| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
Outline:
A. Cloning of DNA fragments:
Recombinant DNA technology and the properties of Type II R.enzymes provided the breakthrough needed to isolate pieces of genomes, including intact genes. This is the process of cloning of DNA fragments.
1. Two essential parts of Cloning: Cloning Vehicle and DNA of interest
a. Cloning Vehicle or Vector: ... [Brown, Tech Notes 3.4, part 2]
a. A DNA molecule with all
elements for Self-Duplication: Replicon
An Origin for DNA Replication ... [Brown, p. 307
ff]
b. means for Selection in a Host: often Antibiotic-Resistance Gene
c. unique R.sites NOT in the
Origin and not in the Selection Gene ...
Example: Polylinker or Multiple Cloning Region (MCR)
... [Brown, Tech Notes 3.4]
Such contains a variety of R.sites suitable for cloning; these
R.sites are present ONLY in the Polylinker or MCR ....
d. often Selection / Screen
for Insert, eg disruption of LacZ assay
in Plasmid vehicles => white colonies on "Xgal" plates
b. DNA of interest ... [Brown, Tech Notes 3.4, part 1]
Examples:
a. R.fragments of complete Genome
b. R.fragments from Chimeric DNA, eg from YAC for Plasmids: SubCloning
c. R.fragments purified from a gel, from a purified chromosome,
etc
d. Genome fragments made by shearing genomic DNA, followed by
creating blunt ends by limited exonuclease digestion.
2. Cloning Methodology: ... [Brown, Tech Notes 3.4, part 1]
1. Cut the Cloning Vehicle
with R.enzyme of choice, eg EcoRI
2. Cut DNA of interest with same R.enzyme
... or R.enzyme yielding same sticky ends, e.g. BamHI and Sau3A
... [Brown, Tech Notes 3.1]
3. Mix the restricted Cloning Vehicle and DNA of Interest R.fragments
together.
4. Ligate fragments together: DNA ligase ... usually
T4 DNA ligase [Brown, Tech Notes 4.2]
NOTE the "sticky ends"
generated by the R.enzyme find each other,
facilitate the ligation ... although "blunt end ligation"
is often used
5. Insert ligated DNA into
host of choice, eg transformation of E. coli
Ca++ treated cells ... electroporation
6. Grow host cells under restrictive
conditions,
e.g. E. coli grown on plates containing Antibiotic
NOTE: The "cloning" of the DNA is the duplication of
the ligated DNA
during growth and "cloning" of the host, e.g. colony
of bacteria ...
All the bacteria within a colony are clones, i.e. the bacteria are identical copies of each other.
Incredible breakthrough:
can create Chimeric DNA molecules !!
can clone DNA from any source into almost any host organism !!!
B. Types of Cloning Vehicles
Examples of Cloning Vehicles:
1. Plasmids: ColE1 based ... pUC vehicles ...
[Brown, Tech Notes 3.4]
commercially available ones, eg pGEM3, pBlueScript
Purpose:
Routine cloning of
relatively small inserts: 100 bp - 15 kb or so
Features:
Plasmid cloning vehicles
must have 1) origin, 2) cloning sites, and 3) selection marker:
1. Plasmids are extrachromosomal
elements or replicons:
They are self-replicating DNA molecules.
The only genetic requirement for a DNA molecule to be self-replicating
is that it contain an origin of DNA replication appropriate for
the host in which it would propogate.
Examples of origins of DNA
replication: ... [Brown,
p. 307 ff]
a. oriC - the E. coli chromosomal origin of DNA replication
b. ori - naturally occuring plasmid origin found on E.
coli ColE1 types, eg pMB9, of plasmids
... commonly used in cloning vehicles which use E. coli as host
c. ARS - Autonomously Replicating Sequences ... sequences
from any eukaryote that function as origins on plasmids propogated
in yeast ... used in YACs
2. For plasmids to function as cloning vehicles or vectors, they must also have:
a. Cloning sites ...
unique restriction sites within the plasmid ... ... [Brown,
Tech Notes 3.4]
Polylinkers or Multiple Cloning Sites (MCS) - region
containing many restriction sites, each of which is present ONLY
in the polylinker, not elsewhere on the plasmid
... commonly found in commercially available plasmid vectors,
e.g. pGEM3, pBlueScript
b. Selection locus ...
