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Home >>Molecular Maps of Animal Genome >>Physical Maps Using Yeast Artificial Chromosome - YAC and In Situ Hybridization - ISH
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Physical Maps Using Yeast Artificial Chromosome - YAC and In Situ Hybridization - ISH - The isolation and characterization of the following three elements essential to replication made it possible to form completely new artificial chromosomes

(i) origin of DNA replication,

(ii) centromeres and

(iii) telomeres. Single copies of each of these elements can be linked to specific DNA segments to form artificial chromosomes, which can be inserted in yeast cells and tested to either functions. Using these artificial chromosomes, molecular map in Drosophila have recently been prepared.

Actually, for large scale physical mapping of a eukaryotic genome, Drosophila is considered a model organism (though not as attractive as yeast or Aspergillus) for the following reasons.

(i) small genome with 165,000 kilobase pairs (kbp)

(ii) the availability of large number of mutations and chromosome rearrangements and

(iii) the use of transposon 'P' for germline transformation. Although a number of localized regions in Drosophila genome have been analyzed by chromosome walking, it is often limited to only few hundred kilobase pairs and requires the use of overlapping lambda (A) or cosmid clones (consult next section for chromosome walking) of the size below 40 kbp it has been estimated that atleast 10,000 cosmid clones will have to be analysed to include all sequences of the genome, so that the task will be formidable.

Since, DNA fragments, several hundred kilobase pairs long, can be cloned in Y AC vectors, a Y AC library of 1500 clones (instead of 10,000 with cosmids) is sufficient for mapping the Drosophila genome. During the years 1989-92, as many as more than 1000 Drosophila Y AC clones (called DY clones) were cytologeally located.
The DY clones were prepared, using the following steps:
(i) high molecular weight (HMW) DNA was isolated from D. melanogaster DNA. using caesium chloride gradient centrifugation;
(ii) molecules larger than 120 kbp were separated by size fractionation in sucrose gradients;
(iii) the large fragments (120kbp) separated as above were ligated onto Y AC vector arms;
(iv) the selected DY clones were used for transformation of yeast cells. When DY clones were to be used for mapping, they were separated from yeast chromosomes by FIGE (field inversion gel electrophoresis) and later in each case, Drosophila DNA fragment was isolated from DY clone by enzyme cleavage.

This DNA is then used for in situ hybridization (ISH) on Drosophila salivary gland chromosomes. This enabled mapping of the entire range of salivary gland chromosomes, although gaps between them do exist.

The fragments used as probes can be used for further detailed restriction mapping or sequencing according to requirements. In humans also detailed Y AC contigs was published in 1992.

The Y AC clones may also be 'independently used for, preparing physical maps of the adjoining regions of a cloned gene in the form of restriction maps. For instance, an effort was made to prepare restriction maps (involving rare cutting restriction enzymes = RE) of some loci in a region of human chromosome 18.

As another example, using cDNA of PAI-2 as a probe (plasminogen activator inhibitor type 2, a gene responsible for heart attacks), three clones were selected from 13000 clones of a Y AC library.

These clones contained, complete or a part of PAI- 2 locus, as shown by colony hybridization. The Y AC clones were digested with fare cutting REs and the resulting fragments were size fractionated, and restriction maps were prepared.

 
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