Restriction enzymes (or) restriction endonucleases

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Restriction endonucleases

[or]

restriction enzymes 

[or] 

molecular scissors 

---Restriction endonucleases these name ends with a suffix ASE which means this an enzyme.
---Endonucleases are proteinitious enzymes
---enzymes are worked as catalysts. We all know about catalysts that they enhance the rate of reaction and reduce time taken for a successive reaction

--- for example all the living organisms are blessed with a life. Right?
But most us ( humans) lead life in routine manner just like a normal chemical reaction.
--- when we add some goals, aims, aspirations to our life, just like a catalyst to reaction this will enhance the efficiency of our life.

Enzymes with somewhat similar activity of restriction endonucleases
Enzymes those perform degradation of DNA similar to endonucleases are as follows
1.  S1 nucleases
2 . Exonucleases    
S1 nucleases act upon only single standard genetic materials without out any recognition and accuracy.

Exonucleases also removes nucleotides from the single standard DNA. Exonuclease is a co enzymes of DNA POLYMERASE 1.  which removes only 1 to few nucleotides, starts its activity either from 5' or from 3' end, not from both ends.

             RestrictionEndonucleases            

History
---once upon a time, before the discovery of R.Eases a technique named SONICATION is used to agitate the DNA.

Restriction enzymes, a Revolutionary discovery in molecular biology.

Why they are called so??

A name RESTRICTION is given because of their ability of restricting (host restriction and modification phenomenon)
restriction enzymes  are found in bacterium. This enzymes acts as defense system, just like our immune system
--- we are infected by bacteria where as bacteria are infected by bacteriophage.
( virus ) when a bacteriophage invade on bacteria and injects its genetic material into the bacterium these restrication enzymes acts as defensive barriers. R.E cuts the viral dna and meanwhile protects own genetic material by methylating ( adding methyle group) specific or recognition sequence.

         This idea of restriction enzymes started as a hypothesis by Werner Arber who noticed that certain bacterial strains fought off bacteriophage infection by chopping off its DNA. But bacterial DNA remains unchanged why?

Arber and meselson hypothesized that bacterial cells produce two types of enzymes: one called a “restriction” enzyme that can identify and cut foreign DNA, and a “modification” enzyme that recognizes the host DNA and protects it from cleavage. This hypothesis was proven in an experiment in which two enzymes were isolated from E. coli. The modification enzyme, methylase, protected DNA of the bacterium, while the restriction enzyme chopped off phage (non-methylated) DNA.

Chronological order of discovery:
1969 Werner Arber and Stewart Linn discovered that most
bacteria contained a class of enzymes which modify the bases in
DNA termed methylases.

The phenomena of restriction and modification were illustrated by experimenting with bacteriophage λ on two E. coli host strains. C and K

In 1970, Hamilton O. Smith, Thomas Kelly and Kent Wilcox isolated
and characterized the first type II restriction enzyme, HindII, from
the bacterium Haemophilus influenzae.

In 1972 Daniel Nathans and Kathleen Danna showed that cleavage of SV40
DNA by restriction enzymes yielded specific fragments which can
be separated using polyacrylamide gel electrophoresis, thus
showing that restriction enzymes cut at specific sites and can be
used for mapping of the DNA.

Arber discovered restriction enzymes.
Smith verified Arber's hypothesis.
Nathans pioneered the applications of restriction enzymes

1n 1978 three of these collectively awarded with noble prize.

Although primarily found in bacterial genomes
and plasmids, restriction endonucleases also exist
in archaea, viruses, and eukaryotes.
As manimum as 7 and maximum of 14  genes are found in bacteria, but some of them are inactive.
Including all types, there are
3500 restric-
tion enzymes found in 10000 speices that recognize 259 different DNA
sequences are now known.
Data of enzymes discovered are updated every month in follow website
www.neb.com
Host restriction modification phenomenon:

Restriction endonucleases were originally named for their ability to restrict the growth of phage in a host bacterial cell by cleavage of the invading DNA. The endonucleases recognize specific sites for action called Restriction sites.
In this manner, they may be acting as bacterial protection systems. The DNA of the host is protected from restriction by the activity of a methylase(s),which recognizes the same sequence as the restriction enzyme and methylates a specific nucleotide (4-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, or 6-methyladenine) on each strandwithin this sequence. Once methylated, the host DNA is no longer a substrate for the endonuclease.
Because both strands of the host DNA are methylated and even hemi-methylated DNA is protected,
freshly replicated host DNA is not digested by the endonucleases.
   
