Agrobacterium tumifaciens

Hello Friends
Topic of discussion is Agrobacterium tumifaciens
It is gram negative bacteria.
The agrobacterium causes crown gall disease and hairy root syndrome.
A. tumifaciens causes crown gall and A. rhizogenes causes hairy root syndrome.
The bacteria enter plant through cuts and wounds.
Most of these bacteria have plasmids. The size of plasmid is variable and the ability to synthesize the opines and virulence is plasmid borne traits.
This plasmid is called the Ti plasmid.
Only a segment (which is called t DNA) of this plasmid enters the plant on infection and integrated into host genome at non specific site.
The ends of tDNA are specialised as they are flanked by 25 bp repeats called as border sequence.
These border sequence are involved in tDNA transfer but they themselves do no get transffered.
The genes which regulate the trasfer of tDNA are present at some other sites in plasmid DNA and are called vir genes.
vir A and vir G are important and constituvely expressed and their product are required for expression of other vir genes.
vir A product is a protein kinase.
The wounded plant also secrete acetosyringone for vir gene expression.
vir G product is soluble protein which is phosphorylated by vir A product.
vir G product is transcription factor required for activation of rest of vir genes.
vir B produces component of pilus.
virD1 encodes for endonuclease which act on border sequence to create nicks and initiate transfer of tDNA.
vir D2 remain attached to processed DNA.
vir C1 and C2 product act on enhancer sequence.
vir E codes a DNA binding protein which provide stability to DNA.The processed DNA is called "Fire Cracker Complex".
The vir D2 product are attached to processed tDNA and protect this DNA from host nucleases.
vir D2 protein also has nuclear localising signal and are required for recombination evennts.
The cell in which tDNA enters have high level of phytohormones, the genes called tms1 and tms2 synthesize auxin, tmr synthesize cytokinin, ocs synthesize octopine and nos synthesize nopaline.
This tDNA is also used as vector by removing tms1, tms2 and tmr region.
It is even used to express in eukaryotic system as promoter sequence and polyadenylation sites of tDNA are very similar to eukaryotic system.
Many monocots are not infected by this bacteria as they do not sectrete acetosyringone.





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Cell cycle Continued

Hello Friends
Free online life science study material. To continue with cell cycle
In the M or mitotic phase the CDK1 cyclin B complex also phosphorylate component of nuclear pore and some protein of inner nuclear membrane.so choromosome is unable to bind to inner nuclear membrane and whole structure gets disorganized.
Protein of nuclear pore are imbibed by endoplasmic reticulum and golgi bodies also get fragmented.
Endoplasmic reticulam remains intact.
Centrosome also gets phosphorylated and repell each other.
Microtubule associated proteins are target of CDk1-cyclin B compex, so microtubule also get disorganized.
Microtubule associated protein (MAP) are present on the cytoplasm periphery initialy prior to cell division.
At metaphase Anaphase promoting Complex (APC) is present, which is a ubiquitin ligase.
If APC is inactive, cell do not proceed to cell division and gets activated only when choromosmes are properly alligned to spindle fibre.
If they are not alligned properly, Mad and Bub protein bind to kinetocore and lead to inhibition of APC.
Active APC have two targets:
1. APC bind ubiquitin to securin and cohesin gets degraded by separase enzyme.The securin subunit is attached to separase enzyme and APC act on securin to detach from separase.
2. Cyclin B is target of APC and gets deactivated by APC.
APC inactivated in late G1 phase by Gi/S CDKs.

CDKs are regulated by phosphorylation and dephosphorylation. It is activating phosphorylation at some sites and activating dephosphorylation on the other sites.
A threonin of CDK at 160 position requires phosphorylation and is phosphorylated by CAK= CDK activating kinase.
At 14 and 15 th position threonina nd tyrosin are phosphorylated respectively, they require dephosphorylation for activation and this is done by cdc25 phosphatase.
At 14 and 15 th position during inactive state they are phophorylated by wee1 kinase to make them inactive.


This ends the topic cell cycle regulation.

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cell cycle

Hello All
to continue with cell cycle
When cyclins are complexed with CDKs they become activated.
The cyclinD CDK 4&6 has a target protein called Rb protein.
Rb is a tumor suppressor protein. It is found to be mutated in cancer patients.
In normal cell Rb protein remains under phosphorylated especialy during G0 and G1 phase.
Its activity can be changed by level of phosphorylation.
It has affinity towards E2F which is a transcription factor in under phosphorylated state.
So binding of Rb to E2F suppress the activity of E2F.
The cyclinD CDK 4&6 complex phosphorylate Rb and its affinity towards E2F decreases and E2F after separating from E2F becomes activated and since it is a transcription factor, after activation it helps expression of genes required in cell cycle progression.
Another target of CDK4&6 and cyclin D complex is Cyclin E.
Cyclin E binds with CDK 2 and target the MCM helicase.
MCM helicases after activation unwinds the DNA for replication.

