Do Bugs have Soluble Adenylyl Cyclase (sAC)?

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Soluble adenylyl cyclase (sAC) is a form of the
mammalian adenylyl cyclase that is distributed
throughout the cytoplasm of cells. Sequence
analysis of the Drosophila genome does not
predict the existence of a fly homolog of sAC,
but biochemical evidence suggests its existence.
We intend to use the short stretches of amino
acid sequence identity between mosquito,
Anopheles gambiae, and human sAC genes, to design
degenerate primers for use in PCR reactions, in
an attempt to identify, clone, and characterize a
Drosophila sAC ortholog. Two degenerate PCR
primers were designed based on distinct regions
of amino acid sequence identity detected by a
BLAST search between the mosquito, Anopheles
gambiae, and human sAC. These primers were used
in PCR reactions containing adult and embryonic
Drosophila cDNA (variables) and rat and human sAC
plasmids (positive controls). The appropriately
sized fragment (estimated 900bp) that was gel
purified, will be cloned, and sequenced to
determine whether it encodes a Drosophila sAC
ortholog. A band approximately 900bp in size was
obtained from the PCR reaction and purified. The
purified PCR product will later be inserted into
a TA plasmid and sent for sequencing. The
sequence obtained will be compared with that of
other sAC enzymes to determine whether the
primers successfully amplified a portion of
Drosophila sAC. NIH Grants GM62328 (J.B.),
HD422060 (J.B.), and HD38722 (L.R.L), Weill
Cornell Medical College
Do Bugs have Soluble Adenylyl Cyclase (sAC)?
Salihah Dick1 Jeanne Farrell2 Lonny R.
Levin2 Jochen Buck2
Weill Medical College of Cornell University2
Barry University1

• The search for insect sAC
• sAC-like biochemical activity observed in
  Drosophila melanogaster
• (Livingstone et al. 1984).
• Drosophila genome revealed no sAC homology
• Human and putative mosquito sAC possess two
  regions of identity.
• Regions used to design degenerate PCR primers

RESULTS

• The Adenylyl Cyclases
• Class of enzymes responsible for synthesizing
  cyclic adenosine
• monophosphate (cAMP) in living cells (Zippin et
  al. 2001 Sunahara and
• Taussig, 2002).
• cAMP is an almost universal second messenger
  molecule (Zippin et al.
• 2001 Sunahara and Taussig, 2002).
• There are two mammalian adenylyl cyclases
  transmembrane adenylyl
• cyclase (tmACs) and soluble adenylyl cyclase
  (sAC) (Zippin et al. 2001).
• sAC is molecularly and biochemically distinct
  from the tmACs
• Catalytic domains resemble the cyanobacterial
  cyclases more than the other mammalian cyclases
  (Wuttke et al. 2001).
• sAC does not respond to G-protein or forskolin
  (Buck et al. 1999).
• The sAC gene has no transmembrane domains
  (reviewed in Zippin et al. 2001).
• sAC is both soluble and particulate (reviewed in
  Zippin et al. 2001).

PURPOSE
To use the degenerate PCR primers to clone and
sequence a Drosophila sAC ortholog.
MATERIALS AND METHODS
Figure 2. PCR reaction with P1239/P1240 primer
mix. Lanes 1 and 2 contained the molecular
markers. Lanes 3-6 contained the DNA templates,
with rat and human sAC plasmids in lanes 3 and 4,
and embryonic and adult cDNA in lanes 5 and 6
respectively. Although the 900bp fragment of
interest was conserved in both variables and
positive controls, bands A-D of lane 5 were
removed, purified and used in one-primer
dependent PCRs since they all fell within the
desired range of 850-1000bp.

• Isolation of Drosophila mRNA
• Obtained embryonic and adult Drosophila poly-A
  mRNA commercially (CLONTECH).
• Generation of cDNA
• Made cDNA by reverse transcription PCR (rtPCR) of
  mRNA using oligo-dT primers.
• cDNA generated according to manufacturers
  protocol using kits from Applied Biosystems and
  GibcoBRL.
• Reverse transcribed 1-2µg of poly-A mRNA per
  reaction.
• 2µL used as template in subsequent PCRs.
• Degenerate PCR primer design
• Performed BLAST search between human sAC and the
  mosquito genome using the Ensembl Mosquito Genome
  Server, Blast View (http//www.ensembl.org).
• Used regions of 100 amino acid sequence
  homology GGDILKF in the first catalytic domain
  and CGIVGHTV in the second catalytic domain to
  design the primers (P1239 and P1240
  respectively).
• Primer 1240 was obtained by reverse complementing
  the derived nucleotide sequence.
• Both primers had 3072 fold degeneracy.
• Obtained both primers from INVITROGEN, Inc.
• PCR with Degenerate Primers
• 1.0 µL of Primer 1239/1240
• 2.5 µL 10x Advantage2 PCR Buffer

B
A
nucleus
1650bp
1000bp
850bp
Juxtanuclear region

• Figure 3. One-primer dependent PCR reaction
  performed on the four purified embryonic cDNA
  fragments. Lanes 3-5 contained the purified
  fragment A, fragment B was in lanes 6-8, fragment
  C in lanes 9-11, and fragment D in lanes 12-14.
  Primers were designed to flank and mark the
  initiation sites of the putative Drosophila sAC
  amplification region therefore no product should
  have been obtained when they were used singly.
  However, the PCR reactions containing P1240
  generated results that were almost identical to
  the P1239/P1240 mix.

C
C
D
Juxtanuclear region
Mitochondria
A
B
D
A
D
Figure 1. Immunofluorescent staining
demonstrating subcellular localization of sAC.
(A, B) Non-overlapping polyclonal N-term
antiserum, reveals a staining pattern (shown in
green) demonstrating sAC localization in the
cytoplasm, mitochondria, and nuclei of COS7
cells. (D) Merged image confirming sAC
co-localized with the mitochondrial marker
Mitotracker (Molecular Probes), shown in red (C),
and the nuclear dye, DAPI, shown in blue (B, D).
sAC was also present in a juxtanuclear region
devoid of mitochondria indicated by the arrow
(reproduced with permission from L. Levin and J.
Buck).
ACKNOWLEDGEMENTS
REFERENCES
Cornell University Lonny R. Levin, Ph.D. Jochen
Buck, M.D./ Ph.D Jeanne Farrell Program Manager
Ms. Ruth Gotian Program Director Olaf S.
Anderson, M.D. NIH Grants
Barry University Dr. Jeremy Montague Dr. Flona
Redway Sr. John Karen Frei, O.P., Ph.D. MBRSRISE
Program
ADDRESS Barry University
11300 NE 2nd Ave. Miami, FL
33161 PHONE [phone] FAX
(305) 899- 4866 EMAIL salihahdick_at_hotmail.c
om

• Buck J, Sinclair ML, Schapal L, Cann MJ, Levin
  LR. 1999. Cytosolic adenylyl cyclase defines a
  unique signaling molecule in mammals. Proc. Natl.
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• Livingstone MS, Sziber PP, Quinn WG. 1984. Loss
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• Sunahara RK, Taussig R. 2002. Isoforms of
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• The Wellcome Trust, Sanger Institute, Ensembl
  Mosquito Genome Server (Blast View). Ensembl
  Genome Browser home page. http//www.ensembl.org.
  Accessed 2002 July 19.
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• Zippin J, Levin L, Buck J. 2001.
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