We report a comprehensive large-scale expression profiling analysis of mammalian male germ cells undergoing mitotic growth, meiosis, and gametogenesis by using high-density oligonucleotide microarrays and highly enriched cell populations. Among 11,955 rat loci investigated, 1268 were identified as differentially transcribed in germ cells at subsequent developmental stages compared with total testis, somatic Sertoli cells as well as brain and skeletal muscle controls. The loci were organized into four expression clusters that correspond to somatic, mitotic, meiotic, and postmeiotic cell types. This work provides information about expression patterns of approximately 200 genes known to be important during male germ cell development. Approximately 40 of those are included in a group of 121 transcripts for which we report germ cell expression and lack of transcription in three somatic control cell types. Moreover, we demonstrate the testicular expression and transcriptional induction in mitotic, meiotic, and/or postmeiotic germ cells of 293 as yet uncharacterized transcripts, some of which are likely to encode factors involved in spermatogenesis and fertility. This group also contains potential germ cell-specific targets for innovative contraceptives. A graphical display of the data is conveniently accessible through the GermOnline database at http://www.germonline.org.

The GATA transcription factors GLN3 and GAT1 activate nitrogen-regulated genes in Saccharomyces cerevisiae. NPR1 is a protein kinase that controls post-Golgi sorting of amino acid permeases. In the presence of a good nitrogen source, TOR (target of rapamycin) maintains GLN3 and NPR1 phosphorylated and inactive by inhibiting the type 2A-related phosphatase SIT4. We identified NPR1 as a regulator of GLN3. Specifically, loss of NPR1 causes nuclear translocation and activation of GLN3, but not GAT1, in nitrogen-rich conditions. NPR1-mediated inhibition of GLN3 is independent of the phosphatase SIT4. We also demonstrate that the E3/E4 ubiquitin-protein ligase proteins RSP5 and BUL1/2 are required for GLN3 activation under poor nitrogen conditions. Thus, NPR1 and BUL1/2 antagonistically control GLN3-dependent transcription, suggesting a role for regulated ubiquitination in the control of nutrient-responsive transcription.In yeast, the quality of the available nitrogen source controls the expression of genes encoding proteins required for the uptake and assimilation of nitrogenous compounds. This regulatory process is mediated by four GATA transcription factors, the activators GLN3 and GAT1/NIL1 and the repressors DAL80 and DEH1/NIL2/GZF3. GLN3 is the major activator of nitrogen-regulated genes (for recent reviews, see Refs. 1 and 2). URE2 is a GLN3-binding protein that retains GLN3 in the cytoplasm to prevent its nuclear translocation and activation during growth under good nitrogen conditions.The two homologous kinases TOR1 1 and TOR2 are central controllers of cell growth, and their inactivation with rapamycin causes physiological changes characteristic of nutrientstarved cells (3, 4). A TOR signaling pathway regulates the expression of nitrogen-regulated genes by inhibiting GLN3 and GAT1 (5). In the presence of a preferred nitrogen source such as NH 4 ϩ or glutamine, TOR promotes complex formation between GLN3 and URE2 by maintaining GLN3 in the phosphorylated state necessary for URE2 binding (5). Upon nitrogen limitation or rapamycin treatment, the TOR-controlled phosphatase SIT4 causes GLN3 dephosphorylation, and GLN3 dissociates from URE2 and translocates into the nucleus to activate target genes (5).NPR1 encodes a Ser/Thr kinase that controls the post-Golgi sorting and degradation of amino acid permeases. NPR1 inversely regulates GAP1, the general amino acid permease, and TAT2, a tryptophan permease (2). In cells grown in poor nitrogen sources, NPR1 is dephosphorylated by the TOR-modulated SIT4 phosphatase to become active (6). In turn, active NPR1 allows GAP1 to reach the plasma membrane and causes the rapid degradation of TAT2. In cells shifted to a good nitrogen source medium, plasma membrane GAP1 is internalized and pre-existing permease is no longer delivered to the plasma membrane, whereas TAT2 is stabilized at the plasma membrane (7-9). GAP1 down-regulation occurs via its ubiquitination by the E3 ubiquitin protein ligase RSP5 in conjunction with the RSP5-binding proteins BUL1 and BUL2 (8,10,11).Here, we ...

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Beta-thymosins are actin monomer-binding polypeptides that are expressed in a neuronal growth-specific manner during embryonic development. Here, we show that regenerating retinal ganglion cells and non-neuronal cells of the optic nerve transiently activate beta-thymosin transcription after optic nerve lesion in the zebrafish. In retinal cell cultures, beta-thymosin is found at highest concentration in growth cones, branching points and varicosities of neurite-extending retinal ganglion cells. These places often exhibit reduced phalloidin staining, indicating that beta-thymosin promotes the disassembly of actin filaments. Beta-thymosin distribution within neurons in culture is distinct from actin, tubulin and the actin-severing protein gelsolin. Ectopic expression of beta-thymosin in a central nervous system (CNS) catecholaminergic cell line leads to alterations in the shape of the cell bodies and neurites. Beta-thymosin-positive cells spread more fully and exhibit an excessive degree of branching. We partially cloned two other actin-binding proteins, profilin and gelsolin, and analysed their expression patterns. Profilin is constitutively expressed in virtually all cells. Gelsolin, like beta-thymosin, is selectively increased in regenerating retinal ganglion cells. During development, however, gelsolin mRNA is not detected in the nervous system. These findings indicate that distinct mechanisms control the actin cytoskeleton in embryonic and regenerating neurons, and that beta-thymosin may be a major regulator of actin dynamics in the zebrafish CNS.