Types of Restriction Endonucleases

Return to Restriction Endonucleases

Restriction enzymes are traditionally classified into four types on the basis of

 subunit composition,
 cleavage position,
 sequence specificity and;
 cofactor requirements.

However, amino acid sequencing has uncovered extraordinary variety among restriction
enzymes and revealed that at the molecular level, there are many more than four different
types.

Type I enzymes are complex, multisubunit, combination restriction-and-modification enzymes

that cut DNA at random far from their recognition sequences. Although common, Type I
enzymes are of considerable biochemical interest, but they have little practical value since they
do not produce discrete restriction fragments or distinct gel-banding patterns.

Type II enzymes cut DNA at defined positions close to or within their recognition
sequences. They produce discrete restriction fragments and distinct gel banding patterns, and
they are the only class used in the laboratory for routine DNA analysis and gene cloning. Rather
than forming a single family of related proteins, Type II enzymes are a collection of unrelated
proteins of many different sorts. Type II enzymes frequently differ so completely in amino acid
sequence from one another, and indeed from every other known protein, that they exemplify the
class of rapidly evolving proteins that are often indicative of involvement in host-parasite
interactions.

The most common Type II enzymes are those like HhaI (NEB #R0139), HindIII (NEB #R0104),
and NotI (NEB #R0189), that cleave DNA within their recognition sequences. Enzymes of this
kind are the principal ones available commercially. Most recognize DNA sequences that are
symmetric, because they bind to DNA as homodimers, but a few, (e.g., BbvCI (NEB #R0601):
CCTCAGC) recognize asymmetric DNA sequences, because they bind as heterodimers. Some
enzymes recognize continuous sequences (e.g., EcoRI (NEB #R0101): GAATTC) in which the
two half-sites of the recognition sequence are adjacent, while others recognize discontinuous
sequences (e.g., BglI (NEB#R0144): GCCNNNNNGGC) in which the half-sites are separated.
Cleavage leaves a 3´-hydroxyl on one side of each cut and a 5´-phosphate on the other. They
require only magnesium for activity and the corresponding modification enzymes require only S-
adenosylmethionine. They tend to be small, with subunits in the 200-350 amino acid range.

The next most common Type II enzymes, usually referred to as ‘Type IIS" are those like FokI
(NEB #R0109) and AlwI (NEB #R0513) that cleave outside of their recognition sequence to one
side. These enzymes are intermediate in size, 400-650 amino acids in length, and they recognize
sequences that are continuous and asymmetric. They comprise two distinct domains, one for
DNA binding, the other for DNA cleavage. They are thought to bind to DNA as monomers for
the most part, but to cleave DNA cooperatively, through dimerization of the cleavage domains of
adjacent enzyme molecules. For this reason, some Type IIS enzymes are much more active on
DNA molecules that contain multiple recognition sites.

Type IIG restriction enzymes, the third major kind of Type II enzyme, are large, combination
restriction-and-modification enzymes, 850-1250 amino acids in length, in which the two
enzymatic activities reside in the same protein chain. These enzymes cleave outside of their
recognition sequences and can be classified as those that recognize continuous sequences (e.g.,
AcuI (NEB #R0641): CTGAAG) and cleave on just one side; and those that recognize
discontinuous sequences (e.g., BcgI (NEB #R0545): CGANNNNNNTGC) and cleave on both
sides releasing a small fragment containing the recognition sequence. The amino acid sequences
of these enzymes are varied, but their organization is consistent. They comprise an N-terminal
DNA-cleavage domain joined to a DNA-modification domain and one or two DNA sequence-
specificity domains forming the C-terminus or present as a separate subunit. When these
enzymes bind to their substrates, they switch into either restriction mode to cleave the DNA, or
modification mode to methylate it.

Type III enzymes are also large combination restriction-and-modification enzymes. They
cleave outside of their recognition sequences and require two such sequences in opposite
orientations within the same DNA molecule to accomplish cleavage; they rarely give complete
digests.

