Shuttle vectors

Certain vectors act like a shuttle. A vehicle carrying people or goods regularly between the two places is known as a shuttle. Similarly, in the molecular world, certain vectors act like the shuttle. The shuttle vectors get transferred into two hosts. They move the desired genes between the organisms. Hence, shuttle vectors survive in more than one cell type. The shuttle vectors often shuttle between E. coli and yeast. Development of a shuttle vector involves plasmid isolation from the organism of interest and identifying its replicon. They have a very high transformation frequency. Recombinant plasmid libraries involve utilization of the shuttle vectors.

Components of a shuttle vector:

A shuttle vector contains separate origins of replication. Hence, these vectors have two origins of replication such as E. coli and yeast origins of replication respectively. There is a reason for having two origins of replication. The prokaryotic origins do not work in that of eukaryotic cells. Also, the required DNA sequence differs. Next, it should have a centromere sequence to allow the right partition of the plasmid in the yeast. It allows correct segregation of shuttle vectors at the time of cell division. The centromere sequence is known as a cen sequence. 
Multiple cloning site in the shuttle vector is nothing but a restriction site capable of getting cleaved with many restriction enzymes. Other genes present in a shuttle vector include antibiotic resistance gene and leucine biosynthesis gene. Selection of a plasmid for a yeast cell requires a leucine biosynthesis gene. The yeast cell starves without the adequate levels of leucine. Hence, the survival of the yeast requires the presence of leucine biosynthesis gene. Transformation of a shuttle vector into the yeast primarily involves rupturing the yeast cell wall using enzymes and calcium chloride. This step is important because the yeast cell wall consists of polysaccharide which creates a hindrance to the uptake of foreign DNA. Culturing the yeast in a suitable medium regenerates the cell wall.

Image: Shuttle vector

Introduction of a shuttle vector into the mammalian cells:

Designing of the shuttle vectors depends on the requirement of the mammalian cells. Later on, they get introduced into the mammalian cells and integrate with the mammalian chromosome. The mammalian shuttle vectors must have specific sequences associated with mammalian DNA. The first cloning experiment in mammals involved SV40 viral vectors. The SV40 viral vectors help the plasmid in replicating in the mammalian cells without integrating into the genome. The mammalian artificial chromosomes (MACs) consist of the telomeric and Centromeric sequences for segregation of markers.

Lambda shuttle vectors:

The transgenic animal experiments involve phage shuttle vectors. Hence, the animal cells readily accept the phage shuttle vectors. The transgenic animal undergoes a treatment with an exogenous agent leading to mutated reporter genes. Hence, the shuttle vector now contains the mutated genes. Detection of a mutated gene requires a single step in vitro packaging reaction. It involves a small amount of DNA prepared from a mouse tissue followed by extraction of shuttle vectors from the host. It gets packed into the infectious phage particle through in vitro packaging reaction. The bacteriophage shuttle vector gets integrated as a multi-copy concatemer. Treatment with a putative mutagen helps to remove the shuttle vectors from the chromosome with the in vitro packaging of the vector DNA into phage particle. The shuttle vector gets packed into the phage because of the presence of cos regulatory elements. Bacteriophage with shuttle vectors infect the bacteria followed by visualization of plaques. Mutated gene analysis involves blue-white screening of the colonies. An X-gal indicator compound distinguishes recombinants and non-recombinants.

Advantages of transgenic shuttle vectors:

They represent a cost-effective test for in vivo mutagenicity. It is easy to detect mutations in any tissue including the sperm and the ova. Shuttle vectors include commercial kits designed for various experiments.

Role of shuttle vectors in gene cloning by complementation of mutations:

A complementation test helps in determining the presence of mutants in a gene or a functional unit. The test distinguishes the wild-type and the mutant phenotypes. Two recessive mutations on the opposite chromosomes indicate a mutant phenotype. Hence, it results in no complementation. Two recessive mutations present in the same chromosome indicate a wild-type phenotype. Hence, complementation occurs. Two mutants present on the same segment arise due to positive complementation. A diploid or a heterokaryon contributes to studying complementation of genes. Gene cloning by complementation of mutations includes a classic example of yeast genes. It includes an example of yeast-E. coli shuttle vector. These vectors replicate autonomously and in the yeast and E. coli cells. The yeast-E. coli shuttle vectors consist of beta-lactamase gene, an ampicillin resistance gene, pUC 18 sequence, and multiple cloning sites. Loss of beta-galactosidase function indicates appropriate DNA insertion in the multiple cloning sites. A genomic library consists of DNA fragments obtained from wild-type yeast. A genomic library transforms a mutant host yeast strain. It consists of two mutations. One mutant helps in selecting the transformants. It is known as ura3. Another mutation helps in searching the wild-type gene mutation. The wild-type gene required for the biosynthesis of arginine is known as ARG1 gene. The mutation in the gene known as arg1 mutation helps in the complementation test. Arginine amino acid plays an important role in yeast cell growth. Yeast cells lacking arginine or enzyme responsible for arginine biosynthesis do not grow well.

