GENE SILENCING

Gurbachan S. Miglani
Visiting Professor

School of Agricultural Biotechnology

Punjab Agricultural University
Ludhiana
 Gene Silencing
• A general term used to describe the regulation of gene
expression (Redberry 2006).
• In particular, it refers to the ability of 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 (Redberry 2006)
• When genes are silenced, their expression is reduced or
suppressed
• Gene silencing is considered a gene knockdown mechanism
since the methods used to silence genes
 Types of Gene Silencing
• Transcriptional Gene Silencing (TGS)
a) Genomic Imprinting
b) Paramutation
c) Transposon silencing
d) Position effect
e) RNA-directed DNA methylation
• Post-Transcriptional Gene Silencing (PTGS)
(f) RNA interference
(g) Nonsense mediated decay
• Meiotic Gene Silencing (MGS)
(h) Transvection
(i) Meiotic silencing by unpaired DNA
 a) Genomic Imprinting
• The epigenetic phenomenon by which certain genes are expressed in
a parent-of-origin-specific manner
o If the allele inherited from the father is imprinted, it is thereby
silenced, and only the allele from the mother is expressed
o If the allele from the mother is imprinted, then only the allele from
the father is expressed
• Involves DNA methylation and histone methylation without altering
the genetic sequence
• These epigenetic marks are established ("imprinted") in
the germline (sperm or egg cells) of the parents and are maintained
through mitotic cell divisions in the somatic cells of an organism
• Appropriate imprinting of certain genes is important for normal
development
 Imprinting mechanisms

