Quantitative analysis of cell-type specific gene expression in the green alga Volvox carteri
© Nematollahi et al; licensee BioMed Central Ltd. 2006
Received: 22 September 2006
Accepted: 21 December 2006
Published: 21 December 2006
The multicellular alga Volvox carteri possesses only two cell types: mortal, motile somatic cells and potentially immortal, immotile reproductive cells. It is therefore an attractive model system for studying how cell-autonomous cytodifferentiation is programmed within a genome. Moreover, there are ongoing genome projects both in Volvox carteri and in the closely related unicellular alga Chlamydomonas reinhardtii. However, gene sequencing is only the beginning. To identify cell-type specific expression and to determine relative expression rates, we evaluate the potential of real-time RT-PCR for quantifying gene transcript levels.
Here we analyze a diversified pool of 39 target genes by real-time RT-PCR for each cell type. This gene pool contains previously known genes with unknown localization of cellular expression, 28 novel genes which are described in this study for the first time, and a few known, cell-type specific genes as a control. The respective gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes. We provide expression data for all these genes.
The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox, which can be extended to a much larger number of genes or to developmental or metabolic mutants. Our expression data also provide a basis for a detailed analysis of individual, previously unknown, cell-type specifically expressed genes.
Although determination of the sequence of every gene in Volvox or any other species allows a better understanding of the organism's physiological potential, it is just the first step of a complete description of how the organism works. One of the next steps should be the determination of mRNA expression levels. Because it is known from many species that much of the transcriptome is compartmentalized and Volvox is particularly suitable for studies of multicellularity and cellular differentiation, it is logical to start with an analysis of cell-type specific gene expression, i.e. somatic cells versus gonidia, in order to provide a basis for disclosing cell-specific functions.
In earlier studies, 19 gonidia-specific and 12 somatic-cell-specific cDNAs have already been identified in wild-type Volvox by a differential screen of cDNA libraries, and abundance of the transcripts has been analyzed in each of the cell types by Northern blots using radiolabeled restriction-digested DNA as probes ; two of these cDNAs/genes have been added to our study as a reference (gon30, gon167). Furthermore, a couple of interesting developmentally-controlled or cell-type specific genes and their gene products have been identified by generating and analyzing mutants or by Mendelian analysis, e.g. the lag gene product (late gonidia), which acts in large pregonidial cells to repress somatic development [4, 7, 8], and the regA gene product (somatic regenerator), which acts on somatic cells to suppress gonidial development . The latter gene was also used as a control gene in our study. Previously, it has also been shown that somatic cells and gonidia display substantially different patterns of both newly synthesized and accumulated major polypeptides , but at that time, it was not possible to obtain discrete sequences of these polypeptides, so their identity remained unknown.
A different approach, which is used in this study, is to investigate the different developmental programs in the two cell types by characterizing the abundance of novel or previously known mRNAs separately for each cell-type by real-time RT-PCR studies. This method should allow a considerable expansion of the number of genes investigated, studies done under different physiological conditions, and repeated experiments using mutant strains.
Here we show a quantitative analysis of a diversified pool of about forty target genes with regard to cell-type specific gene expression and relative expression rate in wild-type individuals of the green alga Volvox carteri. The investigated gene pool contains previously known genes with unknown localization, novel Volvox genes, which are described in this study for the first time, and a few previously characterized genes with known cell-type specific localization as controls. The corresponding gene products are, for instance, part of photosynthesis, cellular regulation, stress response, or transport processes.
Target genes for differential analysis
List of 39 Volvox genes and gene products, including subset classification, comparisons with homologs in other species, and significance of the sequence relationship to these homologs.
V. carteri gene
(• novel Volvox gene)
V. carteri gene product
Significance: percent identity with characterized homolog (# of identical residues/total # of residues)
Gene product and species name of characterized homolog used for sequence comparison
98.0 % (370/377)
[99.0 % (375/377)]
actin Chlamydomonas reinhardtii  [GenBank:D50839]
pherophorin-like ECM-glycoprotein SSG185  [GenBank:X51616]
54.4 % (137/252)
[85.3 % (215/252)]
pherophorin-C3 Chlamydomonas reinhardtii  [GenBank:DQ196109]
somatic regenerator RegA  [GenBank:AF106963]
67.5 % (81/120)
[91.7 % (110/120)]
RegA-like sequence protein RlsA [9, 16] [GenBank:AF106962]
G30 protein [6, 56] [GenBank:AF110790]
85.4 % (380/445)
[91.2 % (406/445)]
low-CO2 inducible protein LciC Chlamydomonas reinhardtii [43, 44] [GenBank:AB168094]
G167 protein  [GenBank:U31955]
99.1 % (210/212)
[99.5 % (211/212)]
recombinase (ORF-C) on retrotransposon kangaroo-1 Volvox carteri  [GenBank:AY137241]
RegA-like sequence protein RlsA [9, 16] [GenBank:AF106962]
67.5 % (81/120)
[91.7 % (110/120)]
somatic regenerator RegA  [GenBank:AF106963]
chloroplast-specific ribosomal protein [GenBank:AY835992]
36.7 % (79/215)
[76.3 % (164/215)]
chloroplast-specific ribosomal protein PSrp-1, 30S subunit Spinacia oleracea  [GenBank:M55322]
C2H2-type zinc finger related protein with arsenite-resistance domain [GenBank:AY850006]
43.0 % (16/37)
[62.0 % (23/37)]
RING finger protein 13 RNF13 Gallus gallus [GenBank:AY787020]
ATP-energized ABC transporter Mrp2 [GenBank:AY835993]
91.4 % (243/266)
[99.6 % (265/266)]
ABC transporter Mrp1 Chlamydomonas reinhardtii  [GenBank:AF442557]
gonidia-specific protein KA_k47 [GenBank:AY835994]
nitrate reductase NitA  [GenBank:X64136]
80.8 % (698/864)
[96.1 % (830/864)]
nitrate reductase Nit1 Chlamydomonas reinhardtii  [GenBank:AF203033]
flagellar α dynein (heavy chain) [GenBank: EF123072]
93.6 % (132/141)
[97.8 % (138/141)]
flagellar α dynein (heavy chain) ODA11 Chlamydomonas reinhardtii [21, 22] [GenBank:L26049]
kinesin-like protein [GenBank: EF123073]
84.8 % (117/138)
[98.5 % (136/138)]
kinesin-like protein FLA10 Chlamydomonas reinhardtii  [GenBank:L33697]
ferredoxin Fer1 [GenBank: EF123074]
74.2 % (95/128)
[93.7 % (120/128)]
ferredoxin PETF (chloroplast) Chlamydomonas reinhardtii  [GenBank:L10349]
nucleic acid binding protein Nab1 [GenBank: EF123075]
87.8 % (216/247)
[97.1 % (239/247)]
nucleic acid binding protein Nab1 Chlamydomonas reinhardtii  [GenBank:AY157846]
