The document provides a historical overview of key discoveries related to DNA as the genetic material:
1) In the early 1900s, chromosomes were shown to carry hereditary information. By the 1940s-1950s, experiments by Avery, Griffith, Hershey and Chase provided evidence that DNA - not protein - was the genetic material.
2) Watson and Crick proposed the double helix structure of DNA in 1953 based on Chargaff's rules and Franklin's X-ray crystallography photos. Their model explained how DNA replicates and hereditary information is passed from parents to offspring.
3) Subsequent work in the 1960s by Nirenberg, Matthaei and others cracked the
CONTENT
-Hybridization Introduction
-Southern Hybridization
Blotting (Transfer Of DNA On A Membrane)
-Application
-DNA Fingerprinting
-DNA Typing
Introduction-Hybridization
The capacity of denatured DNA to reanneal under appropriate ionic strength and temperature is known as Hybridization.
In case of a mixture of DNA molecules, hybridization leads to formation of hybrid molecules.
Often a DNA probe is used in hybridization for detecting or finding a particular DNA sequence from a mixture of DNA molecules.
Hybridization may be done with DNA fragments separated by electrophoresis, dot blot: DNA spots on a membrane or colony blot: hybridization with bacterial colonies containing particular rDNA.
Southern Hybridization
A method of DNA hybridization established by Ed Southern in 1975.
Genomic DNA from an organism is restriction digested and separated by electrophoresis.
The electropherosed DNA is transferred on to a membrane.
The membrane bound DNA is denatured using an alkali.
A DNA probe is radiolabeled and added to the denatured DNA in presence of appropriate buffer for hybridization.
After sufficient time for hybridization the membrane is exposed to X-ray film.
The X-ray film shows signals of hybridization: where the DNA probe has complementary structure on the transferred DNA.
The resulting X-ray film is known as an Autoradiogram and the process Autoradiography.
DNA fingerprinting
Using the method of DNA hybridization with an appropriate DNA probe to identify individuals as done with fingerprinting.
The method was first done by Sir Alec Jeffreys in 1985.
Any biological material such as a drop of blood, saliva, semen, and any body part such as bones, tissue, skull, teeth, hair with root etc found at the scene of crime is used as source DNA.
DNA probe used for DNA fingerprinting may be a VNTR or minisatellite DNA.
The probe is labeled and used to hybridize the source DNA.
An autoradiogram is generated that show different band patterns for different individuals.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
CONTENT
-Hybridization Introduction
-Southern Hybridization
Blotting (Transfer Of DNA On A Membrane)
-Application
-DNA Fingerprinting
-DNA Typing
Introduction-Hybridization
The capacity of denatured DNA to reanneal under appropriate ionic strength and temperature is known as Hybridization.
In case of a mixture of DNA molecules, hybridization leads to formation of hybrid molecules.
Often a DNA probe is used in hybridization for detecting or finding a particular DNA sequence from a mixture of DNA molecules.
Hybridization may be done with DNA fragments separated by electrophoresis, dot blot: DNA spots on a membrane or colony blot: hybridization with bacterial colonies containing particular rDNA.
Southern Hybridization
A method of DNA hybridization established by Ed Southern in 1975.
Genomic DNA from an organism is restriction digested and separated by electrophoresis.
The electropherosed DNA is transferred on to a membrane.
The membrane bound DNA is denatured using an alkali.
A DNA probe is radiolabeled and added to the denatured DNA in presence of appropriate buffer for hybridization.
After sufficient time for hybridization the membrane is exposed to X-ray film.
The X-ray film shows signals of hybridization: where the DNA probe has complementary structure on the transferred DNA.
The resulting X-ray film is known as an Autoradiogram and the process Autoradiography.
DNA fingerprinting
Using the method of DNA hybridization with an appropriate DNA probe to identify individuals as done with fingerprinting.
The method was first done by Sir Alec Jeffreys in 1985.
Any biological material such as a drop of blood, saliva, semen, and any body part such as bones, tissue, skull, teeth, hair with root etc found at the scene of crime is used as source DNA.
DNA probe used for DNA fingerprinting may be a VNTR or minisatellite DNA.
The probe is labeled and used to hybridize the source DNA.
An autoradiogram is generated that show different band patterns for different individuals.
A bacteriophage (informally, phage) is a virus that infects and replicates within a bacterium. The term is derived from "bacteria" and the Greek (phagein), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have relatively simple or elaborate structures. Their genomes may encode as few as four genes, and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are among the most common and diverse entities in the biosphere.
Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface,] and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (see phage therapy). Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection
detail description about different models of DNA helicase. basically 2 types of mechanism involved in the helicase function and it has various models. this information is refer from research paper and some review articles.
its my university task to make a assignment on the brief history of molecular biology i am sure i done it quite well by linking all the information to molecular
Levels of organisation of DNA explains how 2 meters long DNA is compacted into chromatin. Useful self-assessment questions are given in the slides. If you want to know the answer, you can ask in comments.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
Facts about DNA
Eukaryotic chromosomes
Chemical composition of eukaryotic chromosomes
Histones
Non-histone chromosomal protein
Scaffold proteins
Folded fibre model
Nucleosome model
H1 proteins
Histone modification
Chromatosome
Higher order of chromatin structure
Mechanism of DNA packaging
Conclusion
genetic material in organization, Central dogma,transcription in prokaryotes ...Patelrushi11
Historical background of molecular genetics, genetics material in organisams- Experiments, Nucleic acid as genetic material, central dogma, transcription in prokaryotes eukaryotes, genetic codegenetic code and its characteristics, silent feature of genetic codon,wobbal hypothesis
detail description about different models of DNA helicase. basically 2 types of mechanism involved in the helicase function and it has various models. this information is refer from research paper and some review articles.
its my university task to make a assignment on the brief history of molecular biology i am sure i done it quite well by linking all the information to molecular
Levels of organisation of DNA explains how 2 meters long DNA is compacted into chromatin. Useful self-assessment questions are given in the slides. If you want to know the answer, you can ask in comments.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
Facts about DNA
Eukaryotic chromosomes
Chemical composition of eukaryotic chromosomes
Histones
Non-histone chromosomal protein
Scaffold proteins
Folded fibre model
Nucleosome model
H1 proteins
Histone modification
Chromatosome
Higher order of chromatin structure
Mechanism of DNA packaging
Conclusion
genetic material in organization, Central dogma,transcription in prokaryotes ...Patelrushi11
Historical background of molecular genetics, genetics material in organisams- Experiments, Nucleic acid as genetic material, central dogma, transcription in prokaryotes eukaryotes, genetic codegenetic code and its characteristics, silent feature of genetic codon,wobbal hypothesis
DNA caries genetic information of organisms. This presentation covers the discovery of DNA as genetic material, structure of DNA, Nucleotides and nucleosides. Watson and crick DNA model.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
1. • In the mid-1800s; Gregor Mendel; studied the inheritance of various traits in pea
plants.
• In 1920s; P. A. Levene; elucidated basic chemical composition of nucleic acids.
• Fred Griffith; 1928; transfer of virulence in the pathogen Streptococcus pneumoniae
(pneumococcus).
• Between 1941 and 1944; Archer Martin and Richard Synge; invention of paper
chromatography.
• By 1948; Erwin Chargaff; had begun using paper chromatography to analyse the base
composition of DNA from a number of species.
• In 1951; Rosalind Franklin arrived at King’s College, London, and joined Maurice Wilkins
in his efforts to prepare highly oriented DNA fibers and study them by X-ray
crystallography. By the winter of 1952–1953, Franklin had obtained an excellent X-ray
diffraction photograph of DNA.
• 1953; Watson and Crick propose DNA double helix.
• 1962; Watson, Crick, and Wilkins received the Nobel Prize in 1962 for their discoveries.
Historical Highlights
2. The Search for the Genetic Material
1. Some substance must be responsible for passage of traits from
parents to offspring. For a substance to do this it must be:
a. Stable enough to store information for long periods.
b. Able to replicate accurately.
c. Capable of change to allow evolution.
DNA as Genetic Material
3. In the early 1900s, chromosomes were shown to be the
carriers of hereditary information.
DNA and Protein
Most scientists initially believed that protein must be the
genetic material.
4. • Frederick Griffith’s 1928 experiment with Streptococcus
pneumoniae bacteria in mice showed that something passed from dead
bacteria into nearby living ones, allowing them to change their cell
surface.
• He called this agent the transforming principle
7. Avery’s Transformation Experiment
DNA as Genetic Material: The Avery Experiment
1. In 1944, Avery, MacLeod and McCarty published results of a study that
identified the transforming principle from S. pneumoniae.
2. Only the nucleic acid fraction was capable of transforming the bacteria.
3. Critics noted that the nucleic acid fraction was contaminated with
proteins.
