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Yeast Artificial Choromosome

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Yeast Artificial Choromosome

  1. 1. YEAST ARTIFICIAL CHROMOSOME BY: Abhishek Kadam & Rutuja Rokade.
  2. 2. CONTENTS  Introduction  Purpose and features  Yeast Artificial Chromosome Plasmids  Construction YAC  Homologous Recombination  Application  Comparison between YAC And BAC System  Research
  3. 3. Introduction Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone DNA sequences in yeast cells YACS are plasmid shuttle vectors capable of replicating and being selected in common bacterial hosts such as Escherichia coli, as well as in the budding yeast Saccharomyces cerevisiae.
  4. 4. YAC is an artificially constructed chromosome that contains :  Centromere  Telomeres  Autonomous replicating sequence (ARS) element required for replication and preservation of YAC in yeast cells YACS behave like naturally existing chromosomes, provided that they are of the proper size, showing comparable stability.
  5. 5.  Cloning vehicles that propogate in eukaryotic cell hosts as eukaryotic Chromosomes.  Clone very large inserts of DNA :- 100 kb - 10 Mb. Purpose & Features  One arm contains an autonomous replication sequence (ARS), a centromere (CEN) and a selectable marker (trp1). The other arm contains a second selectable marker (ura3).  Insertion of DNA into the cloning site inactivates a mutant expressed in the vector DNA and red yeast colonies appear.  Transformants are identified as those red colonies which grow in a yeast cell that is mutant for trp1 and ura3. This ensures that the cell has received an artificial chromosome with both telomeres (because of complementation of the two mutants) and the artificial chromosome contains insert DNA (because the cell is red).
  6. 6. Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination. Yeast Replicating plasmids (YRp): These vectors contain an Autonomously Replicating Sequence (ARS) derived from the yeast chromosome. As the name suggests, these vectors can replicate independently of the yeast chromosome; however, they tend to be unstable and may be lost during budding. The Four Main Types Of Yeast Plasmids Are:
  7. 7. Yeast Centromere plasmids (YCp): These are considered low copy vectors and incorporate part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as though they are small independent chromosomes and are thus typically found as a single copy Unlike the ARS vectors, CEN vectors are stable without integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors can also be controlled if specific regulatable elements are included.
  8. 8. Construction  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 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.  Ligation of necessary centromeric sequences for mitotic stability.
  9. 9.  Ligation of Autonomously Replicating Sequences (ARS) providing an origin of replication to undergo mitotic replication Allows the plasmid to replicate extrachromosomally, but renders the plasmid highly mitotically unstable, and easily lost without the centromeric sequences.  Ligation of artificial telomeric sequences to convert circular plasmid into a linear piece of DNA.  Insertion of DNA sequence (up to 1000kb).  Transformation yeast colony.
  10. 10. Homologous Recombinantion  In recombinationally-targeted YAC cloning, YACs are assembled in vivo, by recombination, and not by ligation in vitro  Recombination takes place between a target segment of the exogenous DNA, and the YAC vector that contains sequences homologous to these targets  Firstly two YAC vectors arms and the exogenous segment (flanked by desired sequences) are into the yeast cell  Then followed by recombination  Results in formation of desired stable YACS.
  11. 11. Recomentional Targeting cloning with YAC vectors. A yeast strain is transformed with a mixture of the two YAC vector arms and large fragments of DNA. Recombination in vivo results in the formation of a specific YAC clone. The two YAC vector arms are derived from linearized plasmids that contain targeting segments that are homologous to the termini of the DNA segment that is to be cloned.
  12. 12. 2 1 3 4 Application Applications of YACS include generating whole DNA libraries of the genomes of higher organisms To identifying essential mammalian chromosomal sequences necessary for the future construction of specialized mammalian artificial chromosomes (MACS) Another major application of YACS is in the study of regulation of gene expression by cis-acting. controlling DNA elements That are present either upstream or downstream of large eukaryotic genes, after the transfer of these YACS from yeast to mammalian cells
  13. 13. YAC Genomic Libraries  It is possible to construct YACs with megabase-long inserts using the precise homologous recombination  Original DNA sequence of a eukaryotic genome fragment more than 2Mb in size can be maintained in a single YAC vector
  14. 14. Research…!! Human artificial chromosome (H for measuring chromosome inst (CIN) and identification of gene required for proper chromosom transmission . BY : - Natalaykouprina,Mikhail Liskovykh Nikolai Petrov, Vladimir Larionov . Journal name :- Experimental Cell Research
  15. 15.  CHROMOSOMAL INSTABILITY CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole chromosomes. In the vast majority of human tumors the molecular basis of CIN remains unknown.  The development of a conceptually simple colony color sectoring assay that measures yeast artificial chromosome (YAC) loss provided a powerful genetic tool to assess the rate of chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a human artificial chromosome (HAC)-based assay has been recently developed and applied to quantify chromosome mis-segregation events in human cells. Abstract
  16. 16.  This assay allowed identification of novel human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1.  The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR- based libraries to identify the complete spectrum of CIN genes.  This will reveal new insights into mechanisms of chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
  17. 17. References :- https://www.slideshare.net/gurya87/yeast-artificial-chromosomes-yacs-44970900 https://www.slideshare.net/vidhyakalaivani29/yeast-artificial-chromosomes https://www.sciencedirect.com/science/article/pii/S0014482719306937 https://www.sciencedirect.com/journal/experimental-cell-research
  18. 18. CREDITS: This presentation template was created by Slidesgo, including icons by Flaticon, infographics & images by Freepik THANKS !! Thank you for Your Patience , All The BEST…!!

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