ribosome-associated protein (chloroplast-specific) [GenBank: EF123076]
55.0 % (60/109)
[89.9 % (98/109)]
ribosome-associated protein Rap41 (chloroplast-specific) Chlamydomonas reinhardtii  [GenBank:AY177616]
chloroplast fructose-1,6-bisphosphatase [GenBank:EF123077]
61.1 % (196/318)
[86.4 % (275/318)]
chloroplast fructose-1,6-bisphosphatase FBP Brassica napus  [GenBank:L15303]
Calvin cycle protein CP12 [GenBank: EF123078]
78.6 % (81/103)
[97.1 % (100/103)]
Calvin cycle protein CP12 Chlamydomonas reinhardtii  [GenBank:AJ005284]
profilin PrfA [GenBank: EF123079]
59.5 % (78/131)
[90.8 % (119/131)]
profilin PRF1 Chlamydomonas reinhardtii  [GenBank:AF335423]
superoxide dismutase Fsd1 [GenBank: EF123080]
88.7 % (204/230)
[98.7 % (227/230)]
superoxide dismutase FSD1 Chlamydomonas reinhardtii  [GenBank:U22416]
ribosomal protein L37 [GenBank: EF123081]
59.8 % (58/97)
[87.6 % (85/97)]
ribosomal protein L37 (RPL37) Homo sapiens  [GenBank:NM_000997]
glutamate synthase [GenBank: EF123082]
68.4 % (575/841)
[92.4 % (777/841)]
glutamate synthase GltB Spinacia oleracea  [GenBank:AF061515]
heat shock protein 70 B [GenBank: EF123083]
70.9 % (141/199)
[94.5 % (188/199)]
heat shock protein 70 Hsc70-7 Arabidopsis thaliana  [GenBank: AF217459]
hsp40-like heat shock protein [GenBank: EF123084]
34.0 % (34/100)
[70.0 % (70/100)]
heat-shock protein HSJ1 (DnaJ-like) Homo sapiens  [GenBank:NM_001039550]
ubiquitin conjugating enzyme E2 [GenBank: EF123085]
77.9 % (116/149)
[94.0 % (140/149)]
ubiquitin conjugating enzyme E2 (UBC14) gene Arabidopsis thaliana  [GenBank:U33759]
pontin [GenBank: EF123086]
76.7 % (345/450)
[95.3 % (429/450)]
Pontin52 Homo sapiens  [GenBank: AF099084]
retinoblastoma-like protein Mat3 [GenBank: EF123087]
73.6 % (162/220)
[92.3 % (203/220)]
retinoblastoma-like protein Mat3 Chlamydomonas reinhardtii  [GenBank: AF375824]
vacuolar processing enzyme VPE [GenBank: EF123088]
61.0 % (61/100)
[86.0 % (86/100)]
vacuolar processing enzyme VPE-2 Nicotiana tabacum  [GenBank:AB075949]
sulfur deprivation response regulator Sac1 [GenBank: EF123089]
87.1 % (242/278)
[98.6 % (274/278)]
sulfur deprivation response regulator Sac1 Chlamydomonas reinhardtii  [GenBank: U47541]
required-for-cell-differentiation 1 protein Rcd1 [GenBank: EF123090]
69.5 % (196/282)
[90.1 % (254/282)]
required-for-cell-differentiation 1 protein Rcd1 Homo sapiens  [GenBank:NM_005444]
adenylate cyclase [GenBank:EF123091]
63.6 % (98/154)
[86.4 % (133/154)]
adenylate cyclase, type II ADCY28 Chlamydomonas reinhardtii (JGI Chlamydomonas ID 121064)
NaCl-inducible protein [GenBank: EF123092]
58.3 % (49/84)
[86.9 % (73/84)]
NaCl-inducible protein (NIP) Chlamydomonas reinhardtii [GenBank:AU066522]
low-CO2 inducible protein LciB [GenBank:EF123093]
56.3 % (190/339)
[87.4 % (396/339)]
low-CO2 inducible protein LciB Chlamydomonas reinhardtii [43, 44] [GenBank:AB168093]
protein of unknown function [GenBank: EF123094]
protein of unknown function [GenBank:EF123095]
protein of unknown function [GenBank: EF123096]
protein of unknown function [GenBank: EF123097]
protein of unknown function [GenBank: EF123098]
protein of unknown function [GenBank: EF123099]
- Subset A: Known Volvox genes with known status of cell-type specific expression (5 genes: actA, ssgA, regA, gon30, gon167).
- Subset B: Known Volvox genes that have previously been identified via characterized homologs in Volvox and in which a cell-type specific expression is predictable due to the characteristics of the homologous gene (1 gene: rlsA).
- Subset C: Known Volvox genes with putative cell-type specific expression based on preliminary experiments (4 genes: csrp1, ard1, mrp2, gspk47).
- Subset D: Well-known Volvox genes with unknown status of cell-type specific expression (1 gene: nitA).
- Subset E: New Volvox genes that were identified in this project via characterized homologs in other species and in which a cell-type specific expression is predictable due to the characteristics of the homologs (7 genes: dyhA, klpA, fer1, nab1, rap41, fbp1, cp12).
- Subset F: New Volvox genes that were identified in this project via characterized homologs in other species but in which the status of cell-type specific expression is not predictable (15 genes: prfA, fsd1, rpl37, glu1, hsp70B, hsp40A, ubcA, ponA, mat3, vpeA, sac1, rcd1, adcA, nipA, lciB).
- Subset G: New Volvox genes for which no characterized homologs in any other organism have been identified and for which, consequently, the status of cell-type specific expression is unknown (6 genes: upf1, upf2, upf3, upf4, upf5, upf6).
Characteristics of genes within subset A
Subset A contains 5 Volvox genes (Table 1). Each gene has been investigated previously for cell-type specific expression. The actin gene actA, expressed uniformly in somatic cells and gonidia , was used as a reference transcript in several previous studies [12–14] and is therefore used as the reference gene in the real-time experiments described below. The gene coding for the extracellular matrix glycoprotein SSG185, ssgA, is known to be expressed mainly by somatic cells . Similarly, the regA gene, a key gene controlling cell differentiation in Volvox carteri by suppressing reproductive activities in somatic cells, is expressed only in somatic cells . In a previously described search for cell-type specific genes of Volvox carteri , several gonidia-specific genes have been identified. Two of these genes, gon30 and gon167, are included in our study. The gon167 mRNA has been shown to be present in variable but moderate levels in gonidia, with highest expression levels at the beginning of cleavage and during cleavage divisions. In contrast, the gon30 mRNA was at its lowest during cell cleavages and had maximal expression much later. For both gon30 and gon167, the mRNA level in somatic cells has been shown to be low at all stages.
Characteristics of genes within subset B
Subset B contains only one Volvox gene (Table 1), the rlsA gene, which has previously been identified via the homologous Volvox gene regA [9, 16]. Partial sequences of rlsA and regA show extensive similarity, including fully conserved exon/intron boundaries. Since regA is expressed only in somatic cells (see above), a cell-type specific expression of rlsA in somatic cells seems to be probable, but cell-type specific expression has not been investigated so far.
Characteristics of genes within subset C
Subset C contains 4 Volvox genes (Table 1) that have been identified by differential screenings of cDNA libraries of gonidia versus somatic cells in the group of Dr. R. Schmitt (University of Regensburg, Germany) [17, 18] and which have been deposited in GenBank, but experimental details are not available. Two of these genes are described as specific for somatic cells: a gene coding for a putative chloroplast-specific ribosome-associated protein (csrp1, "KSS_k11") and one that codes for a putative arsenite-resistance protein (ard1, "KSS_k05"). Both remaining genes are described as specific for gonidia: a gene coding for a putative ATP-energized ABC transporter (mrp2, "KA_k18/MH_k18") and one that codes for a putative gonidia-specific protein (gspk47, "KA_k47").
