Beruflich Dokumente
Kultur Dokumente
Nucleic Acids
Introduction
Structural Features
of Nucleic Acids
Nucleic acids are polymers containing nitrogenous
bases attached to sugarphosphate backbones. The
common nitrogenous bases of nucleic acids are the
bicyclic purines, adenine and guanine, and the
monocyclic pyrimidines, cytosine, uracil, and
thymine (Fig. V-1).
These purines and pyrimidines join to the
sugarphosphate backbones of nucleic acids
through repeating !-linked N-glycosidic bonds involving the N 9 position of purines and the N1 position of pyrimidines. There are two classes of
nucleic acids: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). DNA and RNA differ in one
of their nitrogenous base components (uracil in
RNA, thymine in DNA) and in their sugar (ribose)
moiety, as indicated in Fig. V-2.
Repeating 3",5"-phosphodiester bonds join the
D-ribose units of RNA and the 2-deoxy-D-ribose
units of DNA. A nucleoside is a nitrogenous base plus
a sugar, while a nucleotide is a nitrogenous base, plus
NH2
N1
2
Numbering
system
3 4
H
N
7
H
Adenine
H
H2N
N3
H
N
H
Guanine
NH2
Numbering
system
1 6
CH3
N
N
Cytosine
Uracil
H
Thymine
304
SECTION V
Nucleic Acids
NH2
N
N
O
H
A nucleotide: any
combination of a base,
a sugar, and a phosphate
NH2
O$
N
H
OH
O
P
NH
N
NH2
NH2
O$
O$
NH
H
O$
CH2
CH3
CH2
O$
O
O
CH2
OH
O
O
CH2
CH2
A nucleoside: any
combination of a base
and a sugar
O$
NH2
OH
O
O
O
NH
O
N
CH2
CH2
O$
OH
O
O
O
CH2
NH2
H
O$
or
A
G
or
C
U
A G C U
bitals of the stacked planar base pairs add to the stability of the polynucleotide (Figs. V-3, and V-4).
Most DNAs contain extensive secondary
structure, existing as large double-stranded helixes containing two separate, hydrogen-bonded
SECTION V
Introduction
Supporting sugar
phosphate backbone
Planar H-bonded
base pairs
H
CH3 O
Overlapping
orbitals
N
N
N
O
Thymine:Adenine
Orbitals
H
N
N
N
H
Guanine:Cytosine
Possible complementary
WatsonCrick base pairing
complementary sequences. These separate primary strands exist together in opposite or antiparallel orientation (one strand running 5" to 3"
and the other running 3" to 5"), while being stabilized by intramolecular base stacking and by intermolecular hydrogen-bonded base pairing. This
yields the long, fibrous double helix characteristic of most DNAs (Fig. V-4). This lengthy structure contributes to the pronounced physical char-
305
306
SECTION V
Nucleic Acids
o
o
Sugarphosphate
backbones
o
o
o
o
o
o o
N
N
H N
o
o
CH2
Y
Cm
A
A
U
Gm
Unpaired free
bases in
anticodon
intramolecular
loop
CH2
o o
CH2
N HN
oo
o o
o
CH2
CH2
oo P
H NN
o
oo o
CH2
oo
o o o
P
oN
H-bonded base
pairing and
base stacking
CH2
CH2
CH2
oo P
o o
oo
CH2
where present in RNA, are generally intramolecular and generate helical hairpin loops within a
primary nucleotide sequence (see Fig. V-4). Thus,
precipitated RNAs are more amorphous than fibrous. Accordingly, most RNAs do not markedly
increase solution viscosity and can be difficult to
crystallize for subsequent x-ray analysis.