The DNA is scanned by ATM and ATR protein for any DNA damage. They are kinases.
ATM is activated if it find double strand damage and ATR activates by single strand damage.
ATM and ATR  phosphorylate p53 protein and phosphorylation of p53 stabilizes it. after stabilization the level of p53 increases and they expresses cip and kip family of protein as p53 is transcription factor for them, which are discussed in earlier blog. Cip and Kip activation leads to cell cycle arrest in G1 phase.

CDK1 and cyclin B target is condensin protein which are required for chromosome activation in the S phase.
Lamins are also phosphoryated by CDK1 and cyclin B and they get depolymerised after phosphorylaton.
Microtubule associated protein are also the targets of this complex and  get depolymerised.
Rest I will discuss in next blog.

Hello Friends

The Regulation of Cell Cycle
The cell cycle is under the control of various cdks and Cyclins.These control the cell cycle progression to varoius phases of cell cycle.
Here I am dealing with the G or growth phase and M.or mitotic Phase.
The cell cycle is divided into four phases
1. G0 Phase and G1
2. S or synthesis phase wherein synthesis of DNA takes place
3 G2 Phase
4. M or mitotic Phase.
There are check points in the cell cycle which requires CDK-cyclin activity to pass them.
One check point at G1 phase. To pass this CDK4&6 and cyclin D is required.
Another check point is at G1-S transition this require CDK2 and cyclin E.
CDK2 and Cyclin E is also required for initiation of DNA replication and chromosome replication.
For progression through S phase CDK2 and cyclin A is required.
Another check point is at S-G2 transition which requires CDK 1 and cyclinA.
For G2 - M transition CDK 1 and cyclin B are required.
Some mechanism regulate CDKs.
Several inhibitors bind to CDK at specific sites for its activation and deactivation.
1. Ink 4 family of protein: These protein are prominent for CDK 4& 6. when they bind to them they stop cell cycle from G1-S progression and the cell will remain in G1 phase.
2. Cip/Kip Family of proteins: Act on CDK 2 and can also bind to cyclin A and E and stop cell cycle in S or G1 phase. These also hep in association of CDK 4& 6 with cyclinD and in G2 M transition phase it also enhance association of CDK 1 with cyclin B.
All external factors such as erk, ras, Raf, MEK signal the cell externally for cell division and these finaly led to the formation of cyclin D.
Cyclin D is only  synthesised as long as external signals are available.
Cyclin D mutation also led to several cancer.


Hope this will help you all. See you all in next blog.
Thnkyou for reading.

Hello Friends

to continue
Lambda repressor is under tight regulation.
Two types of regulation
1. Positive Autoregulation
2.Negative Autoregulation

1. Positive Autoregulation:
It activates its own expression.
It makes level do not reach so low.

2.Negative Autoregulation:
PRM being activated by repressor at OR2 to make more repressor but if concentration increases too high repressor will bind to OR3 also and repress PRM.
This act decreases concentration of repressor.

On new infection the phage has to chose between lytic and lysogeny.
cII and cIII products decide this step.
cII transcribed from PR and cIII from PL.
cII is transcriptional activator it binds to a site called PRE(Repressor Establishment)
PRE stimulate transcription of cI(repressor).
Thus, repressor can be transcribed from PRE and PRM.
PRE is weak promoter so cII product help polymerase bind to PRE.
Once, sufficient repressors are made from PRE, they can bind to OR1 and OR2 and directs their own synthesis from PRM.
Thus repressor synthesis established from one promoter and maintained by other promoter.
Thus, upon infection transcription immediately initiate from PR and PL.
PR synthesise both cro and cII.
cro favours lytic phase. Once cro product is at certain level of concentration it will bind OR3 and block PRM activity.
cII favours lysogeny by directing transcription of repressor gene and for successful lysogeny repressor must bind to OR1 and OR2 and activate PRM before cro inhibit that promoter.
So, phage favous lytic in good bacterial growth environment and lysogeny in poor conditions.

For diagrams you can consult books or diagrams online.

Hope this will help you all. See you all in next blog.
Thnkyou for reading.