8. Oswald Avery with his colleagues set
out to discover which constituent in
the heat-killed virulent pneumococci
was responsible for Griffith’s
transformation.
9. The Hershey-Chase Bacteriophage Experiment
DNA as Genetic Material: The Hershey-Chase Experiment
1. More evidence for DNA as the genetic material came in 1953 with Alfred
Hershey and Martha Chase’s work on E. coli infected with bacteriophage
T2.
2. In one part of the experiment, T2 proteins were labeled with 35S, and in
the other part, T2 DNA was labeled with 32P. Then each group of labeled
viruses was mixed separately with the E. coli host.
3. The 35S-labeled protein was found outside the infected cells, while the
32P-labeled DNA was inside the E. coli.
10. The Hershey-Chase Experiment
(a) When E. coli was infected with a T2 phage containing 35S protein, most of the
radioactivity remained outside the host cell. (b) When a T2 phage containing 32P DNA was
mixed with the host bacterium, the radioactive DNA was injected into the cell and phages
were produced. Thus DNA was carrying the virus’s genetic information.
13. The Composition and Structure of DNA and RNA
• DNA and RNA are polymers composed of monomers called nucleotides.
• Each nucleotide has three parts:
a. A pentose (5-carbon) sugar.
b. A nitrogenous base.
c. A phosphate group.
• The pentose sugar in RNA is ribose, and in DNA it’s deoxyribose. The only
difference is at the 2’ position, where RNA has a hydroxyl (OH) group, while DNA
has only a hydrogen.
Structures of deoxyribose and ribose in DNA and RNA
14. There are two classes of nitrogenous bases:
a. Purines (double-ring, nine-membered structures) include adenine (A) and
guanine (G).
b. Pyrimidines (one-ring, six-membered structures) include cytosine (C),
thymine (T) in DNA and uracil (U) in RNA.
Structures of the nitrogenous bases in DNA and RNA
15. • The structure of nucleotides has these features:
a. The base is always attached by a covalent bond between the 1′ carbon of the
pentose sugar and a nitrogen in the base (specifically, the nine nitrogen in
purines and the one nitrogen in pyrimidines).
b. The sugar-base combination is a nucleoside. When a phosphate is added
(always to the 5′ carbon of the pentose sugar), it becomes a nucleoside
phosphate, or simply nucleotide.
c. Nucleotide examples are shown in Figure 2.11, and naming conventions are
given in Table 2.1.
• Polynucleotides of both DNA and RNA are formed by stable covalent bonds
(phosphodiester linkages) between the phosphate group on the 5′ carbon of one
nucleotide, and the 3′ hydroxyl on another nucleotide. This creates the
“backbone” of a nucleic acid molecule.
• The asymmetry of phosphodiester bonds creates 3′-5′ polarity within the nucleic
acid chain.
17. The Discovery of the DNA Double Helix
• James Watson and Francis Crick published the famous double-helix structure in 1953.
• When they began their work, it was known that DNA is composed of nucleotides, but
how the nucleotides are assembled into nucleic acid was unknown.
• Two additional sources of data assisted Watson and Crick with their model:
a. Erwin Chargaff’s ratios obtained for DNA derived from a variety of sources
showed that the amount of purine always equals the amount of pyrimidine, and
further, that the amount of G equals C, and the amount of A equals T.
b. Rosalind Franklin’s X ray diffraction images of DNA showed a helical structure with
regularities at 0.34 nm and 3.4 nm along the axis of the molecule (Figure 8.9).
20. Watson and Crick’s three-dimensional
model has these main features:
a. It is two polynucleotide chains
wound around each other in a
right-handed helix.
b. The two chains are antiparallel.
c. The sugar-phosphate backbones are
on the outside of the helix, and the
bases are on the inside, stacked
perpendicularly to the long axis like
the steps of a spiral staircase.
21. d. The bases of the two strands are held
together by hydrogen bonds between
complementary bases (two for A-T pairs
and three for G-C pairs). Individual H-
bonds are relatively weak and so the
strands can be separated (by heating, for
example). Complementary base pairing
means that the sequence of one strand
dictates the sequence of the other strand.
22. e. The base pairs are 0.34 nm apart, and one full
turn of the DNA helix takes 3.4 nm, so there
are 10 bp in a complete turn. The diameter of a
dsDNA helix is 2 nm.
f. Because of the way the bases H-bond with
each other, the opposite sugar-phosphate
backbones are not equally spaced, resulting in
a major and minor groove. This feature of
DNA structure is important for protein binding.
