Genetics of Cancer

The incidences of cancer started shooting up in the year 1991. Since a decade the cancer cases increased by a factor of three. There is simply no complete cure to this dreadful condition. It involves changes in the genome leading to uncontrolled cellular proliferation, transformation, invasion, metastasis, apoptosis suppression, and angiogenesis. The environmental factors such as chemicals, radiations, viruses, microbes, and hormones cause cancer. Apart from the above reasons, there are more factors involved. The cell follows a cyclical pattern of division involving different phases. It includes the G1, S, G2, and the M phases respectively. The transition of one phase from the other involves checkpoints. The CDK/cyclin complexes mainly control the cell cycle. The checkpoints determine the damaged DNA. They also help in checking the problems in the cell cycle machinery. Hence, they play a crucial role in permitting normal cells to continue. Problems in the cell cycle checkpoints trigger cancerous conditions. Certain viruses such as retroviruses cause cancer. They increase the oncogenic products. Also, for the normal cells, apoptosis plays a crucial role. The cancer cells do not undergo apoptosis.

Image 1: Cancer cells and normal cells

Cell cycle and cancer:

A cell cycle involves six main checkpoints such as the restriction point, the G1/S DNA damage checkpoint, the S phase DNA damage checkpoint, G2/M checkpoint, centrosome duplication checkpoint, and mitotic checkpoint. The restriction point occurs between the mid to late G1 phase. This point ensures the cell to enter into the S phase after receiving the appropriate signals. The G1/S damage checkpoint occurs at the G1 phase transition. It senses the DNA damage. The S phase DNA damage checkpoint arrests the cell cycle in the later part. It detects the DNA damage or an incomplete replication of DNA. The G2/M checkpoint also detects the damaged DNA. The centrosome duplication checkpoint detects the defects in the centrosome duplication process. This checkpoint also detects centrosome segregation defects. The mitotic checkpoint occurs in the M phase. It checks the formation of mitotic spindles.

The CDK/cyclin complexes control the cell cycle. The cyclin-dependent kinases belong to the class of kinases. The cyclins are known as the regulatory subunits. The CDKs are known as catalytic subunits. The cell cycle checkpoints involve a genetic control. The genes participating in the multiple cell cycle checkpoints are known as gatekeeper genes. These genes prevent the cell cycle progression until the damaged DNA gets repaired.

Phases of the cell cycle	Cyclin-CDK complexes
G1 phase	Cyclin D, CDK-4 Cyclin D, CDK-6
Late G1 phase	Cyclin E, CDK-2
S phase	Cyclin A, CDK-2
G2 phase	Cyclin A, cdc 2
M phase	Cyclin B, cdc 2

Table: All phases of the cell cycle and the associated cyclin-CDK complexes

1.     G1 Phase:

Alterations in the signaling pathways associated with the cyclin-dependent kinases lead to the uncontrolled cell proliferation. Retinoblastoma involves tumor in the retina. It occurs in childhood. The gene responsible for getting mutated and causing the disease is known as the RB gene. It is present on the q arm of the thirteenth chromosome. Deletion or inactivation of both the copies of the RB gene leads to retinoblastoma. The cell loses the protein product pRb.

This phase requires a regulatory protein. It is known as pRb. It gets phosphorylated by cyclin/CDK complex. The pRb binds to the E2F transcription factor and prevents the cell’s entry into the S phase. After the phosphorylation of pRb, it gets inactivated and releases the E2F. Now the cell safely enters the S phase. However, in the retinoblastoma, the cell loses the pRb protein due to RB gene mutation. Hence, the cell enters into the S phase without checking any damaged DNA. Thus, it leads to an unrestrained tumor formation.

2.     G1/S checkpoint:

The tumor suppressor gene known as TP53 gene plays a crucial role in cell cycle arrest and DNA repair. This checkpoint gets invoked due to dsDNA breaks and damage. The product of the TP53 gene is a protein. It is known as p53. It helps in arresting the cell cycle in the G1 phase or the G1/S phase. After the repair of the DNA, the cycle resumes back. However, failure to get repaired leads to apoptosis or cell death. It occurs in the normal cells where p53 gets activated. In the cancer cells, the p53 is not present. Hence, there is no cell cycle arrest and repair of damaged DNA. Thus, the cells form tumors.

3.     G2/M checkpoint:

It is a DNA damage checkpoint. It helps in progressing the cell from the G2 phase to mitosis phase. It maintains the cdc2/ cyclin B1 in an inactive state. The protein p53 also plays a crucial role here.