• Imprinting is a dynamic process

• Imprints are erased and re-established through each
generation so that genes that are imprinted in an adult may
still be expressed in that adult's offspring
• In germline cells the imprint is erased and then re-established
according to the sex of the individual
• in the developing sperm (during spermatogenesis), a paternal
imprint is established
• in developing oocytes (oogenesis), a maternal imprint is
established
 Regulation
• The grouping of imprinted genes within clusters allows them
to share common regulatory elements, such as non-
coding RNAs and differentially methylated regions (DMRs)
• When these regulatory elements control the imprinting of one
or more genes, they are known as imprinting control regions
(ICR)
• Differentially methylated regions are generally segments of
DNA rich in cytosine and guanine nucleotides
• Methylation does not necessarily mean silencing; instead, the
effect of methylation depends upon the default state of the
region
 Imprinted genes in plants
• Angiosperms have double fertilisation
• The 2:1 ratio of maternal to paternal genomes appears to be
critical for seed development (Garnier et al. 2008)
• Some genes are found to be expressed from both maternal
genomes while others are expressed exclusively from the lone
paternal copy (Nowack et al. 2007)
• These imprinted genes are responsible for the triploid
block effect in flowering plants that prevents hybridization
between diploids and autotetraploids (Köhler et al. 2010)
 b) Paramutation
• first observed by the effect it had on the color of corn kernels in
maize plants
• is an interaction between two alleles at a single locus, whereby one
allele induces a heritable change in the other allele (Chandler 2007)
• change may be in the pattern of DNA methylation or histone
modifications (Haring et al. 2010)
• the allele inducing the change is said to be paramutagenic
• the allele that has been epigenetically altered is termed paramutable
• a paramutagenic allele may have altered levels of gene expression,
which may continue in offspring which inherit that allele, even
though the paramutagenic allele may no longer be present
• through proper breeding, paramutation can result in sibling plants
that have the same genetic sequence, but with drastically
different phenotypes
 History of paramutation
• First explained by R.A. Brink in 1950s
• Specific weakly expressed alleles of the red1 (r1) locus in
maize, which encodes a transcription factor that confers red
pigment to corn kernels, can heritably change specific strongly
expressed alleles to a weaker expression state
• The weaker expression state adopted by the changed allele is
heritable and can, in turn, change the expression state of
other active alleles in a process termed secondary
paramutation
• The influence of the paramutagenic allele could persist for
many generations
• Alleles that do not take part in this interaction are called
neutral
• When present together in an organism, the paramutable
allele is converted to the paramutagenic allele, and retains its
paramutagenicity in subsequent generations
• No change in DNA sequence accompanies this transformation,
but instead epigenetic modifications (e.g., DNA methylation)
differentiate the paramutagenic from paramutable alleles
• The paramutable allele is highly transcribed
• The paramutagenic allele that undergoes little to no
transcription
 The r1 locus in maize
• The gene at this locus, when actively transcribed, codes for
a transcription factor that promotes anthocyanin production,
resulting in kernels with a purple color
• One allele at this locus, referred to as B’, is capable of causing
methylation at the other allele, B-I
• This methylation results in reduced transcription and, as a result,
decreased anthocyanin production
• These alleles do not differ in DNA sequence, but they do differ in
their degree of DNA methylation
• The change of the B-I allele to the B’ allele is stable and heritable
• Other, similar examples of paramutation exist at other maize loci,
as well as in other plants such as the model system Arabidopsis
thaliana and transgenic petunias
 Mechanism of paramutation
• Epigenetic modification and RNA-silencing lead to paramutation
• In the case of the r1 locus in maize, DNA methylation of a region
of tandem repeats near the coding region of the gene is
characteristic of the paramutagenic B’ allele
• When the paramutable B-I allele becomes paramutagenic, it too
takes on the same DNA methylation pattern (Belele et al. 2013)
• In order for this methylation to be successfully transferred, a
number of genes coding for RNA-dependent RNA
polymerases and other components of RNA-silencing pathways
are required
• Paramutation is thus mediated via endogenous RNA-silencing
pathways
• The transcription of short interfering RNAs (siRNA) from the
tandem repeat regions supports this
 (c) Transposon silencing
• A form of transcriptional gene silencing (TGS) targeting transposons
• TGS is a product of histone modifications that prevent the
transcription of that area of DNA
• The “jumping” of transposons generates genomic instability and
can cause extremely deleterious mutations
• Transposable element insertions have been linked to many diseases
• The silencing of transposons is therefore extremely critical in the
germline in order to stop transposon mutations from developing
and being passed on to the next generation
• These epigenetic defenses against transposons can be heritable
• Small interfering RNAs are responsible for transposon silencing
 Mechanism of transposon silencing
• Piwi-interacting RNA (piRNA), the largest class of the small RNAs
• piRNAs are between 26 and 31 nucleotides in length
• piRNAs function through interactions with piwi proteins from
the Argonaute protein family (gene silencing proteins)
• Many piRNAs are derived from transposons and other repeated
elements, and therefore lack specific loci
• Some piRNAs map to specific locations clustered in areas near the
centromeres or telomeres
• piRNA clusters make up ~1% of the genome (Khurana and
Theurkauf 2010)
• piRNA-PIWI complexes directly control the activity of transposons
• piRNAs bound to PIWI proteins seem to use post-transcriptional
transcript destruction to silence transposons
• Transposon insertions in introns can escape silencing via the piRNA
pathway
• Transcript destruction by piRNAs occurs after nuclear export.
• Most piRNAs are antisense to mRNAs transcribed from the silenced
transposons, generally associating with Piwi and Aubergine (Aub)
proteins
• Sense-strand piRNAs tend to associate with Argonaute 3 (Ago3) instead
• A cycle called “ping pong” amplification proceeds between the sense
and anti-sense piRNAs involving extensive trimming and processing to
create mature piRNAs
• This process is responsible for the production of most piRNAs in the
germline
 d) Position effect
• It is the effect on the expression of a gene when its location in
a chromosome is changed, often by translocation
• Position effect is also used to describe the variation of
expression exhibited by identical transgenes that insert into
different regions of a genome
• Difference in expression is often due to enhancers that
regulate neighbouring genes
• These local enhancers can also affect the expression pattern
of the transgene
• Since each transgenic organism has the transgene in a
different location each transgenic organism has the potential
for a unique expression pattern
 Position effect in Yeast

Alberts, B. (2002).  Molecular Biology of the Cell (4th ed.).

