Artificial Chromosomes | Zoology Optional Notes for UPSC PDF Download

Introduction

• Efforts to clone and sequence the human genome and genomes of intensively studied organisms such as maize, Arabidopsis thaliana, Drosophila, and yeast require methods of cloning that gives large sized DNA fragments. Even though cosmids are excellent cloning vectors, a cosmid library of the human genome would require over 500,000 clones. Efforts to develop cloning vectors for large DNA vectors started with the extensively studied eukaryote, yeast.
• This organism has many of the useful attributes of E.coli (such as a rapid growth rate, single cells, and a relatively small genome). Yeast has a doubling time about twice that of E.coli, exist as a haploid and diploid, and has a genome only 2½times (12 mb) that of E.coli. Yeast undergoes mitosis and meiosis, has a nucleus, and possesses the enzymes for processing RNA after transcription.
• This last feature is extremely important because E.coli is unable to process RNA in addition, yeast can be transformed easily after treatment with the mixture of snail gut enzymes that degrade the thick, complex yeast cell wall composed of proteins and polysaccharides.

Yeast Artificial Chromosomes (YAC)

• Cloning of DNA fragments much larger than 45 kb became possible in 1987, when D.T. Burke and G.F. Carle developed in the laboratory of M.V. Olson an altogether new type of yeast vector, which they called yeast artificial chromosome (YAC). The development of YAC’s were based on the logic that an eukaryotic linear chromosomes needs for its replication and stability, not only replication origins, but also the centromere and the telomere.
• The centromere sequence would attach to the mitotic spindle during cell division and help in efficient segregation of the chromosomes into the daughter cells. The telomere would preserve the integrity of the ends of the linear chromosomes. Once these elements were provided, the vector could be replicate stably like a chromosome and could accommodate chromosomes sized inserts. Indeed, standard YACs can accommodate around 600 kb DNA inserts, while special type of YACs can accommodate up to 1400 kb DNA inserts.
• Though an YAC vector is meant to be propagated like a chromosome in yeast, it is a circular double stranded DNA that contains a replication origin (colE 1) compatible with E. coli in addition to yeast replication origin or an yeast ARS element. The col El replication origin is useful to a yeast replication origin or an yeast ARS elements. The colEl replication origin is useful for amplification of the vector in E. coli.
• Next to the yeast replication origin is located the centomere of yeast chromosome 4 (Cen 4). The two telomere sequences are from the protozoan Tetrahymenal, which have been found to be functional in yeast. A staffer element containing the His 3 gene of yeast is present between the two telomere sequences through two BamHI restriction endonuclease sites. Three selectable yeast marker genes, Trp 1, Ura 3 and Sup 4 are also present.
• The marker genes Trpl and Ura 3 are on the two sides of the unique restriction endonuclease site SnBl, that produces blunt ends. The SnaB 1 site is used as the cloning site and is located within the Sup4 gene. Sup 4 gene product is a tRNA that suppresses a mutation in the Ade gene of yeast resulting in a change in colour of colonies from red to white in the presence of limiting amounts of adenine in the medium. The YAC vector is digested with BamHI and SnaB I throwing the staffer element out, disrupting the Sup4 gene and yielding two vector fragments termed as left arm and right arm.
• Each of the arms but at its one end a telomere sequence followed by a BamHI overhang, but a blunt flush cut at the other end. The left arm also has the Cen 4 sequence, the ARS1, the ColEl origin, the amp and Trp 1 markers. The right arm contains the Ura 3 marker. These two arms are blunt end ligated with the long chromosomal DNA fragment from any source, thus creating the artificial linear chromosome among other ligation products. Ligation products are introduced into yeast cells that have mutation in the Trpl, Ura3 and Ade loci by lipofection or by fusion with yeast speroplasts.
• The yeast cells are allowed to regenerate the cell walls and plated on medium lacking trytophan and uracil and containing limiting amount of adenine. Only the yeast cells transformed with artificial chromosomes comprising both the left arm and the right arm would grow. The recombinant artificial chromosomes containing the insert would develop into white colonies. The red colonies would represent cells having the linear vector, but no insert. Cells having other ligation products like two left arms or two right arms would not grow.

