This laboratory report summarizes an experiment exploring RNA splicing in Drosophila melanogaster. Genomic DNA and total RNA were extracted from fruit flies and used to study the rngo gene. PCR and RT-PCR were performed on the genomic DNA and cDNA samples. The genomic PCR product was cloned and sequenced. Bioinformatics analysis showed the genomic sequence was longer, containing introns absent from the cDNA, indicating splicing of the rngo pre-mRNA. Future work could investigate other splicing sites and homology to human genes.

1. Simon Fraser University
Laboratory report for MBB 308
Molecular Biology and Biochemistry Laboratory
Its splice to meet you Drosophila
melanogaster
Author:
Elijah Willie
301193627
WEDNESDAY 12-C
Supervisor:
Dr. Peter J unrau
Co-Supervisor:
Dr. Stephanie Vlachos
December 12, 2016
EW
FACULTY OF MOLECULAR BIOLOGY AND BIOCHEMISTRY

2. 1 Introduction
Splicing, or the art of editing pre-Messenger RNA by removing non-coding parts(introns)
and joining together the coding parts(exons) to form a mature Messenger RNA, is one of
the most useful processes for most, if not all eukaryotic cells organisms. This process is
used to regulate gene expression levels of certain genes in parts of an organism. Drosophila
melanogaster, the fuit fly, has over the past few decades become a very versatile organism used
for modelling problems in molecular biology. This is due to the fact they can be produced in
excess with little monetary costs, possess a short life cycle, and can be genetically modified
in many ways.
In the span of five weeks, we will be extracting DNA and RNA from Drosophila melang-
onaster, and by appealing to molecular biology methods such as PCR & RT-PCR, we will
be exploring RNA splicing in Drosophila, by studying primers that maps to the rings lost
(rngo) gene of Drosophila melangonaster. The rngo gene performs molecular functions such
as aspartic-type endopeptidase activity, proteasome binding, ubiquitin binding, and the bi-
ological function of female germline ring canal formation. During oogenesis in Drosophila
melangonaster, there are 16 germline cells that in coalition helps to form a cyst and stay
interconnected.[2] This ring canal that is formed serves to allow for cytoplasmic transport
of proteins, messenger ribonucleic acids, and yolk components from the nurse cells into the
oocyte.[2]
2 Materials and Methods
The materials and methods were followed as described in the MBB 308 fall 2016 laboratory
manual[1](pp 64-69, 71-77, 78-81, 82-85, 86-90). The QIAprep Miniprep Kit was used for
purification during the course of this experiment. The Qiagen DNeasy DNA isolation kit,
and the Illustra RNAspin Mini RNA Isolation Kit was used for extracting DNA and RNA
respectively. Nano Drop readings were taken throughout this experiment for quantification
1

3. of DNA and RNA concentrations as well as protein, and salt contamination. All samples
that required sequencing were sent to the GENEWIZ sequencing facilities.
• Isolation of genomic DNA and total RNA from Drosophila melanogaster.
Adult flies were obtained from teaching faculty, and their genomic DNA, and total
RNA were extracted. Nano Drop readings were taken for the extracted genomic DNA
and total RNA. Extracted genomic DNA were stored on ice, while extracted total RNA
were stored overnight in liquid nitrogen (-80 ◦
C).
• Formaldehyde gel analysis, PCR of gene-specific fragment from genomic
DNA; generation of of cDNA via RT-PCR
A formaldehyde gel analysis was done on the previously extracted total RNA sample
to check it quality. To generate cDNA, a Reverse Transcription(RT) reaction was set
up by diluting 1 µl of total RNA template (1193.9 ng) in to a final volume of 26.25 µl
using RNase-free ddH2O. A RT master mix using 5X buffer ABM, dNTP mix (10 mm),
Oligo dT primer (10 mm, 20-nt long), and RNaseOFF Ribonuclease inhibitor (40 u) in
a total volume of 13.6 µl was generated by the teaching faculty and the diluted total
RNA template was added to this mix. Two separate reactions was then setup including
a control using aliquots from the combined RT master mix and the diluted total RNA.
Reverse Transcriptase (1 u) was added to non control sample, and 1 µl RNase-free
ddH2O was added to the control sample. These samples were then incubated (42◦
C)
for 50 minutes, and heat inactivated (85◦
C) for 5 minutes. Following this, four parallel
PCR reactions were setup, two controls, and one for each the genomic DNA and total
RNA. For this, a master mix was generated as well with the following components:
10X PCR buffer (100 mm Tris − HCl pH 8.3 @ 25◦
C, 500 mm KCl, 15 mm MgCl2),
10X dNTPs, Rngoe2 primer (10 µm), and Rngoe2R primer (10 µm) for a total volume
of 60 µl. 15 µl of this mix was then added to each of the four parallel PCR tubes. For
the genomic PCR reactions, 100 ng of genomic DNA (water for control), Taq DNA
Polymerase (3 u), and ddH20 for a final volume of 50 µl for each of the reactions. For
2