Type IV enzymes recognize modified, typically methylated DNA and are exemplified by the
McrBC and Mrr systems of E. coli.
Cloning Vectors
Details Print

Topic: DB_WIKI

Tags: yac cloning vectors cloning vectors based on e.coli plasmids cloning vectors of m13 bacteriophage
cloning vectors of λ bacteriophage cosmid cloning vectors for higher plants plant viruses as cloning
vectors animal viruses as cloning vectors

Cloning vectors

A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment into
the cloning vector is carried out by treating the vehicle and the foreign DNA with a restriction enzyme that creates the same
overhang, then ligating the fragments together. There are many types of cloning vectors. Genetically engineered plasmids and
bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include
bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Cloning vectors based on E.coli plasmids

Simplest cloning vectors based on small bacterial plasmids are the most widespread in gene cloning experiment. A large number
of different plasmid vectors are available for use with E.coli and these vectors combine ease of purification with desirable
properties such as high transformation efficiency, convenient selectable markers for transformants and recombinants, and the
ability to clone reasonably large pieces of DNA. One of the most popular vector used in gene cloning is pBR322, and it's
nomenclature is shown below:

pBR322 plasmid

p- A plasmid
BR- The laboratory in which the vector was originally constructed(Bolivar and Rodriguez, the two researchers who developed
pBR322).
322- This number distinguishes the plasmid from other plasmids developed in the same laboratory.

Important properties of pBR322 plasmid

 pBR322 is 4363bp in size which helps its purification with ease and recombinant molecules can also be constructed
with it.
 pBR322 carries two sets of antibiotic resistance genes (Figure-1). Either ampicillin or tetracycline resistance can be
used as a selectable marker for cells containing the plasmid, and each marker gene includes unique restriction sites
that can be used in cloning experiments. The great variety of restriction sites(PstI,PvuI, ScaI,BamHI,HindIII) can be
used for insertional inactivation means that pBR322 can be used to clone DNA fragments with any of several kinds of
sticky ends.
 pBR322 has a reasonably high copy number. Normally there are about 15 molecules present in a transformed E.coli
cell, but the number can be increased up to 1000-3000, by plasmid amplification in the presence of a protein
synthesis inhibitor(chloramphenicol).
 Figure 1: Structure of pBR322 plasmid

Other E.coli plasmid cloning vectors

Many other plasmid cloning vectors have been constructed, the majority of which derived from pBR322 plasmid.

pBR327 plasmid

It is a higher copy number plasmid and was constructed by removing a 1089 bp segment from pBR322. This deletion left the
ampR tetR genes intact, but changed the replicative and conjugative abilities of the resulting plasmid.

Comparison between pBR322 and pBR327

 pBR327 has a higher copy number(30-45 molecules) than pBR322. The higher copy number of pBR327 in normal cells
makes this vector more suitable.
 The deletion also destroys the conjugative ability of pBR322, making pBR327 a non-conjugative plasmid that can't
direct its own transfer to other E.coli cells.

pUC8 Plasmid

pUC8 plasmid is derived from pBR322, remaining only the replication origin and the ampR gene. All the cloning sites are
clustered into a short segment of the lacZ' gene carried by pUC8 (Figure-2).
 Figure 2: The restriction site cluster in the lacz' gene of pUC8 plasmid

Advantages with pUC8 plasmid

 The manipulations involved in construction of pUC8 were accompanied by a chance mutation, within the origin of
replication, that results in the plasmid having a copy number 500-700 even before amplification. This has a significant
effect on the yield of cloned DNA obtainable from E.coli cells transformed with recombinant pUC8 plasmids.
 pUC vectors carry different combinations of restriction sites and show greater flexibility in the types of DNA fragment
that can be cloned. Clustering of the restriction sites allows a DNA fragment with two different sticky ends to be
cloned without involving linker attachment. DNA cloned into a member of the pUC series can be transferred directly
to its M13mp counterpart (Figure-3), because of the same restriction site clusters and it can be analysed by DNA
sequencing or in vitro mutagenesis.
 Identification of recombinant cells with pUC8 plasmid, can be achieved in one step by plating on to agar medium
containing ampicillin with X-gal. A cloning experiment with pUC8 can be carried out in half the time needed with
pBR322 and pBR327 because with both pBR322 and pBR327 plasmids, selection of recombinants is a two step
procedure, requiring replica-plating from one antibiotic medium to another.
 Figure 3: Transfer of a DNA fragment from pUC8 to M13mp8

PGEM3Z plasmid

pGEM3Z is having similarity with a pUC vector because of the presence of the ampR genes and lacZ' genes containing a cluster
of restriction sites (Figure-4). The difference is that pGEM3Z has two additional, short pieces of DNA, each of which acts as the
recognition site for attachment of an RNA polymerase enzyme. These two promoter sequences lie on either side of the cluster of
restriction site used for introduction of new DNA into the pGEM3Z molecule.
 Figure 4: Structure of pGEM3Z plasmid

Cloning vectors of M13 bacteriophage

Phage DNA molecules carry several genes that are essential for replication, including genes coding for components of the phage
protein coat and phage-specific DNA replicative enzymes. Alteration or deletion of any of these genes will impair or destroy the
replicative ability of the resulting molecule. So there is only limited scope for modifying the M13 genome and generally phage
cloning vectors are only slightly different from parent molecules.

