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10.2: Plasmid nomenclature - Biology

10.2: Plasmid nomenclature - Biology


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Proper nomenclature is important for distinguishing plasmids. In our experiments:

pYES2.1-MET1 would designate the S. cerevisiae MET1 gene cloned into pYES2.1.

pYES2.1-Met1 would designate the S. pombe Met1 gene cloned into pYES2.1. (Recall from Chapter 6 that S. cerevisiae genes are unusual in using 3 capital letters for their names.)

pYES2.1 -lacZ+ would designate the bacterial LacZ gene cloned into pYES2.1. (Note: bacterial gene names are not capitalized. Plus sign indicates wild type gene.)

Note

The S. pombe ortholog of an S. cerevisiae gene may not share the same gene number. Many of the S. pombe genes received their names due to their homology to previously identified S. cerevisiae genes, but some S. pombe genes had been named before their DNA sequences were known. For example, the ortholog of S. cerevisiae MET2 in S. pombe is Met6.

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10.2: Plasmid nomenclature - Biology

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New nomenclature and classification scheme for the neuronal ceroid lipofuscinoses

We provide a new classification for the neuronal ceroid lipofuscinoses (NCLs) that takes into account recent genetic and biochemical advances. This was originally developed by an international group with clinical, molecular genetic, biological, and morphologic interests, further revised by a panel of world experts in the NCLs, and is now updated in light of recent research findings. The aim is to provide young people, carers, and professionals with a diagnostic label that is informative, leads to effective clinical management of symptoms and in the future perhaps a cure, as well as aiding basic scientific and clinical research. We suggest that clinicians should aim to provide every child and family with detailed diagnostic information at clinical, biochemical, and genetic levels where possible, which the new classification allows in a gene-led hierarchical manner. The robustness and applicability of this updated new classification have been independently audited in the clinical setting using a series of patients previously diagnosed with NCL according to standard ultrastructural, biochemical, or genetic criteria.


Viral Integration

As part of its lysogenic cycle, wild-type AAV integrates into the host genome at a specific site, AAVS1 on human chromosome 19. This site is favored due to the presence of a Rep binding element however, random integrations may occur at a much lower frequency. As a replication-incompetent virus, AAV cannot enter the lytic cycle without help. Another virus, such as adenovirus or herpes simplex virus, or a genotoxic agent such as UV radiation or hydroxyurea, is necessary for lytic cycle activation.

When recombinant AAV (rAAV) is used for research purposes, the Rep protein is supplied in trans , eliminating the ability of rAAV to integrate into its preferred site of genomic integration on human chromosome 19, termed AAVS1. Instead, the rAAV genome is typically processed into a double-stranded circular episome through double stranded synthesis. These episomes can concatemerize, producing high molecular weight structures that are maintained extrachromosomally. rAAV are more likely than wild-type AAV to integrate at non-homologous sites in the genome, and do so at about a 0.1% frequency. Nonetheless, the majority of rAAV particles are thought to be maintained in episomes or concatemers.

Episomes differ profoundly from viral particles produced during a lytic cycle. rAAV episomes can develop chromatin-like organization and persist in non-dividing cells for a period of years without damaging the host cell. In contrast, viral particles produced during a lytic cycle are quickly released through cell lysis. Episomal stability enables long-term transgene expression in non-dividing cells and is a key advantage of rAAV.


Application of methylation in improving plasmid transformation into Helicobacter pylori

Helicobacter pylori is an important gastrointestinal pathogen. Its strains possess different levels of powerful restriction modification systems, which are significant barriers to genetic tools used for studying the role of functional genes in its pathogenesis. Methylating vectors in vitro was reported as an alternative to overcome this barrier in several bacteria. In this study we used two H. pylori-E. coli shuttle plasmids and several single/double-crossover homologous recombination gene-targeting plasmids, to test the role of methylation in H. pylori transformation. According to our results, transformants could be obtained only after shuttle plasmids were methylated before transformation. It is helpful in gene complementation and over-expression although at a low frequency. The frequency of gene-targeting transformation was also increased after methylation, especially for the single-crossover recombination plasmids, the transformants of which could only be obtained after methylation. For the double-crossover recombination targeting plasmids, the initial yield of transformants was 0.3-0.8 × 10 2 CFUs per microgram plasmid DNA. With the help of methylation, the yield was increased to 0.4-1.3 × 10 2 CFUs per microgram plasmid DNA. These results suggest that in vitro methylation can improve H. pylori transformation by different plasmids, which will benefit the pathogenic mechanism research.

Keywords: Helicobacter pylori Methylation Plasmid Transformation.


Methods

Molecular biology

PCR fragments were generated with Phusion DNA Polymerase (Finnzymes) or Taq Plus Precision (Stratagene). Primers and templates are depicted in Supplementary Table S1 and in Supplementary Methods. These fragments, including the regulatory elements, were cloned into the pGEM-T vector (Promega). Fragments were cut from these vectors with SapI (LguI), isolated from TAE agarose gels, purified with the Qiaquick gel extraction kit (Qiagen) and then stored at −20 °C. Donor and acceptor vectors were created in a single ligation step of eight fragments (Supplementary Fig. S1). The consensus sequence and the nomenclature of these plasmids are given in Supplementary Figure S2. The available donor and acceptor vectors are shown in Supplementary Figure S3. Plasmids containing a pUC origin of replication were propagated in TopTen or DH10β cells, and plasmids with an R6Kγ origin of replication in pir+ cells 6 . The genes of interest were cloned into donor and acceptor vectors with T4 DNA ligase or SLIC 12 . Note that existing expression vectors from the pcDNA (Invitrogen) or pEGFP (Clontech) type can be amplified with standard primers and inserted into the desired backbone (Supplementary Fig. S4). The Cre/LoxP recombination reaction was performed at 37 °C for 60 min (New England BioLabs). We recommend using plasmids that were purified with an anion exchange method. Three plasmids can be routinely assembled in a single step. The fourth or fifth plasmid can be inserted in a sequential manner and highly competent cells should be used. Note that there are two possibilities for assembling three plasmids, six possibilities for assembling four plasmids and 24 possibilities for assembling five plasmids. The unpurified Cre reaction mixture was used to transform DH10β or TopTen cells by electroporation. The transformation efficiency of fusions containing four or five plasmids was optimized by culturing the transformed cells overnight at 30 °C in liquid culture containing appropriate antibiotics before plating and/or by using highly competent commercial cells. Plasmids should be analysed by digestion with restriction enzymes. Antibiotics were used in the following concentrations for assemblies of 1–3 plasmids: ampicillin, 50 μg μl −1 kanamycin, 25 μg μl −1 spectinomycin, 50 μg μl −1 chloramphenicol, 4 μg μl −1 and gentamycin, 4 μg μl −1 . Antibiotic concentrations were reduced to 50% when four or five plasmids were assembled. Rearrangement or denaturation of plasmids during growth was eliminated by culturing the bacteria in Luria-Bertani (LB) medium at 30 °C rather than by using 2×YT at 37 °C.

Cell culture

HEK293 and COS cells were maintained at 37 °C with 5% CO2 in DMEM (Amimed) containing 10% fetal calf serum (Amimed), 100 units ml −1 penicillin and 100 μg ml −1 streptomycin (Invitrogen). HAM-F12 (Amimed) was used instead of DMEM for Porcine aortic endothelial cells. Cells were transfected with Fugene HD (Roche) or with calcium phosphate. MultiLabel plasmids were diluted with carrier plasmids (1:3 or 1:5) when the expression level was too high. Stable cell lines were generated with the Flp-In system according to the manufacturer's recommendations (Invitrogen) or by random integration of linearized plasmids. Selection was performed with the following antibiotics and concentrations: G418, 16 μg ml −1 (Calbiochem) hygromycin, 50 μg ml −1 (Roche) zeocin, 100 μg μl −1 (Invitrogen). Homing endonucleases were obtained from New England Biolabs.

Microscopy

Cells were plated on glass coverslips, cultured for 40 h and then fixed with 4% formaldehyde (either directly in the culture medium or in PBS). Samples were washed three times with PBS and then mounted with Citifluor AF1. Imaging was performed on a Leica SP5 laser scanning confocal microscope or on an Olympus IX81 equipped with an Andor iXon EM camera (Supplementary Movie 1). Emission windows of the fluorescent proteins were defined with a construct expressing five non-overlapping fluorescence-tagged subcellular markers. EBFP2 was excited with the 405 nm laser line and emission was collected from 430 to 450 nm (405/430–450). The other fluorescent proteins were analysed as follows: mTFP1 (458/485–510) mCitrine (514/525–545) mCherry (543/585–620) mPLUM (633/640–800). In addition, the spectral mode (xyλ) of the microscope was used to verify the presence of all fluorescent proteins (data not shown). Quantification was performed with LAS AF software (Leica Microsystems). Figures were processed with Photoshop or Imaris software (Bitplane). Gaussian filter, brightness and contrast including gamma adjustment were used to increase the visibility of vesicles. All operations were carried out on the entire set of images.