   Digested                   protected

                                       CH3
                                          |   
5' G| A A T T C 3'   5' G A A T T C 3'
        |________
3' C T T A A |G 5'   3' C T T A A G 5'
                                                |
                                                CH3
   Staggered cut         methylated
                 EcoR1 modification
As so often in biology exceptions exists

Here in some cases instead of methyl group DNA is modified by inserting sulfur.
Recently discovered dnd system illustrate about the insertion of sulfur into the PHOSPHORUS backbone forms a PHOSPHOROTHIOATE group one of the non- bridging oxygen bonds in the phosphate backbone can be replaced by sulfur.
This type of modification are carried out by five types dnd's I.e., dnd A B C D E
or 4 ssp enzymes  I.e., ssp A B C D
L- cysteine desulfarse provides sulfur atoms to dnd A, which is  needed for modification.
dnd's have operons and other activities
This is a very vast Topic (dnd and ssp ) . if you want more data you may work on it by books

Nomenclature:
Restriction enzymes are named according to the proposal of Smith and Nathans. Enzymes are named with three letters in italics are derived from the
first letter of the genus and the first two letters of the microbial species from which the enzyme was
derived. An additional letter without italics may be used to designate a particular strain. This is followed by a roman numeral to signify the first, sec-
ond, and so on, enzyme discovered from the organism.

Enzyme        Source Organism                            Recognition Sequence
HpaII        Haemophilus parainfluenzae               C/CGG    GGC/C
MboI        Moraxella bovis                                      /GATC     CTAG/
NdeII        Neisseria denitrificans                          /GATC      CTAG/
EcoRI       Escherichia coli RY13                           G/AATTC  CTTAA/G
EcoRII      Escherichia coli RY13                      /CC(A or T)GGGG(T or A)CC/
EcoRV      Escherichia coli J62/pGL74               GAT/ATC  CTA/TAG
BamHI      Bacillus amyloliquefaciens                 G/GATCC  CCTAG/G
SauI          Staphylococcus aureus                        CC/TNAGG  GGANT/CC
BglI           Bacillus globigii                                       GCCNNNN/NGGC
                                                                                     CGGN/NNNNCCG
NotI          Nocardia otitidis-caviarum                GC/GGCCGC   CGCCGG/CG
DraII         Deinococcus radiophilus                     RG/GNCCY  YCCNG/GR

/ = position where enzyme cuts; N = any base, R = any purine, Y = any pyrimidine

Isoschizomers

Different types of restriction enzymes isolated from different bacteria which
have same recognition sequence.
Ex. SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each
other

Neoschizomers

Different R. enzymes having same recognition sequence but cutting site (base) is not same.

Working of restriction enzymes in simply in 3 words
Finding           కనిపెట్టుట             recognition
Covering         కాపాడుట             methylation
Cutting            కత్తిరించుట           digestion

Strategies followed by restriction enzymes for recognition:

Cutting of DNA by Restriction Enzymes:

It might seem logical for the DNA to be cut at the recognition site where the restriction enzyme binds. This is often true, but not always. There are two major types of restriction enzyme that differ in where they cut the DNA, relative to the recognitionsite. How restriction enzymes find their binding sites in vast lengths of DNA is considered

When the restriction enzymes diffused to the target DNA, check the site of contact for a match to their recognition sequence, and released the DNA if it did not match. This procedure would take so long that invading viral DNA would be expressed and take over the cell before the restriction enzyme destroyed it.

Two major strategies have been proposed for proteins that must locate target sequences within long DNA molecules in a reasonable time. This applies not only to restriction and modification enzymes but to regulatory proteins that must locate promoters or other specific sites on DNA.
One way is for the protein to slide along the DNA strand without letting go.
The protein monitors the sequence rapidly, but without extreme accuracy, as it moves along the DNA strand. If a possible match is found, the enzyme stops and tests if the match is accurate. The alternative is hopping from one site on the DNA sequence to another that is usually reasonably close but may sometimes be far away . The enzyme does not usually lose contact with the DNA but relies on DNA looping to bring the sites together . After hopping, the protein will prescan the DNA by local sliding for a while before hopping again.