New York: Garland Science. ISBN 0-8153-4072-9
 Position effect in Drosophila

Alberts, B. (2002).  Molecular Biology of the Cell (4th ed.). New

York: Garland Science. ISBN 0-8153-4072-9
 Position effect in Plants
Oenothera

• In an X-ray induced interchange of Oenothera blandina, the

capacity of the gene Ps for pigmenting the flower buds is
reduced
• One of the chromosome breaks is in arm 3 of chromosome
3.4, close to the locus of Ps
• When the translocated Ps gene is transferred to normal
blandina by crossing-over it reçovers its normal activity
• This is the first experimental demonstration of a position
effect in plants (Catcheside 1939)
Maize

• The gene, called Mediator of paramutation1 (Mop1), codes for an

RNA-processing enzyme that is necessary for making the small
RNAs that are responsible for silencing the transposon MuDR
• A second gene, Mu killer (MuK), is then needed to establish
heritable silencing (Gross 2006)
• Muk, produces a long hairpin RNA molecule that can trigger DNA
methylation and heritable silencing of one or many MuDR elements
• Muk-induced silencing involves a directed and heritable change in
gene activity in the absence of changes in DNA sequence
• MuDR elements remain inactive even after Muk segregates
• Muk effectively silences the MuDR element at this position
• Singh et al. (2008) identified a particular position at which the
MuDR element reactivates after Muk is lost
• This is the first position effect that is associated with the reversal of
epigenetic silencing
e) RNA-directed DNA methylation (RdDM)
• An epigenetic process first discovered in plants (Wassenegger
et al 1994)
• During RdDM, double-stranded RNAs (dsRNAs) are processed
to 21-24 nucleotide small interfering RNAs (siRNAs) and
guide methylation of homologous DNA loci
• In plants dsRNAs may be generated from following sources
o Viral replication intermediates
o Products of the endogenous RNA-directed RNA polymerase
o Transcribed inverted repeats
o Transposable elements
• Besides siRNAs, other proteins involved in the establishment
of RdDM are Argonautes, DNA methyltransferases, chromatin
remodelling complexes and the plant-specific Polymerase IV
and Polymerase V
• All these components act in concert to add a methyl-group at
the 5' position of cytosines
• Cytosines at all sequence context (CG, CHG, CHH) may get de
novo methylated in plants
 f) RNA interference
• A biological process in which RNA molecules inhibit gene
expression, typically by causing the destruction of
specific mRNA molecules
• Known by other names: co-suppression/post-transcriptional
gene silencing (PTGS)/ quelling
• Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in
Physiology or Medicine for their work on RNA interference in
the nematode worm Caenorhabditis elegans, which they
published in 1998
• RNAi has immense potential in suppression of desired genes
• RNAi is precise, efficient, stable and better than antisense
technology for gene suppression (Saurabh et al. 2014)
• Found in many eukaryotes
 Central to RNA interference
• Two types of small ribonucleic acid (RNA) molecules
o  microRNA (miRNA)
o small interfering RNA (siRNA)
• These small RNAs are the direct products of genes
• These small RNAs can bind to other specific messenger
RNA (mRNA) molecules
• Result is either increase or decrease their activity
• Two other small RNAs emloyped in RNAi
o piwi-interacting RNA (piRNA)
o repeat associated small interfering RNA (rasiRNA)
 miRNA and siRNAs
• Mature miRNAs are structurally similar to siRNAs produced from
exogenous dsRNA, but before reaching maturity, miRNAs must first
undergo extensive post-transcriptional modification
• A miRNA is expressed from a much longer RNA-coding gene as
a primary transcript known as a pri-miRNA which is processed, in
the cell nucleus, to a 70-nucleotide stem-loop structure called a pre-
miRNA by the microprocessor complex
• This complex consists of an RNase III enzyme called Drosha and a
dsRNA-binding protein DGCR8. The dsRNA portion of this pre-miRNA
is bound and cleaved by Dicer to produce the mature miRNA
molecule
• miRNAs typically have incomplete base pairing to a target and inhibit
the translation of many different mRNAs with similar sequences
• siRNAs typically base-pair perfectly and induce mRNA cleavage only
in a single, specific target
 piwi-interacting RNA (piRNA)