Western blotting

HEK293 cells were plated in 6 cm dishes at a density of 50% 1 day before transfection. Cells were transfected with FugeneHD according to the manufacturer's recommendations (Roche) and lysed 40 h later with lysis buffer (0.5% Triton X-100, 50 mM Tris–HCl, 100 mM NaCl). The supernatant was used for western blotting after sonification and centrifugation. Antibodies used were rabbit anti-EGFR (Neomarkers, RB-1417, diluted 1:1,000 in 3% BSA/TBST) and mouse anti-myc-tag (9E10, diluted 1:5 in 3% BSA/TBST). As secondary antibodies, alkaline phosphatase-coupled goat anti-mouse (Southern Biotech, 1031-04, diluted 1:10,000 in 3% milk powder/TBST) and anti-rabbit (Southern Biotech, 4050-04, diluted 1:10,000 in 3% milk powder/TBST) antibodies were used, followed by chemiluminescene detection.


3 Main Classification of Vectors (With Diagram)

The point is what we are targeting from our gene of interest — its multiple copies or its protein product.

Depending on these criteria vec­tors are of following two types:

1. Cloning Vectors:

We use a cloning vec­tor when our aim is to just obtain numer­ous copies (clones) of our gene of interest (hence the name cloning vectors). These are mostly used in construction of gene libraries. A number of organisms can be used as sources for cloning vectors.

Some are created synthetically, as in the case of yeast artificial chromosomes and bacte­rial artificial chromosomes, while others are taken from bacteria and bacteriopha­ges. In all cases, the vector needs to be genetically modified in order to accommo­date the foreign DNA by creating an in­sertion site where the new DNA will fit­ted. Example: PUC cloning vectors, pBR322 cloning vectors, etc.

2. Expression Vectors or Expression construct:

We use an expression vector when our aim is to obtain the protein prod­uct of our gene of interest. To get the pro­tein we need to allow the expression of our gene of interest (hence the name expres­sion vector) by employing the processes of transcription and translation.

Apart from the three DNA sequences discussed above (origin of replication, selectable markers and multiple cloning sites), the expression vectors have some special additional se­quences as well.

a. A bacterial promoter, such as the lac promoter. The promoter precedes a restriction site where foreign DNA is to be inserted, allowing transcription of foreign sequence to be regulated by adding substances that induce the pro­moter.

b. A DNA sequence that, when tran­scribed into RNA, produces a prokaryotic ribosome binding site.

c. Prokaryotic transcription initiation and termination sequences.

d. Sequences that control transcription initiation, such as regulator genes and operators.

In some types of expression vectors which are specifically used in association with the bacterial host (like E. coli), multiple cloning site is not immediately adjacent to the ribo­some binding sequence, but instead is preceded by a special sequence coding for a bacterial polypeptide.

While using such type of expres­sion vectors the gene of interest is inserted just after the gene for bacterial polypeptide. In this way we fuse two reading frames, producing a hybrid gene that starts with the bacterial gene and progresses without a break into the codons of our gene of interest.

The product of gene expression is therefore a hybrid protein, con­sisting of short bacterial polypeptide fused into amino terminus of our target polypeptide se­quence. This hybrid polypeptide chain consist­ing of two different types of polypeptides is called a fusion protein.

The followings are the reasons for incorporation of a fusion protein before our gene of interest:

(a) The presence of bacterial peptide at the start of fusion protein may stabilize the molecule and prevent it from being de­graded by the host cell. In contrast the for­eign polypeptides that lack a bacterial seg­ment are often destroyed.

(b) The bacterial polypeptide may act as a sig­nal peptide, responsible for transporting our target protein to a specific location from where these are collected. For ex­ample, if the bacterial peptides are derived from a protein that is exported by the cell (e.g. products of ompA genes), then our target polypeptide will simply be transported outside of the host cell straight into the culture media from where these can be collected.

(c) The bacterial polypeptide may also help in purification of the target polypeptide by different purification techniques such as affinity chromatography.

Vector Classification # 2.

On the Basis of Host Cell Used:

After construction of a recombinant DNA these can be introduced into a host cell. So depend­ing on the host cell the vectors are designed and constructed. All the parts of the vectors must be functionally compatible with the host. For example, if we are making a vector for a bacterial host, it must have a suitable origin of replication which will be functional in a bac­terial cell.

Depending on this basis the vectors are classified as under:

1. Vectors for Bacteria:

These are spe­cial bacterial origin of replication and an­tibiotic resistance selectable markers. Bac­teria support different kinds of vectors, e.g. plasmid vectors, bacteriophages vectors, cosmids, phasmids, phagemids, etc.

They have special origin of replication called as autono­mously replicating sequences (ARS), e.g., yeast replicative plasmid vectors (YRp) etc.

3. Vectors for Animals:

These vectors are needed in biotechnology for the synthesis of recombinant protein from genes that are not expressed correctly when cloned in E. coli or yeast, and methods for cloning in humans are being sought by clinical mo­lecular biologists attempting to devise techniques for gene therapy, in which a disease is treated by introduction of a cloned gene into the patient, e.g., P-element, SV40 etc.

The production of genetically modified plants has become possible due to successful use of plant vec­tors. e.g., Ti-plasmid, Ri-plasmid etc.

Vector Classification # 3.

On the Basis of Cellular Nature of Host Cell:

On this basis, the vectors are of two types:

1. Prokaryotic Vectors:

This comprises of all vectors for bacterial cells.

This comprises of all the vectors for yeast, animal and plant cells.

Prokaryotic Vectors (Bacterial Vectors):

The E. coli cell which is frequently used as a prokaryotic host needs specific types of vec­tors which are designed accordingly to func­tion in its cytoplasm. Plasmid based and bacteriophage based vectors are most common prokaryotic vectors. The prokaryotic vectors include plasmid derived vectors, bacteriophage derived vectors, phagemid vectors, plasmid vectors and fosmid vectors.

These are dis­cussed as follows:

These are the most common vectors for the prokaryotic host cells. Bacteria are able to ex­press foreign genes inserted into plasmids (Fig. 4.13). Plasmids are small, circular, double- stranded DNA molecules lacking protein coat that naturally exists in the cytoplasm of many strains of bacteria.

Some of the examples of naturally occurring plasmids are Ti plasmids, F-factors, R-factors, Co/E1 plasmid, etc. Plas­mids are independent of the chromosome of bacterial cell and range in size from 1000 to 200 000 base pairs. Using the enzymes and 70s ribosomes that the bacterial cell houses, DNA contained in plasmids can be replicated and expressed.

The bacterial cells benefit from the pre­sence of plasmids, which often carry genes that express proteins able to confer antibiotic re­sistance. These also protect bacteria by carry­ing genes for resistance to toxic heavy metals, such as mercury, lead, or cadmium.

In addi­tion, some bacteria carry plasmids possessing genes that enable bacteria to break down her­bicides, certain industrial chemicals, or the components of petroleum. The relationship be­tween bacteria and plasmids is endosymbiotic both the bacteria and plasmids benefit from mutual arrangement. Plasmids also possess characteristic copy number.

The higher the copy number, higher is the number of indi­vidual plasmids in a host bacterial cell. If more copies of plasmid exist, more protein will be synthesized because of the larger number of gene copies carried by the plasmid. The num­ber of copies plays a role in phenotypic mani­festation of a gene. For example, the more cop­ies of an antibiotic-resistance gene there are, the higher the resistance to the antibiotic.

It is very important to note that naturally occur­ring plasmids do not have all necessary se­quences which are required by a DNA molecule to act as a profitable vector. Due to this, natu­ral plasmids are extracted and modified by inserting suitable DNA segments and a com­plete vector DNA molecule is made.

Plasmid-cloning vectors are derived from bacterial plas­mids and are the most widely used, versatile, and easily manipulated ones.

The following are different types of plasmid vectors:

This was the first widely used, purpose built plasmid vector. pBR322 has a relatively small size of 4,363 bp. This is important because transformation efficiency is inversely proportional to size and above 10 kbp is very low.

Thus, there is ‘room’ in pBR322 for an insert of at least six kbp. Also this vector has a rea­sonably high copy number (

15 copies per cell), which can be increased 200-fold by treatment with a protein-synthesis inhibitor—chloramphenicol amplification.