method for selection of host cells containing the plasmid
Examples: ... [Brown, Tech Notes 3.4]
1) Antibiotic resistance - genes that confer resistance
to ampicilin (amp), tetracycline (tet), kanamycin
(kan), chloramphenicol (cat), etc ... select by
growing IN PRESENCE of antibiotic
2) Auxotrophic marker - genes that permit growth IN ABSENCE
of metabolite using a host that is auxotrophic (mutant) for the
gene, e.g. TRP or PYR genes in pYAC2 in Journal Article 1, with
use of yeast host that is trp- ura-
2. Viral DNA, eg Phage Lambda derivatives, eg lambda gt10 and lambda
gt11
... [Brown, Tech Notes 4.3]
Purpose:
1. Cloning of same size to larger inserts than plasmids:
2. Advantages of Lambda for cloning:
Features:
Phage lambda is a bacteriophage
or phage, i.e. bacterial virus, that uses E.
coli as host.
Its structure is that
of a typical phage: head, tail, tail fibers, head contains
the genome
Lambda viral genome: 48.5 kb linear DNA with a 12 base ssDNA
"sticky end" at both ends; these ends are complementary
in sequence and can hybridize to each other (this is the cos
site: cohesive ends).
Infection: lambda tail fibers adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host.
Lambda Life Cycle:
Depending on the nutrient environment of the E. coli host, lambda
can enter one of two life cycles:
1. Lytic Phase:
In this phase, DNA
replication occurs and progeny lambda heads and tails are made.
Progeny lambda DNA molecules are inserted into empty lambda heads
and tails are joined, yielding progeny, infective lambda phage
particles.
A phage encoded lysozyme enzymatically lyses the E. coli host
"from within", breaking apart the cell surface, and
releasing the progeny phage
DNA replication first proceeds by a "theta mechanism", in which the circular lambda DNA replicates from a unique origin bidirectionally around the molecule, yielding daughter DNA molecules... [Brown, Fig 12.6]
Late in infection, lambda DNA replication switches to a "rolling circle" mode of DNA replication, during which a single origin is used and long DNA concatemers are produced: long tandem repeats of the lambda DNA genome...[Brown, Box 12.1]
These concatemers are used to fill empty lambda heads during the phage assembly process.
2. Lysogenic Phase:
In this phase, the
lambda DNA genome entering the E. coli host cell is integrated
into the E. coli chromosome at a specific site, the lambda
attachment site.
When so integrated, the lambda
DNA is called the prophage
and the E. coli cell is said to be a lysogenic cell or
"in the lysogenic state".
The lambda prophage will occasionally spontaneously excise from the E. coli chromosome and the lambda DNA will then proceed through the lytic cycle.
This prophage excision can also be induced by UV light.
Lambda as a Cloning Vehicle: ... [Brown, Tech Notes
4.3]
Most lambda cloning experiments require ONLY the lytic phase of
the lambda life cycle.
Hence the lambda genes required for the Lysogenic Phase as "dispensable"
for cloning.
These "dispensable" genes constitute the replaceable
region in lambda cloning.
3. Cosmids: Plasmid Cloning Vehicle with Lambda cos sites ... [Brown, Tech Notes 4.3]
Purpose:
1. Clone large inserts
of DNA: size ~ 45 kb
Features:
Cosmids are Plasmids
with one or two Lambda Cos sites
Presence of the Cos site permits in vitro packaging of cosmid
DNA into Lambda particles ...
Thus, have some advantages
of Phage Lambda as Cloning Vehicle:
1. strong selection for cloning of large inserts: packaging of
"headfuls" of DNA
2. Infection process rather than transformation for entry of chimeric
DNA into E. coli host
3. maintain Cosmids as phage particles in solution in the refrigerator
But Cosmids are Plasmids:
Thus do NOT form plaques ... but rather cloning proceeds via E.
coli colony formation
4. YACs: Yeast Artificial Chromosomes ... Journal Article 1 ... [Brown, Fig 4.12]
Purpose:
1. Cloning Vehicles
that propogate in eukaryotic cell hosts as Eukaryotic Chromosomes
2. Clone VERY large inserts of DNA: 100 kb - 10 Mb
Features:
YAC Cloning Vehicles
are plasmids ...
