Important words and
concepts from Chapter 8, Black, 1999 (3/28/2003):
by Stephen T. Abedon (abedon.1@osu.edu)
for Micro 509
at the Ohio State University
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(1)
Chapter Title: Recombinant DNA and Genetic Engineering
(2)
Horizontal transfer of DNA (bacterial sex)
(a)
While
mutation is the ultimate source of all alleles (i.e., all genetic variation),
sex is a means by which alleles found in one organism can get
together with the alleles found in another organism
(b)
This,
of course, happens all the time in many animal and plant (as well as protozoan
and fungal) species since these organisms undergo meiotic sexual cycles (i.e.,
alternating between haploid and diploid with meiosis generating haploid cells
and fertilization reestablishing the diploid)
(c)
Bacteria,
too, have sex but this sex is not intimately tied to reproduction
(d)
In
addition, while we (for instance) have sex with whole individuals, bacteria
have sex only with parts of individuals (bear with me on this)
(e)
That
is, sex in eucaryotes involves getting DNA from two different parents into the
same cell, whereupon the DNA undergoes a process called molecular recombination
which serves to shuffle together the DNA from each parent
(f)
In
bacteria, DNA from two parents also comes together in a single cell, but with
bacteria one parent supplies both the cell and a complete chromosome (i.e., a
genome) while the other parent supplies only a small subsection of a chromosome
(I like to use the term"snippet" as in "snippet of DNA")
(g)
Nevertheless,
the snippet of DNA from the other parent can recombine into the chromosome of
the parent supplying the cell, thus completing the bacterial sexual act
(h)
The
snippet can carry an allele that differs from the allele (of the same gene)
found in the parent supplying the cell, thus bringing together alleles within
that parent (i.e., the snippet's allele with all of the other alleles already
found on the recipient cell's bacterial chromosome)
(i)
Bacterial
sex can occur as a consequence of three different processes:
(i)
Transformation
(ii)
Transduction
(iii)
Conjugation
(j)
[horizontal transfer,
horizontal gene transfer,
bacteria sex, bacterial sex, “snippet sex bacteria”
(Google Search)]
[bacteria sex (MicroDude)] [index]
(a)
The
cell supplying the snippet of DNA
(a)
The
cell supplying the cell as well as the rest of the bacterial chromosome
(a)
Transformation
is the uptake of DNA directly from an organism's environment
(b)
That
DNA finds its way into the environment particularly when dead bacterial cells
spontaneously fall apart
(c)
Often
bacteria are somewhat discriminatory in the kind of DNA they are willing to
take up, preferring DNA from their own species
(d)
See Figure 8.1, The
discovery of transformation
(e)
The
DNA taken up by a cell is then recombined into the recipient cell's chromosome
(f)
See Figure 8.2, The
mechanisms of bacterial transformation
(g)
[transformation (MicroDude)] [index]
(a)
A
bacterial cell that is capable of being transformed (i.e.,
of taking up DNA directly from the environment) is said to be competent
(b)
Not
all bacteria are naturally competent, though bacteria that are not naturally
competent (e.g., E. coli) often can
be manipulated in the laboratory in such a way that they become able to pick up
environmental DNA
(c)
Once
the DNA is inside the cell, previously existing recombination mechanisms allow
the integration of the DNA snippet into the recipient's chromosome
(d)
[bacteria and competence
(MicroDude)]
[index]
(a)
Transduction
is the transfer of DNA-from a donor cell to a
recipient cell-with the DNA packaged within
a bacteriophage
(b)
(bacteriophages are viruses that infect bacteria)
(c)
The
donor DNA is packaged within a bacteriophage, the bacteriophage is released
into the extracellular environment, and the donor DNA is transferred to the
recipient cell when the bacteriophage infects the recipient cell
(d)
Key
to this whole process is that the bacteriophage has made a mistake so that, rather than packaging its own chromosome, it accidentally
has packaged a portion of the donor cell's (the bacteriophage's host) DNA
(e)
Note
that not all bacteriophage are capable of transducing and that of those that
are capable, some are more efficient (e.g., make more mistakes) than others
(f)
See Figure 8.5, Generalized
transduction
(g)
[transduction (MicroDude)] [index]
(a)
Conjugation
is a mechanism of bacterial sex (or a sex-like mechanism in that it often
involves a transfer of DNA but no recombination following transfer) that occurs
following the docking together of two bacteria, a donor and a recipient
(b)
The
occurrence of conjugation is due to the presence of certain plasmids in the
donor bacteria that posses genes for making the proteins involved in
docking and transfer, and then it is these plasmids that typically are what is
transferred from one bacteria to the other during the conjugative act
(c)
Note
that the donating bacteria is described as being male and that the act of
conjugation (if all goes as planned) serves to convert the recipient bacteria
also to a male (thus, one starts with one male and finishes with two)
(d)
[conjugation (MicroDude)] [index]
(a)
A
plasmid is an "extra-chromosomal" piece of bacterial DNA
(b)
Plasmids
