Sunday, 31 January 2021

What are the Chargaff's rules? & It's importance for DNA

Chargaff's rules were given by Austrian-American biochemist Erwin Chargaff (1905-2002) in 1950. Chargaff's rules are universal. Most forms of life obay this rules. This rules are applicable on the double stranded DNA, in both circular and linear form.

DNA contains four types of Deoxyribonucleotides having  Adenine, Guanine, Cytosine and Thiamine. In DNA structure Adenine pairs with Thiamine and Guanine pairs with Cytosine. So there are two hydrogen bond present between Adenine and Thiamine(A=T) and three hydrogen bond present between Guanine and Cytosine(G≡C).

Studies of Chargaff on DNA chemistry (Chargaff's rules) -

• Rule -1

All DNA possess purine and pyrimidine in equal proportions (1:1 ratio). All DNA have A=T and G=C.
Explanation -
  According to this rule the concentration of Adenine is always equal to the concentration of Thiamine and the concentration of Guanine is always equal to the concentration of Cytosine. (%A=%T & %G=%C)
  Hence, this rule says that total number of Adenine always equal to the total number of Thiamine and the total number of Guanine always equal to the total number of Cytosine.
The total number of Purine ( Adenine & Guanine) will always be equal to the total number of Pyrimidine (Thiamine & Cytosine). This means A+G = T+C, the ratio of Adenine and Guanine is equal to the ratio of Thiamine and Cytosine.

• Rule -2
  DNA from different sources have a characteristic A+T/G+C ratio and AT≠GC.
Explanation -
This rule only the balance of AT pair and GC pairs varies between species, means the percentage of G+C does not necessarily equal to the percentage of A+T.

Some important question about Chargaff's rules

Q1). If a double stranded DNA has 21% of Cytosine, calculate the Adenine.
Q2). In an experiment, DNA was found to have 31% Adenine and 19% Guanine. The quantity of Cytosine shall be
Q3). A segment of DNA has 120 Adenine and 110 Cytosine bases. The total number of nucleotide present in the segment is

Answers-
Ans. 1)  29% Adenine.
Ans. 2)  19 % Cytosine.
Ans. 3)  460.

Saturday, 30 January 2021

Reproduction in Bacteria by Binary Fission (steps)

 

When the bacteria are inoculated in to a nutrient medium and incubated, they grow and Increase in number. Bacteria reproduce mainly by transverse binary fission. However, some bacteria show unusual methods of reproduction.


Reproduction in Bacteria by Binary Fission :

This is the most common mode of reproduction in bacteria. where a single cell divides in to two daughter cells of equal size. A cell just needs to grow to its double the size and then splits in to two daughter cells after development of a transverse septum. The cell division is preceded by chromosome replication, so that each daughter cell gets a copy of genome.

Events leading to new cell formation :

The new cell formation Initiates by the septum formation in the centre of the cell, when the cell reaches to a maximum size and cell mass. This is followed by formation of two daughter cells. The steps leading to binary fission are as under:

1). Septum formation, leading to cell division starts only after the completion of chromosomal replication.
2). Inward growth of cytoplasmic membrane starts, at the middle of the cell. Mesosomes play important role in this event.
3). Now the new cell wall material deposits along the Invigilating cell membrane. This results into septum formation and formation of two daughter cells.
4). In case of cocci, the septum formation starts at the equatorial ridge in the cell wall. In this case, the new cell wall material is approximately one half of the total.
5). In case of gram-positive rods, the septum formation occurs in the same manner. Only 15% of the new wall is dertved from septum. The remainder is formed along the cylindrical part of the cell. This is because the cell grows by elongation.
  In the cell, the new cell wall material is being added as the inner layers. During growth, outer old layers get spread thinly and ultimately destroyed by degradative enzymes.

Septum formation in the gram-negative becteria.
a) Cell wall layers of bacteria. b) Formation of fold on outer membrane at a site where septum will be formed. c) Downward synthesise of cell membrane and peptidoglycan layer, resulting in septum formation. d) Completion of septum formation. e) Invagination of outer membrane, followed by cell division. 

6). In gram-negative bacteria, a fold appears in the outer membrane, at which the septum formation starts. Cell membrane and peptidoglycan layer grow inward and ultimately divides the cell. The outer membrane Invaginates only at the linal stage of cell division.
7). It is proposed that central mesosome plays important role in cell division. Genome of cell remains attached to mesosome. During binary fission, the mesosome divides and the genome replicates. Hence, each daughter cell gets complete genome.

Monday, 25 January 2021

Intron & Exon : Definition, Characteristics, Distribution, Classification Function and significance

  A gene is a section of DNA with information to construct a protein. Most of the potion of a gene in eukaryotes consist of non-coding DNA. That interrupts the relatively short segment of coding DNA. A gene mainly contains two parts one is Introns and another one is Exons.

Exons are the coding sequence
  of the gene.
Introns are the non-coding
  sequence of the gene.

Introns & Exons

The mRNA molecule synthesized from such a gene by transcription is called a primary RNA or pre mRNA transcript.


* Introns :

An intron is any non-coding nucleotide sequence within a gene, which is represented in the primary transcript of the gene but is removed by RNA Splicing in final mRNA product.

Introns are first discovered in 1977 by Phillip Sharb and Richard Roberts independently.

Characteristics :

- The term intron refers to both the DNA sequence within gene and RNA corresponding sequence in primary transcripts.
- Introns are not universal, because they are not present in Prokaryotes but present in most genes of higher eukaryotes.
- Introns range in size from about 50 nucleotides to >100,000 nucleotides.
- Large genes consist of a long string of alternating exons and introns with most of genes consisting of introns.

Distribution :

Introns are rare in Prokaryotic genome and not found in most genes of lower simple eukaryotes such as yeast.
Many genes like interferons, histone genes, ribo nuclease genes, heat shock protein genes, many of G-Protein couppled receptor lack introns. Which indicates the intros are not essential for gene function in eukaryotes.
  Introns are also found in nuclear, mitochondrial & chloroplast genome.