Characteristics of genes within subset D
Only one gene is within subset D (Table 1). It is the gene that codes for nitrate reductase, nitA , which has previously been characterized in much detail. This gene is also the standard selectable marker in Volvox transformation experiments . Nevertheless, to our knowledge, this well-investigated gene has never been analyzed for cell-type specific expression in Volvox.
Characteristics of genes within subset E (novel Volvox genes)
The other five genes were predicted to be expressed only in the gonidia. They are nuclear genes that encode for chloroplast-targeted proteins. fer1 encodes a ferredoxin; this protein family contains Fe-S clusters which plays a key role in electron-transfer during photosynthesis. nab1 codes for an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna . rap41 codes a ribosome-associated protein, which has been identified only in the 70S ribosome of the chloroplast . fbp1 encodes a chloroplast-specific fructose-1,6-bisphosphatase, a key enzyme of the Calvin cycle for photosynthetic CO2 assimilation . cp12 codes for Calvin cycle protein CP12, a small chloroplast protein, which is essential for the assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex . In V. carteri, major metabolic activities of the chloroplast are encoded by nuclear genes that are attenuated in mature somatic cells , which is why the small chloroplasts of somatic cells don't grow in size. Therefore, photosynthesis-related nuclear genes within subset E are expected to be expressed mainly within the gonidia.
Characteristics of genes within subset F (novel Volvox genes)
As in subset E, all fifteen genes within subset F (Table 1) were obtained by searching the Volvox whole-genome shotgun reads at the Chlamydomonas web site of the Joint Genome Institute with sequences of well-known genes from other species (Fig. 2). In contrast to subset E, there is no concrete information or indication about cell-type specific expression for any of these genes. The novel Volvox genes within subset F code for the following proteins (gene names in parentheses): profilin (prfA), an actin binding protein ; superoxide dismutase (fsd1), a key enzyme in the antioxidant defense system ; ribosomal protein L37 (rpl37), a 60S ribosomal subunit protein ; glutamate synthase (glu1), an enzyme that catalyzes the reductive amination of α-ketoglutarate ; heat shock protein 70B (hsp70B), which is involved in folding of nascent polypeptides ; an hsp40-like heat shock protein (hsp40A), which might be a cochaperone protein that regulates complex formation between Hsp70 and client proteins ; a ubiquitin conjugating enzyme E2 (ubcA), which controls specificity of ubiquitin-protein conjugation during selective protein degradation ; pontin (ponA), a transcriptional cofactor which is known to play an essential role in the control of cellular growth and proliferation during development ; a retinoblastoma-like protein (mat3), which is involved in the control of cell division in C. reinhardtii ; vacuolar processing enzyme (vpeA), a vacuolar protease with caspase-1 activity, which is involved in programmed cell death in higher plants ; a sulfur deprivation response regulator (sac1), a protein which is critical for survival of C. reinhardtii during sulfur deprivation and which is involved in control of cysteine biosynthesis [40, 41]; a required-for-cell-differentiation 1 protein (rcd1), which is a transcriptional cofactor that mediates retinoic acid-induced cell differentiation in Homo sapiens ; adenylate cyclase (adcA), the enzyme that catalyzes the conversion of ATP to 3',5'-cyclic AMP and pyrophosphate; an NaCl-inducible protein (nipA), a very small protein that has been identified in the halotolerant C. reinhardtii strain HS-5; and finally, a low-CO2 inducible protein LciB (lciB), which seems to be part of a carbon-concentrating mechanism in C. reinhardtii [43, 44].
Characteristics of genes within subset G (novel Volvox genes)
Subset G contains 6 Volvox genes with unknown function and unknown localization (Table 1). Though we found homologs of these genes in other species, especially in C. reinhardtii, none of the homologous genes or gene products has previously been characterized. We named these genes upf1, upf2, upf3, upf4, upf5, and upf6.
Selection of primer combinations and testing by genomic PCR and RT-PCR
Primers used for quantitative real-time RT-PCR and lengths of cDNA and gDNA products.
Forward Primer (5'→3')
Reverse Primer (5'→3')
Analysis of cell-type specific gene expression by real-time RT-PCR experiments
Volvox spheroids were broken in a Dounce homogenizer, and cell-size based separation of cell types was achieved by successive filtration on screens of different mesh sizes and a centrifugation step (Fig. 1B, C); total RNA was isolated from the separated cell types using the phenol-based TRI Reagent (see Methods).
Results of real-time RT-PCR experiments. Standard deviations are given in parentheses.
ΔCt somatic cells
2-ΔΔCt or 1/2-ΔΔCt
×-fold higher expression in
5.94 (± 0.7)
0.42 (± 0.2)
5.52 (± 0.8)
11.27 (± 0.3)
4.04 (± 0.2)
7.23 (± 0.2)
2.04 (± 0.5)
-0.69 (± 0.5)
2.73 (± 0.3)
-1.62 (± 0.2)
0.92 (± 0.3)
-2.54 (± 0.2)
11.17 (± 0.8)
5.06 (± 0.5)
6.12 (± 0.4)
2.36 (± 1.7)
0.46 (± 0.2)
1.90 (± 2.0)
7.65 (± 0.6)
8.84 (± 0.8)
-1.18 (± 1.4)
2.49 (± 0.1)
-1.55 (± 0.7)
4.04 (± 0.6)
4.59 (± 0.3)
5.40 (± 0.2)
-0.81 (± 0.4)
8.27 (± 0.3)
7.82 (± 0.6)
0.45 (± 0.3)
5.71 (± 0.2)
-1.61 (± 0.2)
7.32 (± 0.4)
4.29 (± 0.6)
-0.13 (± 0.4)
4.42 (± 0.2)
1.52 (± 0.0)
4.47 (± 0.3)
-2.95 (± 0.2)
2.08 (± 0.4)
4.14 (± 0.0)
-2.06 (± 0.1)
3.58 (± 0.0)
5.92 (± 0.4)
-2.33 (± 0.4)
-0.33 (± 0.4)
0.31 (± 0.2)
-0.64 (± 0.5)
-0.82 (± 0.2)
1.18 (± 0.2)
-1.99 (± 0.1)
1.23 (± 0.5)
3.63 (± 0.1)
-2.40 (± 0.4)
2.07 (± 0.2)
3.78 (± 0.4)
-1.71 (± 0.5)
-0.68 (± 0.5)
2.21 (± 0.0)
-2.89 (± 0.5)
3.49 (± 0.2)
2.70 (± 0.2)
0.79 (± 0.3)
0.12 (± 0.3)
0.54 (± 0.0)
-0.43 (± 0.4)
3.24 (± 0.4)
0.54 (± 0.3)
2.70 (± 0.2)
5.84 (± 0.0)
3.69 (± 0.1)
2.15 (± 0.0)
5.22 (± 0.5)
4.31 (± 0.2)
0.91 (± 0.3)
2.31 (± 0.1)
-0.87 (± 0.2)
3.18 (± 0.1)
3.84 (± 0.2)
3.20 (± 0.5)
0.64 (± 0.3)
6.88 (± 0.1)
5.84 (± 0.1)
1.03 (± 0.2)
1.70 (± 0.6)
4.08 (± 0.1)
-2.38 (± 0.7)
6.23 (± 0.3)
1.76 (± 0.6)
4.47 (± 0.4)
-0.45 (± 0.1)
-2.21 (± 0.4)
1.76 (± 0.4)
1.68 (± 0.5)
-0.46 (± 0.6)
2.14 (± 0.1)
7.44 (± 0.4)
6.51 (± 0.2)
0.93 (± 0.6)
1.69 (± 0.1)
2.16 (± 0.2)
-0.47 (± 0.3)
2.14 (± 0.4)
1.50 (± 0.1)
0.64 (± 0.6)
12.50 (± 0.2)
14.64 (± 0.7)
-2.14 (± 0.9)
-3.35 (± 0.1)
-4.67 (± 0.1)
1.32 (± 0.2)
-0.74 (± 0.4)
-3.51 (± 0.1)
2.76 (± 0.3)
The greatest difference in expression rate between both cell types was seen in dyhA which reveals a ~160-fold higher expression in somatic cells than in gonidia.