The nomenclature of pBR 322:

The nomenclature of ‘pBR 322’ can be under­stood with following explanation:

1. ‘p’ indicates as a plasmid

2. ‘BR’ identifies Bo-liver and Rodriguez, the two researchers who developed it

3. �’ distinguishes those plasmids from others (like pBR 325, pBR 327, etc.) developed in the same laboratory.

The Construction of pBR322:

1. Origin of Replication:

It carries a frag­ment of plasmid pMB1 that acts as an ori­gin for DNA replication and thus ensures multiplication of the vector.

It carries two anti­biotic resistance genes—ampicillin and tetracycline.

It carries a number of unique restriction sites. Some of these are located in one of the antibiotic resistance genes (e.g., sites for Pst I, Pvu I, and Sac I are found in Ampr and BamHI and Hind III in Tetr). Cloning into one of these sites inactivates the gene allowing recombi­nants to be differentiated from non-recombinants known as insertional inactivation.

By pedigree we understand the origin of pBR322. pBR322 is not a naturally occurring plasmid. It is manufactured by following cer­tain steps which are outlined in (Fig. 4.15). It is important to note that pBR322 comprises DNA derived from three different naturally occur­ring plasmids.

The amp R gene originally re­sided on the plasmid R1 (a naturally occur­ring antibiotic resistant plasmid in E. coli), the tet R is derived from R6-5 (a second antibiotic resistant plasmid) and the origin of replica­tion is derived from pMB1, which is closely related to the Colicin producing plasmid ColE1.

Recombinant selection with pBR322 – in- sectional inactivation of an antibiotic resis­tance gene.

When we have introduced our recombinant DNA (vector + gene of interest) into the host cell (by a process called transformation and the host cells that takes up the recombinant DNA are called transformed host cells) then we have to screen the entire host population in order to select the transformed cells (with recombi­nant DNA) from the non-transformed one (without recombinant DNA).

Every vector has some mechanism associated with it for this screening.

Here we will discuss what is the mechanism followed by the pBR322 vector in this regard. pBR322 has several unique restriction sites that can be used to open up the vec­tor before insertion of a new DNA fragment. BamHl, for example, cuts pBR322 at just one position, within the cluster of genes that code for resistance to tetracycline.

A recombinant pBR322 molecule, one that carries an extra piece of DNA in the BamHl site is no longer able to confer tetracycline resistance on its host, as one of the necessary genes is now dis­rupted by the inserted DNA. Cells containing this recombinant pBR322 molecule are still resistant to ampicillin, but sensitive to tetra­cycline (amp R tef s ).

Screening for pBR322 re­combinants is performed in the following way. After transformation the cells are plated onto ampicillin medium and incubated until colo­nies appear [Fig. 4.16(a)].

All of these colonies are trans-formants (remember, untransformed cells are amp s and so do not produce colonies on the selective medium), but only a few con­tain recombinant pBR322 molecules: most con­tain the normal, self-ligated plasmid. To iden­tify the recombinants the colonies are replica plated onto agar medium that contains tetra­cycline [Fig. 4.16(b)].

After incubation, some of the original colonies regrow, but others do not [Fig. 4.16(c)]. Those that do grow consist of cells that carry the normal pBR322 with no inserted DNA and, therefore, a functional tet­racycline resistance gene cluster (amp R tet R ).

The colonies that do not grow on tetracycline agar are recombinants (amp R tef s ) once their positions are known, samples for further study can be recovered from the original ampicillin agar plate.

It is widely used as a cloning vector. In addi­tion to this, it has been widely used as a model system for study of prokaryotic transcription and translation.

Advantages of pBR322:

4.4 kb) enables easy purifi­cation and manipulation.

2. Two selectable markers (amp and tet) al­low easy selection of recombinant DNA.

3. It can be amplified up to 1000-3000 copies per cell when protein synthesis is blocked by the application of chloramphenicol.

Disadvantages of pBR322:

1. It has very high mobility i. e it can move to another cell in the presence of a conjugative plasmid like F-factor. The nic-bom (bom=basis of mobility) region of pBR322 is responsible for this feature. Due to this, the vector may get lost in a population of mixed host cells.

2. There is a limitation in the size of the gene of interest that it can accommodate.

3. Not a very high copy number is present as is expected from a good vector.

4. Although insertional inactivation of an antibiotic resistance gene provides an ef­fective means of recombinant identifica­tion, the method is made inconvenient by the need to carry out two screenings, one with the antibiotic that selects for trans-formants, followed by the second screen, after replica plating, with the an­tibiotic which distinguishes recombinants.

This makes the screening process time- consuming and laborious.

Another vector pBR327 was derived from pBR322, by deletion of nucleotides between 1,427 to 2,516. These nucleotides are deleted to reduce the size of the vector and to elimi­nate sequences that were known to interfere with the expression of cloned DNA in eukaryotic cells. pBR327 still contains genes for re­sistance against two antibiotics (tetracycline and ampicillin).

pBR327 has following two ad­vantages over pBR322:

1. pBR327 has high copy number (30-45 cop­ies per cell).

pUC are obtained by modifying the pBR322 vector. pUC vectors are smaller than pBR322 of being only

2.7 kb. But comparatively they have a high copy number. A mutation within the origin of replication produces 500 to 600 copies of the plasmid per cell without amplification.

The Nomenclature of pUC Vectors:

The nomenclature of ‘pUC’ can be understood with the following explanation:

1. ‘p’ indicates the plasmid.

2. ‘UC’ stands for university of California where it was first developed by J. Mess­ing et al.

We also see many numbers after this like pUC8, pUC18, pUC19 and so on. They are just the series of pUC and have been named just to separate from each other.

The construction of pUC vectors:

1. Origin of Replication: It is derived from the origin of replication of pBR322.The ColE1origin of replication of pBR322 has been modified by carrying out a chance mutation so that each transformed E. coli cell has 500-600 copies of the plasmid.

2. Selectable Marker: It has an ampicil­lin resistant gene. The transformed host cells can grow on media having ampicillin whereas non-transformed cells die.

3. lac Z’ gene having MCS.

4. The lac Z’ is incorporated into this vector codes for the enzyme beta-galactosidase which acts on a chromogenic substrate called X-gal (present in bacterial culture media). The expression of lac Z’ gene is in­duced by another compound present in the same media called Iso-propyl-thiogalactoside (IPTG).

When the enzyme substrate reaction takes place, then the X- gal is converted from white to a blue com­pound. Now the lac Z’ gene itself has MCS. Hence, when the gene of interest has been introduced into the lac Z’ gene, then it fails to code for beta-galactosidase and thus in this case the substrate (X-gal) is never converted to any other colour. This type of screening is called blue-white screen­ing.

Pedigree of pUC Vectors:

During the construction of pUC the only se­lectable marker that is kept out of pBR322 is ampicillin resistant gene. But all the MCS are removed from the amp R by carrying out chance mutations. The ColE1 origin of replication is also modified by the same process so that it can smoothly carry out the process of replica­tion again and again ultimately increasing the copy number of the vector.

Along with this a lac Z’ sequence coding for beta galactosidase is also inserted. Similarly after this by the pro­cess of chance mutation we create MCS within the lac Z’ sequence (Fig. 4.19).

Screening of Transformed Host Cells us­ing pUC Vectors:

After transforming the host cells we carry out their screening to select the transformed cells from non-transformed ones. pUC8 [Fig. 4.20(a)], which carries the ampicillin resistance gene and a gene called lac Z’, which codes for part of the enzyme beta-galactosidase.

Cloning with pUC8 involves insertional inactivation of the lac Z’ gene, with recombinants identified because of their inability to synthesize beta-galactosidase [Fig. 4.20(b)].

Beta-Galactosidase is one of the series of enzymes involved in breakdown of lactose to glucose plus galactose. It is normally coded by the gene lac Z, which resides on E. coli chromosome. Some strains of E. coli have a modi­fied lac Z gene, one that lacks the segment re­ferred to as lac Z’ and coding for the a-peptide portion of beta-galactosidase [Fig. 4.21(a)].

These mutants can synthesize the enzyme only when they harbour a plasmid, such as pUC8, that carries the missing lac Z’ segment of the gene.

A cloning experiment with pUC8 involves selection of trans-formants on ampicillin agar followed by screening for beta-galactosidase ac­tivity to identify recombinants. Cells that harbour a normal pUC plasmid are ampR and able to synthesize beta-galactosidase [Fig. 4.21(a)] recombinants are also ampR but un­able to make beta-galactosidase [Fig. 4.21(b)].

Screening for beta-galactosidase presence or absence is, in fact, quite easy. Rather than assay for lactose being split to glucose and galactose, we test for a slightly different reac­tion that is also catalysed by beta-galactosi­dase.

This involves a lactose analogue called X-gal (5-bromo-4-chloro-3-indolyl-beta-D- galactopyranoside) which is broken down by beta-galactosidase to a product that is coloured deep blue.

