A minichromosome is a small chromatin-like structure resembling a chromosome and consisting of centromeres, telomeres and replication origins [1] but little additional genetic material. [2] [ self-published source? ] They replicate autonomously in the cell during cellular division. [3] Minichromosomes may be created by natural processes as chromosomal aberrations or by genetic engineering. [1]
Minichromosomes can be either linear or circular pieces of DNA. [3] By minimizing the amount of unnecessary genetic information on the chromosome and including the basic components necessary for DNA replication (centromere, telomeres, and replication sequences), molecular biologists aim to construct a chromosomal platform which can be utilized to insert or present new genes into a host cell. [3]
Producing minichromosomes by genetic engineering techniques involves two primary methods, the de novo (bottom-up) and the top-down approach. [1]
The minimum constituent parts of a chromosome (centromere, telomeres, and DNA replication sequences) are assembled [4] by using molecular cloning 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 [5] ) into a functional chromosome. This approach has been attempted for the introduction of minichromosomes into maize for the possibility of genetic engineering, but success has been limited and questionable. [6] In general, the de novo approach is more difficult than the top-down method due to species incompatibility issues and the heterochromatic nature of centromeric regions. [5]
This method utilizes the mechanism of telomere-mediated chromosomal truncation (TMCT). This process is the generation of truncation by selective transformation of telomeric sequences into a host genome. This insertion causes the generation of more telomeric sequences and eventual truncation. [3] 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. [7] The limitation of this approach is that it is labor-intensive.
Unlike traditional methods of genetic engineering, minichromosomes can be used to transfer and express multiple sets of genes onto one engineered chromosome package. [8] Traditional methods which involve the insertion of novel genes into existing sequences may result in the disruption of endogenous genes [1] 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, [9] 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.
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. [10] In plants, the telomere sequence is conserved, which implies that this strategy can be utilized to successfully construct additional minichromosomes in other plant species. [1]
In 2007, scientists reported success in assembling minichromosomes in vitro using the de novo method. [6]
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. [11]
Minichromosomes have also been successfully inserted into yeast and animal cells. These minichromosomes were constructed using the de novo approach. [3]
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that benefit the survival of the organism and confer selective advantage such as antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain only additional genes that may be useful in certain situations or conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via transformation. Synthetic plasmids are available for procurement over the internet.
A transposable element is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Transposition often results in duplication of the same genetic material. In the human genome, L1 and Alu elements are two examples. Barbara McClintock's discovery of them earned her a Nobel Prize in 1983. Its importance in personalized medicine is becoming increasingly relevant, as well as gaining more attention in data analytics given the difficulty of analysis in very high dimensional spaces.
A telomere is a region of repetitive nucleotide sequences associated with specialized proteins at the ends of linear chromosomes. Telomeres are a widespread genetic feature most commonly found in eukaryotes. In most, if not all species possessing them, they protect the terminal regions of chromosomal DNA from progressive degradation and ensure the integrity of linear chromosomes by preventing DNA repair systems from mistaking the very ends of the DNA strand for a double-strand break.
Molecular evolution is the process of change in the sequence composition of cellular molecules such as DNA, RNA, and proteins across generations. The field of molecular evolution uses principles of evolutionary biology and population genetics to explain patterns in these changes. Major topics in molecular evolution concern the rates and impacts of single nucleotide changes, neutral evolution vs. natural selection, origins of new genes, the genetic nature of complex traits, the genetic basis of speciation, the evolution of development, and ways that evolutionary forces influence genomic and phenotypic changes.
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. The vector contains features that allow for the convenient insertion of a DNA fragment into the vector or its removal from the vector, for example through the presence of restriction sites. The vector and the foreign DNA may be treated with a restriction enzyme that cuts the DNA, and DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of bacterial artificial chromosome
In genetics, an insertion is the addition of one or more nucleotide base pairs into a DNA sequence. This can often happen in microsatellite regions due to the DNA polymerase slipping. Insertions can be anywhere in size from one base pair incorrectly inserted into a DNA sequence to a section of one chromosome inserted into another. The mechanism of the smallest single base insertion mutations is believed to be through base-pair separation between the template and primer strands followed by non-neighbor base stacking, which can occur locally within the DNA polymerase active site. On a chromosome level, an insertion refers to the insertion of a larger sequence into a chromosome. This can happen due to unequal crossover during meiosis.
Subtelomeres are segments of DNA between telomeric caps and chromatin.
P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.
A genomic library is a collection of overlapping DNA fragments that together make up the total genomic DNA of a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. In order to construct a genomic library, the organism's DNA is extracted from cells and then digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. Next, the vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.
A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6–10 megabases (Mb) in size instead of 50–250 Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, including disease defense.
In molecular biology, insertional mutagenesis is the creation of mutations in DNA by the addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for research purposes in the lab.
In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.
Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.
Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.
Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.
James A. Birchler is an American biologist who is currently Curators' Professor at University of Missouri where he studies gene dosage, polyploidy, and cytogenetics in both maize and drosophila. In 2002 he was named a fellow of the American Association for the Advancement of Science. and in 2011 he was elected to the National Academy of Sciences. In 2017 he was named the SEC Professor of the Year.
A linear chromosome is a chromosome which is linear in shape, and contains terminal ends. In most eukaryotic cells, DNA is arranged in multiple linear chromosomes. In contrast, most prokaryotic cells generally contain a singular circular chromosome.