4. the RT PCR reactions, 5 µl of RT reactions (both control and non control), Taq DNA
Polymerase (3 u), and ddH20 for a final volume of 50 µl for each of the reactions.
Both the genomic and RT PCR reactions were subjected to the thermocycler with
initial denaturing temperature of 95◦
C for 3 minutes, 95◦
C for 30 seconds, annealing
temperature of 55◦
C for 30 seconds, extension temperature of 72◦
C for 60 seconds.
This was repeated for 24 cycles. After staying at temperature of 72◦
C for 10 minutes,
the samples were incubated overnight at 4◦
C.
• Gel Electrophoresis of genomic PCR,RT-PCR products, Purification of
gene-specific fragment from genomic PCR; cloning of genomic PCR into
pGEM-T Easy vector.
A 0.1% agarose gel was run for analysis of the success of the genomic and RT-PCR
reaction products. The genomic and RT-PCR reaction products were then purified
and their Nano Drop readings were taken. A ligation reaction for the genomic PCR
reaction was setup (partner performed a similar one for RT-PCR) with the following
conditions: 5X ligation buffer, pGEM-T Easy vector (50 ng), genomic PCR product
(25 ng of ), T4 DNA Ligase (3 u), ddH2O for a total reaction volume of 10 µl. This
ligation reaction was then transformed into JM109 competent cells, streaked on to
two LB/amp/X-Gal/IPTG plates (100 mm IPTG, 20 mg ml−1
), and left for growth
overnight.
• Purification of of pGEM-T Easy genomic PCR, diagnostic restriction digest,
and gel electrophoresis cloned genomic product and cycle sequencing of
genomic clone (a digested RT-PCR sample was obtained from a partner.)
Two white colonies, one from each of the streaked plates from the week before were
inoculated the day before. On the day of, these inoculated cultures were purified,
and Nano Drop readings were taken. A digestion reaction was then setup for one of
the purified plasmid with the following conditions: plasmid DNA (210.9 ng), 10X Fast
3

5. Digest buffer (2 µl), EcorI-FD* (5 u), and ddH2O for a total reaction volume of 20 µl.
After digestion, a 0.7% agarose gel was run to visualize the success of the digestion
reaction. lastly, an aliquot of the purified genomic PCR product was prepared and
sent for sequencing (a partner sent a purified RT-PCR sample as well).
• Bioinformatics analysis of sequenced products
Bioinformatics analysis of the sequenced products was done using online tools such as
ClustalX2, BLAST, and FlyBase.
3 Results
In an effort to explore splicing patterns within Drosophila melanogaster, genomic DNA and
total RNA were extracted from adult flies. Table 1 shows the concentration of the genomic
DNA and total RNA extracted. The concentration of genomic DNA and total RNA was
measured to be 44.7 ng µl−1
, and 1193.9 ng µl−1
respectively. From Table 1 we also see that
the absorbance ratios for the genomic DNA and total RNA fall within acceptable ranges
(see appendix) which indicates very little contamination by salt and protein respectively.
This part of the experiment was generally a success as genomic DNA, and total RNA was
successfully extracted while containing little to no protein and/or salt contamination. Figure
1 shows a formaldehyde gel image of the total RNA sample. We are interested in lanes 10 and
11 which represents the total RNA sample with RNase present, and not present respectively.
Since RNase degrades RNA, we would expect not to see any bands in the lane with the
RNase treated sample as seen in lane 10 of Figure 1. The double bands in lane 10 represent
the 28Sβ, and 28Sα, 18S respectively. After the formaldehyde gel analysis, PCR of a
gene specific fragment from the genomic DNA, and generation of cDNA via RT-PCR was
performed using the genomic DNA, and total RNA samples respectively. Figure 3 shows the
results of 0.1% agarose gel for qualitative analysis of the success of the PCR and RT-PCR
reactions. Ideally we would expect to see bands for both the RNA sample and the DNA
4

