Northern blotting technique

Electrophoretically separated RNA molecules get transferred from the gel to the absorbent sheet, immersed in a labeled probe for hybridization. Thus, the northern blotting follows the same steps of Southern blotting. However, RNA instead of DNA gets blotted. The standard Northern blotting procedures help to compare the quantities of the transcripts from different tissues. The technique’s sensitivity increases to a 100 fold using an mRNA or a messenger RNA. The technique analyses many types of RNAs such as micro RNA, small nuclear RNAs or snRNAs, small interfering RNAs or siRNAs, and mRNA. The technique is helpful in RNA interference studies or RNAi technology. It also involves studying the expression of oncogenes. Northern blotting includes human RNAs, plant, and animal RNAs. The technique has placed itself very well in genetic research and engineering.

Image: Northern blotting

The description of the blotting procedure is as follows:

Step 1: Isolation of mRNAs:

RNA extraction uses a homogenized tissue sample. Oligo (dT) cellulose chromatography helps in isolating mRNA with a poly (A) tail. We know the mechanism of polyadenylation well. It protects the RNA from degradation by adding polyadenine nucleotides to the RNA molecule. Widely used methods include phenol-chloroform extraction or trizol method.

Step 2: Electrophoresis:

Once the RNA isolation gets completed, the loading of the samples in the wells helps to obtain the bands. Separation of the RNA molecules involves agarose gels most of the times. Fragmented RNA or micro RNA separation may include polyacrylamide gels. Formaldehyde acts as a denaturing agent in the electrophoresis. It limits the RNA secondary structure. Fragment sizes comparison uses a ladder RNA in another well. Electrophoresis involves staining the gel with ethidium bromide. Handling ethidium bromide requires caution because of its carcinogenicity and neurotoxicity. Ethidium bromide intercalates the RNA, thereby acting as an intercalating agent.

Step 3: Using a nylon membrane:

After separation of RNA molecules through gel electrophoresis, the samples get transferred to a nylon membrane through a capillary system. Following is the apparatus for northern blotting:

The first step involves filling a tray with a buffer solution consisting of formamide. It helps in lowering the annealing temperature of the probe-RNA interaction thereby preventing RNA from getting degraded. Then the RNA gets immobilized to the membrane through a covalent linkage. UV light or heat facilitates this property. The blotting paper used in this apparatus plays a role in capillary action by carrying the buffer solution through the gel. Once the RNA interacts with the buffer, it gets transferred to the membrane. A stack of paper towels and weight kept on the membrane enhances the imprint of the bands on the membrane. Now the RNA molecules present on the membrane exactly resemble as they were on the gel.

Step 4: Hybridization with the labeled probe:

Exposure of the membrane to the probe ensures hybridization of RNA molecules. Northern blotting procedure involves probes composed of complementary sequences of RNA. It consists of at least 25 complementary bases. Northern blotting uses cDNA as a probe. Radioactive labeling of the probe includes a radioactive isotope P³². Alternative labeling involves non-radioactive techniques such as chemiluminescence labeling. The chemiluminescence technology involves the breakdown of the chemiluminescence substrates through enzymes such as alkaline phosphatase or horseradish peroxidase. The substrates produce a detectable emission of light.

Step 5: The procedure of chemiluminescent labeling:

The probe (cDNA) gets attached to the enzyme alkaline phosphatase or horseradish peroxidase. Alternatively, labeling of the probe may involve a ligand. The ligand gets attached to the enzyme. Determination of the efficiency of the hybridization includes ionic strength, viscosity, duplex length, and base composition. Non-hybridized probes get removed by washing the membrane filter gently.

Step 6: Exposure to the X-ray film:

An X-ray film detects the signals generated with the chemiluminescent-labeled probes hybridized to RNA. A quick and sensitive signal generation occurs through chemiluminescent labels.

Step 7: Quantification of RNA through densitometry:

The process of densitometry involves a quantitative measurement of the optical density in the light-sensitive material.

Following is the summary of the northern blotting procedure:

1.     Isolation of RNA from a tissue sample.
2.     Loading the samples and the markers in the electrophoretic wells.

3.     Fragments get separated based on their sizes. Molecules with larger sizes are near the wells. Molecules with smaller sizes move faster. The fragments appear in the form of bands.