A quantitative analysis of cell-type specific gene expression by real-time RT-PCR requires a quick, efficient, and quantitative method for the physical removal of one cell type from another. After mechanical disruption of a multicellular organism, existing separation methods in other species take advantage of cell size, density, surface charge, hydrophobic surface properties, and antigen status in order to separate cell types. These methods are quite complicated in the majority of cases and include sedimentation, centrifugal elutriation, partitioning in aqueous two-phase systems, flow cytometry, immuno methods (including magnetic, column, and panning techniques), and free flow electrophoresis . In contrast, viable Volvox cells can be separated quite easily (see Methods) (Fig. 1B, C).
To identify divergent transcriptional activities in the two morphologically and functionally distinct cell types of Volvox carteri, we analyzed a pool of 39 genes; these genes were grouped into 7 subsets.
Gene subsets A-C: comparison of the obtained real-time RT-PCR results with the expectations
Four out of five mRNAs of subset A were localized just as expected: actA was expressed uniformly in somatic cells and gonidia and was used as a reference . ssgA and regA were expressed mainly in somatic cells, as previously described [9, 15]; ssgA showed a ~46-fold higher expression in somatic cells than in gonidia and expression of regA was ~150-fold higher in somatic cells. The gonidia-specific gene gon167  showed a ~6-fold higher expression in gonidia, just as expected. Only the gonidia-specific gene gon30  didn't meet the expectations at first sight, since there was a higher expression in somatic cells as compared to gonidia. However, gon30 is a very late "gonidial" gene , which has its maximal expression after cell cleavages, and the mRNAs for our experiments were isolated at the very beginning of cell cleavages. In contrast to gon30, expression of gon167 peaks at the beginning of cleavages. In the case of gon30, we obviously compared only the minimal mRNA level in gonidia with that of somatic cells; at this point, expression of gon30 is higher in somatic cells than in gonidia.
The mRNA of the sole member of subset B, rlsA, was localized as expected. Similar to the homologous gene regA, the rlsA gene is strongly expressed in somatic cells; there was a ~69-fold higher expression in somatic cells than in gonidia.
Subset C contained 4 genes with putative cell-type specific expression based on preliminary experiments [17, 18]. As expected, csrp1 was expressed mainly in somatic cells, but our result with this particular gene was not so clear due to high standard deviations for the ΔCt and ΔΔCt values (see above). gspk47 was expressed mainly in gonidia, as expected, although the ΔΔCt value was rather low. We couldn't confirm the localization of ard1 and mrp2: in our hands ard1 was expressed mainly in gonidia, and mrp2 was expressed mainly in somatic cells, with a ~16-fold higher expression in somatic cells. These results contradict previous preliminary results. The discrepancies between earlier results and our results within subset C might follow from differences in the experimental approach: we used a wild-type Volvox carteri strain for preparation of both gonidia as well as somatic cells. In the group of Dr. R.Schmitt [17, 18] a wild-type strain was used only for preparation of somatic cells, whereas the gonidia were isolated from a regA-mutant strain in which the somatic cells de-differentiated to gonidia because in this way it is much easier to isolate large amounts of RNA from this cell type. In the light of our results, it seems questionable that these secondary gonidia of regA-mutants show exactly the same expression pattern as wild-type gonidia. Another problem might be that it is not possible to synchronize regA-mutants (in contrast to wild-type algae); therefore every RNA preparation from regA-mutants is more or less heterogeneous with respect to the developmental stage and might even contain RNA from somatic cells that have not begun to de-differentiate.
Gene subsets D-G: validation of the obtained real-time RT-PCR results
Subset D contained only a single, well-known Volvox gene with a previously unknown status of cell-type specific expression. Here we show that this gene, nitA, is expressed more or less uniformly in somatic cells and gonidia (there is only an insignificant, 1.36-fold higher expression in somatic cells). The gene product of nitA, nitrate reductase, plays a central role in nitrate acquisition because it is the first enzyme in the pathway, and it is required for growth when nitrate is the sole nitrogen source. Therefore, it makes sense that both cell types express this gene similarly.
Subset E covered 7 novel Volvox genes, dyhA, klpA, fer1, nab1, rap41, fbp1, and cp12, which were identified via their characterized homologs from other species and in which, therefore, a cell-type specific expression seemed to be predictable due to the characteristics of the homologs. The motility-related genes dyhA and klpA showed, respectively, a ~160-fold and ~21-fold higher expression in somatic cells. This is a reasonable result because in V. carteri only somatic cells have flagella. Likewise, all five putative chloroplast/photosynthesis-related Volvox genes, fer1, nab1, rap41, fbp1, and cp12, were shown to be expressed predominantly in gonidia. Since chloroplast/photosynthesis-related metabolic activities are known to be localized mainly in the huge chloroplasts of gonidia and only to a minor degree in the small chloroplasts of somatic cells, the obtained expression rates are logical.
All 15 new Volvox genes within subset F have been identified via characterized homologs in other species, but, in contrast to the genes within subset E, the status of cell-type specific expression of these genes was not predictable. Four genes, prfA, fsd1, rpl37, and rcd1, were shown to be expressed predominantly in gonidia, and another seven genes, hsp40A, ubcA, mat3, sac1, adcA, nipA, and lciB, were mainly expressed in the somatic cells. The last four genes, hsp70B, glu1, vpeA, and ponA, were more or less uniformly expressed in somatic cells and gonidia; hsp70B showed a slightly higher expression level in gonidia, whereas glu1, vpeA, and ponA expression was somewhat higher in somatic cells.
Within subset G, which contains 6 new Volvox genes for which no characterized homologs in any other organism have been identified and the status of cell-type specific expression was unknown, one gene, upf4, was mainly expressed in gonidia, and two others, upf5 and upf6, were shown to be expressed predominantly in somatic cells. upf2 showed only a somewhat higher expression in gonidia, and expression of upf1 and upf3 was just slightly higher in somatic cells; these three genes were more or less uniformly expressed in the different cell types. Future experiments will have to reveal the concrete functions of the genes within subset G.
Taking into account all the gene subsets, it can be stated that if a prediction for expression in a specific cell-type was possible, this prediction came true, except for two genes within subset C. However, the discrepancy within subset C can possibly be explained by differences in the experimental approach (see above). Our findings with respect to the gon30 gene serve as a warning to any future investigators who employ these methods that it will be important to pay not only close attention to the spatial aspects of differential gene expression but also to temporal aspects; in addition, environmental conditions (light, temperature, culture medium etc.) should be kept in mind.