6. sample. However, from Figure 3, we only see a band for the DNA sample, and none for the
RNA sample. This deviation from expectation can likely be contributed to lack of adequate
amount of RNA in the sample loaded in said lane. RNA as a molecule is very unstable and
very easily degradable so much care needs to be taken when working with samples containing
RNA. The lack of careful handling of the RNA sample in this experiment likely led to RNA
degradation and thus inadequate amount for agarose gel analysis.
For the remainder of the experiment, focus was adverted to genomic sample as my partner
obtained adequate results for the RNA sample. The purified genomic sample was cloned
into a pGEM-T Easy vector, transformed into JM109 competent cells, plated onto two
LB/Amp/X-Gal/IPTG plates, and left for overnight growth. Figure 2 shows the results
of the cloning procedure. Ideally, one would expect to see little to no blue colonies in the
transformed plates. This is because the gene-specific fragment should ideally be inserted into
the LacZ gene, thus preventing expression of Lac-Z, and hence only white colonies produced.
However for Figure 2a, and 2b, we observe a 3:1 ratio of white colonies to blue colonies which
shows that the cloning was for the most part a success. However, the observed blue colonies
is indicative of plasmids where the fragment failed to successfully insert within the Lac-Z
gene. Before preparing the genomic sample for thermocycle sequencing, inoculated colonies
of cloned cells were purified and a restriction digest using EcoRI was done for qualitative and
quantitative purposes. Qualitatively, it was done in order to check that the appropriate clone
was obtained, and quantitatively to check the observed sizes of the products with the sizes of
the expected products. Figure 4 show the results of running digested products as well as the
undigested products. The bands for both the digested and undigested products shows that
cloning was a success. We would also expect the digested products to have sizes around 2.8
- 3.0 Kbp which is also observed. After thermocycle sequencing, a series of bioinformatics
tools were used to analyze the sequencing results. Figures 6 through 20 show the results of
of these bioinformatics tools. These analyses showed that primers used for this experiment
mapped to a genomic, and mRNA(cDNA) segment of Drosophila melangonaster, with the
5

7. genomic being a portion of the X chromosome, and the cDNA being a portion of the rings
lost (rngo) gene.
4 Discussion and Future Work
Alternative splicing is one of the most regulated process in most eukaryotes as it serves
to provide a means of genetic control by organisms, thus it is a crucial mechanism for
control of gene expression.[3] In relation to humans, alternative splicing can be beneficial or
detrimental to humans well being depending on how it is regulated. Even though alternative
splicing is vital for the development of most human gene functions, its misregulation can lead
to various diseases.[3] Alternative splicing is also regulated in Drosophila melangonaster.
Regulation of alternative splicing finds its prominence in the germ cells, muscle and the
central nervous system where it modulates the expression of various proteins including cell-
surface molecules and transcription factors.[4] Previous studies involving splicing patterns
in Drosophila melangonaster has helped to establish the various effects of regulation on
splicing. Regulation of splicing in Drosophila melangonaster helps to promote tissue and
stage-specific protein isoforms which all have different functions during development.[4]
Other studies pertaining to various types of flies have in many ways given researchers
numerous insights into splicing in general. This five weeks study of splicing in Drosophila
melangonaster was for the most part a success. We were successfully able to extract genomic
DNA, and total RNA from the fruit-fly Drosophila melangonaster, and using these, we were
able to explore splicing patterns by sending DNA and RNA samples for sequencing and ob-
serving a significant difference in the lengths of the sequenced products. Some observations
that became apparent through the bioinformatics assessments of the genomic and cDNA
sequenced data is the length difference between the two, with the genomic DNA having a
longer sequence length. When the genomic DNA is aligned to the cDNA (Figure 6), we see
gaps present which is indicative of introns that were spliced from the genomic DNA. This
6

