Germ cells are distinct from somatic cells in their immortality, totipotency, and ability to undergo meiosis. Candidates for components that guide the unique germline program are the distinctive granules observed in germ cells of many species. We show that a component of germ granules is essential for fertility in C. elegans and that its primary function is in germline proliferation. This role has been revealed by molecular and genetic analyses of pgl-1. PGL-1 is a predicted RNA-binding protein that is present on germ granules at all stages of development. Elimination of PGL-1 results in defective germ granules and sterility. Interestingly, PGL-1 function is required for fertility only at elevated temperatures, suggesting that germline development is inherently sensitive to temperature.

Two components of the germ-line-specific P granules of the nematode Caenorhabditis elegans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stages of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event.Embryos of the free-living soil nematode Caenorhabditis elegans generate distinct founder cells via a series of asymmetric cell divisions. At each division, the germ-line daughter cell inherits distinctive non-membrane-bound particles, called P granules (1-3). P granules are partitioned to the primordial germ cell P 4 of the 16-to 24-cell embryo and become perinuclear. P granules persist around the nuclei of all germ cells, until gametogenesis, at which point they are excluded from mature sperm and become dispersed within the cytoplasm of mature oocytes in preparation for cytoplasmic localization in the embryo. Although the distribution pattern of nematode P granules has been well-studied, the identity and function of P-granule components have yet to be determined.Germ granules are found in many species (4, 5). The germ-line-specific polar granules of Drosophila melanogaster have been well-studied, with a number of different genes identified that are required for polar granule assembly and germ-cell formation, including vasa, staufen, valois, oskar, tudor, mago nashi, and germ-cell-less (6-15). With the exception of vasa, these genes encode novel proteins. Vasa, however, is a member of a family of proteins with recognizable motifs and predictable function. Vasa is an RNA helicase of the DEAD-box family (8, 9) whose ATP-dependent RNA helicase activity has been demonstrated in vitro (16). As polar granules contain RNA as well as protein (11,12,15,17), a germ-linespecific RNA helicase may function to bind and unwind RNAs necessary for germ-line development. Several potential vasa homologues have been cloned, including glh-1 (germ-line helicase 1) from Caenorhabditis, Xvh (Xenopus vasa homologue), mvh (mouse vasa homologue), and rvh (rat vasa homologue) (18)(19)(20)(21). glh-1 in C. elegans is unique among RNA helicase genes reported, including vasa, in that its predicted product contains four retroviral-like zinc fingers (18). We have identified a second C. elegans germ-line RNA helicase gene, glh-2, that also encodes zinc fingers. Immunolocali...

Here we show that emb-30 is required for metaphase-to-anaphase transitions during meiosis and mitosis in Caenorhabditis elegans. Germline-specific emb-30 mutant alleles block the meiotic divisions. Mutant oocytes, fertilized by wild-type sperm, set up a meiotic spindle but do not progress to anaphase I. As a result, polar bodies are not produced, pronuclei fail to form, and cytokinesis does not occur. Severe-reduction-of-function emb-30 alleles (class I alleles) result in zygotic sterility and lead to germline and somatic defects that are consistent with an essential role in promoting the metaphase-to-anaphase transition during mitosis. Analysis of the vulval cell lineages in these emb-30(class I) mutant animals suggests that mitosis is lengthened and eventually arrested when maternally contributed emb-30 becomes limiting. By further reducing maternal emb-30 function contributed to class I mutant animals, we show that emb-30 is required for the metaphase-toanaphase transition in many, if not all, cells. Metaphase arrest in emb-30 mutants is not due to activation of the spindle assembly checkpoint but rather reflects an essential emb-30 requirement for M-phase progression. A reduction in emb-30 activity can suppress the lethality and sterility caused by a null mutation in mdf-1, a component of the spindle assembly checkpoint machinery. This result suggests that delaying anaphase onset can bypass the spindle checkpoint requirement for normal development. Positional cloning established that emb-30 encodes the likely C. elegans orthologue of APC4/Lid1, a component of the anaphase-promoting complex/cyclosome, required for the metaphase-to-anaphase transition. Thus, the anaphase-promoting complex/cyclosome is likely to be required for all metaphase-to-anaphase transitions in a multicellular organism.

The Vasa DEAD-box helicases are widespread markers of germ cells across species, and in some organisms have been shown to be essential for germ-cell formation and development. In contrast to the single Vasa gene in most systems analyzed, Caenorhabditis elegans has four Vasa family members, the germline helicases GLH-1, GLH-2, GLH-3, and GLH-4. Our analysis of deletion alleles of each glh gene demonstrates that GLH-1 is the key member of the family: loss of GLH-1 function causes sterility that is mainly maternal effect, is manifested predominantly at elevated temperature, and is due to reduced germcell proliferation and impaired formation of both sperm and oocytes. The other GLHs are not essential. However, GLH-4 serves redundant roles with GLH-1: loss of both genes' function causes glh-1-like sterility at all temperatures. Molecular epistasis analysis demonstrates that GLH-1 and GLH-4 are required for proper association of the PGL family of proteins with P granules, suggesting a pathway of P-granule assembly in which the GLHs are upstream of the PGL proteins and the mRNA cap-binding protein IFE-1. While loss of some P-granule components causes worms to be defective in RNA interference, loss of GLH-1 and GLH-4 does not compromise RNAi. Thus, RNAi likely does not require intact P granules but instead relies on particular P-granule factors. We discuss the evolution of the Vasa/GLH genes and current views of their functions and the assembly and roles of germ granules among species.

Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF II s), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAF II 48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF II 110) and human TAF4 (hTAF II 135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved Cterminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperaturesensitive mutations in the yTAF4 HFD ␣2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF II 68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the ␣3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide ␣3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD ␣3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the ␣3 helix is separated from the ␣2 helix by an extended loop containing a conserved functional domain.Accurate transcription initiation at protein-coding genes by RNA polymerase II requires the assembly of a multiprotein complex around the mRNA start site (1). Transcription factor TFIID is one of the general factors involved in this process. TFIID comprises the TATA binding protein (TBP), 1 responsible for specific binding to the TATA element found in many RNA polymerase II promoters, and a set of TBP-associated factors (TAF II s) (2, 3). A subset of TAF II s are present not only in TFIID but also in the SAGA, PCAF, STAGA, and TFTC complexes that lack TBP but are involved in RNA polymerase II transcription (4 -8). A subset of TAF II s are also found in a macromolecular complex containing Drosophila polycomb group proteins (9).

An important goal is to identify the direct activation domain (AD)-interacting components of the transcriptional machinery within the context of native complexes. Toward this end, we first demonstrate that the multisubunit TFIID, SAGA, mediator, and Swi/Snf coactivator complexes from transcriptionally competent whole-cell yeast extracts were all capable of specifically interacting with the prototypic acidic ADs of Gal4 and VP16. We then used hexahistidine tags as genetically introduced activation domain-localized cross-linking receptors. In combination with immunological reagents against all subunits of TFIID and SAGA, we systematically identified the direct AD-interacting subunits within the AD-TFIID and AD-SAGA coactivator complexes enriched from whole-cell extracts and confirmed these results using purified TFIID and partially purified SAGA. Both ADs directly cross-linked to TBP and to a subset of TFIID and SAGA subunits that carry histonefold motifs.

We have performed a systematic structure-function analysis of Saccharomyces cerevisiae TAF25, an evolutionarily conserved, single-copy essential gene which encodes the 206-amino-acid TAF25p protein. TAF25p is an integral subunit of both the 15-subunit general transcription factor TFIID and the multisubunit, chromatinacetylating transcriptional coactivator SAGA. We used hydroxylamine mutagenesis, targeted deletion, alaninescanning mutagenesis, high-copy suppression methods, and two-hybrid screening to dissect TAF25. Temperature-sensitive mutant strains generated were used for coimmunoprecipitation and transcription analyses to define the in vivo functions of TAF25p. The results of these analyses show that TAF25p is comprised of multiple mutable elements which contribute importantly to RNA polymerase II-mediated mRNA gene transcription.mRNA gene transcription is mediated by RNA polymerase II working in concert with multiple general transcription factors (GTFs). The basal mRNA gene transcription machinery, as originally defined in vitro, is comprised of the GTFs TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH, which function with RNA polymerase II to promote preinitiation complex (PIC) formation in vitro and in vivo (see references 29 and 55 for reviews). GTFs contribute to PIC formation in multiple ways, either acting sequentially in a stepwise fashion (7) to form a PIC or acting as a single unit comprising the RNA polymerase II holoenzyme (35,39). Although the composition of the basal transcription machinery and its possible modes of action have been fairly well characterized, the mechanisms of transcriptional activation are still poorly understood (44). Transactivator proteins have been shown to interact with a variety of targets, including the protein components of the RNA polymerase II transcription machinery as well as chromatin constituents and activities which modify chromatin. Among these putative transactivator targets are the GTF TFIID and the SAGA (Spt-Ada-Gcn5 acetylase) histone acetyltransferase complex. Both of these multisubunit complexes have been extensively studied in yeasts and metazoans (see references 1, 4, 18, and 66 for recent reviews).Yeast TFIID is composed of 14 TATA box DNA binding protein-associated factors (TAFs) exhibiting molecular masses ranging from 150 to 17 kDa (61). Although the identifies of TFIID subunits are known, the exact stoichiometry of these multiple subunits within the complex is not. With the exception of TAF30p (30), all TFIID subunits are encoded by single-copy essential genes, and all display a high degree of sequence conservation among eukaryotes. One of these subunits, yeast TAF130p (also known as TAF145p) (58), and its metazoan counterparts (human and Drosophila TAF250p) contain intrinsic enzymatic activities that contribute to transcription (15,16,47,49,53,54,56).Genetic and biochemical experiments have indicated that direct interactions between the activation domains (AD) of transcriptional activators and the subunits of TFIID play key roles in transactivation (1...