Honours / Core Course (CC)

MOLECULAR BIOLOGY
NUCLEIC ACIDS
Salient features of DNA

Q.What do you mean by highly repeated DNA sequences?

This type of sequence present in at least 106 copies per genome, constitute anywhere from about 1 to 10 % of the total DNA. Highly repeated sequence are typically short and present in clusters in which the given sequence repeats itself over and over again without interruption. Highly repeated sequences fall into several overlapping categories, including satellite DNA, minisatellite DNAs and microsatellite DNAs.

Q.What is satellite DNA?

These DNA consists of short sequences (from about 5 to 100 base pairs in length) that are repeated many times to form very large clusters containing upto 100 million base pairs of DNA. The name "satellite DNA" refers to the phenomenon that repetitions of a short DNA sequence tend to produce a different frequency of the bases adenine, cytosine, guanine and thymine, and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient. Microsatellites are thought to have originated by polymerase slippage during DNA replication. This comes from the observation that microsatellite alleles usually are length polymorphic; specifically, the length differences observed between microsatellite alleles are generally multiples of the repeat unit length.

 

Q.Mention a staining procedure to identify DNA in cell specimens.

Feulgen stain is a staining technique discovered by Robert Feulgen (1924) and used in histology to identify chromosomal material or DNA in cell specimens. It is darkly stained. It depends on acid hydrolysis of DNA, therefore fixating agents using strong acids should be avoided. Neither RNA nor any cell substance will give the precise DNA type of staining reaction. Hydrolysis permits Schiff’s reagent to react with aldehyde groups in the sugar deoxyribose. BY measuring the amount of light transmitted through the chromosome, quantitative estimation of DNA can be made.

Robert Feulgen (1924)

Chargaff’s Rule

Q.If double stranded DNA has 14% G (guanine), what percent A (adenine), T (thymine) and C (cytosine) would you expect?

As double stranded DNA has 14%G, it mean there will be 14%C (According to Chargaff's rules states that DNA from any cell of all organisms should have a 1:1 ratio Base Pair i.e A is equals to T and G is equals to C or Purine=pyimidine )

Total G+C in Double stranded DNA= 14+14= 28%

Total A+T in double stranded DNA will be = 100-28= 72%

Total % of A= 72/2=36%

Total %of T= 36% (As percentage of A=% of T According to Chargaffs Rule)

Hence in given Double stranded DNA :

A=36%, T=36%,G=14% and C=14%

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Hypo and Hyperchromic shift

Q.What do you mean by Hypechromatic effect?

Heat or other stresses can cause a complex molecule like DNA to denature or lose its function due to the unfolding of the 3-dimensional structure. In the denaturation of double-stranded DNA, the hydrogen bonds of the duplex structure break, the helix unwinds and the strands separate. However no covalent bonds break. The viscosity of the DNA decreases and both the UV absorption and buoyant density increase. The increase in UV absorption of heated DNA in solution, called the hyperchromatic shift, is the easiest change to measure. This effect is illustrated in figure1. When absorption at 260nm is monitored and plotted against temperature during heating, a melting point of the DNA is obtained. The midpoint of this point at which 50% of the strand are unwound or denatured. When the curve plateaus at its maximum optical density, denaturation is complete and only single strands exist. Analysis of melting profiles provides a characterization of DNA and an alternative method of estimating its base composition.

Watson and Crick Model of DNA

Q.State the main features of Watson and Crick’s double helix model of DNA on the basis of X-ray crystallography.

1.The DNA molecule consists of two polynucleotide chains wound around each other in a right – handed double helix.

2.The two chains are antiparallel (show opposite polarity), i.e., the two strands are oriented in the 5’ to 3’ way and the other strand oriented 3’ to 5’.

3.The sugar-phosphate backbones are on the outsides of the double helix, with the bases oriented toward the central axis. The bases of both chains are flat structures oriented perpendicularly to the long axis of the DNA.

4.The bases in each of the two polynucleotide chains are bonded together by hydrogen bonds, which are relatively weak chemical bonds. The specific pairing observed are A bonded with T (two hydrogen bonds) and G bonded with C (three hydrogen bonds).

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RNA
Types

Q.State important features of tRNA.

i)A 5'-terminal phosphate group is present.

ii)The acceptor stem is a 7- to 9-base pair (bp) stem made by the base pairing of the 5'-terminal nucleotide with the 3'-terminal nucleotide (which contains the CCA 3'-terminal group used to attach the amino acid).

iii)The CCA tail is a cytosine-cytosine-adenine sequence at the 3' end of the tRNA molecule. The amino acid loaded onto the tRNA by aminoacyl tRNA synthetases, to form aminoacyl-tRNA, is covalently bonded to the 3'-hydroxyl group on the CCA tail.

iv)The D arm is a 4- to 6-bp stem ending in a loop that often contains dihydrouridine.

v)The anticodon arm is a 5-bp stem whose loop contains the anticodon.

vi)The T arm is a 4- to 5- bp stem containing the sequence TΨC where Ψ is pseudouridine, a modified uridine.

Q.What is the importance of CCA sequence found in tRNA?