If X-gal (plus an inducer of the en­zyme such as Iso-pro-pylthiogalactoside, IPTG) is added to the agar, along with ampicillin, then non-recombinant colonies, the cells of which synthesize beta-galactosidase, will be coloured blue, whereas recombinants with a disrupted lac Z’ gene and unable to make p-galactosidase, will be white.

This system, which is called Lac selection, is summarized in [Fig. 4.21(b)]. Note that both ampicillin resistance and the presence or absence of p-galactosidase is tested on a single agar plate. The two screen­ings are, therefore, carried out together and there is no need for the time-consuming replica-plating step that is necessary with plas­mids such as pBR322.

Uses of pUC Vectors:

pUC vectors can be used both as cloning vec­tor and expression vector. When used as an expression vector its sequences are slightly modified to meet necessary requirements.

Advantages of pUC Vectors:

The pUC vectors offer following major advan­tages over pBR322 vectors:

(a) High copy number of 500-600 copies per cell.

(b) Easy and single step selection.

(c) The unique restriction sites used for clon­ing are clustered within the MCS. This allows cloning of a DNA fragment having two different sticky ends.

Disadvantages of pUC:

It cannot accommodate a gene of interest larger than 15kb.

These are similar to cosmids but are based on the bacterial F-plasmid. The cloning vec­tor is limited, as a host (usually E. coli) can only contain one fosmid molecule. Fosmids are 40 kb of random genomic DNA. Fosmid library is prepared from a genome of target organism and cloned into a fosmid vector.

Low copy num­ber offers higher stability than comparable high copy number cosmids. Fosmid system may be useful for constructing stable libraries from complex genomes.

Bacteriophage Derived Vectors:

Bacteriophages, or phages as they are com­monly known, are viruses that specifically in­fect bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or occasionally ribonucleic acid (RNA)) molecule carrying a number of genes, includ­ing several for replication of the phage, sur­rounded by protective coat or capsid made up of protein molecules (Fig. 4.22).

The general pattern of infection, which is the same for all types of phage, is a three-step process:

1. The phage particle attaches to the outside of bacterium and injects its DNA chromo­some into the cell.

2. The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes on the phage chromosome.

3. Other phage genes direct synthesis of pro­tein components of capsid, and new phage particles are assembled and released from the bacterium.

With some phage types the entire infection cycle is completed very quickly, possibly in less than 20min. This type of rapid infection is called a lytic cycle, as release of the new ph­age particles is associated with lysis of the bacterial cell.

The characteristic feature of a lytic infection cycle is that phage DNA repli­cation is immediately followed by synthesis of capsid proteins, and the phage DNA molecule is never maintained in a stable condition in the host cell. In contrast to a lytic cycle, lysogenic infection is characterized by reten­tion of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions.

Fred Blatter and his colleagues were the first to develop a bacteriophage as vector.

The following are different types of plasmid vectors:

I. Bacteriophage M13 vectors:

The M13 family of vectors is derived from bac­teriophage M13. This is a male specific (infects E. coli having f. pili), lysogenic filamentous phage with a circular single-stranded DNA genome about 6,407 bp (6.4 kb) in length. Once inside the host-cell the single-stranded DNA of M13 phage acts as the template for synthe­sis of a complementary strand, resulting in normal double-stranded DNA [Fig. 4.23(a)].

This molecule is not inserted into the bacte­rial genome, but instead replicates until over 100 copies are present in the cell [Fig. 4.23(b)]. When the bacterium divides, each daughter cell receives copies of the phage genome, which continues to replicate, thereby maintaining its overall numbers per cell.

As shown in [Fig 4.23(c)], new phage particles are continuously assembled and released, about 1000 new ph­ages being produced during each generation of an infected cell.

The Attraction of M13 as a Cloning Vector:

Several features of M13 make this phage at­tractive as the basis for a cloning vector. The genome is less than 10 kb in size, well within the range desirable for a potential vector. In addition, the double-stranded replicative form (RF) of the M13 genome behaves very much like a plasmid, and can be treated as such for experimental purposes.

It is easily prepared from a culture of infected E. coli cells and can be reintroduced by transfection. Most importantly, genes cloned with an M13-based vec­tor can be obtained in the form of single- stranded DNA. Single-stranded version of cloned genes are useful for several techniques, notably DNA sequencing and in vitro mutagen­esis.

Using an M13 vector is an easy and reli­able way of obtaining single-stranded DNA for this type of work.

Construction of M13 vectors:

The first step in the construction of M13 clon­ing vector is to introduce the lac Z’ gene into the inter-genic sequence. This gives rise to M13 mp1 which forms blue plaques on X-gal agar (Fig 4.25(a)) M13 mp1 does not progress any unit restriction site in the lac Z’ gene.

It however, contains a hexanucleotide sequence GGATTC near the start of the gene. A single nucleotide change (by using in vitro mutagen­esis) would make this GAATTC, which is an EcoR1 site.

This results in the formation of M13 mp2. M13 mp2 has a slightly altered lac Z’ gene but the beta-galactosidase enzyme pro­duced by cells infected with M13 mp2 is still perfectly functional. M13 mp2 is the simplest M13 vector. DNA fragments with ECoR1 sticky ends can be inserted into the cloning site and recombinants are distinguished as clear plaques on X-gal agar.

We go for further modifications of M13 mp2 resulting in the production of another M13 vector called M13 mp7. In the generation of M13 mp7 first of all we synthesize a short oli­gonucleotide called poly-linker that consists of a series of restriction sites and has EcoR1 sticky ends. This poly-linker is inserted into the EcoRI site of M13 mp2 to generate M13mp7.

This poly-linker also provides as many as four possible cloning sites (ECoRI, BamHl, SaiI and PstI) to the new vector. It is very important to note that the poly-linker is designed so that it does not totally disrupt the lac Z’ gene: a read­ing frame is maintained throughout the poly-linker, and a functional, though altered, beta-galactosidase enzyme is still produced.

Screening of transformed host cells us­ing bacteriophage M13 vectors:

Insertion of new DNA almost invariably pre­vents beta-galactosidase production. So recombinant plaques are clear on X-gal agar. Alter­natively, if the poly-linker is reinserted, and the original M13mp7 reformed, then blue plaques result.

Uses of bacteriophage M13 Vectors:

For a long time the most important application of M13 clon­ing was in DNA sequence determination by the Sanger method, also called the dideoxy or chain-termination method. This relies on synthesis of DNA in the presence of chain terminating inhibitors, the 2′, 3′- di-deoxynucleoside triphosphates (ddNTPs). The method is now a very standard tool of molecular biology.

2. Phage Display Vectors:

An important use of filamentous phage is in phage dis­play systems. Here, coding sequences are inserted into one of the coat protein genes. The result is that the phage are generated with a hybrid form of this protein, which is a fusion of the normal protein sequence and the protein product of the inserted se­quence (assuming the inserted sequence has the same reading frame as the coat protein gene).

The phages are secreted from the cell, with this extra material ‘displayed’ on the outside. These display vectors have many uses, e.g., in screening libraries by panning and for vaccine production.

Some protocols for site-directed mutagenesis also use single- stranded DNA, which can be obtained with vectors based on filamentous phages. Single-stranded DNA is also of particular use in generating probes for RNA analy­sis.

Probes can be prepared that are spe­cific for RNA transcripts from either strand of DNA. The latter applications are out­side the scope of this book, but more infor­mation can be obtained from specialized laboratory manuals.

Advantages of bacteriophage M13 vector:

1. Advantages over Lambda Phage Vec­tor:

M13 is an example of a filamentous phage and is completely different in struc­ture from lambda. Furthermore, the M13 DNA molecule is much smaller than the lambda genome, being only 6407 nucle­otides in length. It is circular and is un­usual in that it consists entirely of single- stranded DNA.

The smaller size of the M13 DNA molecule means that it has room for fewer genes than the lambda genome. This is possible because the M13 capsid is cons­tructed from multiple copies of just three proteins (requiring only three genes), whereas synthesis of the lambda head- and-tail structure involves over 15 differ­ent proteins.

In addition, M13 follows a simpler infection cycle than lambda , and does not need genes for insertion into the host genome. Injection of an M13 DNA molecule into an E. coli cell occurs via the pilus, the structure that connects two cells during sexual conjugation.

M13-based vectors are that they contain the same poly-linker and alpha-peptide fragments as the pUC series and recombinants can be selected by the blue → white colour test. Also the size of the genome is below 10 kb and so is easy to handle.

Disadvantages of bacteriophage M13 vec­tors:

The following are the disadvantages of bacte­riophage M13 vectors:

1. Gene of interest more than 2kb cannot be cloned.

2. It has low yield of DNA.

3. The phage produce many toxins in high concentration.

II. Lamda Phage Vectors:

This is a widely used vector for the cloning of very large pieces of genes.