4.     Exposure of the gel to a buffer solution and a membrane filter.

5.     RNA gets transferred from the gel to the membrane.

6.     RNA hybridization using probes.

7.     Signal detection

8.     RNA quantification

Applications of northern blotting:

Gene expression study includes the pattern of gene expression in the tissues, organs and developmental stages. Northern blotting applies in studying the overexpression of oncogenes, the upregulation or downregulation of oncogenes and tumor-suppressor genes. It may help to find a gene function. Northern blotting helps to check the cloned DNA. Especially it checks the cloned cDNA since the technique uses cDNA as a probe to detect specific RNA. Analysis of micro RNAs becomes easy with this technique. A high resolution northern blotting monitors RNA expression. Micro RNAs are short and non-coding regulatory molecules. Post-transcriptional regulation of genes involves the role of micro RNA. The technique is readily available. Hence it is used in micro RNA analysis. The probes used in the technique are known as locked nucleic acid modified oligonucleotide probes. They are extremely sensitive and specific in detecting mature micro RNAs.

Immuno-northern blotting detects RNA modification through antibodies. The RNA gets separated through electrophoresis and transferred to the membrane. Immuno-blotting involves antibodies. It reveals antibody cross-reactions, characterization of antibodies, and modified nucleosides. It is a highly specific technique. The northern blotting technique also characterizes the RNA interference reagents. The comparison of the data derived from deep sequencing of micro RNAs with endogenous and exogenous RNAs is possible with the northern blotting technique. A gene silencing phenomenon is known as RNAi or RNA interference. The double-stranded RNAs get processed into small interfering RNAs (siRNA). A siRNA acts like a guide and enables cleavage of a homologous RNA. It occurs mainly in the RNA induced silencing complex (RISK). High-resolution northern blotting efficiently detects the length heterogeneity of the RNAi technology reagents.

A blot-base is an online database used to publish northern blots. It is a database used in genome sequencing, determination, and the protein structure.

Reverse northern blotting:

It is a variant of northern blotting technique. In this, the DNA fragments get hybridized with the RNA probes labeled radioactively. It enables gene expression profiling.

Virtual northern blotting:

It involves a comparison of relative amounts of transcripts in different tissues. The comparison is between small quantities of total RNA and full-length cDNA.

Advantages and disadvantages of northern blotting:

Detection of RNA size and observation of alternate spliced products become easy with this method. The technique is applicable for quantitative as well as qualitative analysis. It has a high specificity. With so many advantages, there are disadvantages too. As compared with RT-PCR, northern blotting has a low sensitivity. Analyzing thousands of genes at a time is not possible. The RNases are always ready to degrade the sample. Hence the technique always requires RNase inhibitors and proper sterilization of glassware. Chemicals used in northern blotting may be risky.

References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field

[2] Ana Techniques in Biotechnology, Goutam Bhowmik

[3] Gene Cloning and DNA Analysis, T.A. Brown
[4] Molecular Plant Biology: A Practical Approach, Volume 2, Philip M. Gilmartin, P. M. Gilmartin, Dr. Chris Bowler

                                  © Copyright, 2018 All Rights Reserved.

Translation in prokaryotes

The process of protein synthesis from a mRNA molecule is known as translation. It occurs mainly in the ribosomes. Consider the messenger RNA structure like a tape. As soon it attaches to the ribosome, the process of protein synthesis starts. The movement of the RNA molecule starts creating a lengthy polypeptide chain. First, the leading end on mRNA (the 5' end) emerges from the first ribosome. Next, it attaches to the second ribosome for synthesizing the next polypeptide chain. Once the RNA moves from a ribosome, it gets vacated for the new set of instructions. The amino acids start getting assembled into a polypeptide chain from the amino terminus. The process of assembly finishes at the carboxylate terminus. During the process of translation, an RNA molecule passes on the amino acid molecule to the growing polypeptide chain. Hence, it is known as a tRNA molecule. The transfer RNAs belong to a class of smallest biologically active molecules. The transfer RNAs attach to the amino acids at their 3' end. 

The translation of mRNA occurs from 5’ to 3’ direction. An amino acid binds to its specific tRNA. The codon of mRNA binds to the anticodon of tRNA through a complementary base pairing. The mRNA is specific in recognizing the anticodon. An enzyme known as aminoacyl tRNA synthetase attaches a correct amino acid to the tRNA. This process is known as aminoacylation or charging. Let us consider the example of valine tRNA. The first step involves binding of amino acid and ATP to a specific aminoacyl tRNA synthetase. Hence, the reaction leads to the loss of two phosphate groups from ATP. It leads to the formation of aminoacyl AMP. The next step involves binding of uncharged tRNA to the enzyme. As a result, the enzyme transfers the amino acid to tRNA. Hence, the aminoacyl tRNA releases from the enzyme. A factor known as transfer factor I participates in binding the charged tRNA to the ribosome. The transfer factor II is also known as translocase. It is a protein capable of forming a complex with the GTP and the ribosome. When the charged tRNA gets translocated from its ribosomal entrance site to the peptidyl site, it leads to the hydrolysis of GTP to GDP, thereby releasing the translocase.

