BIL 250 Chapter 17 review Part 2

Teh LV regions are located about

6 kilobases away from thE joining region, a region that contains five possible J encoding exons

Although researchers examined only 1 percent of the genome, they identified more than

1000 direct physical interactions between promoters and distal regulatory elements such as enhancers in each cell type explained Approimatley 60 percent of these interactions were specific to only one cell type

The drosophila genome contains about

13000 gens but dscam gene can produce 2.5 times that many proteins

The binding sites include approximately

40000 enhancers elements and 100000 promoters

each of the light and heavy chains contains

a constant region at the C terminus and a variable region at the N terminus

The next gene in the cascade, doublesex is

a critical control point in the development of sexual phenotype It produces a functional mrNA and protein in both females and males However, the pre mRNA is processed in a sex psecific matter to produce differnt trasnciprts and hence differnt DSX protiesn

In this case,the term gene would encompass

a higher order concept that includes all the senses coding RNAS that contribute to a specific phenotype

The presence of active p53 triggers

a negative feedback loop that creates more mdm2 protein, which rapidly returns p53 to its rare and inactive state

ubiquitin

a small protein that tags other proteins for degradation by proteolytic enzymes.

In females, the functional TRA protein acts as

a splicing factor that binds to the dsx pre mRNA and directs splicing in a female specific pattern

ARE

a stretch of nucleotides that consist of A and U ribonucleotides These AU rich elements are usually located in the 3' untranslated regions of mrNAs that have short, regulated half lives These ARE containing mRNAs encode proteins that are involved in cell growth or transcription control and need to be rapidly modulated in abundance

The sum of the sxl gene acts as

a switch that selects the pathway of sexual development by controlling splicing of the dsx transcript

scientists have discovered that

abnormal activities of miRNAs can tigbe to the occurrence of cancers, diabetes and heart disease

Alternative splicing is found in

all metazoans but is especially common in vertebrates, including humans

The researchers speculate that

all of these multiple interactions between enhancer snp rotors likely do not occur simultaneously within the same cell but reflex the sum of interactions that are occurring within a population of cells that as assaye dodge in individual experiments

short RNAs act in the nucleus to

alter chromatin structure and bring about repression of transcription

The abundance of elements as well as the discovery of the extent of therin eitneraitons promises to

alter our definition of a gene and our under naidn of how the information in DNA is transformed into phenotype

Only about 7 percent of looping interactions occur between

an enhancer and the enares promoter

Adjacent to the J region is

an enhancer seunde that stimulates transcription from any primer located nearby and a single C exon that encodes the constant region of the k light chain molecule

In unstressed cells, p53 is bound by

another protein called Mdm2

It is estimated that

approximately 10 percent of mammalian mrnas contain the instability elements

This is in contrast to the

approximately 20000 protein coding genes that occupy less than 2 percent of the genome

Within the 125 cell types explained

approximately 30 percent of these DNA sites are bound by transcription factors in any one cell type Only 3700 DNA sites are found by transcription factors in all cell types

Approximately two thirds of protein coding genes express

at least two different splicing variants, with an average of four alternative transcripts per locus

Scientists estimate that

at least two thirds of protein coding genes in the human genome can undergo alternative splicing Thus, humans can produce several hundred thousand different points from the appxomiatley 20,000 genes in the haploid genome

75 percent of the genome can

be transcribed

Mdm2 protein

binds to the transcriptional activation domain of the p53 molecule, blocking its ability to induce transitions In addition, Mdm2 acts as a ubiquitin ligase, adding ubiquitin residues onto the p53 protein

The female DSX protein and the male portion are

both transcription factors, but u act in different ways

The presence in females or the absence in males of xl proteins begins

cascades of pre mRNA splicing events that are specific for females or males

DM2

caused by an expansion of the repeat sequence CCTG within the first intron of the ZNF9 gene Affected individuals may have up to 11,000 copies of the repeat sequence in the ZNF9 intron In DM2, the severity of symptoms is not related to the number of repeats

DM1

caused by the expansion of the trinucleotide repeat CTG in the 3' untranslated region of the DMPK gene In unaffected individuals, the DMPK gene contains between 5 and 35 copies of the CTG repeat sequence whereas in DM1 patients the gene contains between 150 and 2000 copies The severity of the symptoms is directly related to the number of copies of each repeat sequence