This sequence is important for the recognition of tRNA by enzymes and critical in translation. In prokaryotes, the CCA sequence is transcribed in some tRNA sequences. In most prokaryotic tRNAs and eukaryotic tRNAs, the CCA sequence is added during processing and therefore does not appear in the tRNA gene.

Q.What is antisense RNA?

Messenger RNA (mRNA) is a single stranded molecule that is used as the template for protein translation. It is possible for RNA to form duplexes, similar to DNA, with a second sequence of RNA complementary to the first strand. This second sequence is called antisense RNA. The formation of double stranded RNA can inhibit gene expression in many different organisms including plants, flies, worms and fungi.

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Formation of antisense RNA blocks translation

Function
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DNA REPLICATION
Mechanism of DNA Replication in Prokaryotes
Experimental proof -
Semi-conservative, bidirectional and discontinuous

Q.Why both the strands of DNA do not act as template for RNA synthesis?

1. Both the strands of DNA are with different sequence. Proteins thus formed, due to presence of different amino acids, will differ.

2. Due to formation of two different proteins from one DNA will complicate the genetic information and transfer mechanism.

3. If two strands of RNA are formed from one DNA molecule simultaneously, being complementary, RNA will become double stranded. It will stop the translation step and protein formation will not be there. Thus, transcription step will not be of any use for protein synthesis.

 

Q.What is the basic principle of Taylor’s experiment?

Autoradiography experiment in Vicia faba. Autoradiography was also utilized by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells of Vicia faba. Results of such experiments were first published in 1957. After incorporation of tritiated thymidine, when root tips were transferred to unlabellcd medium, in the first generation of duplication both chromatids were labelled (interpreted as one DNA double helix in each chromatid, and only one of the two strands labelled). In the second cycle of duplication, in each chromosome, one of the two chromatids was found to be labelled. This was interpreted as showing semi-conservative mode of duplication

RNA priming
Replication of telomeres
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TRANSCRIPTION
Mechanism of Transcription —
In prokaryotes
In eukaryotes
Transcription factors
Difference between prokaryotic and eukaryotic transcription
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TRANSLATION
Genetic code
Degeneracy of the genetic code

Q.What do you mean by degeneracy of amino acid?

This phenomenon gives rise to: multiple codes for a given amino acid (degeneracy), possible suppression of point mutation in the 3rd base of the codon. There are three amino acids encoded by six different codons: serine, leucine, and arginine. Only two amino acids are specified by a single codon. One of these is the amino-acid methionine, specified by the codon AUG, which also specifies the start of translation; the other is tryptophan, specified by the codon UGG.

Two different leucine codons (CUC , CUU ) can be read by the same leucine tRNA molecule, con-trary to regular base-pairing rules

Wobble Hypothesis

Q.What do you mean by wobble hypothesis? [Crick (1966) proposed this hypothesis]

According to this hypothesis, the base in first position of anti-codon on tRNA is usually an abnormal base like inosine, pseudouridine, tyrosine etc. These abnormal bases are able to pair with more than one type of nitrogenous base in the third position of the codon on mRNA.Example- Inosine (I) can pair with A, C or U, this base is called Wobble base or fluctuating base. Wobble occurs at position 1 of the anti-codn and position 3 of the codon.

Mechanism of protein synthesis in prokaryotes.
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Q.What are the major types of aminoacyltRNA synthetase?

There are two classes of aminoacyl tRNA synthetase—

(i)Class I has two highly conserved sequence motifs. It aminoacylates at the 2'-OH of a terminal adenosine nucleotide on tRNA, and it is usually monomeric or dimeric (one or two subunits, respectively).

(ii)Class II has three highly conserved sequence motifs. It aminoacylates at the 3'-OH of a terminal adenosine on tRNA, and is usually dimeric or tetrameric (two or four subunits, respectively). Although phenylalanine-tRNA synthetase is class II, it aminoacylates at the 2'-OH.

Q.How termination of protein synthesis occurs in prokaryotes?

In presence of termination codons like UAG ("amber"), UAA ("ochre"), UGA ("opal"/umber), releasing factors (RFs)along with energy rich molecules initiate the dissociation of ribosomal complex, growing PPC ,m-RNA etc. Experimental evidences suggest the complete dissociation procedure need appearance of another initiation codon (i.e.AUG for methionine) in the m-RNA.(For more information follow contact us)

POST TRANSCRIPTIONAL MODIFICATIONS AND PROCESSING OF EUKARYOTIC RNA
Capping and Poly A tail formation in mRNA
Split genes
Concept of introns and exons
Splicing mechanism
Alternative splicing and RNA editing
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GENE REGULATION
Regulation of Transcription in prokaryotes
lac operon
trp operon
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Regulation of Transcription in eukaryotes
Activators
Enhancers
Silencer
Repressors
miRNA
Mediated gene silencing
Epigenetic Regulation
DNA Methylation
Histone Methylation & Acetylation
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DNA REPAIR MECHANISMS
Types of DNA repair mechanisms
RecBCD model in prokaryotes
Nucleotide and base excision repair
SOS repair
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MOLECULAR TECHNIQUES
PCR
Western and Southern blot
Northern Blot
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