Identi’ication and Characterization of the Drosophila retinal degeneration B 205 (rdgB205) Gene as a Phosphoinositol Transfer Protein. ABSTRACT The retinal degeneration B 205 (rdgB205) gene in Drosophila causes retinal degeneration of photoreceptor cells in the eye in response to light. We aimed to identify and characterize a mutation in the phosphatidylinositol transfer protein (PITP) domain as a factor resulting in retinal degeneration in rdgB205 mutants. Chromosome and deletion mapping resulted in the determination of the rdgB205 gene being located in the 12BC region of the X chromosome. A complementation test resulted in the identiFication of rdgB205 as an allele of the known gene rdgB. Mutations to the genes norpA and ninaE caused the suppression of the rdgB205 phenotype, suggesting that rdgB205 functions downstream of these two genes in the phototransduction cascade. The PITP domain was ampliFied using PCR and the product was electrophoresed to ensure that the selected 567 base pair fragment of the 834 base pair rdgB205 gene was properly cloned. Sequencing of this fragment resulted in the discovery of a point mutation at base pair 175 which caused adenine to be replaced by guanine, resulting in a nonconservative, missense mutation at amino acid 58 where glutamic acid was replaced by glycine. We also compared rdgB in Drosophila to the rat brain PITP domain and determined the 58th amino acid was not evolutionarily conserved. Immunolocalization in Drosophila head sections revealed that the location of expression between wild type and rdgB205 mutants had not changed, suggesting a loss of function producing the rdgB205 phenotype rather than a lack of expression. These results suggest that rdgB205 functions as a PITP domain due to its allelism with rdgB and similar expression of their respective proteins.
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Identi'ication and Characterization of the Drosophila retinal degeneration B 205 (rdgB205)
Gene as a Phosphoinositol Transfer Protein.
ABSTRACT
The retinal degeneration B 205 (rdgB205) gene in Drosophila causes retinal degeneration
of photoreceptor cells in the eye in response to light. We aimed to identify and characterize a
mutation in the phosphatidylinositol transfer protein (PITP) domain as a factor resulting in
retinal degeneration in rdgB205 mutants. Chromosome and deletion mapping resulted in the
determination of the rdgB205 gene being located in the 12B-‐C region of the X chromosome. A
complementation test resulted in the identiFication of rdgB205 as an allele of the known gene
rdgB. Mutations to the genes norpA and ninaE caused the suppression of the rdgB205 phenotype,
suggesting that rdgB205 functions downstream of these two genes in the phototransduction
cascade. The PITP domain was ampliFied using PCR and the product was electrophoresed to
ensure that the selected 567 base pair fragment of the 834 base pair rdgB205 gene was properly
cloned. Sequencing of this fragment resulted in the discovery of a point mutation at base pair 175
which caused adenine to be replaced by guanine, resulting in a non-‐conservative, missense
mutation at amino acid 58 where glutamic acid was replaced by glycine. We also compared rdgB
in Drosophila to the rat brain PITP domain and determined the 58th amino acid was not
evolutionarily conserved. Immunolocalization in Drosophila head sections revealed that the
location of expression between wild type and rdgB205 mutants had not changed, suggesting a
loss of function producing the rdgB205 phenotype rather than a lack of expression. These results
suggest that rdgB205 functions as a PITP domain due to its allelism with rdgB and similar
expression of their respective proteins.
INTRODUCTION
The retinal degeneration B 205 (rdgB205) gene in Drosophila encodes a
phosphatidylinositol transfer protein (PITP) that is essential to proper functioning of the
phototransduction cascade and prevention of retinal degeneration (Milligan 1997). The
Drosophila eye is composed of about 800 ommatidia, which contain eight photoreceptor cells
each. Photoreceptor cells contain a cell body and a rhabdomere, which is Filled with the
membrane protein rhodopsin. The rdgB205 protein is localized adjacent to the rhabdomeres, in
the subrhabdomeric cisternae (SRC) (Vihtelic 1993). Retinal degeneration is caused by the death
of these photoreceptor cells that will lead to blindness in the Flies.
In Drosophila, several genes involved in the phototransduction cascade are known to
cause the failure of photoreceptor cells leading to retinal degeneration. One of these genes, ninaE,
is characterized by a failure to produce functional rhodopsin in the photoreceptor cells R1-‐6
(Vihtelic 1991). Another gene, norpA, encodes the phototransduction effector molecule
phospholipase C (VIhtelic 1991). Suppression of the rdgB phenotype in the presence of a
mutated ninaE or norpA gene suggests that rdgB functions downstream of phospholipase C in the
phototransduction cascade (Vihtelic 1993). Further evidence suggests that rdgB does not
function directly downstream of phospholipase C, but instead is mediated by a mechanism
related to the regeneration of phosphatidylinositol 4,5-‐diphosphate (PIP2) (Vihtelic 1993). The
current proposed mechanism involves the hydrolyzation of PIP2 into inositol 1,4,5-‐triphosphate
(IP3) and diaglycerol (DAG), followed by a multistep process that results in DAG being converted
back into PIP2 (Wang and Montell 2007). During this multistep process, PITP (encoded by rdgB)
promotes the transfer of phosphoinoside (PI) from the SRC back into the rhabdomeres for
phosphorylation of PI into PIP2 (Wang and Montell 2007). A lack of being able to transport PI
back into the rhabdomere for phosphorylation is a proposed effect of the rdgB mutation leading
to retinal degeneration. This model is consistent with rdgB’s localization to the SRC membrane,
but PI transfer has not been proven to be the critical activity in producing rdgB-‐type retinal
degeneration (Paetkau 1999).
