Plant Genome Organization and Structure


Bacteriophage Lambda Vectors


Yeast Artificial Chromosomes (YACs)

Bacterial Artificial Chromosomes (BACs)

Library Screening and Gene Sequencing

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Bacteriophage Lambda Vectors

We have talked about plasmids as vectors for cloning small pieces of DNA. The limitation of this vector is the size of DNA that can be introduced into the cell by transformation. This presents problems when you are trying to create a genomic library of a large genome such as with plants. A genomic library contains all of the DNA found in the cell of the plant (or any organism). If you digest plant DNA to completion with a restriction enzyme, ligate those fragments into a plasmid vector and transform bacterial cells, only a portion of those fragments will be represented in the final transformation products. If a gene of interest is located on a large fragment then you will not be able to isolate that gene from a plasmid library. But what can be done to increase the probability of obtaining a clone which contains the entire gene. First you need to use a vector that can accept large fragments of DNA. Examples of these are bacteriophage and cosmid vectors and more recently yeast artificial chromosomes.

Bacteriophage lambda vectors were developed because several observations were made that suggested that they could complete their life cycles even if foreign DNA was inserted into a portion of its genome. This suggested that certain regions of the virus were not essential. Let's first discuss the life cycle of lambda.

  1. Adsorption - the phage particle binds at a maltose receptor site of the bacterial cell; growing the cell in the presence of the sugar increase the number of receptor sites
  2. Penetration - DNA is injected into the cell; at this point it can enter one of two pathways;
    • Lysogenic pathway - the phage DNA becomes integrated into the genome and is replicated along with the bacterial DNA; it remains integrated until it enters the lytic pathway
    • Lytic pathway - large scale production of bacteriophage particles that eventually leads to the lysis of the cell; base pairing at the cos site leads toa circular molecule
  3. Early transcription - transcription proceeds from the pL and pR promoters, through the N and cro genes and stops at terminators tL and tR1; a low level of transcription through the O and P genes occurs and terminates at tR2; the N product is an antitermination factor that is important for the next stage of transcription
  4. Delayed early transcription - the N product binds to RNA polymerase and transcription proceeds past the tL, tR1 and tR2 terminators; genes to the left of N, involved in recombination, to the right of cro, involved in replication, are expressed at this point; another protein expressed from the Q gene is used for antitermination of later transcription
  5. Replication - early replication is through a theta form initiated from a single origin of replication site; later replication is via rolling circle replication; this produces long concatamers of the phage DNA that are cleaved at the cosL and cosR sites
  6. Late transcription - the protein product of the cro gene builds up to a critical level and then binds to the oL and oR to stop early transcription; another protein, a product of the Q gene, has built up and activates transcription at the p'R promoter by antitermination; transcription terminates with in the b region; this transcription results in the production of the proteins required for the head and tail of the mature phage particle and those required for bacterial cell lysis
  7. Assembly - a prophage head is produced; a unit length DNA is placed into the head by the action of the Nu1 and A proteins; the DNA is locked into place by the D protein and ter function of the A protein clips the DNA at the cosL and cosR sites; the concatamer is released, the tail is added and the mature phage particle is completed
Packaging of the DNA into the head does not require a complete length of wild type lambda. It has been determined that a lambda molecule that is between 78% and 105% of wild type length can be packaged. This is from 37 to 53 kb in length.

Two important developments suggested that lambda may be suitable as a cloning vector. First it was determined that the gene products between the J and N genes could be removed and the life cycle could be completed. Second, restriction enzyme sites could be eliminated which permitted the development of a vector with a single site for insertion of foreign DNA. Two types of vectors have been developed:

Insertional vector - DNA is inserted into a specific site
Replacement vector - foreign DNA replaces a piece of DNA (stuffer fragment) of the vector

Let's talk about a specific vector EMBL 3 and EMBL 4. One important concern when cloning with lambda vectors is that you want to maximize the number of resulting phage particles that contain foreign DNA. Or said another way you want to minimize the number of wild type particles. One approach is through spi selection. This refers to sensitivity to P2 interference.

Bacteriophage Phenotypes for Growth on P2 Lysogens

Phenotype Growth on P2 lysogens? (bacterial strain)
spi+ (red+gam+)
spi- (red-gam-)

EMBL 3/4 vectors have placed the red and gam genes in the stuffer fragment. Thus only those particles from which the stuffer has been replaced can grow well in a P2 lysogen bacterial cell.

Cloning in Lambda Vectors

  1. Make a partial digest of DNA.
  2. Ligate the DNA to the arms of the vector.
  3. Package the DNA into phage particles using premade mixes.
  4. Screen and then amplify.
  5. Store the library for future use as a plate lysate.
Copyright © 1998. Phillip McClean