Frequency :
The frequency of intron within different genomes is observed to very widely across the spectrum of biological organisms.
Examples:
a). Extremely common in nuclear genome of vertebrates specially human and mice. In many vertebrates protein coding genes contain multiple introns.
b). Introns are rare in nuclear genes of bakers yeast.
c). Mitochondrial genome of eukaryotic microorganisms contain many introns, while mitochondrial genome of vertebrates does not contain introns.

• Longest eukaryotic intron found in Drosophila, located on dhc-7 gene, the length of intron is >3.6Mb.
• The shortest intron found in human, located on MST1L gene which is 30 bp long.

Classification of introns :
  Introns are classified into four types depending upon their splicing machanism and location.

Type 1 ( AU-AG or AU-AC Introns)
  This type of introns are found in nuclear eukaryotic pre mRNA.
  This type of intron is non self splicing. It requires spliceosome for splicing.

Type 2 (group-I introns)
  This type of introns are found in nuclear, eukaryotic pre rRNA genes and organelles RNAs.
  This types of introns are self splicing.

Type 3 ( group-II introns)
  This type of introns are found in chloroplast, mitochondrial genes and some Prokaryotic RNAs.
  This type of introns are self splicing.

Type 4 ( tRNA introns)
  This type of introns are found in eukaryotic nuclear pre rRNA.
  These introns are non self splicing and they requires enzymes for splicing.

Introns usually do not code for proteins. However certain introns of group I and II class contain ORF (open reding fragment) whose expression allows the protein to be mobile.

Significance of introns :

1) Alternate splicing

  Alternate splicing isThe processing of an RNA transcript into different mRNA molecules, and a singal gene might encode many proteins. Thus aquisition of introns have been positively selected as a source of functional diversity.

2) Regulating gene expression
- Introns contain functional elements like regulatory elements and alternative promoters.
- First introns provides binding site for transcription factor or may act as transcriptional enhancers/repressors.
- Introns act as internal pramoter to produce alternate RNA.
- Introns are required for RNA editing (5' cap & 3' tail).
- sequence introns serves as guide for the chemical alteration of exonic nucleotides by RNA editing.

3) gene splicing by mi RNA &
si RNA
- Introns releases trans acting factors such as micro RNA (mi RNA) and small nucleolar RNA (sno RNA).
- mi RNA targets include transcription factors and genes involved in stress response, hormone signalling and cell metabolism.

4) Exon shuffling
  Introns play important role in evolution by facilitating recombination exons of different genes.


* Exons

Exons is any part of a gene that
encode a part of the final mature RNA. Produced by that genes (RNA) after introns have been removed by RNA splicing.

Introduction :

- The term exon is derived from expressed region and coined by Walter Gilbert in 1978.
- The term exon refers to both the DNA sequence with in a gene and corresponding sequence in RNA transcript.
- In RNA splicing introns are removed and exons are covalently joined to generate mature mRNA.
- The entire set of exons is called exome, just like gene and genome.
- In human genome only 1.1% genome is exon, 24% introns and 75% of genome is intragenic DNA.

Structure and Size of Exons :
- In protein coding genes, the exons include both protein coding sequence and 5' and 3' untranslated regions (UTR).
- Mostly first exon includes both the 5' UTR and dirt part coding sequence. rarely UTRs may contain introns.
- Some non-coding RNA transcript also have exons and introns.

Functions of Exons :
- Exons are piece of coding DNA that encode proteins.
- Different exons encode different domains of a protein. These domain may be encode by a single exon or multiple exons spliced together.
- Presence of exons and introns allows greater molecular evolution by exon shuffling.
• Exon shuffling - exons on sister chromatids are exchanged during recombination.

Alternative splicing :
  This process allows the exons to be arranged in different combinations, when introns are removed.
  Exons also allows for multiple proteins to be translated from same gene.
  Alternate splicing and deftect in splicing can result in disease,   example- Alcoholism and cancer,
Human slo gene - The gene consist of 35 exons, which combine to form over 500 mRNA. The different mRNAs control which sound frequencies can be heard.

Sunday, 24 January 2021

Difference between Translation & Transcription

  Translation and Transcription both are the process in which genetic information which is stored in DNA is converted into Protein via RNA. Transcription and translation both are important part of the central dogma. Here we are comparing Translation and Transcription.

Difference between Transcription & Translation :

• Transcription is the process which takes place inside the Nucleous of the cell in Eukaryotes and Cytoplasm in Prokaryotes. /
Translation is the process which takes place in the Cytoplasm of the cell in Eukaryotes as well as in Prokaryotes.

• Transcription is the process where our cell will create RNA molecule using DNA templet. /
Translation is the process of building a Protein from mRNA strand which is synthesise in Transcription.

• In Transcription process RNA-polymerase enzyme splits the hydrogen bond between double helix structure of DNA and move ahed. /
In Translation Ribosome reads mRNA strand codon and move ahed (Ribosome starts to read from AUG codon).

• For the Transcription process row materials are used is four types of ribonucleotide molecules - ATP, GTP, CTP and UTP. /
During Translation the raw material used which are 20 types of amino acids.

• The Transcription process forms three types of RNAs, which are rRNA, tRNA and mRNA. /
While these all three types of RNAs take part in Translation process.

• The Transcription process requires RNA-polymerase enzyme and some other Transcription factors. /
Transcription requires Ribosome and initiation factor, elongation factor and translocase factors.

• For the Transcription process there is no requirement for any adaptor molecule. /
During Translation process Adaptor(tRNA) molecules bring amino acids over the temple.

• The Transcription process is ends with the Splicing machanism of mRNA by removal of Introns by using spliceosome. /
While there is no any Splicing machanism present in Translation process, it uses spliced mRNA.

• In the series of reaction first Transcription occurs and than Translation occurs.