• The 1962 Nobel Prize in Physiology or Medicine
was awarded to Francis Crick, James Watson and
Maurice Wilkins (the head of the lab in which
Franklin worked). Franklin had already died, and
so was not eligible.
24. The Genetic Code
• The final step in the expression of genes that encode proteins is translation.
• The mRNA nucleotide sequence is translated into the amino acid sequence of a
polypeptide chain.
• Protein synthesis is called translation because it is a decoding process.
• The information encoded in the language of nucleic acids must be rewritten in the
language of proteins.
• There is code degeneracy. i.e., there are up to 6 different codons for a given amino acid.
• Only 61 codons, the sense codons, direct amino acid incorporation into protein.
• The remaining 3 codons (UGA, UAG, and UAA) are involved in the termination of
translation and are called stop or nonsense codons.
25. • The 5′ nucleotide in the anticodon can vary, but generally, if the nucleotides in the second
and third anticodon positions complement the first two bases of the mRNA codon, an
aminoacyl-tRNA with the proper amino acid will bind to the mRNA-ribosome complex.
• This pattern is evident on inspection of changes in the amino acid specified with variation
in the third position (see table).
• This loose base pairing is known as wobble and relieves cells of the need to synthesize so
many tRNAs.
• Wobble also decreases the effects of DNA mutations.
26. • Genetic codons Discovered by Marshall Nirenberg ,Heinrich
Matthaei,philip,Leder and Har Govind Khorana in 1968.
• Nirenberg and Khorana the Nobel prize.
History and definition of Genetic Code
• Genetic code is a triplet of neiutides that coded that coded the
specific amino acid.
• Ex. AUG-met
• UUU-Phe
The amino acids encoded
by all 64 possible codons
were determined.
27. The genetic code required determining how 4 nucleotides (A, T, G, C) could
encode more than 20 amino acids.
Francis Crick and Sydney Brenner determined that the DNA is read in sets of 3
nucleotides for each amino acid.
Each codon consists of three bases (triplet). There are 64 codons.
They are all written in the 5' to 3' direction.
61 codons code for amino acids. The other three (UAA, UGA, UAG) are stop
codons (or nonsense codons) that terminate translation.
There is one start codon (initiation codon), AUG, coding for methionine. Protein
synthesis begins with methionine (Met) in eukaryotes, and formylmethionine
(fmet) in prokaryotes.
The code is unambiguous.
The Genetic Code
28. • The code is degenerate. More than one codon can specify a single
amino acid.
• All amino acids, except Met and tryptophan (Trp), have more than one
codon.
• For those amino acids having more than one codon, the first two bases
in the codon are usually the same. The base in the third position often
varies.
• The code is almost universal (the same in all organisms). Some minor
exceptions to this occur in mitochondria and some organisms.
• The code is commaless (contiguous).
• There are no spacers or "commas" between codons on an mRNA.
• Neighboring codons on a message are non-overlapping.
29. reading frame: the series of nucleotides read in sets of 3 (codon)
• only 1 reading frame is correct for encoding the correct sequence UU of amino
acids
UUC GAG
UUU
AUG
UCG AAU
U UCU CGA UGU UUG AGA
UU
AU
CUC GAU GUU UGA GAA U
Reading frame 1
Reading frame 2
Reading frame 3
31. stop codons: 3 codons (UUA, UGA, UAG) in the genetic code used to
terminate translation.
start codon: the codon (AUG) used to signify the start of translation.
31
Types of codons
32. Unambiguous
Each codon specifies a particular amino acid, the codon ACG codes for the
amino acid threonine, and only threonine.
Non overlapping
This means that successive triplets are read in order. Each nucleotide is part of
only one triplet codon.
Commanelss which mean there is no punctuation witbeen two
codans
32
Characteristics of the Genetic Code
34. • The first two bases of the codon make normal (canonical) H-bond pairs
with the 2nd and 3rd bases of the anticodon
• At the remaining position, less stringent rules apply and non-canonical
pairing may occur
• The rules: first base U can recognize A or G, first base G can recognize U
or C, and first base I can recognize U, C or A (I comes from deamination
of A)
• Advantage of wobble: dissociation of tRNA from mRNA is faster and
protein synthesis too
The Wobble Hypothesis
35. • It is a triplet code.
• – Each three-nucleotide codon in the mRNA specifies one amino acid
• • It is comma free.
• – mRNA is read three bases at a time without skipping any bases.
• • It is non-overlapping/non-ambiguous.
• • It is almost universal.
