Gene Cloning Vectors: BAC & YAC Learning Objectives: by the End of This Section, Student Will Be Able To

Gene Cloning Vectors: BAC & YAC

Learning Objectives:

By the end of this section, student will be able to:

• Identify the need for the design of higher order molecular vectors • Explain the problems encountered while cloning the large insert DNA segments • Appreciate the applications of the vectors for human welfare • Illustrate the construction of BAC and YAC • Differentiate between BAC and YAC and also other vectors

The world has been in the grip of Covid-19 pandemic and its causative infectious agent SARS-Cov-2 virus since December, 2019. Across the globe, the people are looking towards the research community and research establishments of molecular Biotechnology for the right vaccine that can potentially give protection against the most contagious virus, SARS-Cov-2. The private research establishments like “Add gene” are preparing and making available plasmids containing SARS-Cov-2 sequences for research or vaccine development.

Investigating the aforementioned virus needs viral genome sequencing, the expression of viral proteins in a controlled expression ecosystem, preparation of appropriate host systems, DNA/RNA sequences for integration into vectors, right kind of vectors, sequences of human proteins involved in viral infection in the humans etc. All these require suitable techniques and products of molecular biotechnology.

Some of the important requirements needed for the processes to be carried out by the molecular biologists are the suitable gene cloning vectors. What’s cloning? When we hear about cloning, cloning of Dolly immediately flashes in our mind. Cloning is of different types. They are:

1. Gene Cloning 2. Reproductive Cloning 3. Therapeutic Cloning

Gene Cloning: Producing multiple, identical copies of a gene or a piece of DNA is gene cloning. It is, otherwise, called molecular cloning.

Background of Gene/Molecular Cloning:

Herbert Boyer and Stanley Cohen first demonstrated the complete molecular cloning process in 1973 when they successfully cloned genes from the African clawed frog (Xenopus laevis) into a bacterial plasmid which was then introduced into the bacterial host E.coli. Gene cloning is a set of methods carried out in series to construct a recombinant DNA and transform it into a host organism. This entire procedure makes use of many molecular tools that are derived from microorganisms.

Tool Box for Gene Cloning:

1. Gene of Interest

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

2. Cloning Vector 3. Restriction Endonucleases 4. DNA Ligase 5. Host cell/organism

Cloning Vector

Vector is, actually, a vehicle to carry something. Gene Cloning Vector is a piece of DNA that can be stably and independently maintained as an extra-chromosomal element in a host cell and which can carry a foreign/passenger gene for cloning purposes. There are other kinds of vectors which are employed for various purposes in molecular biology. They are: expression vectors and shuttle vectors.

Gene Cloning vectors employed in Genetic Engineering

❖ Plasmids ❖ Phage DNA ❖ Cosmids/Fosmids ❖ Bacterial Artificial Chromosome ❖ Yeast Artificial Chromosome ❖ Human Artificial Chromosome

The aforementioned list of gene cloning vectors includes both naturally occurring and designed or engineered vectors.

Characteristics of an ideal Gene Cloning Vector

❖ Origin of Replication ❖ Cloning Sites ❖ Genetic Marker ❖ High Copy number ❖ Easy for transformation ❖ Stable maintenance in host

The gene cloning vectors, like plasmids, occur naturally in most of the genera of bacteria and also yeast. But those plasmids usually lack some of the properties like unique cloning site for restriction enzyme and selectable marker. This is why the molecular biologists started designing the vectors by securing various elements available in different vector sources in order to suit their needs.

Designed/Engineered Cloning Vectors:

The natural vectors lack one or few of the ideal properties that the researchers require to suit their needs. In such a case, they started designing to integrate features like high copy number, MCS, reporter element, marker genes etc. this has led to the production of many vectors since the first designed plasmid vector, pBR322 was made, almost five decades ago. Still the engineering keeps going on for the design and synthesis of new vectors.