Image 1: Translation initiation and peptide bond formation: (1) The first image depicts the binding of the fMet tRNA to the initiation site. (2) The serine tRNA binds in the A site. (3) Peptide bond formation (4) The uncharged tRNA moves to the E site and the tRNA consisting of amino acids linked through peptide bond moves to the P site.

Initiation of translation:

The mRNA with an AUG initiation codon gets involved in the process of translation. The mRNA ribosome binding site (also known as Shine Dalgarno sequence) also contributes to the process of translation. The 30S ribosomal subunit binds to AUG start codon. Later on, the initiator tRNA binds to this site. Since, AUG start codon codes for methionine amino acid, the newly made proteins start with amino acid methionine. In prokaryotes, the methionine gets modified to formylmethionine (fMet). The fMet tRNA has a 5’-CAU-3’ anticodon. Binding of fMet tRNA to the start codon releases an IF3. Hence, it forms a 30S initiation complex. It consists of mRNA, 30S subunit, fMet tRNA, IF1, and IF2. The next step involves binding of the 50S ribosomal subunit, leading to the hydrolysis of GTP. This reaction releases the IF1 and IF2 factors. It finally leads to the formation of the 70S initiation complex. A site-P site hypothesis describes the ribosomal sites. Following are the three main binding sites for the aminoacyl tRNA:

·        The exit site is also known as the E site.

·        The peptidyl site is also known as the P site.

·        Aminoacyl site is also known as A site.

The fMet tRNA binds to the mRNA at the P site. A site accepts the incoming aminoacyl tRNA. For transferring the peptide group, it requires translocation of tRNA. Hence, the tRNA gets translocated from the A site to the P site.

Elongation:

The amino acids get added to the polypeptide chain thereby allowing it to grow till the required amount. It involves three main steps. Primarily, the aminoacyl tRNA comes in contact with the ribosome and binds to it. Secondly, it leads to the formation of the peptide bond through an enzymatic reaction. The third step involves the movement of the ribosome (translocation) along the mRNA. It considers one codon at a time. The peptidyl site of the ribosome comes in contact with the AUG codon. It is suitable for the fMet tRNA to bind to the mRNA. The tRNA anticodon helps in binding to the mRNA codon. Next step involves binding of the aminoacyl tRNA (for example ser tRNA) in the A site. Binding of an aminoacyl tRNA to the codon in the A site releases the elongation factor known as EF-Tu. The reaction involves the hydrolysis of GTP. Note that the elongation factors EF-Tu get recycled for the next aminoacyl tRNA.  Now, the fMet tRNA and aminoacyl tRNA come close to each other. Since these two amino acids are adjacent to each other, a peptide bond forms between the two amino acids. In this case, a peptide bond forms between the formylmethionine and serine amino acids. The reactions get catalyzed by peptidyl transferase.

The amino acids linked by a peptide bond get attached to the aminoacyl tRNA situated at the A site. The tRNA is now known as peptidyl tRNA. Next step involves the process of translocation. The ribosome moves from one codon to another codon. This step again involves EF-G factors and GTP. Hence, the peptidyl tRNA moves from A site to the P site, leaving the A site empty. Similarly, the uncharged tRNA moves to the E site from the P site. An uncharged tRNA is a tRNA that has given its amino acid for peptide chain elongation.

The tRNA without an attached amino acid gets released. Next, the ribosome starts preparing for the next elongation cycle. The empty A site gets occupied by another aminoacyl tRNA with a specific anticodon. The above process gets repeated till the sufficient proteins get synthesized.

Image 2: Elongation and termination of translation: (5) Next aminoacyl tRNA binds to the A site. (6)Peptide chain elongation. (7) Termination of the process due to the activity of the release factor. (8) Dissociation.