Image 2: Cell cycle

Cellular proliferation:

Signal transduction involves extracellular growth factors. They regulate cell growth and differentiation. The genes encoding the growth factors or the growth factor receptors may get mutated. Hence, they lead to oncogenic properties. A gene encodes for the signal transducing protein. It is known as ras gene. The transcription factor gets encoded by another gene. It is known as the Myc gene. Mutations in both the genes also cause cancer.

Genes, Viruses, and Cancer:

Cancer involves mutations in three main gene classes. They include proto-oncogenes, tumor suppressor genes, and mutator genes. The products of proto-oncogenes stimulate cell proliferation. The mutant ones are known as oncogenes. They are the active forms of cancer genes. The oncogenes stimulate unregulated cellular proliferation. The RNA viruses also replicate via DNA intermediate. These viruses are known as retroviruses. Upon the retroviral infection, the RNA genome of the viral particle synthesizes a kind of cDNA. It is known as proviral DNA. The viruses also have oncogenes. They are known as viral oncogenes. When they occur in the host cell, these genes are known as cellular oncogenes. The host DNA sequences homologous to that of the virus are known as proto-oncogenes. These genes get activated to oncogenes. Three main methods do this. The first method involves increasing the amount of proto-oncogene product. The second method involves mutations in the coding sequences. Chromosomal translocation also leads to activation of oncogenes.

Apoptosis and Cancer:

The cell death or apoptosis gets triggered in the case of unrepaired damaged DNA or any other unwanted cellular conditions. The failure of the checkpoints in stopping the cell cycle progression also triggers cancer. Cancer also occurs due to the activation of anti-apoptotic genes such as Bcl_2. Thus, many such factors contribute to cancer. 

References:
[1] Human Genetics, 3/e, Gangane
[2] Molecular Genetics of Cancer, John Cowell
[3] The Genetics of Cancer: Genes Associated with Cancer Invasion, Metastasis, Gajanan V. Sherbet, M. S. Lakshmi
[4] API Textbook of Medicine, Ninth Edition, Two Volume Set, Y P Munjal, Surendra K Sharma

© Copyright, 2018 All Rights Reserved.

Bacteriophage lytic and lysogenic pathways

The bacterial viruses or bacteriophages are quite selfish entities that totally depend on the bacterial cells for their replication. The nature of the bacteriophage is such that it uses the genetic machinery of the bacteria, takes the shelter of the bacterial cells, grows and reproduces in these cells and lyses the bacterial cells to release its hundreds of progenies. Thus a bacteriophage utilizes the bacterial cells and makes them non-functional later on. From the research point of view, the bacteriophages are easy to study since they require bacteria to grow. The discovery of triplet gene code was possible using the bacteriophage. Francis Crick and his colleagues used these entities for exploring wild-type and mutant phenotypes. Gene mapping is possible with bacteriophages. Recombinant DNA technology uses bacteriophages for performing genetic crosses, construction of chromosome maps, and for intergenic and intragenic mapping. Most of the phages infect E. coli for their survival.

Max Delbruck was the first scientist to initiate genetic studies in Bacteriophages. The structure of a bacteriophage is studied using an electron microscope. The design of a bacteriophage is such that it can sit on the bacterial cell and inject its genetic material into the cell. They consist of two basic structural components such as the head and the filamentous tail. The protein coat forming the head is called a capsid. The phage genome is present in its capsid. The bacteriophage has a filamentous tail. It has additional spider-leg like structures. These typical structures facilitate the landing of the bacteriophages on the bacterial cells thereby helping in the attachment.

The capsid is icosahedral with a three-dimensional structure surrounding the nucleic acid. Sometimes the capsid may have a filamentous or helical structure. Bacteriophages may have DNA or RNA as their genetic material. Lambda bacteriophages infect E. coli and have a double-stranded DNA. ϕX174 phages have a single-stranded circular DNA. MS2 phages have a single-stranded linear RNA. Thus there are different types of phages with different genome structures. In some phages the genomes are segmented, meaning that different RNA molecules carry different genes. ϕX174 phages have extra information in the form of overlapping genes that share nucleotide sequences and code for different gene products.

Studying replication cycles in phages helps us in understanding the life cycle of these entities. There are two types of life cycles observed in bacteriophages such as lytic and lysogenic cycles.

