Artificial chromosomes

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 Artificial Chromosomes,

 Artificial chromosomes are laboratory constructs that contain DNA sequences and that perform the critical functions of natural chromosomes. They are used to introduce and control new DNA in a cell, to study how chromosomes function, and to map genes in genomes.

 Bacterial artificial chromosomes (BACs)

  •     Bacterial artificial chromosomes (BACs) are simple plasmid which is designed to clone very large DNA fragments ranging in size from 75 to 300 kb.
  •     BACs basically have marker like sights such as antibiotic resistance genes and a very stable origin of replication (ori) that promotes the distribution of plasmid after bacterial cell division and maintaining the plasmid copy number to one or two per cell.
  •     BACs are basically used in sequencing the genome of organisms in genome projects (example: BACs were used in human genome project).
  •     Several hundred thousand base pair DNA fragments can be cloned using BACs.

Common gene components in BAC 
  •  RepERepE: for plasmid replication and regulation of copy number. 
  •  parA and parBparA and parB: for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. 
  •  Selectable markerSelectable marker: for antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection. 
  •  T7 & Sp6T7 & Sp6: phage promoters for transcription of inserted genes.
Applications of BAC 
  •  Contribution to models of disease: Inherited diseaseContribution to models of disease: Inherited disease 
  •  BACs are now being utilized in modeling genetic diseases, often alongside transgenic mice. 
  •  BACs have been useful in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a gene's expression level. 
  •  BACs have been used to study neurological diseases such as Alzheimer's disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers.
Yeast artificial chromosomes (YACs)

  •     YACs are yeast expression vectors.
  •     A very large DNA fragments whose sizes ranging from 100 kb to 3000 kb can be cloned using YACs.
  •     Mostly YACs are used for cloning very large DNA fragments and for the physical mapping of complex genomes.
  •     YACs have an advantage over BACs in expressing eukaryotic proteins that require post translational modifications.
  •     But, YACs are known to produce chimeric effects which make them less stable compared to BACs.



  • Applications of YAC 

    •  Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require post translational modification.
    •  By being able to insert large fragments of DNA, YACs can be utilized to clone and assemble the entire genomes of an organism. With the insertion of a YAC into yeast cells, they can be propagated as linear artificial chromosomes, cloning the inserted regions of DNA in the process. With this completed, two process can be used to obtain a sequenced genome, or region of interest:
    • ➢ Physical Mapping
    • ➢ Chromosome Walking
    Limitations of using YAC vectors 
    •  Very large DNA molecules are very fragile and prone to breakage, leading to problem of rearrangement. 
    •  High rate of loss of the entire YAC during mitotic growth. 
    •  Difficult to separate YAC from the other host chromosomes because of their similar size. 
    •  Separation requires sophisticated pulse-field gel electrophoresis (PFGE). 
    •  Yield of DNA is not high when the YAC is isolated from yeast cells. 
    •  Clones tend to be unstable, with their foreign DNA inserts often being deleted.
    Human artificial chromosomes (HACs)

    •     Human artificial chromosomes (HACs) or mammalian artificial chromosomes (MACs) are still under development.
    •     HACs are microchromosomes that can act as a new chromosome in a population of human cells.
    •     HACs range in size from 6 to 10 Mb that carry new genes introduced by human researchers.
    •     HACs can be used as vectors in transfer of new genes, studying their expression and mammalian chromosomal function can also be elucidated using these microchrosomes in mammalian system.
    Mammalian Artificial Chromosomes

    • Mammalian artificial chromosomes (MACs) are conceptually similar to YACs, but instead of yeast sequences they contain mammalian or human ones. In this case the telomeric sequences are multimers (multiple copies) of the sequence TTAGGG, and the commonly used centromeric sequence is composed of another repeated DNA sequence found at the natural centromeres of human chromosomes and called alphoid DNA.

    • Because the alphoid DNA is needed in units of many kilobases, these MAC DNAs are grown as YACs or, more recently, as BACs. When added to suitable cell lines, these MAC DNAs form chromosomes that mimic those in the cell, with accurate segregation and the normal complement of proteins at telomeres and centromeres. Their primary use is not in genome mapping but as vectors for delivery of large fragments of DNA to mammalian cells and to whole animals for expression of large genes or sets of genes. They are still in development, and although gene expression has been demonstrated they have not been used in a practical application.


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