RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
RAPD markers are decamer DNA fragments.
RAPD is a type of PCR reaction.
as the name suggest it is a fast method when compared to the traditional PCR medthod.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
Techniques based on the principle of selectively amplifying a subset of restriction fragments from a complex mixture of DNA fragments obtained after digestion of genomic DNA with restriction endonucleases.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
INTERFERENCE means the act of interfering with something, here, with RNA. RNAi is an evolutionarily conserved mechanism triggered by dsRNA molecules, to prevent the expression of specific genes or the translation, causes sequence-specific degradation of the targeted mRNA molecules of that particular gene. It was also known as CO-SUPPRESSION, POST TRANSCRIPTIONAL GENE SILENCING [PTGS] in plants and QUELLING in fungi.
RNA interference (RNAi) is a mechanism that inhibits gene expression at the stage of translation or by hindering the transcription of specific genes.
RNAi targets include RNA from viruses and transposons.
Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research.
Biological screening of herbal drugs: Introduction and Need for
Phyto-Pharmacological Screening, New Strategies for evaluating
Natural Products, In vitro evaluation techniques for Antioxidants, Antimicrobial and Anticancer drugs. In vivo evaluation techniques
for Anti-inflammatory, Antiulcer, Anticancer, Wound healing, Antidiabetic, Hepatoprotective, Cardio protective, Diuretics and
Antifertility, Toxicity studies as per OECD guidelines
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The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
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The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
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June 3, 2024 Anti-Semitism Letter Sent to MIT President Kornbluth and MIT Cor...Levi Shapiro
Letter from the Congress of the United States regarding Anti-Semitism sent June 3rd to MIT President Sally Kornbluth, MIT Corp Chair, Mark Gorenberg
Dear Dr. Kornbluth and Mr. Gorenberg,
The US House of Representatives is deeply concerned by ongoing and pervasive acts of antisemitic
harassment and intimidation at the Massachusetts Institute of Technology (MIT). Failing to act decisively to ensure a safe learning environment for all students would be a grave dereliction of your responsibilities as President of MIT and Chair of the MIT Corporation.
This Congress will not stand idly by and allow an environment hostile to Jewish students to persist. The House believes that your institution is in violation of Title VI of the Civil Rights Act, and the inability or
unwillingness to rectify this violation through action requires accountability.
Postsecondary education is a unique opportunity for students to learn and have their ideas and beliefs challenged. However, universities receiving hundreds of millions of federal funds annually have denied
students that opportunity and have been hijacked to become venues for the promotion of terrorism, antisemitic harassment and intimidation, unlawful encampments, and in some cases, assaults and riots.
The House of Representatives will not countenance the use of federal funds to indoctrinate students into hateful, antisemitic, anti-American supporters of terrorism. Investigations into campus antisemitism by the Committee on Education and the Workforce and the Committee on Ways and Means have been expanded into a Congress-wide probe across all relevant jurisdictions to address this national crisis. The undersigned Committees will conduct oversight into the use of federal funds at MIT and its learning environment under authorities granted to each Committee.
• The Committee on Education and the Workforce has been investigating your institution since December 7, 2023. The Committee has broad jurisdiction over postsecondary education, including its compliance with Title VI of the Civil Rights Act, campus safety concerns over disruptions to the learning environment, and the awarding of federal student aid under the Higher Education Act.
• The Committee on Oversight and Accountability is investigating the sources of funding and other support flowing to groups espousing pro-Hamas propaganda and engaged in antisemitic harassment and intimidation of students. The Committee on Oversight and Accountability is the principal oversight committee of the US House of Representatives and has broad authority to investigate “any matter” at “any time” under House Rule X.
• The Committee on Ways and Means has been investigating several universities since November 15, 2023, when the Committee held a hearing entitled From Ivory Towers to Dark Corners: Investigating the Nexus Between Antisemitism, Tax-Exempt Universities, and Terror Financing. The Committee followed the hearing with letters to those institutions on January 10, 202
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An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
2. Contents
Introduction
Types of gene silencing
-Transcriptional gene silencing
-Post transcriptional gene silencing
RNA i (RNA Interference) pathway
-Si RNA and Mi RNA
-Mechanism
Application
Conclusion
Previously asked questions
Bibliography
3. Gene silencing is a technique that aims to reduce or eliminate the production of
a protein from it’s corresponding gene.
It generally describe the “switching off” of a gene by a mechanism other than
genetic modification.
That is, a gene which would be expressed (“turned on”) under normal
circumstances is switched off by machinery in the cell.
It occurs when RNA is unable to make a protein during translation.
Gene silencing is same as gene knock down but is totally different from gene
knock out.
When genes are knock down ,there expression is reduced , where in contrast
when genes are knocked out, they are completely erased from organism’s
genome and thus have no expression.
Introduction
4. 1990 Jorgensen:
To deepen the pigmentation in petunias Introduction of transgenes
homologous to endogenous genes often resulted in plants with both gene
suppressed called co suppression.
Resulted in degradation of the endogenous and transgene mRNA.
1995 Guo and Kemphues:
injection of either antisense or sense RNAs in the germline of C.elegans was
equally effective at silencing at homologous target genes.
