3 Key excerpts on "Bacterial Artificial Chromosome (BAC)"

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  eBook - ePub

  Introduction to Plant Biotechnology (3/e)

  • H S Chawla(Author)
  • 2011(Publication Date)
  • CRC Press

    (Publisher)

  R ). The advantage of this kind of vector is that only few clones are required to cover a particular genome because YAC can accommodate large inserts of DNA.

  BACTERIAL ARTIFICIAL CHROMOSOME (BAC)

  BAC is another cloning vector system in E. coli developed by Mel Simon and his colleagues as an alternative to YAC vectors. BAC vectors are maintained in E. coli as large single copy plasmids that contain inserts of 50–300kb. BAC vectors contain F-plasmid origin of replication (oriS), F plasmid genes that control plasmid replication (repE) and plasmid copy number (parA, B, C), and the bacterial chloramphenicol acetyltransferase (CMR ) gene for plasmid selection. The methods used to construct a BAC library are essentially the same as those for a standard plasmid library, except that the insert DNA must be prepared by preparative pulse field gel electrophoresis. pBeloBAC 11 is an example of a BAC vector developed in the Simon lab (Fig. 16.9 ). High molecular weight DNA is partially digested with the enzyme HindIII, size fractionated by PFGE, and ligated to the HindIII digested and phophatase-treated pBeloBAC 11 vector. E. coli cells are electroporated with the ligated material and white colonies (DNA insertion into lacZ α subunit gene) are isolated on the basis of chloramphenicol resistance. The insert DNA can be excised with the rare cutting enzyme NotI (8 bp recognition site). The plasmid can also be linearized at the cosN site by lambda terminase to facilitate end labeling for restriction enzyme mapping. The bacteriophage T7 and SP6 transcriptional promoter sequences flanking the HindIII cloning site provide a means to generate end specific RNA probes for library screening. DNA segments from the bithorax gene of Drosophila have already been cloned using this vector.

  Fig. 16.8:

  A schematic diagram of pYAC4 vector and cloning of genomic DNA. Concept of the figure taken from Miesfeld: Applied Molecular Genetics.

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  eBook - ePub

  From Genes to Genomes

  Concepts and Applications of DNA Technology

  • Jeremy W. Dale, Malcolm von Schantz, Nicholas Plant(Authors)
  • 2011(Publication Date)
  • Wiley

    (Publisher)

  tel sequences at each end, so that the yeast transformant can use these sequences to build functional telomeres. The titans amongst vectors, YACs are routinely used to clone 600 kb fragments, and specialized versions are available that can accommodate inserts close to 2 Mb, which is approximately one thousand times more DNA insert than in a plasmid. As such, they will not only easily accommodate any eukaryotic gene in its entirety, but also the gene will be within its framework of three-dimensional structure and distant regulatory sequences. They have therefore been very useful in the production of transgenic organisms (see Chapter 11).

  Figure 2.31 Structure and use of a yeast artificial chromosome vector.

  However, YACs have problems with the stability of the insert, especially when working with very large fragments as these can be subject to rearrangement by recombination. Furthermore, apart from the fact that many laboratories are not set up for the use of yeast vectors, the recombinant molecules are not easy to recover and purify. Thus, larger bacterial vectors are used more than YACs even though their capacity is lower. These include vectors based on bacteriophage P1, which are able to accommodate inserts in excess of 100 kb, and bacterial artificial chromosomes (BACs), which are based on the F plasmid and can accommodate 300 kb of insert. These vectors are more stable than YACs, and have played an important role in genome sequencing projects (see Chapter 8).

  2.13 Summary

  In this chapter we have described the basic technology needed for cloning pieces of DNA. Further topics such as the use of expression vectors for optimising product formation, and vectors for eukaryotic cells, are described in Chapter 7. One essential aspect of the basic techology has not been dealt with, namely how do you use these methods to get hold of a clone carrying a specific gene? In the next chapter, we will look at the construction and screening of gene libraries, which formed the central part of the earlier achievements in this field. Nowadays, with the amount of information readily available from genome sequencing projects, for many purposes it is often possible to bypass the use of gene libraries and use the polymerase chain reaction to amplify the required gene. We will deal with this in Chapter 4.

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  eBook - ePub

  Plant Genome Analysis

  Current Topics in Plant Molecular Biology

  • Peter M. Gresshoff(Author)
  • 2020(Publication Date)
  • CRC Press

    (Publisher)

  Chapter 11

  Plant Yeast Artificial Chromosome Libraries and Their Use: Status and Some Strategic Considerations

  Roel P. Funke and Alexander Kolchinsky

  Plant Molecular Genetics and Center of Legume Research, The University of Tennessee, Knoxville, TN 37901-1071, USA.

  Introduction

  Many genes that are now being pursued by plant scientists cannot be cloned by conventional procedures. These include disease resistance loci (e.g., Martin et al, 1992, 1993) and genes that are involved in hormonal responses and developmental pathways (Chang et al, 1993). The time and location of expression of these genes is usually not known, and often they are identified by mutational analysis (e.g., nitrate tolerant symbiosis (nts) in soybean, Carroll et al, 1985, and the auxin resistance gene AXR1 in Arabidopsis, Leyser et al, 1993). The genes may be characterized and placed on a genetic or RFLP map (e.g., Landau-Ellis et al, 1991).

  It is hoped (and in at least three cases it has been demonstrated; Leyser et al, 1993; Martin et al, 1993; Chang et al, 1993) that many of these genes will be amenable to positional or map-based cloning (Wicking and Williamson, 1991). Once they have been localized on a chromosome or linkage group by co-segregation with genetic or RFLP markers, it is envisaged that the genes may be approached by chromosome walking (Gibson and Somerville, 1992).

  Yeast artificial chromosomes (YACs; Burke et al, 1987) have promised to be the vehicle of choice for such expeditions. This is because the nearest flanking marker may be several hundred kilobases (kb) from the locus of interest, and stretches of DNA as large as this may be stably propagated in yeast. It was hoped that YACs could bridge regions of repetitive DNA that would foil a chromosome walk in E. coli

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