The genetics and evolution of centromere assembly in vertebrates and fission yeast: We are using a set of engineered mini-chromosomes to investigate the genetics and evolution of centromere assembly in vertebrates and fission yeast. Our work has led us to develop several new techniques for chromosome engineering and we are also applying these to the construction of a plant artificial chromosome.
Human mini-chromosome engineering
Centromeres mediate the segregation of chromosomes at cell division; a function that is conserved throughout eucaryotic evolution. However centromeric DNA is amongst the most rapidly evolving in the genome and thus poses a paradox which we should like to resolve. In order to do this we need to understand how centromeric DNA is recognized. We are using vertebrate cells and fission yeast to address this question. In both organisms centromeres are comprised of long stretches of DNA. These sequences are hard to manipulate experimentally and so much of our effort has been devoted to developing methods for manipulating such long tracts of DNA. In our early work we used telomeres as in vivo reagents to fragment human chromosomes. This worked well and generated a set of mini-chromosomes that have been useful in subsequent experiments and told us that the minimum size for a centromere in human cells was about 100kb. More recently we have developed a new type of site specific recombinase for use in both vertebrate cells. At the moment we are using this type of enzyme to swap the existing centromere sequence on a yeast or human mini-chromosome with a defined tract of sequence whose function we wish to test.
Our technology for manipulating chromosome structure is of general applicability and we are also using it to build artificial plant chromosomes. Our approach is described below.
Engineering the Human Y chromosome by telomere directed chromosome breakage
Chromosome engineering and centromere function
Sunir Malla PhD student, Felix Dafnis postdoc and William Brown
Outline: We are developing new methods for engineering chromosome structure and using them to study the mechanism and sequence specificity of centromere assembly in chicken and human cells.
1. We are establishing a method for assembling very large (100kb -1Mb) tracts of transgenic DNA on vertebrate chromosomes
2. We are establishing a method for irreversibly deleting DNA sequences of arbitrary length from human chromosomes.
3. We are using these methods to engineer human and other mini-chromosomes with centromeric DNA of defined length and sequence and then studying how these different centromeres work in chicken, mouse and human cells.
Chromosome engineering and mouse centromere function
Outline: I am studying the mechanism and sequence specificity of centromere assembly in mouse cells.
1. In collaboration with William and Sunir I am establishing a method for assembling very large (100kb -1Mb) tracts of transgenic DNA on vertebrate chromosomes
2. I am using this method to build centromeres on mini-chromosomes that are unstable in mouse cells with the object of identifying the features of the sequence necessary for centromere function in mouse cells.
Chromosome engineering and yeast centromere function
Engineering a rice artificial chromosome
Zhang Yao Xu, Leverhulme Trust funded post-doc
Outline: I am using the methods of chromosome engineering established in our group to convert a human mini-chromosome into a plant artificial chromosome. The long term goal is to develop an artificial chromosome vector for this important crop plant.
I am taking one of William's mini-chromosomes and adding to it a variety of cis-acting sequences that will enable it to function as a chromosome in plant cells. These sequences include, of course, centromeric DNA, a selectable marker gene and telomeres. I will then introduce the modified mini-chromosome into plant protoplasts by somatic cell fusion techniques. This work is carried out in collaboration with Plant Sciences at the Sutton Bonnington Campus.
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