Certain RNAs also possess substantial amounts
of tertiary structure. Tertiary structure in RNA
refers to the folding of secondary structural elements, such as double helical regions or hairpin
stem-loops, into discrete three-dimensional structures. Forces involved in stabilizing such interactions are diverse, involving hydrogen-bonding, base
SECTION V
Introduction
Two
chromatids
(2 x 10 coils)
One coil
(30 rosettes)
One rosette
(6 loops)
Nuclear
scaffold
One loop
(50 x 106 bp)
30 nm Fiber
Beads-ona-string
form of
chromatin
Histones
DNA
307
308
SECTION V
Nucleic Acids
Prokaryotic ribosome
50S subunit
MW = 1.8 % 106
30S subunit
MW = 1 % 106
70S overall
Eukaryotic ribosome
60S subunit
MW = 2.5 % 106
40S subunit
MW = 1.5 % 106
80S overall
SECTION V
Chemical Properties
of Nucleic Acids
Action of Alkali
The N-glycoside linkages of DNA and RNA are
stable in mildly alkaline conditions. However, the
phosphodiester linkages of DNA and RNA demonstrate markedly different chemical properties in
mild alkali. For example, 0.3 M KOH (37C)
rapidly (!1 hr) catalyzes the cleavage of all phos-
Introduction
O
CH2
H
OH
O
CH2OH O
$
OH
CH2
H
O$
O$
H2O
&
CH2OH O
Cyclic-2",3"-phosphonucleosides
OH
OH
$
Nucleoside-3"-phosphates
P
O
O$
Base
Base
Base
Base
Base
H
OH
O
O$
O$
Nucleoside-2"-phosphates
309
SECTION V
Nucleic Acids
phosphodiester bond cleavage occurs during acidcatalyzed depurination. Even more phosphodiester
cleavage occurs during acid-catalyzed depyrimidination.
Physical Properties
of Nucleic Acids
Several of the physical properties of nucleic acids
have already been mentioned in the discussion of
nucleic acid structure. The following properties
play important roles in the isolation, detection, and
characterization of nucleic acids.
UV Absorption of Nucleic Acids
All nucleic acids, nucleosides, and nucleotides
strongly absorb ultraviolet (UV) light. This UV absorption arises from the nitrogenous bases, each of
which possesses its own unique absorption spectrum (Fig. V-8). The sum of these individual nitrogenous base spectra generates the overall UV
spectrum of DNA and RNA. Several features of
these spectra deserve further comment. First, the
nucleoside spectra are a function of pH. This property reflects the titration of specific functional
groups within the nitrogenous bases. Such pH-dependent spectral shifts provide an additional tool
for characterizing unknown solutions containing
these nucleosides. Second, the ratios of the absorption at two different wavelengths (e.g.,
A280 /A260) at any pH provide criteria for identification of individual nitrogenous bases or nucleosides, and for the estimation of the relative proportions of individual nucleosides in an
oligonucleotide. Third, a nucleic acid spectrum is
the sum of the individual nitrogenous base spectra
present in the nucleic acid. Such summation yields
a spectrum with a 'max of 260 nm. It follows, then,
that the absorption of a solution containing a nucleic acid is proportional to the nucleic acid concentration. In general, a 1-mg/ml solution of
single-stranded DNA (lacking any degree of secondary structure) has an A260 of 25 at neutral pH
in a 1-cm light path (260 ( 25 (mg/ml)$1 cm$1).
Extensive secondary structure, as found in native
tRNA, rRNA, and DNA, reduces the UV absorption of the stacked, hydrogen bonded bases so that
Adenosine
1M
pH 7.0 E1cm
= 14,900
260
pH 7.0 280/260 = 0.15
1.0
0
Guanosine
1M
pH 7.0 E1cm
260 = 11,700
pH 7.0 280/260 = 0.66
1.0
Absorbance of 1 % 104 M solution in a 1-cm cuvette
310
0
Cytidine
1M
pH 7.0 E1cm
260 = 7500
1.0
0
Uridine
1M
pH 7.0 E1cm
260 = 9900
1.0
0
Thymidine (with
deoxyribose)
pH 7.0 E1M
1cm260 = 8700
pH 7.0 280/260 = 0.74
1.0
220
260
300
Nanometers
DNA
20
10
220
260
Nanometers
300
SECTION V
Introduction
RNA
20
10
220
260
Nanometers
300
Figure V-9 UV characteristics, right, of single-stranded RNA and, left, of DNA. () ( pH 7.0,
( ) ( pH 1.0, ( ) ( pH 13.0. The denaturation of double-stranded DNA at pH 1
and 13 causes an increase in UV absorbance, an illustration of the hyperchronic effect.