As expected, gonidia and somatic cells clearly differ in the composition of their mRNA pools, since it is this difference in cell-type specific gene expression which finally accounts for the different phenotypes of the two cell types. A cell-type specific gene expression does not necessarily result from a cell-type specific activation of these genes but can also come from a cell-type specific inhibition in the other cell-type. For example, it is known that the regulatory protein RegA acts on somatic cells to suppress gonidial development by inhibiting genes whose products are required for chloroplast biogenesis .
The results show that quantitative real-time RT-PCR is a favorable approach to analyze cell-type specific gene expression in Volvox carteri. Our approach not only provides a basis for a detailed analysis of individual, previously unknown Volvox genes of the investigated set of genes but also allows for future analysis of the same set of genes (by using the same primers and other RT-PCR conditions) with respect to inducibility by stress, wounding, deficiency or abundance of nutritional compounds, or response on the presence of the sex-inducer (the trigger of sexual development in V. carteri). Furthermore, it allows a characterization of the transcription of these genes in the life cycle (probably without separating cell-types because a separation of cell types from embryos or juveniles can not be achieved earlier than 18–20 h after the onset of embryogenesis, and embryonic and parent somatic cells can't be separated from each other even later . It is also possible to repeat these experiments using developmental or metabolic mutants instead. Finally, this approach can also be extended to a much larger number of genes. We hope that our analysis of cell-type specific expression of almost 40 genes was able to stimulate discussion about the application of a genome-wide expression analysis in Volvox in order to reveal the complete germ-soma program of this fascinating green alga.
The wild-type Volvox carteri f. nagariensis strain EVE (female) was obtained from D.L. Kirk (Washington University, St. Louis, MO) and was described previously . Cultures were grown in Volvox medium  at 30°C in an 8 h dark/16 h light (10000 lux) cycle .
Sequence analysis and homology search
The sequences of cDNA and genomic DNA fragments were compared with each other to exclude duplicates and to identify overlapping sequences which belong to the same gene by using DNASIS software (version 7.00; Hitachi Software Engineering, South San Francisco, CA). Homology searches with cDNA and genomic DNA fragments in different sequence databases were performed using BLAST . Initially, Volvox carteri f. nagariensis whole-genome shotgun reads at the Chlamydomonas reinhardtii web site (version 3.0) of the Joint Genome Institute (JGI)  were screened using BLASTn and blocks substitution matrix 62 (BLOSUM62)  for pairwise sequence alignment with a cut-off expectation value (E-value) of 10-5 and a word size of 3 (filtering disabled). Subsequently, the sequence databases of the National Center for Biotechnology Information (NCBI)  were searched for homologous protein sequences using tBLASTx with an expect threshold of 10, a word size of 3, gap costs of 11 for opening a gap, and gap costs of 1 for extending a gap (filtering disabled). Finally, the Chlamydomonas EST database at the Chlamydomonas reinhardtii web site (version 3.0) of JGI  was screened using tBLASTx and the BLOSUM62 scoring matrix with a cut-off E-value of 10-5 and a word size of 11 (filtering disabled).
Oligonucleotide primers for all PCR, standard RT-PCR or real-time RT-PCR were designed using the primer analysis software Oligo 6 (Molecular Biology Insights, Cascade, CO), Primer Express (Applied Biosystems, Foster City, CA), or DNASIS software (version 7.00; Hitachi Software Engineering, South San Francisco, CA). The primers used for real-time RT-PCR experiments are listed in Table 2.
Large-scale separation of cell types
Shortly before the onset of cell cleavage of reproductive cells (gonidia), 10-liter cultures of synchronously grown V. carteri spheroids were harvested by filtration on a 100-μm mesh nylon screen, and the concentration of organisms was brought to ~1000 spheroids/ml with Volvox medium. To obtain gonidia, the spheroids were broken up in a 50 ml Dounce homogenizer with a tight-fitting pestle (B. Braun, Melsungen, Germany) by moving the pestle up and down twice. The cell suspension was filtered through a 100 μm nylon screen, and the flow-through was filtered through a 40 μm nylon screen. Only free gonidia, single somatic cells, and small cell sheets containing several ECM-embedded somatic cells can pass the 40 μm nylon screen, whereas larger cell sheets, hemispheres of the spheroids, or spheroids which have only been slit remain on the nylon screens. The gonidia were separated from most residual somatic cells and ECM fragments by centrifuging for 5 min at 350 g in 7% (v/v) Percoll (Sigma-Aldrich, St. Louis, MO). Individual somatic cells that remained after this procedure were removed from the gonidia by filtering through a 10 μm nylon screen. Single somatic cells pass this screen, in contrast to gonidia. The gonidia were washed on the screen three times with 100 ml Volvox medium each and were used for the gonidial RNA preparation.
To obtain somatic cells, spheroids were broken up in the 50 ml Dounce homogenizer with a tight-fitting pestle as described above, exept the pestle was moved up and down seven times. The obtained cell suspension was diluted with medium to the two-fold volume and kept at room temperature for 20 min. Gonidia and larger fragments of spheroids that contain gonidia settled during this time by unit gravity. By contrast, somatic cell sheets without gonidia floated to the surface, were drawn off, and were used for the somatic RNA preparation.
Isolation of total RNA
Extraction of total RNA was done using 1 g frozen gonidia or somatic cells, 10 ml of the phenol-based TRI Reagent (Sigma-Aldrich, St. Louis, MO), and 3 ml trichloromethane. RNA was precipitated from the aqueous phase with isopropanol. RNA pellets were washed twice with 75% ethanol, air dried, and dissolved in RNase-free (DEPC treated) distilled water. RNA quantitation and purity check were done by agarose-formaldehyde gel electrophoresis and by measuring absorption at 260 and 280 nm with an Ultrospec 2100 pro UV/Visible Spectrophotometer (GE Healthcare, Uppsala, Sweden).
Isolation of genomic DNA
Genomic DNA was isolated from Volvox algae using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). DNA quantitation and purity check were done by agarose gel electrophoresis and UV-photometry using an Ultrospec 2100 pro UV/Visible Spectrophotometer (GE Healthcare, Uppsala, Sweden).
Genomic PCR was carried out in a total volume of 25 μl containing ~100 ng of genomic DNA, 1 μM of each primer, 0.1 mM dNTP, and 2 units Taq DNA polymerase in 1× PCR reaction buffer (20 mM Tris-HCl pH 8.8, 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 1 % Triton X-100, 1 mg/ml BSA). Forty cycles (95°C, 20 s; 55°C, 30 s; 72°C, 1 min) were performed with a T3 Thermocycler PCR system (Biometra, Göttingen, Germany). The lengths of PCR products were determined by comparison with DNA size markers (100 bp DNA marker, Fermentas, St. Leon-Rot, Germany and 1 kb DNA ladder, Invitrogen, Carlsbad, CA). Products of PCR amplification were cloned into the pBluescript II SK vector (Stratagene, La Jolla, CA) and sequenced.