Overall, the vast majority of transposition in a cell do not

code for points and remain within the nucleus

Each of these steps can be regulated to

control the quantity of a protein product In addition, the stability, modifications and activities of the protein product can be regulated

Treatment of these tumors with synthetic mirANs may be able to

correct these defects and reeves cancer phenotypes

antigens

defined as molecules, usually proteins, which bring about an immune response

All mRNA molecules are

degraded at some point after their synthesis, but the lifetime of an mrNA, defined in terms of its half life, can vary widely between different mRNAs and can be regulated in response to the needs of the cell

siRNAs

derived from longer RNA molecules that are linear, double stranded and located in the cell cytoplasm In nature, these siNRA precursors arise within cells as a result of virus infection or the expression of transposons - both of which synthesize double stranded rNA molecules as part of their life cycles

miRNAs

derived from single stranded rNAS that are transcribed within the nucleus from the cells' own neon and that contain a double stranded stem loop structure Nuclease enzymes within the nucleus recognize these stem loop structures and cleave them from the longer single stranded rNA The stem loop RNA fragments are exported from the nucleus into the cytoplasm where they are further processed by the dicer complex into shorts linear, double stranded miRNAS

Alternative splicing can generate

different forms of mRNA from identical pre mRNA molecules, sot hat expression of one gene can give rise to a number of proteins with similar or different functions

So far, more than 1500 mirNAS have been

discovered in the human genome This is probably an underestimate and scientists speculate that eukaryotic genomes may contain thousands of genes that are transcribed into short noncoding rNAS which may regulate the expression of more than half of all protein coding genes

Scientists have shown that

each neuron expresses a different subset of dscam protein isoforms

In addition, RNA molecules can be

edited and bases can be modified

In 2003, the US national human genome research institute launched the

encyclopedia of DNA elements or ENCODE project

ENCODE data show that

enhancers can influence the transcription of several different promoters and not necessarily the closest one

Hyper Mutation introduces

even more vaition into the LV Legion's sequence

Teh mature messenger RNA is then

exported to the cytoplasm, where it is translated and degraded

Splicing variants expressed by each gene are

expressed at different levels with one variant being the predominant one

Amplification of specific genes, such as rRNA genes in xenopus oocytes, allows

expression of these geese at times during development when high levels of these gene products are required

Recent studies show that the

expression profiles the miRNA genes are characteristic of each tumor type This observation may lead to more precise method so diagnosing tumors, predicting their course and planning treatments

Under normal conditions, the levels of p53 are

extremely low in cells and p53 that is present is inactive

In the presence of sxl

female splicing patterns are expressed which override the default male splicing patterns

When the ratio is 1.0

females are reduced

The four viable regions, when combined together in toe the immunoglobulin molecule

form a unique structure that recognizes one specific antigen

these data likely suggest that

genes and their regulatory elements likely interactions. complex and fluid networks

Except for the occurrence of sequence alterations due to mutations

genomic DNA in most organisms remains remarkably stable However, despite this genomic stability, some significant empanel of gene regulation by DNA rearrangement exist

The discovery of small RNA molecules that control gene expression has

given rise to a new fold of research

clinical trials

have also been started to test RNAi in the rtermane of hepatitis B and respiratory syncytial virus infections Other clinical trials are planned for siRNA treatment of influenza and hepatitis C virus infections

In addition, V regions are susceptible to

high rate so frandom somatic muatiotn - hypthermauttion - during B cell deeloment

Mammals are capable of producing

hundreds of millions of different types of antibodies in response to the presence of a wide variety of angels - more types of antibodies than their agnes in techgnome

Two of these polypeptides are

identical light chains and two are identical heavy chains Together, they form a Y shaped immunoglobulin structure

A total of 128000 transcripts were

identified and more than 70000 for these arise either from DNA in intergenic region sor from antisense transcription within protein coding genes More than 7000 small rNAS were identified including small nuclear RNAS, small nucleolar RNAS, miro rNAS, and transfer RNAS

The goal of ENCODE is to

identify all the functional DNA sequences that lie within the 3.1 billion nucleotides of the human genome and to temerin who these elements act to regulate gene expression

