There are listed things to do.
1. Please write the discussion part, it should be comparing my results (Provided in the lab), and to others (Journal Articles), This section should be 1000 words. = Do not for get the in text citations.
2. Please add the in text citations in the introduction, part written.
3. After you done for the above two things, write a 200 words abstract, for the whole lab, then add a paragraph to the conclusion.
ABSTRACT
INTRODUCTION
For mutation to occur in CHMP2B gene (a form of gene which has homologue CG4618 gene) ,
it is usually as a result of the chromosomal 3-linked “fronto-temporal dementia”(FTD3). This
mutation (of CHMP2B gene which do act as the acceptor site for “Gene Splicing for the sixth
exon”),do leads to the “35 aa at terminus of carboxyl group” of protein, to become truncated. As
a result of this mutation(in CHMP2B gene) , diverse “cellular physiological havocs”( like
impairment of the “endosomal-lysosomal pathway” and severe “accumulation of ubiquitin” ,
“autophagosomes” , “p62-positive inclusions”, “receptor misregulation” and “axonal swelling”)
expressed and observed in primary nerve cells, different cell lines, animals, human and even fruit
flies(Drosophila melanogaster)
Scientifically, due to the overexpression or knock-outs of the wild type gene (CHMP2BWT) in
the genome animals like mice and the fruit flies inclusive, there has been
understatement/hypothesis regarding the fact that, any defects that are as a result of the mutant
gene(CHMP2BIntron 5), such defects will certainly have a “Gain of Function Effect”(this is a
genetical effect which helps in enhancing diverse biological functions of genetic products.
In the eye of fruit flies(Drosophilia), when the CHMP2BIntron 5 become expressed by the GMRGAL4( a driver which that helps in ) the Toll Receptors and the Signalling Pathways will be
misregulated.
Due to the fact that the expressed receptor and its activity are being regulated by
Spatiotemporary Method, we have to make a researches so as to determine whether the
disruptive effect (as a result of CHMP2B mutant gene;CHMP2BIntron 5) during the different
developmental stages of the Drosophila eye, will result into varieties of receptor misregulation ,
also phenotypical differences. Therefore, ey-Gal4 protein act as driver for the Upstream
activating sequence(UAS) a system of expression, which facilitate the control of the “transgene
spatiotemporal expression”. At the embryonic stage of Drosophila, the expression of the
ey>Gal4, become initiated until reaching the larva cells of the eye disc which will precedes the
morphogenetic hole/fissure formation, as result of specified definition to determine and
differentiate photoreceptor cells of Drosophila in different case scenario.
Furthermore, after the “first initiation of ey-Gal4,” the GMR-Gal4 become then become
expressed in the second initiation process, which lead to the cellular development of the eye cells
that are posteriorly located to the morphogenetic hollow/fissure. Also, the expression of “UASFlag-CHMP2B gene complex” (which is usually driven by the ey-Gal4) is very significant in
determining the effect of “dysfunctionated ESCRT III complexes”. Hence, this dysfunction can
finally be traced down to the CHMP2B gene which enhances the regulation of “Notching
Activity/Signaling” in the mechanism of eye morpho-genetic process.
MATERIALS AND METHODS
Fly-Base function is used as a database, selected a sub-deficiency
(Df(2L)BSC183/CyO) and two mutation genes(Heat Shock Protein 60C (Hsp 60C), Blue cheese
(bchs)) based on the position found within our original deficiencies(Df(2L) ED284). All
Drosophila strains were ordered from “Bloomington Drosophila Stock Centre”( Indiana, USA)
except for GMR-Gal4;UAS-CHMP2BIntron 5/CyO,GMR-Gal4/CyO and wild-type(WT)were
provided by Dr Sean T . Sweeney at the University of York. The full detail of fly stocks that we
used. Moreover, our flies that we ordered provide a balancer chromosome such as CyO or SM6a.
The balancer chromosomes are responsible for maintaining the homozygous lethal
mutation to avoid being lost from their population and prevents multiple alleles found on the
same chromosome, avoiding separation by meiotic recombination. Additionally, many balancers
carry a recessive visible mutation which will be helpful to identify and select from the crosses.
For example, Cy-O was likely most popular used as second chromosome balancer which the fly
will show the curly wings.
Fly Maintenance:
Fruit flies(Drosophila) were raised on a standard yeast diet at temperature of 25OC ,and stock
were flipped into new food vial once a week. Fly food was ordered from a biological food
company.
Setting Up Crosses:
The virgin Fruit flies used in this experiment were GMR-Gal4: UAS-CHMP2BIntron 5/CyO,
GMR-Gal4/CyO and the wild type(CHMP2BWT). In order to collect virgin flies , selection is
done for the vials that had many pupas(checked for the one with blackly-coloreds; meaning it
will be hatched soon) and then flipped them into new food vials. The old vial was left(which is
containing pupas) for a period of 5-6hours at 25oC before then collecting the flies from these
vials. Although , assumption was made, that these flies were still virgins because they were still
in their young stage. Then, the virgin flies were transferred from the old vials into the empty vial;
an icebox is then used to immobilise them.
