plasmids

PLASMID
An extra chromosomal DNA which can replicate independently without any relation to chromosomal DNA is called a plasmid
Plasmids thus does not have any genetic relationships with the organism from which of is collected
Usually plasmids are found in naturally occurring bacteria but are sometimes found in eukaryotic organism like sacchromyces cervicea.
Plasmids are usually considered as transferrable agents or a means of transport too.
Size of a plasmid usually ranges from 1 to over 1000 kb.
Plasmids play a role in the process of bacterial conjugation as well horizontal gene transfer.
Plasmid can only uptake the genetic material through the process of transformation.
Plasmids can aid bacteria in the process of nitrogen fixation too.
Plasmids are the stable genetic elements found not only in bacteria but also in the mitochondria of some plants
Plasmids can be composed of DNA or RNA double stranded or single stranded linear or circular.
Plasmids are used to produce high amounts of proteins as such the plasmids with antibiotic resistant gene is introduced into an organism..
The organisms thus produces the protein which acts against gene thus releasing lots of required proteins.
TYPES OF PLASMIDS
Basically there are two types of plasmids based on the process of replication.
A)Non integrating plasmids B)Integrating plasmids.
NON INTEGRATING PLASMIDS
There are the plasmids which replicate with in the host cell but does not integrate with the bacterial DNA
INTEGARTING DNA
This type of plasmids usually integrate with the bacterial DNA and then starts the process of replication.
Based on the ability to transfer to other bacteria the plasmids are classified into two types.
A)Conjugative plasmids B)Non conjugative plasmids.
CONJUGATIVE PLASMIDS
These plasmids contain a type of genes called tra-genes which perform the process of conjugation.
NON CONJUGATIVE PLASMIDS
These are usually non integrating they cannot initiate the process of conjugation and hence they can only be transferred with the assistance of conjugative plasmids.
BASED ON THE FUNCTION THERE ARE DIFFERENT TYPES OF PLASMIDS
A)Fertility/F-plasmids B)Resistance/R plasmids C)Col plasmids D)Degradative plasmids E)virulence plasmids.
F-PLASMIDS
Contain tra-genes thus help in the process of conjugation.
R-PLASMIDS
Contain genes that are against antibiotics / poisons thus help bacteria to produce pili that forms a bridge during the process of conjugation
COL-PLASMIDS
Contain the genes that code for the bacteriocins and proteins.
DEGRADATIVE PLASMIDS/METABOLIC PLASMIDS
Enables the digestion of unusual substances . EX:Salicylic acid ,toluene etc.,
VIRULENCE OF PLASMIDS
Taurns bacteria into a pathogen .
One or more major classification includes yeast plasmids .
They are of two types A)Yeast integrative plasmid B)Yeast replicative plasmid.
YEAST INTEGRATIVE PLASMID
They rely on the first integrating into host chromosome .They help in studying of solo gene.
YEAST REPLICATIVE PLASMID
They carry the sequence of chromosome with origin of replication into host .This is usually less stable in nature.
TRANSFER OF PLASMIDS
The process of transfer of plasmids involves four major steps they are A)Identification B)Classification C)Purification D)Transfer .
IDNETIFICATION.
The process of identification of the plasmids is based on the digestion with specific endonucleases.
CLASSIFICATION
After digesting with endonucleases plasmids are subjected to get electrophoresis which helps in the process of fragmentation patterns obtained .
PURIFICATION
Purified plasmids DNA can be obtained by the process of centrifugation.
TRANSFER OF PLASMIDS
The last step in transferring the plasmid with the desirable gene into the host .
PROCESS OF PLASMID TRANSFER /PLASMIDS ACTING AS TRANSFERING AGENTS .
Plasmid vectors are one of the best approaches that gave successful stories of gene therapy.
GENE THERAPY.
It is the process in which the gene with required qualities is inserted into the plasmid DNA and then the plasmid DNA is introduced into the host organism where the process of gene expression starts thus yielding good results.
PROCESS OF CLONING INTO PLASMID .
Step-1: Plasmid with gene for antibiotic resistance are selected.
Treat the plasmid with restriction endonucleases i.e., ecori
Same restriction endonucleases is used to cleave the foreign DNA too.
Then forming a wick on plasmid DNA and collecting the required gene sequence from foreign DNA,
HYBRIDIZATION
The foreign DNA sequence are the hybridized at sticky ends of plasmid DNA .
LIGASE
The enzyme ligase is then used to form the sticky ends or opened ends of plasmid DNA ,
Thus forming the recombinant DNA .
DNA INSERTION
Plasmid DNA is then inserted into bacterial cell.
CLONING
The plasmid DNA is then subjected to gene amplification process then producing number of copes.