Teh SXL protein is produced only

in embryos with an X:A ratio of 1

Alternative splicing increases

increases the number of genes that can be made from each gene As a result, the number of proteins that can organism can make - its roteome - is not the same as the number of genes in the genome and protein diversity can exceed gene number by an order of magnitude

An added level of control is that p53 is a transcription factor that

induces the transcription of the mdm2 gene

The immune system protects organisms against

infection and the presence of forgien substance that may enter blood or body tissue

Fire and Mello experimental protocol

injected roundworm cells with either single stranded or double stranded rNA molecules - both containing sequences complementary to the mRNA of the unc-22 gene

ENCODE has examined the

interactions between enhancers and promoter a

Intermediate ratios produce

intersexes

This massive endeavor which isc opening more than 400 scientists around the world

is spontaneously half completed

As regulatory roles are being discovered for many of the small noncoding RNAS as well as some long noncoding RNAs

it is still possible the function will be discovered for more of these RNAS

Another consequence of the ENCODE data is that

it will be necessary to alter our concept of a gene as a discrete unit of inheritance

The steady state level of an mrNA is

its amount in the cells determined by a combination of the rate at which the gene is transcribed and the rate at which the mrna is degraded

RNAi was first discovered during

laboratory research, in studies of plants and animal gene expression

These data also indicate that

major differences exist between cell types as to who their gnomes are regulated with each cell type using different combinations of regulatory elements and binding proteins in order to determine the cells' gene expression phenotype

When the ratio is 0.5

males are produced even when no y chromosome is present

Several dozen scientific supply companies now

manufactured synthetic siRNA molecules of specific ribonucleotide sequence of ruse in research These molecules can be introduced into cultured cells to knock out specific gene products

Changes in splicing patterns can have

many different effects on the translated protein Even small changes can alter the protein's enzymatic activity, receptor binding capacity or protein localization in the cell

Another observation is that genes express

many placing variants simultaneously

Regulation of gene expression occurs at

many points along the pathway from DNA to protein

RNAi may be the

method by which cells recognize these double stranded rNAS and inactive them, protecting the organism from external to internal assaults

In addition, certain cancers appear to have defects in

miNRA gene expression

One of the most surprising discoveries of ENCODE research is that

more than 80 percent of the human genome contains some type of regulatory element or participates in active transcription

Many of these small RNAS originate within

mrAS and other long nocing rnAS

DNA encoding the immunoglobulin gene is not fixed but undergoes

multiple programmed changes, includign deletionsm trnaslaitocatiosn nd random mtuaitons

Besides gene amplifications, several other types of programmed DNA rearrangements

occur during developmental regulation for some eukaryotic genes

There are 70 to 100 different L leader and V variable regions about half

of which are functional and can encode the variable V portion of the K light chain molecule

Appromately 50 percent of promoters physically interact with more than

one distal regulatory element, and some have as many as 20 different interactions Similarly, approximately 10 percent of enhancers interact with more than one promoter an dosm times with as many as ten different promoters

During development of a mature B cell

one of the LV regions along with its promoter is randomly joined by a recombination event to one of the ive J regions This recombination event deletes the entire intervening DNA on chromosome 2

Each B cell synthesizes only

one type of immunoglobulin

in the case of ZNF9

only the CCUG sequence repeat itself accumulates in the nucleus, as the remainder of the intron is degraded after splicing of a gene It appears that the accumulated RNAs bind to and sequester proteins that would normally be involved in regulating the alternative splicing patterns of a large number of other RNAs These rNAs include those whose products are required for the proper functioning of muscle and neural tissue

Scientists have discovered

over 20 genes that are inappropriately spliced in dM1 muscle, ehrat and brain

In theory, any disease caused by

over pexion of a specific gene or even normal expression of an abnormal gene product could be attacked by therapeutic rNAi

Finally,some cancers are characterized by

overexpression or abnormal expression of one or several key proto oncogenes

The presence of ubiquitin on p53 results in

p53 degradation

A more extreme example is provided by

para, another gene expressed in the nervous system of drosophila,

Antibody diversity results in

part from the random recombination often do 35 of 50 different functional LV regions with anyone for five different J regions

In addition, rearrangements that lead to loss of some DNA sequences occur only in

particular types of somatic cells in higher eukaryotes Hence these changes are not passed on to progeny

