Restriction enzymes (RE) have developed into one of the primary tools in
molecular biology. They can be employed to cut DNA molecules precisely into
fragments of a predictable size. A more accurate name for this class of enzymes
is restriction endonuclease, because they break DNA
molecules at internal (endo) positions. Enzymes that degrade DNA by digesting
the molecule from the ends of the DNA strand are called exonuclease (exo =
"outside").
Restriction endonucleases are frequently named using the following convention:
The first italicized letter indicates the genus of the organism from which the
enzyme was isolated. The second and third italicized letters indicate the
species. An additional letter indicates the particular strain used to produce
the enzyme. The Roman numerals denote the sequence in which the restriction endonuclease enzyme from that particular genus, species, and strain of bacteria have been isolated. See Table I.
| Table I |
|
EcoR I
|
E = genus Escherichia
|
|
|
co = species coli
|
|
|
R = strain RY 13
|
|
|
I = first RE to be isolated from this species
|
|
BamH I
|
B = genus Bacillus
|
|
|
am = species amyloliquefaciens
|
|
|
H = strain H
|
|
|
I = first RE to be isolated from this species
|
|
Hind III
|
H = genus Haemophilus
|
|
|
in = species influenzae
|
|
|
d = strain Rd
|
|
|
III = third RE to be isolated from this species
|
Some restriction endonucleases cut cleanly through the DNA molecule by
cleaving both of the complimentary strands of the DNA molecule at the same
nucleotide position within the recognition sequence. The recognition sequences
are generally 4 to 6 basepairs long (refer to Table II).
| Table II |
|
Some Restriction Endonucleases and Their Recognition Sequences
|
|
Enzyme Source
|
Abbreviation
Recognition
|
Sequence
|
|
Bacillus amyloliquefaciens H
|
BamH I
|
G/GATCC
CCTAG/G
|
|
Brevibacterium albidum
|
Bal I
|
TGG/CCA
ACC/GGT
|
|
Escherichia coli RY13
|
EcoR I
|
G/AATTC
CTTAA/G
|
|
Haemophilus aeqyptius
|
Hae III
|
GG/CC
CC/GG
|
|
Haemophilus influenzae Rd
|
Hind III
|
A/AGCTT
TTCGA/A
|
|
Haemophilus parainfluenzae
|
Hpa II
|
GTT/AAC
CAA/TTG
|
|
Haemophilus parainfluenze
|
Hha II
|
C/CGG
GGC/C
|
|
Providencia stuartii 164
|
Pst I
|
CTGCA/G
G/ACGTC
|
|
Streptomyces albus G
|
Sal I
|
G/TCGAC
CAGCT/G
|
These 4-8 nucleotide recognition sites are also termed palindromic sequences,
because both strands have the same sequence running in opposite directions.The
RE scans the length of the DNA molecule and stops to cut the molecule only at
its particular recognition site. For example, the endonuclease Hind III will
cut a double strand of DNA in the following way (see page 2):
The Hind III enzyme recognizes the sequence
AAGCTT
TTCGAA
and will cut the DNA as shown below whenever it encounters that sequence.
Some restriction endonucleases (Hae III, Bal I) cut cleanly through the DNA
molecule by cleaving both of the
complimentary strands of the DNA molecule at the same nucleotide position
within the recognition sequence. These enzymes produce a blunt-end cut (Figure
1).
Bal I cuts at the center of the restriction site, leaving blunt ends.
Other RE's (Hind III) cleave the complimentary strands at a different point
within the recognition sequence, which results in a staggered cut. A staggered
cut exposes single stranded regions of the molecule. These single stranded
regions ("sticky ends") are especially useful in making
recombinant-DNA molecules (Figure 2).
Hind III makes staggered cuts in DNA leaving cohesive
"sticky" ends.
DNA restriction fragments produced by the same RE can be spliced together. The
sticky ends produced as a result of the staggered cut by the restriction enzyme
allow complimentary regions in the sticky ends to recognize one another and
pair up. The enzyme ligase, ATP, magnesium ions, and proper pH and temperature
are required to reform DNA bonds that have been broken by a restriction enzyme
that makes a sticky end cut.
Lambda is a bacteriophage which infects E. coli cells. The viral DNA is linear,
double-stranded molecule of 48,502 basepairs (bp) [48.5 Kbp]' with a molecular
weight of about 3 x 10 7 and codes for approximately 50 different phage
proteins. The first 12 bases at both 5' ends are single stranded and can
hybridize to each other to form circular molecules (Figure 3).
Every nucleic acid chain has a direction defined by the orientation of its
sugar-phosphate backbone. The end terminating with the 5' carbon atom is called
the 5' end, while the end terminating with the 3' carbon atom is called the 3'
end. All RNA and DNA chains grow in the 5' --> 3' direction. By convention,
the first base at the left (5'-end) of the linear DNA is nucleotide number 1.
The base sequence of these terminal regions, which are known as cos (cohesive
sticky ends), are complimentary to each other. Thus, by forming base pairs
between the cohesive ends, linear DNA molecules will circularize as shown in
Figure 3.
The circularization of lambda DNA also occurs in an infected
bacterial cell. When lambda phage infects an E. coli cell host, the phage DNA
is injected into the cell and cohesive ends anneal or stick together to form a
circular molecule. Transcription and replication of the phage DNA occurs, and
more phage particles are made within the host bacterium. Later, the bacterial
cell breaks apart (lyses), releasing new infective phages, and the bacterial
cell dies. Less common is the integration of phage DNA into the bacterial
chromosome. Following integration, the bacterial cells behave normally, and
both phage DNA and bacterial chromosomal DNA is replicated together. If the
bacterial cell is subjected to a negative outside environmental insult
(ultraviolet light), the lambda DNA is excised from the bacterial chromosome
and begins its normal viral replicative activities.
The DNA of phage lambda may be divided into three regions (see figure 4). The
left-hand region includes all the genes
(A through J) whose products are necessary to produce phage head and tail
proteins and to package the DNA into the virus. The central region contains
genes involved in the integration of the DNA into the E. coli chromosome. The
remaining portion of the viral genome includes the major control region for
transcription and replication as well as the genes for cell lysis.
Some of the restriction endonucleases that cleave lambda are listed in Table
III below. The positions in the table refer to the 5' base of the recognition
sequence. Site position(s) read left to right (5' --> 3' position).
| Table III |
No. of
Enzyme
|
Sites
|
Position of Sites
|
|
|
|
1
|
2
|
3
|
4
|
5
|
6
|
7
|
|
BamH I
|
5
|
5505
|
22346
|
27972
|
34499
|
41732
|
|
|
|
EcoR I
|
5
|
21226
|
26104
|
31747
|
39168
|
44972
|
|
|
|
Hind III
|
7
|
23130
|
25157
|
27479
|
36895
|
37459
|
37584
|
44141
|
For example, a restriction map for phage lambda cut by
Bam H I is presented above (Figure 5).
Beginning at left, each of the numbered restriction sites was plotted along the
length of the phage genome. The number of nucleotides (the fragment size) to
the left of each restriction site were then calculated:
|
(6)
|
16,841
|
|
|
7,233
|
|
|
6,770
|
|
|
6,527
|
|
|
5,626
|
|
|
5,505
|
Any restriction enzyme that cuts linear DNA N times will yield N+1 fragments.
Because smaller DNA fragments migrate faster down a gel, the order in which you
would expect to observe fragments ranges from highest to lowest base pair
number.
In this experiment, you will study the effects of EcoR I, Hind III, and EcoR I/
Hind III double digests on lambda DNA, and by analyzing fragment patterns,
construct a restriction map of these restriction sites.
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