Lytic cycle:

A T₄ bacteriophage is used to study the lytic cycle. This bacteriophage has a double-stranded linear DNA. The first step of the lytic cycle involves the landing of the bacteriophage on the bacterial cell surface. The spider-leg like structures on the phage tail facilitate in phage landing and attachment. The phage particle interacts with the receptor protein present in the bacterial cell. The receptor of the T₄ phage is known as Omp C or an outer membrane protein. It is a type of porin that forms membrane channels for uptake of nutrients. The next step is to inject the DNA into the bacterial cells. The structure of the phage facilitates an easy injection of the genetic material into the bacteria. Once the phage DNA enters the cell, the bacteria stops synthesizing own materials.

Image 1: Bacteriophage lytic pathway

The phage genome replication starts as soon as the bacterial replication stops. The replication of the phage DNA begins within a minute. It requires only five minutes depolymerizing the phage DNA. The nucleotides help in replication of the phage genome. New phage capsids start developing after twelve minutes. Various phage particles get assembled within the bacterial cell. The genome or the phage DNA gets inserted into the capsid heads. Cell lysis enables release of the phages. It takes twenty minutes to release the progeny. Each lytic cycle produces 200 to 300 bacteriophages. The progeny phages are further capable of infecting the other bacteria. This experiment was carried out by Ellis and Delbruck in 1939. One step growth curve represents the results. The latent period lasts from 0 to 22 minutes revealed that there was no change in the number of cells during the first twenty-two minutes of the infection. The phages are mixed with the bacteria and plated on a culture medium. An entire lawn of bacteria is allowed to grow on the medium. Each phage infects the bacterium on the plate and the phages released from one infection attack the adjacent bacteria. The cycle continues in this way. The result of lysis depicts a clear patch in the lawn of bacteria. The clear patch is known as a plaque. Most of the phages undergo a lytic cycle. Some phages undergo a lysogenic cycle.

Lysogenic cycle:

Image 2: Lysogenic pathway

The lysogenic cycle of a lambda phage is studied. A specialty of a lysogenic cycle is that the phage genome gets integrated into the host DNA. The striking feature of the lysogenic cycle helped researchers to use lambda phage as cloning vectors. A quiescent form of a bacteriophage known as prophage occurs due to the entry of phage DNA into the cells. The phage DNA and the bacterial DNA undergo site-specific recombination. The integration occurs between identical 15 base pair sequences so that the lambda DNA integrates into the same position within the E. coli DNA. The phage may switch over to the lytic cycle in response to physical or chemical stimuli. A second recombination event begins. This event excises the phage genome from the host DNA. Thus the phage genome replication starts. A Lysogeny is a phenomenon in which the phage genome gets integrated into the bacterial genome. These phages are known as temperate phages. The one carrying a temperate phage in a prophage state is known as a lysogenic bacterium.  Lysogenization is an experimental production of a lysogenic bacterium by exposing them to temperate phage. The bacteria change their phenotype, sometimes in their morphology, pathogenic capability or synthetic properties accompanying a lysogeny. This phenomenon is known as lysogenic conversion. Hence, through lysogenic conversion, the temperate phage can spread virulence factors such as exotoxins and exoenzymes.

     References:
[1] Microbial Genetics, Keya Chaudhary
[2] Molecular Genetics of Bacteria, Jeremy W. Dale, Simon F. Park
[3] Genetics, G. Ivor Hickey

                                                           
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A review on Bacteriophages

Viruses capable of infecting bacteria are known as bacteriophages. These viruses infect the bacterial cells and multiply their genome. They infect various strains of Escherichia coli. The viral properties stop the bacterial replication process. They use the bacterial genetic machinery for their survival and replication. Different institutes worked on bacteriophages and used these viruses as model organisms. It is possible to map the genes with the help of phage genome studies. The bacteriophages follow two types of reproductive cycles such as lytic and lysogenic cycle. Different types of phages exist in the world of microbes.

Bacteriophage consists of a protein coat and a genome. The bacteriophage genetic material consists of DNA or RNA. The bacteriophage structure is made up of capsid head and a filamentous tail. Phage capsid involves proteinaceous coat. The icosahedral capsid is common among bacteriophage. The genome resides in the phage head. During the phage particle assembly, the genetic material gets packaged into the phage head.

There are various classes of bacteriophages. Some of the bacteriophages have a non-enveloped contractile tail whereas some others have an enveloped and rod-shaped tail. Some of them have isometric, ovoid, bottle-shaped, and lemon-shaped structures. It involves variation in the genomes. Some of the bacteriophages have a linear and double-stranded DNA. Examples include T4, Mu, PBSX, and P2 phages. M13 phages have a circular single-stranded DNA. Some other bacteriophages have a circular double-stranded DNA. Linear and segmented RNAs exist in bacteriophages.