Hence, arg1 mutant strains consist of an inactive enzyme for arginine biosynthesis. The shuttle vector helps in preparing the genomic library. It consists of URA3 as a selectable marker and yeast DNA consisting of the wild-type gene (ARG1 gene). The mutant yeast cells consist of ura3 arg1 mutant genes. Due to the transforming cells, some cells lacking the normal ARG 1 gene receive it from the genomic library having ARG 1 gene (which is wild-type). Such transformants express ARG 1 gene and synthesize arginine in their cells. Hence, they grow on a minimal medium in the absence of arginine in spite of having the defective arg1 gene. The ARG 1 gene overcomes the mutant arg1 gene by complementation. Hence, yeast-E. coli shuttle vectors helped in preparing genomic libraries for identifying complementation.

Yeast Integrative Plasmid (YIP 5) as a shuttle vector:

YIP 5 yeast vector consists of E. coli plasmid with a copy of URA3 gene. Hence, it is a shuttle vector used in both E. coli and yeast. It is difficult to clone the yeast cells and generate a large number of clones. Hence, the YIP 5 shuttle vectors solve the problem. The YIP 5 or a Yeast Integrative Plasmid consists of the ampicillin resistance gene (Amp^R gene), a tetracycline resistance gene (Tet^R gene), URA3 yeast gene and E. coli origin of replication. The size of the YIP 5 vector is 5.5 kb. Since there is an E. coli origin of replication, it is possible to construct recombinant YIP 5 molecules, before transferring them into the yeast cells. Without the origin of replication, the vector does not propagate independently inside the yeast cells but survives upon integration into a yeast chromosome. Homologous recombination between the URA3 gene in the vector and the chromosomal copy of the same gene results into the integration. Hence, it replicates along with the host chromosome.

Yeast episomal plasmid vector:

A high copy number plasmid gets combined with another plasmid part known as pBR322 for constructing a shuttle vector. A yeats episomal vector consists of segments from pBR322 carrying origin of replication, ampicillin resistance gene, a selectable marker gene of yeast, and REP gene for replication.

Animal viral vector:

The growth of the basic animal vector involves an E. coli cell because of its convenience. The mammalian vectors are shuttle vectors having prokaryotic sequences for the propagation of the vector in E. coli and expression units of eukaryotes (mammals). 

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[3] Gene Cloning and Manipulation, Christopher Howe

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.

[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh
[6] IGenetics, Peter Russell

© Copyright, 2018 All Rights Reserved.

A Review on Restriction Enzymes

The enzymes involved in cleaving the specific regions in the genome are known as restriction enzymes. They are also known as molecular scissors. They are used widely in molecular biology. A restriction site is a specific region in the DNA consisting of bases cleaved by these enzymes. The process in which the restriction enzyme cleaves at a particular site is known as restriction digestion. The products in the form of the nucleic acid fragments are known as the restriction digests. They are known to play a crucial role in mapping, sequencing, and cloning. Most of the restriction enzymes belong to the class of endonucleases. It is an enzyme cleaving or hydrolyzing the phosphodiester bond within a DNA. The knife and fork model of DNA replication represents a triangular structure. This triangular structure is a sign of an endonuclease enzyme. The DNA molecule consists of internal phosphodiester bonds which get cleaved through restriction endonucleases.

In somatic tissue or cells, these enzymes are capable of hydrolyzing DNA by introducing double-strand breaks. Naturally occurring restriction endonucleases are known as the cell protectors. They protect the cells from viral infections. For research and analysis purpose, the extraction source of the enzymes involves microorganism or chemical synthesis.

Properties of restriction endonucleases:

A restriction enzyme recognizes a restriction site at 3’ carbon and phosphate group of a phosphodiester bond. The resultant fragments have 5’ phosphate group and 3’ hydroxyl group. Bacteria and green algae chlorella have naturally occurring restriction enzymes. Bacteria undergo a process of methylation and modify the restriction sites. Hence, the bacterial cells get protected from the action of restriction enzymes. Arbor, Nathans, and Smith received a Nobel prize in discovering the restriction enzymes. Many restrictions enzymes select sites having an axis of symmetry. The number of cuts made by restriction enzymes gets determined by the frequency of occurrence of the restriction sites. The restriction sites occur in a randomly distributed base pair. The formula (1/4)ⁿ indicates the probability of the occurrence of the restriction site with a 50% GC content. (Where n indicates the no. of nucleotide pairs in the recognition sequence).

Image: Restriction digestion (Treatment of the DNA sample with restriction endonucleases leads to the synthesis of fragments of different sizes.

Nomenclature:

The nomenclature of the restriction enzymes involves the organisms from which they were isolated. The first letter belongs to the genus and the second and third letter from the species. It involves an italic or underlined font followed by Roman numerals. For example, EcoRI is an example of a restriction enzyme obtained from E. coli strain RY13. Thus, ‘Eco’ belongs to E. coli, and RI belongs to the strain RY13. There are four classes of the restriction enzymes based on the recognition sequences.