• piRNAs represent the largest class of small non-coding RNA

molecules expressed in animal cells, deriving from a large
variety of sources, including repetitive DNA and transposons
• piRNAs appear to act both at the post-transcriptional and
chromatin levels
• piRNAs are distinct from miRNA due to at least an increase in
terms of size and complexity
• Repeat associated small interfering RNA (rasiRNAs) are
considered to be a subspecies of piRNA
 Mechanism of RNA Silencing
  Large single-stranded RNA with inverted repeats
hairpin/panhandle constructs
transported from the nucleus to
the cytosol through exportin-5
dsRNA

Dicer

small (21-28 nt = nucleotides long) strands of miRNAs/siRNAs

Argonaute
RISC
rasiRNA have a role in guiding
chromatin modification
Destruction of target mRNA

Basic mechanistic flow for RNA Silencing

 Mechanism of RNAi
• RNAi is initiated by the enzyme  Dicer, which cleaves
long double-stranded RNA (dsRNA) molecules into short
double-stranded fragments of ~20 nucleotide siRNAs
• Each siRNA is unwound into two single-stranded RNAs
(ssRNAs), the passenger strand and the guide strand
• The passenger strand is degraded and the guide strand is
incorporated into the RNA-induced silencing complex (RISC)
• Guide strand pairs with a complementary sequence in a
messenger RNA molecule and induces cleavage by Argonaute,
the catalytic component of the RISC complex
• A valuable research tool, both in cell culture and in living
organisms
• synthetic dsRNA introduced into cells can selectively and
robustly induce suppression of specific genes of interest
• RNAi may be used for large-scale screens that systematically shut
down each gene in the cell
• RNAi pathway is also used as a practical tool
in biotechnology, medicine and insecticides (Kupferschmidt 2013)
• When the dsRNA is exogenous (coming from infection by a virus with
an RNA genome or laboratory manipulations), the RNA is imported
directly into the cytoplasm and cleaved to short fragments by Dicer
• Initiating dsRNA can also be endogenous (originating in the cell), as in
pre-microRNAs expressed from RNA-coding genes in the genome
• The primary transcripts from such genes are first processed to form
the characteristic stem-loop structure of pre-miRNA in the nucleus,
then exported to the cytoplasm (Figure)
• The two dsRNA pathways, exogenous and endogenous, converge at
the RISC
The stem-loop secondary structure of a pre-microRNA  from Brassica oleracea
(from Wikipedia)
 dsRNA cleavage
• Endogenous dsRNA initiates RNAi by activating
the ribonuclease protein Dicer
• Dicer binds and cleaves double-stranded RNAs (dsRNAs) to produce
double-stranded fragments of 20–25 base pairs with a 2-nucleotide
overhang at the 3' end
• These short double-stranded fragments are called small interfering
RNAs (siRNAs)
• These siRNAs are then separated into single strands and integrated
into an active RISC complex
• After integration into the RISC, siRNAs base-pair to their target mRNA
and cleave it, thereby preventing it from being used as
a translation template
 Three prime untranslated regions (3'UTRs)
and microRNAs
• The 3'-UTR often contains microRNA response elements (MREs).
• MREs are sequences to which miRNAs and regulatory proteins bind
• By binding to specific sites within the 3'-UTR, miRNAs can decrease
gene expression of various mRNAs
• A single miRNA can reduce the stability of hundreds of unique mRNAs
• A single miRNA may repress the production of hundreds of proteins
• The active components of RISC are endonucleases called argonaute
proteins
• Argonaute cleave the target mRNA strand complementary to their
bound siRNA
• The guide strand of siRNA binds the argonaute protein and directs
gene silencing
 RNA-induced transcriptional silencing (RITS)
• Modification of histones downregulates genes pre-transcriptionally
• Process is carried out by a complex of proteins called the RITS complex