Termination:

None of the tRNAs possess anticodon for a stop codon. A protein group known as termination factor or release factor (RF) help the ribosome in recognizing a stop codon. There are three types of release factors in E. coli such as RF1, RF2, and RF3. Each RF is a single polypeptide. The role of RF1 involves recognition of UAA and UAG codons. The RF2 involves recognition of UAA and UGA. The RF3 does not recognize any stop codon. The ribosome recognizes a chain termination codon (UAG). Then the polypeptide chain present on the peptidyl tRNA (present on the P site) gets released. The ribosomal subunits get dissociated thereby separating the remaining components.

References:

[1] Principles of genetics, Gardner, M. J. Simmons, D. P. Snustad, eighth edition.

[2] Biology of the Prokaryotes, edited by Joseph W. Lengeler, Gerhart Drews.
[3] Genetics, G. Archunan
[4] Genetics, Daniel Hartl, Maryellen Ruvolo

© Copyright, 2018 All Rights Reserved.

Mammalian RNAs Show Striking Edits

The discovery of RNA editing in the mid-1980s involved mitochondrial mRNAs of trypanosomes. This protozoan causes sleeping sickness. The nucleotides get inserted or deleted post-transcriptionally through RNA editing. Alternatively, it leads to the conversion of one base to another. After completion of the transcription, the uridines get inserted into mRNA. It inserts cytidines and deletes a few bases. RNA editing also uses a group of RNAs known as guide RNAs. An RNA molecule consists of uracil, adenine, guanine, and cytosine. The uracil gets incorporated into an RNA molecule through the RNA editing process.

Image: RNA editing

RNA editing in humans:

RNA editing occurs in human mRNA for apolipoprotein B. The gene encoding the protein also codes for an amino acid polypeptide known as apolipoprotein B48. The intestinal cells synthesize this protein. Deamination of a cytosine causes conversion of cytosine to uracil. Thus, a CAA codon gets converted into a UAA codon. RNA editing plays an important role in creating diverse antibodies. It also plays an important role in controlling the HIV infection cycle. Adenosine deaminase acts on RNA and carries out deamination of adenosine to inosine. The adenosine deaminases are known as Adenosine Deaminases Acting on RNAs (ADARs).

Other examples of RNA editing in mammals include tissues such as intestinal, muscular tissues, testis, tumors, B lymphocytes, and brain cells. The changes occurring due to the conversion of the cytosine to uracil involve apolipoprotein B mRNA. There exist other target RNAs too. The mRNA in muscle is known as alpha-galactosidase mRNA. It changes uridine to adenine. It converts a phenylalanine codon to a tyrosine codon. Wilm’s tumor mRNA is an example of target RNA for testicular tumor involving the conversion of uridine to cytidine. Hence, a leucine codon gets converted to a proline codon. B-lymphocytes consist of immunoglobulin mRNAs transcribed into immunoglobulins or antibodies. Changes in the mRNA lead to antibody diversity. ADAR editing occurs before intron splicing. Some nucleotides within the introns also undergo the process of editing. RNA editing inspires molecular diversity and hence classified under the epigenetic modification in some cases.

A nonclassic conversion of G to A occurs in hnRNPK in malignant colorectal samples. Originally the RNA editing system must be an evolutionary event in animals.

What is gRNA?

Guide RNAs or gRNAs guide in the insertion or deletion of uridine nucleotide residues into mitochondrial mRNAs. They boost the process of RNA editing. Guide RNAs not only guide RNA editing but also guide DNA editing. Guide RNA editing is uncommon in humans and mostly seen in trypanosomes.

Human transcriptomes reveal abundant A to I editing sites:    

Previous bioinformatics methods were not so efficient in determining A to I sites. These methods possessed a risk of mistakenly identifying false single nucleotide polymorphisms (SNPs) as RNA editing sites. However, sophisticated algorithms with regulated accuracies for sequence alignment and comparison addressed this problem. The comparison studies included two main elements. The genomes alignment consisted of human expressed sequence tags (ESTs) and cDNA. Do you know what an expressed sequence tag is? An EST is a unique DNA sequence derived from a cDNA library or a sequence transcribed into a tissue. The element order depended on the sequence similarity. The designing of the algorithm helped in removing single nucleotide polymorphisms, random mismatches, and mutations, sequencing errors, and searched only for reverse complement alignments. The algorithms revealed many A to I editing sites in thousands of genes.

RNA editing in Alu elements:

An ALU family involves a dispersed intermediately repetitive DNA sequence found in the human genome. About three thousand copies of these elements constitute a genome. Many human A to I RNA editing sites exist.

Is RNA editing a cure for diseases?