You may also read

 Intron & Exon : Definition, Characteristics, Distribution, Classification Function and significance

DNA vs RNA - structure, difference and comparison





Saturday, 23 January 2021

Classification of Microorganisms Based on Requirements for Molecular Oxygen

Classification of Microorganisms based on requirements for molecular oxygen 


Oxygen is essential for energy metabolism of the organisms, However, bacteria differ in their requirement for molecular Oxygen. Accordingly, they can be classifled in to four groups. 

 1. Aerobes 

 2. Anaerobes 

 3. Facultative anaerobes

 4. Microaerophiles 


1. Aerobes 

   These are the bacteria which can grow only in the presence of free molecular oxygen. When grown in broth media, these bacteria, usually give pellicle growth. This is because of their aero tactic movement. 

   Bacteria move towards surface medium due to the surface tension. Hence, they grow on the surface of medium to give pellicle growth, e.g. Bacillus.


2. Anaerobes 

   Anaeroble bacteria are those which do not require molebular oxygen for growth. They can be further classifled as

a). Non stringent or tolerant anaerobes and

b). Stringent or strict unaerobes.


- Non stringent anaerobes are those, which can tolerate low levels of molecular oxygen. 

- Stringent anaerobes are those, which cannot tolerate even low levels of gaseous oxygen and are killed on exposure to oxygen in air.

e.g. Certain Clostridia. In broth media, they grow at the bottom of tube. 

Patterns of growth show by different oxygen types of bacteria in broth media. (1) Pellicle growth where oxygen is maximum available. (2) Growth beneath the surface where oxygen availability is limiting. (3) Dispersed growth through the medium. (4) Growth at the bottom of the tube where anaerobic conditions exist.


3. Facultative anaerobes 

   The bacteria, which can grow both in presence or absence of molecular oxygen or air, are called facultative anaerobes. They can grow in absence of oxygen. But if it is available, they can use molecular O₂ during their respiratory metabolism and grow well. 

e.g. E. coli. In broth media, their growth develops uniform turbidity. 


4. Microaerophiles 

   These are the bacteria which require low levels of oxygen for growth. However, they cannot tolerate high concentration of O₂ present in air and are killed. In broth media, they grow beneath the surface, where oxygen availability is limiting. 



Friday, 22 January 2021

Ames test Principle

   Chemical carcinogens can induced mutation which can leads to cancer or tumor. AMES test is uses bacteria for the detection of these potential mutagens which are carcinogenic agents. Ames test is the simple process, it is inexpensive and it is indirect assay for testing of mutagens.

Ames test is given by Brush Ames in 1970 in university of california. In Ames test becteria Salmonella typhimurium (Histidine auxotroph) is used.

In this experiment Salmonella typhimurium culture is used, which is an auxotroph for histidine, which means that can not synthesise histidine its own. To grow this organisms you need to provide histidine in medium. 
   Another thing that add into culture is rat liver enzyme so that the test organisms can be metabolised into the metabolites products. 

Principle:

In Ames test there are two plates are prepared one is test plate and another is control plate.

1). In the Test plate you add a possible mutagen and incubate this plate. Now the bacteria are spread on an agar plate with a small amount of histidine. This small amount of histidine in the growth medium allows the bacteria to grow for an initial time and have the opportunity to mutate. 
  When the histidine is depleted, only bacteria that have mutated to gain the ability to produce their own histidine will survive. After 48 hours of incubation, the mutagenicity of a substance is proportional to the number of colonies observed.
 
2)  The control plate is without the possible mutagens. Mutation can be random or induced so in a condition where you are not adding a possible mutagens. In that condition also there are chances that the test culture will show you some amount of mutation. That's why control plate is prepared, so that you can check for the presence of natural revertants
  Natural revertants are basically mutants which are having a mutation, which will change their histidine auxotroph back to the wild type form. 

Interpretation :

If the test compound is not mutagenic the number of colonies on the control plate, it will be approximately to the control plate. but if the test compound is mutagenic it will induce a large amount of mutation in the test organisms in the salmonella typhimurium culture and you will obtain a high number of colonies which will indicate a really high number of revertants 

Conclusion :
 If the number of colonies on the test plate is more compared to the control plate in that condition you can conclude that a test compound is actually a mutagen.





 

Saturday, 16 January 2021

Structure and Difference Between MHC Class 1 and MHC Class 2 molecules

  MHC (major histocompatibility complex) is a tightly linked complex of genes. These genes will be encoding for proteins that are expressed on the cell surface and this perticular cell surface proteins are really important for Antigen presentation and rapid graph rejection. These MHC genes are present on Chromosome 6.

MHC genes encodes MHC molecules which are expressed on cell surface. In case of a foreign antigen, which is comming inside the cell will broke it down into smaller peptides. These peptides then bind to the MHC molecules and these MHC molecules present a small peptide or a small fragment of this antigen on the cell membrane for recognition by respective
T-cell.


Structure of MHC Class-I Molecule :

  MHC (Major Histocompatibility Complex) molecules are present on the cell membrane.
MHC Class I molecule contains one alpha(α) chain and one beta(β) chain
Alpha chain is of 45 kDa. This alpha chain is divided in 3 segments -
  1) Extracellular segment,
  2) Transmembrane segment,
  3) Cytoplasmic tail segment.

The Extracellular segment consist of 3 domain that is α1, α2, and α3 domains. Each of these domain contains approximately of 90 amino acids. The alpha chain is inserted into the cell membrane through a Transmembrane domain of 25 amino acid. The entire structure has a cytoplasmic tail of 30 amino acids.

The α3 domain is invariant and it contains binding site for the cytotoxic T-cell (CD8+). The peptide binding cleft is present between alpha-1 and alpha-2 domain and it is closed both ends.

Along with the α chain MHC Class-1 molecules show the presence of a small β2 microglobin chain of 12 kDa. This
β2 microglobin has a non covalent interaction with the α3 is very essential for the expression of MHC Class 1 molecule on the cell surface. If β2 and α3 are not interacting then no active MHC molecule can be formed. After a peptide is bound to the peptide binding cleft, the MHC Class 1 structure is transported to the cell membrane and the antigen is presented to the respective T-cell.