Although transcriptional control exerts the primary effect on gene regulation in eukaryotes

posttranscriptional regulation plays an equal if not more significant role

In addition to its use in laboratory research, RNAi is being developed as

potential pharmaceutical agent

The recombination event that joins tan LV region to a J region is not

precise and can occur anywhere with a region containing several base pairs

In males, no TRA protein is

present and splicing of the dsx pre mRNA results in a male specific mRNA and protein

Introns were seen to be spliced in

primary transcripts that were still itn process of being transcribed

Prior to translation, eukaryotic mRNA transcripts can be

processed by the removal of non coding introns, the precise splicing together of the remaining exons, eh addition of a cap at the mrNA 5' end and the synthesis of a poly A tail at its 3' end

In these cases, these DNA rearrangements are

programmed and essential for normal expression of the genes involved

Recently, scientists have discovered that DM1 and DM2 are caused not by changes in the protein products of the DMPK or ZNF9 genes, but by

the toxic effects of their repeat containing RNAs

ENCODE scientists propose that

the transcript be considered the bsid unity inheritance, rather than the DNA from which it is transcribed

If RNAi method can target these specific gene products

they might help us treat cancers that have become resistant to other methods such as radiation or chemotherapy

if sxl is absent

tra pre mRNAs are spliced into such a way that a translation stop codon remains in the mature mRNA Translation of this mRNA results in a truncated nonfunctional protein

When sxl protein is present,

tra pre mRNAs are spliced out prepuce mature mNRAs that are translated into functional TRA protein

each LV region is preceded by

transcription initiation sequences

Through alternative splicing

two peptide hormones with different structures, locations and functions are synthesized from the same gene

Because enhancer interactions often skip the nearest gene promoter

unknown mechanism must exist to allow regulatory elements to target the appropriate gene at the right time and places to allow appropriate gene expression

Although the immune system has many parts

we will focus on humoral immunity which involves the production of proteins called immunoglobulins or antibodies that bind directly to antigens

Transcription factors bound to enhancer and silencer elements are thought to

work by interacting with other transcription proteins located at promoters These interactions result in the formation of DNA loop supt o 1 million base pairs in length

Fire and Mello results

Although they expected that the single stranded antisense RNA molecules would suppress unc-22 gene expression b y being to the endogenous sense MNRA, they were surprised to discover that one injection for double stranded unc-22 RNA was 10 to 100 fold more powerful in repressing expression of the unc22 mRNA They studied the phenomenon further and published their results in Nature They reported that the presence of double stranded rNA acts to degrade the mrnA if the mNRA is scompletelnary in sequence to one strnad of the double stranded RNA Only a few molecules of double stranded RNA are needed to bring about the degradation of large amounts of mRNA

similarities between siRNA and miRNA

Both types of RNA are short double stranded molecules between 20 and 25 ribonucleotides long

pathways an mRNA can be degraded Third, an mRNA may be cleaved internally by an endonuclease, having unprotected ends at which encon claes degradation may proceed

Examples of endonucleolytic cleavages are those that occur during nonsense mediated decay and those triggered by RNA interference Nonsense mediated mRNA decay occurs when translation terminates at premature stop codons Endonucleases attack the mRNA near the stop codon leaving unprotected ends that are then degraded by exonuclease Nonsense mediated decay is an important mechanism for removing mutated mRNA molecules that could, if efficiently translated, result in accumulation of aberrant protein products

pathways in which mRNA can be degraded

First, an mRNA may be targeted for degradation by enzymes that shorten the length of the poly A tail Second, decapping enzymes can remove the 7 methylguanosine cap, which also renders the mRNA unstable Third, an mRNA may be cleaved internally by an endonuclease, having unprotected ends at which encon claes degradation may proceed

steps of the RNAi pathway commonalities

First, siRNA or miRNA molecules associate with an enzyme complex called the rNA induced silencing complex or RISC Second, within the RISC, the shorts double stranded rNA s denature and the sense strand is degrade Third, the rNA/RISC complex becomes a functional and highly specific agent of rNAi, seeking out mRNA molecules that are complementary to the antisense rNA contained in the RISC At this point, RNAi can take one of two pathways

