteins, but all of DNA is
transcribed
40% of human genome is Transposons &
repeat genetic elements.
Evolutionary Origins? may have been
self-splicing mobile genetic elements
that inserted themselves into host genomes
Advent of Spliceosomes: catalytic RNA/protein
complexes
that snip RNAs out of mRNAs,
would encourage introns to proliferate, mutate, evolve
fall 2005 skip this material Role of Introns? Not Junk, but
rather
Genetic Control Elements
[figure*] Micro RNAs -
derived from introns? - occur in plants, animals, & fungi
a) help control timing of developmental processes as cell proliferation,
apoptosis, and stem cell maintenance
b) help tag chromatin with methyl and acetyl groups
c) may help in alternative splicing mechanisms
COMPLEXITY: to build a complex structure one must have
bricks & mortar,
as well as an architectural plan. DNA,
therefore should contain both - the materials and the
plan:
a) component molecules - proteins, carbs, lipids, and nucleic acids:
all known living organism use the same bricks and mortar
b) the difference between Man & Monkey is the architectural plan.
Where is the Architectural Information? we've always assumed in the
regulatory proteins
Maybe it's in the non-coding mirco-RNAs (intronic elements)
Thus the greater proportion of the genome of complex organisms, the introns,
isn't junk,
but rather, it is functional RNA that regulates time dependent complexity?