Lambda is a typical example of a head-and-tail phage. The genetic material is DNA which is present in the polyhedral head structure and the tail serves to attach the phage to the bacterial surface and to inject the DNA into the cell. The lambda DNA molecule is 49 kb in size.

It is a temperate phase and this can carry out lytic and lysogenic cycles. The positions and identities of most of the genes on the lambda DNA molecule are known (Fig. 4.28).

Lambda phage can have both linear and circular forms of DNA. The molecule shown in (Fig. 4.28) is linear, with two free ends, and represents the DNA present in the phage head. This linear molecule consists of two comple­mentary strands of DNA, base paired accord­ing to the Watson-Crick rules.’ However, at either end of the molecule is a short 12-nucleotide stretch in which the DNA is single-stranded [Fig. 4.30(a)].

The two single strands are complementary, and so can base pair with one another to form a circular, completely double-stranded molecule [Fig. 4.30(b)]. Comple­mentary single strands are often referred to as ‘sticky’ ends or cohesive ends, because base pairing between them can ‘stick’ together the two ends of a DNA molecule (or the ends of two different DNA molecules).

The lambda cohesive ends are called the cos sites and they play two distinct roles during the lambda in­fection cycle. First, they allow the linear DNA molecule that is injected into the cell to be cir­cularized, which is a necessary prerequisite for insertion into the bacterial genome (Fig. 4.29).

The second role of the cos sites is rather different, and comes into play after the pro-ph­age has excised from the host genome. At this stage a large number of new lambda DNA molecules are produced by the rolling circle mechanism of replication [Fig. 4.30(c)], in which a continuous DNA strand is rolled off the template molecule. The result is a catenane consisting of a series of linear A genomes joined together at the cos sites.

The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual lambda genomes. This endonuclease, which is the product of gene A on the lambda DNA mol­ecule, creates the single stranded sticky ends, and also acts in association with other proteins to package each lambda genome into a phage head structure.

The cleavage and packaging processes recognize just the cos sites and the DNA sequences to either side of them. Chang­ing the structure of the internal regions of the lambda genome, for example, by inserting new genes has no effect on these events so long as the overall length of the A genome is not al­tered too greatly.

Problems associated with naturally oc­curring lambda phage to be used as clon­ing vectors:

Two problems have to be solved before lambda- based cloning vectors could be developed:

1. The lambda molecule can be increased in size by only about 5%, representing the ad­dition of only 3kb of new DNA. If the total size of the molecule is more than 52kb, then it cannot be packaged into the lambda head structure and infective phage par­ticles are not formed. This severely limits the size of a DNA fragment that can be inserted into an unmodified lambda vec­tor.

2. The lambda genome is so large that it has more than one recognition sequence for virtually every restriction endonucleases. Restriction cannot be used to cleave the normal lambda molecule in a way that will allow insertion of new DNA, because the molecules would be cut into several small fragments that would be very unlikely to reform a viable lambda genome on relega­tion.

Due to these reasons the DNA of naturally occurring lambda phage cannot be used as a cloning vector. To solve this issue we modify the lambda’s genome and make it suitable to be a successful vector.

Solving the Problems:

From research it has been found out that large segment in the central region of the lambda DNA molecule can be removed without affecting the ability of the phage to infect E. coli cells. Removal of this non­essential region between positions 20 and 35 on the map decreases the size of the lambda genome by up to 15kb.

This makes a room for as much as 18kb of new DNA which can be added to it to form a recombinant molecule.

This non-essential genes thus removed are involved in integration and excision of the lambda pro-phage from the E. coli chromosome. A deleted lambda genome is, therefore, non-lysogenic and can follow only the lytic infec­tion cycle. This in itself is desirable for a clon­ing vector as it means that we can get plaques (a visible structure formed within a cell cul­ture, such as bacterial cultures within some nutrient medium).

We can remove un­necessary restriction sites by carrying out in vitro mutagenesis. For example, an ECoRI site, GAATTC, could be changed to GGATTC, which is not recognized by the enzyme.

Types of Lambda Vectors:

There are two types of lambda cloning vectors.

(a) Lambda Insertion Vectors:

In this case a large segment of the non-essential re­gion has been deleted, and the two arms ligated together. An insertion vector pos­sesses at least one unique restriction site into which new DNA can be inserted. The size of the DNA fragment that an indi­vidual vector can carry depends on the extent to which the non-essential region has been deleted, e.g. lambda-gtl0, lambda- ZAP11.

(b) Lambda Replacement Vectors:

These vectors have two recognition sites for the restriction endonucleases. These sites flank a segment of DNA that is replaced by the DNA to be cloned [Fig.4.34(a)]. Of­ten the replaceable fragment (or stuffer fragment) carries additional restriction sites that can be used to cut it up into small pieces so that its own reinsertion during a cloning experiment is very unlikely.

Re­placement vectors are generally designed to carry large pieces of DNA than inser­tion vectors can handle e.g., lambda- EMBL, lambda-GEMll, etc.

Cloning experiments with lambda inser­tion or replacement vectors:

A cloning experiment with a lambda vector can be carried out by following the similar method that we followed for a plasmid vector—the lambda DNA molecules are digested with suit­able restriction endonuclease enzyme, the gene of interest is added, the mixture is ligated and the resulting recombinant DNA is introduced into E. coli host cell (by a process called trans­fection) [Fig. 4.35(a)].

This type of experiment requires that the vector be in its circular form, with the cos sites hydrogen bonded to each other.

The transfection process which requires a circular lambda DNA molecule is not particu­larly efficient. To obtain a greater number of recombinants we can introduce some refine­ments in the lambda genome. In this regard we can prefer a linear form of the vector. When the linear form of the vector is digested with the relevant restriction endonuclease, the left and right arms are released as separate frag­ments.

A recombinant DNA can be constructed by mixing together our gene of interest with the vector arms [Fig. 4.35(b)]. Ligation results in several molecular arrangements, including catenae’s comprising left arm-DNA-right arm repeated many times. Recombinant ph­age thus produced in the test tube can be used to infect an E.coli culture.

Visualization of Phage Infection after the Process of Transfection:

The entry of recombinant DNA in the host cell is followed by the lytic cycle which eventually results in the lysis of the host cell. The lysed host cell can be located on the agar medium as plaques on a lawn of bacteria. Each plaque is a zone of clearing produced as the phages lyse the cell and move on to infect the neighbouring bacteria.

Screening of transformed host cells us­ing bacteriophage lambda vectors:

A variety of ways could be employed to distin­guish between recombinant plaques from non- recombinant ones.

The methods are as follows:

(a) Insertional Inactivation of Lac Z’ Gene Carried by the Lambda Phage Vector:

Insertion of our gene of interest into the lac Z’ gene inactivates beta- galactosidase synthesis. Recombinants are distinguished by plating cells on X-gal agar where the recombinant plaques are clear whereas non-recombinant plaques are blue in colour.

(b) Insertional Inactivation of Lambda Cl Gene:

Several lambda cloning vectors have restriction site in the cl gene. Insertional inactivation of cl gene cause a visible change in the plaque morphology. Normal plaques appear turbid (hazy) whereas re­combinant plaques with disrupted cl gene are clear.

(c) Selection using Spi Phenotype:

P2 ph­age is a relative of lambda phage, lamda phages cannot infect E. coli cells that al­ready has an integrated P2 phage in its genome. Due to this, lamda phage is said to be Spi- + (sensitive to P2 pro-phage in­fection). Some lambda cloning vectors are designed so that insertion of new DNA causes a change from Spi- + to Spi- – , en­abling the recombinant to infect cells that carry P2 pro-phages.

Such cells are used as host for cloning experiments with these vectors. In this case recombinants are Spi-, so they are able to form plaques.

(d) Selection on the Basis of Lambda Ge­nome Size:

We know this from the begin­ning that any gene of interest which is less than 37kb or more than 52kb cannot be packed in the head of lambda phage. Many lambda vectors have been constructed by deleting large segments of the lambda DNA molecule and so are less than 37kb in length.

These can only be packaged into mature phage particles after our gene of interest has been inserted. This brings the total genome size up to 37kb or more. Hence, with these vectors only recombi­nant phages are able to replicate.

Uses of Bacteriophage Lambda Vectors:

The main use of all lambda based vectors is to clone DNA fragments that are too long to be handled by plasmid or M13 vectors. A replace­ment vector such as lambda-EMBL4 can carry up to 20kb of our gene of interest. This com­pares with a maximum insert size of about 8kb for almost all plasmids and less than 3kb for M13 vectors.