M13mp1 and M13mp2

The construction of a M13 cloning vector involves introduction of the lacZ' genes into the intergenic sequence. This results in
production of M13mp1, which forms blue plaques on X-gal agar. M13mp1 (Figure-5) contains the hexanucleotide sequence
GGATTC, near the start of the lacZ' gene, a single nucleotide change in which would make this GAATTC, an EcoRI site.

Figure 5: Construction of M13mp1 from M13 cloning vector

The alteration is carried out using in vitro mutagenesis, resulting in M13mp2 (Figure-6), which has a slightly altered lacZ' gene.
M13mp2 is the simplest M13 cloning vector.

Figure 6: Construction of M13mp2 from M13mp1 cloning vector

M13mp7

Introduction of additional restriction sites into the lacZ' gene of M13 vectors is the basis of construction of a M13mp7 vector. A
short oligonucleotide, called a polylinker (Figure-7(a)) having a series of restriction sites and EcoRI sticky ends is synthesized in
the test tube and inserted into the EcoRI site of M13mp2, to give M13mp7 (Figure-7(b)), which is a more complex vector with
four possible cloning sites(EcoRI, BamHI, SalI and PstI).The short oligonucleotide sequence is known as a polylinker and it is so
designed that it doesn't totally disrupt the lacZ' gene; a reading frame is maintained throughout the polylinker, and a functional,
though altered, β-galactosidase enzyme is still produced.

Figure 7: Construction of M13mp7

Complex M13 vectors

The complex M13 vectors have more complex polylinkers inserted into the lacz' gene. M13mp8 is a complex vector and it has
the ability to take DNA fragments with two different sticky ends. M13mp9 is another vector having the same polylinker but in
the reverse orientation. A DNA fragment cloned into M13mp8, if excised by double restriction, and then inserted into M13mp9,
will now itself be in the reverse orientation, which is important in DNA sequencing, in which the nucleotide sequence is read
from one end of the polylinker into the inserted DNA fragment.

Cloning vectors of λ bacteriophage

The first two types of vectors to be produced were λ-insertion and λ-replacement vectors.
Insertion vectors

An insertion vector possesses at least one unique restriction site into which new DNA can be inserted. λgt10 and λZAPII are two
popular insertion vectors. λgt10 can carry up to 8kb of new DNA, inserted into a unique EcoRI site located in the cI gene.
Insertional activation of this gene distinguishes recombinants as clear rather than turbid plaques.

Replacement vectors
A λ replacement vector has two recognition sites for the restriction endonuclease used for cloning. These sites flank a segment
of DNA that is replaced by the DNA to be cloned. The replaceable fragment often carries additional restriction sites that can be
used to cut it up into small pieces. Replacement vectors are generally designed to carry larger pieces of DNA than insertion
vectors can handle. λWES.λB' and λEMBL4 are two popular replacement vectors.

Cosmid

A cosmid is a plasmid that carries a cos site the sequence yielding cohesive ends (Figure-8). Cosmids are hybrids between a
phage DNA molecule and a bacterial plasmid, and are designed in such a way that the enzymes that package the λ DNA molecule
into the phage protein coat need only the cos sites in order to fuction. A typical cosmid has replication origin, unique restriction
sites and selectable markers from the plasmid; therefore selection strategy for obtaining the recombinant vectors is based on that
for the contributing plasmid. Cosmid vectors are constructed using recombinant DNA techniques.

Figure 8: A cosmid vector

Cloning experiment with a cosmid

The cosmid vectors are opened by the appropriate restriction enzyme at a unique site, are then mixed with DNA inserts prepared
by using the same enzyme and annealed. These DNA fragments are produced usually by partial digestion with a restriction
endonuclease, as total digestion produce too small fragments to be cloned with a cosmid. Ligation is carried out so that
concatamers are formed (Figure-9). In vitro packaging will cleave the cos sites and place the recombinant cosmids in mature
phage particles. These λ phages are then used to infect E.coli culture, infected cells are plated on to a selective medium and
antibiotic-resistant colonies are grown.

