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Mitochondrial Genome Sequencing of a
Calcareous Sponge, Scypha ciliata
Wang Yijing
2005-12-17
Introduction
Department of Molecular Evolution, EGS, EBC
October 31st - November 23rd Supervisor: Mikael Thollesson
Laboratory Introduction
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
Work hours: 9:00 a.m.- 6:00 p.m. The whole day at the bench Breaks:
coffee breaks at 10 am and 3 pm cakes on Friday afternoons!
Seminars: 4:00-5:00 every Wednesday afternoon.
Project Introduction
A typical metazoan mitochondrial genome
16kbp circular molecule 37 genes coding proteins, rRNAs
and tRNAs compactly arrayed conservatively ordered
Project Introduction
A metazoan mtDNA 16kbp circular molecule 37 genes coding proteins, rRNAs
and tRNAs compactly arrayed conservatively ordered
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? 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
Hexactinellida (glass sponges)
Demospongiae (demosponges)
Calcarea (calcareous sponges)
Project Introduction
Project Introduction
Project Introduction
Choanocyte Choanoflagellate - Collar Cell in Sponges -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
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
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
DNA degrading in the specimen? Little information on the mtDNA sequence
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
The 16S rRNA gene sequence alignment by Clusal X 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
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
We got the amplification products from PCR. The sequencing of CO I Gene failed. Reasons:
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
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