Unlike DNA editing, RNA editing helps in tweaking the gene expression instead of interfering with the genome and making permanent changes to it.

© Copyright, 2018 All Rights Reserved.

Transcription in Prokaryotes

DNA is a special part of the cell. Without DNA, there would not have been life, evolution, survival, and existence. The base of synthesizing RNA starts from a DNA strand. Hence, DNA has a strict impact on the processes occurring in the body. DNA gets transcribed into RNA. Studying transcription helps to investigate how mutations affecting transcription cause inherited diseases. The first step involved in gene expression is transcription. Interested in cloning or treating a genetic disorder with DNA? Then keeping updates regarding transcription processes is helpful. RNA is a key nucleic acid involved in synthesizing proteins. Watson and Crick proposed the central dogma. It is a two-step process denoted as DNA leading to transcription of RNA and RNA leading to translation of the protein. Not all genes encode for proteins. Hence, not all DNA gets transcribed to RNA.

A gist of DNA replication:

Initiation of replication starts at a point where the unwinding of DNA takes place. Enzymes synthesize an RNA primer and the fragments so that new nucleotides get added. The segments get elongated through a process of elongation followed by removal of the primers. The unjoined fragments get ligated to end the process of replication. Using DNA as a template, the transcription starts.

RNA synthesis:

Genes are known as ordered sequences of nucleotide bases encoding polypeptide chains via RNA molecules. They are nothing but the pieces of DNA that consist of specific information for making a particular protein. Each gene associates with regulatory sequences known as gene regulatory elements. They are involved in regulation of transcription. The process of transcription starts with denaturation of the DNA double helix. The enzyme known as RNA polymerase catalyzes the process of transcription. In prokaryotes, RNA polymerase is responsible for unwinding. The process of unwinding in eukaryotes occurs with the help of other proteins. The DNA starts unwinding next to the gene involved the process. Hence, RNA polymerase starts catalyzing the RNA synthesis. The RNA gets synthesized from 5’-3’ direction along the 3’-5’ template strand. Out of the two DNA strands, only one strand participates in the process of transcription. Total four nucleotide phosphates act as precursors for transcription. They include ATP, GTP, CTP, and UTP. Recall that DNA synthesis requires RNA primers. However, RNA synthesis does not require primers. RNA polymerases are efficient in initiating the synthesis of new polynucleotide chains without any primers. Participant molecules in RNA synthesis are DNA strand, RNA polymerases, nucleoside triphosphates, elongation and termination factors. Also, there is a requirement of specific gene sequences for the initiation of transcription. In both prokaryotes and eukaryotes, the process of transcription occurs in three consecutive steps such as initiation, elongation, and termination. The eukaryotic transcription needs an understanding of prokaryotic transcription first. 
A prokaryotic gene responsible for transcription needs the three following sequences:

·    The promoter sequence is an upstream sequence present at the start of the RNA coding gene sequences.

·   RNA coding gene sequences are known as DNA sequences capable of getting transcribed to RNA.

·        The terminator specifies the destination point of transcription.

The genes specifying the initiation process have two promoter sequences known as -35 and -10. Each promoter has a specific consensus sequence.

Promoter region	No. of base pairs upstream	Consensus sequence
-35	35	5’-TTGACA-3’
-10	10	5’-TATAAT-3’

Table: Promoters -35 and -10 respectively.

Initiation:

RNA synthesis gets initiated by the recruitment of RNA polymerase holoenzyme. It binds to the promoter region. The holoenzyme consists of the core enzyme form of RNA polymerase. It has four polypeptides such as two α, one β, and one β’ polypeptides bound to a sigma factor. It recognizes both the promoter regions. It first recognizes -35 region where the DNA is a close promoter complex. Then the holoenzyme unwinds the DNA. The untwisted form of the promoter is known as an open promoter complex. The RNA polymerase orients itself to the Pribnow box for further process. Thus, the process of initiation includes two main steps. First, RNA polymerase gets attached to its core promoter. Second, the closed promoter gets converted into an open promoter complex. Then the RNA gets synthesized. For a successful initiation, RNA polymerase now moves away from the promoter region.