8. is expected as cDNA is typically shorter in length than genomic DNA due to processing of
mRNA. We also see conserved regions at these gaps, namely the 5’-GT , 3’-AG (GT/AG) A
possible explanation for this could be that these sites serve as recognition sites for spliceo-
somes. Futheer bioinformatics analsysis of the rngo gene has shown presence of various
homologs in humans. The most prominent ones being the Human Dna Damage-inducible
Protein, and the Retroviral-like Protease (Rvp) Domian Of Human Ddi1. Future studies
may be concentrated on other possible donor and acceptor sites within spliced regions of
total RNA extracted from Drosophila melangonaster when compared to its genomic DNA,
and the resulting genes homology to other organisms including humans.
7

9. 5 Appendix
Nano Drop readings for PCR and Plasmid products
sample A260
A230
A260
A280
Concentration(
ng µl−1
)
Genomic DNA 0.87 1.84 44.7
Total RNA 2.30 2.33 1193.9
Purified genomic
PCR
1.38 2.05 15.6
Purfied RT-PCR 1.22 2.06 10.8
Purfied genomic
plasmid
2.04 1.89 210.9
**Purfied RT
plasmid**
0.77 1.8 7.8
Table 1: Nano Drop readings for PCR and plasmid products. ** represents values for sample
borrowed from a laboratory colleague.
8

10. Figure 1: Formaldehyde gel of total RNA. Lane 1: RNA ladder. Lane 10: RNase-. Lane 11:
RNase+
9

11. (a) 200 µl Plating
(b) 50 µl Plating
Figure 2: Cultures after transforming into JM109 competent cells and plating onto
LB/Amp/X-Gal/IPTG plates. Note we see about four times more cultures in (a) as com-
pared to (b)
10

12. Figure 3: Gel electrophoresis of genomic PCR and RT-PCR DNA samples. Lane 1: DNA
ladder, Lane 2: G+, Lane 3: G-, Lane 4: RT+, lane 5: RT-
11

13. Figure 4: Gel of digested genomic PCR, RT-PCR. Lane 1: ladder, lane 2: GT+ PCR, lane
3: GT undigested, lane 4: G+ digested, lane 5: RT+ PCR, lane 6: RT+ undigested, lane 7:
RT+ digested,
12

14. Figure 5: The pGEM-T Easy Vector used for ligation. Note: this vector contains 3’-T
overhangs used for ligation with Taq polymerase PCR products, and insertion site in the
LacZ gene used for blue/white screening.
13

15. Figure 6: Raw sequenced received from sequencing. This sequence has not been pre-processed
for manual alignment as seen by the sequence of N’s. This sequence also contains the forward
and reverse primers used.
14

16. Figure 7: Chromatographic sequence of genomic sequence using the universal primer T7
generated using FinchTV
Figure 8: Chromatographic sequence of the reverse complement of the genomic sequence
using the universal primer SP6 generated using FinchTV
15

17. Figure 9: Chromatographic sequence of cDNA sequence using the universal primer T7 gen-
erated using FinchTV
Figure 10: Chromatographic sequence of the reverse complement of the cDNA sequence
using the universal primer SP6 generated using FinchTV
16