When the DNA to be cloned is exceptionally large, the vectors got to be designed in such a way that they harbor a large chunk of the sequence. When the genomic libraries were to be prepared for higher

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

organisms, the existing natural and designed vectors could not be suitable. This led to the design of the following new vectors:

1. BAC 2. YAC

BACTERIAL ARTIFICIAL CHROMOSOME (BAC)

One of the recently designed plasmid vectors is BAC. It finds so many applications ranging from molecular biology research to drug and vaccine development. BAC is an engineered version of F’plasmid of E.coli. The F’ Plasmid is incompatibility group involved in E.coli conjugative ability and chromosomal transfer, which can exist as an extra-chromosomal element. BAC is a circular ultra-low copy number plasmid. The elements used in the construction of BAC have been sourced from different organisms. The original BAC vector, pBAC108L, is based on a mini-F plasmid, pMBO131 of E.coli strain ER2420S. The original BAC vectors are 7.4 kb while others are 8-9kb in length. Artificial chromosomes are designed in vitro from defined constituents obtained from various natural sources. BAC insert size ranges from 50 kb to as large as 300 kb.

Construction of BACs:

The following are the important constituents of most of BACs:

➢ The Ori S and rep E were sourced from the E.coli F’plasmid. The genes Ori S and repE are unidirectional self-replicating genes. The gene rep E acts as an initiation factor for the assembly of replication complex at Ori S. ➢ The genes par A, par B and par C part of sop ABC partitioning system carry out the function of segregation of plasmids during cell division. They ensure that each daughter cell gets one plasmid. ➢ The cloning region includes the following features: unique cloning sites BamHI, SphI and HindIII in a lacZα gene allowing for insert screening by α-complementation i.e. Blue/White Screening. ➢ Some BACs have Chloramphenicol (CMR) constituent as selectable marker ➢ The vector was added with MCS flanked by the universal promoters T7 and SP6. This whole sequence is flanked on either side by GC rich RE sites for insert excision. T7 and SP6 phage promoters help in the transcription of inserted genes ➢ cos N has been introduced from phage Lambda. It is the site at which the enzyme terminase makes a staggered cut for packaging

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

➢ loxP sites were taken from P1 bacteriophage. The Cre recombinase acts on this site to permit linearization of the plasmid for convenient restriction mapping.

Since the inception of BAC cloning system, the BACs have undergone constant modifications intended for increasing ease-of-use and for specific purposes and systems. The following are different BAC vectors designed chronologically.

The basic BAC: pBAC108L

This was, first, designed in 1992 by Melsimon and his colleagues. It is capable of cloning and stably maintaining DNA fragments >300 kb in E.coli, but lacks selectable marker for recombinants. Thus, the recombinant clones in pBAC108L must be identified by colony hybridization, which is not well-suited for library development. But the other genes for self-replication; copy number and segregation of daughter plasmids are present. pBeloBAC11:

This was derived from pBAC108L by replacing the cloning site region of pBAC108L with a DNA fragment containing the lacZ gene and cloning sites embedded into lacZ for colony color selection, blue for non- recombinants and colorless for recombinants. pBeloBAC11 has two EcoRI sites, one in the lacZ gene and the other in the transformant selection marker chloramphenicol (CMR) resistance gene. However, pBeloBAC11 is still a low-copy number plasmid from the presence of parA and parB. This is of 7.4 kb size.

pECBAC1:

The two widely used BAC vectors are pBeloBAC11 and pECBAC1. The EcoRI site in the chloramphenicol resistance gene of pBeloBAC11 was destroyed to facilitate cloning large segments of DNA. Therefore, pECBAC1 is capable of cloning DNA fragments generated with EcoRI, in addition to those generated with HindIII and BamHI as pBeloBAC11. pBAC e 3.6:

The latest BAC i.e. pBACe3.6 is also based on pBAC108L but is more highly modified than pBeloBAC11. In this BAC, low copy number issue has been addressed. An insert from p UC19 was introduced for high copy number. A different selection process was added by inserting sacB gene into which the cloning sites were carved. The size is 11.49 kb. Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

Applications of BAC Vectors:

1. Quicker production, validation and release of new vaccines through reverse genetic approaches. 2. Creation of physical mapping of large genomes 3. Preparation of genomic libraries of higher organisms 4. The cloning and study of genomes of several large DNA and RNAviruses 5. BACs act as infectious clones which made possible the study of Human Cyto Megalo Virus, Herpes viruses, Corona viruses etc. 6. Genome sequencing of higher organisms as was done in Human Genome Project.

7. BACs are very useful in the making of transgenic animals with segments of human DNA 8. Applications in investigation of genetic disorders and gene therapy.