Image: Transcription in prokaryotes

Elongation and termination

 The RNA polymerase approximately includes a 30 base pair of DNA. It includes a transcription bubble of 12-14 base pairs. The RNA gets attached to the template strand of the DNA. RNA-DNA base pairs assist the RNA in the attachment. The main feature of RNA polymerase observed during the elongation process does not constantly synthesize the transcript. Instead, it follows a discontinuous synthesis. The process of elongation although rapid gets interspersed by brief pauses. During brief pauses, the active site of the polymerase undergoes a slight structural conformation or gets rearranged. A pause lasts for a few milliseconds and accompanies backtracking thereby occurring randomly. A pause helps in termination of the transcript synthesis. The termination of transcription follows a signal given by terminator sequences. An important protein involved in the process of termination is known as rho (ρ). Hence, there are two types of terminator sequences known as Rho-dependent and Rho-independent terminators. The Rho-dependent terminators are known as type II terminators. They lack A-T string and avoid forming hairpin loops. Rho has RNA binding ATPase domain. Rho-independent terminators, on the other hand, form hairpin loops and consist of inverted repeat sequences. A string of AT base pairs transcribes a string of Us. When the inverted palindrome (with a hairpin loop) gets transcribed, there arises RNA-RNA base pairing. Studies revealed the presence of a flap structure on the surface of RNA polymerase which mediates in termination.

Anti-termination:

It is a process beyond termination. There is an anti-termination process too common in prokaryotes. Anti-termination occurs when RNA polymerase ignores a terminator signal and continues to elongate the transcript until the next signal. Anti-termination provides a mechanism through which few genes at the end of the operon get switched off. Anti-termination protein attaches to the DNA and transfers to the RNA polymerase. However, the reason behind switching off those genes remains unclear.

Premature termination and attenuation:

The primary transcript produced by RNA polymerase is known as a mature mRNA. The bacteria involve coupled transcription and translation. It allows a special type of control known as attenuation. It is a process in which the expression of amino acid biosynthesis gets regulated. Some of the bacteria get bacteriophage infection. Bacteriophages transcribe their genomes using bacterial RNA polymerases.

References:

[1] Transcription, William M. Brown, Philip M. Brown
[2] Molecular Biology of the Cell, Bruce Alberts

© Copyright, 2018  All Rights Reserved

The Discovery of the Genetic Material

Geneticists knew the hereditary factor that helped to pass on the necessary information from the parent to the offspring. The progeny show similar characteristics of the parents. Hence, it was necessary to know the reason or the factor behind passing on the characteristics from the parent to the offspring. The experimenters working on the hereditary material looked for three main factors. The first factor involved the stability of the information. Unless the information required for the growth, development, and reproduction is stable, it won’t get passed to the next generation. The second factor considered was the accuracy of the hereditary factor in replication. Unless it replicates accurately, the hereditary factor won’t be able to pass on the same information to the next generation. The third factor plays a crucial role in variation.

Fredriech Mischer’s discovery of nuclein:

Fredriech Mischer isolated the pus cells from the waste bandage. He studied these cells in detail and found the hereditary material in the nucleus of the cells. Nuclein or the material obtained from the nucleus revealed carbon, nitrogen, oxygen, hydrogen, and phosphorus. It was a biochemical test for detection. After forty years of Mischer’s research work, scientists came up with chromosome structure. It revealed nucleic acid and protein.

Griffith’s Transformation Experiment:

Griffith worked with the hereditary material in the year 1928. He used Streptococcus pneumonia for his experiments. It is a pathogenic bacteria leading to pneumonia disease. The pathogenic strains of this bacteria show the presence of sugary coat. It helps in spreading the disease and increasing the virulence of the bacterium. Griffith worked with two main strains such as the S strain and the R strain. The S strain forms smooth and shiny colonies. These strains are highly infectious. They have a well-defined polysaccharide coat. Griffith used two types of S strains such as IIS and IIIS. The R strain forms rough colonies. It is a relatively harmless strain. It lacks the polysaccharide coat. Hence, it is not a virulent strain. The S strain occasionally mutates to R. The IIS strains got mutated to IIR strains. The mice injected with the mutated strain survived. Next, the experimenter tried injecting the IIIS strain in the mice. The mice died. The reason behind the death of these mice included the infection due to virulent IIIS strain. Next, the experimenters used heat-killed IIIS strains. The mice survived the infection. The heat treatment killed the bacterial cells. Hence, the infection did not spread. Finally, the experimenter tried injecting a combination of heat-killed virulent IIIS strain with IIR strain. The mice died. The probable reason for the death of the mice included the interaction between the IIR and the IIIS bacterial strains. Hence, Griffith concluded the concept of transforming principle. Something got transferred from the dead cells to the live cells. Griffith’s transformation experiment paved the way for other experiments determining the main transforming principle. 