Figure 9: A cosmid vector is used to clone large fragments of DNA

Yeast vectors

The yeast Saccharomyces cerevisiae is one of the most important organisms in biotechnology. Development of cloning vectors
for yeast has been stimulated greatly by the discovery of a plasmid that is present in most strains of S. cerevisiae. The 2µm circle,
as it is called is one of only a very limited number of plasmids found in eukaryotic cells.

Yeast plasmids

Vectors derived from the 2µm circle are called yeast episomal plasmids or YEps. YEps may contain the entire 2µm plasmid, or
include just the 2µm origin of replication. YEp13 illustrates several general features of yeast cloning vectors. It is a shuttle
vector. It contains the 2µm origin of replication and the selectable LEU2 gene, along with the entire pBR322 sequence (Figure-
10), and can therefore replicate and be selected for in both yeast and E. coli.

Figure 10: A yeast episomal plasmid

The standard cloning procedure in yeast (Figure-11) is to perform the initial cloning experiment with E.coli and to select
recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced
into yeast. YEps may integrate into a yeast chromosome by homologous recombination with the defective genomic copy of the
selection gene.
 Figure 11: Cloning with a yeast episomal plasmid

Yeast Integrative plasmids or YIps

Yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying
the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3, that codes an enzyme related to
the biosynthesis of pyrimidine nucleotides (T, C).

Yeast Replicative plasmids or YRps

Yeast replicative plasmids which transport a sequence of chromosomal DNA that includes an origin of replication. These
plasmids are less stable, as they can "get lost" during the budding.

Yeast Artificial Chromosome(YAC)

Artificial chromosomes can be used to clone huge pieces of DNA in yeast. First described in 1983 by Murray and Szostak, a
YAC is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences named
autonomous replicating sequence needed for replication and preservation in yeast cells (Figure-12). A YAC is built using an
initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is then used to
ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA. The
construction of the YAC clone is similar to that for cosmids, in that two end fragments are ligated with target DNA to yield the
complete chromosome, which is then introduced into yeast cells. YAC vectors can accommodate genomic DNA fragments of
more than 1 Mb, and hence can be used to clone entire human genes, such as the cystic fibrosis gene, which is 250 kb in length.
 The yeast sequences that have been included on pYAC3 are as follows: TEL represents a segment of the telomeric DNA
sequence, which is extended by the telomerase enzyme inside the yeast cell. CEN4 is the centromere sequence for chromosome 4
of S.cerevisiae. The ARS (autonomously replicating sequence) functions as a yeast origin of replication.TRP1 and URA3 are
yeast selectable markers, one for each end, to ensure that only properly reconstituted YACs survive in the yeast cells. SUP4,
which is insertionally inactivated in recombinants (Figure-12), is a gene which is the basis of a red-white color test, which is
analogous to blue-white screening in E.coli.

Figure 12: pYAC3

Cloning with pYAC3

The vector is first digested with a combination of BamHI and SnaBI, cutting the molecule into three fragments. The BamHI
fragment is removed, leaving two arms, each bounded by one TEL sequence and one SnaBI site. The DNA to be inserted is
ligated between the two arms producing the artificial chromosome (Figure-13). The DNA must have blunt ends as SnaBI is a
blunt end cutter. Then the artificial chromosome is introduced into S. cerevisiae by protoplast transformation. The yeast strain
that is used is a double auxotrophic mutant, trp1- and ura3- which will be converted to trp1+ and ura3+ by the two markers on the
artificial chromosome.
 Figure 13: Cloning with a YAC vector

Selection in S.cerevisiae

Saccharomyces cerevisiae selectable markers do not normally confer resistance to toxic substances, as in E.coli plasmids, but
instead enable the growth of yeast on selective media lacking specific nutrients. Transformants are selected by plating on to
minimal medium, on which only cells containing a correctly constructed artificial chromosome will able to grow. If any cells
transformed with an incorrect artificial chromosome, will not be able to grow on minimal medium as one of the markers will be
absent.

Cloning vectors for higher plants

There are important potential benefits of gene cloning using higher plants as the host organisms. Three types of vector system
have been used with varying degrees of success with higher plants:
  Vectors based on naturally occuring plasmids of Agrobacterium.
 Direct gene transfer using DNA fragments not attached to a plant cloning vector.
 Vectors based on plant viruses.