RISC pathway

If the antisense rNA in the RISC is perfectly complementary to the mRNA, the RISC will cleave the mRNA The cleaved mRNA within the RISC is not exactly complementary to the mRNA, the RISC complex stays bound to the mRNA, interfering with the ability of ribosomes to translate the mRNA RNAi can silence gene expression by affecting either mrNA stability or translation

RITS pathway

In addition to repressing mRNA translation and triggering mRNA degradation, siRANs and mirNAS can also repress the transcription of specific genes and larger regions of the genome They do this by associating with a different complex - the RNA induced transcriptional silencing complex RITS The antisense RNA strand within the RITZ targets the RITS complex to specific gene promoters or larger regions of chaotic RITS then recruits chromatin modifying enzymes of these regions These enzyme methylates histones and DNA, resulting in heterochromatin formation and subsequent transcriptional silencing As a result of their effects on chromatin mediated gene silencing, miRNA molecules are thought to be involved in epigenetic phenomena such as gene imprinting and X chromosome inactivation

sxl in females and males

In females, transcription factor encoded by genes on the X chromosome are thought to activate transcription of the scl gene In males, the lower concentration of these transcription factors is not sufficient to activate transcription of sxl As a result of this differentiation regulation, the sxl protein is expressed only in female embryos

pathways an mRNA can be degraded First, an mRNA may be targeted for degradation by enzymes that shorten the length of the poly A tail

In newly synthesized mRNAs, the poly A tail is about 200 nucleotides long and binds a protein known as poly A binding protein The binding of the protein to the poly A tail helps to stabilize the mRNA If the poly A tail is shortened to less than 30 nucleotides, the mRNA becomes unstable and acts as a substrate for exonucleases that degrade the RNA in a 3' to 5' or 5' to 3' direction

There are many ways by which the quantity and activity of a gene product can be regulated

In some cases, the translation of an mRNA can be regulated to produce the correct quantity of protein product In other cases, the stability of a protein can be modulated or the protein can be modified after translated to change its structure and affect its activity

splicing of the CT/CGRP primary transcript thyroid cells

In thyroid cells, the CT/CGRP primary transcript is spliced in such a way that the mature mRNA contains the first four exons only In these cells, the exon 4 polyadenylation signal is used to process the mRNA and add the poly A tail This mRNA is translated into the calcitonin peptide, a 32 amino acid peptide hormone that is involved in regulating calcium

Recent studies are demonstrating that

RNA induced gene silencing mechanisms operate during normal development and control the expression of baterins of ngos involved in tissue specific cellular differentiation

ENCODE experiment demonstrate that

RNA splicing occurs during transcription

In thyroid cells, the CT/CGRP primary transcript is

spliced in such a way that the mature mRNA contains the first four exons only

Such defects in the regulation of RNA splicing are known as

spliceopathies

As a consequence, p53 proteins are

stabilized, the levels of p53 increase and the protein in able to act as a transcription factor

In animal models, siRNA molecules have

successfully treated virus infections, eye diseases, cancers and inflammatory bowel disease

Immunoglobulins are

synthesized by a type of blood cell known as a B lymphocyte or B cell which undergoes development and maturation in the bone marrow immunoglobulin molecules consist of four polypeptide chains, held together by disulfide bonds

Viral infections are an obvious

target and science have had promising results using RNAi in tissue cultures to reduce the severity of infection by several types of viruses such as HIV, influenza and polio

Sex in drosphila is determined by

teh ratio fo x chrosomes to sets of autosomes X:A

One well studied mRNA stability element is

that adenine uracil rich element ARE - a stretch of nucleotides that consist of A and U ribonucleotides

in vitro studies have shown

that each dscam protein isoform rna bind to the same dscam protein isoform but not to others Even a small change in amino acid sequence reduces or eliminates the binding between two dscam molecules

An important example of posttranslational regulation is

that of the p53 protein

splicing of the CT/CGRP primary transcript brain and peripheral nervous system

the CT/CGRP primary transcript is spliced to include exons 5 and 6 but not exon 4 In these cells, the exon 6 polyadenylation site is recognized The CGRP mRNA encodes a 37 amino acid peptide with hormonal activity in a wide range of tissues