PLATTING
Bacteria then platted with the antibiotic in the medium only those hybridized plasmids can same and all other will die thus helps in the selection of recombinant.
CULTURING
After the process of selection it is subjected to culturing thus producing required DNA /clones.
HOST RESTRICTION IN PLASMID TRANSFER
After the process of plasmid identification ,purification,and insertion of the required DNA into the plasmid the plasmid must be transferred into any of the bacterial host.
Transfering into the host then continuous the further process of cloning and replication thus increasing the number /quality.
Common host that are used for plasmid transfer are Ecoli and salmonella.
Some times this transfer is restricted some host enzymes thus restricting the plasmid from entering into the host.
Other than those hosts the plasmids cannot be transferred into other hosts as they show the host restriction.
EXAMPLES /CARE STUDY
Genome degradation
In the process of host restriction genome degradation is the important step where the plasmid DNA material may be lost due to the enzymes of restriction released by other hosts that cannot host the plasmid .
ex:
Host restricted salmonella serotypes usually cause the genome degradation of the plasmid DNA is restricted into them.
Hosts even have restriction factor Which prevent the process of replication of plasmid DNA then the process of gene expression prevented in the hosts that restrict the plasmid DNA .
EUKARYOTIC GENE REGULATION.
As the complexity of organisms is increasing the mode of gene regulationmust be complex to control the process of gene expression.
For the process of cell specialization and cell regularity it needs the process of gene regulation for eukaryotic organisms.
The process of gene expression itself is very complex in case of eukaryotic organisms.
Transcription and translation are considered as the basic steps in the process of gene expression.
In eukaryotes because of the presence of nucleus seperates transcription from translation in a way not seen in prokaryotes.
Regulation occur at the following steps thus regulating the process of gene regulation in eukaryotes if included.
A)At the time of transcription B)While RNA processing C)During the process of m RNA longetivity D)Translation.
These are the major steps when the eukaryotic regulation occur.
REGULATION OF TRANSCRIPTION /RNA PROCESSING PROCESS
Steps involved mRNA processing are A)Addition of 5?cap B)Addition of a3?poly(A)tail C)Removal of introns .
Usually the regulation process occur in any of these steps.
The regulation at this step of transcription /RNA processing is divided into two types. A)Whether RNA must process B)Which exons are retained m RNA.
Regulation step at RNA process indicates/ determines whether the m RNA gets translated/ not .
During this type of regulation of RNA does not get processed it will be transported out of nucleus and will not be translated forever.
In the second type of regulation i.e., which exons are retaived affects the function of the protein produced.< br/> Exon shuffling is a process in which some genes have exons that can be exchanged thus by this process the polypeptide produced could have a different function lather than the actual function , this production may some times even cause the premature stopin g of cooling.
REGULATION OF RNA LONGETIVITY
Longetivity is the step of degradation of RNA after being processed i.e., the life time of RNA .
The regulation at this step results in the required gene product to be formed rather than other gene product .
For example of two mRNA?s of same langetivity are present and one is made to degrade an hour late than first one by following the process of gene regulation it results in the production of the required ploypeptide more in amount than the other.
REGULATION OF TRANSLATION
This is one of the major regulatory step in eukaryotes in which although the mRNA is ready because of this step of regulation the expression of the product cannot occur.
ex: Although mRNA is ready in the egg it does not get expressed until it gets matured.
REGULATION OF TRANSCRIPTION
Instead of repressors and operon system that are present in prokaryotes activation /other wise transcriptional factors paly a major role in transcription control /regulation.
Regulation can be brought by
A)Altering the rate at which RNA transcripts processed.
B)Altering the rate at which mRNA gets degraded.
C)Altering the efficiency of translation.
PROMOTER AND ENHANCER ELEMENTS
Transcriptional factors are the important protein complexes that help RNA polymerase in binding to DNA.
There are five major types of transcription factor that palys a role in the gene expression like zinc finger proteins , helix turn helix protein , lucin zipper proteins-binding DNA ,helix loop helix-usually then get dimerized.
Steroid receptors In ligand binding thus regulating the activity.
These transcriptional factors which are named as activators ,promoters, enhancers paly a major role in the regulation of the eukaryotic gene expression.
ENHANCERS
The transcriptional factors whose binding increases the rate of transcription of the gene are called enhancers .
Usually these are located thousand of box pairs away from the gene they control.
Enhancers are usually located upstream ,down stream or even with in the gene they control.
Enhancers binding proteins in addition to their DNA binding site have sites that bind to transcription factors assembled the proteins of the gene .
The length of these enhancer is very short and these enhancers bind to the protein to enhance the transcription levels of genes.
Enhances donot directly act on the promoter region but are bound by activator proteins which usually help in transcribing of genes.
EXAMPLE AND ROLE OF A GENE ENHANCER:
HACNS contributed in evolution keeps in modification in the ankle and foot thus allows human to walk.