Agrobacterium tumefaciens-Ti plasmid

A. tumefaciens is a soil microorganism that causes crown gall disease in many species of dicotyledonous plants. Crown gall
occurs when a wound on the stem allows A. tumefaciens to invade the plant. After infection the bacteria cause a cancerous
proliferation of the stem tissue in the region of the crown. The ability to cause crown gall disease is associated with the presence
of the Ti(Tumour inducing) plasmid (Figure-14) within the bacterial cell. Ti plasmid is a large circular plasmid (greater than
200kb) that carries numerous genes involved in the infection process.

Figure 14: The Ti plasmid

The Ti plasmid is lost when Agrobacterium is grown above 28°C. Such cured bacteria do not induce crown galls, i.e. they
become avirulent. The infection part of the plasmid is integrated into the plant chromosomal DNA is called the T-DNA, is
between 15 and 30kb in size, depending on the strain. It is maintained in a stable form in the plant cell and is passed on to
daughter cells as an integral part of the chromosomes (Figure-15). The T-DNA contains eight or so genes that are expressed in
the plant cell and are responsible for the cancerous properties of the transformed cells. These genes also direct synthesis of
unusual compounds, called opines, that the bacteria use as nutrients. A.tumefaciens genetically engineers the plant cell for its own
purposes.
 Figure 15: Integration and expression of T-DNA in plant genome

Production of transformed plants with the Ti plasmid

Generally plant cells and protoplasts are infected with Ti plasmid. Plant cells and protoplasts can be plated on to a selective
medium in order to isolate transformants (Figure-16). A mature plant regenerated from transformed cells will contain the cloned
gene in every cell and will pass the cloned gene to its offspring. Regeneration of a transformed plant will occur only if the Ti
vector has been disarmed so that transformed cells do not display cancerous properties. Disarming is possible because the cancer
genes, all of which lie in the T-DNA are not needed for infection process, infectivity being controlled mainly by the virulence
region of the Ti plasmid. The only parts of the T-DNA that are involved in infection are two 25 bp repeat sequences found at the
left and right borders of the region integrated into the plant DNA. Any DNA placed between these two repeat sequences will be
treated as T-DNA and transferred to the plant. It is therefore possible to remove all the cancer genes from the normal T-DNA,
and replace them with an entirely new set of genes, without disturbing the infection process.
 Figure 16: Transformation of cultured plant cells by recombinant Agrobacterium tumefaciens

The binary vector pBIN19 is a disarmed cloning vector in which the left and right T-DNA borders flank a copy of the lacZ' gene,
containing a number of cloning sites, and a kanamycin resistance gene that functions after integration of the vector sequences into
the plant chromosome. Ti vectors such as pBIN19 have recently been supplemented by related vectors based on the Ri plasmid
of Agrobacterium rhizogenes. Ri plasmid and Ti plasmids are very similar, the main difference being that transfer of the T-DNA
from an Ri plasmid to a plant results not in a crown gall but in a hairy root disease, typified by a massive proliferation of a highly
branched root system. The possibility of growing transformed roots at a high density in a liquid culture is being explored by
biotechnologists as a potential means of obtaining large amounts of protein from genes cloned in plants.

Direct gene transfer

Genes may be transiently or permanently introduced into cultured eukaryotic cells without the use of a vector in the strict sense.
A eukaryotic gene on a bacterial plasmid, may transiently express its product when transfected into a cell line, even if the plasmid
doesn't replicate in that type of cell. Alternatively DNA introduced by transfection or microinjection may become stably
integrated into the cell's chromosomal DNA. This process normally require significant sequence similarity between the incoming
DNA and the genome in animal cells but, in plant cells any supercoiled plasmid can randomly integrate into the genome (Figure-
17). Such stably transfected cells can be selected by the presence of a drug resistance gene in much the same way as bacterial
transformants and can continue to express protein from foreign genes through many cell divisions.
 Figure 17: Direct gene transfer

Plant viruses as cloning vectors

Most plants are subjected to viral infection and vast majority of plant viruses have genomes of RNA. RNA viruses are not so
useful as potential cloning vectors because manipulations with RNA are rather more difficult to carry out. Two classes of DNA
virus are known to infect higher plants, the caulimoviruses and geminiviruses, but neither is identically suited for gene cloning.
Caulimovirus vector could only be used to clone very short pieces of DNA. Geminiviruses at first appear more promising as they
naturally infect important crops such as maize and wheat. But during the infection cycle the geminivirus genome undergoes
rearrangements and deletions, which could scramble up any additional DNA that had been inserted, an obvious disadvantage for
a cloning vector.

