Chromosomes, Artificial

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.

Natural Chromosome Function

DNA, which constitutes the genome of a cell, is always packaged with a variety of proteins, and together these make up the chromosomes. A chromosome serves to compact the DNA and protect it from the damage, while at the same time allowing the genes it contains to be available for transcription into RNA. In addition to these functions, extra ones are necessary when the cell divides. Prior to cell division the DNA must be copied and these copies separated (segregated) and delivered to different parts of the cell, ensuring that each of the new cells receives only a single copy.

To ensure correct segregation, chromosomes have to have distinct components that are composed of specific DNA sequences and associated proteins. Bacterial chromosomes, (plasmids ) which are circular, have a single site at which DNA replication originates, and attachment to the cell membrane results in segregation. Artificial bacterial chromosomes (BACs) mimic this using appropriate origin sequences.

In organisms with multiple linear chromosomes (eukaryotic organisms) the process is more complicated. The ends of the chromosomes must be protected from degradation and from the mechanisms that the cell uses to protect itself against broken DNA. Telomeres , which provide these functions, are arrays of short, repeated sequences with complexes of specific proteins attached. To ensure segregation complexes of other proteins, DNA sequences known as kinetochores form at sites known as centromeres . These contain molecular motors, systems to monitor correct segregation, and sites for attachment of microtubules . Chromosomes will contain one or more origins of replication.

Yeast Artificial Chromosomes

Artificial chromosomes for use in yeast and mammalian cells aim to replicate these components on a single DNA molecule. In bakers yeast (Saccharomycescerevisiae ), telomeres, centromeres, and origins of replication have all been defined using genetics and have been cloned. When assembled they can be grown as a small chromosome in bacteria and form a vector capable of incorporating up to a million bases of other DNA as a chromosome in yeast (a YAC).

This technology has been used to investigate the properties of yeast chromosomes but has been most extensively used in the early phases of genome mapping projects. By cloning complete representations of the human genome into large YACs, the order of these YACs could be deduced by a number of methods and overlapping ones assembled conceptually into a representation of regions of the human genome. These are useful for finding genes from information about the inheritance of genetic diseases. They have also been useful for testing the function of genes in mice. Because of the size of YACs, they frequently contain all of the DNA needed to control the expression of genes with the correct developmental and tissue specificity. When injected into developing mouse eggs, they can fully correct mutations. More recently YACs have been largely supplanted by BACs, because the latter are easier to manipulate and prepare in the laboratory.

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.

see also Chromosome, Eukaryotic; Human Genome Project; Mapping; Telomere.

Howard Cooke

Bibliography

Grimes, B., and H. Cooke. "Engineering Mammalian Chromosomes." Human Molecular Genetics 7, no. 10 (1998): 1635-1640.

Willard, H. F. "Genomics and Gene Therapy: Artificial Chromosomes Coming to Life." Science 290 (2000): 1308-1309.

artificial chromosome A type of cloning vector that has some features of true chromosomes and is used to clone relatively large fragments of DNA. Bacterial artificial chromosomes (BACs) are based on the F (fertility) plasmid found naturally in E. coli bacteria (see sex factor). They can accommodate inserts of foreign DNA up to about 300 kilobase (kb) in length. Also included are several bacterial genes necessary for replication of the plasmid by the host cell and a gene (usually for resistance to an antibiotic) that allows selection of BAC-containing cells. Larger DNA fragments are cloned using yeast artificial chromosomes (YACs). These are linear vectors derived from a circular plasmid found naturally in baker's yeast (Saccharomyces cerevisiae) and capable of accommodating DNA inserts of up to 1000 kb. YACs have a centromere, enabling them to attach to the mitotic spindle of their yeast host and undergo normal segregation during cell division. They are also engineered with telomeres, the DNA sequences that cap either end of a chromosome. Thus YACs behave like mini-chromosomes. They are used for cloning eukaryotic genes or gene segments, for making DNA libraries of organisms with large genomes (e.g. mammals), and for studying gene function.