This is post no. 2 under the main topic.
| 04-24-2014, 08:36 AM
(This post was last modified: 05-02-2014, 02:02 PM by zemaxe7.)
Unusual chromosomes and their properties
Apart from other unusual chromosomes that occur „regularly“ in the nature (polytene, lamp brush and B chromosomes), there is also the fourth type of unusual chromosomes – Artificial chromosomes. As their name suggests, they are man-made, synthetic chromosomes, constructed with the intention to be used in genetic engineering on the chromosomal level (manipulation of whole chromosomes). This means that they contain fragments of target DNA inserted into the carrier chromosome, which can then be inserted into the host cell where they will be expressed along with the target DNA (very useful in cloning experiments/applications).
When comparing artificial chromosomes with other vector molecules used in genetic transformations, the major difference (and advantage) of artificial chromosomes is the size of the foreign target DNA they can contain. While other vectors (like plasmids and phages, or hybrid ones like cosmids and phagemids) can contain only up to 50 kb of foreign DNA (in the best case; the number is usually around 10 kb), artificial chromosomes can contain up to 1000 kb of target DNA. This makes them a lot better targets for cloning of larger genes, or for the creation of genomic libraries, e.g.
When we say artificial chromosomes (focusing on human artificial chromosomes), we mean exactly that – they are very similar to the normal chromosomes. In fact, when observed structurally, they are identical, because in order for the artificial chromosome to be functional, it must have all of the parts like the regular chromosomes do. These include centromeres, telomeres, protein scaffold, origin of replication, etc.
The main application of artificial chromosomes is like of any other vector molecule – it can be used to study specific DNA fragments. They can be inserted into the host cells (usually bacteria) which will propagate and the fragment of interest will be transcribed first, and then translated later on. The product in the form of protein can then be analyzed. The same process can also be used to study the gene expression of specific organisms, or to induce a new gene into the target organism. Moreover, artificial chromosomes are great for the creation of genomic libraries (since they can contain a lot more foreign DNA than the regular vector molecules), which effectively reduces the number of vectors needed to store genomic DNA of one organism.
This is post no. 3 under the main topic.
| 04-25-2014, 06:53 AM
Types of artificial chromosomes
YAC – Yeast artificial chromosome
YAC is the first artificial chromosome produced and it was the major breakthrough when compared to the previous vector molecules as it could contain up to 1000 kb of foreign DNA (which is about 20 times more than the hybrid vectors like cosmids and phagemids, and hundreds of times more than some regular plasmid vectors). As the name suggests, this chromosome was produced using the Yeast DNA.
YAC contains both parts of the chromosome and parts of the vector molecule. These include centromere, telomeres, origins of replication and selectable markers. The main problem with the YAC is the fact that it is unstable (this is the main reason why it was replaced with BAC – bacterial artificial chromosome during the Human Genome Project). However, its stability was improved to a certain degree by the group of scientists during the 1980s. They have accomplished this through the discovery of autonomously replicating sequences (ARS).
Autonomously replicating sequences are basically origins of replication which can also affect the plasmid stability. They are found in Yeast and the interesting thing about them is the fact that they don’t have to be used in every cell cycle.
Advantages and applications of YAC
One of the best applications of yeast artificial chromosome is its usage to express eukaryotic proteins. Regular bacterial plasmids or phages cannot be used as vectors for eukaryotic genes since eukaryotes possess specific posttranslational modifications not found in prokaryotes.
Moreover, since YACs can contain a lot of foreign DNA, they can be used in the creation of genomic libraries (genomic library contains the whole genome of one organism). This can be achieved in two ways: random fragmentation (which is better for the creation of gene bank) and partial restriction enzyme digestion (which is better for the creation of genome bank of an organism). Once the genomic library is generated, it should be maintained in the stable form.
This is post no. 4 under the main topic.
| 05-01-2014, 02:29 PM
Types of artificial chromosomes
BAC – Bacterial artificial chromosome
This is another popular artificial chromosome used in genetic engineering. The main difference between it and the yeast artificial chromosome is that YAC can contain up to three times more DNA, but the BAC is more stable.
This characteristic makes it the preferable choice when doing some genetic experiments like sequencing; especially since bacterial artificial chromosome can still contain a lot of DNA, up to around 300 kb), which is a lot more than regular plasmids or virus-based vectors. It was used during the genome projects, like in the case of Human Genome Project, which was started with YAC, but scientists decided to shift to BAC after the yeast artificial chromosome proved to be unstable. The basic idea behind the genome project is to have the organism’s whole sequence. This can be accomplished by sequencing the organism’s genome – amplifying it using BAC vectors (containing inserts of organism’s DNA) and PCR. The final sequence is ordered and produced using computers.
Bacterial artificial chromosome is based on the F-plasmid (fertility plasmid). This is important since F-plasmid contains parA and parB partition genes which contribute to the overall stability of the BAC, plus they partition the vector DNA to daughter cells during the cell division, resulting in even distribution of plasmid after the division.
Additional parts of the bacterial artificial chromosome are selectable markers, of course, like the ones for antibiotic resistance or blue/white selection. Also repE region, which is responsible for the replication initiator protein whose role is also to regulate the number of copies of BAC. It also contains the target sites where restriction enzymes will cut and where the DNA of interest will be inserted, and it has T7 and Sp6 regions, which are phage promoters, sites of the RNA pol binding and the initiation of transcription (RNA production).