311
312
Table V-1 Titration Properties of Nitrogenous Bases and Phosphates of Nucleic Acids and Their
Subunits
Name of Ionizing Group
Primary Ionization
Phosphodiesters of
internucleotide
linkages of RNA
and DNA
Secondary Ionization
CH2
O
O
pKa1 ( 1.5
OH
) H&
O$
P
O
O
O
CH2
Phosphomonoesters
of nucleotides or
terminal phosphates
CH2
CH2
O
R
O
OH
pKa1 ( 0.9
) H&
OH
Adenine in adenosine,
adenylic acids, AMP, etc.,
and RNA and DNA
) H&
&
O$
NH2
N
O$
OH
NH3
N
O
pKa2 ( 6.0
NH2
&
pKa1 ( 3.6
&
N
R
)H
R
Guanine in guanosine,
guanylic acids, GMP, etc.,
and RNA and DNA
O
H
H2N
N&
N
N
pKa1 ( 2.3
) H&
H2N
NH2
&N
pKa1 ( 4.3
) H&
N
R
O
N
O
pKa1 ( 9.2
O
CH3
N
R
pKa1 ( 9.8
) H&
N
O
) H&
O$
N
R
O$
CH3
N
R
N
R
NH2
Thymine in thymidine,
thymidylic acids, TMP, etc.,
and DNA
H2N
H
O
Uracil in uridine,
uridylic acids, UMP, etc.,
and RNA
) H&
pKa2 ( 9.2
R
Cytosine in cytidine,
cytidylic acids, GMP, etc.,
and RNA and DNA
O
H
CH3
N
O
N
R
SECTION V
Introduction
Southern blotting
Molecular weight
size standards Sample
Restriction
endonuclease
Chromosomal DNA
Agarose-gel
electrophoresis
Incubate with
single-stranded,
radiolabeled DNA
molecule (probe)
(
)
Hybridization
Expose
to film
Film
Northern blotting
Molecular weight
size standards Sample
Isolate RNA
Agarose-gel
electrophoresis
Cell
Expose
to film
Incubate with
single-stranded,
radiolabeled RNA or
DNA molecule (probe)
(
)
Hybridization
313
314
SECTION V
Nucleic Acids
SECTION V
Introduction
Selectable genetic
marker that allows the
determination of which
host cells took up the
plasmid following
transformation
Figure V-11 Features found on many popular plasmids used for different molecular biology applications.
lated, it can be introduced into and faithfully propagated (cloned) by the host organism.
This process of inserting genes into plasmids is
the cornerstone of molecular biology. Once a gene
of interest has been separated from the chromosome and inserted into a plasmid, the gene can be
subjected to a number of different manipulations
that provide insight into its biological function. In
biochemical studies, it is often necessary to express
the gene carried on the plasmid, isolate the protein
that it encodes, and study its function. To aid in this
process, many recombinant plasmids have been engineered to include powerful promoter sequences
5" to the MCS. Genes cloned into the MCS downstream (3") to the promoter can be expressed at high
315
316
SECTION V
Nucleic Acids
Table V-2 Some Commonly Used Antibiotics, Their Modes of Action, and
Their Inactivation by Resistant Host Cells
Antibiotic
Chloramphenicol
Ampicillin
Erythromycin
Tetracycline
Kanamycin and
Neomycin
Streptomycin
Mode of Action
Inactivation
levels in the host cell, and the proteins that they encode can be purified. Often, you would like to control the expression of genes cloned into plasmids.
This becomes critical if the high level expression of
a particular gene product is found to be lethal to
the host cell. Many of the promoter elements that
flank the MCS, therefore, are able to be induced
through the addition of a small molecule or some
other change in the condition of the growth
medium. In this way, the host strain can be grown
in culture, and the gene of interest can be expressed
at any particular time during the growth cycle. A
list of inducible promoters found in recombinant
plasmids and their means of induction are listed in
Table V-3.
Expression of a gene of interest is by no means
the only manipulation that can be carried out once
the gene of interest has been inserted into a plasmid. Current techniques in molecular biology allow you to make virtually an unlimited number of
mutations in the gene-carrying recombinant plasmid: 1 specific base pair can be changed, 2 specific
base pairs can be changed, 50 base pairs can be
deleted, 20 base pairs can be added, and so forth.