Structure of MHC Class-II Molecule :


  MHC Class-II molecule consist of an alpha (α) chain and a beta (β) chain both of which are embedded into the cell membrane. Both the alpha chain and beta chain consist of three segment which are -
  1) Extracellular segment,
  2) Transmembrane segment,
  3) Cytoplasmic tail segment.

Alpha chain is of 33 kDa, and it has two external domains
α1 and α2. Where β chain is of 28 kDa. and it has two external domains β1 and β2.

In MHC Class 2 molecule β2 domain contains the binding site for T-helper cell. In MHC Class 2 molecule peptide binding cleft is presented between α1 and β2 domain and this is open at both ends. This peptide binding cleft can accomodate peptide of the size 13 to 18 amino acids in length.


Difference Between MHC Class 1 and MHC Class 2 Molecules :

- MHC Class 1 molecule is Expressed on Nucleated cells. while, MHC Class 2 molecule Expressed on Antigen presenting cells(APCs)

- External domain of MHC Class 1 molecules contains membrane bound glycoprotein (α chain of 45 kDa) associated non-covalently with β2 microglobulin (12 kDa).
While, external domain of MHC class 2 molecule contains 2 different glycoproteins α chain (33kDa) and β chain (28kDa).

- MHC Class 1 molecule α chain has 3 external domain.
MHC Class 2 molecule α and β chain have 2 external domain each.

- MHC Class 1 molecules are Encoded by B, C and A genes located at telomeric end on chromosome 6 in human.
MHC Class 2 molecule are Encoded by DP, DQ and DR genes located at centromeric end on chromosome 6 in human.

- In MHC Class 1 molecule Peptide binding is located between α1 and α2 domain.
while, In MHC Class 2 molecule peptide binding is located between α1 and β1 domain.

- Peptide binding cleft closed at both the end in MHC Class 1 molecule and it is open in MHC Class 2 molecules.

- Nature of bound peptide in MHC Class 1 molecule is Extended structure in which both ends interact with MHC cleft but middle arches up away from MHC molecules. While in MHC Class 2 molecule Extended structure is held to a constant elevation above the floor of MHC cleft.

- MHC Class 1 molecule generate Cell mediated immune response, while MHC Class 2 molecules generate Humoral immune response.

- The source of Peptide in MHC Class 1 molecule is Endogenous origin. While in MHC Class 2 molecule it is Exogenous origin (self or non self).

- MHC Class 1 molecules are present antigen to Cytotoxic Tc (CD8+) cells.
MHC Class 2 molecules are present antigen to Helper Th(CD4+) cells.

- Binding site of respective T cells in MHC Class 1 molecule is at α3 domain. while in MHC Class 2 binding site of respective T cell in β1 domain.


Tuesday, 12 January 2021

Difference Between Enveloped Viruses & Non-Enveloped Viruses

 Despite being exposed to billions of viruses per day, only some viruses actually cause infection indicating that viruses affect humans in different ways. here, we will discus how the characteristics of viruses correlate with their structure.


Virion

Virus partical also known as the virion, which is the extra cellular  form of a virus used to spread from one cell or organisms to another. in contrast to bacteria or fungi, viruses are complex molecular structures. however they don't count as living organisms as they dont have their own metabolism.

  Viruses come in many shapes ans sizes such as bullet, icosahedral and sphere. A virion consists of a viral genome which can be either DNA or RNA, and is enclosed in a protein capsid, that provides protection. These viruses are referred to as non-enveloped viruses. This is in contrast to viruses surrounded by a biological membrane, known as an envelope which contains lipids and proteins. As its name suggests, these viruses are also called enveloped viruses.

A virion is only infectious is fully assembled. If the envelop of virus is destroyed it is no longer infectious. Viral genome don't encode the full set of proteins required for independent metabolism. however they only encode certain proteins that link the virus to the metabolic pathways of the cell they infect, such as energy metabolism or translation machinery.

There fore viruses are considered obligate intracellular parasites and the cells they infect are referred to as host cells. After entry into the cell, the viral genome is released from its protective shell and depending on whether it has a DNA or RNA genome, interferes with host transcription and translation processes.


Capsid and Envelop

  Transfer of the viral genome and its release into the cell requires the viral capsid to be sufficiently stable to protect the genome while being labile enough to be released or uncoated inside the host cell. Structures with such opposite properties are called metastable.

A capsid consists of identical subunits made up of structural proteins connected to each other in a process termed self assembly. As the individual proteins assemble to form a large capsid, their surface charge and polarity is minimised while their contact region is maximised. This leads to a decrease in the energy of the capsid system and provides the driving force for capsid self assembly. At the same time, the energy stored in the order of the system increases, which can act as a driving force for viral uncoating. As capsid subunits are held togather by weak non-covalent bonds, dissociation can be initiated by exposing the capsid to thermal energy.

Viral uncoating can also be initiated by mechanical traction or a pH change. however, the trigger releasing the viral genome depends on the virus and the host cell. Contrary to what one would expact, an envelope increases viral sensitivity to physical influencing factors as biological  membranes are relatively fragile structures.


Enveloped virus vs non enveloped viruses

• Consequently, enveloped virus can't survive the extreme acid of the stomach and don't usually enter through the gastrointestinal  tract in the host. While non-enveloped viruses are less sensitive to extreme pH and can easily enter via gastrointestinal  tract.

• Enveloped viruses are also more sensitive to heat dryness and disinfectants such as ethanol or propanol, making them an easy target of the hygienic measures. While the non enveloped viruses are less sensitive to heat, dryness and simple disinfectants.

• Advantage of viral envelop is the biological membrane that forms the envelop is drived from the host cell, originating from compartments such as the endoplasmic reticulum or golgi apparatus, or from the plasma membrane. This provides a shields protecting the virus particle more efficiently against the attack of the host immune system. although the viral envelop is embedded with viral proteins. While non-enveloped viruses are easy target for the host's immune system.