For standard RT-PCR, first strand cDNA synthesis was performed using Moloney murine leukemia virus (MMLV) reverse transcriptase lacking ribonuclease H activity (H minus) (Promega, Madison, WI). 1 μg total RNA was incubated with 10 pmol of a specific reverse primer in a total volume of 14 μl at 70°C for 5 min and cooled immediately on ice for 5 min. After addition of 5 μl MMLV RT 5× reaction buffer (250 mM Tris-HCl pH 8.3 at 25°C, 250 mM KCl, 20 mM MgCl2, 50 mM DTT), 1.25 μl 10 mM dNTPs, and 200 units MMLV RT (H minus), cDNA synthesis was performed at 50°C for 60 min in a total volume of 25 μl. PCR was subsequently carried out using 10 μl of the reverse transcription mixture, 1 μM of each primer, 0.2 mM dNTPs, and 2.5 units Taq DNA polymerase in 1× PCR reaction buffer in a total volume of 50 μl. PCR was performed on a T3 Thermocycler PCR system (Biometra, Göttingen, Germany) using the following cycling conditions: 45 cycles of 95°C for 20 s, 55°C for 30 s, and 72°C for 40 s. Products of RT-PCR amplification were cloned into pSPT18/pSPT19 vectors (Roche, Penzberg, Germany) and sequenced.
1 μg total RNA was treated with 5 units DNaseI (Promega, Madison, WI) in DNase-I buffer (20 mM Tris, pH 8.4, 2 mM MgCl2, 50 mM KCl) in a total volume of 10 μl at 37°C for 10 min to remove contaminating DNA within the RNA preparation. The reaction was stopped by the addition of 1 μl 25 mM EDTA and incubation at 65°C for 10 min. Real-time quantification of RNA targets was done using the QuantiTect SYBR Green RT-PCR Kit (Qiagen, Hilden, Germany). Use of 2× QuantiTect SYBR Green RT-PCR Master Mix together with the QuantiTect RT Mix allows both reverse transcription and PCR to take place in a single tube. The components of 2× QuantiTect SYBR Green RT-PCR Master Mix include HotStarTaq DNA Polymerase, QuantiTect SYBR Green RT-PCR buffer, the fluorescent dye SYBR Green I, and the passive reference dye ROX. The QuantiTect RT Mix contains Omniscript and Sensiscript Reverse Transcriptases. Reactions contained 300 ng DNase-I-treated template RNA, 0.8 μM of each primer, 12.5 μl 2× QuantiTect SYBR Green RT-PCR Master Mix, and 0.25 μl QuantiTect RT Mix in a total volume of 25 μl. Reverse transcription occured at 50°C for 30 min. The subsequent incubation at 95°C for 15 min denatured the cDNA template, deactivated the reverse transcriptases, and activated the HotStarTaq DNA Polymerase. After this, 40 cycles of PCR amplification (95°C, 20 sec; 55°C, 30 sec; 72°C, 40 sec) followed. All real-time RT-PCR reactions were performed using a DNA Engine Opticon Continuous Fluorescence Detection System (MJ research, Waltham, MA). This system excites fluorescent dyes with absorption spectra in the 450 to 495 nm range, like SYBR Green I, and sensitive optics detect fluorophores with emission spectra in the 515–545 nm range (like SYBR Green I). Results were analyzed using OpticonMonitor software (version 1.06, MJ research, Waltham, MA). All real-time RT-PCR experiments were carried out in triplicate together with RT minus (RTM) and no template controls (NTC). The final products of all real-time RT-PCR reactions were visualized by agarose gel electrophoresis to assure amplification of a single product and to verify the size of the cDNA products by comparison with a 100 bp ladder (100 bp Marker, Fermentas, St. Leon-Rot, Germany).
Analysis of gene expression by using the 2-ΔΔCt method
The expression level of a given target gene in gonidia versus somatic cells was analyzed using real-time RT-PCR and the 2-ΔΔCt method [52, 53]. The Volvox actin gene, which is known to be similarly expressed in both cell types , was used as an internal control in all real-time RT-PCR experiments. In order to apply the 2-ΔΔCt method [52, 53], the results of real-time RT-PCRs were represented as cycle threshold (Ct) values. The Ct value was defined as the cycle at which a sample crosses a threshold which is significantly above the background fluorescence and within the exponential phase of the amplification. The average from three Ct measurements was calculated for both the given target gene and the actin gene. ΔCt was determined as the mean of the triplicate Ct values for the target genes minus the mean of the triplicate Ct values for the actin gene. For each target gene, ΔCt measurements were performed separately for each cell type. The ΔΔCt represented the difference between the two cell types for a given target gene, more precisely ΔΔCt = ΔCt (gonidia) - ΔCt (somatic cells). The ×-fold higher expression of a given target gene in gonidia compared to somatic cells was calculated as 2-ΔΔCt. If expression of a given target gene was lower in gonidia as compared to somatic cells, the expression was calculated by 1/2-ΔΔCt.
GenBank accession numbers
All 28 novel sequences described in this study have been deposited in GenBank under the following accession numbers:
dyhA [GenBank: EF123072], klpA [GenBank:EF123073],
fer1 [GenBank:EF123074], nab1 [GenBank:EF123075],
rap41 [GenBank:EF123076], fbp1 [GenBank: EF123077],
cp12 [GenBank: EF123078], prfA [GenBank: EF123079],
fsd1 [GenBank: EF123080], rpl37 [GenBank: EF123081],
glu1 [GenBank: EF123082], hsp70B [GenBank: EF123083],
hsp40A [GenBank: EF123084], ubcA [GenBank: EF123085],
ponA [GenBank: EF123086], mat3 [GenBank: EF123087],
vpeA [GenBank: EF123088], sac1 [GenBank: EF123089],
rcd1 [GenBank: EF123090], adcA [GenBank: EF123091],
nipA [GenBank: EF123092], lciB [GenBank: EF123093],
upf1 [GenBank: EF123094], upf2 [GenBank: EF123095],
upf3 [GenBank: EF123096], upf4 [GenBank: EF123097],
upf5 [GenBank: EF123098], and upf6 [GenBank: EF123099].
basic local alignment search tool
expressed sequence tag
polymerase chain reaction
reverse transcription-polymerase chain reaction
To obtain initial sequence information of several Volvox genes, Volvox whole-genome shotgun reads were kindly provided by Daniel Rokhsar (US Department of Energy Joint Genome Institute) . We wish to thank Rüdiger Schmitt (University of Regensburg, Germany) and his group for providing copies of finished diploma theses. We also want to thank Kordula Puls for technical assistance. This work was supported by a scholarship of the Studienstiftung des deutschen Volkes to GN.