● ---- protein or enzyme reading DNA by sliding or by hopping

 
Classification of restriction enzymes:
Type 1
Type 2 (orthodox)
Type 3
Type 4
Type 1 enzyme :
It is the first identified enzyme in two strains of E. coli (K-12 and B).

Type I enzyme cuts at a site about 1000 bp away from the recognition site.

The recognition site is composed of two specific portions: one with 3 to 4 nucleotides and other with 4 to 5 nucleotides which is separated by 6 to 8 nucleotides which is non specific and this site is asymmetrical.

It posses both restriction digestion and modification methylase activities.

Their cofactor includes adenosine triphosphate (ATP), S-Adenosyl methionine (AdoMet) and magnesium (Mg2+).

Type I enzyme involves three subunits: HsdR for restriction digestion,
HsdM for adding methyl groups (methyltransferase activity) and
HsdS for recognition of DNA binding site (specificity).

Some of the examples of Type I enzymes are EcoB and EcoK.

Type 2 restriction enzyme :

the common Type II endonucleases
are homodimers (most between 25 and 35 kDa for
the monomeric subunit), require only Mg2-, and
cleave within palindromes, partial palindromes, or interrupted palindromes. Despite dissimilar primary sequence,

Type II enzyme cleave within a short specific distance of about 4 to 8 nucleotides in length and the sites are usually undivided.

They cleave the phosphodiester bond of DNA either as a blunt end or sticky end.

They do not use ATP or AdoMet but require Mg2+ as a cofactor.

Type II enzyme is a homodimeric enzyme and are most commonly used enzymes.

After cutting two types of ands are formed by the restriction enzymes.

1 . Sticky or overhang or cohesive ends
3' over hang
5' overhang

2 . Blunt ends

There are seven subtypes of type II enzyme with different characteristics:
Type IIS (Eg: Fok I)
Type IIE (Eg: Nae I)
Type IIF (Eg: NgoM IV)
Type IIT (Eg: Bpu10 I)
Type IIG (Eg: Eco 571)
Type IIB (Eg: Bcg I)
Type IIM (Eg: DPNI-RO)


Type IIS
Cleavage at a defined distance from the recognition site
Asymmetric recognition site
Fok I

Type IIE
Interact with two recognition sites
One serves as the allosteric effector and the other site is for cleavage
Nae I

Type IIF
Interact with two recognition sites
Both are required for cleavage
Homotetrameric enzyme
NgoM IV

Type IIT
Contain different subunits with restriction and modification activities
Bpu10 I

Type IIG
Comprised of a single polypeptide chain with restriction and modification activity
Eco571

Type IIB
Cleave both sides of the recognition site
Bcg I

Type IIM
Recognize methylated sites DPNI-RO


Star activity of an enzyme :

Under extreme conditions e.g. elevated pH or low ionic strength, restriction endonucleases are capable of cleaving sequences which are similar but not identical to their defined recognition sequence.This altered specificity is known as star activity.

To avoid  star activity one can have to maintain optimum environment of reaction



Type 3 restriction enzyme :
Type III enzyme recognizes two separate non-palindromic sequences and cleaves DNA at the site of 25 to 27 bp from the recognition site.

Type II enzymes are composed of two subunits: Res and Mod.

The Res subunit is required for restriction digestion and the Mod subunit is for the recognition of DNA sequence and methyltransferase modification. Therefore, Type III enzymes are multifunctional and hetero-oligomeric.

Type III enzyme requires ATP and Mg2+ cofactor for their role in DNA methylation and restriction digestion.

Type III enzyme methylate only one strand of DNA at the N-6 position of adenosine which is enough to protect it against restriction digestion.

Some of the examples of Type III enzymes are EcoP I and Hinf III.

Type 4 restriction enzymes:

Type IV enzyme cleaves near or close to the recognition sequence.

It targets methylated DNA, hydromethylated DNA and glucosyl hydromethylated DNA which are all modified types of DNA.

Some of the examples of Type IV enzymes in the systems of E. coli are McrBC and Mrr.

There are also other type of restriction enzyme that are artificial restriction enzyme.