From Wikipedia
• In fission yeast, RITS complex contains argonaute,
a chromodomain protein Chp1, and a protein called Tas3 of
unknown function
• Induction and spread of heterochromatic regions requires the
argonaute and RdRP proteins
• RITS forms a complex with siRNAs complementary to the local
genes and stably binds local methylated histones, acting co-
transcriptionally to degrade any nascent pre-mRNA transcripts
that are initiated by RNA polymerase
• The formation of such a heterochromatin region is dicer-
dependent
 Major differences between
plant and animal gene silencing
• In plants, RNAi is thought to propagate by the transfer of siRNAs
between cells through plasmodesmata (channels in the cell walls
that enable communication and transport) (Lodish et al 2004)
• Heritability comes from methylation of promoters targeted by RNAi
• The new methylation pattern is copied in each new generation of
the cell
• Distinction between plants and animals lies in the targeting of
endogenously produced miRNAs
• in plants, miRNAs are usually perfectly or nearly perfectly
complementary to their target genes and induce direct mRNA
cleavage by RISC
• in animals' miRNAs tend to be more divergent in sequence and
induce translational repression which may be produced by
inhibiting the interactions of translation initiation factors with the
messenger RNA's polyadenine tail
 Chromatin-dependent gene silencing
(CDGS) pathways of RNAi

• Involves the assembly of small RNA complexes on nascent

transcripts
• Regarded as encompassing mechanisms of action which
implicate transcriptional gene silencing (TGS) and co-
transcriptional gene silencing (CTGS) events (Bühler 2009)
• This is significant at least because the evidence suggests
that small RNAs play a role in the modulation of chromatin
structure and TGS (Gonzalez et al 2008; Kim et al 2005)
Applications of RNAi

• Gene knockdown
• Functional genomics
• Medicine
• Antiviral therapies
• Cancer
• Safety
• Genome-scale screening
• Insecticide
• Biotechnology
• Foods
• Transgenic plants
 RNAi in Crop Improvement
• RNAi has developed for having nicotine-free tobacco,
decaffeinated coffee, nutrient fortified and hypoallergenic
crops
• Arctic apples were produced by RNAi suppression of PPO
(polyphenol oxidase). These apple varieties that will not
undergo browning after being sliced
• RNAi can be used for improving in plants stress tolerance and
enhanced nutritional level
• RNAi may be useful in inducing early flowering, delayed
ripening, delayed senescence, breaking dormancy, stress-free
plants, overcoming self-sterility, etc.
• RNAi has been used to produce cotton stocks whose seeds contain
reduced levels of delta-cadinene synthase, a key enzyme in
gossypol production. This makes them unsuitable for human
consumption
• Reduction of the cyanogenic natural product linamarin in cassava
plants
• Reduction in the levels of allergens in tomato plants
• Fortification of plants such as tomatoes with dietary antioxidants
• The Flavr Savr tomato and two cultivars of ringspot-resistant papaya
exploited the RNAi pathway
 Transgenic plants expressing small RNAs

• Transgenic crops have been made to express small bits of RNA,

carefully chosen to silence crucial genes in target pests
• Small RNAs exist that affect only insects that have specific genetic
sequences
• In 2009, RNAs were identified that could kill any one of four fruit
fly species while not harming the other three
• The International Potato Center in Lima, Peru is looking for genes
to target in the sweet potato weevil, a beetle whose larvae
ravage sweet potatoes globally
• Researchers are trying to silence genes in ants, caterpillars and
pollen beetles
• Monsanto will likely be first to market, with a transgenic corn seed
that expresses dsRNA based on gene Snf7 from the western corn
rootworm
• Silencing Snf7 stunts larval growth, killing them within days
 g) Nonsense-mediated decay (NMD)  pathway 