Hello Friends
To continue with the Lysogenic and Lytic cycle of Lambda Phage..
PRM transcribes only cI and it is a weak promoter and efficient only when activator is bound just upstream.
PL and PR are stong promoter and do not require any activator.

For lytic phase PL and PR remains on PRM is off and for lysogeny PRM is on and  PL and PR are off.
cI encodes Lambda repressor which is dumbbell shaped and join to DNA as a dimer.
Lambda repressor and cro can each bind with 6 operators. 3 operator on left and 3 on right side of cI.
OR1 OR2 OR3 are three operators on the right side of cI. PR overlaps OR1 and OR2 and PRM overlaps OR2 and OR3.

For lytic cycle single cro dimer bind to OR3 which overlap PRM and repress it. No repressor and no cro on OR1 and OR2 therefore PR binds RNA polymerase and transcribes lytic genes. so does  PL.
For lysogeny PRM is on. Repressor is bound cooperatively at OR1 and OR2. RNA Polymerase bind at PR and its transcription is repressed.

Induction to lytic phase.

Eg. E. coli sense and respond to DNA damage by activating Rec A protein. Rec A cleaves Lex A protein during DNA damage repair mechanism. Lambda repressor resemble Lex A hence Lambda repressor is also cleaved by Rec A protein.The cleavage of repressor removes C terminal domain of repressor and its dimerization and cooperativity is lost.
Loss of dimer triggers transcription PR and PL which induces lytic phase.
I will continue with it in the next blog.
Thank you for reading.

Overview of Lambda Phage genome

Hello Friends

Free online life science study material.

The topic I am going to brief about is Lambda Phage lysogenic and lytic phase gene regulation introduction.

It is a Bacteriophage which is consist of a capsid, a tail and tail fiber.

It has double stranded, linear DNA as genetic material, 50Kb genome with 50 genes.

The linear genome has 12bp cohesive ends which are complementary and it gets circularize as soon as it infects the bacterium.

Now, gene regulation of lytic and lysogenic phase takes place with the help of regulatory proteins.

A region of genome of Lambda has sequence like this

                                                                                                   

PL

cI

PRM

PR

cro

 All other genes are outside this region except one and are transcribed directly from P_L and P_R or from other promoters whose activities are controlled by their products.

P_RM transcribes only cI . It is repressor maintenance promoter. It is weak promoter and efficient only when activator bind just upstream.

P_L and P_R are strong promoters and do not require activator.






 P_{L and} P_RM  codes towards left and P_{R codes towards right.}

For lytic phase  P_L and P_R are switched on and P_RM  is switched off and for lysogenic phase P_RM and rest two are off.

cI encodes Lambda repressor which can act as both repressor and activator.

As a repressor it s target is σ subunit of RNA Ploymerase , the adjacent to the part of σ which recognize -35 region on DNA.

As activator it works like CAP as in Lac operon.

The cI and cro are functional in dimeric form.

Rest I will discuss in next blog.

          

Hope this will help you all. See you all in next blog.

Thankyou for reading.

Hello Friends

I am Sakshi Garg,  I would like to share my notes with you which I have prepared for preparation. These might be useful for you.

I would also like to know your views regarding each topic, any queries regarding the notes and also if you would like to share any information with me I will be highly thankful.I will be writing breifly and in a very simple language.

Today, the topic that I am going to discuss is Tranduction.



Transduction is transfer of bacterial genes by virus.

Transduction are of two types:
 1. Generalized Transduction
2.Specialized Transduction


Generalized Transduction:

This occurs during lytic cycle of virulent and temperate phage and can transfer any part of bacterial genome.

During packaging of capsids in lytic phase random fragments of bacterial DNA can also be packed by mistake in some capsids.

Since viral capsid can contain limited amount of DNA, the viral DNA is left behind. These virus can inject DNA of bacteria into other bacteria after infection but does not initiate lytic cycle.

Double stranded DNA remain Double stranded during transfer and integrates into host bacterium or endogenote.

Specialized Transduction:

also known as restricted Transduction.

When prophage leaves the bacterial genome the cleavage or excision of viral DNA is improper.

Phage may contain portion of bacterial DNA next to integration sites.

This type of transducing phage is defective and cannot reproduce without assistance.

This can be explained with this example: phage att site or attachment site and bacterial att site are similar and can recombine with each other.
Since viral att site are next to bacterial gal and bio sites these can be carried by lambda phage due to mistake in excising.

Stable transduction may arise through recombination of bacterial genome nad phage chromosome.

Hope this will help you all. See you all in next blog.
Thnkyou for reading.