typically are stably maintained within bacterial cells, replicating fast enough
that they are passed on to bacterial progeny as the bacteria divide
(c)
Like
bacterial chromosomes, plasmids are circular,
double-stranded DNA
(d)
The
major difference between chromosomes and plasmids is that plasmids are much
smaller than chromosomes plus tend to carry genes that are not essential except
in certain environments
(e)
[plasmids (MicroDude)] [index]
(a)
One
category of genes found on plasmids code for resistance to antibiotics
(b)
These
resistance or R plasmids often contain more than one bacterial-resistance gene
(c)
A
bacterium containing an R plasmid that expresses the appropriate antibiotic
resistance gene can survive when exposed to the antibiotic, whereas a bacterium
lacking the resistance gene will not
(d)
Because
plasmids may be readily transferred from cell to cell (e.g., just as snippets
of DNA may be transferred from cell to cell), bacteria are capable of acquiring
resistance to multiple antibiotics simply by acquiring a single resistance
plasmid
(e)
Put
another way, it means that antibiotic resistance can evolve in one kind of
bacteria, and then that resistance can be wholly transferred (e.g., via conjugation),
intact, to a new bacterium, including to bacteria that otherwise can cause
disease
(f)
[R plasmids (MicroDude)] [index]
(a)
Genetic
engineering involves the transfer of DNA to a recipient cell using artificial
techniques (i.e., something other than or in addition to sex)
(b)
Often
this DNA is manipulated in the test tube prior to its transfer
(c)
There
are various means of manipulating DNA and there are various means of
transferring DNA to a recipient cell (e.g., transformation, transduction)
(d)
Additionally,
there are various things that one can do with the DNA that has been transferred
to a recipient cell
(e)
Note
that the transferred DNA may be from the same species or from a different
species than the recipient
(f)
Such
successfully transferred DNA is said to be cloned
(g)
[genetic engineering (MicroDude)] [index]
(12)
Recombinant DNA technology
(a)
Recombinant
DNA technology represents a number of methods employed to
(i)
manipulate DNA outside of cells
(ii)
place manipulated DNA back into cells
(iii)
manipulate that DNA following its incorporation back into
cells
(b)
[genetic engineering (MicroDude)] [index]
(13)
DNA manipulation outside of cells (restriction endonuclease)
(a)
The
key to manipulating DNA outside of cells is the existence of enzymes known as
restriction endonucleases
(i)
The
restriction part of the name derives
from the actual use of these enzymes by the bacteria that make them:
restricting the replication of bacteriophages (by chewing up the bacteriophage
DNA)
(ii)
The
nuclease part of the name means these
enzymes cut DNA
(iii)
The
endo part of the name means that they
cut DNA in the middle of double helix strands (rather than chewing DNA up from
the ends, i.e., as do exonucleases)
(b)
Restriction
endonucleases cut DNA only at specific nucleotide sequences and thus are tools
by which DNA may be cut at specific locations
(c)
Thus,
a specific gene may be cut out of an organism's genome
(d)
Further
techniques allow one to specifically change the nucleotide sequence of the
isolated gene
(e)
See Figure 8.15, Producing
recombinant DNA
(f)
[DNA technology, restriction enzymes (MicroDude)] [index]
(14)
DNA transfer to recipient cell (vector)
(a)
To
transfer manipulated DNA back into a cell, one typically first inserts the DNA
into a vector
(b)
A
vector may be a plasmid (transformation) or
a bacteriophage chromosome (transduction) or both
(c)
The
vector or plasmid are opened up (cut) using specific restriction endonucleases
(d)
The
isolated gene is then inserted into this opening
(e)
An
additional enzyme, DNA ligase, then covalently attaches the gene into the
vector, thus making gene and vector into one double helix
(f)
The
vector may then be transduced or transformed into a recipient cell
(g)
Within
that cell the vector is allowed to replicate
(h)
Often
these vectors also contain antibiotic-resistance genes which, in the presence
of the appropriate antibiotic, allow only those cells that have successfully
received the vector to replicate
(i)
See Figure 8.15, Producing
recombinant DNA
(j)
[cloning vector, expression vector (MicroDude)] [index]
(15)
DNA manipulation within the recipient cell
(a)
Once
the DNA is in a recipient cell, things can be done with it
(b)
One
thing that can be done is to allow the introduced gene to express (e.g.,
produce a new protein), thus changing the phenotype of the recipient cell
(c)
A
second thing that can be done is the gene product (a protein) can be overly
expressed so that the resulting relatively high concentration of protein can be
purified and either used for a specific purpose or employed for the
characterization of the protein (which often is far easier given a relative
abundance of protein)
(d)
A
third thing that can be done is the inserted gene may be sequenced using DNA
sequencing techniques; sequencing permits further characterization as well as
further manipulation of the gene
(e)
The
inserted gene may serve as a source of DNA for further cloning of the gene
(e.g., to place in vectors having different properties, so that relatively
large concentrations of the gene sequence may be manipulated outside of the
cell, etc.)