- Starr RC: Control of differentiation in Volvox. Dev Biol Suppl. 1970, 4: 59-100.Google Scholar
- Green KJ, Kirk DL: Cleavage patterns, cell lineages, and development of a cytoplasmic bridge system in Volvox embryos. J Cell Biol. 1981, 91: 743-755. 10.1083/jcb.91.3.743.PubMedView ArticleGoogle Scholar
- Starr RC: Structure, reproduction and differentiation in Volvox carteri f. nagariensis Iyengar, strains HK9 & 10. Arch Protistenkd. 1969, 111: 204-222.Google Scholar
- Kirk DL: Volvox: Molecular-genetic Origins of Multicellularity and Cellular Differentiation. 1998, Cambridge: Cambridge University PressGoogle Scholar
- JGI Chlamydomonas reinhardtii genome v.3.0 portal. [http://genome.jgi-psf.org/Chlre3/Chlre3.home.html]
- Tam LW, Kirk DL: Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle. Dev Biol. 1991, 145: 51-66. 10.1016/0012-1606(91)90212-L.PubMedView ArticleGoogle Scholar
- Kirk DL: The ontogeny and phylogeny of cellular differentiation in Volvox. Trends Genet. 1988, 4: 32-36. 10.1016/0168-9525(88)90063-7.PubMedView ArticleGoogle Scholar
- Kirk DL, Kaufman MR, Keeling RM, Stamer KA: Genetic and cytological control of the asymmetric divisions that pattern the Volvox embryo. Dev Suppl. 1991, 1: 67-82.PubMedGoogle Scholar
- Kirk MM, Stark K, Miller SM, Müller W, Taillon BE, Gruber H, Schmitt R, Kirk DL: regA, a Volvox gene that plays a central role in germ-soma differentiation, encodes a novel regulatory protein. Development. 1999, 126: 639-647.PubMedGoogle Scholar
- Kirk DL, Kirk MM: Protein synthetic patterns during the asexual life cycle of Volvox carteri. Dev Biol. 1983, 96: 493-506. 10.1016/0012-1606(83)90186-0.PubMedView ArticleGoogle Scholar
- Cresnar B, Mages W, Müller K, Salbaum JM, Schmitt R: Structure and expression of a single actin gene in Volvox carteri. Curr Genet. 1990, 18: 337-346. 10.1007/BF00318215.PubMedView ArticleGoogle Scholar
- Amon P, Haas E, Sumper M: The sex-inducing pheromone and wounding trigger the same set of genes in the multicellular green alga Volvox. Plant Cell. 1998, 10: 781-789. 10.1105/tpc.10.5.781.PubMedPubMed CentralView ArticleGoogle Scholar
- Hallmann A: The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales. Plant J. 2006, 45: 292-307. 10.1111/j.1365-313X.2005.02627.x.PubMedView ArticleGoogle Scholar
- Hallmann A, Amon P, Godl K, Heitzer M, Sumper M: Transcriptional activation by the sexual pheromone and wounding: a new gene family from Volvox encoding modular proteins with (hydroxy)proline-rich and metalloproteinase homology domains. Plant J. 2001, 26: 583-593. 10.1046/j.1365-313x.2001.01059.x.PubMedView ArticleGoogle Scholar
- Ertl H, Mengele R, Wenzl S, Engel J, Sumper M: The extracellular matrix of Volvox carteri: molecular structure of the cellular compartment. J Cell Biol. 1989, 109: 3493-3501. 10.1083/jcb.109.6.3493.PubMedView ArticleGoogle Scholar
- Duncan L, Nishii I, Howard A, Kirk D, Miller SM: Orthologs and paralogs of regA, a master cell-type regulatory gene in Volvox carteri. Curr Genet. 2006, 50: 61-72. 10.1007/s00294-006-0071-4.PubMedView ArticleGoogle Scholar
- Schmidt K: Gonidien- und somazell-spezifisch exprimierte Gene bei Volvox carteri. Diploma Thesis. 2004, Regensburg, Germany: University of RegensburgGoogle Scholar
- Amann K: Identifizierung neuer regA-kontrollierter Gene bei der grünen Kugelalge Volvox carteri. Diploma Thesis. 2002, Regensburg, Germany: University of RegensburgGoogle Scholar
- Gruber H, Goetinck SD, Kirk DL, Schmitt R: The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics. Gene. 1992, 120: 75-83. 10.1016/0378-1119(92)90011-D.PubMedView ArticleGoogle Scholar
- Schiedlmeier B, Schmitt R, Müller W, Kirk MM, Gruber H, Mages W, Kirk DL: Nuclear transformation of Volvox carteri. Proc Natl Acad Sci USA. 1994, 91: 5080-5084. 10.1073/pnas.91.11.5080.PubMedPubMed CentralView ArticleGoogle Scholar
- Mitchell DR, Brown KS: Sequence analysis of the Chlamydomonas alpha and beta dynein heavy chain genes. J Cell Sci. 1994, 107 (Pt 3): 635-644.PubMedGoogle Scholar
- Mitchell DR, Brown KS: Sequence analysis of the Chlamydomonas reinhardtii flagellar alpha dynein gene. Cell Motil Cytoskeleton. 1997, 37: 120-126. 10.1002/(SICI)1097-0169(1997)37:2<120::AID-CM4>3.0.CO;2-C.PubMedView ArticleGoogle Scholar
- Walther Z, Vashishtha M, Hall JL: The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. J Cell Biol. 1994, 126: 175-188. 10.1083/jcb.126.1.175.PubMedView ArticleGoogle Scholar
- Kozminski KG, Beech PL, Rosenbaum JL: The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. J Cell Biol. 1995, 131: 1517-1527. 10.1083/jcb.131.6.1517.PubMedView ArticleGoogle Scholar
- Mussgnug JH, Wobbe L, Elles I, Claus C, Hamilton M, Fink A, Kahmann U, Kapazoglou A, Mullineaux CW, Hippler M, et al: NAB1 is an RNA binding protein involved in the light-regulated differential expression of the light-harvesting antenna of Chlamydomonas reinhardtii. Plant Cell. 2005, 17: 3409-3421. 10.1105/tpc.105.035774.PubMedPubMed CentralView ArticleGoogle Scholar
- Yamaguchi K, Beligni MV, Prieto S, Haynes PA, McDonald WH, Yates JR, Mayfield SP: Proteomic characterization of the Chlamydomonas reinhardtii chloroplast ribosome. Identification of proteins unique to the 70 S ribosome. J Biol Chem. 2003, 278: 33774-33785. 10.1074/jbc.M301934200.PubMedView ArticleGoogle Scholar
- Rodriguez-Suarez RJ, Wolosiuk RA: Sequence of a cDNA encoding chloroplast fructose-1,6-bisphosphatase from rapeseed. Plant Physiol. 1993, 103: 1453-1454. 10.1104/pp.103.4.1453.PubMedPubMed CentralView ArticleGoogle Scholar
- Wedel N, Soll J: Evolutionary conserved light regulation of Calvin cycle activity by NADPH-mediated reversible phosphoribulokinase/CP12/glyceraldehyde-3-phosphate dehydrogenase complex dissociation. Proc Natl Acad Sci USA. 1998, 95: 9699-9704. 10.1073/pnas.95.16.9699.PubMedPubMed CentralView ArticleGoogle Scholar
- Stark K, Schmitt R: Genetic control of germ-soma differentiation in Volvox carteri. Protist. 2002, 153: 99-107. 10.1078/1434-4610-00088.PubMedView ArticleGoogle Scholar
- Kovar DR, Yang P, Sale WS, Drobak BK, Staiger CJ: Chlamydomonas reinhardtii produces a profilin with unusual biochemical properties. J Cell Sci. 2001, 114: 4293-4305.PubMedGoogle Scholar
- Chen H, Romo-Leroux PA, Salin ML: The iron-containing superoxide dismutase-encoding gene from Chlamydomonas reinhardtii obtained by direct and inverse PCR. Gene. 1996, 168: 113-116. 10.1016/0378-1119(95)00691-5.PubMedView ArticleGoogle Scholar