1998 Mello and Fire:
Extension of above experiments, combination of sense and antisense
RNA(=dsRNA) was 10 times more effective than single strand RNA.
Short history of gene silencing
5. The discovery of the mechanism of RNA interference by ds RNA by prof.
Andrew Fire and prof. Craig Mello in 1998, gave them the Nobel prize in
2006.
HOW does it works?
1. This is accomplished by binding a specific strand of RNA to an existing
m-RNA strand.
2. The m-RNA creates a copy of DNA strand.
3. By binding the RNA to the m-RNA, m-RNA is prevented from replicating
that portion of the DNA.
4. Specific genes can be targeted and prevented from replicating in to new
DNA strands.
6. genes are regulated at either the transcriptional level or post-transcriptional level,
therefore silencing can be induced either at transcriptional level or post-
transcriptional level.
There are mainly two types of gene silencing
1. Transcriptional gene silencing
2. Post transcriptional gene silencing
Types of Gene silencing
Transcriptional gene silencing Post transcriptional gene silencing
Promoter silenced Promoter active
Genes hypermethylated in
promoter region
Genes hyper methylated in coding
region
Purpose – viral immunity Purpose - viral immunity
7. is the result of histone modifications, creating an
environment of heterochromatin around a gene that
makes it inaccessible to transcriptional machinery (RNA
polymerase, transcription factors, etc.).
Genomic imprinting:
genomic imprinting is a genetic phenomenon by which
certain genes are expressed in a parent-of-origin-specific
manner. It is an inheritance process independent of the
classical Mendelian inheritance.
genomic imprinting have been demonstrated in insects,
mammals and flowering plants.
Transcriptional gene silencing
8. Paramutation:
paramutation is an interaction
between two alleles of a single
locus, resulting in a heritable
change of one allele that is
induced by the other allele.
Paramutation was first
observed by the effect it had on
the colour of corn kernels in
maize plants.
9. Position effect:
Position effect is the effect on the expression of a gene when its location in
a chromosome is changed, often by translocation. This has been well
described in Drosophila with respect to eye colour and is known as
position effect variegation (PEV).
10. RNA Directed DNA Methylation:
RNA-directed DNA methylation is
an epigenetic process first
elucidated in plants whereby small
double-stranded RNAs (dsRNA's)
are processed to guide methylation
to complementary DNA loci. In the
model plant organism Arabidopsis
thaliana
11. Transposon silencing:
Transposon silencing is a form of
transcriptional gene silencing
targeting transposons.
Transcriptional gene silencing is a
product of histone modification
that prevent the transcription of
that area of DNA.
The “jumping” of transposon
generates the genomic instability
and cause the extremely
deleterious mutations.
Transposable element insertion
have been linked to many disease
including haemophilia ,SCID and
predisposition to cancer.
12. Transgene silencing:
Unfortunate insertion of
transgene in to a
transcriptionally inactive part of
genome. When an insertion of
any transgene it does not show
activity as per desire and this is
because of it’s instability.
The lose of transgene stability is
because of gene silencing.
E.g. slow fruit softening tomato,
by reducing expression of
polygalactouronase enzyme.
13. The ability of exogenous or sometimes endogenous RNA to supress the
expression of the gene which corresponds to the m-RNA sequence.
RNA i (RNA interference):
it is a post transcriptional process triggered by the introduction of double
stranded RNA (ds RNA) which leads to the gene silencing in a sequence
specific manner.
First evidence came from studies on nematode caenorhabditis elegans. further
analysis in fruit fly drosophila melanogaster.
It is also known as post transcriptional gene silencing / co suppression and
quelling.
Post transcriptional gene silencing
14. over view of RNA i:
During RNA i Double-stranded RNAs cut into short double-stranded RNAs,
s(small) i(interfering) RNAs, by an enzyme called Dicer. These then base pair
to an mRNA through a dsRNA-enzyme complex. This will either lead to
degradation of the mRNA strand
Highly specific process
Very potent activity
So far only been seen in eukaryotes
Evidence 30% of genome is regulated by RNA I
RNA i pathway guided by,
Si RNA (small interfering RNA):
Small interfering RNAs that have an integral role in the phenomenon of RNA
interference (RNA i), a form of post- transcriptional gene silencing
RNA i: 21-25 nt fragments, which bind to the complementary portion of the
target mRNA and tag it for degradation
15. A single base pair difference
between the Si RNA
template and the target
mRNA is enough to block
the process.
Each strand of Si RNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’
overhangs
16. Mi RNA (micro RNA):
mi RNA Originate from capped &
polyadenylated full length precursors (pri-miRNA)
Hairpin precursor ~70 nt (pre-mi RNA) Mature
mi RNA ~22 nt (mi RNA)
mi RNA originates with SS RNA that forms a
hairpin secondary structure.
Mi RNA regulates post-transcriptional gene
expression and is often not 100% complementary
to the target.
And also mi RNA help to regulate gene
expression, particularly during induction of
heterochromatin formation serves to down
regulate genes pre- transcriptionally (RNA
induced transcriptional silencing or RITS) RITS.
