Channelrhodopsins (CHR1 and CHR2) are light-gated ion channels acting as sensory photoreceptors in Chlamydomonas reinhardtii. In neuroscience, they are used to trigger action potentials by light in neuronal cells, tissues, or living animals. Here, we demonstrate that Chlamydomonas cells with low CHR2 content exhibit photophobic and phototactic responses that strictly depend on the availability of CHR1. Since CHR1 was described as a H þ -channel, the ion specificity of CHR1 was reinvestigated in Xenopus laevis oocytes. Our experiments show that, in addition to H þ , CHR1 also conducts Na þ , K þ , and Ca 2þ . The kinetic selectivity analysis demonstrates that H þ selectivity is not due to specific translocation but due to selective ion binding. Purified recombinant CHR1 consists of two isoforms with different absorption maxima, CHR1 505 and CHR1 463 , that are in pH-dependent equilibrium. Thus, CHR1 is a photochromic and protochromic sensory photoreceptor that functions as a light-activated cation channel mediating phototactic and photophobic responses via depolarizing currents in a wide range of ionic conditions.

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Stable nuclear transformation of Volvox carten was achieved using the cloned V. carteni itAr gene (which encodes nitrate reductase) to complement a nitA mutation. Following bombardment of mutant cells with plasmid-coated gold particles, putative transformants able to utilize nitrate as a nitrogen source were recovered with an efficiency of -2.5 x 10-5. DNA analysis indicated that the plasmid integrated into the genome, often in multiple copies, at sites other than the nitA locus. Cotransformants were recovered with a frequency of 40-80% when cells were cobombarded with a selected and an unselected marker. Thus, V. cartie becomes one ofthe simplest multicellular organisms that Is accessible to detailed molecular studies of genes regulating cellular differentiation and morphogenesis.Volvox carteri is a multicellular organism with a complete division of labor between somatic and reproductive cells (1, 2). Genetic analysis (1-4) has led to the hypothesis that a small number of loci act to cause differentiation of these two cell types (5), and patterns of cell-type-specific gene expression in wild-type and mutant embryos are consistent with that hypothesis (6). However, detailed molecular analysis ofthese putative regulatory loci has awaited a method for transforming the organism with exogenous DNA.We repeatedly tried to transform V. carteri with various bacterial or plant selectable markers that were introduced by microinjection, electroporation, particle bombardment (7), UV-laser microbeam irradiation (8), agitation with glass beads (9), etc. As with the related unicellular alga, Chlamydomonas reinhardtii, reproducible transformation with heterologous selectable markers was not achieved, possibly because of an inability of these algae to express heterologous genes. Again in parallel with C. reinhardtii (9-12), success in transforming V. carteri has come with the availability of a homologous selectable marker: here we report use of the recently cloned nitrate reductase-encoding gene of V. carteri, nitA (13), to complement a nitA mutation. MATERIALS AND METHODSRecipient Strains. Strains used as DNA recipients were F1 female progeny of HB11A, a previously described, multiply marked strain of V. carteri f. nagariensis (4). All of these strains inherited from HB11A a stable mutant allele (reversion rate, <10-8) that confers resistance to chlorate, abolishes the ability to utilize nitrate as a nitrogen source, maps to the nitA locus, and is therefore inferred to be a stable loss-of-function mutation of nitA, the gene encoding nitrate reductase (13). Strain 153-81 was given the mnemonic Gls/ Reg ("gonidialess/regenerator") because it inherited from HB11A a regA mutation that causes somatic cells to redifferentiate as gonidia (asexual reproductive cells) and also has a spontaneous mutation at the gis ("gonidialess") locus that results in an absence of any "true" gonidia (6). GIs/Reg was used in initial studies because it has only one type of cells, all of which can reproduce; thus it provides a homogeneous populati...

The doublecortin (DCX) gene encodes a 40-kDa microtubuleassociated protein specifically expressed in neuronal precursors of the developing and adult CNS. Due to its specific expression pattern, attention was drawn to DCX as a marker for neuronal precursors and neurogenesis, thereby underscoring the importance of its promoter identification and promoter analysis. Here, we analysed the human DCX regulatory sequence and confined it to a 3.5-kb fragment upstream of the ATG start codon. We demonstrate by transient transfection experiments that this fragment is sufficient and specific to drive expression of reporter genes in embryonic and adult neuronal precursors. The activity of this regulatory fragment overlapped with the expression of endogenous DCX and with the young neuronal markers class III b-tubulin isotype and microtubule-associated protein Map2ab but not with glial or oligodendroglial markers. Electrophysiological data further confirmed the immature neuronal nature of these cells. Deletions within the 3.5-kb region demonstrated the relevance of specific regions containing transcription factor-binding sites. Moreover, application of neurogenesis-related growth factors in the neuronal precursor cultures suggested the lack of direct signalling of these factors on the DCX promoter construct.

We have cloned and characterized a single copy C. reinhardtii gene containing an open reading frame of 333 nucleotides encoding a 12.7 kDa protein. The novel protein, DIP13, exhibits 60% identity with two mammalian proteins, human NA14 and an unnamed mouse protein. Homologous sequences are also present in several protozoan, trematode and fish genomes, but no homologs have been found in the completed genomes of yeast, Drosophila, C. elegans and A. thaliana. By using a specific antibody we have localized DIP13 to microtubule structures, namely basal bodies, flagellar axonemes and cytoplasmic microtubules. Anti-DIP13 antibody also specifically recognized human NA14 by immunofluorescence and stained basal bodies and flagella of human sperm cells as well as the centrosome of HeLa cells. Expression of the DIP13 open reading frame in antisense orientation in Chlamydomonas resulted in multinucleate, multiflagellate cells,which suggests a role for this protein in ensuring proper cell division. Thus,DIP13/NA14 could represent the founding members of a new class of highly conserved proteins that are associated with microtubule structures.