Chloramphenicol acetyl
transferase acetylates the
antibiotic
!-lactamase cleaves the ring
in the antibiotic
An enzyme methylates a
specific adenine residue in the
rRNA so that it is unable to bind
the antibiotic
A membrane protein actively
expels or transports the
antibiotic out of the cell
Aminoglycoside phosphotransferase phosphorylates
the antibiotic
A deletion of the S12
ribosomal protein prevents
binding of the antibiotic
317
Plasmid
Digest plasmid within the multiple
cloning site with restriction endonuclease
Plasmid 1
Plasmid 2
Plasmid 3
Clone 1
Clone 3
Clone 2
318
SECTION V
Nucleic Acids
Inducer
lac
tac
'
BAD
MMTV
(mouse mammary
tumor virus)
GAL1
AOX1
MT
(metallothionein)
*HSP
Isopropylthiogalactoside (IPTG)
Isopropylthiogalactoside (IPTG)
Increased temperature (42C)
Arabinose
Dexamethasone
Galactose
Methanol
CuSO4
Muristerone A
excised and replaced with any foreign DNA fragment that is 10 to 20 kb in size (Fig. V-13). Once
the DNA fragment of interest has been inserted into
the bacteriophage ' chromosome, the chimeric
chromosome can be packaged in vitro into phage
particles that will deliver the DNA into the E. coli
host. Since the chimeric genome still contains the
genes required for replication, phage assembly, and
cell lysis, the DNA fragment that is inserted into
the chromosome will be replicated (cloned), packaged, and used to infect neighboring E. coli cells.
The plaque produced when cells infected with the
recombinant phage are plated on a lawn of uninfected E. coli cells contains progeny (clones) of a single phage. Since bacteriophage ' is able to package
only DNA between 40 and 50 kb into the phage
head, DNA inserts smaller than 10 kb and larger
than 20 kb in size cannot be cloned using this vector. Other E. coli bacteriophages, such as the singlestranded M13 phage, have smaller chromosomes
that can accept DNA fragments smaller than 10 kb
in size.
Cosmids
The cosmid is another variation of the plasmid and
viral DNA vectors that has been developed to allow the cloning of DNA fragments that range from
40 to 50 kb. As the linear bacteriophage ' DNA is
SECTION V
Introduction
Nonessential genes
Chromosomal DNA
es
nti
se
es
al
n
ge
Restriction
endonuclease
Restriction
endonuclease
n
No
Ligate
s
A
gi
DN
gin
a
k
ert
c
s
e
in
pa
l if siz
ro
vit
sfu b in
s
n
I
e 0k
cc
su 102
is
Infect E. coli
and plate on a lawn
of uninfected cells
Plaques containing
progeny (clones) of
a single recombinant
bacteriophage
319
320
SECTION V
Nucleic Acids
cos site
Selectable
genetic
marker
Chromosomal DNA
Cosmid
Multiple
cloning
site
Selectable
genetic
marker
Ligate
cos site
is
ng
agi
NA
k
c
rt D
pa
e
s
o
r
n
it
if i
In v
h
ful
ess enoug
c
c
su rge
a
is l
SECTION V
Introduction
321
322
SECTION V
Nucleic Acids
CH3
DNA methylase
GAATTC
GAATTC
NH2
N
HN
N
N
NH2
Adenine
CH3
&
CH2
CH2
CH2
&
H3N
CH
OH
OH
CH3
N
Ribose
N -methyladenine
COO$
NH2
S-adenosylmethionine
N
S
CH2
CH2
CH2
&
H3N
CH
OH
OH
COO$
S-adenosylhomocysteine
CH3
DNA methylase
CCCGGG
NH2
N
O
HN
H
N
Cytosine
N
O
CH3
N 4-methylcytosine
Figure V-15 The DNA methylase component of the restriction modification system methylates a specific
cytosine or adenine residue, making it incapable of being acted on by the companion restriction endonuclease.