• – In nearly all organisms, most codons have the same amino acid meaning.
• – Of 20 amino acids, 18 are encoded by 2 or more codons.
• • The code has start and stop signals.
• – AUG is the usual start signal and defines the open reading frame.
Points to remember:
36. • • Stop signals are codons with no corresponding tRNA
• – the nonsense or chain-terminating codons.
• – generally three stop codons: UAG, UAA, and UGA.
• More than one codon can code for each amino acid.
• Each amino acid can be coded for by more than one codon
38. The Organization of DNA in Chromosomes
1. Cellular DNA is organized into chromosomes. A genome is the
chromosome or set of chromosomes that contains all the DNA of an
organism.
2. In prokaryotes the genome is usually a single circular chromsome.
In eukaryotes, the genome is one complete haploid set of nuclear
chromosomes; mitochondrial and chloroplast DNA are not included.
39.
40. Viral Chromosomes
1. A virus is nucleic acid surrounded by a protein coat. The nucleic acid
may be dsDNA, ssDNA, dsRNA or ssRNA, and it may be linear or
circular, a single molecule or several segments.
2. Bacteriophages are viruses that infect bacteria. Three different types
that infect E. coli are good examples of the variety of chromosome
structure found in viruses.
41. T-even phage
3. The T-even phages (T2, T4 and T6) have similar structures; all have
dsDNA genomes composed of a one linear DNA molecule surrounded
by a protein coat.
ΦX174 phage
4. ΦX174 is a small, simple virus with one short ssDNA chromosome. In 1959,
Robert Sinsheimer found that the DNA of ΦX174 has a base composition
that does not fit the complementary base-pair-rules. single strand DNA
rather than dsDNA
42. λ phage
5. Bacteriophage λ is somewhat like the T-
even phages in structure. However, its
chromosome changes form. A linear
molecule of dsDNA is packaged inside the
protein head, but after the virus infects its
host the chromosome becomes circular due
to base-pairing of complementary 12-base
single-stranded regions at the ends of the
linear molecule (Fig. 2.18)
43. Fig. 2.18 chromosome structure varies at stages of lytic infection of E. coli
44.
45. Prokaryotic Chromosomes
1. The typical prokaryotic genome is one circular dsDNA chromosome, but
some prokaryotes are more exotic, with a main chromosome and one or
more smaller ones. When a minor chromosome is dispensable to the life
of the cell, it is called a plasmid. Some examples:
a. Borrelia burgdorferi (Lyme disease in humans) has a 0.91-Mb linear
chromosome, plus an additional 0.53-Mb of DNA in 17 different linear
and circular molecules.
b. Agrobacterium tumefaciens (crown gall disease of plants) has a 3.0-Mb
circular chromosome and a 2.1-Mb linear one.
46. 2. Archaebacteria also vary in chromosomal organization, but only circular
forms have been found. Examples:
a. Methanococcus jannaschii has three chromosomes of 1.66-Mb, 58-kb
and 16-kb.
b. Archaeoglobus fulgidus has one 2.2-Mb circular chromosome.
3. Both Eubacteria and Archaebacteria lack a membrane-bounded nucleus,
hence their classification as prokaryotes. Their DNA is densely arranged in
a cytoplasmic region called the nucleoid.
4. In an experiment where E. coli is gently lysed, it releases one 4.6-Mb
circular chromosome, highly supercoiled (Figures 2.20). A 4.6-Mb double
helix is about 1mm in length, about 103 times longer than an E. coli cell.
DNA supercoiling helps it fit into the cell.
48. 5. A molecule of B-DNA, with 10bp/turn of the helix, is in relaxed conformation. If turns
of the helix are removed and the molecule circularized, the DNA will form
superhelical turns to compensate for the added tension.
6. Either overwinding or underwinding DNA will create a structure where 10bp/turn of
the helix is not the most energetically favored conformation, and supercoils will be
induced. Both positive and negative supercoils will condense the DNA.
7. All organisms contain topoisomerase enzymes to supercoil their DNA.
8. Prokaryotes also organize their DNA into looped domains, with the ends of the
domains held so that each is supercoiled independently (Figure 2.22).
9. The compaction factor for looped domains is about 10-fold. In E. coli there are
about 100 domains of about 40kb each.
51. • In addition to the genetic material present in the nucleoid, many procaryotes (and
some yeasts and other fungi) contain extrachromosomal DNA molecules called
plasmids.