Since rdgB encodes an integral membrane protein that may bind calcium (Vihtelic 1993),
it has been proposed that the gene may play a role in maintaining the functionality of the
rhabdomere by transporting membrane proteins to the rhabdomere and acting as a source of the
Ca2+ release following phototransduction (Vihtelic 1993). The functionality of the SRC
membrane as an intracellular Ca2+ store has led to experimentation that suggests that either the
rdgB protein may alter intracellular Ca2+ levels or is regulated by Ca2+ (Paetkau 1999). Further,
a mutation to the light activated calcium channel called trp resulted in slowed rdgB degeneration
(Paetkau 1999). Another study showed that blocking the calcium channels slowed the rdgB-‐type
degeneration, suggesting that an increase in intracellular Ca2+ via the calcium channels may lead
to rdgB-‐type degeneration (Sahly 1992). This is in contrast to inaC (encoded by protein kinase C,
PKC), which does not slow rdgB-‐type retinal degeneration (Paetkau 1999). This data supports
that PKC is not directly upstream of rdgB and that Ca2+ entry into photoreceptors stimulates
rdgB degeneration (Paetkau 1999).
Our study aims to identify and characterize the function of an EMS-‐induced mutant
allele of rdgB, rdgB205. We know that rdgB-‐type retinal degeneration is involved with the PITP
domain, so we targeted that area of the rdgB gene for examination. PCR ampliFication of a 567
base pair fragment of the PITP domain of rdgB205 as well as DNA sequencing allowed for
comparison to the wild-‐type rdgB gene resulting in the discovery of a nonconservative missense
mutation in the amino acid sequence. The mutation was compared with the rat brain PITP
domain and was determined to be not evolutionarily conserved. Immunolocalization, which
showed the location of the protein expression in the mutant rdgB205 head section versus a wild
type, showed that the rdgB205 protein was expressed in the same location as wild-‐type, but
suggests a loss of functionality due to the mutation. RdgB205’s allelic nature to rdgB as well as its
sequence identity with the rat brain PITP (Vihtelic 1993) serve to suggest its function as a PITP.
MATERIALS AND METHODS
Deep Pseudopupil Analysis: By placing Flies under a light microscope, it is possible to
fully examine the individual rhabdomeres within the ommatidia of the compound eye. The deep
pseudopupil itself represents virtual images of the rhabdomere patterns from several ommatidia
that are observed head on. A wild-‐type deep pseudopupil exhibits a trapezoidal shape, composed
of seven dots which indicate seven of the eight individual rhabdomeres of the ommatidia, one of
which always being distal to the observer and therefore unable to be seen. Deep pseudopupils
showing signs of retinal degeneration have blurry, scattered, dull, or fewer than seven
rhabdomeres visible.
Chromosome Mapping: rdgB205 females were mated to balancer males (X/Y; SM1/Sco;
TM2/Sb). After 14 days, the F1 generation was examined for phenotypic markers Scutoid (Sco)
and Stubble (Sb), which were then examined for retinal degeneration via deep pseudopupil
analysis.
Deletion Mapping: rdgB205 males were mated to deFicient bar eye females (Df/FM6;
+/+; +/+). Four different deFiciencies were utilized: Df 1-‐ 966, breakpoints 11C – 11F; Df 2-‐ 967,
Transfer Protein Domain of Drosophila Retinal Degeneration B Protein Is Essential for
Photoreceptor Cell Survival and Recovery from Light Stimulation
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Drosophila retinal degeneration B Suppressors.
Sahly, I., S. Bar Nachum, E. Suss-‐Toby, A. Rom, A. Peretz, T. Byk, Z. Selinger, B. Minke. 1992.
Calcium channel blockers inhibit retinal degeneration in the retinal-‐degeneration-‐B mutant of
Drosophila.
Vihtelic, T.S., D.R. Hyde, and J.E. O’Tousa. 1991. Isolation and characterization of the Drosophila
retinal degeneration B (rdgB) gene.
Vihtelic, T.S., M. Goebl, S. Milligan, J.E. O’Tousa, and D.R. Hyde. 1993. Localization of Drosophila
retinal degeneration B, a membrane-‐associated phosphatidylinositol transfer protein.
Wang, T., C. Montell. 2007. Phototransduction and retinal degeneration in Drosophila.
Whaley, M. 2014. Laboratory Manual: Classical and Molecular Genetics
Grant: Link more experiments into the discussion and in future experiments. Beefs it up and gives it validity.
1) Abstract -‐ molecular defects -‐ nucleotide changes in the PITP domain?, Amino acid changes? Non-‐conservative changes? Characterize by what our mutant actually is.
2) USE missense -‐ its there it just cant carry out function, loss of function.
3) ASK: Abstract: molecular defects: nucleotide changes in the PITP domain that would
result in alterations to the amino acid sequence, causing conservative/non-‐conservative
changes to the protein. Conservative is more important? Should we only be looking for
conservative since it is the part that clearly has a close like to effect?
4) We know that the mutation doesn’t affect expression location due to IF just the function.
Propose that the folding is changed? Glutamic acid → glycine (negative to polar
uncharged) base pair 175. Altering the binding of PI or active site of transfer.