• Enveloped viruses can also exit the host cell, without disrupting the host cell membrane. In contrast, non-enveloped viruses exit the cell by lysis, which is a highly immunogenic event. enveloped viruses are usually less immunogenic than non-enveloped viruses. In other words, the envelope helps the virus to evade the host immune response.



Saturday, 9 January 2021

DNA vs RNA - Structure, Differences and Comparison

  Deoxyribonucleic acid (DNA) and Ribonucleic acid (RNA) are the most important molecules in biology, responsible for the storage of genetic information that underpins all life. They are both linear polymers, consisting of Penrose sugars, phosphates and Nitrogenous bases, but there are some key differences which separate the DNA & RNA. Let's learn about that difference.

1). Chemical Differences between DNA and RNA :

• DNA stands for Deoxyribonucleic acid mean one oxygen atoms is messing on the second carbon OH group, only Hydrogen atom is present.
   While, RNA stands for Ribonucleic acid means a complet ribose sugar is present with OH group at the second number of carbon.

• DNA has for basic Nitrogenous bases which are Adenine (A), Guanine (G), Cytosine (C) and Thiamine (T).
  While, RNA also has four bases, forth base as Uracil (U) insted of Thiamine (T).
- The difference between Thiamine and Uracil lies in the methyl group, Thiamine has an external methyl group while Uracil don't have methyl group.

DNA is a Double Stranded molecule, while RNA is a Single Stranded molecule.
- DNA is a double helical molecule mean double helix and these two strands are twisted on each other to form a spiral arrangements.
  While, RNA is a single helix and can wrap with each other to fold.

DNA is a stable molecule, while RNA is and unstable molecule and the life of RNA is very short as compare to DNA.

• DNA molecule is very long molecule up to 3 billion base pairs in humans.
While the strands of RNAs are very short as compared to DNA, largest RNA may be few thousand
bases only.

Structure of Nucleic acids DNA & RNA


2). DNA & RNA Type Differences

• DNA has three basic types :
  - A Type DNA,
  - B Type DNA,
  - Z Type DNA.
While, RNA has more than 30 valid known types like
Transcriptional RNAs :
    mRNAs,
    rRNAs,
    tRNAs.
Regulatory RNAs,  Post- modification RNAs and Viral RNAs etc.


3). DNA & RNA Structural Difference :

• DNA structure is consisted of Exons (Coding sequence) and Introns (non-coding sequence) both portions.
While, RNA strand contains only Exons, Introns are removed after splicing machanism

• DNA is wrapped around Histone proteins, although histones are not part of DNA but still histones are compulsory for coiling of DNA and stability.
  Similarly RNA don't have any kind of histone proteins.

• DNA strand can coil around itself to condense.
  While, RNA strand fold by interaction with it's own chain into secondary and tertiary structure.

• DNA has transposable elements  which can jump from one locus to  other. Similarly RNA don't have such elements.


4). DNA & RNA Functional difference :

• DNA have it's own information in terms of genes which encode into RNA and then into proteins to build or regulate function in organisms.
  While, RNA is formed from DNA and them function as massenger or regulator element.

• DNA can not function directly, while some RNAs like regulatory RNAs function directly without going into proteins.


5). DNA & RNA Location Difference :

• In Eukaryotes DNA is present in nucleolus only. In eukaryotes RNA is formed in Nucleous then migrate to cytoplasm.
- In Prokaryotes DNA is in specific area in cytoplasm called as nucleoid, while in Prokaryotes RNA moves freely in cytoplasm.


6). Evolutionary Difference between DNA & RNA :

•  DNA is considered as secondary molecule with respect to evolution an it is thought as evolved from RNAs.
  While the primary genetic molecule which is thought to be made from raw during origin of living things is RNA. So RNA evolved First

Monday, 4 January 2021

Apoptosis : Mechanism and Morphologic Changes

  Apoptosis, also called "programmed cell death" is the process where the cell regulates its own death through the production of certain enzymes. These enzymes cause degradation of nuclear and cytoplasmic material and the cell breaks into fragments called apoptotic bodies. These apoptotic bodies are then removed by the process of phagocytosis.

  Apoptosis could be triggered by factors such as infections, especially viral infections, misfolding of proteins due to mutations and DNA damage due to mutation, radiation, hypoxia and free radicals.

  Apart from pathologic factors inducing programmed cell death, apoptosis is also a homeostatic mechanism, where cells that are not needed are killed, thereby maintaining a steady state population of cells.

Cell undergo Apoptosis

Morphological changes during Apoptosis


  During apoptosis, the cell undergoes certain morphologic changes that can be seen in light and electron microscopy.
• The cell shrinks and becomes smaller in size.
• The cytoplasm and organelles become tightly packed.
• The nuclear chromatin shrinks and becomes condensed at the center or at the periphery, a process called pyknosis.

Following this, chromatin material undergoes  karyorrhexisi.e it disintegrates and becomes fragmented. Under the microscope, a cell undergoing apoptosis would appear shrunken, with a dense eosinophilic cytoplasm and small clumps of hematoxophilic chromatin material.

Further, the cell starts to form blebs on its surface and starts to break off into small fragments called apoptotic bodies. These apoptotic bodies have portions of cytoplasm, organelles and nuclear fragments of the cell.

Apoptosis does not elicit inflammation

It is important to understand that apoptosis does not elicit inflammation, unlike another form of cell death called necrosis. This is because; apoptotic bodies have an intact plasma membrane and prevent any content from leaking out into the interstitial space. Also, apoptotic bodies are quickly recognised by phagocytes and removed from the environment.

Pathways of inducing cell death

  A cell can undergo apoptosis due to several reasons and depending on the etiologic factors may have 3 different pathways of initiating cell death. Apoptosis could be initiated by signals from
- The intrinsic pathway,
- The extrinsic pathway and
- The perforin /granzyme pathway.