Bacterial Artificial Chromosomes (BAC)

• Bacterial artificial chromosomes (BAC) were developed by Mel Simmons and coworkers in the early 1990s and are based on the fertility factor (F factor) of Escherichia coli. The F plasmid, a ~ 100 kb circular double stranded DNA, is present is an E. coli cell in only 1-2 copies. The synthetic BAC vectors, which are only ~7.5 kb double stranded DNA circles contain the replication origin oriS and the gene repE of the F plasmid that are responsible for initiation and proper orientation of replication of the BAC vector.
• The parA and parB genes of the F plasmid ensure efficient segregation of the F factor into the daughter E. coli cells after its replication are also incorporated in the BAC vector. The BAC vectors also contain multiple cloning sites (mcs), a selectable marker in the form of antibiotic resistance and colour based identification (lac Z complementation system) of recombinants carrying inserts.
• The naturally occurring F2 factors consist of up to 25% of the E. coli genome integrated into the basic F factor and are very stable. This characteristic of the F factor contributes to the ability BACs to accommodate very large amount of external DNA to the extent of 300kb.
• The recombinant BACs have been found to exhibit a lower level of rearrangement and chimerism of the cloned DNA sequence than exhibited by YACs. The cloning of DNA in BACs is done as is done in a plasmid, by linearising the vector with a restriction endonuclease, treating with phosphatase and then ligating with the DNA fragments to be cloned. E. coli has to be transformed by electroporation because of the large size of the recombinant BAC.

Mammalian Artificial Chromosomes (MACs)

• The YACs, the BAC and the PACs (plasmid artificial chromosomes) have found regular use for cloning large genomic DNA fragments in various genome sequencing projects. Another potential application of artificial chromosomes is in gene therapy of human. For gene therapy, we need to have gene in human DNA fragments including their promoters and all the control elements. This would have to be introduced into the target cells efficiently and would have to be stably maintained inside the nucleus, generation after generation through unlimited number of divisions.
• The DNA would have to be expressed properly without interfering with the function of other resident. Such large artificial human chromosome (aptly termed mini-chromosomes), if 1-10 mb size have been claimed to be stable for more than 100 cell generations. Satellite DNA based mini-chromosomes of 20-30 mb have also been reported.

Artificial Human Chromosomes

• Researchers at the School of Medicine and Athersys, Inc. have created the first artificial human chromosome. The synthetic chromosomes represent a breakthrough in medical research and provide scientists with a powerful new tool for the study of human genetics. Artificial chromosomes may also offer a new approach to gene therapy and the treatment of a broad range of genetic diseases. A report of the research was published in the April 2007 issue of Nature Genetics.
• “This opens the door to a whole new avenue of research in chromosome biology and gene therapy,” said Huntington F. Willard, chairman of genetics at the School of Medicine and University Hospitals of Cleveland. “While it’s been known since the early years of this century that chromosomes carry genes, until now the complexity and size of normal chromosomes has limited our ability to analyze their structure and function. The synthetic micro-chromosome system now allows us to perform detailed studies on the nature of chromosomes – essentially the next phase of the Human Genome Project which is to move from just mapping genes to actually understanding how they work and influence human disease.”
• In this study, the research team created artificial chromosomes from normal human material. The researchers first synthesized arrays of alpha satellite DNA, and then introduced the resulting centromeric material into human cells in conjunction with telomeres and genomic DNA.
• Inside the cells, the independent elements assembled to form miniature chromosomes, or synthetic micro-chromosomes, that were structurally similar to human chromosomes, but contained less genetic material. Analysis of the newly introduced artificial chromosomes demonstrated normal centromeric activity, genetic stability, and continued gene expression through repeated rounds of the cell cycle.