Image 1: Griffith’s experiment: It describes the experimental procedure carried out by Griffith. He first injected the mouse with rough strain (IIR) to check for their survival. The mouse survived. Next, he injected smooth strain IIS. The mouse did not survive. Third, he injected heat-killed smooth strain. The mouse again survived. Finally, he mixed both rough strain and heat-killed smooth strain and injected into the mouse. The mouse survived.

Avery’s experiment:

Avery and colleagues tried identifying the transforming principle. The experiment initiated in 1930 and worked till 1940’s. The experimenters worked on the same S. pneumoniae bacterium with virulent IIIS strains. They lysed the IIIS cells with a detergent and subjected to centrifugation. The separation of cellular components gives rise to the cell extract excluding the debris. The extract culture mixed with the culture of IIR got plated on a suitable medium. The IIIS colonies grew on the medium. The scientists knew that the transforming principle could be DNA, RNA, protein or the polysaccharide. Hence, they treated the cells with various enzymes. Due to the enzymatic treatment, the polysaccharides and the proteins got degraded. The experimenters again checked for the transforming principle using the IIIS strains. Hence, they found out that DNA or RNA could be the genetic material. To confirm which of the two could be the transforming principle, they treated the RNA with a nuclease such as RNase. Through the repeated experiments, the experimenters realized DNA to be the transforming principle. 

Image 2: Avery’s experiment: It describes two main steps such as treatment of the sample containing a mixture of DNA and RNA first with the RNase and then with the DNase enzymes. After treating the mixture with the RNase, and culturing, the researchers plated the mixture on a suitable medium and observed the growth. In this case, the colonies grew. Next, they treated the mixture with DNase and conducted the same procedure. This time, the colonies did not grow. 

Hershey Chase experiment:

Hershey and Chase studied T2 bacteriophage life cycle and correlated with DNA in the year 1953. It follows a lytic pathway. First, the bacteriophage infects the bacterial cell and injects its genome inside. Upon the entry of the phage genome, the bacteria stops synthesizing its DNA. The phage diverts the bacterial machinery for the replication of its own genome. The progeny phages get released from the bacterial cells. The bacterial cells get lysed. Hence, the bacteriophage uses the bacterial machinery to proliferate. Hershey and Chase tried studying the DNA through this experiment. They used two types of radioisotopes such as ³²P and ³⁵S. The experimenters mixed the phages with radioisotopes. Next, the radioactively labeled T2 progeny infected the E coli cells. The infection of the bacteria with phages having ³²P radioisotope gave rise to progeny having the same. The phages having ³⁵S radioisotope did not pass on the isotope to the progeny phages. The ³⁵S radioisotope involves radiolabelling of the proteins. From the above experiment, the researchers concluded the DNA to be the genetic material. 

Image 3: Hershey-Chase experiment: It describes the discovery of DNA as the genetic material using radioactive isotopes such as ³⁵S (the first step) and ³²P (the second step).

Discovery of the RNA as the genetic material:

An example of an RNA virus includes the tobacco mosaic virus. It produces lesions on the tobacco leaves. It consists of an RNA molecule and a protein coat. Both have a spiral configuration. The protein molecule surrounds the RNA and protects it from the degradation by nucleases. 

References:

[1] Advanced Biology - Page 396, Michael Kent, 2000 Preview
[2] Genetics 101 - Page 35, Michael Windelspecht, 2007 ‎Preview

© Copyright, 2018 All Rights Reserved

What is a gene?

Introduction to genes:

The basic unit of the hereditary material is known as a gene or a cistron. It is an ordered sequence of nucleotides. These sequences encode polypeptide chain via mRNA molecule. Both DNA and RNA are known as nucleic acids. Genetics is the study of structure, function, and the regulation of genes. Studying genes helps us to know more about proteins, cellular functions, and disorders associated with them. In humans, gene targets help to study mutations and target them through advanced drug discovery and therapeutics. In bacteria, genes are manipulated to obtain the desired product. The genetic engineering or recombinant DNA technology helps to manipulate the genes. The desired gene can be integrated into a vector to obtain the desired product.

Modification of plant genes improves the quality of the crop and improves the yield. The genes play an important role in the growth and reproduction of an organism. Slight gene mutations, if harmful, lead to drastic changes in the cell and cause genetic disorders. Genetics plays an important role in preventing genetic disorders through prenatal testing, cytogenetic and molecular genetic techniques. Genes form the main basis of inheritance. We all have some traits obtained from our ancestors. The genes get passed on from generation to generation. We look similar to our parents, yet appear different. Helpful mutations in the genes lead to genetic diversity and variation. That is why we all look different from each other. The complete set of chromosomal and extrachromosomal genes of an organism is known as the genome. It consists of the complete genetic composition of an organism. 