Intrinsic pathway

  In the intrinsic pathway, mitochondria become leaky and ooze out proteins called cytochrome C, which initiate apoptosis.

  Usually the cytoplasm and mitochondrial membrane harbour proteins called Bcl-2 and Bcl-x which are anti-apoptotic and preserve the integrity of the mitochondrial membrane, preventing apoptotic proteins like cytochrome C from leaking into the cytoplasm.

However, in the absence of a growth signal, or insults due to radiation or protein misfolding, stress proteins called “BH3 only” proteins are stimulated. “BH3 only” proteins comprising of Bim, Bid and Bad  proteins block the function of Bcl-2 and Bcl-x.

These proteins further activatetwo pro-apoptotic effectors called Bax and Bak, which create channels in the mitochondrial membrane, allowing intra-mitochondrial proteins like cytochrome C to leak into the cytoplasm.

  Cytochrome C, in the cytoplasm, binds with a protein called Apaf - 1 (Apoptosis activating factor 1) to form a complex called “apoptosome”.

Apoptosome complex binds with Caspase 9 and begins to cleave and activate adjacent caspase-9 molecules.
  Caspase 9 is an initiator caspase and activated Caspase 9 molecules activate executioner Caspases like caspase 3 and caspase 6 leading to apoptosis of the cell.

Causes Apoptosis

Cytotoxic T-lymphocytes or CD8 T cells cause apoptosis of infected cells or tumor cells by Fas Ligand and Fas/death receptor interaction. Many cell types express a receptor called the Fas receptor and cytotoxic T cells express Fas Ligand. These Cytotoxic T cells bind to the Fas receptor on tumor cells or infected cells through their Fas Ligands.

This interaction may produce apoptotic signals through two pathways -
-  Extrinsic pathway
-  Granzyme/Perforin pathway.

Extrinsic pathway

The Extrinsic Pathway involves binding of an adapter protein called Fas-associated death domain (FADD) to the cytoplasmic end of at least 3-4 Fas ligands. This then binds with caspase 8, another initiator caspase, which gets cleaved to become active.

  The active caspase 8 further activates other caspase 8 molecules. These in turn activate executioner Caspases 3 and 6 leading to apoptosis of the cell.

Granzyme/Perforin pathway

At times the Granzyme/Perforin pathway is initiated on FasL/Fas receptor interaction. T cells release perforins, which form trans-membrane pores on the cells, through which granzymes, another protein secreted by T-cells, enter.

Granzymes, could either directly activate executioner caspase molecules or cause DNA cleaving, leading to apoptosis.

Execution of Apoptosis

Executioner caspases 3 and 6 cause degradation of
chromosomal DNA and also degradation of cytoskeletal proteins, which cause the morphological changes such as nuclear fragmentation and cellular shrinkage respectively. However, it is not yet known what causes changes like cellular blebs and apoptotic bodies.

Apoptotic bodies are coated with a phospholipid called  phosphotidylserine, which is recognised by phagocyte receptors. Also, apoptotic bodies may be coated with opsonins like antibody IgG or complement proteins like C3b which are recognised by phagocytes thus facilitating rapid phagocytosis of apoptotic bodies. 

Nitrogen Cycle Steps : Proteolysis, Ammonification, Nitrification Denitrification and Nitrogen Fixation and diagram

  Cycling of nitrogenous materials makes life on the Earth possible . The sequence of changes from free atmospheric nitrogen to fixed inorganic nitrogen, to simple organic nitrogen to complex nitrogeneous compounds in tissue of plants , animals and microorganisms and the eventual release of the nitrogen back to atmosphere is called Nitrogen cycle .

  Each process involves reduction or oxidation of nitrogen and each step is mediated by specific organisms having specific enzymes.

  The nitrogen cycle is due to the activities of decomposers and nitrogen bacteria. The nitrogen bacteria are grouped into three categories based on their roles as :
  • Nitrogen - fixing bacteria  
  • Nitrifying bacteria
  • Denitrifying bacteria

I). Nitrogen fixation :

   Thousands of tonnes of nitrogen is present in atmosphere, but this free N2 cannot be directly used by plants or animals.

Nitrogen fixation is a process in which atmospheric nitrogen is converted into ammonia which is then used in the biosynthesis of organic nitrogenous compounds, conversion of a limited amount of nitrogen to ammonia is done chemically during lighting strikes.

         N₂ + 3H₂➞ 2NH3

  The major amount of nitrogen fixation is biological and It is uniquely a procaryotic process.

  The nitrogen-fixing bacteria fix 70% of about 225 million metric tons of nitrogen annually. The cyanobacteria and bacteria that fix nitrogen are diverse and found in normal soils, deserts, hot-springs, marine as well as fresh water, Antartic regions, etc.

• Nitrogen fixation can be carried out under oxic and anoxic conditions. The nitrogen fixers can be grouped in two main groups:
(a) Non-symbiotic nitrogen fixers. (b) Symbiotic nitrogen fixers.

a). Non-symbiotic nitrogen fixing bacteria live freely and independently in the soil. The aerobic bacteria belonging to this group Include organisms like Azotobacter, Beijerinkia and cyanobacteria, and Trichodesmium.

  Cyanobacteria are procaryotic, free living algae that can fix nitrogen by obtaining hydrogen from hydrogen sulfide (H₂S) in sulfurous environments. Azotobacter are free-living soil bacterium and a heterotroph. The methylotrophic bacteria can also fix nitrogen when grown on methane, methanol or hydrogen containing substrates.

  Some genera of facultatively anaerobic bacteria that can carry out nitrogen fixation include Bacillus, Citrobacter, Enterobacter, Klebsiella, etc. Klebsiella pneumoniae can also fix nitrogen in the intestines of humans and other animals. The obligate anaerobes that carry out nitrogen fixation include the phototrophic bacteria belonging to the genera Rhodospirillum and chromatium, and other bacteria namely Clostridium, Desulfotomaculum, Desulfovibrio, etc.