After this somatic recombination event

the LV promoter is activated by the nearby presence of the immunoglobulin enhancer result gin transcription of a gene The resulting rNA is spliced to remove the introns between the L and V regions and between the J and C regions After translocation of the mature mRNA, the [polypeptide is processed by removal of the ladder manio acis The rearranged K gene is then matintaine and passed on to progeny of that B cell

The steady state level determines

the amount of mrNA that is available for translation

The changes in the levels and activity of p53 are due to

the combination of increased protein stability and modifications to the protein

Because alternative splicing is far more common in vertebrates

the combinations of protein that can be produced from human genome may be astronomical

The regulatory gene at teh beginning of thsi cascade is

the gene sex lethal, which codes an RNA binding protein

The mechanisms of DNA rearaurants described here

the human K light chain genes also occur during the formation of the gene that encodes another type of light chain gene as well as the gen eth encodes the immunoglobulin heavy chains

When cells suffer DNA damage or metabolic stress

the level of p53 protein increase dramatically in addition, p53 becomes an active transcription factor

proteome

the number of proteins that can organism can make

These data suggest

the potential for large amounts of antisense and overlapping transcription initiation through protein coding and noncoding genes

Scientists are particularly enthusiastic about

the potential uses of RNAi in the diagnosis and treatment of cancers

One of the targets of the sxl protein is

the pre mRNA encoded by the transformer gene

The ultimate end point of gene expression is

the presence of an active gene product

Because of this imprecision

the recombination between any particular pair of LV and J regions still shows considerable variation

Two major types of short RNA molecules are involved in RNA induced gene silencing

the smallinterfing rNAs or siRNAS and the micrRNAs or miRNAS

SXL alters

the splicing pattern of tra pre mRNA by binding to the pre mRNA and altering recognition of splicing signals by the splicing machinery

In these studies

the synthetic siRNA molecules were applied to the surfaces of mucous membrane or into eyes, brian tissues or the lower intestine

The human k light gne is assembled during

B cell development from multiple dNA regions located along chromosome 2

Often the fetal splicing patterns occur in

DM1 and DM2 patients by the normal transactions ot adult spelling patterns are lacking

DSX F vs DSX M

DSX F represses the transcipton fo genes whose proudcts contorl male sex deelopment, whereas DSX M activates teh tranciton fp gens whose proudcts contorl male sexual development In addition, DSX M represses the transcription of genes that control female gsexual development

Given the existence of alternative splicing, how many different polypeptides can be derived from the same pre mRNA? Research on the dscam gene in drosophila melanogaster

During development, cells of the nervous system most accurately connect with one another Even in drosophila, with only about 250000 neurons, this is a formidable task Neurons have cellular processes called axons that form connections with other nerve cells The drosophila dscam gene encodes a protein that guides axon growth, ensign that neurons are correctly wired together In dscam pre MRNA, exons 4,69 and 17 eac consist of an array of possible alternatives These are spliced into the mature mRNA in an exclusive fashion, so that each exon is represented by no more than one of its possible alternatives There are 12 alternatives to exon 4, 48 for exon 6, 33 for exon 9 and 2 for exon 17 The number of possible combinations could be forme suggests that theoretically dscam gene can produce 38016 proteins

Within the 80 percent of the genome once considered "junk DNA"

ENCODE has created thousands of enhancers promoters, sequences encoding RNA transcripts and sites of specific chromatin modifications

By examining chromatin features such as DNase hypersensitivity and histone modifications that are charceirisc of DNA involved in regulatory factor binding

ENCODE researchers discovered more than 1 million bidding sites for gene regulation proteins, including transcription factors that bind to enhancers and promoters

enhancer RNA

Enhancer elements often bond tain bonda finde rotmer elements that initiate transcription from the enhancer element and extend outward for several kilobases The most active enhancers in a cell type produces the most enhancer RNA

When cells are stressed

Mdm2 and p53 are modified by phosphorylation and acetylation, resulting in the release of mdm2 form p53

DNA rearrangements

One type of arrangement results in the creation of new genes from gene fragments located at various positions in the genome Another type of rearrangement result isn't a switch in expression of genes as a result of a reaction or DNA copying event

What are the mechanisms by which mRNA stability can be regulated?