• This phenomenon exists in all eukaryotes

• Reduces errors in gene expression by eliminating mRNA
transcripts that contain premature stop codons (Baker and
Parker 2004)
• Translation of these aberrant mRNAs could, in some cases,
lead to deleterious gain-of-function or dominant-negative
activity of the resulting proteins (Chang et al 2007)
• NMD was first described in human cells and in yeast in 1979
• NMD limits the translation of abnormal proteins
 Pathway of nonsense-mediated decay
• In Saccharomyces cerevisiae (yeast), three up-frameshift (UPF) proteins
main factors make NMD conserved core pathway
o UPF1
o UPF2
o UPF3
• In humans, NMD conserved core pathway has
o UPF1
o UPF2
o UFP3A
o UFP3B

 UPF2 and UPF3 are part of the exon-exon junction complex (EJC) bound to

mRNA after splicing

 All UPF proteins are trans-acting elements

 Other proteins of NMD pathway

• Other proteins, eIF4AIII, MLN51, and the Y14/MAGOH

heterodimer, also function in NMD
• UPF1 phosphorylation is controlled by the proteins SMG-1,
SMG-5, SMG-6 and SMG-7
• During the first round of translation, the ribosome removes
the exon-exon junction complexes bound to the mRNA after
splicing occurs
• If, after this first round of translation, any of these proteins
remain bound to the mRNA, NMD is activated
 After NMD is activated …
• Ribosome is released before reaching them EJC
• Termination of translation leads to the assembly of a complex
composed of UPF1, SMG1 and the release factors, eRF1 and
eRF3, on the mRNA
• If an EJC is left on the mRNA because the transcript contains a
premature stop codon, then UPF1 comes into contact with
UPF2 and UPF3, triggering the phosphorylation of UPF1
• The phosphorylated UPF1 then interacts with SMG-5, SMG-6
and SMG-7, which promote the dephosphorylation of UPF1
• SMG-7 is thought to be the terminating effector in NMD, as it
accumulates in P-bodies
• P-bodies are cytoplasmic sites for mRNA decay
 h) Transvection
• An epigenetic phenomenon that results from an interaction
between an allele on one chromosome and the corresponding
allele on the homologous chromosome
• Transvection can lead to either gene activation or repression. It can
also occur between nonallelic regions of the genome as well as
regions of the genome that are not transcribed (Lewis 1954)
• Phenotype of a given genotype can be altered solely by disruption
of somatic (or meiotic) pairing
• Such disruption is accomplished by introduction of a heterozygous
rearrangement that disrupts pairing in the relevant region
• Transvection appears to be dependent upon chromosome pairing
• Enhancers of one allele activate the promoter of a paired second
allele
i) Meiotic silencing by unpaired DNA (MSUD)
• In meiotic silencing by unpaired DNA (MSUD), DNA unpaired
in meiosis causes silencing of all DNA homologous to it,
including genes that are themselves paired (Shiu et al 2001)
• A semidominant Neurospora mutant, Sad-1, fails to perform
MSUD.
• MSUD may provide insights into the function of genes
necessary for meiosis, including genes
• It may also contribute to reproductive isolation of species
within the genus Neurospora
• The wild-type allele, sad-1(+), encodes a putative RNA-
directed RNA polymerase
 Mechanism of MSUD
• Shiu and Metzenberg (2002) describe in detail the isolation
of a mutation, Sad-1UV, that suppresses the dominance of
various ascus-dominant mutations
• Additional dominant, semidominant, and recessive Sad-
1 alleles have been generated by repeat-induced point (RIP)
mutation
• The DNA of the dominant RIP alleles becomes methylated,
but dim-2-dependent methylation is not necessary for
silencing
• Meiotic silencing is confined to the ascus in which DNA is
unpaired, and silencing does not spread to neighboring asci
in a fruiting body of mixed genetic constitution
References
 Alberts B (2002). Molecular Biology of the Cell (4th ed.). New York: Garland Science.
Baker KE and Parker, R. (2004). Nonsense-mediated mRNA decay: Terminating
erroneous gene expression. Curr Opin Cell Biol 16 (3): 293–9.
Belele CL, Sidorenko L, Stam M, Bader R, Arteaga-Vazquez MA, and Chandler, and Vicki
L (2013). Specific tandem repeats are sufficient for paramutation-induced trans-
generational silencing. PLOS Genet 9 (10):
e1003773. doi:10.1371/journal.pgen.1003773.
Catcheside DG (1939) A position effect in Oenothera. J Genet. 38: 345. doi:10.1007/
BF02982179.
Chandler V (2007). Paramutation: from maize to mice. Cell 128 (4): 641–5.
Chang YF, Imam JS, and Wilkinson MF (2007). The nonsense-mediated decay RNA
surveillance pathway. Annu Rev Biochem 76: 51–74.
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, and Mello CC (1998). Potent and
specific genetic interference by double-stranded RNA in Caenorhabditis
elegans. Nature 391 (6669): 806–11.
Garnier O, Laoueillé-Duprat S,; and Spillane C (2008). Genomic imprinting in
plants. Epigenetics 3 (1): 14–20.
Gross, L (2006). Transposon silencing keeps jumping genes in their place. PLoS Biol 4:
e353. doi:10.1371/journal.pbio.0040353.
Haring M, Bader R, Louwers M, Schwabe A, van Driel R and Stam M (2010). The role of
DNA methylation, nucleosome occupancy and histone modifications in
paramutation. Plant J. 63 (3): 366–378.
Hood E (2004). RNAi: What's all the noise about gene silencing? Env Health
Perspec 112 (4): A224–9.
Khurana J, and Theurkauf W (2010). piRNAs, transposon silencing, and Drosophila
germline development. JCB 191 (5): 905–13.
Köhler, C; Mittelsten SO, and Erilova A (2010). The impact of the triploid block on the
origin and evolution of polyploid plants. Trends Genet 26 (3): 142–8.
Kupferschmidt K. (2013). A Lethal Dose of RNA. Science 341 (6147): 732–3.
Lewis EB (1954). The theory and application of a new method of detecting chromosomal
rearrangements in Drosophila melanogaster.  Amer Natural 88 (841): 225–39.
Meister G, and Tuschl T (2004). Mechanisms of gene silencing by double-stranded
RNA. Nature 431 (7006): 343–9.
Mocellin S, and Provenzano M (2004). RNA interference: learning gene knock-down
from cell physiology. J. Transl. Med. 2 (1): 39. doi:10.1186/1479-5876-2-39. 
Nowack MK, Shirzadi R, Dissmeyer N, Dolf A, Endl E, Grini PE, Schnittger A (2007).
Bypassing genomic imprinting allows seed development". Nature 447 (7142): 312–5.
Redberry G (2006). Gene Silencing : New Research. New York: Nova Science Publishers.
Saurabh S, Vidyarthi AS, and Prasad D (2014). RNA interference: concept to reality in
crop improvement. Planta 239 (3): 543–64.
Shiu PKT, and Metzenberg RL. 2002. Meiotic silencing by unpaired DNA: properties,
regulation and suppression. Genetics 161 (4): 1483-95.
Shiu PKT, Raju NB, Zickler D, and Metzenberg RL (2001) Meiotic silencing by unpaired
DNA. Cell 107 (7): 905-16.
Singh J, Freeling M, and Lisch D (2008) A position effect on the heritability of
epigenetic silencing. PLoS Genet 4 (10): e1000216.
doi:10.1371/journal.pgen.1000216.
Wassenegger M, Heimes S, Riedel L, Sänger HL (1994). RNA-directed de novo
methylation of genomic sequences in plants. Cell 76 (3): 567–76.
Woodhouse MR, Pedersen B, and Freeling M (2010). "Transposed genes in Arabidopsis
are often associated with flanking repeats". PLoS Genet. 6:
e1000949. doi:10.1371/journal.pgen.1000949.