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The multicellular alga Volvox carteri possesses only two cell types: mortal, motile …


Biology Articles » Protistology » Phycology » Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri » Figures

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- Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri

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Figure 1 Phenotype of Volvox carteri and appearance of separated cell types. A) Wild-type phenotype of an asexual female of Volvox carteri f. nagariensis containing ~2000 small, terminally differentiated somatic cells at the surface and ~16 large reproductive cells (gonidia) in the interior. More than 95% of the volume of such a spheroid consists of a complex but transparent extracellular matrix. B) Isolated somatic cell sheets of V. carteri. C) Isolated gonidia of V. carteri.

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Figure 2 Pairwise sequence alignment of amino acid sequences. Alignment of sequences, deduced from Volvox genes used in this study, with previously known proteins characterized in other species. Identical residues are given as white letters on a dark blue background. Similar residues are given as black letters on a light blue background. The sequence alignment was done using DNASIS/PROSIS Software. With the exception of NIP/NipA, which are shorter, a section of 100 residues is shown. Species names: A.t., Arabidopsis thaliana; B.n., Brassica napus; C.r., Chlamydomonas reinhardtii; H.s., Homo sapiens; N.t., Nicotiana tabacum; S.o., Spinacia oleracea; V.c., Volvox carteri.

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Figure 3 Comparison of gene expression of six target genes in gonidia versus somatic cells by quantitative real-time RT-PCR. Amplification curves for A) actA (internal control for the 2-ΔΔCt method), B) gon167, C) ssgA, D) rpl37, E) fer1, and F) regA. The target-specific fluorescence signal of SYBR Green fluorescence emission (detection range 515–545 nm) is plotted against the number of PCR cycles. Curves of gonidial RT-PCRs are given in red, somatic RT-PCRs in blue. All real-time RT-PCR experiments were carried out in triplicate, and a mean amplification curve was generated for each cell-type. The threshold level is given by a broken, horizontal line. The cycle at which the mean amplification curve of gonidial or somatic real-time RT-PCRs crosses the threshold (Ct value) is indicated by a broken, vertical line.

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Figure 4 Visualization of final products after real-time RT-PCRs of six target genes. The following sizes have been predicted for the amplified cDNA fragments of the corresponding mRNAs: A) actA, 104 bp; B) gon167, 112 bp; C) ssgA, 130 bp; D) rpl37, 138 bp; E) fer1, 116 bp; and F) regA, 101 bp. RT minus and no template controls were free of any DNA product as expected. The lanes on the agarose gels were loaded with: M, 100 bp size marker; Gon, reaction product from gonidia; Soma, reaction product from somatic cells; Ntc, no template control; Rtmc, RT minus control.

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Figure 5 Sequencing of real-time RT-PCR products of six target genes. A) actA, B) gon167, C) ssgA, D) rpl37, E) fer1, and F) regA. The positions of PCR primers are indicated in bold. Positions of introns within these cDNA-fragments are indicated by arrowheads. All mRNAs have been spliced as predicted, and the cDNA fragments, which have been obtained by the above mentioned real-time RT-PCRs, showed the expected sequences.

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Figure 6 Visualization of ΔCt values of 38 genes from V. carteri somatic cells and gonidia. Blue quadrangles: values from somatic cells; red circles: values from gonidia. Each pair of ΔCt values for a single gene is connected by a vertical black bar; the length of this bar corresponds to the ΔΔCt value. actA was used as a reference and thus defines the zero line.

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Figure 7 Cell-type specific gene expression of 38 genes from V. carteri determined by the 2-ΔΔCt method. The gene names are given at the end of the horizontal expression bars. The length of the expression bar illustrates the ×-fold higher expression of a given target gene within the given cell type with respect to the other cell type. Blue: higher expression in somatic cells; red: higher expression in gonidia.

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