Enzymes with no. of recognition sequences in base pairs	Property
4	Symmetrical sequences
6	Symmetrical sequences
8	Symmetrical sequences
spacer sequence	Non-specific sequences

Table: Enzymes and their properties 

Constructing a restriction map:

A restriction map is a kind of a physical map. It consists of a piece of DNA with restriction sites specific for endonucleases. The number of bases decides the sites of separation. Restriction enzymes create two types of ends such as sticky or blunt ends. A staggered cut in the restriction site with symmetrical nucleotide sequence generates sticky ends. There are two types of ends such as 5’ or 3’ overhanging ends. When a restriction enzyme cuts at a symmetrical nucleotide sequence of a restriction site between the two base pairs, the blunt ends get created. When the DNA gets digested with the restriction enzymes, and the resultant fragments get separated on a gel, the banding patterns are visible after staining. They are known as restriction fragment length polymorphisms (RFLPs). Sometimes the restriction sites vary in different individuals. A restriction enzyme cleavage site present in an individual may be absent in another individual. RFLPs help in mapping the genes or polymorphic sites. However, the non-polymorphic sites get missed. Restriction mapping solves this problem. A single restriction enzyme or combination of restriction enzymes cleave the DNA efficiently. Thus we obtain the fragments of different sizes measured in kilobases. The electrophoretic gel separates the fragments based on their sizes. Autoradiography helps to visualize the bands and cut the desired portion of the gel to take the DNA fragment.

Cloning Vectors:

A cloning procedure primarily uses a piece or a fragment of DNA obtained from the restriction digestion. The fragment of DNA gets inserted into the vector. The technique involves two main steps. First, the plasmid gets cleaved at a unique restriction site with an appropriate restriction enzyme. The second step involves the insertion of a piece of DNA cut with the same enzyme. The procedure further involves mixing of the cloning vector and a DNA fragment. It results into base pairing and annealing the two single-stranded ends. An enzyme known as DNA ligase acts as a sealing agent. It seals the gaps formed due to a phosphodiester bond.

Applications of restriction enzymes:

1.     DNA typing:

DNA fingerprinting or DNA typing determines the paternity and maternity of an individual. It helps in crime investigation. The establishment of variability in the ethnic groups involves DNA typing. Endangered species determination and genetic variability studies involve DNA typing techniques. Forensic medicine largely involves DNA fingerprinting techniques. Initially, the DNA obtained from the suspect individual gets a treatment with the restriction enzymes. The restriction digests obtained from the procedure include DNA fragments of different sizes. The fragments get separated as per their sizes in an electrophoretic apparatus. Using a Southern blot and a probe hybridization technique, visualization of the DNA bands of the suspect involves autoradiography.

It is not possible to separate the DNA fragments without the restriction enzymes. Hence, obtaining the information of the suspect invites difficulties. In the detection of paternity or maternity cases, the DNA obtained from the child and his alleged parents have to undergo restriction enzyme treatment. Autoradiogram shows a banding pattern of each sample as a DNA fingerprint.

2.     DNA molecular testing:

Genetic testing such as disease gene detection requires restriction enzyme treatment. The requirement of the restriction enzymes using PCR determines the presence of microsatellites. RFLP analysis and restriction mapping utilize the restriction enzymes. Detection of genes associated with diseases such as sickle cell anemia, phenylketonuria (PKU), and others become easy to detect.

3.     Gene Cloning:

The isolated DNA gets cleaved through RE treatment. The fragments obtained through RE treatment mix with the cloning vector and get integrated into the restriction sites in the cloning vectors. The cloning vector gets transformed into the host and allowed to replicate. The process is known as molecular cloning. Identical copies of DNA of interest obtained from the above process are known as clones. Expression of genes, synthesis of protein products, gene therapy, and many other important applications require gene cloning.

4.     Preparing recombinant vectors:

Restriction enzyme treatment targets multiple cloning sites or the restriction sites in a plasmid or any other vector. The fragment of interest gets inserted into the multiple cloning sites or a polylinker and gets transformed into the host. The host (bacterial cells) plated on a suitable medium show the growth of the recombinant colonies.

5.     Industrial applications:

It is possible to grow plant hybrids showing resistance to herbicides, pesticides, and drought tolerant varieties. Restriction digests obtained through restriction enzyme treatment get inserted into a vector. Then the DNA gets microinjected into the animal’s pronucleus to obtain transgenic animals. Hence, it is possible to produce a recombinant protein product using a transgenic animal. Example, the gene of interest expressed in a mammary tissue helps protein secretion in the milk.

6.     Genomic and cDNA libraries:

 The recombinant DNA libraries consist of a collection of clones with the desired DNA sequence. The fragments subjected to restriction enzymes get cloned into a vector to obtain a collection of clones known as genomic or cDNA libraries. 

References:
[1] Essential Genetics: A Genomics Perspective, Hartl, Elizabeth W. Jones
[2] Molecular Biology, David P. Clark, Nanette J. Pazdernik
[3] Genetic Engineering, Verma P.S. & Agarwal V.K.
[4] Molecular Biology and Genomics, Cornel Mulhardt

© Copyright, 2018 All Rights Reserved.