Animal viruses as cloning vectors

The first eukaryotic DNA virus was SV40, for which a complete nucleotide sequence and a detailed understanding of
transcription were available. The genome of SV40 contains very little non-essential DNA so it is necessary to insert the foreign
gene in place of essential viral genes and to propagate the recombinant genome in the presence of a helper virus. This virus is
capable of infecting several mammalian species, following a lytic cycle in some host and a lysogenic cycle in others. The genome
is 5.2kb in size and contains two sets of genes, the early genes, expressed early in the infection cycle and coding for proteins
involved in viral DNA replication, and the late genes, coding for viral capsid proteins (Figure-18). However, all work using SV40
virions to propagate recombinant DNA molecules is severely constrained by the facts that the viral genome is small, 5.24 kb, and
that the packaging limits are strict. Such systems can not, therefore, be used for the analysis of most eukaryotic genes.
 Figure 18: The SV40 genome

Adenoviruses, are a group of viruses which enable larger fragments of DNA to be cloned than it is possible with an SV40
vector. Adenoviruses are more difficult to handle because the genomes are bigger.

Papillomaviruses, which also have a relatively high capacity for inserted DNA, have the important advantage of enabling a
stable transformed cell line to be obtained. Papillomavirus transformed cells don't contain integrated viral DNA rather they
contain between 50 and 300 copies of unintegrated, circular viral DNA although some proportion of these viral genomes exists as
concatamers and/or catenates. Bovine papillomavirus(BPV), which causes warts on cattle, has an unusal infection cycle in mouse
cells, taking the form of a multi copy plasmid with about 100 molecules present per cell. It doesn't cause the death of the mouse
cell, and BPV molecules are passed to daughter cells on cell division. Shuttle vectors consisting of BPV and pBR322 sequences,
and capable of replication in both mouse and bacterial cells, are therefore of great value in animal cell biotechnology.

Retroviruses, though have single-stranded RNA genomes but provides perhaps the most promising vector system of all.
During the process of reverse transcription, sequences from the termini of viral RNA are duplicated to generate long terminal
repeats(LTRs). These long terminal repeats contain both the promoter and the polyadenylation signal for the transcription of viral
mRNAs. The specificity of proviral DNA integration is also determined by the long terminal repeats. Although retroviruses can
integrate at many sites within the cellular genome, integrative recombination always occurs at particular sites at the ends of the
LTRs. The sequences appropriately inserted between the two LTRs will be integrated intact which contrasts sharply with the
integration of papovavirus or adenovirus DNA, during which extensive rearrangements of the integrated viral sequences are
commonplace. A further great advantage of retroviruses is that they are natural transducing viruses.

Baculoviruses, enable large amounts of proteins to be obtained from genes cloned in insect cells. One of the major proteins
encoded by the virus genome is polyhedrin, which accumulates in very large quantities in the nuclei of infected cells, since the
gene has an extremely active promoter. The same promoter can be used to drive the over expression of a foreign gene engineered
into the baculovirus genome, and large quantities of protein can be produced in infected insect cells in culture. This method is
being used increasingly for large-scale culture of proteins of animal origin, since the insect cells can produce many of the post-
translational modifications of animal proteins which a bacterial expression system.

References

1. Brown, T.A. (1998). Cloning vectors for E.coli, Cloning vectors for organisms other than E.coli. Gene cloning an introduction,
3rd Edn. Stanley Thornes (Publishers) Ltd.
2. Lewin, B. 1994. genes V. Oxford University Press, New York

3. Singh, B.D.(2003). Biotechnology. Kalyani Publishers, New Delhi

4. Turner Phil, McLennan Alexander, Bates Andy and White Mike (2005). Cosmids,YACs and BACs, Eukaryotic vectors.
Instant notes(138-143), Molecular Biology. Taylor& Francis Group
Plasmid
From Wikipedia, the free encyclopedia

(Redirected from Plasmid vector)

Illustration of a bacterium showing chromosomal DNA and plasmids. Not to scale.

A plasmid is a small DNA molecule within a cell that is physically separated from a
chromosomal DNA and can replicate independently. The term was coined by Lederberg and
Hays and shortly discovered by Tatum. They are most commonly found in bacteria as small
circular, double-stranded DNA molecules; however, plasmids are sometimes present in archaea
and eukaryotic organisms. In nature, plasmids often carry genes that may benefit the survival of
the organism, for example antibiotic resistance. While the chromosomes are big and contain all
the essential genetic information for living under normal conditions, plasmids usually are very
small and contain only additional genes that may be useful to the organism under certain
situations or particular conditions. Artificial plasmids are widely used as vectors in molecular
cloning, serving to drive the replication of recombinant DNA sequences within host organisms.

Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a
suitable host. However, plasmids, like viruses, are not generally classified as life.[1] Plasmids can
be transmitted from one bacterium to another (even of another species) via three main
mechanisms: transformation, transduction, and conjugation. This host-to-host transfer of genetic
material is called horizontal gene transfer, and plasmids can be considered part of the mobilome.
Unlike viruses (which encase their genetic material in a protective protein coat called a capsid),
plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for
transfer to a new host. However, some classes of plasmids encode the conjugative "sex" pilus
necessary for their own transfer. The size of the plasmid varies from 1 to over 200 kbp,[2] and the
number of identical plasmids in a single cell can range anywhere from one to thousands under
some circumstances.

The relationship between microbes and plasmid DNA is neither parasitic nor mutualistic,
because each implies the presence of an independent species living in a detrimental or
commensal state with the host organism. Rather, plasmids provide a mechanism for horizontal
gene transfer within a population of microbes and typically provide a selective advantage under a
given environmental state. Plasmids may carry genes that provide resistance to naturally
occurring antibiotics in a competitive environmental niche, or the proteins produced may act as
toxins under similar circumstances, or allow the organism to utilize particular organic
compounds that would be advantageous when nutrients are scarce.[3]

Contents

 1 Properties and characteristics

 2 Classifications and types
 3 Vectors
o 3.1 Cloning
o 3.2 Protein production
o 3.3 Gene therapy
o 3.4 Disease models
 4 Episomes
 5 Plasmid maintenance
 6 Yeast plasmids
 7 Plasmid DNA extraction
 8 Conformations
 9 Software for bioinformatics and design
 10 Plasmid collections
 11 See also
 12 References
 13 Further reading
o 13.1 Episomes
 14 External links

Properties and characteristics

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as
with the top instance, whereas episomes, the lower example, can integrate into the host chromosome.

The American molecular biologist Joshua Lederberg first introduced the term plasmid in 1952 -
originally to describe any bacterial genetic material that exists in an extrachromosomal state for
at least part of its replication cycle.[4] Later in 1968, it was decided that the term plasmid should
be adopted as the term for extrachromosomal genetic element,[5] and to distinguish it from
viruses, the definition was narrowed to genetic elements that exist exclusively or predominantly
outside of the chromosome and can replicate autonomously.[6]

In order for plasmids to replicate independently within a cell, they must possess a stretch of
DNA that can act as an origin of replication. The self-replicating unit, in this case the plasmid, is
called a replicon. A typical bacterial replicon may consist of a number of elements, such as the
gene for plasmid-specific replication initiation protein (Rep), repeating units called iterons,
DnaA boxes, and an adjacent AT-rich region.[7] Smaller plasmids make use of the host replicative
enzymes to make copies of themselves, while larger plasmids may carry genes specific for the
replication of those plasmids. A few types of plasmids can also insert into the host chromosome,
and these integrative plasmids are sometimes referred to as episomes in prokaryotes.[8]

Plasmids almost always carry at least one gene. Many of the genes carried by a plasmid are
beneficial for the host cells, for example: enabling the host cell to survive in an environment that
would otherwise be lethal or restrictive for growth. Some of these genes encode traits for
antibiotic resistance or resistance to heavy metal, while others may produce virulence factors that
enable a bacterium to colonize a host and overcome its defences, or have specific metabolic
functions that allow the bacterium to utilize a particular nutrient, including the ability to degrade
recalcitrant or toxic organic compounds.[6] Plasmids can also provide bacteria with the ability to
fix nitrogen. Some plasmids, however, have no observable effect on the phenotype of the host
cell or its benefit to the host cells cannot be determined, and these plasmids are called cryptic
plasmids.[9]

Naturally occurring plasmids vary greatly in their physical properties. Their size can range from
very small mini-plasmids of less than a 1 kilobase pairs (Kbp), to very large megaplasmids of
several megabase pairs (Mbp). At the upper end, little can differentiate between a megaplasmid
and a minichromosome. Plasmids are generally circular, however examples of linear plasmids
are also known. These linear plasmids require specialized mechanisms to replicate their ends. [6]

Plasmids may be present in an individual cell in varying number, ranging from one to several
hundreds. The normal number of copies of plasmid that may be found in a single cell is called
the copy number, and is determined by how the replication initiation is regulated and the size of
the molecule. Larger plasmids tend to have lower copy numbers.[8] Low-copy-number plasmids
that exist only as one or a few copies in each bacterium are, upon cell division, in danger of
being lost in one of the segregating bacteria. Such single-copy plasmids have systems that
attempt to actively distribute a copy to both daughter cells. These systems, which include the
parABS system and parMRC system, are often referred to as the partition system or partition
function of a plasmid.
Classifications and types

Overview of bacterial conjugation

Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop.

