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Artificial chromosomes - YAC and BAC
- 1. Bacterial and yeast artificialchromosomes PRESENTATION BY N.CHITRA. M.Sc.,Microbiology ( 2nd year ) ST.PETER’S INSTITUTE OF HIGHER EDUCATION AND RESEARCH, AVADI, CHENNAI – 54.
- 2. Yeast Artificial Chromosomes(YACs) Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast. It is a human-engineered DNA molecule used to clone DNA sequences in yeast cells. They are the products of a recombinant DNA cloning methodology to isolate and propagate very large segments of DNA in a yeast host. By inserting large fragments of DNA, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. The amount of DNA that can be cloned into a YAC is, on average, from 200 to 500 kb. However, as much as 1 Mb (mega, 106) can be cloned into a YAC.
- 4. Structure A yeast artificial chromosomecloning vector consists of two copies of a yeast telomeric sequence (telomeres are the sequences at the ends of chromosomes), a yeast centromere, a yeast ars (an autonomously replicating sequence where DNA replication begins),and appropriate selectable markers.
- 6. Working Principle ofYeast Artificial Chromosomes The yeast artificial chromosome, which is often shortened to YAC, is an artificially constructed system that can undergo replication. The design of a YAC allows extremely large segments of genetic material to be inserted. Subsequent rounds of replication produce many copies of the inserted sequence, in a genetic procedure known as cloning. The principle is similar to that for plasmids or cosmids. The experimenter introduces some typical elements that are necessary for correct replication. In the case of YACs, the replication origins are the centromeres and telomeres of the yeast chromosomes, which must be inserted into the DNA being cloned. The constructs can be transformed in yeast Spheroplast and are then replicated there. In contrast to the vectors, YACs are not circular; they are made of linear DNA.
- 9. Process YAC vectoris initially propagated as circular plasmid inside bacterial host utilizing bacterial ori sequence. The circular plasmid is cut at a specific site using restriction enzymes to generate a linear chromosome with two telomere sites at terminals. The linear chromosome is again digested at a specific site with two arms with different selection marker. The genomic insert is then ligated into YAC vector using DNA ligase enzyme. The recombinant vectors are transformed into yeast cells and screened for the selection markers to obtain recombinant colonies.
- 11. Advantages of YeastArtificial Chromosomes Yeast artificial chromosomes (YACs) provide the largest insert capacity of any cloning system. Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs (yeast episomal plasmids), have advantageous over bacterial artificial chromosomes (BACs). They can be used to express eukaryotic proteins that require post-translational modification. A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unstable, underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones. It is possible to reintroduce YACs intact into mammalian cells where the introduced mammalian genes are expressed and used to study the functions of genes in the context of flanking sequences.
- 12. Uses of YeastArtificial Chromosomes Yeast artificial chromosomes (YACs) were originally constructed in order to study chromosome behavior in mitosis and meiosis without the complications of manipulating and destabilizing native chromosomes. YACs representing contiguous stretches of genomic DNA (YAC contigs) have provided a physical map framework for the human, mouse, and even Arabidopsis genomes. YACs are extremely popular for those trying to analyze entire genomes.
- 13. Limitations of YeastArtificial Chromosomes A problem encountered in constructing and using YAC libraries is that they typically contain clones that are chimeric, i.e., contain DNA in a single clone from different locations in the genome. YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of the cloned DNA. As a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA have occurred. The efficiency of cloning is low (about 1000 clones are obtained per microgram of vector and insert DNA). YACs have been found to be less stable than BACs. The yield of YAC DNA isolated from a yeast clone containing a YAC is quite low. The YAC DNA is only a few percents of the total DNA in the recombinant yeast cell. It is difficult to obtain even 1 μg of YAC DNA. The cloning of YACs is too complicated to be carried out by a lone researcher.
- 14. Schematic representation of retrofittinga circular YAC into a BAC using BRV vectors.
- 15. Bacterial Artificial Chromosomes BAC vectors are plasmids constructed with the replication origin of E. coli F factor, and so can be maintained in a single copy per cell. These vectors can hold DNA fragments of up to 300 kb Recombinant BACs are introduced into E. coli by electroportation Once in the cell, the rBAC replicates like an F factor
- 17. Characteristic features ofBAC vectors The original BAC vector, pBAC108L, is based on a mini-F plasmid, pMBO131 (Figure 1) which encodes genes essential for self-replication and regulates its copy number inside a cell. The unidirectional self-replicating genes are oriS and repE while parA and parB maintain copy number to one or two for each E. coli genome. Multiple cloning sites is present, flanked by “universal promoters” T7 and SP6, all flanked by GC-rich restriction enzyme sites for insert excision. Presence of cosN and loxP sites(cloned in by bacteriophage l terminase and P1 Cre recombinase, respectively) permits linearization of the plasmid for convenient restriction mapping. There is a chloramphenicol resistance gene for negative selection of non- transformed bacteria. Vector is 6900 bp in length and is capable of maintaining insert DNA in excess of 300 kilobases (kb)
- 18. Other BAC Vectors There have been many modifications done to increase the ease-of-use as well as for use in specific systems and situations. pBeloBAC11 2 and pBACe3.6 modified BAC vectors based on pBAC108L They are commonly used as a basis for further modification.