Final chimeric DNA is a linear DNA molecule with Telomeric ends:
Artificial Chromosome
Need on the Vector:
1. ARS - Autonomously
Replicating Sequence: eukaryotic origin of DNA replication
2. Selection markers, e.g. trp and ura genes, for each "arm"
of the YAC
3. CEN - CENtromeric sequences: provide stability of YAC segregation
during mitosis
Operationally:
Cloning vehicle is
Circular since it is a Plasmid
Thus, need to use two R.enzymes during cloning, to generate
cloning site and to expose the Telomeric ends: this generates
the two "arms" of the YAC
Need then to select for BOTH arms of the YAC
Additional features:
1. often have a selection
for an insert
2. YAC cloning vehicles often have a bacterial origin of DNA replication
(ori) and a selection marker for propogation of the YAC
through bacteria.
Result: the YAC can use both yeast and bacteria as a Host
5. Other Cloning Vehicles: PACs and BACs ...[Brown, p. 75]
a. PACs - P1-derived Artificial Chromosomes
E. coli bacteriophage P1 is similar phage lambda in that it can exist in E. coli in a prophage state. However, unlike lambda, prophage P1 exists in the E. coli cell as a plasmid, NOT integrated into the E. coli chromosome. The size of P1 in this prophage plasmid state can be very large, and P1 cloning vehicles have been constructed that permit cloning of large DNA fragments, with cloning and propogation of the chimeric DNA as a P1 plasmid inside E. coli cells. These plasmid PACs can contain DNA inserts of size up to a few hundred kb of DNA.
b. BACs - Bacterial Artificial Chromosomes
These chimeric DNA molecules use a naturally-occurring low-copy number bacterial plasmid origin of replication, such as that of F-plasmid in E. coli, in the cloning vehicle. As such the cloning vehicle that can be cloned as a plasmid in a bacterial host, and its natural stability generally permits that cloning of large pieces of insert DNA, i.e. up to a few hundred kb of DNA.
C. Genome Types of Cloning Experiments:
1. Gene Isolation - Reverse Genetics
Assays:
i) Complementation of Host Mutant deficient in Gene Activity
ii) "Probe" with radioactive short DNA molecule of sequence
from the Gene
Obtain short DNA sequence, eg, from Amino Acid Sequence of purified Protein
Thus: go from Protein to Gene
with no previously isolated Mutations ...
Reverse Genetics ... this is the reverse of standard genetics,
in which mutants are isolated and characterized (gene -> protein)
Often use such a short DNA
oligonucleotide Probe to partially purify
R.fragments for further cloning:
Southern Gels: ... [Brown, Tech Notes 2.1]
Run R.fragments out on a Gel
Transfer DNA from Gel to Nitrocellulose membrane
Hybridize the fragments in Bands on the Gel to the Probe
Detect correct bands via Radioactivity and film
Purify R.fragments from this region of the Gel to use in Cloning
Characterize Gene physically: Restriction Map ... Sequence
Can also use Hybridization
of a Probe for a Gene to:
1. detect DNA spotted and denatured on filter paper: dot blots
2. disrupted Lambda phage from a Lambda cDNA library on plates
3. lysed cells on a plate with DNA transferred to a nitrocellulose
membrane ...
in situ Hybridization
2. Genomic Cloning - Genome
Libraries
... [Brown, Section
3.3, 3.4]
Purpose: obtain library of complete genome
Usually use Phage Lambda ... see advantages above
Use of Isoschizomer R. enzymes:
... [Brown, Tech
Notes 3.1]
same sticky ends from cleavage
Example: BamHI and Sau3A ... Sau3A is a "4-cutter" and
hence has many more sites in a genome than does BamHI, which is
a "6-cutter"
Partial restriction with Sau3A yields large fragments, whose ends
are from sites at many locations on the genome ... thus get overlapping
fragments ...
Much less likely to get good overlapping fragment pattern with
BamHI due to lack of sufficient restriction sites ...
3. Mapping from Cloning:
Sequence Tagged Sites (STSs)
... [Brown, Section
3.3]
Sequence Tagged Sites (STSs) are short DNA sequences (usually < 500 bp) determined from unique sequences in a given genome, and then mapped to that genome.
These then provide methodology for high-resolution physical mapping of a genome.
a. Sources of DNA for STSs:
1. ESTs - Expressed Sequence Tags
These are DNA sequences determined from expressed regions of the
genome, or genes
If a given expressed gene is present uniquely on the
genome, the sequences of the EST is a good STS sequence.