(f)
[analysis of cloned DNA, DNA sequencing, subcloning, biotechnology links
(MicroDude)]
[index]
(16)
Hybridomas (monoclonal antibodies)
(a)
A
hybridoma is a fusion of a cancer cell (a myeloma) with a clone of an
antibody-producing cell (a B cell)
(b)
Hybridomas
are immortal (i.e., can divide indefinitely, a property that is not true for
most mammalian cells) and produce
antibodies
(c)
Only
a single type of antibody can be produced by a given hybridoma clone (an
antibody is a protein with high specificity for binding to specific other
molecules such as to other proteins)
(d)
The
antibody produced by a hybridoma is called a monoclonal antibody:
(i)
mono
meaning one
(ii)
clonal
meaning that the hybridoma is derived (asexually) from a single cell and all
progeny cells greatly resemble that single ancestral cell
(iii)
antibody
because it is an antibody that is produced
(e)
Monoclonal
antibodies are wonderful because they bind only to specific proteins (or other
molecules) thus allowing
(i)
the
identification of the specific protein (as in disease diagnosis)
(ii)
or
in the specific binding to specific organisms or tissues in the treatment of
disease (a long-time dream of applied immunology)
(f)
Note
that hybridomas and monoclonal antibodies, as typically produced/employed have
nothing to do with gene cloning by
molecular techniques (e.g., are not a product of recombinant DNA technology)!!!
Don’t let the word clonal fool you…it means “product of asexual replication” in
the context of monoclonal antibody
(g)
See Figure 17.12, Production
of monoclonal antibodies
(h)
See accompanying text on
pages 473-474 of your text
(i)
[monoclonal antibodies
(Google Search)]
[monoclonal antibody production
(graphic) (Access Excellence)]
[index]
(17)
Polymerase chain reaction (PCR)
(a)
Polymerase
chain reaction is a means by which an amount of DNA (e.g., a single gene) may
be increased enormously without first having to clone the DNA
(b)
To
PCR a gene requires "primers" which are pieces of DNA that are
complementary to the ends of the gene (one primer for each end)
(c)
The
primers allow the initiation of DNA replication (which is how the gene is
copied, thereby increased in number)
(d)
After
replication that gene, primers, and DNA polymerase (the enzyme that replicates
the DNA) are heated up to 60ºC to cause the DNA double helix (half of which was
just synthesized) to unwind into two single-stranded pieces of DNA
(e)
60ºC
is hot enough to denature an ordinary DNA polymerase; however the DNA
polymerase used in PCR comes from Thermus
aquaticus (Tac for short) which
lives in hot springs and consequently is a DNA polymerase that is adapted to
high temperatures
(f)
Lowering
temperatures allows the primers to initiate additional DNA synthesis of both
the original template strand (one primer) and the just-synthesized strand (the
other primer)
(g)
Repeat
often enough and one makes one heck of a lot of DNA
(h)
PCR
allows the production of huge quantities of DNA starting with as little as a
single copy of a gene
(i)
See Figure on page 193
(18)
Vocabulary [index]
(a)
Bacterial sex
(b)
Competence
(c)
Conjugation
(d)
DNA manipulation
outside of cells
(e)
DNA manipulation
within the recipient cell
(f)
DNA transfer to recipient cell
(g)
Donor cell
(i)
Horizontal transfer
of DNA
(j)
Hybridomas
(m)
Plasmids
(o)
Recipient cell
(p)
Recombinant
DNA technology
(s)
Transduction
(t)
Transformation
(u)
Vector