Artificial restriction enzymes :

In addition to the many natural restriction enzymes isolated from bacteria and archaea, it is now possible to synthesize artificial restriction enzymes that cut DNA at any desired sequence. Examples:

zinc-finger nucleases

TALENs

CRISPR RNA molecules

APPLICATIONS

1. Restriction fragment length polymorphism (RFLP)

2. Restriction mapping

3. Molecular cloning

4. Restriction enzyme mediated integration

5. DNA or gene sequencing

6. Pulse field gel electrophoresis etc.,

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Glyoxisomes:-

               Glyoxisomes are specialized peroxisomes found in plants and fungi, especially in fat storage tissues in the germinating seeds and also in filamentous algae. Their main function is to convert fatty acids to carbohydrates. These glyoxisomes take part in photorespiration and nitrogen metabolism in root nodules. It was first discovered by 'Beevers' and 'Breidenbach' and they called these new organelles 'glyoxisomes'.

Structure:-

               Glyoxisomes are very small spherical bodies with a single unit membrane. Their internal content ie, the matrix is finely granular. In glyoxisomes, fatty acids are hydrolyzed to acetyl-CoA by peroxisomal β oxidation enzymes.  In sucrose gradient centrifugation, they have a high equilibrium density. It contains many enzymes such as isocratic lyase, malate synthetase, glycolate oxidase, etc. The enzymatic reactions can be explained below;

Glyoxalate cycle:- 

                    These Glyoxisomes play an important role in the glyoxylate cycle. This cycle is a tricarboxylic acid cycle that converts acetyl-CoA to succinate for the synthesis of carbohydrates. It is an anaerobic pathway found mainly in plants, bacteria, protists, and fungi but not in animals. These glyoxisomes also play a vital role in gluconeogenesis. This pathway allows cells to obtain energy from fat.The cycle can be described below;

• Courtesy by google images

 Peroxisomes:-

                                       Peroxisomes are small and single membrane-bound organelles containing oxidative enzymes, which are found in eukaryotes. These oxidative enzymes are involved in the metabolic activity of the body and the digestive enzymes break down the toxic materials in the cell. The marker enzymes help to distinguish it from the other cell organelles. These peroxisomes are considered an important part of the microbody as they are involved in lipid production. They also convert reactive oxygen species into safer molecules by an enzyme 'catalase' and detoxifies hydrogen peroxide. For eg;

Hydrogen peroxide(  H2O2  )➡️water( H2O ) + oxygen( O2 )

peroxisome or microbody                     

                         

                                    These peroxisomes were first identified by "Christian de Duve" in 1967 as cell organelles. Human cells may contain about 100 peroxisomes depending upon the type of cell. These also play a role in the breakdown of fatty acids in animal cells. Usually, these peroxisomes exist as individual ones (fibroblasts) but sometimes they are found in interconnected tubules such as the peroxisome reticulum in liver. 

Structure of peroxisome:-

                          Peroxisomes are single membrane-bound vesicles found in all eukaryotes ie, both plants and animals. They usually vary in shape, size, and number depending upon the energy requirements of the cell. These are made up of phospholipids which are usually synthesized from smooth ER. They generally range from 0.2-1.5μm. A granular matrix encloses the limiting membrane of proteins and lipids, and the matrix consists of a crystalloid structure containing enzymes.

       There are app. 60 enzymes found in the matrix of peroxisomes which help in the metabolism of the cellular organism. The enzymes involved in the lipid metabolism are synthesized on the free ribosomes and then selectively imported to the peroxisome containing one of the signalling sequences. Some of the other enzymes found in peroxisomes are urate oxidase, D-amino oxidase, catalase. 

Functions of peroxisomes:-

              Some of the functions of peroxisomes are described below

• Peroxisomes involve in various oxidative processes such as hydrogen peroxide metabolism, fatty acid oxidation, lipid biosynthesis.
• They also take part in the catabolism of D-amino acids, polyamines, and bile acids.
• The enzymes present in the peroxisomes help in their metabolic activity like conversion of reactive molecules or their elimination etc.
• Peroxisomes help in the germination of plant seeds by converting storage fatty acids to carbohydrates, which plays a critical role in the growth of germinating plants.
• Peroxisomes also help in the photorespiration in the green plants apart from chloroplasts.
• These help in the degradation of purines by carrying out catabolism of purines, polyamines, and amino acids by uric acid oxidase.
• Peroxisomes of fireflies help in bioluminescence by the presence of luciferase enzyme which helps in finding it's mate or meal. 
• In plants, peroxisomes prevent loss of energy during photosynthesis.