Advantages of Bacteriophage Lambda Vec­tors

Following are the advantages of lambda vec­tors:

1. Storage of phage particles is comparatively much easier than that of plasmid based vectors.

2. The shelf-life of phage particles is infinite.

3. Transfection of E. coli is much easier with phage particles.

Disadvantages of Bacteriophage Lambda Vectors:

If you have isolated a clone, it is frequently quite difficult to isolate large quantities of DNA. In practice, many problems are encoun­tered that do not occur with plasmids. There is still no truly rapid, reliable protocol for the production of very clean lambda-DNA.

The most successful method is to use anion exchange columns. The most frequent problem is that the preparation contains dirt that makes further processing, such as a restric­tion digestion, difficult or impossible.

Even in the replacement vectors, almost two thirds of the DNA is made up of vector sequences. If possible, you should clone the sections that are of interest using plasmids. LambdaZAP banks can save work, because the plasmid portions are cut out in vivo, along with inserted DNA. That process is highly efficient, requires only a relatively few work steps, and lasts only 1 to 2 days.

It is the most sophisticated type of lambda based vector. Cosmids are the hybrids between the phage DNA molecule and bacterial plas­mid. Their design centres on the fact that the enzymes that package the lambda DNA mol­ecule into the phage protein coat need only the cos sites in order to function.

Construction of Cosmid Vectors:

A cosmid is basically a plasmid that carries a cos site. It also needs a selectable marker, such as ampicillin resistant gene, and a plasmid origin of replication. This is important to note that as cosmid lacks all the lambda genes, so at does not produce plaques. Instead colonies are formed on the selective media just as with plasmid vectors.

Cloning Experiment with Cosmid Vectors:

This is carried out as follows. The cosmid is opened and its unique restriction site and our gene of interest is inserted. These fragments are usually produced by partial digestion with a restriction endonuclease, as total digestion almost invariably results in fragments that are too small to be cloned with a cosmid.

Ligation is carried out so that catenanes are formed. These lambda phages are then used to infect an E. coli culture. All colonies are recombinant colonies as non-recombinant lambda phages cannot be packaged into the head of the lambda bacteriophage.

Uses of Cosmid Vectors:

Cosmids are used for construction of genomic libraries of eukaryotes since these can be used for cloning large fragments of DNA.

Advantages of Cosmids:

Followings are advantages of cosmid vectors:

1. These can be used to clone gene of inter­est up to 40 kb.

2. As the lambda phage will insert the recom­binant DNA into the host cell, an extra step of inserting the recombinant DNA into the host cell is not performed.

3. Easy screening method is found.

Although M13 vectors are very useful for the production of single-stranded versions of cloned genes they do have one disadvantage. There is a limit to the size of DNA fragment that can be cloned with an M13 vector, with 1500bp generally being looked on as the maxi­mum capacity.

To get around this problem a number of novel vectors have been constructed which are the hybrids of plasmids and M13 vectors. We call them phagemids (‘phage’ from M13 bacteriophage and ‘mid’ from plasmid).

Construction of Phagemid Vector:

A typical phagemid has following parts:

1. Phage M13 origin of replication.

2. A portion of lac Z’ gene driven by lac pro­moter.

3. A multiple cloning site (MCS) with lac Z’ gene.

4. Phage T7 and T3 promoter sequences flanking the MCS sequences.

5. ColE1 origin of replication.

Plasmids that carry the M13 replication origin in addition to a conventional origin of dsDNA synthesis can be replicated either as dsDNA from the latter or as single-stranded DNA from the M13 origin. Replication from the M13 origin requires the appropriate pro­teins (such as gene II protein) to be provided from a helper phage also replicating within the cell.

Replication generates single-stranded DNA which can then be packaged into phage coats. Examples of phagemids are the vectors pUC118, 119 and 120.

They are replicated as plasmids until the cell containing them is co-infected with a helper phage, such as M13K07, which provides the proteins for single-stranded DNA synthesis and packaging. M13K07 is an M13 phage that has been modified, most im­portantly by the incorporation of a plasmid replication origin.

Replication from this origin allows the helper phage to be present in a high copy number per cell and, therefore, to pro­vide the larger quantities of the proteins that are required to replicate and package the phagemid molecule.

M13K07 also contains a kanamycin resistance gene to allow for selec­tion for the presence of the helper phage. (Of course, it is possible that the M13K07 helper phage may be packaged too, but in practice the packaged phagemid molecules are found to be in a 100-fold excess over the helper phage.)

Another example of these vectors is the pBluescript series, such as pBluescriptIIKSÞ, shown in Fig. 4.39. This series of plasmids con­tains, in addition to features already described, promoters from the E. coli bacteriophages T3 or T7, which are useful for expressing cloned sequences.

Uses of Phagemid Vectors:

This vector is a multipurpose vector as it can serve as following:

Advantages of Phagemid Vectors:

The main advantage of the phagemid system is that it can be used to provide single-or double-stranded material without any re-cloning.

Phasmids are truly plasmids with phage genes. These are linear duplex DNAs whose ends are lambda segments that contain all the genes required for a lytic infection and whose middle-portion is linearized. Both the lambda and the plasmid replication functions are intact.

Nor­mally, plasmid vectors carry a lambda attach­ment site. Once inside E. coli cell, the phas­mid can replicate like a phage and form plaques in the normal way. However, if the vector contains the gene that encodes lambda repressor, then the plasmid replicates as a plasmid rather than as a phage.

Depending upon the functioning or non-functioning of cl- Protein (coded by repressor), the phasmid can replicate as plasmid (cl-Protein inactive) or phage when cl-protein is active. The activity of cl-protein can be inactive by growing the E.coli culture at 40°C.

Plasmids may be used in variety of ways. For example, DNA may be cloned in the plasmid vector in a conventional way and then the recombinant plasmid can be lifted onto the phage. Plasmids are easy to store, they have an effectively infinite shelf life and screening phages by molecular hybridiza­tion gives cleaner results than screening bac­terial colonies.

Most cloning experiments are carried out with E. coli as the host, and the widest variety of cloning vectors are available for this organism. E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of molecular biology such as gene structure and function. However, under some circumstances it may be desirable to use a dif­ferent host for a gene cloning experiment.

But when the aim of the RDT experiment is not just to study a gene but to use cloning to con­trol or improve synthesis of an important meta­bolic product (e.g., a hormone such as insu­lin), or to change the properties of the organ­ism (e.g., to introduce herbicide resistance into a crop plant), then we take a host cell which is more advanced and capable of meeting an advanced level of metabolism.

Due to this, many times we consider eukaryotic vectors for our cloning experiments. Yeast, animal and plant vectors are all considered as eukaryotic vectors.

The yeast (Saccharomyces cerevisiae) is one of the most important organisms in biotechnol­ogy. Its role is also very important in brewing and bread making. Yeast has been used as a host organism for the production of important pharmaceuticals from cloned genes. Develop­ment of cloning vectors for yeast has been stimulated greatly by the discovery of plasmid that is present in most strains of S. cerevisiae.

Various yeast vectors have been designed, once the ability and utility of yeast is confirmed. All of them have three features in common.

1. All of them contain unique target sites for a number of restriction endonucleases.

2. All of them can replicate in E. coli often at high copy number.

3. All of them employ markers that can be used to select recombinant yeasts, e.g., Hi53, leo2, trpl and ura3.

Types of Yeast Vectors:

All the yeast vectors can be divided into three types:

1. Yeast cloning vectors (or Yeast plasmid vectors)

2. Yeast expression vectors

3. Yeast artificial chromosomes (YAC)

Yeast Cloning Vectors (or Yeast Plasmid Vectors):

These vectors are used to clone (make several duplicate copies) our gene of interest in the yeast host cell. All the cloning vectors have been engineered from 2fx plasmid which is the naturally occurring plasmid in the yeast cell.

They are of following types:

1. Yeast Episomal Plasmids (YEps):

It is 6,318 bp long and has a copy number of 70-200. Most of the YEps are shuttle vec­tors (can be used as vectors both in prokaryotic hosts and eukaryotic hosts) and thus have been engineered accord­ingly. An example of YEps is Yep13.

It have following parts:

(a) Origin of replication derived from 2m plas­mid.

(b) amp R and tet R region from pBR322. This selectable marker region is helpful for the screening of recombinants host cells when the vector is used in a prokaryotic cell.

(c) LEU2 region which is derived from yeast chromosome and could be used as a selectable when the vector is used in an eukary­otic cell. LEU2, which codes for beta-iso-propyl-malate dehydrogenase, one of the enzymes involved in the conversion of pyruvic acid to leucine.

This is very important to note that when we are taking yeast cells for YEps then we have to take only leu2 yeasts which must be auxo­trophic mutants having non-functional LEU2 gene.

