Telomere mediated minichromosome production

A minichromosome is a small

chromatin Chromatin is a complex of DNA and protein found in eukaryotic cells. The primary function is to package long DNA molecules into more compact, denser structures. This prevents the strands from becoming tangled and also plays important roles in r ...

-like structure resembling a

chromosome A chromosome is a long DNA molecule with part or all of the genetic material of an organism. In most chromosomes the very long thin DNA fibers are coated with packaging proteins; in eukaryotic cells the most important of these proteins are ...

and consisting of

centromere The centromere links a pair of sister chromatids together during cell division. This constricted region of chromosome connects the sister chromatids, creating a short arm (p) and a long arm (q) on the chromatids. During mitosis, spindle fibers ...

telomere A telomere (; ) is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Although there are different architectures, telomeres, in a broad sense, are a widespread genetic feature mos ...

s and

replication origin The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...

s but little additional genetic material. They replicate autonomously in the cell during

cellular division Cell division is the process by which a parent cell divides into two daughter cells. Cell division usually occurs as part of a larger cell cycle in which the cell grows and replicates its chromosome(s) before dividing. In eukaryotes, there are ...

. Minichromosomes may be created by natural processes as chromosomal aberrations or by genetic engineering.

Structure

Minichromosomes can be either linear or circular pieces of DNA. By minimizing the amount of unnecessary genetic information on the chromosome and including the basic components necessary for

DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all living organisms acting as the most essential part for biological inheritanc ...

(centromere, telomeres, and replication sequences), molecular biologists aim to construct a chromosomal platform which can be utilized to insert or present new

gene In biology, the word gene (from , ; "... Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a b ...

s into a

host cell In biology and medicine, a host is a larger organism that harbours a smaller organism; whether a parasitic, a mutualistic, or a commensalist ''guest'' (symbiont). The guest is typically provided with nourishment and shelter. Examples include a ...

Production

Producing minichromosomes by genetic engineering techniques involves two primary methods, the '' de novo'' (bottom-up) and the top-down approach.

''De novo''

The minimum constituent parts of a chromosome (centromere, telomeres, and DNA replication sequences) are assembled by using

molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their DNA replication, replication within Host (biology), host organisms. The use of the word ''cloning'' re ...

techniques to construct the desired chromosomal contents ''in vitro''. Next, the desired contents of the minichromosome must be transformed into a host which is capable of assembling the components (typically yeast or mammalian cells) into a functional chromosome. This approach has been attempted for the introduction of minichromosomes into

maize Maize ( ; ''Zea mays'' subsp. ''mays'', from es, maíz after tnq, mahiz), also known as corn (North American and Australian English), is a cereal grain first domesticated by indigenous peoples in southern Mexico about 10,000 years ago. The ...

for the possibility of genetic engineering, but success has been limited and questionable. In general, the ''de novo'' approach is more difficult than the top-down method due to species incompatibility issues and the

heterochromatic Heterochromia is a variation in coloration. The term is most often used to describe color differences of the iris, but can also be applied to color variation of hair or skin. Heterochromia is determined by the production, delivery, and concentra ...

nature of centromeric regions.

Top-down

This method utilizes the mechanism of

-mediated chromosomal truncation (TMCT). This process is the generation o
truncation
by selective transformation of telomeric sequences into a host genome. This insertion causes the generation of more telomeric sequences and eventual truncation. The newly synthesized truncated chromosome can then be altered through the insertion of new genes for desired traits. The top-down approach is generally considered as the more plausible means of generating extra-numerary chromosomes for the use of genetic engineering of plants. In particular it is useful because their stability during cell division has been demonstrated. The limitation of this approach is that it is labor-intensive.

Role in genetic engineering

Unlike traditional methods of genetic engineering, minichromosomes can be used to transfer and express multiple sets of genes onto one engineered chromosome package. Traditional methods which involve the insertion of novel genes into existing sequences may result in the disruption of endogenous genes and thus negatively affect the host cell. Additionally, with traditional gene insertion methods, scientists have had less ability to control where the newly inserted genes are located on the host cell chromosomes, which makes it difficult to predict inheritance of multiple genes from generation to generation. Minichromosome technology allows for the stacking of genes side-by-side on the same chromosome thus reducing likelihood of segregation of novel traits.

Plants

In 2006, scientists demonstrated the successful use of telomere truncation in maize plants to produce minichromosomes that could be utilized as a platform for inserting genes into the plant genome. In plants, the telomere sequence is conserved, which implies that this strategy can be utilized to successfully construct additional minichromosomes in other plant species. In 2007, scientists reported success in assembling minichromosomes ''in vitro'' using the ''de novo'' method. The use of minichromosomes as a means for generating more desirable crop traits is actively being explored. Major advantages include the ability to introduce genetic information which is highly compatible with the host genome. This eliminates the risk of disrupting various important processes such as cell division and gene expression. With continued development, the future for use of minichromosomes may make a huge impact on the productivity of major crops.

Other organisms

Minichromosomes have also been successfully inserted into yeast and animal cells. These minichromosomes were constructed using the ''de novo'' approach.

References

{{reflist Genetics