SECTION V
Introduction
Restriction Endonuclease
Recognition Sequence
Enzyme
Activity
BamHI
5"-G!GATTC-3"
3"-CCTAA"G-5"
5"-A!GATCT-3"
3"-TCTAG"A-5"
5"-AT!CGAT-3"
3"-TAGC"TA-5"
5"-G!AATTC-3"
3"-CTTAA"G-5"
5"-GAT!ATC-3"
3"-CTA"TAG-5"
5"-A!AGCTT-3"
3"-TTCGA"A-5"
5"-GGTAC!C-3"
3"-C"CATGG-5"
5"-CA!TATG-3"
3"-GTAT"AC-5"
5"-GC!GGCCGC-3"
3"-CGCCGG"CG-5"
5"-GAGCT!C-3"
3"-C"TCGAG-5"
5"-G!TCGAC-3"
3"-CAGCT"G-5"
5"-!GATC-3"
3"-CTAG"-5"
5"-CCC!GGG-3"
3"-GGG"CCC-5"
5"-AAT!ATT-3"
3"-TTA"TAA-5"
5"-C!TCGAG-3"
3"-GAGCT"C-5"
Deoxyribonuclease I
(DNaseI)
Exonuclease III
BglII
ClaI
EcoRI
EcoRV
HindIII
KpnI
NdeI
NotI
SacI
SalI
Sau3AI
SmaI
SspI
XhoI
component in four different DNA repair mechanisms: base-excision repair, base mismatch repair,
nucleotide excision repair, and recombinational
(SOS) repair. Homologous recombination, or exchange of DNA fragments within the genome that
have similar sequences, also requires DNA ligase.
This latter type of recombination, which occurs frequently during meiosis, is responsible for the genetic and phenotypic diversity seen within populations of higher eukaryotes.
Although DNA ligase is essential for numerous
in vivo processes, it has also proven to be a very
powerful tool in the growing field of recombinant
DNA technology. As discussed earlier in this section, a gene of interest can be excised from the
Lambda
exonuclease
Nuclease P1
Nuclease S1
Mung bean
nuclease
Snake venom
phosphodiesterase I
Ribonuclease H
Ribonuclease I
Polynucleotide
kinase
Alkaline
phosphatase
chromosome with restriction endonucleases, isolated, and inserted into a vector. The ligation of the
DNA fragment of interest into the vector is catalyzed in vitro by DNA ligase (Fig. V-16). The reaction catalyzed by DNA ligase is energy-requiring.
The enzyme first attaches an adenyl group (from
ATP or NAD&) to the 5"-phosphoryl group present on one of the two DNA strands. The phosphoamide adduct produced in this reaction is then
subject to nucleophilic attack at the 5" phosphoryl
group by the 3" hydroxyl group present on the other
323
324
SECTION V
Nucleic Acids
T4 DNA Ligase
NH2
O
DNA
Lys
ligase
NH2 & O
O
P
$O
O
P
$O
CH2
$O
OH
OH
NH2
N
O
&
DNA
Lys
ligase
NH2
CH2
$O
H
O
O
OH
OH
O
&
O
O
$O
O$
P
$O
Pyrophosphate
HO
O$
Adenylation
NH
N
N
N
N
O
O
O
O
O
O$
O
O$
CH
H
H
HO
OH
&
DNA
Lys
ligase
NH2
OH
NH2
N
O
$
&
CH2
$O
H
Strands linked via a
phosphodiester bond
OH
OH
N
N
SECTION V
C
CH2
NH2 &
O
$
&
OH
OH
CH2
H
DNA
Lys
ligase
OH
OH
&
NH2
O$
CH2
NH2
O
NH2
NH2
OH
OH
N
N
&
O
C
&
325
DNA
Lys
ligase
Introduction
NH2
HO
CH2
O$
Adenylation
2
NH
&
OH
OH
N
N
N
N
O
O
O
O
O$
2
CH
H
H
HO
OH
&
DNA
Lys
ligase
NH2
OH
NH2
N
O
$
&
CH2
$O
H
Strands linked via a
phosphodiester bond
Figure V-16 (continued) The reaction catalyzed by DNA ligase (E. coli).