• courtesy by google images.

 Vacuoles:-

                              Vacuoles are the membrane-bound organelles found in all plant cells, bacterial, fungal, and animal cells too. It is a storage structure in a cell. These are the empty space organelles found in the cytoplasm and filled with watery fluid that contains various substances. These vacuoles are the important cell organelles in the cell as they help in the storage of nutrients required by a cell to survive and can also store the waste from the cell thereby preventing the cell from contamination. Plant cells store nutrients, metabolites, and waste in their vacuoles and also use them for transporting from one cell to another. The vacuoles in plants are larger than the animal cells.

Structure of vacuole:-

             It is a membrane-bound structure present in the cellular matrix of a cell. Generally, there is no basic shape or size for this vacuole. The vacuoles are usually small during immature or undividing stages and arise initially to become a large ones. Actually plant cells have larger vacuoles than animal cells as they require more water, organic and inorganic components for the proper functioning of cells.  A vacuole is surrounded by a membrane called "tonoplast" or "vacuolar membrane". 

                      This tonoplast separates the vacuolar contents from the cell's cytoplasm. This membrane mainly involves in the regulation of ions in the cell. It also helps in isolating the particles that are thought to be a threat to the cell. The components of the vacuole is known as 'cell sap' that differs entirely different from the cytoplasm. There may be several vacuoles in a cell and this tonoplast helps to separate from the cytoplasm. These vacuoles are relatively similar to lysosomes as they also contain a wide range of hydrolytic enzymes.

Types of Vacuoles:- 

                  There are various types of vacuoles present in the cells and some of them are:-

(i)Sap vacuoles:- These sap vacuoles are found mostly in plant cells and consist of a number of transport systems for the passage of different substances. A large central vacuole is present in higher plants. The fluid present in this vacuole is known as 'sap'. 

(ii)Contractile vacuoles:- These are found in freshwater algal cells and some protists such as paramoecium. These contractile vacuoles take part in osmoregulation and excretion.

(iii)Food vacuoles:- It is formed by the fusion of lysosome and phagosome and it consists of digestive enzymes by which food and the other nutrients are digested. These food vacuoles are found in protists, protozoa, and other higher animals, etc.

(iv)Gas vacuoles:- These are also called as 'air vacuoles' and store gases. Apart from storing gases, they also help in providing buoyancy, mechanical strength, and protection from harmful radiations. These vacuoles are found in prokaryotes.

Gas vacuoles

Functions of vacuoles:-

• Storage:- Vacuoles help in the storage of salts, minerals, organic acids, and other proteins within the cell. A large number of lipids are stored in the vacuoles. Some waste products are also stored here.
• Transportation in plant cells:- Proteins found in the tonoplast control the flow of water in and out of the vacuole through active transport and also pump potassium ions in and out of the vacuolar interior. It also helps in endocytosis and exocytosis of various substances and lysosomes are the vesicles that intake food and digest it.
• Turgor pressure:- Vacuoles are completely filled with water by which it exerts a force on the cell wall. This is known as turgor pressure. Also, the salts present in vacuole add to the osmotic activity of the vacuole thereby contributing to turgor pressure. This pressure helps in cell elongation, withstand extreme conditions, supporting plants in an upright position, and also provides a shape to the cell.
• The vacuole pushes up all the contents of the cell's cytoplasm against the cellular membrane thus keeping the chloroplasts closer to the light so that the light-absorbing efficiency is improved.
•  The pH of the plant vacuoles may be as 9 to 10 due to large quantities of alkaline substances or as low as 3 due to the accumulation of quantities of acids.
• In fungal cells, vacuoles are involved in many processes such as homeostasis of cell pH, osmoregulation, the concentration of ions and degradative processes, etc.