During the period when they undergo immobilization, they become transferred into a small petri
dish containing ice, for easy identification of the female flies that will be done under a
microscope, before then placing them into empty vials , which is of about 3-5 virgin females. In
otherwise, for the collection of male flies, there is no need for the male to be of virginity
standard.
Therefore, one vial is chosen in our fly stocks from original deficiency, sub-deficiency and
interested gene vials. Then, identification of the male flies is also carried out microscopically, by
making the flies to be immobilizing.
Crosses of Drophila Flies:
Crosses:
For genetic interaction studies, GMR-Gal4:UAS-CHMP2BIntron 5CyO, GMR-Gal4/CyO and the
wild-type(CHMP2BWT) virgins were crossed with sub-deficiency and mutant genes. Setting up
by putting 3-5 virgin females with three male flies per cross, and every cross had three repeat
crosses to set up. These crosses took 10-12days to hatch. However, the parents need to be
separated into new food vials when they already have larvae. Consequently, it was carried out to
distinguish the parents from their offspring and reproduce the number of new progenies from the
original parents.
Fly Analysis and Fly Imaging:
So as to quantify the mutant gene CHMP2BIntron 5 for eye phenotype, offspring from each cross
were put onto the ice petri dish from which the straight-winged flies were selected to analyse in
all crosses. Arbitrary classification was done for the eye phenotype, based on size of black spots
surface of their eyes. Therefore, there is a need for eye phenotype to be categorized into 4
groups: non(normal eyes), low (+), medium(++) and high(+++). Low severity was charactetised
as a few black tiny spots on their eyes( with < 15% by total coverage). The medium severity was
characterized as a few tiny black spots with/without larger black spots on their eyes(15-45% by
total coverage), while the high severity contained a large proportion of black spots more than a
half of their eyes. An example of fly eye images in each categorized provided. However, we
chose left eye side to analyze in every cross. After the flies analysis, a microscopic camera called
the “Dino-Eye,” was used to take picture of their eyes.
Statistical Analysis:
Chi-Square tests was used here, to calculate the P values so as to find the significant difference
between the control and our experimental groups in the R program. On the other hand, R
program is used to create the bar chart which depicts the illustration of the data for the fly
analysis.
Design Primers:
Provision of PCR was done, in order to confirm the region of the original
deficiency(Df(2L)ED284). The PCR process was then carried out by using “3-D confirmation
method”; where-which “FASTA sequence of Df(2L)ED284” was used as a template, provided
that the available two specific primers are the (i) “Custom A (TGCTGAGCTATTGTCCTCCG)”
and (ii) “Custom B (GGTGCGGGTGTGGATTAGAT)”. Also, the PRY4 Sequence
(CAATCATATCGCTGTCTCACTCA),W7500D (GTCCGCCTTCAGTTGCACTT) and
W11678U(TCATCGCAGATCAGAAGCGG).
Setting up PCR:
Extraction of the total of DNA from a whole fly body was done using “Squishing buffer”. PCR
was then set to into three different tubes including; (1) PRY 4 with Custom A, (2) PRY 4 with
Custom B and (3) W7500D with W11678U. Preparation of the PCR mixture in each reaction,
including One Taq 2X Master Mix with Standard Buffer, forward and reversed Primer DNA
Template, Nuclease-Free Water.” Then, PCR performed by the use of “Biorad PCR machine”, in
which two of “” based on the Tm point of each of the primers. However, the reaction of
thermocycler programs the PCR setting, thermocycler program, Squishing and Standard Buffer
preparation will be furtherly explained.
Electrophoresis:
The analysis of PCR products are determined by electrophoresis. About 1% of agarose gel is
performed with SYBR Safe in a BioRad chamber exactly at 100v for 45mins. Then examination
of the electrophoresis gel imagery is done through a “UV trans-illuminator” by the aid of
“Biorad gel-doc go-System”.
RESULT:
The genetic screening done is used to identify the loss of the Df(2L)ED284/SM6a;
which is not a modifying agent of CHMP2BIntron 5 neurotoxicity.CHMP2BInron 5is used as model
of flight for the FTD-associated mutant of CHMP2B, which is known to be driven by the action
of “Gal4-UAS complex” system. This latterly then results into degeneration of phenotypic traits
that are responsible for black eyes pigmentation in the flies eye. With this, the expression of the
phenotypic character is confirmed in the flies exhibiting CHMP2BIntron 5 gene; but no phenotypic
character expression in Df(2L)ED284/SM6a fly. Therefore ,investigation is done to affirm that
Df(2L)ED284 is whether a candidate modifier of CHMP2BIntron 5 toxicity, by crossing the
Df(2L)ED284/SM6a together with GMR-Gal4; while the UAS CHMP2BIntron 5 is under the
control of Df(2L)ED284/+.
1). Flies Eye Imagery at Low Resolution
2) Flies Eye Imagery at Medium Resolution
3) Flies eyes with a High Resolution
4) Flies Normal Eye Imagery Resolution
Hence, this result shows that the offsprings of the experimented flies did not show a significant
enhancement of CHMP2BIntron 5 toxicity compare with CHMP2BIntron 5/+.