18. Figure 11: Manual alignment of sequenced genomic DNA, and cDNA. Exons are presented
in red, while introns are presented in blue. Note the donor acceptor splice sites present.
(GT/AG). Aligned using ClustalX2
17

19. Figure 12: Splice sites showing donor and acceptors for genomic DNA for Drosophila melang-
onaster. Obtained from FlyBase
18

20. Figure 13: Primer Blast search results for forward and reverse primer. There were results
for both the genomice and mRNA (cDNA).The genomic DNA sequence is predicted to be a
part of Drosophila melangonaster chromosome X, and the cDNA sequence predicted to be
part of Drosophila melangonaster rings lost (rngo) gene, with sizes 623 and 496 base pairs
respectively. Obtained from primer Blast 19

21. Figure 14: Blast results of genomic DNA using FlyBase. Top hits containing the lowest
E-scores are shown. E-scores are indicative of match quality. The lower the score, the better
the match. Obtained from BLAST
20

22. Figure 15: FlyBase Blast search on the genomic DNA sequence. These results are consistent
with the sequence belonging to chormosome X of Drosophila melangonaster which can be
seen by all 623 base pairs matching with an E-value of 0. Obtained from FlyBase
21

23. (a) Chromosome map of the rngo gene that the genomic DNA maps to. The green arrow rep-
resentative of the gene and orange are representative transcripts and the thinner gray lines are
representative of regions containing introns. The direction of the arrows indicates transcription
directionality. Obtained from FlyBase
(b) Summary of the functionality of the rngo gene. Obtained from FlyBase
Figure 16: Gene information for genomic sequence obtained from FlyBase
22

24. Figure 17: allelic and phenotypic information for rngo being accessed obtained from FlyBase
23

25. Figure 18: Amino acid translations for sequenced DNA. Obtained from FlyBase
Figure 19: rngo gene homology in humans. Obtained from BLASTp
24

26. Figure 20: Alignment of homology in humans to the rngo gene obtained from BLASTp
25

27. • Primers Sequence
Rngoe2: GAT TGC CGA AGA GAT CAA GCA G
Rngoe2R: ATC CTC CGA GTT TCC TGT CAG
• Restriction Enzymes Sites
EcoRI : : 5’...C/TCGAG....3’
• Materials acquisition
All enzymes used during the course of this experiment was acquired by
the teaching staff.
• Figures acquisition
Figure 1 was annotated by Taylor Blue, Wed-12-D. This figure was used
because both samples were run on the same gel.
Figure 5 was acquired from the MBB 308 2016 teaching slides.
• Acceptable Ranges for Nano Drop
DNA: A260
A280
1.8
RNA: A260
A280
2.0
• Bioinformatics tools used
FlyBase: http://flybase.org
BLAST: https://blast.ncbi.nlm.nih.gov/Blast.cgi
ClustaX: http://www.clustal.org/clustal2/
FinchTV: http://www.geospiza.com/ftvdlinfo.html
26

28. References
[1] Z. Ding, B. Honda, A. Kim, J. Lum, S. MacLean, F. Pio, D. Sinclair, M. Syrzycka,
P.J. Unrau, S. Vlachos and S. Wang. Fall 2016 MBB308-3 Molecular Biology
and Biochemistry Laboratory Manual Department of Molecular Biology and
Biochemistry. Simon Fraser University, Burnaby, BC. 2016
[2] Tobias Morawe, Mona Honemann-Capito, Walter von Stein, Andreas Wodarz Loss of
the extraproteasomal ubiquitin receptor Rings lost impairs ring canal growth
in Drosophila oogenesis. The Journal of Cell Biology Apr 2011, 193 (1) 71-80; DOI:
10.1083/jcb.201009142
[3] Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights
from molecular and genomics approaches.Nature reviews Molecular cell biology.
2009;10(11):741-754. doi:10.1038/nrm2777.
[4] Venables JP, Tazi J, Juge F. Regulated functional alternative splicing in
Drosophila. Nucleic Acids Research. 2012;40(1):1-10. doi:10.1093/nar/gkr648.
27