 

 Nucleus:-

                                The nucleus is an oval-shaped membrane-bound organelle present in all eukaryotic cells. It is present both in plant and animal cells. It is large and present in the center of a cell. It is a structure that contains the cell's hereditary information and helps in controlling the growth and reproduction of the cell. It is the most integral component of the cell and contains DNA which controls the growth and function of the cell and helps in coordinating the cell activities. The nucleus here is similar to the brain in it's functions and hence known as the "Brain of the cell"

                 It was first discovered by "Robert Brown" in 1831 when he was scrutinizing the epidermis in a collection of orchids with his microscope.  He identified a small opaque spot in the cell and later he noted that this spot can be observed in the early stages of pollen formation. He then further realized that this part is an important component in the cell and named it as "Nucleus". 

Structure of Nucleus:-

                      It is a typically membrane-bound structure and the most prominent organelle in a cell. It is present in all eukaryotic cells except in few cells like mammalian cells etc. The nucleus may be round, oval, or disc-shaped depending on the type of cell. Structurally, the cell nucleus consists of the following parts. They are as follows;-

1.  Nuclear membrane
2. Nucleoplasm
3. Nucleolus
4. and chromosomes 

1.Nuclear membrane:-

•  It is a double layered membrane that surrounds the nucleus and protects it from any mechanical injuries.
•  As it encloses the nucleus, it is also called a "Nuclear envelope". 
• It helps in the entry and exit of material into the nucleus and also separates the nucleus from the other parts of the cell.
•  A liquid-filled space is present between the two layers of the membrane called the 'perinuclear space'.
• The nuclear membrane is perforated with numerous pores called "nuclear pores". 
• The "Nuclear pores" are the sites for the exchange of large molecules between the nucleus and cytoplasm.

2.Nucleoplasm:-

•  The liquid-filled matrix present in between the two layers of the membrane is called as 'nucleoplasm'.
•  It is also called the  'karyoplasm'

3.Nucleolus:-

• It is a small, spherical-shaped structure found in the nucleus of both plant and animal cells.
• It plays a vital role in the synthesis of RNA and in the formation of ribosomes.
  
• Some of the eukaryotic cells have nucleus consisting of more than 4 nucleoli.
• The nucleolus disappears when a cell undergoes cell division and again reforms after the cell is formed.

4. Chromosomes:-

• Chromosomes consist of DNA that contains hereditary information and promotes cell growth, cell development, and reproduction.
• These are self-reproducing thread-like structures packed with DNA located in the nucleus.
• When a cell is resting, ie, not dividing the chromosomes are organized into long structures called 'chromatin'.

    (for more details on this topic, refer to the chromosomes and chromatin page in the blog)

Nucleus in plant cell:-

                                  The nucleus in the plant cells can be different in different plants. The various forms of nucleus present in the plant can be given in the following way;-

• Uninucleate cell: Plant cell which contain only a single nucleus and also referred to as monokaryotic cell
• Binucleate cell: Plant cell which contains two nuclei at a time and also referred to as dikaryotic cell. For eg; paramoecium
• Multinucleate cell: Plant cell which contains more than 2 nuclei at a time and also referred as polynucleate cell. For eg; latex cells in plants, bonemarrow cells in animals
• Enucleate cell: Plant cell without a nucleus. For eg; mature sieve tubes of phloem, RBCs of mature mammals.

Functions of Nucleus:-

1. The nucleus is responsible for cell division, growth, differentiation, and protein synthesis too.
2. It helps in the exchange of DNA and RNA between the nucleus and the other parts of the cell.
3. It is the control centre of the organism as it regulates the integrity of the gene and gene expression.
4. It controls the hereditary characters of a cell organism.
5. The nucleus is the site of transcription where mRNA is produced for protein synthesis.

Robert brown

• Courtesy by google images 

 Chromatin:-

                                     In eukaryotes, the genetic material ie, DNA is complexed with proteins in a specialized structure called as "Chromatin". This chromatin consists of double-stranded DNA to which large amounts of protein and a small amount of RNA are added. This chromatin usually engages with functions like repair, replication, and recombination, etc.  The dynamic structure of this chromatin influences it to work functionally in the genome. 