17. Dicer:
RNAse III-like dsRNA-specific ribonuclease
• Enzyme involved in the initiation of RNA i.
• It is able to digest dsRNA into uniformly sized small RNAs (si RNA)
Dicer family proteins are ATP- dependent nucleases.
RNAse III enzyme acts as a dimer
Loss of dicer→loss of silencing processing in vitro
Dicer homologs exist in many organisms including C.elegans, Drosophila, yeast
and humans (Dicer is a conserved protein)
DICER’s domain:
Dicer is a ribonuclease (RNAse III family) with 4 distinct domains:
1) Amino-terminal helicase domain
2) Dual RNAse III motifs in the carboxyl terminal segment
3) dsRNA binding domain
4) PAZ domain (110-130 amino-acid domain present in protein like Argo, Piwi..);it
is thought to be important for protein-protein interaction
19. RISC (RNA Inducing Silencing Complex):
RISC is a large (~500-kDa) RNA-multi protein complex, which triggers mRNA
degradation in response to Si RNA
Unwinding of double- stranded Si RNA by ATP independent helicase.
The active components of an RISC are endonucleases called argonaute
proteins which cleave the target mRNA strand.
20. Steps of the RNA i pathway :
I. ds RNA is sliced by an ATP-dependent
ribonuclease (Dicer) into short
interfering RNAs (si RNAs).
duplexes of 21 23 nucleotides bearing
two-nucleotide 3' overhanging ends.
II. Si RNAs are transferred to a second
enzyme complex, designated RISC for
RNA i -induced silencing complex.
The si RNA guides RISC to the target
mRNA, leading to its destruction.
the antisense strand of the Si RNA is
perfectly complementary
21. RNA i PATHWAY:
dsRNA are chopped into short
interfering RNAs (siRNA) by Dicer.
The si RNA-Dicer complex recruits
additional components to form an
RNA-Induced Silencing Complex
(RISC). The siRNA unwinds.
The unwound siRNA base pairs
with complementary mRNA, thus
guiding the RNAi machinery to the
target mRNA.
The target m RNA is effectively
cleaved and subsequently
degraded – resulting in gene
silencing.
22.
23. Non sense Mediated Decay:
Nonsense mediated decay
(NMD) is a cellular mechanism
of mRNA surveillance that
functions to detect nonsense
mutations and prevent the
expression of truncated or
erroneous proteins.
NMD is triggered by exon
junction complexes (EJCs)
(components of the assembled
RNP) that are deposited during
pre-mRNA processing.
24. Anti sense RNA technology:
It blocks the activity of the m
RNA in a stoichiometric
manner.
Antisense RNA has the opposite
sense to m RNA.
The presence of complimentary
sense and antisense RNA in the
in the same cell can lead to the
formation of a stable duplex
which interferes with gene
expression at the level of RNA
processing or possible
translation.
This technology widely used in
plants for gene inhibition.
25. Advantages of gene silencing:
Downregulation of gene expression simplifies "knockout" analysis.
Easier than use of antisense oligonucleotides. Si RNA more effective and
sensitive at lower concentration.
Cost effective
High Specificity middle region 9-14 are most sensitive With Si RNA, the
researcher can simultaneously perform experiments in any cell type of interest
Can be labelled Ease of transfection by use of vector
blocking expression of unwanted genes and undesirable substances.
Inducing viral resistance
Powerful tool for analysing unknown genes in sequenced genomes.
Useful approach in future gene therapy.
Oligonucleotides can be manufactured quickly, some within one week; the
sequence of the mRNA is all that is needed
26. Disadvantages of gene silencing:
”High pressure injection” and electroporation can cause significant
injection damage to the integrity of the normal tissues and organs and
thus preclude the utilisation in a clinical set-up.
Liposomes/cationic encapsulated Si RNA may also be toxic to the host and
may cause severe host immune responses.
Other emerging strategies includes chemical modification of Si RNA
molecules and encapsulated with different molecules are still in their
infancy and need to be thoroughly investigated before used in therapeutic
applications.
27. Application of Gene silencing
Specific gene silencing using RNA i in cell culture.
Cancer treatments
RNA interference has been used for applications in biotechnology, particularly
in the engineering of food plants that produce lower levels of natural plant
toxins.
Such techniques take advantage of the stable and heritable RNA i phenotype
in plant stocks. For example, cotton seeds.
Modulation of HIV-I replication by RNA i.
Small RNA and it’s application in andrology and urology.
Developing technologies for epigenomic analysis and clinical application of
molecular diagnosis.
Currently there are at least six oligonucleotide drugs inducing RNA i for illness
including cancer.
28. it is the epigenetic regulation of gene expression and widely used in
agriculture and in biotechnology.
Besides the all types of gene silencing the RNA i is the important post
transcriptional gene silencing.
conclusion
29. Molecular biology of the cell 5th edition by whatson, baker , bell, gann,
levinn, losick.
Molecular biology by David p. Clark ELSEVIER.
RNA i guide to gene silencing by Gregory J. Hannon.
Gene silencing theory, techniques and applications by Anthony J. catalano
Bibliography