Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG).poly(dC), poly(dA).poly(dT) and poly(dAT).poly(dAT), but not with poly(dGC).poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

Intracellular Localization of DNA Polymerases in the Oocyte of Starfish, Asterina pectinifera: (starfish/DNA polymerase α/DNA polymerase ß/germinal vesicle/enucleation).

Oocytes of the starfish, Asterina pectinifera, were separated into karyoplasts and cytoplasts by centrifugation in a sucrose density gradient in the presence of cytochalasin B. More than 90% of the DNA polymerase activity of whole oocytes was recovered in the karyoplasts, and less than 10% in the cytoplasts. DNA polymerase α was specifically localized in karyoplasts. Of the DNA polymerase ß activity of whole oocytes, 70-80% was found in the karyoplasts, and about 15% in the cytoplasts. The problem of whether DNA polymerases are localized within germinal vesicles in starfish oocytes is discussed.

Intracellular Localization and Sedimentation Coefficient of DNA Ligase in Oocytes of the Starfish, Asterina pectinifera: (starfish/DNA ligase/germinal vesicle).

When immature oocytes of Asterina pectinifera were separated into karyoplasts (nuclear fragments) and cytoplasts (anuclear fragments) by cytochalasin B treatment and centrifugation in a sucrose gradient, almost all their DNA ligase activity was recovered in the karyoplasts. Thus, DNA ligase seems to be localized in the germinal vesicles or their vicinity in starfish oocytes. No ontogenic change in the activity of DNA ligase per oocyte, egg or embryo, or in its sedimentation coefficient (4.1 S) was observed during oocyte maturation, fertilization, or early development.

DNA Replication in Permeabilized Cells of Sea Urchin Embryos: (DNA replication/permeabilized embryonic cells/sea urchin).

For study of the regulation of DNA replication in sea urchin embryos during the early stages of development, an embryonic cell system that was permeable to exogenously supplied nucleotides was established. Embryos were permeabilized by incubating them in hypotonic buffer containing 0.3 M glucose. The permeabilized embryonic cells maintained their morphological integrity, and synthesized DNA when supplied with exogenous dNTPs. DNA synthesis in these permeabilized embryonic cells required the presence of ATP and three other deoxyribonucleoside triphosphates in addition to labeled dTTP. DNA synthesis was almost completely inhibited by N-ethylmaleimide, and proceeded in a discontinuous fashion. Only cells permeabilized during the S phase could incorporate nucleoside triphosphates into DNA: cells permeabilized during other phases did not synthesize DNA. During a 60 min-incubation period, over 10% of the genomic DNA was replicated under the experimental conditions used.

Detection and characterization of the cis-element in the first intron of the Ars gene in the sea urchin.

Cis-active elements that are related to temporal regulation of the Ars gene expression in embryos of the sea urchin, Hemicentrotus pulcherrimus, are localized in the region between -252 b and +38b. This promoter region, however, can only determine the onset of gene transcription at a defined period in sea urchin development, and cannot determine the level of the Ars gene transcription. The element that enhances Ars promoter activity is found in the first intron of the Ars gene. The first intron element of H. pulcherrimus shows a similar level of activity when introduced in embryos of another species of sea urchin, Temnopleurus toreumaticus. The size of the first intron element is determined to be 229 b.

cis-Elements and Protein Factors Related to Regulation of Transcription of Arylsulfatase Gene during Sea Urchin Development: (sea urchin development/Ars gene/cis-elements/enhancer/protein factors).

Eight restriction fragments (I-VIII) were prepared to cover a whole span of the enhancer region in the upstream of the Ars gene of the sea urchin, Hemicentrotus pulcherrimus, and their abilities to influence on the Ars gene expression were estimated by CAT assay. Only three fragments (III, IV and V) encompassing a 0.6 kb region between -2.8 kb and -2.2 kb stimulated CAT expression. By mobility shift assays, it was found that the Ars enhancer region is composed of multiple cis-acting elements that interact with nuclear proteins in a sequence-specific manner. Among them, two sequences, a G-string and a GATCTCCCC, were determined by DNA footprinting as sites of protein-DNA interaction. The DNA-binding factor prevalence changed ontogenically in three different patterns. Possible activation of DNA-binding proteins through their modification is discussed.

Developmental Timing of Synthesis and Translation of Arylsulfatase mRNA in Sea Urchin Embryo: (sea urchin embryo/arylsulfatase/transcription/actinomycin D/emetine).

In the sea urchin (Hemicentrotus pulcherrimus) embryo, the arylsulfatase activity is known to increase dramatically after the mesenchyme blastula stage. In the present experiment, we have studied the mechanism underlying this increase in the arylsulfatase activity by using the inhibitors of transcription and translation. Inhibition of transcription by actionmycin D before hatching prevented the increase in the arylsulfatase activity, while inhibition of transcription after hatching failed to prevent the increase in the enzyme activity. Inhibition of translation by emetine always reduced the arylsulfatase activity. These results have been interpreted as that the arylsulfatase activity in the blastula embryo is supported by the pre-synthesized mRNA, and that the marked increase in the enzyme activity which is observed from the mesenchyme-blastula stage to the prism stage is due to the transcription of the arylsulfatase gene which occurs during the development from hatching to the mesenchyme-blastula stage.