Most of the enhancers are located upstream.
Some more enhancers include ECOLI
It is usuallu located 120 bp from the start site contain sites for nitrogen regulatory protein C.
Yeast GAL1 and Gal0 genes
These are some of the enhancers which usually enhance the process of transcription.
SILENCERS:
Quite Opposite factors to enhances are silencers usually on binding of a transcriptional factor to these of causes the gene expression to be repressed .
PROMOTER
These are the regulatory regions located upstream towards 5?region of a gene.
Promoter usually regulates where ,when,and to what level of gene is to be expressed.
Promoter are the regions where the RNA polymerase binds these allowing the process of gene expression to proceed.
In eukaryotes RNA polymerase cannot recognize the promoter by itself then it needs the help of some transcriptional factors which aid in binding to the DNA .
These promoter usually contain specific DNA sequences that are recognized by protein known as transcription factors.
Promoters are processed in both prokaryotes and eukaryotes.
Prokaryotic promoter are the short sequences at -10and -35 positions
These are present upstream from the transcription start site.
Sequence of promoter which is at -10 is called the box with TATAAT nucleotides.
Where are sequence at -35 usually consists of six nucleotides TTGACA.
Eukaryotic promoters cause the DNA to bend back on itself which allows for placement of regulatory sequences.
TATA box lies very close to transcriptional sole in eukaryotes .
Promoters are on broad classified into two types .They are core promoters and proximal promoters
CORE PROMOTER This is placed approximately at -34 ,core promoter acts as a binding site RNA polymer.
PROXIMAL PROMOTER Uusally this is upstream of the gene that tends to contain primary regulatory elements .
It is situated approximately at -250 and is specific for transcription factor binding sites.


REPITITIVE DNA
DNA sequences that are repeated are called repetitive DNA sequences.
These repetitive DNA does not code for any proteins.
Repititive DNA counts 5-1000 nucleotides repeated 100 times .
ex:GGTTA GGTTA GGTTA???????????..
Repititive DNA are classified into three different types
A)Highly repetitive B)Moderately repetitive C)Single copy or (very low copy number)
HIGHLY REPITITIVE
10-15%of DNA of highly repetitive.
These are usually present adjacent to each other and the DNA can integrate very soon.
A-T-T-C-G-A-T-T-C-G- - - - - - - - - - - -
repeats are again classified into A)Satellite DNA B)Mini satellite C)Micro satellite.
satellite DNA : They tend to produce a different frequency of the nucleotides adenine,cytosine ,guanine,and thymine.
mini satellite DNA: Mini satellite usually contain the G-C rich DNA upto a length of 10 bp to 100 bp
micro satellite: These are also called as simple sequence repeats .
Micro satellites are typically neutral and co-dominant .
These are usually used as molecular markers.
USES OF REPEATS
: These are used in finding out the kinshop and parental hereditary paternity
moderately repetitive: Roughly 25-40%of DNA fragments usually repeat.
This include repeats also known as mobile elements/transposable elements
Interspered repeats are broadly classified into two types
A)Short B)Long .
SHORT NUCLEAR ELEMENTS: These are the short DNA sequences that represent the reverse transcribed RNA molecules transcribed by RNA polymerase III into tRNA ,rRNA .
The most common types of SINES are alu sequence
These 280 bp long which does not have any coding sequences .
SINES account for 13% and usually represented as junk DNA.
LONG NUCLEAR ELEMENTS: These are found in large numbers in eukaryotic genomes .
These are transcribed to an RNA using RNA polymeraseIII.
LINES code for enzyme like reverse transcriptase
LINES also code for endonucleases
SINGLE COPY
: This class accounts for 50-60%of mammalian DNA
Gene rearrangement
: A structural ordered of a chromosome because of recombination of genes that causes a change in the order of its loci and results in the formation of new arrangement of sequence of gene is called gene rearrangement.
TYPES OF REARREAGEMENTS INCLUDE
: Deletions,duplications ,inversions are considered as the significant source of gene variation.
deletion: Certain sequences are usually deleted thus in this type of mutations .
Usually one or more nucleotides are removed from the DNA thus alters the reading frame of the DNA
Duplication
: Duplication also called chromosomal duplication /gene amplification no.ofcopies of a single functional sequence DNA is produced using this duplication process.
Inversion
: In an inversion usually the entire section of DNA is reversed.
Deletions and duplications usually result in specific susceptibility to rearrangements and then genomic instability.
It was found that many discase traits consist of genomic rearrangements rather than point mutations of single genes.
Some abnormalities and disregulations result due to gene rearrangements.
DNA rearrangements occurs both by homologoes and non homologous recombination mechanisms .
Homologous recombination is found to be the best way of gene rearrangements .