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Yeast artificial chromosome

From Wikipedia, in a visual modern way
This article is about Chromosomes derived from yeast DNA. For Yeast created from synthetic DNA, see Synthetic DNA.

Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae[1], 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 [2]

The bakers' yeast S. cerevisiae is one of the most important experimental organisms for studying eukaryotic molecular genetics.[1]

Beginning with the initial research of the Rankin et al., Strul et al., and Hsaio et al., the inherently fragile chromosome was stabilized by discovering the necessary autonomously replicating sequence (ARS);[2] a refined YAC utilizing this data was described in 1983 by Murray et al.[3]

The primary components of a YAC are the ARS, centromere [3] , and telomeres [4] from S. cerevisiae. Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector.[4]

This is a photo of two copies of the Washington University Human Genome YAC Library. Each of the stacks is approximately 12 microtiter plates. Each plate has 96 wells, each with different yeast clones.
This is a photo of two copies of the Washington University Human Genome YAC Library. Each of the stacks is approximately 12 microtiter plates. Each plate has 96 wells, each with different yeast clones.

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Saccharomyces cerevisiae

Saccharomyces cerevisiae

Saccharomyces cerevisiae is a species of yeast. The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have been originally isolated from the skin of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism behind the most common type of fermentation. S. cerevisiae cells are round to ovoid, 5–10 μm in diameter. It reproduces by budding.

Human Genome Project

Human Genome Project

The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a physical and a functional standpoint. It started in 1990 and was completed in 2003. It remains the world's largest collaborative biological project. Planning for the project started after it was adopted in 1984 by the US government, and it officially launched in 1990. It was declared complete on April 14, 2003, and included about 92% of the genome. Level "complete genome" was achieved in May 2021, with a remaining only 0.3% bases covered by potential issues. The final gapless assembly was finished in January 2022.

Bacterial artificial chromosome

Bacterial artificial chromosome

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

Autonomously replicating sequence

Autonomously replicating sequence

An autonomously replicating sequence (ARS) contains the origin of replication in the yeast genome. It contains four regions, named in order of their effect on plasmid stability. The A-Domain is highly conserved, any mutation abolishes origin function. Mutations on B1, B2, and B3 will diminish, but not prevent functioning of the origin.

Centromere

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 attach to the centromere via the kinetochore.

Construction

A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using restriction enzymes; DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a single large, circular piece of DNA.[3] [5] The basic generation of linear yeast artificial chromosomes can be broken down into 6 main steps:

  1. Ligation of selectable marker into plasmid vector: this allows for the differential selection of colonies with, or without the marker gene. An antibiotic resistance gene allows the YAC vector to be amplified and selected for in E. coli by rescuing the ability of mutant E. coli to synthesize leucine in the presence of the necessary components within the growth medium. TRP1 and URA3 genes are other selectable markers. The YAC vector cloning site for foreign DNA is located within the SUP4 gene. This gene compensates for a mutation in the yeast host cell that causes the accumulation of red pigment. The host cells are normally red, and those transformed with YAC only, will form colorless colonies. Cloning of a foreign DNA fragment into the YAC causes insertional inactivation of the gene, restoring the red color. Therefore, the colonies that contain the foreign DNA fragment are red.[5]
  2. Ligation of necessary centromeric sequences for mitotic stability[6]
  3. Ligation of Autonomously Replicating Sequences (ARS) providing an origin of replication to undergo mitotic replication. This allows the plasmid to replicate extrachromosomally, but renders the plasmid highly mitotically unstable, and easily lost without the centromeric sequences.[4][7]
  4. Ligation of artificial telomeric sequences to convert circular plasmid into a linear piece of DNA [8]
  5. Insertion of DNA sequence to be amplified (up to 1000kb)
  6. Transformation yeast colony[9]

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Plasmid

Plasmid

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.

Restriction enzyme

Restriction enzyme

A restriction enzyme, restriction endonuclease, REase, ENase or restrictase is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class of the broader endonuclease group of enzymes. Restriction enzymes are commonly classified into five types, which differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another. To cut DNA, all restriction enzymes make two incisions, once through each sugar-phosphate backbone of the DNA double helix.

DNA ligase

DNA ligase

DNA ligase is a type of enzyme that facilitates the joining of DNA strands together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-strand breaks in duplex DNA in living organisms, but some forms may specifically repair double-strand breaks. Single-strand breaks are repaired by DNA ligase using the complementary strand of the double helix as a template, with DNA ligase creating the final phosphodiester bond to fully repair the DNA.

Escherichia coli

Escherichia coli

Escherichia coli, also known as E. coli, is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes (EPEC, ETEC etc.) can cause serious food poisoning in their hosts, and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains do not cause disease in humans and are part of the normal microbiota of the gut; such strains are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones). For example, some strains of E. coli benefit their hosts by producing vitamin K2 or by preventing the colonization of the intestine by pathogenic bacteria. These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship — where both the humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh faecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards.

Leucine

Leucine

Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α-amino group (which is in the protonated −NH3+ form under biological conditions), an α-carboxylic acid group (which is in the deprotonated −COO− form under biological conditions), and a side chain isobutyl group, making it a non-polar aliphatic amino acid. It is essential in humans, meaning the body cannot synthesize it: it must be obtained from the diet. Human dietary sources are foods that contain protein, such as meats, dairy products, soy products, and beans and other legumes. It is encoded by the codons UUA, UUG, CUU, CUC, CUA, and CUG.