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography: (acethylcholinesterase/purification/Sea Urchin, Hemicentrotus pulcherrimus).

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage. Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 105 as determined by Sepharose CL-6B column chromatography.

Melittin, a Component of Bee Venom, Activates Unfertilized Sea Urchin Eggs: (melittin/activation/sea urchin egg).

Melittin, which is known to stimulate phospholipase A, in many cells, caused as much elevation of fertilization membranes and increase in respiration of unfertilized eggs of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus as normal fertilization. In melittin-activated eggs, amino acid transport was decreased to less than that of unfertilized eggs, nucleoside transport was only slightly, activated, protein synthesis was rather inhibited and neither DNA synthesis nor cleavage was observed. It is concluded that although melittin induces the cortical reaction and activation of respiration in unfertilized eggs, its cytotoxicity prevents any "late changes".

Effects of Aphidicolin on Arylsulfatase Gene Expression in Sea Urchin Embryos: (arylsulfatase/aphidicolin/gene expression/sea urchin embryo).

Expression of the arylsulfatase (Ars) gene in sea urchin embryos begins just before hatching and ceases at the pluteus stage. Initiation of the Ars gene expression is inhibited by aphidicolin, which inhibits DNA synthesis without arresting the total RNA synthesis. Based on these finding it is supposed that DNA replication is a prerequisite for initiation of the Ars gene expression in developing sea urchin embryos.

Cis-Acting Elements for Proper Ontogenic Expression of Arylsulfatase Gene of Sea Urchin Embryo.

To define the cis-acting regulatory elements required for proper ontogenic expression of the Arylsulfatase gene in the sea urchin (Hemicentrotus pulcherrimus) embryo, we have constructed the fusion genes composed of bacterial CAT gene and various regions from the 5' flanking sequence of the Ars gene, injected them into unfertilized sea urchin eggs and monitored their expression during development. A wild type construct containing the 5' flanking region of the Ars gene spanning from -2 to -3160 was expressed under proper temporal regulation following injection into unfertilized eggs. By deletion analysis the transcriptional enhancers were localized between -1280 and -2680 of the Ars gene. In addition, sequences acting as a negative regulator as well as a minimal promoter for expression of the Ars gene were detected by internal deletion analysis.

Spatial Patterns of Arylsulfatase mRNA Expression in Sea Urchin Embryo: (arylsulfatase/sea urchin embryo/gene expression).

Previously we showed that the arylsulfatase (Ars) activity increases markedly after the mesenchyme blastula stage, and that the increase in its activity is due to initiation of the Ars gene transcription. In order to detect cells responsible for Ars mRNA production in the sea urchin embryo, we carried out in situ hybridization with the use of 35 S-labeled Ars RNA probe transcribed from Ars cDNA in a vector pT7T3-19U. The Ars gene is uniformly expressed in the cells of blastula, and the expression becomes restricted to aboral ectoderm during development by gastrula stage.

Histochemical Detection of Arylsulfatase Activity in Sea Urchin Embryos: (arylsulfatase/sea urchin embryo/histochemistry/electron microscopy).

Localization of arylsulfatase activity in the sea urchin embryo was determined histochemically by light and electron microscopy. Histochemical observations by light microscopy revealed that the arylsulfatase activity appears after the gastrula stage and that it is restricted to the cells of the aboral ectoderm. The enzyme activity is mainly located in the apical cellular cytoplasm and is associated with lysosome-like structures that are frequently fused with yolk granules. Intense activity is also detected in the region of the endoplasmic reticulum and Golgi apparatus. No enzyme activity is found in the extracellular spaces of embryos.

Corrected Structure of the 5' Flanking Region of Arylsulfatase Gene of the Sea Urchin, Hemicentrotus pulcherrimus: (5' flanking sequence/sea urchin/arylsulfatase gene/G-string).

The nucleotide sequence of the 5' flanking region of the arylsulfatase (Ars) gene of the sea urchin, Hemicentrotus pulcherrimus, is extensively corrected. The one previously reported from our laboratory (ref. 7, Develop, Growth & Differ., 34, 719-729, 1992) contains many sequencing errors and should be replaced with that presented here. Correction includes the addition of a 294 nucleotide (nt) sequence between -728 and -1,021. In the previous sequencing, this fragment was missed by overlooking one Hindlll site during subcloning. In addition to a cluster of direct repeats between -2,592 and -3,440, an inverted repeat is detected between -217 and -476. A possible role of the inverted repeat in the regulation of transcription of the Ars gene is discussed.

Expression of Na+ , K+ -ATPase α-Subunit in Animalized and Vegetalized Embryos of the Sea Urchin, Hemicentrotus pulcherrimus.