2. Genomic Sequences
Any genomic sequence that is present uniquely on the genome
can be used as an STS
Good examples of such are DNA sequences previously determined
and deposited in a DNA database such as GenBank.
b. Mapping of STS Sequences:
Once one has a set of DNA sequences to be used
as STSs, they must be mapped to the genome.
This can be done in many ways, including the following two general
procedures:
1. Cloning and use of a Clone Library
2. Radiation Hybrids and use of a Radiation Hybrid Panel
The STS markers, DNA sequences of length 500 bp or less, are nearly always assayed using Polymerase Chain Reaction (PCR) amplification of the STS region. PCR is amenable to automation and hence so-called "High ThroughPut (HTP)" methodology (highly automated, robotic methodology) can be used.
4. cDNA Cloning - Expressed Sequence Tags (ESTs) ... [Brown, Sect 3.3.1]
Purify a messenger RNA of interest
...
"Reverse transcribe" the RNA into DNA, get a "complementary
DNA strand":
cDNA ... [Brown, Fig 3.11]
Degrade the mRNA and synthesize the second DNA strand
Clone the DNA so synthesized ...
Often use lambda cloning
vehicles for cloning cDNA species ...
can thereby maintain an entire cDNA library as lambda phage in
liquid at high density:
> 1012 phage per ml
cDNA library ... same thing but with ALL mRNAs from given tissue
This provides a library of all mRNA sequences expressed in this tissue
Such cDNA libraries can provide the DNA for sequencing to map sequence tagged sites (STSs) to a given genome or chromosome from a genome ... see Brown, Research Brief 3.1
D. Radiation Hybrids
Radiation Hybrids provide a methodology for high resolution mapping of DNA physical markers such as STSs on chromosomes of higher organisms such as human and mouse.
For human chromosomes, Radiation Hybrid
Panels are created by:
1. irradiating human cells with x-rays, thereby fragmenting the
chromosomes in these cells into fragments. The fragments are smaller
when the x-ray dose is higher.
2. creating a human-hamster fusion cell by fusing the irradiated
human cells with mutant thymine-requiring hamster cells. Fusion
can occur between these cells when mediated with chemicals or
Sendai virus.
3. creating a hybrid cell line by growing the fused cells
in HAT media, selecting for human chromosome fragments containing
a thymine gene. Cells in this fused cell line contain the hamster
chromosomes plus some human chromosome fragments (as much as about
35% of the human genome).
The Radiation Hybrid Panel consists of several such hybrid cell lines, all created in one fusion experiment. Typically, such a Panel has 50-100 different cell lines. Specific Radiation Panels, eg the G3 panel, have been used to construct a high resolution STS map of the human genome.
1. Mapping STSs from Radiation Hybrids
Radiation Hybrids can be used similarly to Clones in a Clone Library for high resolution mapping of STS markers. The principles for doing this are as follows:
1. each Hybrid Cell Line in a given Radiation
Hybrid Panel contains many fragments of human chromosomes.
2. STSs that are close together will tend to be present together
in the same human chromosome fragment in a given hybrid cell line;
those that are far apart or on different chromosomes will not
tend to be found in the same hybrid cell lines.
3. this tendency is quantitated by recording what percent of the
time two markers are found in the same cell line. This is almost
always the same as the two markers being present on the same human
chromosome fragment.
4. Doing this yields the following type of data:
Data from Human Chromosome 21q:
S16 (8) S48 (9) S46 (22) S4
with S16 (19) S46 and S48 (29) S4
Thus, STS markers S16 and S48 appear on the
same human chromosome fragment 92% of the time, ie the distance
between the markers is 8 units.
Similarly, STS markers S16 and S46 appear on the same human chromosome
fragment 81% of the time, ie the distance between the markers
is 19 units.
Note that the distance between S16 and S48, i.e. 8 units, plus the distance between S48 and S46, i.e. 9 units, is close to the distance between S16 and S46, i.e. 19 units.
Radiation Hybrid and Clone mapping of STS markers
are widely used to provide high resolution physical maps of chromosomes
and genomes... [Brown, Research Brief 3.1]
These STS markers also provide a framework of mixed markers on the genome for complete Genome Sequencing. ... [Brown, Chap 4]
E. Other Types of Cloning Experiments:
1. Site Specific Mutagenesis
Introduce specific Point Mutations
(single base changes) into Cloned Gene
Move back into Chromosome via Generalized Recombination ...
2. Protein Production:
Overexpress Gene Product, eg Insulin, in Bacteria ... Bacterial Factories
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith