Plasmid Classification of Plasmid Replication and Conjugation in Plasmid Importance of Plasmid.

MOLECULAR GENETICS

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Plasmids are double stranded, closed circular covalently bonded DNA

molecules, which exist in the cell as extra chromosomal units. They are self-replicating

and found in a variety of bacterial species. e.g. E. coli, Shijella, Erwinia, Streptococcus,

Vibrio, Salmonella, Agrobacterium, Enterobactor, Rhizobium, etc.

British Genetist Joshua Lederberg (1952) first use this term.

Plasmid Host Cell

•PACYC177 Escherichia Coli

•PBR322 Escherichia Coli

•PBR324 Escherichia Coli

•PMB9 Escherichia Coli

•PRK646 Escherichia Coli

•PC194 Staphylococcus aereus

•PSA0501 Staphylococcus aereus

•PBS161-1 Basillus Subtilis

•PWWO Pseudomonas Putida

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Features of plasmid

• Plasmids are most often found in cytoplasm of bacterial cell.

• It is a circular, double-stranded DNA molecule.

• Most of the plasmids are small in size and contain a few numbers of genes.

• Plasmids genes are replicated, Transcripted and translated with a chromosomal gene independently.

• The atomic weight of plasmid is 1×106 to 200×106

• Plasmid has a 200-10000 bp and contains1-50 genes.

• It easily separates from the bacterial chromosome.

• It may be transferred from one cell to other cell and also one species to other species of bacteria.

• As like as nuclear DNA plasmids are attached with histon.

• It has a replication ori.

• Anti-resistance genes of plasmids are used as a selectable marker.

• Replication system of plasmid is unidirectional or bidirectional.

• They exists separately from main chromosome. Some plasmids can integrate with chromosome and exists along with it. This type of plasmids are called episomes. For example F factor of E.coli is an episome. 3

Classification of Plasmid

Types of plasmids by their ability to transfer to other bacteria – two types1. Conjugative plasmids: Plasmids contain ‘tra’ genus (genes necessary for

non-sexual transfer of genetic material) which perform the complex process of conjugation the transfer of plasmids to another bacterium. E.g. All F and F’ plasmids may R plasmids and some col plasmid are conjugative.

2. Non-conjugative plasmids: These plasmids are incapable of initiating conjugation hence they can only be transferred with the assistance of conjugative plasmids.

Types of plasmids by their function- five types 01. F & F’ plasmids: Which sex factor are responsible for transfer genetic

material from one strain to another strain in bacteria is called F & F’ plasmid.

F = Sex Factor F’ = Fertility Factor

02. R Plasmid :These plasmids contain genes that can build a resistance against antibiotics or persons. This was discovered in Japan in 1956. When a strain of bacteria called Shigella was shown to acquire resistance against several drugs.

03. Col Plasmids: Colicins are toxic proteins, which are produced by bacteria (Escherichia, Shigella, and Salmonella) and which kill bacteria other than those. This now popularly known as col plasmids.

04. Degradative Plasmids: Which enable the digestion on unusual substances as like salicylic acid. Eg. Pseudomonas putida carry Tol plasmids. 4

Some types of plasmids are often related to yeast cloning vectors that include-

Yeast Integrative Plasmid (YIP), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the function of a solo gene or when the gene is toxic.

Yeast Replicative Plasmid (YRP), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can ‘get lost’ during the badding.

05.Virulence Plasmids: Which turn the bacterium into a pathogen. Eg. Ti Plasmids in Agrobacterium tumefaciens.

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Replication of DNA

As a carrier of genetic information’s DNA has following two important functions- Heterocatalytic function: When DNA directs the synthesis of chemical molecules

other than itself (e.g. synthesis of RNA, proteins etc.) then such functions of DNA are called heterocatalytic functions.

Autocatalytic Function: When DNA directs the synthesis of DNA itself are called autocatalytic functions.

J. Cairns (1963) was the first molecular biologist who visualized replicating chromosome of E. Coli by autoradiography and suggested that its DNA replication is semiconservative. Cairn’s studies also indicated that DNA replication seemed to initiate at a fixed position on the chromosome, which he called origin and proceeded sequentially and unidirectionally around the circular structure (mitochondria and some other cellular plasmids).

More recent studies by M. Masters and P. Broda (1971) have shown that the replication of the E. Coli and small virus chromosome is more commonly bidirectional. That is it progresses in both the directions from the origin.

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Whether unidirectional or bidirectional the circular appearance of the initial parental DNA in organisms such as E.Coli and Bacillus subtilis therefore changes into a Ɵ(theta) shaped structure.

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Rolling circle Replication

Rolling circle model of DNA replication was initially proposed by W. Gilbert and D. Dressler. Rolling circle replication occurs in a number of organisms, including the late stages of growth of phage (lambda), transfer of the ƛ E. coli sex factor, replication of particular DNA sections in Xenopus amphibians that are specifically associated with production of ribosomal RNA, and replication of some single stranded DNA phages such as Ø X174.

Phage Ø X174 and Rolling circle replication: Bacteriophage ØX174 is representative of a group of small viruses, both bacterial and eukaryotic, that store their genetic information in a single stranded circular molecule of DNA. When these viruses infect a host cell. E. coli in the case of Ø X174, the single stranded viral DNA replicate by three stages-

Stage1. Production of replicative form (RF)

Stage2. Replication of the parental replicative form (RF)

Stage3. Production of progeny positive strands.

Stage 1. Production of replicative form

1. Conversion of the single stranded chromosome to a double stranded parental replicative (RE) form.

2. Synthesis of the complementary ‘negative’ (-) strand is initiated by the synthesis of a short RNA primer. 9

Fig: Production of replicative form

Primase

DNA poly-merase III

DNA ligase

+ Strand

RNA Primer

DNA- Strand

+Strand

3. This reaction is catalysed by premise and requires the activity of a complex of at least six different priming proteins, this complex is sometimes called the ‘Primosome’.

4. DNA polymerase III next catalyzes the covalent addition of deoxyribonuclectides to the 3’ end of the RNA primer.

5. Synthesis of the complementary negative strand then takes place discontinuously until the positive (+) strand template is exhausted.

6. The primosome appears to travel around the circular template strand, pausing to initiate the synthesis of each new Okazaki fragment.

7. Excision of the RNA primers and gap filling appear to be catalysed by DNA Polymerase-I.

8. DNA ligase then catalyzes the formation of a covalent linkage between the adjacent 3’OH and 5’-PO4 groups, to produce the closed, double stranded parental RF. 10

Fig: Replication of the parental replicative form

Stage-II. Replication of the parental replicative form

1. Rolling circle replication of the parental RF occur to produce a population of progeny RF’s.

2. The positive strand of the parental RF is cut at the origin by the sitespecific endonuclease (nickase) activity of the ØX174 gene A protein nicks nte parental RF only at the origin.

3. During the nicking event, the gene a protein becomes covalently attached to the 5’-phosphoryl group of the positive strand and it remains linked to the 5’- terminus until a complete progeny positive strand has been synthesized.

4. The 5’ end of the positive strand is displaced from the negative strand and deoxyribonucleotids are adden to the free 3’- OH as the circle rotates about its axis.

5. Once a new positive strand origin has been synthesized, the gene a protein cleaves the nascent origin and simultaneously ligates the 3’ and 5’ end to produce a covalently closed circular positive strand.

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6. Synthesis of the complementary negative strand then takes place discontinuously as in stage I.7. The parental RF continues to replicate by the rolling circle mode until a population of about 60 progeny RF’s are produced.

Stage-III. Production of progeny positive strand

1. Rolling circle replication of progeny, RF’s occurs just as for parental RF’s in stage II. 2. Except the, negative strands are not synthesized.

3. Instead, the positive strands are packaged in progeny virus.

4. Preventing positive strand from serving as a template for negative strand synthesis the positive strand binds with newly synthesized viral coat proteins.

5. Maturation of the progeny virion completes the phage ØX174 life circle. Approximately 500 progeny virions are produced per infected cell.

Fig: Production of progeny positive strand12

Displacement loop DNA replication:

This mode of replication occurs in mammalian mitochondria, the mitochondrial chromosome in circular. One strand of the chromosome in denoted as H strand, while its complementary strand is named L strand. Replication begins at a specific origin, but only one strand, the H strand, is replicated, the other strand (the L strand) of the chromosome, is displaced forming a loop, called displacement loop or D loop. When replication of (the H strand) has progressed up to about 2/3 of the chromosome the replication of the displaced single strand (L strand) begins. This replication begins at a different origin and replication proceeds in the opposite direction. Thus both the strands of DNA are replicated in a continuous manner, their replications begin at different origins, and replication of one strand (H strand) begins much earlier than that the other (L strand). In this case, replication is not only unidirectional, but the replication of only one of the two strands takes place at each of the replication forks. The same is the case with rolling circle replication.

D loops are often not associated with replication and are maintained in both mtDNAs and cpDNAs. In some mtDNAs, e.g. in Xenopus laevis, a single but longer D loop is found, while in others several loops, e.g. upto 6 in linear mtDNA of Tetrahymena, are reported. In the cpDNA of higher plants, two D loops are found. The D loops occur as opening of about 500-600 bp in the mammalian.

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Importance of plasmids

1. Medical uses: Plasmid contains some genes advantages to the bacterial host by

genetic engineering.

A. Plasmids can code for antibiotics: Some bacteria become immune to antibiotics

because they have plasmids that make them immune. They can also give their

plasmids to other bacteria through transformation.

EsistanceKanamyicin resistanceChloramphenicol resistanceB-galactosidase resistanceGentamycin resistance

B. Production of antibiotics-

Eg- Ampicillin, Tetracycline, kanamycine, Bleomycin, Hygromycin B, Chloramphenicol.

C.Degradation of complex organic compounds.

D. Production of insulin

E. Production of colchicines.

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2. Agricultural uses:

a. Herbicide resistant plants

b. Pest inside resistant plants

c. Virus resistant plants

e. Stress resistant plants

f. Improvement of crops quality

3. Transgenic animals.

4. Production of industrial & GMO foods.

5. DNA technology in Bioconservation.

6. Recombinant DNA technology

7. Cloning vectors.

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