- 19. pBAC108L The first BAC vector. Hasa set of regulatory genes, OriS, and repE which control F- factor replication, and parA and parB which limit the number of copies to one or two. pBAC108L lacked a selectable marker for recombinants. Thus clones with inserts had to be identified by colony hybridization
- 20. pBeloBAC11 The primarycharacteristic of this vector is the addition of a lacZ gene into the multiple cloning site 2 of pBAC108L. Plates supplemented with X-gal/IPTG, an intact lacZ gene encodes b-galactosidase which catalyses the supplemented substrate into a blue substance. Successful ligation of insert DNA into the vector inactivates lacZ, generating white colonies, indicating the presence of a successful vector-insert ligation. It is still a low-copy number plasmid due to presence of parA and parB. Size of vector is 7507 bp in length.
- 22. pBACe3.6 This vectoris based on pBAC108L but is more highly modified than pBeloBAC11. In order to overcome the issue of low plasmid copy numbers, the P1 replicon in F’ was deleted and a removable high copy number replicon originating from an inserted pUC19 was introduced. This vector contains 2.7 kb pUClink stuffer fragment which is flanked by two sets of six restriction sites within a sacB region. Levansucrase, a product of sacB gene, which converts sucrose (sup- plemented in the media) to levan, which is toxic to E. coli host cells. Hence, if the vector is re-ligated without an insert, the functional sacB produces levansucrase and the cells die before forming colonies. Successful ligation of an insert into the vector increases the distance from the promoter to the coding region of sacB, disrupting toxic gene expression in the presence of sucrose.
- 25. In additionto this vectors, there are many specialized BAC vectors carrying a variety of different combinations of drug resistance genes. Besides, many different selection mechanisms and markers are available. Modifications of cloning sites (unique restriction endonuclease sites) are also common as per the addition of genes and promoters specific to different strains of bacteria
- 26. Development of BAC vector Bacterialplasmid is cut within lacZ gene with restriction enzymes. Gene of Interest is cut out using restriction enzyme. F+ Plasmid and gene of interest ligate. BAC is electroporated into E.coli cells. Cells are plated on medium. White colonies are composed of transformed lacZ cells.
- 28. Advantages of BACVectors The large size of BACs help to minimize site of integration effects, a phenomenon which has been defined as endogenous sequences (such as gene coding regions and distal regulatory elements) to be disrupted, and to produce potentially undesirable phenotypes in gene cloning technology. Endogenous gene expression more accurately than other cloning systems. The human genome BACs consist of the full gene structure(which play very important role in gene regulation). Therefore the human genome BACs will ensure full mRNA processing and splicing when genes are transcribed, and produce the full complement of protein isoforms once mRNAs are translated. It can be transfected and expressed in mammalian cell lines even if transfection efficiency and copy numbers are low.
- 29. Disadvantages of BACvectors A construct containing a large genomic fragment is likely to contain non-related genes which may lead to indirect, non-specific gene expression and unanticipated changes in the cell phenotype. Recombinant BAC constructs can be time- consuming and labor-intensive. The large size BAC DNA constructs are more easily degraded and sheard during manipulation before transfection.
- 31. Applications of BACvectors BACs are useful for the construction of genomic libraries but their range of use is vast. It spans from basic science to economically rewarding industrial research, and fields as prosaic as animal husbandry. In genomic analyses, it helps in determining phylogenetic lineage det between species. Helps in study of horizontal gene transfer and since bacterial genes are usually clustered, the ability of BAC vectors to accommodate large inserts has allowed the study of entire bacterial pathways. By isolating DNA directly from soil or from marine environments, the “metagenomes” of those organisms which are either uncultureable or are termed viable but uncultureable can be cloned into BAC vectors and indirectly studied.
- 32. In industrialresearch fields where BAC vectors are invaluable tools in cataloguing novel genomes is in the discovery of novel enzymes. Work has been done on identifying enzymes that are involved in biopolymer hydrolysis or even radioactive waste management. BAC vectors have been instrumental in studying large double stranded DNA viruses both from an academic point of view and as a tool to develop improved vaccines. In genomic research, high throughput determination of gains and losses of genetic material using high resolution BAC arrays and comparative genomic hybridization (CGH) have been developed into the new tools for translational research in solid tumors and neurodegenerative disorders.
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- 34. YAC is agenetically engineered chromosome with the use of yeast DNA for the purpose of cloning. Gender YACs were designed to clone large fragments of genomic DNA into yeast. Insert Length YACs can contain megabase-sized genomic inserts.(1000 kb – 2000 kb). Construction YAC DNA is difficult to purify intact and requires high concentration for generating YAC vector system. Chimerism YACs are often chimeric. Stability YAC is unstable. Modifications Yeast recombination is very feasible and always remains active. Hence it can generate deletions and other rearrangements in a YAC. Maintenance Manipulating recombinant YACs usually requires YAC to be transferred into E. coli for subsequent manipulation. Hence, it is a laborious process. BAC is a genetically engineered DNA molecule using E. coli DNA for the purpose of cloning. Gender BACs were developed for cloning large genomic fragments into Escherichia coli. Insert Length BACs can carry inserts of 200–300 kb or less. Construction BAC is easy to purify intact and can be easily constructed. Chimerism BACs are rarely chimeric. Stability BAC is stable. Modifications E. coli recombination is prevented and is turned on when required. Hence, it reduces the unwanted rearrangements in BACs. Maintenance BAC modification occurs directly in E. coli. So there is no need for DNA transfer. Hence, the process is not laborious. DIFFERENCE BETWEEN YAC AND BAC VECTORS
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