- Barnard GF, Staniunas RJ, Puder M, Steele GD, Chen LB: Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain. Biochim Biophys Acta. 1994, 1218: 425-428.PubMedView ArticleGoogle Scholar
- Dincturk HB, Knaff DB: The evolution of glutamate synthase. Mol Biol Rep. 2000, 27: 141-148. 10.1023/A:1007107909619.PubMedView ArticleGoogle Scholar
- Sung DY, Vierling E, Guy CL: Comprehensive expression profile analysis of the Arabidopsis hsp70 gene family. Plant Physiol. 2001, 126: 789-800. 10.1104/pp.126.2.789.PubMedPubMed CentralView ArticleGoogle Scholar
- Cheetham ME, Brion JP, Anderton BH: Human homologues of the bacterial heat-shock protein DnaJ are preferentially expressed in neurons. Biochem J. 1992, 284 (Pt 2): 469-476.PubMedPubMed CentralView ArticleGoogle Scholar
- van Nocker S, Walker JM, Vierstra RD: The Arabidopsis thaliana UBC7/13/14 genes encode a family of multiubiquitin chain-forming E2 enzymes. J Biol Chem. 1996, 271: 12150-12158. 10.1074/jbc.271.21.12150.PubMedView ArticleGoogle Scholar
- Bauer A, Huber O, Kemler R: Pontin52, an interaction partner of beta-catenin, binds to the TATA box binding protein. Proc Natl Acad Sci USA. 1998, 95: 14787-14792. 10.1073/pnas.95.25.14787.PubMedPubMed CentralView ArticleGoogle Scholar
- Umen JG, Goodenough UW: Control of cell division by a retinoblastoma protein homolog in Chlamydomonas. Genes Dev. 2001, 15: 1652-1661. 10.1101/gad.892101.PubMedPubMed CentralView ArticleGoogle Scholar
- Hatsugai N, Kuroyanagi M, Yamada K, Meshi T, Tsuda S, Kondo M, Nishimura M, Hara-Nishimura I: A plant vacuolar protease, VPE, mediates virus-induced hypersensitive cell death. Science. 2004, 305: 855-858. 10.1126/science.1099859.PubMedView ArticleGoogle Scholar
- Davies JP, Yildiz FH, Grossman A: Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation. EMBO J. 1996, 15: 2150-2159.PubMedPubMed CentralGoogle Scholar
- Ravina CG, Chang CI, Tsakraklides GP, McDermott JP, Vega JM, Leustek T, Gotor C, Davies JP: The sac mutants of Chlamydomonas reinhardtii reveal transcriptional and posttranscriptional control of cysteine biosynthesis. Plant Physiol. 2002, 130: 2076-2084. 10.1104/pp.012484.PubMedPubMed CentralView ArticleGoogle Scholar
- Hiroi N, Ito T, Yamamoto H, Ochiya T, Jinno S, Okayama H: Mammalian Rcd1 is a novel transcriptional cofactor that mediates retinoic acid-induced cell differentiation. EMBO J. 2002, 21: 5235-5244. 10.1093/emboj/cdf521.PubMedPubMed CentralView ArticleGoogle Scholar
- Asamizu E, Miura K, Kucho K, Inoue Y, Fukuzawa H, Ohyama K, Nakamura Y, Tabata S: Generation of expressed sequence tags from low-CO2 and high-CO2 adapted cells of Chlamydomonas reinhardtii. DNA Res. 2000, 7: 305-307. 10.1093/dnares/7.5.305.PubMedView ArticleGoogle Scholar
- Miura K, Yamano T, Yoshioka S, Kohinata T, Inoue Y, Taniguchi F, Asamizu E, Nakamura Y, Tabata S, Yamato KT, et al: Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling a carbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol. 2004, 135: 1595-1607. 10.1104/pp.104.041400.PubMedPubMed CentralView ArticleGoogle Scholar
- Fisher D, Francis GE, Rickwood D: Cell Separation: A Practical Approach. The Practical Approach Series. Edited by: Rickwood D, Hames BD. 1998, Oxford: Oxford University Press, 193:Google Scholar
- Adams CR, Stamer KA, Miller JK, McNally JG, Kirk MM, Kirk DL: Patterns of organellar and nuclear inheritance among progeny of two geographically isolated strains of Volvox carteri. Curr Genet. 1990, 18: 141-153. 10.1007/BF00312602.PubMedView ArticleGoogle Scholar
- Provasoli L, Pintner IJ: Artificial media for freshwater algae: problems and suggestions. The Ecology of Alga. Edited by: Tyron CA, Hartman RT. 1959, Pittsburgh, PA: Pymatuning Laboratory of Field Biology, Special Publication no. 2, University of Pittsburgh, 84-96.Google Scholar
- Starr RC, Jaenicke L: Purification and characterization of the hormone initiating sexual morphogenesis in Volvox carteri f. nagariensis Iyengar. Proc Natl Acad Sci USA. 1974, 71: 1050-1054. 10.1073/pnas.71.4.1050.PubMedPubMed CentralView ArticleGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1006/jmbi.1990.9999.PubMedView ArticleGoogle Scholar
- Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA. 1992, 89: 10915-10919. 10.1073/pnas.89.22.10915.PubMedPubMed CentralView ArticleGoogle Scholar
- NCBI BLAST. [http://www.ncbi.nlm.nih.gov/blast/index.shtml]
- Bustin SA: Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol. 2000, 25: 169-193. 10.1677/jme.0.0250169.PubMedView ArticleGoogle Scholar
- Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 2001, 29: e45-10.1093/nar/29.9.e45.PubMedPubMed CentralView ArticleGoogle Scholar
- DOE Joint Genome Institute (JGI). [http://www.jgi.doe.gov/index.html]
- Sugase Y, Hirono M, Kindle KL, Kamiya R: Cloning and characterization of the actin-encoding gene of Chlamydomonas reinhardtii. Gene. 1996, 168: 117-121. 10.1016/0378-1119(95)00711-3.PubMedView ArticleGoogle Scholar
- Meissner M, Stark K, Cresnar B, Kirk DL, Schmitt R: Volvox germline-specific genes that are putative targets of RegA repression encode chloroplast proteins. Curr Genet. 1999, 36: 363-370. 10.1007/s002940050511.PubMedView ArticleGoogle Scholar
- Duncan L, Bouckaert K, Yeh F, Kirk DL: kangaroo, a mobile element from Volvox carteri, is a member of a newly recognized third class of retrotransposons. Genetics. 2002, 162: 1617-1630.PubMedPubMed CentralGoogle Scholar
- Johnson CH, Kruft V, Subramanian AR: Identification of a plastid-specific ribosomal protein in the 30 S subunit of chloroplast ribosomes and isolation of the cDNA clone encoding its cytoplasmic precursor. J Biol Chem. 1990, 265: 12790-12795.PubMedGoogle Scholar
- Im CS, Grossman AR: Identification and regulation of high light-induced genes in Chlamydomonas reinhardtii. Plant J. 2002, 30: 301-313. 10.1046/j.1365-313X.2001.01287.x.PubMedView ArticleGoogle Scholar
- Fernandez E, Schnell R, Ranum LP, Hussey SC, Silflow CD, Lefebvre PA: Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA. 1989, 86: 6449-6453. 10.1073/pnas.86.17.6449.PubMedPubMed CentralView ArticleGoogle Scholar
- Stein M, Jacquot JP, Miginiac-Maslow M: A cDNA clone encoding Chlamydomonas reinhardtii preferredoxin. Plant Physiol. 1993, 102: 1349-1350. 10.1104/pp.102.4.1349.PubMedPubMed CentralView ArticleGoogle Scholar
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