The leu2 − yeast host cells are unable to syn­thesize amino acid leucine and can survive, only if this amino acid is supplied as a nutri­ent in the growth medium [Fig. 4.41(a)]. Se­lection is possible because trans-formants host cells contain a copy of YEp (having LEU2 gene) and are quite able to grow in the absence of amino acid.

In a cloning experiment, cells Eire plated out onto minimal medium, which con­tains no added amino acids. Only transformed cells are able to survive and form colonies [Fig. 4.41(b)].

YEp may get inserted into the yeast chro­mosome by a process of homologous recombi­nation between the plasmid LEU2 gene and the yeast mutant LEU2 gene. The plasmid may remain integrated, or a later recombination event may result in it being excised again.

2. Yeast Integrative Plasmids (YIps):

These are basically bacterial plasmids car­rying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene.

3. Yeast Replicative Plasmids (YRps):

These are able to multiply as independent plasmids because they carry a chromo­somal DNA sequence that includes an ori­gin of replication. An example is YRp7.

Yeast Expression Vectors:

These vectors are used when our aim is to ex­press our gene of interest in the yeast cell. Yeast expression vectors will employ promoter and terminator sequences in addition to the gene of interest. Apart from these we have genetic tags like the gene for green fluores­cent protein (GFP) for tracking the location of the protein after its biosynthesis.

Following are few examples:

High copy yeast expression vector carrying the aminoglycoside phosphotransferase gene for selection in yeast using G418. Inserts are expressed from the strong TEF promoter.

Low copy yeast expression vector carrying the aminoglycoside phosphotransferase gene for selection in yeast using G418. Inserts are expressed from the weak CYC1 promoter.

Yeast expression vector for secreted proteins. A strong TEF1 promoter drives constitutive expression of a cDNA fused to the pre-pro leader sequence of mating factor alpha to ensure secretion of the protein product into the medium.

Yeast Artificial Chromosomes:

Yeast artificial chromosomes (YACs) are synthetic double-stranded linear constructs containing the elements necessary for replica­tion as independent chromosomes in yeast. See artificial chromosomes for more details.

The followings are some examples of animal vectors:

Baculovirus in­fects insects. This virus is rod shaped with a large double-stranded genome. During normal infections, baculovirus produces nuclear inclusion bodies which consist of virus particles embedded in a protein ma­trix.

This protein matrix is called polyhedrin and it accounts for 70% of total pro­tein encoded by the virus. Genetic manipu­lation of the viral DNA is not possible as it has a very large DNA with many restric­tion sites for a single enzyme.

Hence, the gene of interest is cloned into the small recombination transfer vector and co- transfected into insect cell lines along with the wild type of virus in the cell. Homolo­gous recombination takes place between the polyhedrin gene and our gene of inter­est.

Thus, our gene of interest will be trans­ferred from the vector plasmid into the wild type of virus and polyhedrin gene will be transferred from the virus on the plas­mid.

This is something like displacement reaction. This displacement of gene will not affect the replication of virus, as polyhedrin gene is not required for repli­cation. The recombination virus replicates in the cells and generates characteristic plaques (without inclusion bodies).

Nor­mally the virus is cultured in the insect cell line of Spodopterafrugiperda. The gene of interest is expressed during the infection and very high yields of protein can be achieved by the time the cell lyses.

2. Bovine Papilloma Virus Vector:

Bo­vine papilloma virus (BPV) causes warts (uncontrolled epithelial proliferation) in cattle.

BPV normally infects terminally differen­tiated squamous epithelial cells.

BPV has a capsid protein surrounding a circular double-stranded DNA of size 79 kb. 69% of this genome is important for viral function, whereas 31 % of the genome can be replaced by any foreign DNA sequence like our gene of interest.

The recombinant BPV is constructed by ligating our gene of interest and BPV vector (69%) onto the pBR 322 plasmid, thus generating the shuttle vector containing plasmid ori site and virus replication sequences. These shuttle vectors are multiplied in E. coli cells first and then they are transformed into mouse cell line.

The major advantage of BPV is the generation of permanent cell line. As the infected cells are not killed, a stable plasmid number is found even when the insert is of large size. The selection of transform-ants is very easy as they form a pile of cells on the transferred monolayer of cells called “Focus”.

The transformed cells are then selected by the presence of marker gene which is mostly the neomy­cin phospho transferase gene coding for resistance against G418. Example of this type of vector is p3.7LDL.

3. SV40 Virus based Vectors:

SV 40 is a spherical virus with double-stranded cir­cular DNA of size 5.2 kb. The viral protein contains three viral coded proteins. VP1 is the major protein present in the capsid with a size of 47000 kDa. Two more pro­teins VP2 and VP3 are also present.

The DNA of virus is associated with the four histones (H4, H2A, H2B and H3) proteins. The viral DNA can be segmented into five precise segments coding for five different proteins small T, large T, VP1, VP2 and VP3. VP1 coding region overlaps VP2 and VP3 in a different translation reading frame. SV 40 virus infects monkey kidney cell lines.

The virus travels to the nucleus and gets uncoated. Then both the T-genes located near the origin are translated in the clockwise direction. The large T pro­tein is important for virus DNA replica­tion and starts after the translation of large T -protein.

Replication starts at the origin and is bi-directional. It terminates when two replication forks meet. About 105 molecules of duplex DNA are synthe­sized per cell. Along with DNA replication, VP1, VP2 and VP3 proteins are synthe­sized.

Then packing of DNA occurs to form new virions, which are released by the ly­sis of cell. The entire process can also be initiated by transfection with naked SV 40 DNA. SV 40 vectors are constructed simi­lar to phage vectors. Portions of the viral genome are removed and replaced by other DNA segments. There are three types of SV 40 vehicles each of which have a distinct advantage or disadvantage among themselves.

(a) SV40 Passive Transforming Vectors:

These vectors neither replicate nor pro­duce virions, but simply integrate the DNA segments into the cellular DNA. These transformed cells replicate the new DNA as an integral part of their own genomes. These plasmids are also shuttle vectors and include selective markers like herpes virus, thymidine kinase or neo genes.

Apart from the selective markers, they include transcriptional regulator signals and polyadenylation sites.

(b) SV40 Trans-ducting Vectors:

These vec­tors are capable of replicating and pack­ing into virion particles. Transducing vec­tors contain a segment of 300 bp which functions as the origin of replication and provides the transcriptional regulatory sig­nals for the synthesis of mRNAs.

This type of vector takes an insert of size 3.9 to 4.5 kb. These plasmids do not have the genes that code for VP1, VP2 and VP3. As no DNA can be added to SV 40 DNA without removing any DNA from the genome, to add the insert, the genome DNA that is not required is removed.

The functions of the DNA that are lost by these deletions are supplied by using a helper virus or by inserting the SV 40 deleted genes into the host DNA. Normally the recombinant SV 40 vectors (usually consist of DNA of in­terest and replication sequence and gene for coding VP1, VP2 and VP3) are trans­formed into the cos cell line.

Cos cell line is a kidney cell line of the African green monkey kidney. It has the T-protein gene incorporated in the genome. So when the vector is transfected into these cells, virion particles are yielded with the help of helper virus.

(c) SV40 Plasmid Vectors:

These vectors multiply in the monkey cell line but are not packed as the virions. These vectors usually contain origin of replication se­quences and larger T-protein gene but do not contain VP1, VP2 and VP3 genes. They are shuttle vectors, and have the ability to multiply both in E. coli and monkey cell line.

Cloning vectors for higher plants were devel­oped in 1980s and their use has led to the ge­netically modified (GM) crops that are in the headline today.

We will examine the details of plant vec­tors and the genetic modification of crops . Here we look at the cloning vectors and how they are used.

Three types of cloning sys­tem have been used with varying degrees of success with higher plants:

1. Vectors based on naturally occurring plas­mids of Agrobacterium (e.g., Ti plasmids from A. tumifaciens and Ri plasmid from A. rhizogens).

2. Direct gene transfer using various types of plasmid DNA. (e.g., using of supercoiled plasmids).

3. Vectors based on plant viruses (e.g., Caulimo virus vectors and Gemini virus vectors).

Shuttle vectors:

Shuttle vectors are those which can multiply into two different unrelated species. Shuttle reactors are designed to replicate in the cells of two species, as they contain two origins of replication, one appropriate for each species as well as genes that are required for replication and not supplied by the host cell, i.e., it is self-sufficient with the process of its replication.

The shuttle vectors are of following types:

1. Eukaryotic – Prokaryotic Shuttle Vec­tors:

Vectors that can propagate in eukaryotes and prokaryotes. e.g., YEp vec­tors can be propagated in yeast (fungi) as well as in E. coli (bacteria).