The gene is a heritable determinant of a trait showing the property of segregation. With this reference, the way genes pass within the family are studied. The genes in a family may or may not have diverged from each other. Gene family involves a set of genes that descend from a common ancestor. Genes express themselves in several generations. They also interact with each other. Several genes can collaborate with each other and give rise to one phenotypic trait. The genes may be present in the cell in a particular dosage. It is important to know the frequency of a gene. The genes are present in a specific place on a chromosome, described as gene loci. Gene mapping through annotation of DNA sequences is possible based on gene loci information. DNA sequence annotation with regulatory element sites, coding regions, non-coding regions, and mutations accelerates the mapping. Sometimes many copies of a gene may be present in a chromosome, known as gene redundancy. Thus genes are vast and diversified.

Image 1: Basic genetic structure

HUGO gene nomenclature

It is a standard for gene nomenclature decided by the Human Genome Organisation (HUGO) committee. It is a meaningful naming of a gene. Names accompany useful symbols. A gene symbol is a unique abbreviation of the name of a gene. It consists of uppercase letters in italics, letters in Latin and numbers in Arabic. A putative gene name is locus based.

Here are the naming guidelines:

1.     The symbols must be unique and prohibited to use elsewhere.

2.     The gene symbols must have Latin letters and Arabic numbers

3.     Punctuation marks are not allowed in gene symbols.

4.     The gene symbols should not contain any references of species.

5.     The nomenclature of genes must evolve with the latest technology rather than follow age-old rules.

Structure of a gene:

A gene includes regions preceding and following the coding and non-coding regions. The preceding region is known as the leader sequence which is at 5’ position. It is the untranslated region. There is a coding region known as Exon. An exonic region specifies for an amino acid sequence. These exons are interrupted by non-coding regions known as introns. The untranslated trailer sequences follow them. The untranslated trailer sequences are at 3’ end. The spliced RNA molecule consists of only exons as the non-coding regions or introns for splicing out. Although DNA is a double-stranded molecule, only one strand encodes for RNA synthesis. The sense strand runs from 5’ to 3’ direction and encodes specific molecules. The gene has an open reading frame which is an indication of sense strand direction. The extremities of the gene consist of regulatory sequences. Plus the gene also consists of promoters, enhancers, silencers and other regions.

Image 2: Gene structure

Gene Expression- The Central Dogma

Expression of the genes involves the conversion of the genes coded information into the structures present and operating the cells. The genes are expressed to initiate the synthesis of the mRNA molecule and translated into a protein. Other examples of RNA include rRNA and tRNA. The tRNA and rRNA genes remain untranslated. The gene expression also involves a phenotypic manifestation by a process known as gene action. Differential gene expression studies involve gene expression at different levels. They express differently under different experimental conditions. The central dogma is a two-step process describing the gene expression. Francis Crick proposed the central dogma after the discovery of nucleic acids. DNA undergoes a process of transcription to synthesize RNA which further undergoes translation to form proteins. Not all genes express proteins. Thus, not all genes are transcribed to get RNA. Originally through central dogma, it was postulated that the genetic information is transferred only from nucleic acid to nucleic acid and from nucleic acid to protein. Thus, the genetic information gets transferred from DNA to DNA, DNA to RNA and from RNA to protein. The genetic information never transfers from protein to nucleic acid. The cis-trans test determines the functionality of genes. It determines whether the independent mutations occur in a single gene or several genes.

Cis-trans Complementation test

It helps to determine whether the two mutant sites are in the same functional unit or a gene. It is an allelism test and determines whether two different recessive mutations on the opposite chromosome of a diploid complement each other. The same two mutations in a diploid or a partial diploid show a wild-type phenotype. Cis mutations exhibit a wild-type phenotype. There is no genetic complementation when the mutations are in trans. The term cistron indicates gene.

The gene characterization is possible with sequence, transcription, and homology if it does not contribute to a phenotype. A gene is a functional and physical unit of heredity. The gene code is said to be a triplet. There are total 64 codons that code for 20 different amino acids. 

References:

[1] What is a gene?- Genetics Home Reference - NIH

[2] Gene – Wikipedia
[3] HUGO Gene Nomenclature Committee

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