(b) Symbiotic Nitrogen Fixers :
  Symbiotic nitrogen fixation is a process in which atmospheric nitrogen is fixed into ammonia by a mutualistic association between plants and bacteria, neither of which can fix nitrogen independently.

  The best studied nitrogen-fixing bacteria is Rhizobium the symbiont of roots of leguminuous plants. They can fix 150-200 kg of nitrogen per hectare of land annually.

  It is an endosymbiotic relationship between roots of leguminuous plants like clover, lupins, peas, beans, alfa-alfa and a nitrogen fixing bacterium Rhizobium. These bacteria establish themselves in the cells of root tissues of the host plant.

After developing infection thread on root hair, the infected plant grows abnormally with increased rate of growth forming nodule on the root system. In the nodule the rhizobla dwell and fix atmospheric nitrogen into NH4+ which is made available to plants.

These other symbiotic nitrogen fixing systems include actinomycetes and frankia-which can fix nitrogen symbiotically with many types of woody shrubs.

The genera Azospirillum fixes nitrogen association with grass/cereals. The cyanobacteria- Trichodesmium and Anabaena sp. fixes nitrogen in association with water fern Azolla.

  They are found in the pores of the leaves of Azolla and are used as biofertilizers in rice fields in Asia.

  The symbiotic nitrogen fixers has great nitrogen amount of nitrogen fixing ability and responsibe for large amount of nitrogen fixed globelly.

  The amount of nitrogen fixed by free living bacteria contributes to a small fraction of total nitrogen

Enzymes Involved in nitrogen fixation :
• The genetic information for nitrogen fixation is found in the 'nif' genes of nitrogen fxing microbes.
• The 'nif' genes encodes for the formation of an enzyme complex called nitrogenase system composed of nitrogenase and nitrogenase reductase.

The N, fixation reaction occurs as under :
N₂ + 8e¯ + 8H+ +  16MgATP ➞ 2NH3 + H₂+  16MgADP + 16Pi.

The nitrogenase system has two co-proteins, a MoFe cofactor containing molybedenum as well as iron, and Fe¯ protein containing only Iron.

The active nitrogenase is associated.with Fe, Mo - cofactor. At this site reduction of N₂ to NH3 occurs.

The electrons are transferred through ferredoxin or flavodoxin to nitrogenase (Fe-protein) reductase and then to nitrogenase.

  N₂ fixation requires large amount of ATP and H₂ production also occurs during this process.

  Only some strains of Rhizobium and Bradyrhizoblum have dehydrogenase to utilize the hydrogen. Other N₂ fixing bacteria evolve hydrogen gas and are often colonized by hydrogen oxidizing Acinetobacter strains.

Nitrogenase is very sensitive to oxygen and is irreversibly Inactivated by even low concentration of O₂.

Nitrogen fixation Is therefore restricted to habitats in which nitrogenase is protected from exposure to molecular to molecular oxygen.

Both symblotic and  non symblotic N₂ fixers can be used as blofertillizer to increase soil fertility.

The product of N₂ fixation is ammonia, which is immediately incorporated into organic matter as an amine (glutamine). These amino N atoms are converted into amino acids and proteins, nucleic acid and other biomolecules in plants, animals and microorganisms.

The N₂ cycle continues with the degradation of these molecules into NH4+ within many microbes through many pathways including proteolysis.

II). Proteolysis :

Plants use ammonia produced by symbiotic nitrogen fixers, nonsymbiotic nitrogen fixers, and ammonia available through assimilatory reduction of nitrates to synthesize amino acids and eventually plant proteins.

  When plants are consumed by animals the plant protein are converted to animal proteins. This immobilized nitrogen in animal and plant proteins and other nitrogenous compounds can be released only when plants and animals die.

These proteins and other nitrogenous compounds are decomposed in the soil.

The process of enzymatic breakdown of proteins is called proteolysis.

Proteolysis is carried out by microbes which produce extracellular enzymes called Proteases.

The proteins are converted to smaller molecules called peptides, which are further decomposed to amino acid by enzyme peptidase.

Proteinases are produced by many fungi and bacteria including Clostridium, Bacillus, Pseudomonas, Proteus, Aspergillus, Mucor, spp. etc. Peptidases are produced by many bacterial genera. The process is also known as dissimilation of organic nitrogen.

III). Ammonification or Amino Acid Degradation :

The end product of proteolysis are amino acids which are further degraded by soil microorganisms for use as nutrients.

• Ammonification is the process in which release of ammonia takes place from complex organic nitrogenous compounds. It usually occurs under aerobic condition. The microorganisms responsible are Peus, Bacillus, Microccus, etc.

• The ammonia produced has different fates :
(1) As it is volatile, it may leave the soil.
(2) It may be solubilized in water and ammonium lons are produced.
(3) Ammonium lons can be utilized both by plants and microorganisms.
(4) It may be oxidized to nitrates by a process called nitrification.

Putrefication is the anaerobic degradation of amino acid resulting into formation of amines. This is mainly carried out by anaerobic bacterlum belonging to genus Clostridium.

IV). Nitrification :

This is a two step process carried out by chemolithotrophs.

The first step is sometimes described as Nitrosofication where NH4+ is first oxidized to nitrite by bacterial genera Nitrosomonas, Nitrosobacter, Nitrosolobus, Nitrosococcus, etc.

The reaction occurs as follows:
NH4+ + O ➞ NO₂¯ + H₂O + 2H+ + energy
* NH4+ ammonium lons
* NO₂¯ Nitrite

These bacteria are chemolithotrophic, gram negative, aerobic, motile, rod-shaped bacteria, sensitive to acidity.

The second step described as Nitrification is carried out by nitrite oxidizing bacteria as under :
NO₂¯ + O ➞ NO3¯(nitrate) + energy

The nitrite produced in the first step is toxic to plante the nitrification of nitrite to nitrate is very useful for agriculture.