2. Prokaryotic – Prokaryotic Shuttle Vec­tors:

Vectors that can be propagated in two unrelated prokaryotic host cells, e.g., RSF1010 vectors can be propagated both in bacteria as well in spirochetes.

The common features of such shuttle vec­tors or eukaryotic vectors are the following:

(a) They are capable of replicating into two or more types of hosts including prokaryotic and eukaryotic cells.

(b) They replicate autonomously, or integrate into host genome and replicate when the host cell multiplies.

(c) These vectors are commonly used for transporting genes from one organism to another.

The presence of two replication origins some­times poses special problems, one portion of replication origin of one species is totally un­related to another and interferes with the rep­lication of other host. Hence, in a shuttle vec­tor various types of replication origins are to be inserted and checked before experimenting.

Artificial Chromosomes:

Artificial chromosomes are synthetically de­signed DNA molecules of known structure, which are assembled in vitro (in the labora­tory) from specific DNA sequences that acts like a natural chromosome. Artificial chromo­somes are circular or linear vectors that are stably maintained in, usually, 1-2 copies per cell.

They are huge in size in comparison to other vectors but can clone very large segments of chromosomes (even an entire chromosome). Before seeing different types of artificial chro­mosomes the three key components of an eukaryotic chromosomes can be seen which are necessary for its stable maintenance inside a cell.

1. The centromere, which is required for the chromosome to be distributed correctly to daughter cells during cell division.

2. Two telomeres, the structures at the ends of a chromosome, which are needed for the ends to be replicated correctly in order and which also prevent the chromo­some from being nibbled away by exonu­cleases.

3. The origin of replication, which are the positions along the chromosome at which DNA replication initiates, similar to the origin of replication of a plasmid.

Once we have defined the chromosomal structure of an eukaryotic organism (like hu­mans and yeast), then we can isolate the key components of their chromosomes and join them together to form an artificial chromo­some. Then into this artificial chromosome we can insert our gene of interest which can be subsequently cloned in its respective cell.

Fol­lowing are the types of artificial chromosomes:

A. Yeast Artificial Chromosomes (YAC):

A YAC can be considered as a functional arti­ficial chromosome, since it includes three spe­cific DNA sequences that enable it to propa­gate from one yeast cell to its offspring:

The telomere which is located at each chromosome end, pro­tects the linear DNA from degradation by Nucleases.

The centromere which is the attachment site for mitotic spindle fibres, “pulls” one copy of each du­plicated chromosome into each new daugh­ter cell.

3. Origin of Replication(OR):

Replication origin sequences which are specific DNA sequences that allow the DNA replication machinery to assemble on the DNA and move at the replication forks.

Selectable markers that allow easy isolation of yeast cells that have taken up artificial chromosome.

Recognition Site for two restriction en­zymes EcoRI and BamHl.

Cloning experiment using a YAC vector:

1. Large DNA fragments are obtained by car­rying out restriction digestion using EcoRI.

2. The YAC is digested by two restriction enzymes EcoRI and Bam HI.

3. Those two elements recombine at the EcoRI sites and are covalently linked by DNA ligase.

4. A recombinant YAC vector, a yeast artifi­cial chromosome with genomic DNA inserted, is produced. This vector can be used to infect yeast cells and generate an un­limited number of copies.

1. YAC can be used to study various aspects of chromosome structure and behaviour for instance, to examine the segregation of chromosomes during meiosis.

2. YAC cloning system can take DNA insert greater than l00kb. Due to this they can be used to study the functions and modes of expression of genes that had previously been intractable to analysis by recombi­nant DNA techniques.

3. YACs can be propagated in mammalian cells, enabling the functional analysis to be carried out in the organism in which the gene normally resides. Thus by using them we can learn about the true form of gene expression in vivo conditions.

4. Yeast artificial chromosomes are very helpful in the production of gene libraries. E. coli vectors can take DNA insert maxi­mum up to 300kb. Due to this some 30000 clones are needed for a human gene library if we use them as cloning vector.

However, YAC vectors are routinely used to clone 600 kb fragments, and special types are able to handle DNA up to 1400 kb in length, the latter bringing the size of a human gene library down to just 6500 clones.

Sometimes YAC is seen with problem of lacking insert stability, the cloned DNA some­times becoming rearranged by intra-molecular recombination. Nevertheless, YACs have been of immense value in providing long pieces of cloned DNA for use in large scale DNA sequencing projects. Example of YAC is pYAC3.

2. Bacterial Artificial Chromosomes (BAC):

Bacterial Artificial Chromosomes (BACs) are cloning vectors based on the extra-chromosomal plasmids of E.coli, called F factor or fertility factor. These vectors enable the construction of artificial chromosomes, which can be cloned in E.coli.

This vector is useful for cloning DNA fragments up to 350 kb, but can be handled like regular bacterial plasmid vectors, and is very useful for sequencing large stretches of chromosomal DNA.

Like any other vector, BACs contain ori sequences derived from E. coli plasmid F factor, multiple cloning sites (MCS) having unique restriction sites, and suitable selectable markers. The genomes of several large DNA viruses and RNA vi­ruses have been cloned as BACs. These con­structs are referred to as “infectious clones”, as transfection of the BAC construct into host cells is sufficient to initiate viral infection.

The infectious property of these BACs has made the study of many viruses such as herpes viruses, poxviruses and coronaviruses more ac­cessible. BACs are now being utilized to a greater extent in modelling genetic diseases, often alongside transgenic mice.

BACs have been used in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a gene’s expression level. BACs have been used to some degree of success with mice when studying neurological diseases such as Alzheimer’s dis­ease or as in the case of aneuploidy associated with Down’s syndrome.

Examples of BAC are pBACe3.6, pBeloBAC11 etc.

3. Human Artificial Chromosomes (HAC):

A human artificial chromosome (HAC) is a mini-chromosome that is constructed artifi­cially in human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 mega-bases in size, and able to carry new genes introduced by human researchers.

Us­ing its own self-replicating and segregating systems, a HAC can behave as a stable chro­mosome that is independent of the chromo­somes of host cells.

The essential elements for chromosome maintenance and transmission are the follow­ing three regions:

1. The “replication origin,” from which the du­plication of DNA begins,

2. The “centromere,” which functions in proper chromosome segregation during cell division, and

3. The “telomere,” which protects the ends of linear chromosomes.

They are useful in expression studies as gene transfer vectors and are tools for eluci­dating human chromosome function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six months.

4. Pl-Derived Artificial Chromosome (PAC):

These are DNA constructs which are derived from the DNA of PI bacteriophage. They can carry large amounts (about 100-300 kilo-bases) of other sequences for a variety of bio-engineering purposes. It is one type of vector used to clone DNA fragments (100- to 300-kb insert size average, 150 kb) in Escherichia coli cells.


WHO Updates the Nomenclature of SARS-CoV-2 Variants

Lisa Winter
Jun 1, 2021

T he naming of variants of SARS-CoV-2 has been a bit slapdash. Different databases that share the sequences of the virus have different nomenclature norms. For instance, the variant that emerged in the United Kingdom is called B.1.1.7 on the Pango platform, but is called 20I/S:501Y.V1 on Nextstrain. Yesterday (May 31), the World Health Organization (WHO) announced that SARS-CoV-2 variants of interest (VOI) and variants of concern (VOC) will be named based on the Greek alphabet for purposes of public discourse.

As B.1.1.7 was the first VOC designated by WHO, it is called Alpha under the new naming system. B.1.351, which originated in Brazil, is now called Beta. The two other VOCs are P.1, the variant first identified in Brazil and now referred to as Gamma, and B.1.617.2 that originated in India, now called Delta. The six VOIs designated by WHO take up Epsilon through Kappa in the Greek alphabet. The full list will be maintained on WHO’s website.

“These [Greek] labels do not replace existing scientific names (e.g. those assigned by GISAID, Nextstrain and Pango), which convey important scientific information and will continue to be used in research,” WHO’s statement reads.

According to WHO, the technical variant names are too confusing for the general public and so “people often resort to calling variants by the places where they are detected, which is stigmatizing and discriminatory.”

The new naming system comes long after the first variants were described. WHO officials say the decision came after a great deal of discussion on which naming convention would be best. Reuters reports that the group considered other possibilities including portmanteaus, fruits, or Greek deities.

According to STAT, the group behind the decision was made up of many of the same people who are on the International Committee on the Taxonomy of Viruses. Although the organization named SARS-CoV-2, variant nomenclature is beyond its official scope, and so the task was left to WHO.

“I heard it’s sometimes quite a challenge to come to an agreement with regards to nomenclature,” Frank Konings, the leader of the working group, tells STAT. “This was a relatively straightforward discussion in getting to the point where everybody agreed.”


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Watch the video: Gene transfer in plants using Ti plasmid (November 2022).