Further characterization was done for the “Structural and Functional,” aspects of the
e-y>CHMP2BIntron5 phenotype. The longitudinal sections of the wild-type eyes, showed
arrangement of photoreceptor cell nuclei with the normal curvature for the eye in the retina. Ey>CHMP2BIntron5 flies showed eye defects, in the patterning and curvature mechanism of the
eye. However, the eye might be enlarged but has “ommatidia”(additional eye units) which
appears to have cells for normal photoreceptor, which is indicated by a localized pattern of a
comparable rhodopsin and the content found in the flies that contains ey> to those that have wild
type gene. Furthermore, the retinal ey>CHMP2BIntron5 always appeared much more better than
GMR>CHMP2BIntron5 retina. When this happens ,it apparently shows degeneration in the
retina, due to the fact that it shows the defects depth in the retina layer of the flies eyes, that has
become degenerated. Hence , the density off the photoreceptor nuclei is due to the reduced
rhodopsin content.
Images showing the normal(wild type),enlarged and ablated(driven by ey-Gal4)
A countercurrent distribution phototactic assay was also carried out in order to,
determine whether CHMP2BIntron5-mediated structural defect (found in the eye) are associated
with physiological defects. The wild-type flies are were seen showing a strong preference for
light; wherewhich about 96% that are favored for illumination are 2times ≥ in 3 trials. Severe
defects in phototactic index is usually shown in GMR>CHMP2BIntron5 flies,in which retinal
degeneration. In related to wild-type and ey>CHMP2BWT flies, the mutant flies are shown to
also have phototactic. According to this finding, the expression of the gene CHMP2BIntron5 helps
to determine the physiological and structural defects that has occur in Drosophila eyes. Also, the
mutant gene(CHMP2B Intron5 )for eye phenotype, has a similarity to a condition of
tumorigenesis(inflammation that affect the eye)in the flies that shows Notch Activity and those
flies have mutated ESCRT complexes. Hence, investigation was carried out , in order to confirm
if any eye deformities in flies that has mutated gene (ey>CHMP2BIntron5) was as a result of “UpRegulation mechanism” that enhances Notch Activity.
When the longitudinal section of the mutant eye gene become enlarged punctae via
immunolocalization, it helps to show the rate at which Notch accumulation in the wild type gene
for Drosophila eyes.For us to be able to identify the vesicles. Enlarged positive forms of
endosomes(especially the Rab5 and Rab7) enhances Notch positive, which colocalized with the
gene for wild type eyes.
Also, the build of of the Notch inside the endosomal components was checked for
genetically , if it facilitate Up-Regulation mechanism resulting into signaling of the Notch. The
Notch signaling enhances the genes which in their promoter entails the element for the Notch
Activity.
So as to discover whether possibly the changes which occurs in repressors, has
caused the elevation in the signaling pathway of Notch; the level of numbness was examined if
its actually a typical negative gene regulator that controls signaling of Notch. In summary, it was
indicated that the mutant gene expression (CHMP2BIntron5)caused the accumulation of Notch
in signaling and endosomes that are elevated and enlarged respectively.
The R-Studio Result Data
The illustration above was as a result of the analysis of experiment done; with the
aid of R-Studio Application. In the representation, the relationship among the three genes Sn(
“Sn x 6/Intron5”), BSC111(DF(2L)BSC111/Intron5), BSC200(DF(2L)BSC2OO/Intron5), were
shown.
The R-Studio analysis helps to show the “extent of enhancement of the phenotypic
character” at which each of the genes (Sn,BSC111 &BSC200) are being expressed in
Drosophila Flies. Flies that has “Sn gene” are seen to have the “highest percentage for the
enhancement” of the phenotypic character, compares to flies with BSC111 and flies with
BSC200 expression. Apparently based on this result obtained, there is a great tendency that those
flies having highest percentage of enhancement of the phenotype(ey>CHMP2BIntro5), will be
prone to having “structural defects”. These defect can likely be “photactic defect or retinal
degeneration”, in the eye of Drosophila flies.
DISCUSSION
CONCLUSION:
Conclusively, the obtained result help to show and support the fact that the
disruption of both pathways for endosomes and lysosomes“endosomal-lysosomal pathway”was
caused by the “CHMP2BIntron5mutation.” Different types of receptors and their signaling
mechanism are usually misregulated due to the spatiotemporal attribute that results from
“CHMP2BIntron5gene expression”. Similarly, just like the ESCRT mutants, the “endosomallysosomal pathway” blockage is “CHMP2BIntron5 mediated”, so as to form several
transmembrane proteins.
Regardless, the results obtained from the presently definitely contributed to how cellular and
molecular defects were understood.The strongest phenotype determined is due to the most active
receptor that is sensitive to “misregulation” whenever the “CHMP2BIntron5” gene is expressed.
However, identification of additional receptors that are also being misregulated by
“CHMP2BIntron5” in the eyes of Drosophila flies, likewise in their central nervous system neurons
also.
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