• In some cases, numerous different plasmids within a single species have been
identified. (B. burgdorferi, carries 12 linear and 9 circular plasmids)
• Plasmids-
small
double-stranded DNA molecules
can exist independently of the chromosome
replicate autonomously
mostly circular
• Linear plasmids possess special structures or sequences at their ends to prevent their
degradation and to permit their replication.
• Plasmids have relatively few genes, generally less than 30. Their genetic information
is not essential to the host, and cells that lack them usually function normally.
52. • However, many plasmids carry genes that confer a selective advantage to their hosts
in certain environments.
• Episomes: Plasmids which are able to integrate into the chromosome and are thus
replicated with the chromosome.
• Plasmids are not always equally apportioned into daughter cells and sometimes are
lost. The loss of a plasmid is called curing.
• Curing can be induced by acridine mutagens, UV and ionizing radiation, thymine
starvation, antibiotics, and growth above optimal temperatures.
• Conjugative plasmids have genes for the construction of hair-like structures called
pili and can transfer copies of themselves to other bacteria during conjugation.
• F plasmid (F factor or fertility factor) of E. coli, was the first conjugative factor to be
described; contains genes that direct the formation of sex pili that attach an F+ cell
(a cell containing an F plasmid) to an F- cell (a cell lacking an F plasmid).
• F factor is also an episome.
53. • R plasmids (Resistance factors or R factors) give antibiotic resistance on the cells that
contain them.
• R factors typically have genes that code for enzymes capable of destroying or
modifying antibiotics.
• Bacteriocin-encoding plasmids may give the bacteria that harbor them a competitive
advantage in the microbial world. Bacteriocins are bacterial proteins that destroy
other bacteria.
• Col plasmids contain genes for the synthesis of bacteriocins known as colicins, which
are directed against E. coli. Example; cloacins kill Enterobacter species.
• Virulence plasmids encode factors that make their hosts more pathogenic. For
example, enterotoxigenic strains of E. coli cause traveler’s diarrhea because they
contain a plasmid that codes for an enterotoxin.
• Metabolic plasmids carry genes for enzymes that degrade substances such as
aromatic compounds (toluene), pesticides (2,4-dichlorophenoxyacetic acid), and
sugars (lactose).
• Metabolic plasmids even carry the genes required for some strains of Rhizobium to
induce legume nodulation and carry out nitrogen fixation.
54.
55. UNIT 4
Replication of DNA-
• Rolling circle model
Replication of RNA-
• Reverse transcriptase
56.
57. A process of producing two identical copies from one original DNA molecule
Three basic steps:
i) Initiation
ii) Elongation
iii) Termination
i) Initiation:
During initiation, some enzymes like helicase, cut the hydrogen bonds or produce
the nick in the strands of DNA to make it available for the enzymes to carry out further
process of replication. Single strand binding proteins restrict the rebinding of hydrogen
bonds. Enzymes & proteins reach to the nick; make the complex and replication starts.
DNA Replication a general account
58. Real difference is present at the step of elongation.
ii) Elongation:
Polymerase proceeds on the DNA and formation or elongation starts.
In Eukaryotes, the DNA is much larger so the replication forms a linear structure often.
But in some Prokaryotes, specially in bacteria who possess the plasmid, a unique type of
replication occurs known as “Rolling circle Replication”.
59. • Replication in eukaryotes is bidirectional, this type is unidirectional (i.e. in prokaryotes)
• Ideal example of this type is the circular plasmid of bacteria, as it happens only in
circular genomes.
Initiation
• Initiates by the production of nick on one of the two strands producing free 3́-OH and 5́
phosphate ends, by the action of:
• Helicase
• Topoisomerases
• Single stranded binding proteins (SSBPs).
Helicase
Bacterial Plasmid
SSBPs
Rolling Circle Model of DNA Replication
60. Elongation
• For Elongation, the DNA polymerase III binds to the 3́-OH group of broken strand, using
the unbroken strand as a template.
• The polymerase will start to move in a circle for elongation, due to which it is named as
Rolling circle model. As the elongation proceeds, the 5́end will be displaced and will
grow out like a waving thread.
DNA Polymerase III
Broken Strand
Origin Point
61. Termination
• At the point of termination, the linear DNA molecule is cleaved from the circle,
resulting in a double stranded circular DNA molecule and a single-stranded linear DNA
molecule.
• The linear single stranded molecule is circularized by the action of ligase and then
replication to double stranded circular plasmid molecule.
Origin Point
62. Example of Rolling Circle Model: The conjugation between F+ and F- Bacteria