A marked increase in the Na+ , K+ -ATPase activity of sea urchin embryos occurred following an elevation of its mRNA level, revealed by Northern blotting analysis, in developmental period between the swimming blastula and the late gastrula stage. cDNA clone of Na+ , K+ -ATPase α-subunit, obtained from γgt10 cDNA library of sea urchin gastrulae, was digested with EcoRl ad Hindlll. The obtained 268 bp cDNA fragment, hybridized to a 4.6 Kb RNA, was used as probe for Northern blotting analysis. The level of Na+ , K+ -ATPase mRNA was higher in embryo-wall cell fraction isolated from late gastrulae (ectoderm cells) than the level in the bag fraction, containing mesenchyme cells (mesoderm cells) and archenteron (endoderm cells). The activity of Na+ , K+ -ATPase and the level of its mRNA were higher in animalized embryos obtained by pulse treatment with A23187 for 3 hr, starting at the 8-16 cell stage and were considerably lower in vegetalized embryos induced by 3 hr treatment with Li+ than that in normal embryos at the post gastrula corresponidng stage. Augmentation of Na+ , K+ -ATPase gene expression can be regarded as a marker for ectoderm cell differentiation at the post gastrula stage, which results from determination of cell fate in prehatching period.

aproctous, a locus that is necessary for the development of the proctodeum in Drosophila embryos, encodes a homolog of the vertebrate Brachyury gene.

The proctodeum of the Drosophila embryo originates from the posterior end of the blastoderm and forms the hindgut. By enhancer-trap mutagenesis, using a P-element-lacZ vector, we identified a mutation that caused degeneration of the proctodeum during shortening of the germ band and named it aproctous (apro). Expression of the lacZ reporter gene, which was assumed to represent expression of the apro gene, began at the cellular blastoderm stage in a ring that encompassed about 10-15% of the egg's length (EL) and included the future proctodeum, anal pads, and posterior-most part of the visceral mesoderm. In later stages, strong expression of lacZ was detected in the developing hindgut and anal pads. Expression continued in the anal pads and epithelium of the hindgut of larvae; the epithelium of the hindgut of the adult fly also expressed lacZ. The spatial patterns of the expression of lacZ in various mutants suggested that the embryonic expression of apro was regulated predominantly by two gap genes, tailless (tll) and huckebein (hkb): tll is necessary for the activation of apro, while hkb suppressed the expression of apro in the region posterior to 10% EL. Cloning and sequencing of the apro cDNA revealed that apro was identical to the T-related gene (Trg) that is a Drosophila homolog of the vertebrate Brachyury gene. apro appears to play a key role in the development of tissues derived from the proctodeum.

Utilization of a particle gun DNA introduction system for the analysis of cis-regulatory elements controlling the spatial expression pattern of the arylsulfatase gene (HpArs) in sea urchin embryos.

We applied a particle gun method to introduce DNA into fertilized sea urchin eggs for the analysis of cis-regulatory elements responsible for spatial gene expression during development. We introduced HpArs (sea urchin arylsulfatase gene) -GFP and HpArs-LacZfusion constructs into the fertilized eggs and obtained high expression levels of the fusion genes. Using this assay system, we demonstrated that a fragment of HpArs (-3,484 to +4,636) is sufficient for aboral ectoderm-specific expression, and that the region in the first intron from +406 to +1,993 contains the control elements responsible for the repression of the HpArs promoter activity in secondary mesenchyme cells.

A new G-stretch-DNA-binding protein, Unichrom, displays cell-cycle-dependent expression in sea urchin embryos.

We report the identification and characterization of Unichrom, a gene encoding a new G-stretch-DNA-binding protein in the sea urchin embryo. The derived amino acid sequence of Unichrom contains plant homeodomain (PHD) finger and high mobility group (HMG) motifs as well as motifs required for cell-cycle-dependent degradation. The expression of a Unichrom-green fluorescent protein (GFP) fusion protein in sea urchin embryonic cells indicates that Unichrom protein accumulates in nuclei during interphase and disperses into the cytoplasm at mitosis. Overexpression of dominant negative Unichrom, which contains the DNA binding domain lacking the motif for cell-cycle-dependent degradation, causes impairment of chromosome segregation. These results suggest that Unichrom binds to genome DNA at G-stretch and that degradation of Unichrom is required for segregation of chromosomes.

T-brain homologue (HpTb) is involved in the archenteron induction signals of micromere descendant cells in the sea urchin embryo.

Signals from micromere descendants play a crucial role in sea urchin development. In this study, we demonstrate that these micromere descendants express HpTb, a T-brain homolog of Hemicentrotus pulcherrimus. HpTb is expressed transiently from the hatched blastula stage through the mesenchyme blastula stage to the gastrula stage. By a combination of embryo microsurgery and antisense morpholino experiments, we show that HpTb is involved in the production of archenteron induction signals. However, HpTb is not involved in the production of signals responsible for the specification of secondary mesenchyme cells, the initial specification of primary mesenchyme cells, or the specification of endoderm. HpTb expression is controlled by nuclear localization of beta-catenin, suggesting that HpTb is in a downstream component of the Wnt signaling cascade. We also propose the possibility that HpTb is involved in the cascade responsible for the production of signals required for the spicule formation as well as signals from the vegetal hemisphere required for the differentiation of aboral ectoderm.