Electron micrograph of a bacterial DNA plasmid (chromosome fragment).

Plasmids may be classified in a number of ways. Plasmids can be broadly classified into
conjugative plasmids and non-conjugative plasmids. Conjugative plasmids contain a set of
transfer or tra genes which promote sexual conjugation between different cells.[8] In the complex
process of conjugation, plasmid may be transferred from one bacterium to another via sex pili
encoded by some of the tra genes (see figure).[10] Non-conjugative plasmids are incapable of
initiating conjugation, hence they can be transferred only with the assistance of conjugative
plasmids. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes
required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency
only in its presence.
Plasmids can also be classified into incompatibility groups. A microbe can harbour different
types of plasmids, however, different plasmids can only exist in a single bacterial cell if they are
compatible. If two plasmids are not compatible, one or the other will be rapidly lost from the
cell. Different plasmids may therefore be assigned to different incompatibility groups depending
on whether they can coexist together. Incompatible plasmids (belonging to the same
incompatibility group) normally share the same replication or partition mechanisms and can thus
not be kept together in a single cell.[11][12]

Another way to classify plasmids is by function. There are five main classes:

 Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the
expression of sex pili.
 Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or
poisons. Historically known as R-factors, before the nature of plasmids was understood.
 Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other
bacteria.
 Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and
salicylic acid.
 Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups.

Vectors
Further information: Vector (molecular biology)

Artificially constructed plasmids may be used as vectors in genetic engineering. These plasmids
serve as important tools in genetics and biotechnology labs, where they are commonly used to
clone and amplify (make many copies of) or express particular genes.[13] A wide variety of
plasmids are commercially available for such uses. The gene to be replicated is normally inserted
into a plasmid that typically contains a number of features for their use. These include a gene that
confers resistance to particular antibiotics (ampicillin is most frequently used for bacterial
strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a
suitable site for cloning.
A schematic representation of the pBR322 plasmid, one of the first plasmids to be used widely as a
cloning vector. Shown on the plasmid diagram are the genes encoded (amp and tet for ampicillin and
tetracycline resistance respectively), its origin of replication (ori), and various restriction sites (indicated
in blue).

Cloning
Main article: Cloning vector

Plasmids are the most-commonly used bacterial cloning vectors.[14] These cloning vectors contain
a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker
which has several commonly used restriction sites to which DNA fragments may be ligated.
After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called
transformation. These plasmids contain a selectable marker, usually an antibiotic resistance gene,
which confer on the bacteria an ability to survive and proliferate in a selective growth medium
containing the particular antibiotics. The cells after transformation are exposed to the selective
media, and only cells containing the plasmid may survive. In this way, the antibiotics act as a
filter to select only the bacteria containing the plasmid DNA. The vector may also contain other
marker genes or reporter genes to facilitate selection of plasmid with cloned insert. Bacteria
containing the plasmid can then be grown in large amounts, harvested, and the plasmid of
interest may then be isolated using various methods of plasmid preparation.

A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbp.[15] To clone
longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial
chromosomes, or yeast artificial chromosomes are used.

Replicon (genetics)
A replicon is a DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates
from a single origin of replication.

Prokaryotes

For most prokaryotic chromosomes, the replicon is the entire chromosome. One notable
exception found comes from archaea, where two Sulfolobus species have been shown to contain
three replicons. Examples of bacterial species that have been found to possess multiple replicons
include: Rhodobacter sphaeroides (2), Vibrio cholerae,[1] and Burkholderia multivorans (3).
These "secondary" (or tertiary) chromosomes are often described as a molecule that is a mixture
between a true chromosome and a plasmid and are sometimes called "chromids". Various
Azospirillum species possess 7 replicons, Azospirillum lipoferum, for instance, has 1 bacterial
chromosome, 5 chromids, and 1 plasmid.[2] Plasmids and bacteriophages are usually replicated as
single replicons, but large plasmids in Gram-negative bacteria have been shown to carry several
replicons.[3]
Eukaryotes
For eukaryotic chromosomes, there are multiple replicons per chromosome. In the case of mitochondria
the definition of replicons is somewhat confused, as they use unidirectional replication with two
separate origins.