One way that an mRNA's half life can be altered is through specific RNA sequence elements that recruit degrading or stabilizing complexes

para gene

Para not only has at least 6 sites of alternative splicing, leading to 48 possible different mRNA variants, but also undergoes another post translational modification called RNA editign, at 11 positions RNA editing involves base substitutions made after transcription and splicing With both alternative splicing and editing, the para gene can theoretically produced more than 1 million different transcripts

studies phase II and III

Phase II and III efficacy trials have begun to test the use of siRNAS directed again the product of the vascular endothelial growth factor gene, as a drug to treat age related macular degeneration

RNAi mediated gene silencing is relatively

specific and inexpensive and it allows scientists to rapidly analyze gene function

studies phase I

Research into RNAi pharmaceutical has made rapid progress in the phase I toxicity stages of the first human clinical trials have been completed

source of siRNA molecules research lab

Scientists are only able to introduce double stranded RNAs into cells for research or therapeutic purposes In the cytoplasm, double stranded RNA molecules are recognized by an enzyme complex known as Dicer and is cleaved by dicer into SiRNAs

The functional relevance of this enormous amount of trwanciptin throughout the genome is intriguing but onctrovsersal

Some scientists argue that it may be simply "noise" without significance to cellular phenotype If this is the case, substantial regulation of gene expression must occur at post transcriptional levels in eukaryotes

p53 protein

The p53 protein is essential to protect normal cells from the effects of dNA damage and other stresses It is a transcription factor that increases the transcription of a number of genes whose products are involved in cell cycle arrest, DNA repair and programmed cell death

RNAi technology allows investors to

specifically create single gene defect without having to induce inherited gene mutations

DM1 and DM2 repeat containing RNAs

These RNAs accumulate and form inclusions within the nucleus

transformer gene

This pre mRNA is transcribed in both male and female cells

RNAi pathways are also able to repress transcription in indirect ways

Transcription factor mRNS are frequent targets for RNAi induced silencing When the levels of specific transcription factors are reduced in a cell, transcription of genes whose expression depends on these factors i slap repressed

Approximately 30000 of the promoter regions found by examining chromatin features were located within

protein coding genes and 17000 of these were oriented in an antisense direction

myotonic dystrophy

provides an example of how defects in alternative splicing can lead to a wide range of symptoms Myotonic dystrophy is the most common form of adult muscular dystrophy, affecting 1 in 8000 individuals It is an autosomal dominant disorder that occurs in two forms DM1 and DM2 Both of these diseases show a wide range of symptoms, including muscle wasting myotonia (difficulty relaxing muscles), insulin resistance, cataracts testicular atrophy, behavioral and cognitive deficits, cardiac muscle problems and hair follicle tumors

In most somatic cells, as well as in germ cells, these DNA regions are

recognized as shown

It does this by

recognizing molecules on the surface of these forigen substances and by physically bind it to them in a kind of lock and key configuration - a phenomenon referred to as antigen recognition

First discovered in plants, short RNA molecules are known to

regulate gene expression in the cytoplasm of plants, animals and fungi by repressing translation and triggering the degradation of mrNAs This form of sequence specific post transcriptional regulation is known as RNA interference or RNAi

In cells not growing or

require low levels of gene expression, specific complexes bind to the AREAS of these mRNA molecules, bringing about shortening of the poly A tail and rapid mRNA degradation

Failure to control the spacing the dsx transcript in a female model

results in the default splicing of the transcript in a mal model, leading to the production of a male phenotype

These RNAs are transcribed from

sequences within the introns of teacher protein coding genes or form their own promoters located within noncoding regions of the genome

Mutations that affect regulation of splicing contribute to

several genetic disorders

Three major genes in this pathway are

sex lethal, transfer and doublesex

RNA induced gene silencing

short RNAs in the nucleus alter chromatin structure to repress transcription

in vivo studies

show that cells expressing the same isoforms of dscam interact with each other Therefore, it appears that the diversity of dscam protein isoforms in neurons prides a kind of molecular identity tag for each neuron, helping to guide it the correct target and preventing the tagline of extensions from different neurons

Over the last few years, scientists have discovered that

significant amounts of eukaryotic genomes are transcribed by RNAP II into rNA products that contain no open reading frames and are not translated into protein products

Chromosomal ratios are intrepid by a

small number of genes that initiate a cascade of splicing events, resulting in the production of male o female somatic cells and the corresponding male or female phenotypes