OH
OH
N
N
326
SECTION V
Nucleic Acids
SECTION V
Chromosomal DNA
Chromosomal DNA
Digest with
BamHI (G GATCC)
7&&
Introduction
Digest with
BglII (A GATCT)
&&7
7&7
7&7
7
&&
7&&
Ligate
Ligate
&
&& 7
7
&
BamHI sites
still intact
7&&
7
7&
7&&
7
&&
&& 7
7
&
microgram of DNA) to take up the vector. It is extremely important when using this technique to remove
all of the salt from the solution. If this is not done, the
solution will arc (a spark will appear), heat, and kill
all of the host cells. Even with a very-low-ionicstrength solution, a mortality rate of up to 80% is
not uncommon with electroporation. Its major ad-
vantage is the nearly 10,000-fold increase in transformation efficiency (number of transformed cells
produced per microgram of vector DNA) compared
to the traditional E. coli transformation method described earlier.
Evidence indicates that transformation efficiency can be improved with yeast transformation
327
328
1.
2.
3.
4.
5.
6.
7.
8.
9.
Yeast
1. Grow 300 ml of yeast cells to an OD600 of 0.5.
2. Harvest the cells by centrifugation and resuspend the cell pellet in 10 ml of distilled water.
3. Harvest the cells by centrifugation and resuspend the cell pellet in 1.5 ml of Tris/EDTA/lithium acetate:
10 mM Tris, pH 7.5
1 mM EDTA
100 mM lithium acetate
4. Add 15 ,g of plasmid DNA plus 100 ,g of single-stranded carrier (salmon sperm) DNA to a sterile 1.5-ml
microcentrifuge tube.
5. Add 200 ,l of the cells prepared in step 3 to the same tube.
6. Add 600 ,l of the following solution to the same tube:
40% wt /vol polyethylene glycol (PEG-8000)
10 mM Tris, pH 7.5
1 mM EDTA
100 mM lithium acetate
7. Incubate the cells at 30C for 30 min.
8. Add 90 ,l of dimethyl sulfoxide (DMSO), and heat shock the cells at 42C for 15 min.
9. Harvest the cells by centrifugation and resuspend the cell pellet in 1 ml of Tris/EDTA buffer (10 mM Tris, pH 7.5, 1
mM EDTA).
10. Plate cells on selective media.
SECTION V
Introduction
329
330
SECTION V
Nucleic Acids
AmpR
BamHI
site
pBR322
(4361 bp)
TetR
Chromosomal DNA
ori
TetR
Ligate
AmpR
TetR
ori
TetR
AmpR
TetR
Transform E. coli
and plate on ampicillincontaining media.
AmpR
&
ori
ori
TetR
Figure V-18 Insertional inactivation of the tetracycline resistance gene following cloning in pBR322.
ampR ( ampicillin-resistant; tetR ( tetracycline-resistant; ori ( origin of plasmid replication
SECTION V
the plasmid at the BamHI site, the tetracycline resistance gene will be disrupted (insertional inactivation), and cells harboring the plasmid will be unable
to grow in the presence of tetracycline. Therefore,
if ampicillin-resistant colonies are replica plated on
a tetracycline containing plate and do not grow,
then they contain the pBR322 plasmid with an insertion (most likely of the gene of interest) at the
BamHI site. Replica plating is the process of lifting
individual colonies from one plate and transferring
them to the exact same position on a new plate. For
a plate that contains a large number of colonies, this
can be performed by placing a sterilized felt patch
across the surface of the plate and pressing firmly
to allow the felt to come into contact with the bacteria. This felt patch can then be placed over the
surface of a fresh plate and pressed firmly to obtain
an exact replica of the original plate.
A similar type of insertional inactivation screening process for recombinant vectors is described in
the introduction to Experiment 21. Many modern
E. coli cloning vectors contain multiple cloning sites
within the lacZ .-peptide coding sequence. The
LacZ .-peptide consists of the first 150 amino acids
of !-galactosidase. It is known that the LacZ .peptide can be removed (cleaved) from the enzyme
and added back to the C-terminal domain of !galactosidase to produce the fully active enzyme. If
this plasmid is introduced into an E. coli strain in
which the chromosomal copy of the lacZ gene has
the region of the .-peptide deleted, the host strain
will be complemented (restored) for !-galactosidase
activity. If this same plasmid has a gene inserted in
the multiple cloning site within the lacZ .-peptide
sequence, the lacZ .-peptide reading frame will be
disrupted, and the host cell will not display any !galactosidase activity. Any cell that is not complemented for !-galactosidase activity (contains a plasmid with an insertion at the multiple cloning site)
will give rise to white colonies on an IPTG/ Xgal
plate, while cells that received the unmodified plasmid will give rise to blue colonies. This screening
process involving . complementation is described in
greater detail in Experiment 21.
