Everything you need to know about plasmids. Some of the frequently asked questions are as follows:-
Q.1. What is a Plasmid ?
Ans. The plasmids have been reported from prokaryotic as well as eukaryotic cells. A plasmid may be defined as extra-chromosomal genetic element which is not essential for the growth of the cell and has no extra cellular form. In prokaryotes a plasmid is typically a relatively short usually circular double stranded DNA molecule but in eukaryotes may be short circular or linear double stranded DNA molecule. The plasmids are distinct from chromosomal DNA may be mitochondrial DNA (mt DNA) or cytoplasmid DNA (ct DNA).
The plasmids as circular, supercoiled DNA molecules are present in most species or bacteria may be absent in some strains. They are small ranging from about 0.2 to 4 per cent that of the bacterial chromosome. In size they range from 1.5 kb to 300 kb and MWt (molecular weight) 106 to 2 × 108. Many prokaryotes contain more than one plasmids in addition to their chromosome. They make bacterial cell adaptable to the environment, e.g. R plasmid provides resistance against an antibiotic or heavy metal.
They are valuable to a geneticist for two reasons:
(1) To construct partial diploids and
(2) To use in gene cloning in genetic engineering by using plasmids as gene vectors in recombinant DNA technology.
Q.2. How is the nomenclature of plasmids done?
Ans. The plasmids are designated in various ways. The Colicin plasmids are designated according to the colicins they encode, e.g. Col E1-K30 encodes colicin El (lead to membrane changes) Col V I-K 94 encodes colicin V and la. Many antibiotic resistance plasmids are designated by the letter ‘R’ followed by the number, e.g., R1, R4 etc.; others include R6K, RK2, RP1. The recombinant plasmids are indicated by a prefix ‘p’ e.g. pML31.
A number of plasmids arc given trivial names (e.g. Δ), phage plasmid λ dv for λ genes (lambda genes) cro, cl, O, P and the names of some indicate particular functions e.g., Ti plasmid tumour inducing plasmid and TOL plasmid. Some individual plasmids are also known as COLEI plasmid, delta plasmid, PBR 322 for high copy number, pBR 345 for Col El-type replication, PMB1, PMB31, psc 101, R1 plasmid, R100 plasmid and Rp1 plasmid.
Q.3. How are plasmids detected?
Ans. The plasmids are detected by two methods:
1. By conducting genetic experiments. The first plasmid was discovered for F factor in Escherichia coli. A strain of E. coli with genotype Met– Bio– Thr+ Bio+ was mixed with second strain Met+ Bio+ Thr– Leu– and the mixture was placed on minimal agar New colonies appeared on minimal agar at a frequency of about 10-7 which possessed the phenotype Met+ Bio+ Thr+ Leu+ being a recombinant.
2. By conducting physical experiments with bacterial extracts. The plasmids are detected electrophoretically. For it a single colony is taken from a plate and is lysed by lysozyme- detergent procedure and subjected to gel electrophoresis.
The bacterial chromosome being very large cannot penetrate the gel but the plasmid DNA being very short can penetrate Plasmid DNA form a narrow band at a position in the gel characteristic of its molecular weight. The band can be visualized by staining the gel with ethidium bromide, which binds tightly to the DNA and fluoresces while irradiated with UV light.
Q.4. How is the plasmid DNA purified?
The purification of plasmid DNA is done in the following stages:
Ans. (i) The cell lysate which is a translucent solution is made from plasmid containing bacteria by lysozyme-detergent treatment.
(ii) On centrifugation of the cell lysate, the bacterial chromosome complex containing proteins and RNA settle at the bottom of centrifuge tube while the plasmid DNA remains in the clear supernatant known as cleared lysate.
(iii) The chromosomal DNA that might have been left in the cleared lysate is removed by adding CsCl and ethidium bromide and centrifugation.
(iv) The plasmid DNA being covalently circular shows higher density than linear chromosomal fragments and gets separated.
Q.5. The four stages in bacterial conjugation are:
Ans. (i) Formation of specific donor (F+) and recipient (F–) pairs which is known as effective contact.
(ii) Preparation for transfer of DNA from donor to recipient cell called mobilization.
(iii) Transfer of DNA.
