VECTORS

VECTORS::
• Plasmids, bacteriophages and other viruses, and cosmids are used as vectors.
• They can replicate within a host cell while carrying foreign DNA and possess phenotypic traits that allow them to be detected.
• There are four major types of vectors: plasmids, bacteriophages and other viruses, cosmids, and artificial chromosomes.

1. TYPE-------Plasmid (E. coli)
VECTOR----pBR322
Restriction Sequences Pr esent-- BamHI, EcoRI, HaeIII, HindIII, PstISalI, XorII
Features- Carries genes for tetracycline and ampicillin resistance

2. TYPE---- Plasmid (yeast–E. coli hybrid)
VECTOR------- pYe(CEN3)41
Restriction Sequences Present--- BamHI, BglII, EcoRI, HindIII, PstI, SalI
FEATURE--- Multiplies in E. coli or yeast cells
3. TYPE------Cosmid (artificially constructed E. coli plasmid carrying lambda cos site)
VECTOR- pJC720
Restriction Sequences Present--- HindIII
FEATURE----- Can be packaged in lambda phage particles for efficient introduction into bacteria; replicates as a plasmid; useful for cloning large DNA inserts.

4. TYPE----YAC (yeast artificial Chromosome)
VECTOR---- pYAC SmaI, BamHI
Restriction Sequences Present ----SmaI, BamHI
FEATURE -----Carries gene for ampicillin resistance; multiplies in Saccharomyces cerevisiae.
5. TYPE----BAC (bacterial artificial chromosome)
VECTOR----pBAC108L
Restriction Sequences Present ---HindIII, BamHI, NotI, SmaI, and others
FEATURE---Modified F plasmid that can carry 100–300 kb chromosome) fragments; has a cosN site and a chloramphenicol resistance marker.
6. TYPE--- Virus
VECTOR---Charon phage
RESTRICTION SEQ. PRESENT---- EcoRI, HindIII, BamHI, SstI
FEATURE---Constructed using restriction enzymes and a ligase, having foreign DNA as central portion, with lambda DNA at each end; carries β-galactosidase gene; packaged into lambda phage particles; useful for cloning large DNA inserts.
7. TYPE—Virus
VECTOR--- Lambda 1059
RESTICTION SEQ. PRESENT----BamHI
FEATURE----Will carry large DNA fragments (8–21 kb); recombinant can grow on E. coli lysogenic for P2 phage, whereas vector cannot
8. TYPE--- Virus
VECTOR----M13
RESTRICTION SEQ. PRESENT---EcoRI
FEATURE----Single-stranded DNA virus; useful in studies employing single-stranded DNA insert and in producing DNA fragments for sequencing

9. TYPE---- Plasmid
VECTOR----Ti
RESTRICTION SEQ. PRESENT---- SmaI, HpaI
FEATURE---- Maize plasmid

• Plasmids are the easiest to work with; rDNA phages and other viruses are more conveniently stored for long periods; larger pieces of DNA can be cloned with cosmids and artificial chromosomes.

• Besides these major types, there also are vectors designed for a specific function.

• For example, shuttle vectors are used in transferring genes between very different organisms and usually contain one replication origin for each host.

• The pYe type plasmids reproduce in both yeast and E. coli and can be used to clone yeast genes in E. coli.

• All vectors share several common characteristics. They are typically small, well-characterized molecules of DNA. They contain at least one replication origin and can be replicated within the appropriate host, even when they contain “foreign” DNA.

Plasmids

• They are easy to isolate and purify, and they can be reintroduced into a bacterium by transformation.
• Plasmids often bear antibiotic resistance genes, which are used to select their bacterial hosts.
• A recombinant plasmid containing foreign DNA often is called a chimera, after the Greek mythological monster that had the head of a lion, the tail of a dragon, and the body of a goat. One of the most widely used plasmids is pBR322.
• Plasmid pBR322 has both resistance genes for ampicillin and tetracycline and many restriction sites.
• Several of these restriction sites occur only once on the plasmid and are located within an antibiotic resistance gene. This arrangement aids detection of recombinant plasmids after transformation .
• For example, if foreign DNA is inserted into the ampicillin resistance gene, the plasmid will no longer confer resistance to ampicillin. Thus tetracycline-resistant transformants that lack ampicillin resistance contain a chimeric plasmid.



Phage Vectors(Bacteriophage)
• Both single- and double-stranded phage vectors have been employed in recombinant DNA technology. For example, lambda phage derivatives are very useful for cloning and can carry fragments up to about 45 kilobases in length.
• The genes for lysogeny and integration often are nonfunctional and may be deleted to make room for the foreign DNA.
• The modified phage genome also contains restriction sequences in areas that will not disrupt replication.
• After insertion of the foreign DNA into the modified lambda vector chromosome, the recombinant phage genome is packaged into viral capsids and can be used to infect host E. coli cells .
• These vectors are often used to generate genomic libraries. E. coli also can be directly transformed with recombinant lambda DNA and produce phages.
• However, this approach is less efficient than the use of complete phage particles.
• The process is sometimes called transfection.
• Phages other than lambda also are used as vectors. For example, fragments as large as 95 kilobases can be carried by the P1 bacteriophage.


Cosmids
• Cosmids are plasmids that contain lambda phage cos sites and can be packaged into phage capsids.
• The lambda genome contains a recognition sequence called a cos site (or cohesive end) at each end.
• When the genome is to be packaged in a capsid, it is cleaved at one cos site and the linear DNA is inserted into the capsid until the second cos site has entered. Thus any DNA inserted between the cos sites is packaged.
• Cosmids typically contain several restriction sites and antibiotic resistance genes.
• They are packaged in lambda capsids for efficient injection into bacteria, but they also can exist as plasmids within a bacterial host. As much as 50 kilobases of DNA can be carried in this way.

Artificial Chromosomes
• Artificial chromosomes are popular because they carry large amounts of genetic material.
• The yeast artificial chromosome (YAC) is one of the most widely used.
• YACs are stretches of DNA that contain all the elements required to propagate a chromosome in yeast: a replication origin, the centromere required to segregate chromatids into daughter cells, and two telomeres to mark the ends of the chromosome.
• They will also have restriction enzyme sites and genetic markers so that they can be traced and selected.

• Cleavage of a YAC with the proper restriction enzyme such as SmaI will open it up and allow the insertion of a piece of foreign DNA between the centromere and a telomere.
• In this way YACs containing DNA fragments between 100 and 2,000 kilobases in size can be placed in Saccharomyces cerevisiae cells and will be replicated along with the true chromosomes.
• The bacterial artificial chromosome (BAC) is an increasingly popular alternative cloning vector.
• BACs are cloning vectors based on the E. coli F-factor plasmid.
• They contain appropriate restriction enzyme sites and a marker such as chloramphenicol resistance.
• The modified plasmid is cleaved at a restriction site, and a foreign DNA fragment up to 300 kilobases in length is attached using DNA ligase.

• The BAC is reproduced in E. coli after insertion by electroporation.
• This vector is easy to reproduce and manipulate and does not undergo recombination as readily as YACs (which means that the insert is less likely to be rearranged). Because they can carry such large fragments of DNA, artificial chromosomes have been particularly useful in genome sequencing.


Ti Plasmid::
 Cloned genes can be inserted into plant as well as animal cells.
 Presently a popular way to insert genes into plants is with a recombinant Ti plasmid (tumor-inducing plasmid) obtained from the bacterium Agrobacterium tumefaciens .
 It also is possible to donate genes by forming plant cell protoplasts, making them permeable to DNA, and then adding the desired rDNA. The advent of the gene gun will greatly aid the production of transgenic plants.