Transcript Mitochondrial Genome Sequencing of a Calcareous Sponge

Mitochondrial Genome
Sequencing of a
Calcareous Sponge,
Scypha ciliata
Wang Yijing
2005-12-17
Introduction
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Department of Molecular Evolution, EGS,
EBC
October 31st - November 23rd
Supervisor: Mikael Thollesson
Laboratory Introduction
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Alpha-proteobacteria: the evolution of
symbiotic and parasitic relationships,
evolution of mitochondrion and modeling of
evolution process
Hyperthermophilic archaeas: the evolutionary,
regulatory and mechanistic aspects of cell
cycles
Micro arrays, flow cytometry, MegaBace
capillary sequencer etc.
Laboratory Introduction
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Work hours: 9:00 a.m.- 6:00 p.m.
The whole day at the bench
Breaks:
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coffee breaks at 10 am and 3 pm
cakes on Friday afternoons!
Seminars: 4:00-5:00 every Wednesday
afternoon.
Project Introduction
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A typical metazoan
mitochondrial genome
16kbp circular molecule
37 genes
coding proteins, rRNAs
and tRNAs
compactly arrayed
conservatively ordered
Project Introduction
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A metazoan mtDNA
16kbp circular molecule
37 genes
coding proteins, rRNAs
and tRNAs
compactly arrayed
conservatively ordered
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A unicellular mtDNA
about 80 kbp
encodes 1.5 times
more genes
uses a minimally
derived genetic code
encodes bacterial-like
tRNAs and rRNAs.
Project Introduction
Why Sponges?
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the most primitive metazoans evolved from
choanoflagellate-like protist ancestors
at the bottom root of the metazoan phylogenetic tree
based on both morphological studies and molecular
sequences
Thus the mtDNA comparisons between sponges,
unicellular ancestor and other animals will provide
the insights into the mtDNA evolution and
phylogenetic reconstruction.
Project Introduction
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Hexactinellida (glass
sponges)
Demospongiae
(demosponges)
Calcarea (calcareous
sponges)
Project Introduction
Project Introduction
Project Introduction
Choanocyte
- Collar Cell in Sponges
Choanoflagellate
-Protist Ancestor
Project Introduction
mtDNA of Demosponges
 resembles most
features of metazoan
mtDNA
 Yet contains some extra
genes encoding
bacterial-like rRNAs
and tRNAs and use a
minimally derived
genetic code
Project Introduction
However,
the Calcarea are
thought to be more
closely related to
higher metazoan
organisms than the
other two groups.
Scypha ciliata
Strategy
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The aim of the project is to obtain the
complete mitochondrial genome sequence for
a calcareous sponge, Scypha ciliata, by using
XL-PCR, nebulization and cloning, and
sequencing of the clone library.
However, we need to use general primers to
get some sequences that we could use to
design specific primers for XL-PCR since
there are no sequences for the mtDNA of this
species published ever.
Methods
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Total DNA extracted using CTAB extraction
CO I gene amplified using general primers
HCO, LCO at 47℃
16S rRNA gene amplified using 16S-ARL,
16S-BRH at 50℃
Sequencing using MegaBace capillary
sequencer
The results were checked against the
GenBank database using blastn.
Difficulties
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DNA degrading in the specimen?
Little information on the mtDNA sequence
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How fit the primers are to the template?
Touch-down PCR
Contamination
Nuclear homolog to mitochondrial 16S
Sequence primers
Luckily I got the mitochondrial 16S
sequence on the last day!
Results- 16S
Results-16S
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The 16S rRNA gene sequence alignment by
Clusal X
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Scypha ciliata
Calcareous sponges: Grantia, Leucilla, Clathrina
Demosponges: Microciona, Cliona, Tethya,
Halichondria and Geodia
Choanoflagellate: Monosiga
Cnidaria: Urticina, Palythoa and Zoanthus
A phylogenetic tree based on these
alignments by PAUP
Results-16S
Results-16S
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However, 16S rRNA gene of mtDNA is
relatively conserved in its sequence during
the evolution, thus it is not very suitable for
phylogenetic analysis.
But we can use the sequence to design
specific primers for XL-PCR.
Results- CO I
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We got the amplification products from PCR.
The sequencing of CO I Gene failed.
Reasons:
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The products of PCR were not pure enough?
The primers were not suitable to the sequence
reactions.
So we propose to clone the PCR products of
COI gene before sequencing it.
Future Perspectives
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Order other general primers to amplify other
regions
XL-PCR
Shearing
A Clone Library
Sequencing
Assembling
Phylogenetic Analysis
Acknowledgement
Many thanks to everyone in the lab
Especially thanks to Mikael
Anna-Sofie
Olga
Guan Na
Johan Viklund
Yin Jun