REFERENCES
Balbas, P., et al. (1986). Plasmid Vector pBR322 and Its
Special-Purpose DerivativesA Review. Gene 50:3.
Introduction
Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res 15:1311.
Collins, J., and Hohn, B. (1978). Cosmids: A Type of
Plasmid Gene-Cloning Vector That Is Packageable
in Vitro in Bacteriophage ' Heads. Proc Natl Acad
Sci 75:4242.
Dagert, M., and Ehrlich, S. D. (1979). Prolonged Incubation in Calcium Chloride Improves the Competence of Escherichia coli Cells. Gene 6:23.
Davidson, J. N. (1972). The Biochemistry of Nucleic Acids,
7th ed. New York: Academic Press.
Davies, J., and Smith, D. I. (1978). Plasmid Determined Resistance to Antimicrobial Agents. Annu
Rev Microbiol 32:469.
Doerfler, W. (1983). DNA Methylation and Gene Activity. Annu Rev Biochem 52:93.
Dugaiczyk, A., Boyer, H. W., and Goodman, H. M.
(1975). Ligation of EcoRI Endonuclease-Generated
Fragments into Linear and Circular Structures. J
Mol Biol 96:171.
Friedberg, E. C. (1985). DNA Repair. New York: Freeman.
Gerard, G. F. (1983). Reverse Transcriptase. In: Jacob,
S. T., ed. Enzymes of Nucleic Acid Synthesis and Modification: Volume I. DNA Enzymes. Boca Raton, FL:
CRC Press.
Hohn, B. (1979). In Vitro Packaging of ' and Cosmid
DNA. Methods Enzymol 68:299.
Hung, M. C., and Wensink, P. C. (1984). Different Restriction Enzyme-Generated Sticky DNA Ends Can
Be Joined in Vitro. Nucleic Acids Res 12:1863.
Ish-Horowicz, D., and Burke, J. F. (1981). Rapid and
Efficient Cosmid Cloning. Nucleic Acids Res 9:2989.
Kornberg, T., and Kornberg, A. (1974). Bacterial DNA
Polymerases: In: Boyer, P. D., ed. The Enzymes,
3rd ed. Vol. 10, pp. 119144. New York: Academic
Press.
Lehninger, A. L., Nelson, D. L., and Cox, M. M.
(1993). Nucleotides and Nucleic Acids. In: Principles of Biochemistry, 2nd ed. New York: Worth.
Mathews, C. K., and van Holde, K. E. (1990). Nucleic
Acids. In: Biochemistry. Redwood City, CA:
Benjamin/Cummings.
McClelland, M., and Nelson, M. (1987). The Effect of
Site-Specific DNA Methylation on Restriction Endonucleases and DNA Modification MethyltransferasesA Review. Gene 74:291.
Messing, J. (1983). New M13 Vectors for Cloning.
Methods Enzymol 101:20.
Minton, N. P., Chambers, S. P., Prior, S. E., Cole, S.
T., and Garnier, T. (1988). Copy Number and Mobilization Properties pf pUC Plasmids. Bethesda Res
Lab Focus 10(3):56.
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SECTION V
Nucleic Acids
Sykes, R. B., and Mathews, M. (1976). The BetaLactamases of Gram-Negative Bacteria and Their
Role in Resistance to Beta-Lactam Antibiotics.
J Antimicrob Chemother 2:115.
Ullmann, A., Jacob, F., and Monod, J. (1967). Characterization by in Vitro Complementation of a Peptide Corresponding to an Operator-Proximal Segment of the !-Galactosidase Structural Gene of
Escherichia coli. J Mol Biol 24:339.
Watson, J. D., Hopkins, N. H., Roberts, J. W., Steitz,
J. A., and Weiner, A. M. (1987). Molecular Biology of
the Gene, 4th ed. Menlo Park, CA: Benjamin /Cummings.