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*
- the letters of the genetic alphabet...
are the nucleotides
A, T,
G,
& C
of DNA
- the unit of information is
CODON = genetic 'word'
a triplet sequence of nucleotides
'CAT'
in a polynucleotide
3 nucleotides = 1 codon (word) = 1 amino
acid in a polypeptide
- the
definition of (codon) word =
amino acid
- Size of Human Genome:
≈ 3,000,000,000
base pairs or 1.5b in single strand
of DNA genes ≈ 500,000,000
possible
codons (words or
amino acids)
- average page your textbook = approx
850 words
thus, human genome is equal to
588,000 pages or
470 copies of bio text book
reading at
3
bases/sec it would take you about 47.6 years @ 8h/d
- 7d/w WOW... extreme nanotechnology
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.
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 is bacterial with
DNA polymerase III...
several
enzymes*
form a Replication Complex (Replisome) & include:
helicase - untwists DNA
topoisomerase [DNA gyrase] - removes supercoils,
single strand binding proteins - stabilize replication fork,
Primase - makes RNA primer
POL III - synthesizes new DNA strands
DNA polymerase I - removes RNA primer 1 base
at a time, adds DNA bases
DNA ligase repairs Okazaki fragments (seals
lagging strand 3' open holes)
Concept
Activity - DNA Replication Review
Structure of DNA polymerase III*
copies
both strands simultaneously, as DNA is Threaded
Through a
Replisome*
a "replication
machine", which may be stationary by anchoring in nuclear matrix
Continuous & Discontinuous replication occur simultaneously
in both strands
EVENTS:
1. DNA pol III binds
at the origin of replication site in the template
strand
2. DNA is unwound by
replisome complex using
helicase & topoisomerase
3. all polymerases require a preexisting DNA
strand (PRIMER) to start replication,
thus
Primase adds a single short primer to
the LEADING strand
and adds many primers to the
LAGGING strand
4. DNA pol III is a
dimer adding new nucleotides to both strands
primers
direction of reading is 3' ---> 5' on template
direction of synthesis of new strand is 5" ---> 3'
rate of synthesis is substantial 400 nucleotide/sec
5. DNA pol I removes
primer at 5' end replacing with DNA bases, leaves 3' hole
6. DNA ligase seals 3'
holes of Okazaki fragments on lagging strand
the sequence of
events
in detail*
and
DNA
Repair*
Rates of DNA synthesis:
myDNAi movie of replication*
native polymerase: 400 bases/sec with 1
error per 109 bases
artificial:
phophoramidite method (Marvin
Caruthers, U.Colorado); ssDNA synthesis
on polystyrene bead @ 1 base/300 sec with error rate of 1/100b
GENE Expression
the Central Dogma of Molecular Biology depicts flow of genetic information
Transcription - copying of
DNA sequence into RNA
Translation - copying of
RNA sequence into protein
DNA sequence -------> RNA sequence ----->
amino acid sequence
TAC
AUG
MET
triplet sequence in DNA --> codon in mRNA ----> amino acid in protein
Information : triplet sequence in DNA is the genetic word [codon]
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)
- srpRNA is a component of the protein-RNA complex
that recognizes the signal sequence of
polypeptides
targeted to the ER -
figure*
small nucleolar RNA (snoRNA)
- aids in processing of pre-rRNA transcripts for
ribosome subunit formation in the nucleolus
micro RNA's (micro-RNA) - also called
antisense RNA & interfereing RNA;
c7-fig 19.9
short (20-24 nucleotide) RNAs
that bind to mRNA inhibiting it.
figure*
present in
MODEL eukaryotic organisms as: roundworms, fruit flies, mice, humans, & plants (arabidopsis);
seems to help regulate gene expression by controlling the timing of
developmental events via mRNA action
also inhibits translation of target mRNAs.
ex:
siRNA -->
[BARR
Body*]
TRANSLATION - Making a Protein
process of making a protein in a
specific amino
acid sequence
from a
unique mRNA sequence... [
E.M. picture*
]
polypeptides are built on the
ribosome (pic) on a
polysome
[
animation*]
Marshall Nirenberg
(1968 Nobel)
-
synthetic mRNA's used in an in vitro
system
5'-UUU-3' =
phe U
+ C --> UUU, UUC, UCC, CCC
UCU, CUC, CCU, CUU
the Genetic CODE* - 64 triplet codons [61
= aa & 3 stop codons]
universal
(but some
anomalies), 1
initiator codon (AUG),
redundant
but non-ambiguous, and exhibits "wobble*".
GENETIC CHANGE
- a change in DNA nucleotide sequence
(= change in mRNA)
- 2
significant ways mutation &
recombination
[glossary]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 - 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 - 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