Image: Bacteriophage

1.     Bacteriophages as model organisms:

Bacteriophages help in genetic analysis. They serve as vectors in DNA cloning and genetic engineering. It is possible to study genetic recombination using bacteriophages. Bacteriophages infecting bacterial cells produce plaques upon lysis. Hence, it is easy to culture them. Bacteriophages serve as cloning vectors since they exhibit DNA transferring properties.

2. Lytic and lysogenic cycles:

There are two cycles of bacteriophage replication such as lytic and lysogenic cycles. The lytic cycle involves complete lysis of the bacterial cells. The first step involves the infection of bacterial cells. The phage particle lands on the bacterial cell and gets attached to it. Then it interacts with the cell surface receptor and injects its genome into the bacterial cells. Once the phage DNA gets injected into the cell, bacterial replication stops and phage replication process gets initiated. Bacteriophage uses bacterial enzymes for replication. It takes 22 minutes for completing the phage cycle. After completing the replication process, phage particles get assembled and release through the cell. The lysogenic cycle involves the genomic integration into the bacterial chromosome. Depending on the environmental conditions the phage switches over to lytic cycle.

3. Transducing phages:

These phages possess transducing properties. The transducing phages are known as defective phages. During phage genome replication, the defective bacteriophage takes up the bacterial genome. It transfers it to the other bacterium. Hence, the genome gets transferred from the donor bacterium into the recipient bacterium. Two types of transducing phages exist. Generalized transducing phages undergo generalized transduction. Specialized transducing phages undergo specialized transduction. Generalized transduction does not follow any special pattern of transduction.  Any part of the bacterial genome gets transduced. Specialized transduction involves transduction of specific gene or segment of a bacterial chromosome. The defect in the phage becomes an advantage for the bacteria to transfer genes. Transduction does not involve sex pilli of the bacterial cells. It is a phage-mediated process. Bacterial conjugation involves the transfer of F plasmid through sex pill. However, the transduction process directly involves the role of the defective phages.

4. Bacteriophage gene mapping studies:

Studies involving the mutant and wild-type bacteriophage strains helped in mapping the genes. Fine structure and deletion mapping contributed majorly to the field of genome mapping. The idea of overlapping genes came from the bacteriophage gene mapping. Separate mapping of intragenic and intergenic recombinants is possible.

5. Advantages of Bacteriophages:

The bacteriophages are ubiquitous, meaning they exist everywhere. They are highly specific in their action. It is easy to study them using bacterial cultures. Hence, they serve as tools for detecting the pathogenic bacteria. For example, bacteria resistance to specific antibiotics get detected using phage infection. Bacteriophages play a role in therapeutics for tuberculosis. It is possible to improve the TB-vaccine using genetic engineering and bacteriophages.

6. Applications of phages in recombinant DNA technology:

·    Cosmids: A cosmid vector consists of a lambda cos site. Apart from being vectors cosmids serve as probes. They play an important role in FISH and chromosome painting, where cosmid probes get involved. Genomic library preparation using cosmids helps in mapping the genes.

·        For the construction of a cosmid library, we require the phage packaging extract. Here comes the direct role of bacteriophages. The phage packaging extract consists of phage proteins for multiplication, phage lysate, empty phage heads, and unattached phage tails.

·    Phasmids: They are the vectors based on the bacteriophages. A molecular biology technique known as phage display involves the role of these special types of vectors.

·        It is used to study protein-protein interactions and DNA-protein interactions. Phage display involves M13 and filamentous phage.

·   Phage Therapy: Bacteriophages act as anti-bacterial agents since they lyse the bacterial cells. The antibiotic discovery and phages as therapeutic agents still require research and clinical trials, though scientists are working on the same.

·  The role of bacteriophages in the food industry: USFDA approved various bacteriophage products. For example, LMP-102 Intralytix treated ready to eat meat and poultry products. FDA approved LISTEX. It employed bacteriophages for killing the Listeria monocytogenes on the cheese.

·   In vitro diagnostics: The MRSA/MSSA blood test employed bacteriophages for detecting the S aureus and antibiotic-resistant cultures.

·   Contribution in sanitation: Bacteriophages help in sanitation and disinfection of contact surfaces. 
References:
[1] Microbial Genetics, Keya Chaudhari
[2] Molecular Genetics of Bacteria, Jeremy W. Dale, Simon F. Park
[3] Genetics, G. Ivor Hickey

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