Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.The functional analysis of genes (mostly protein-coding genes) transcribed by eucaryotic RNA polymerase II has revealed a number of genetic transcriptional control regions. These include proximal promoter elements, distal promoter elements, and enhancer elements (for reviews, see references 19, 45, and 60). The most common proximal element is the so-called TATA motif, which in higher eucaryotes is generally located around positions -25 to -30 relative to the initiation site (for a review, see reference 7). Results of in vitro (12, 13, 28, 30, 35, 46, 56, 66, 67, 71) and in vivo (5, 9, 15, 27, 29, 49) studies have indicated that this is a critical element both for promoter activity and for determining the exact point of initiation. Moreover, for some promoters the TATA element (sometimes with its sequences near the initiation site) is the only DNA sequence that is absolutely required for a low level of accurate initiation in vitro (13,30,35,56,66) and for mediating transcription responses to viral immediate early proteins in vivo (10, 26, 60a, 70) and in vitro (1, 2). This indicates that these sequences represent core promoter elements and that there exist cellular factors with an intrinsic capacity for functional interactions therewith. At the same time the activities of these core promoters can be greatly increased not only by viral immediate early proteins (above) but also by the action of distal elem...

The Pax6 gene plays crucial roles in eye development and encodes a transcription factor containing both a paired domain and a homeodomain. During embryogenesis, Pax6 is expressed in restricted tissues under the direction of distinct cis-regulatory regions. The head surface ectoderm-specific enhancer of mouse Pax6 directs reporter expression in the derivatives of the ectoderm in the eye, such as lens and cornea, but the molecular mechanism of its control remains largely unknown. We identified a Pax6 protein-responsive element termed LE9 (52 bp in length) within the head surface ectoderm-specific enhancer. LE9, a sequence well conserved across vertebrates, acted as a highly effective enhancer in reporter analyses. Pax6 protein formed in vitro a complex with the distal half of LE9 in a manner dependent on the paired domain. The proximal half of the LE9 sequence contains three plausible sites of HMG domain recognition, and HMG domain-containing transcription factors Sox2 and Sox3 activated LE9 synergistically with Pax6. A scanning mutagenesis experiment indicated that the central site is most important among the three presumptive HMG domain recognition sites. Furthermore, Pax6 and Sox2 proteins formed a complex when they were expressed together. Based on these findings, we propose a model in which Pax6 protein directly and positively regulates its own gene expression, and Sox2 and Sox3 proteins interact with Pax6 protein, resulting in modification of the transcriptional activation by Pax6 protein.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

No abstract

Mouse A-type lamin genes were isolated. Structural analyses revealed that all the three known mouse A-type lamins (A, C and C2) were coded in a single genomic locus in a 22 kilobase DNA segment. The three lamins were coded in 12, 10 and 10 exons for A, C and C2, respectively, and shared 8 exons among them. Primer extension analyses identified possible transcription start sites for both AIC and C2 genes suggesting that the locus is under the control of two separate promoters, that is a somatic cell-acting promoter (for A and C) and a testis-specific promoter (for C2) which resides in the first intron of the MC gene. Sequence characteristics of the possible promoter regions are discussed. Divergence of the two somatic cell-type iamins (A and C) is formally accounted for by differential selection of poly(A) sites together with lamin A-specific splicing.

The FILAMENTOUS FLOWER protein has a zinc finger domain, hydrophobic region, proline-rich region, and a HMG box-like domain. We have reported that zinc release at the zinc finger is probably facilitated by the non-canonical cysteine residue at position 56, and that EDTA causes the structural change and enhances the self-assembly of the protein (Kanaya, E., Watanabe, K., Nakajima, N., Okada, K., and Shimura, Y. In higher plants, all lateral organs exhibit abaxial-adaxial asymmetries, and common genetic mechanism is likely to work to specify organ polarity (1-6). The FILAMENTOUS FLOWER (FIL) gene is thought to be a member of a gene family whose role appears to be pivotal in specifying the abaxial cell fate of lateral organs (1-3). In this gene family, FIL, YABBY2(YAB2), and YABBY3(YAB3) are expressed abaxially in all developing lateral organs and are proposed to redundantly promote the abaxial fate of all lateral organs (1-3). The protein encoded by FIL consists of 229 amino acid residues, and has the aminoterminal zinc finger domain between positions 10 and 60, the hydrophobic region between positions 60 and 100, the prolinerich region between positions 127 and 145, and the carboxylterminal HMG 1 box-like domain between positions 145 and 180 as shown in Fig. 1a. The presence of these domains, in particular the HMG box-like domain, suggests that the FIL gene may function as a transcriptional regulator, and the FIL protein is likely to work as a DNA-binding protein (1).The HMG box is a conserved domain of about 80 amino acid residues, which mediates DNA binding by the HMG box protein (7). The solution structure of the HMG domain consists of a global fold of three ␣-helices (helix I, helix II, and helix III) stabilized in an L-shaped configuration (8, 9). The HMG proteins are divided into two classes. The HMG box proteins in the first class are transcription factors and bind to DNA in a sequence-specific manner, such as the human sex-determining factor SRY (10), the lymphoid enhancer binding factor LEF1 (11, 12), and the T-cell factor TCF-1 (13). The HMG box proteins in the second class are more abundant and bind to DNA in a non-sequence-specific manner. Although their biological functions are currently not clear, they are thought to participate in DNA recombination and repair, activation and repression of transcription, and nucleosome assembly and disassembly. Examples include HMG1 and 2 (HMG1/2) proteins from higher eukaryotes (14, 15), NHP6A and NHP6B from Saccharomyces cerevisiae (16), and HU from Escherichia coli (17). The HMG1/2 proteins strongly distort DNA upon binding, and stabilize bent and supercoiled DNA. NHP6A induces a large bend of DNA upon binding. HU exists in the cell as homo-and heterodimers and seems to participate to wrap the oriC sequence (18).The amino acid sequence of the HMG box-like domain between positions 145 and 180 of the FIL protein which contains the putative helix 3 and helix 4 (Fig. 1b), shows a homology to those of the regions containing helix I and helix II of both the HMG domain ...

We identified MRG-1, a Caenorhabditis elegans chromodomain-containing protein that is similar to the human mortality factor-related gene 15 product (MRG15). RNA-mediated interference (RNAi) of mrg-1 resulted in complete absence of the germline in both hermaphrodite and male adults. Examination of the expression of PGL-1, a component of P granules, revealed that two primordial germ cells (PGCs) are produced during embryogenesis in mrg-1(RNAi) animals, but these PGCs cannot undergo mitotic proliferation, and they ultimately degenerate during post-embryonic development. Zygotic RNAi experiments using RNAi-deficient hermaphrodites and wild-type males demonstrated that MRG-1 functions maternally. Moreover, immunoblot analysis using mutant animals with germline deficiencies indicated that MRG-1 is synthesized predominantly in oocytes. These results suggest that MRG-1 is required maternally to form normal PGCs with the potential to start mitotic proliferation during post-embryonic development.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.