YEAST ARTIFICIAL CHROMOSOME (YAC)

Yeast Artificial Chromosomes are genetically engineered special linear DNA cloning vectors that resemble normal Yeast chromosomes. The host cells are Yeast cells. Basic structural features of YACs were developed from Yeast Centromere shuttle plasmids i.e. YCp series. These YACs can clone DNA insert of up to 2000 kbp. These are Yeast DNA vectors of small size (12kb). They can be cloned and selected in host cells like E.coli and Baker’s Yeast, Saccharomyces cerevisiae. So they are shuttle vectors. They are circular in E.coli and linear in budding yeast cells. It was first described by Murray and Szotack in 1983.

Construction of YAC:

➢ b-lactamase gene for screening (AmpR) and pMB1 origin of replication were taken from bacterial sources ➢ The vector includes Autonomously Replicating Sequence (ARS1) that contains Origin of Replication from Yeast source ➢ CEN4 DNA sequence was integrated into the vector from Centromere region of Chromosome 4 of Yeast. This sequence behaves like Yeast centromere genetically and it confers mitotic stability to the vector ➢ URA3 Selectable Marker for selection on media deficient in Uracil/Uridine ➢ Yeast HIS3 is flanked by Telomere sequences which are adjacent to two cloning sites for Bam H1 ➢ SUP4 Suppressor gene for Tyrosine tRNA taken from Yeast contains a recombination site for insert for color screening. Normal cells with suppressor activity don’t appear red and those with vector are red in color

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

Mechanism of Cloning with YAC:

(httpscommons.wikimedia.orgwikiFileConstruction_of_a_YAC_chem114A.jpg)

➢ The plasmid YAC are, first, propagated in E.coli and purified subsequently ➢ First the digestion is made with Bam HI that results in a linear long fragment with two telomeres at the ends ➢ Second digestion happens in SUP4 gene for integrating the insert to be cloned. This creates 2 fragments, one on left and the other on right. The left one contains marker trp1, ARS1 and CEN4 from left to right with telomere on the extreme left. The right fragment has URA3 marker and Telomere from left to extreme right.

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

➢ Then the insert is integrated such that it is flanked by CEN4 on left and URA3 on right side. This linear recombinant vector is transformed into the Yeast cells.

APPLICATIONS OF YAC:

1. Genomic library preparation for higher organisms 2. Identification of essential mammalian chromosomal sequences for future construction of Mammalian Artificial Chromosomes 3. Study of regulation of gene expression by transferring YACs from Yeast to Mammalian cells 4. 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. 5. 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.

LIMITATIONS OF YACs:

➢ Large DNA molecules are fragile and prone to breakage, leading to problems of rearrangements ➢ Higher rate of loss of entire YAC during mitosis ➢ Difficult to separate from other host chromosomes due to similarity in size. It requires sophisticated Pulse Field Gel Electrophoresis (PFGE) ➢ Yield of DNA is not high when YAC is isolated from Yeast cells ➢ Chromosomes are unstable

DIFFERENCES BETWEEN BAC & YAC

YAC BAC Designed with Yeast DNA Designed with E.coli DNA Host cell is Yeast Host cell is Bacterium Insert size 1-2 kb Insert size 100-300kb Difficult to purify, needs high concentration Easy to purify and sophisticated PFGE Chimerism chance up to 40% Rarely chimeric Unstable in host Stable Chance of deletions and rearrangements Unwarranted rearrangements are reduced Propagation in E.coli, first, and transfer to It is done directly in E.coli. Hence not Yeast cells subsequently is laborious laborious

References:

1. https://nptel.ac.in/courses/102103017/pdf/lecture%2035.pdf 2. https://www.nature.com/subjects/genetic-vectors 3. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/vector-molecular- biology 4. https://www.britannica.com/science/recombinant-DNA-technology#ref964476 5. https://en.wikipedia.org/wiki/Vector_(molecular_biology)

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki Gene Cloning Vectors: BAC & YAC

6. Vectors: A Survey of Molecular Cloning Vectors and Their Uses by Raymond L. Rodriguez, David T. Denhardt: Butterworth Publishers, 1988. Shero, J. H., M. K. McCormick, S. E. Antonarakis, and P. Hieter. “Yeast artificial chromosome vectors for efficient clone manipulation and mapping.” Genomics.U.S. National Library of Medicine, June 1991. Web. 25 Mar. 2017. 7. Ramsay, M. “Yeast artificial chromosome cloning.” Molecular biotechnology. U.S. National Library of Medicine, Apr. 1994. Web. 25 Mar. 2017

Dr Karumanchi Bhanu Prakash, Lecturer in Zoology, GDC Addanki