Nucleosome:                                     

                  The fundamental unit of chromatin is called as 'Nucleosome'. It comprises of DNA, RNA, and some basic proteins such as histones and non-histones (acidic proteins). The amount of RNA and non-histone proteins is variable depending on different chromatin structures whereas there are fixed proportions of DNA and histone proteins in a 1/1 ratio. The histones that are attached to the DNA act as 'anchors' that help in the winding of the components. The non-histones are very heterogenous as they vary in different tissues and include DNA and RNA polymerases among other enzymes. The nucleosome is composed of two main parts:

• the core particle and
• the linker region that adjoins the core particles.

      The core particle is highly conserved and composed of 146 base pairs of DNA wraps around the histone octamer (8 octa core proteins).

       The linker histone links the entry or exit of the DNA strand on the nucleosome.

                  

        Histones:-

                        Histones are the basic proteins present in the DNA of chromatin. There are of five types each one present in large amounts. Histones are small proteins that are basic because of their high content of basic amino acids such as arginine and lysine. Now besides being basic,  they help in binding tightly to DNA, which is an acid. The four main histones are H2A, H2B, H3 and H4. 

             These four histones are present in equimolar units, but H1 is not conserved. It is present only once per 200 basic pairs. It is loosely attached to the chromatin and is not a component of the nucleosome core unit ie, DNA- histone structure. It is bound to the linker segments of DNA that joins the neighbouring nucleosomes. Under the electron microscope, the nucleosome of eukaryotic nuclei it was found that the chromatin had the repeating structure of beads about 10nm of diameter connected by a string. It appeared as "beads-on-string" structure.  

                                                         

Types of Chromatin:-

     There are mainly two types of Chromatin. They are explained below:

1. Heterochromatin:-

•   It is a tightly packed form of chromatin silencing gene transcription ie, the genes or transcription sequences present in them are inactive. 
• Heterochromatin is usually present in the nucleus towards the periphery,
• This heterochromatin is difficult to analyze because of it's condensed state and repetitive DNA sequences.
• It is characterized by intense stains when stained with nuclear stains.
• It is a structure that does not alter in its condensation throughout the cell cycle and it is much more condensed than the euchromatin.
• The tightly packaged DNA in the heterochromatin prevents the chromosomes from various protein factors that lead to the binding of DNA.
• It also helps to prevent the inaccurate destruction of chromosomes by endonucleases.
• Heterochromatin has various functions such as gene regulation, chromosomes integrity etc,
• Telomeres, centromeres, bar bodies, genes 1,9,16 of human beings are some examples of heterochromatin.
• There are mainly two types of heterochromatin. They are as follows:

                     a)Constitutive heterochromatin

                      b)Facultative heterochromatin

Constitutive heterochromatin usually contains and packages the same sequences of DNA in all the same species. It is also repetitive and usually coincides with the structural regions such as telomeres and centromeres. The genes of this constitutive heterochromatin might affect the genes of the tightly-packed ones.                                                                                For eg: In humans, the Y-chromosome in men constitutes this constitutive heterochromatin

Facultative heterochromatin is that region of the chromosome that is heterochromatic in some cells and euchromatin in other cells. It is composed of transcriptionally active genes that adopt the structural and functional characteristics of heterochromatin, but is not as repetitive as the constitutive one.                                                                                               For eg: In humans, one of the X chromosomes in women is inactivated as facultative heterochromatin while the other is expressed as euchromatin. 

2.Euchromatin:-

• It is less condensed and contains the most actively transcribed genes. It is lightly packed DNA that is characterized by less intense staining.
• This is present in the interior of the nucleoplasm and the DNA sequences are transcriptionally active.
• The DNA in euchromatin is unfolded to form a beaded structure, unlike heterochromatin to which histone proteins are folded.
• In euchromatin, the DNA is lightly bound and the genes are active or will be active during the growth.
• Euchromatin forms a more significant part of the genome. It makes 90-92% of the genome in the human being.
• Euchromatin is found in both eukaryotes and prokaryotes. and it exists only in one form ie, constitutive form.
• There are various other chromosomes other than heterochromatin which are examples of  euchromatin 

Significance of Chromatin:

                                Chromatin is meant for efficiently packaging of DNA into small volumes to fit into the nucleus of a cell and protect the DNA structure and its sequence. Packaging DNA into chromatin allows for cell divisions and prevent chromosome breakage besides controlling gene expression and DNA replication.

• courtesy by google images