(iv) Formation of replicative functional plasmid.
Q.6. Choose the correct answers (more than one option may be correct).
Ans. (i) The conjugative plasmid carry genes for effective contact function in bacterial conjugation.
(ii) The preparation for transfer of DNA in conjugation is made by mobilizable plasmid.
(iii) A self transmissible plasmid, e.g., F plasmid is conjugate as well as mobilizable.
(iv) The sex pilus is present 011 the recipient bacterial cell.
(v) The sex pilus is present on the donor bacterial cell.
(vi) The F pilus comprises of a very hydrophobic protein known as pilin.
(vii) The purified pili can bind to recipient bacterial cells.
(viii) Some strains of Streptococcus faecalis have a self-transmissible plasmid.
(ix) In some strains of Streptococcus faecalis plasmid free recipient produce a mating protein analogous to the pheromones of the female insect while such mating protein is not formed in the plasmid containing donor cells.
(x) In the bacterial strain Streptococcus faecalis mating protein or the pheromones lead the donor cells to make a protein known as adhesin which coats the donor cell enabling cells to make donor-recipient pairs.
(xi) Looped rolling-circle mechanism is not used by phage фX174.
(xii) Looped rolling-circle mechanism is used by phage фX174.
(xiii) Looped rolling-circle mechanism explains transfer of F plasmid DNA from an F+ donor to F– recipient.
(xiv) Fertility inhibition is a common feature in R plasmids.
(xv) Most R plasmids never have any active fin repressor.
Ans. (i), (ii), (iii), (v), (vi), (vii), (viii), (ix), (x), (xii), (xiii), (xiv), (xv) Correct; (iv) and (xi) Wrong.
Q.7. What is tra operon?
Ans. The genes which are essential to cause transfer are called tra operon or the tra genes of F.
Q.8. Name the genes for pili formation.
Ans. These are tra A, tra B, tra C, tra E, tra F, tra G, tra H, tra J, tra K, tra L, tra U, tra V, tra W.
Q.9. How does a plasmid replicate?
Ans. A plasmid replicates inside the host cell. Therefore, all plasmids native to the same host species show same mode for replication. It is interesting that like phages there exist much variation in both the enzymology mechanics of plasmid DNA replication. The polymerase III is the major replication protein in E. coli plasmids. The replication generally has been reported to occur by butterfly or rabbits ear mode. In it partially replicated molecules have untwisted replicated portions like Ɵ replication and a supercoiled un-replicated part.
Q.10. What are stringent and relaxed plasmids?
Ans. The stringent plasmids have low copy number in which number per cell may be 1 to 2 while in relaxed plasmid there is high copy number ranging from 10 to 100 per cell.
Q.11. What is amplification of plasmid? What is its use?
Ans. It is an increase in number of plasmids. On addition of chloramphenicol which is an inhibitor of protein synthesis initiation of replication of chromosome DNA is inhibited but replication of plasmid DNA continues and the number of plasmids per cell increases to 1000 or more even. Thus amplification can be used to increase the amount of plasmid DNA.
Q.12. Name the inhibitors of plasmid replication.
Ans. Acridines like pro-flavin acridine orange inhibit the replication of plasmids without inhibiting replication of chromosomal DNA. This sort of inhibition causes the loss of plasmids known as acridine curing.
Q.13. What is integrative suppression?
Ans. Integration of the F into certain bacterial mutants bearing defects in DNA gives rise to the phenomenon called integrative suppression.
Q.14. What is RTF?
Ans. Most of the R-plasmids consist of two contiguous segments of DNA. One of the segments is known as RTF or resistance transfer factor. It carries genes that regulate replication of DNA, copy number, transfer genes and sometimes gene for tetracycline resistance called tet and bears a molecular weight of 11 × 106.
Q.15. What are colicinogenic or Col plasmids?
Ans. These are found in Escherichia coli and produce colicins, the proteins that inhibit the growth of susceptible bacterial strains that lack Col plasmid.
Q.16. What is lacuna?
Ans. When a colicin producing cell is placed in a lawn of sensitive bacterial cell the colicin producing bacterium inhibits the growth of sensitive cells and form a clear zone called lacuna.