Transformation (genetics)

Transformation (genetics)

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Full chromosome III

Chromosome III is the third smallest chromosome in S. cerevisiae; its size was estimated from pulsed-field gel electro- phoresis studies to be 300–360 kb[10]

This chromosome has been the subject of intensive study, not least because it contains the three genetic loci involved in mating-type control: MAT, HML and HMR.[11] In March 2014, Jef Boeke of the Langone Medical Centre at New York University, published that his team has synthesized one of the S. cerevisiae 16 yeast chromosomes, the chromosome III, that he named synIII.[12][13] The procedure involved replacing the genes in the original chromosome with synthetic versions and the finished synthesized chromosome was then integrated into a yeast cell. It required designing and creating 273,871 base pairs of DNA - fewer than the 316,667 pairs in the original chromosome.

Uses in biotechnology

Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require posttranslational modification. By being able to insert large fragments of DNA, YACs can be utilized to clone and assemble the entire genomes of an organism.[14] With the insertion of a YAC into yeast cells, they can be propagated as linear artificial chromosomes, cloning the inserted regions of DNA in the process. With this completed, two processes can be used to obtain a sequenced genome, or region of interest:

  1. Physical Mapping [6]
  2. Chromosome Walking[15]

This is significant in that it allows for the detailed mapping of specific regions of the genome. Whole human chromosomes have been examined, such as the X chromosome,[16] generating the location of genetic markers for numerous genetic disorders and traits.[17]

Schematic of the pBR322 plasmid generated in the Boyer Lab at UC San Francisco by Bolivar and Rodriguez in 1972. It is one of the first and most widely utilized vectors, and the foundation for the YACs created by Murray and Szostak in 1983 The plasmid contains ampicillin and tetracycline resistance genes, and a suite of restriction enzyme target sites for inserting DNA fragments.
Schematic of the pBR322 plasmid generated in the Boyer Lab at UC San Francisco by Bolivar and Rodriguez in 1972. It is one of the first and most widely utilized vectors, and the foundation for the YACs created by Murray and Szostak in 1983 The plasmid contains ampicillin and tetracycline resistance genes, and a suite of restriction enzyme target sites for inserting DNA fragments.

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Plasmid

Plasmid

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.

Bacterial artificial chromosome

Bacterial artificial chromosome

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

Gene mapping

Gene mapping

Gene mapping describes the methods used to identify the locus of a gene and the distances between genes. Gene mapping can also describe the distances between different sites within a gene.

Primer walking

Primer walking

Primer walking is a technique used to clone a gene from its known closest markers. As a result, it is employed in cloning and sequencing efforts in plants, fungi, and mammals with minor alterations. This technique, also known as "directed sequencing," employs a series of Sanger sequencing reactions to either confirm the reference sequence of a known plasmid or PCR product based on the reference sequence or to discover the unknown sequence of a full plasmid or PCR product by designing primers to sequence overlapping sections.

The Human Genome Project

In the United States, the Human Genome Project first took clear form in February of 1988, with the release of the National Research Council (NRC) report Mapping and Sequencing the Human Genome.[18] YACs are significantly less stable than BACs, producing "chimeric effects" : artifacts where the sequence of the cloned DNA actually corresponds not to a single genomic region but to multiple regions. Chimerism may be due to either co-ligation of multiple genomic segments into a single YAC, or recombination of two or more YACs transformed in the same host Yeast cell.[19] The incidence of chimerism may be as high as 50%.[20] Other artifacts are deletion of segments from a cloned region, and rearrangement of genomic segments (such as inversion). In all these cases, the sequence as determined from the YAC clone is different from the original, natural sequence, leading to inconsistent results and errors in interpretation if the clone's information is relied upon. Due to these issues, the Human Genome Project ultimately abandoned the use of YACs and switched to bacterial artificial chromosomes, where the incidence of these artifacts is very low. In addition to stability issues, specifically the relatively frequent occurrence of chimeric events, YACs proved to be inefficient when generating the minimum tiling path covering the entire human genome. Generating the clone libraries is time consuming. Also, due to the nature of the reliance on sequence tagged sites (STS) as a reference point when selecting appropriate clones, there are large gaps that need further generation of libraries to span. It is this additional hindrance that drove the project to utilize BACs instead.[21] This is due to two factors:[22]

  1. BACs are much quicker to generate, and when generating redundant libraries of clones, this is essential
  2. BACs allow more dense coverage with STSs, resulting in more complete and efficient minimum tiling paths generated in silico.

However, it is possible to utilize both approaches, as was demonstrated when the genome of the nematode, C. elegans. There majority of the genome was tiled with BACs, and the gaps filled in with YACs.[21]

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Human Genome Project

Human Genome Project

The Human Genome Project (HGP) was an international scientific research project with the goal of determining the base pairs that make up human DNA, and of identifying, mapping and sequencing all of the genes of the human genome from both a physical and a functional standpoint. It started in 1990 and was completed in 2003. It remains the world's largest collaborative biological project. Planning for the project started after it was adopted in 1984 by the US government, and it officially launched in 1990. It was declared complete on April 14, 2003, and included about 92% of the genome. Level "complete genome" was achieved in May 2021, with a remaining only 0.3% bases covered by potential issues. The final gapless assembly was finished in January 2022.

Bacterial artificial chromosome

Bacterial artificial chromosome

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

Sequence-tagged site

Sequence-tagged site

A sequence-tagged site is a short DNA sequence that has a single occurrence in the genome and whose location and base sequence are known.

Source: "Yeast artificial chromosome", Wikipedia, Wikimedia Foundation, (2023, March 16th), https://en.wikipedia.org/wiki/Yeast_artificial_chromosome.

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Further Reading

Plasmid

Schizosaccharomyces pombe

Cloning vector

Expression vector

Transformation (genetics)

Origin of replication

Library (biology)

Artificial gene synthesis

CTF8

Molecular cloning
See also
References
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