This is post no. 5 under the main topic.
| 05-02-2014, 02:21 PM
(This post was last modified: 05-02-2014, 02:22 PM by zemaxe7.)
Types of artificial chromosomes
HAC – Human artificial chromosome
Human artificial chromosome was constructed from scratch during the 1990s in human fibrosarcoma cell line (fibrous connective tissue containing tumor). This was accomplished by adding alpha-satellite DNA to telomeric and genomic DNA, which resulted in a completely new 47th microchromosome. Its length can go up to 10,000 kb (including the inserted DNA of interest) making it smaller than half of regular human chromosomes (out of haploid chromosome number – 23).
The good thing about human artificial chromosome is that it is completely independent. Unlike yeast and bacterial artificial chromosomes which are based on plasmids and integrate themselves into the genome, human artificial chromosome is constructed de novo, with all the regular parts of a chromosome (centromeres, telomeres, etc.). This means that it won’t disrupt the existing genetic material when inserted in the cell, which is important since YACs are the least stable, while BACs are also less stable than HACs, as their integration can lead to unpredictable expression levels or some other problems when the host cell’s genome is interrupted with their DNA.
Scientists are creating HACs in two ways. One of them is de novo, but it poses some problems because making a chromosome from scratch has some limitations – some specific elements like centromeres might not be constructed correctly, or some DNA fragments might not successfully integrate into them.
Another way of creating HACs is by using one already present normal human chromosome. Namely, existing chromosome is modified by cutting off some of its parts, and then some additional DNA is added at some specific sites (also known as Cre-Lox recombination).
Human artificial chromosomes can be used for a number of different things; for example, as gene transfer vectors in expression studies, for determining the function of human chromosomes, for annotating of the human genome, or for doing some specific things in the cell by carrying additional specific genes that would help in fighting some diseases, for example.
This is post no. 6 under the main topic.
| 06-15-2014, 07:39 PM
How about the entire genome?
Instead of creating only one artificial chromosome, scientists have actually managed to synthesize the entire genome of bacteria. A team of scientists from the J. Craig Venter Institute (JCVI) led by Craig Venter has accomplished this by combing two already existing techniques to transfer new DNA material into bacterial cells. The first one is actually the synthesis of a new genome, and the second one is its transfer into the cell (using nuclear transfer techniques from in vitro fertilization). In the end, scientists have produced the bacteria with synthetic genome capable of self-replication, which is awesome.
The bacterium used is Mycoplasma mycoides. Venter’s team has used its genome as a base to create the new one. It does not sound that it is synthetic at first, but they have actually added a lot of different DNA sequences into the genome, while some others were deleted, and the transplant worked in the end. Moreover, the whole process was done synthetically because, in the words of Craig Venter: “…the cell is totally derived from a synthetic chromosome, made with four bottles of chemicals on a chemical synthesizer, starting with information in a computer…”. The recipient bacterium used was Mycoplasma capricolum, and the resulting genome was around 1000 kb long.
The idea of building artificial genome has actually started some 15 years before, when the scientists have started developing the strategy to build the synthetic genome of M. genitalium, a bacterium with the smallest complement of genes capable of independent growth. Not only that it contains less than 500 protein-coding genes, but more than 100 of them can actually be removed if disrupted one at a time. They have accomplished this by assembling small 6 kb pieces of DNA into a larger molecule. The process was done both in vitro using enzymes and in vivo in yeast (for recombination).
All of this research is leading to the new era of synthetic biology, that will give rise to new ways of, for example, faster vaccine production, usage of light energy, water cleaning, etc.
This is post no. 7 under the main topic.
| 06-15-2014, 09:55 PM
The New Yeast Artificial Chromosome - SynIII
Scientists have managed to synthesize completely new chromosome from one of yeast’s 16 chromosomes. Namely, the team from the Langone Medical Centre at New York, led by Jef Boeke, has used yeast’s chromosome III and made the new one synIII (synthetic III).
The new chromosome had a lot of stuff removed. These were the things scientists thought are not necessary for the proper functioning of yeast. They are mostly some non-coding DNA (also called junk DNA or introns), some jumping genes (transposons) and some repetitive sequences (like subtelomeric repeats).
Boeke and his team have also managed to insert some new sequences as well. They were able to introduce a new function into the chromosome named “chemical switch”. It basically simplifies further genetic modifications by allowing scientists to get different variations of the same chromosome through the genome scrambling. This is important since it allows them “to generate millions of variant daughter genomes, and screen them for interesting properties,” as the leader of the project Jef Boeke said.
They have achieved this by first synthesizing shorter sequences of the DNA and then merging them together. The end result was astonishing. The original chromosome had more than 315 kb, while the new one has around 270 kb, which is around 45 kb less, or around 15% less of genetic material. Even so, the new yeast cells were able to perform all the regular activities like grow and divide, ferment sugar, etc.
This whole project is very important since it moves us one step closer to modifying our human genomes. Yeast cells are eukaryotic, and they are much more similar to ours than bacterial cells are, even though our genome is a lot larger and more complicated. Once we improve our technology in this field even more, we could do all sorts of things. For example, we could improve our immune system, fight cancer easier, slow down the effects of aging, etc. But we could also do something more fancy, like boost our memory or add the ability to see ultraviolet light. The possibilities are endless…