Microorganisms involved in Nitrification:
Bacterial genera Nitrobacter, Nitrospira and Nitrococcus. They are chemolithotrophic gram negative, nonmotile bacteria, rods, sensitive alkaline conditions.
  In addition Nitrosomonas eutropha has been found to oxidize ammonium ion anaerobically to nitrite in a denitrification related reaction.

V). Assimilatory nitrate reduction :

The production of nitrate by nitrification is important because it can be reduced and incorporated into organic nitrogen by plants.

The use of nitrate as a source to synthesize organic nitrogen is called assimilatory nitrate reduction.

Many heterotrophic bacteria can also reduce nitrate to ammonia to organic nitrogen by assimilatory reduction.
NO3¯ + 8e¯ + 9H+ ➞ NH3 + 3H₂0

The ammonia formed is used for the synthesis of amino acids ➞ proteins ➞ other nitrogenous compounds. This process occurs under aerobic condition, in water-logged soil. The oxygen of nitrates serves as an acceptor o electrons and hydrogen.

VI). Denitrification :

In this process nitrates are reduced to nitrous oxide (N₂O) or molecular nitrogen by certain soil bacteria. The process occurs under condition of oxygen limitation.  

The sequence of reactions are as follows:
2NO3¯➞ 2NO₂¯➞  2NO ➞ N₂O  ➞ N₂↑
Nitrate ➞ Nitrite ➞ Nitric Oxide ➞ Nitrous Oxide ➞ free Nitrogen

This process results in a net loss of N₂ from soil into the atmosphere.

The microorganisms responsible for Denitrification:
Achromobacter, Agrobacterium, Alcaligenes, Bacillus, Micrococcus, Pseudomonas, Thiobacillus, Vibrio etc.

Several anaerobic bacteria in soil carry out dissimilative nitrate reduction in which nitrate is reduced to ammonia as follows:
  NO3¯+ H₂ ➞ NH3 ➞ N₂O

The process is enhanced by :
(1) Presence of organic matter
(2) Temperature between  
      25-60°C.
(3) Neutral to alkaline pH.
(4) Anoxic condition.

The major products of denitrification include N₂ gas and nitrous oxide, although nitrite also can accumulate. Nitrite is of environmental concern because it can contribute to the formation of carcinogenic nitrosamines.

Finally nitrate can be transformed to ammonia in dissimilatory reduction by a variety of bacteria including Geobacter metallireducers, Desulfovibrio spp., Clostridium spp., etc.

A recently Identified form of nitrogen conversion is called the anammox reaction (anoxic ammonium oxidation process). In this process anaerobic, chemolithotrophs use ammonium ion as electron donor and nitrite as the terminal electron acceptor.

The marine planctomycete bacteria oxidize large amounts of NH4+ ion to N₂ through anammox reaction.

Denitrification removes nitrogen from soil, thereby reducing fertility of soil.





Friday, 1 January 2021

Difference Between Mitosis & Meiosis

Cell cycle is one of the most breathtaking processes that helps the cells to not only grow but also divide. Cell cycle has two major phases :
1) Interphase, which helps in the preparation of the cells for the division.
2) M(Mitosis/Meiosis) phase, this phase carries out the division of the cell.

Major differences between Mitosis & Meiosis :

First and kye difference between Mitosis and Meiosis is -
Mitosis is occurs in the somatic or body cells, while Meiosis is occurs in exclusively in the reproductive cells.
- That means Mitosis occuring irrespective of whether the individual is a male or a female give rise to somatic cells, which are exact copies of the parent cells.
- On the other hand Meiosis occurs in males and females giving rise to haploid sperm and egg cells respectively.
  The process of Meiosis however has different names in both the sexes, due to the different cells produced as an end result, Meiosis is also referred to as over genesis that is the process of oveum production in females and spermatogenesis that is the sperm production process in males.

•  Another major difference is that Mitosis is used as a mode of asexual reproduction in lower organisms, while Meiosis helps in forming gametes for sexual reproduction.

•  Mitosis has only one cell division while Meiosis involves two successive cell divisions. That simply means Mitosis is a single step cell division, while Meiosis is a two step cell division, and this results Meiosis having two phases namely Meiosis I and Meiosis II.

Mitosis produces two daughter cells, while Meiosis produces four daughter cells after completion of cell division.

• One of the major difference between Mitosis & Meiosis is the chromosomal number of the daughter cells.
- In Mitosis daughter cells are diploid with two sets of chromosomes just like parent cell,
- In Meiosis daughter cells produced are produced haploid with only one set of chromosome. These daughter cells of Meiosis are called gametes. Gametes have exactly half the number of chromosome from the parent cells.

• The two daughter cells that are produced by Mitosis are genetically similar to each other. While the all four daughter cells produced by Meiosis are genetically different from each other.

• Mitosis process has four stages of cell division which are:
  - Prophase,
  - Metaphase
  - Anaphase,
  - Telophase.
While Meiosis has two round of cell division and the process has a total eight stages of cell division which are -
- Prophase I,    - Prophase II, 
- Metaphase I, - Metaphase II,
- Anaphase I,   - Anaphase II,
- Telophase I,  - Telophase II.

• Mitosis occurs in all organisms except viruses, while Meiosis occurs only in Plants, Animals and Fungi.

Deeper comparison between Mitosis & Meiosis :

• The first stage of Meiosis one that is Prophase I is very long phase consisting of other sub stages. On the other hand Prophase of Mitosis is comparatively shorter than Prophase I.

Crossing over does not occur in Prophase of Mitosis, while crossing over definitely occurs in Prophase I of Meiosis.

Independent assortment is technically absent in the Metaphase of Mitosis, while it occurs in Metaphase I of Meiosis.

• The separation of chromatids takes place during Anaphase of the Mitosis, and on the other hand the separation of homologous chromosomes takes place during Anaphase I and the separation of sister chromatids during Anaphase II of Meiosis.

Important tasks for these two processes perform in our body -
- Mitosis helps in the healing and repairing of the cells and tissues of our body. It's also responsible for the growth and the development of the body.
- Meiosis however helps exclusively introducing gametes, this ensures genetic diversity.