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Y Gruenbaum

Publications and source records attributed to Y Gruenbaum.

At least 37 records · Page 2Linked to original sources
PubMed1997

Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope.

Otefin is a 45-kDa nuclear envelope protein with no apparent homology to other known proteins. It includes a large hydrophilic domain, a single carboxyl-terminal hydrophobic sequence of 17 amino acids, and a high content of serine and threonine residues. Cytological labeling located otefin on the nucleoplasmic side of the nuclear envelope. Chemical extraction of nuclei from Drosophila embryos revealed that otefin is a peripheral protein whose association with the nuclear envelope is stronger than that of lamin. Deletion mutants of otefin were expressed in order to identify regions that direct otefin to the nuclear envelope. These experiments revealed that the hydrophobic sequence at the carboxyl terminus is essential for correct targeting to the nuclear envelope, whereas additional regions in the hydrophilic domain of otefin are required for its efficient targeting and stabilization in the nuclear envelope.

Animals↗
PubMed1997

Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system.

A Drosophila cell-free system was used to characterize proteins that are required for targeting vesicles to chromatin and for fusion of vesicles to form nuclear envelopes. Treatment of vesicles with 1 M NaCl abolished their ability to bind to chromatin. Binding of salt-treated vesicles to chromatin could be restored by adding the dialyzed salt extract. Lamin Dm is one of the peripheral proteins whose activity was required, since supplying interphase lamin isoforms Dm1, and Dm2 to the assembly extract restored binding. As opposed to the findings in Xenopus, okadaic acid had no effect on vesicle binding. Trypsin digestion of the salt-stripped vesicles eliminated their association with chromatin even in the presence of the dialyzed salt extract. One vesicles attached to chromatin surface, fusion events took place were found to be sensitive to guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). These chromatin-attached vesicles contained lamin Dm and otefin but not gp210. Thus, these results show that in Drosophila there are two populations of nuclear vesicles. The population that interacts first with chromatin contains lamin and otefin and requires both peripheral and integral membrane proteins, whereas fusion of vesicles requires GTPase activity.

Animals↗
PubMed1997

Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development.

Otefin is a peripheral protein of the inner nuclear membrane in Drosophila melanogaster. Here we show that during nuclear assembly in vitro, it is required for the attachment of membrane vesicles to chromatin. With the exception of sperm cells, otefin colocalizes with lamin Dm0 derivatives in situ and presumably in vivo and is present in all somatic cells examined during the different stages of Drosophila development. In the egg chamber, otefin accumulates in the cytoplasm, in the nuclear periphery, and within the nucleoplasm of the oocyte, in a pattern similar to that of lamin Dm0 derivatives. There is a relatively large nonnuclear pool of otefin present from stages 6 to 7 of egg chamber maturation through 6 to 8 h of embryonic development at 25 degrees C. In this pool, otefin is peripherally associated with a fraction containing the membrane vesicles. This association is biochemically different from the association of otefin with the nuclear envelope. Otefin is a phosphoprotein in vivo and is a substrate for in vitro phosphorylation by cdc2 kinase and cyclic AMP-dependent protein kinase. A major site for cdc2 kinase phosphorylation in vitro was mapped to serine 36 of otefin. Together, our data suggest an essential role for otefin in the assembly of the Drosophila nuclear envelope.

Animals↗
PubMed1996

Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state.

The nuclear matrix maintains specific interactions with genomic DNA at sites known as matrix attachment regions (M/SARs). M/SARs bind in vitro to lamin polymers. We show that the polymerized alpha-helical rod domain of lamin Dm0 provides by itself the specific binding to the ftz M/SAR. In contrast, unpolymerized rod domain does not bind specifically to this M/SAR. Non-specific binding to DNA is also observed with Dm0 containing a point mutation that impairs its ability to polymerize or with the isolated tail domain. These data suggest that the specific binding of lamins to M/SARs requires the rod domain and depends on the lamin polymerization state.

Amino Acid Sequence↗
PubMed1995

Expression of the novel murine homeobox gene Sax-1 in the developing nervous system.

We have isolated the novel murine Sax-1 gene, a member of the NK-1 class of homeobox genes, and report its expression pattern in the developing central nervous system (CNS) in comparison to two other homeobox genes, Evx-1 and Pax-6. Sax-1 was found to be transiently expressed in the developing posterior CNS. First seen in the ectoderm lateral to the primitive streak, the signal later encompassed the neural plate. Posteriorly, the expression domain overlapped with the Evx-1 expression in the streak, while anteriorly it was delimited by the Pax-6 signal in the neural tube. This early phase starting at day 9.5 pc, Sax-1 was expressed in distinct areas of spinal cord, hindbrain and forebrain. Particularly strong signals were detected in rhombomere 1 and in the pretectum. In these areas, subsets of neurons may be marked and specified. In addition to the normal pattern of Sax-1 during development, the expression in different mouse mutants was analysed. In Brachyury curtailed homozygotes, the expression of Sax-1 was found to be reduced during neurulation and even lost at day 9.0 pc. Ventral shift and finally loss of the signal in the ventral spinal cord was observed in Danforth's short tail homozygotes.

Amino Acid Sequence↗
PubMed1994

How is it that microsatellites and random oligonucleotides uncover DNA fingerprint patterns?

Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspersed tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

A role for CdxA in gut closure and intestinal epithelia differentiation.

CdxA is a homeobox gene of the caudal type that was previously shown to be expressed in the endoderm-derived gut epithelium during early embryogenesis. Expression of the CDXA protein was studied during intestine morphogenesis from stage 11 (13 somites) to adulthood in the chicken. The CDXA protein can be detected during all stages of gut closure, from stage 11 to 5 days of incubation, and is mainly localized to the intestinal portals, the region where the splanchnopleure is undergoing closure. In this region, which represents the transition between the open and closed gut, the CDXA protein is restricted to the endoderm-derived epithelium. At about day 5 of incubation, the process of formation of the previllous ridges begins, which marks the beginning of the morphogenesis of the villi. From this stage to day 11 expression of CDXA is localized to the epithelial lining of the intestine. In parallel, a gradual increase in CDXA protein expression begins in the mesenchyme that is close in proximity to the CDXA-positive endoderm. Maximal CDXA levels in the mesenchyme are observed at day 9 of incubation. During days 10 and 11 CDXA levels in the mesenchyme remain constant, and by day 12 CDXA becomes undetectable in these cells and the epithelium again becomes the main site of expression. From day 12 of incubation until adulthood the CDXA protein is present in the intestinal epithelium. Until day 18 of incubation expression can be detected along the whole length of the villus with a stronger signal at the tip. With hatching the distribution along the villi changes so that the main site of CDXA protein expression is at the base of the villi and in the crypts. The transient expression of CDXA in the mesenchyme between days 5 and 11 may be related to the interactions taking place between the mesenchyme and the epithelium that ultimately result in the axial specification of the alimentary canal and the differentiation of its various epithelia. The main CDXA spatial distribution during morphogenesis suggests a tight linkage to the formation and differentiation of the intestinal epithelium itself. CDXA appears to play a role in the morphogenetic events leading to closure of the alimentary canal. During previllous ridge formation the CDXA protein is transiently expressed in the mesenchymal cells thought to provide instructive interactions for the regionalization and differentiation of the gut epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

The spatial and temporal dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.

Sax1 (previously CHox3) is a chicken homeobox gene belonging to the same homeobox gene family as the Drosophila NK1 and the honeybee HHO genes. Sax1 transcripts are present from stage 2 H&H until at least 5 days of embryonic development. However, specific localization of Sax1 transcripts could not be detected by in situ hybridization prior to stage 8-, when Sax1 transcripts are specifically localized in the neural plate, posterior to the hindbrain. From stages 8- to 15 H&H, Sax1 continues to be expressed only in the spinal part of the neural plate. The anterior border of Sax1 expression was found to be always in the transverse plane separating the youngest somite from the yet unsegmented mesodermal plate and to regress with similar dynamics to that of the segregation of the somites from the mesodermal plate. The posterior border of Sax1 expression coincides with the posterior end of the neural plate. In order to study a possible regulation of Sax1 expression by its neighboring tissues, several embryonic manipulation experiments were performed. These manipulations included: removal of somites, mesodermal plate or notochord and transplantation of a young ectopic notochord in the vicinity of the neural plate or transplantation of neural plate sections into the extraembryonic area. The results of these experiments revealed that the induction of the neural plate by the mesoderm has already occurred in full primitive streak embryos, after which Sax1 is autonomously regulated within the spinal part of the neural plate.

Animals↗
PubMed1993

Isolation and characterization of target sequences of the chicken CdxA homeobox gene.

The DNA binding specificity of the chicken homeodomain protein CDXA was studied. Using a CDXA-glutathione-S-transferase fusion protein, DNA fragments containing the binding site for this protein were isolated. The sources of DNA were oligonucleotides with random sequence and chicken genomic DNA. The DNA fragments isolated were sequenced and tested in DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By electrophoretic mobility shift assays it was shown that the different target sequences isolated bind to the CDXA protein with different affinities. The specific sequences bound by the CDXA protein in the genomic fragments isolated, were determined by DNase I footprinting. From the footprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/G. The CAUDAL binding site in the ftz promoter is also included in this consensus sequence. When tested, some of the genomic target sequences were capable of enhancing the transcriptional activity of reporter plasmids when introduced into CDXA expressing cells. This study determined the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in culture.

Amino Acid Sequence↗
PubMed1993

The chicken CdxA homeobox gene and axial positioning during gastrulation.

The chicken homebox containing gene, CdxA (formerly CHox-cad), was previously shown to be expressed during gastrulation. Localization of CdxA transcripts by in situ hybridization to tissue sections revealed that, during gastrulation, expression of this gene exhibits a posterior localization along the primitive streak. The transcripts are localized to epiblast cells in the vicinity of the primitive streak, to cells of the primitive streak itself and in the definitive endoderm as it replaces the hypoblast. In order to study in greater detail the pattern of expression of the CdxA gene during gastrulation, we expressed the full-length CdxA protein as a fusion protein in E. coli and generated monoclonal antibodies against it. Chicken embryos at different stages of gastrulation were processed for whole-mount immunohistochemical localization of the protein using anti-CdxA antibodies. Once the pattern of expression in the whole embryo was determined, the same embryos were sectioned to determine the identity of the cells expressing the CdxA protein. Detailed analysis of the CdxA protein in embryos, from the onset of primitive streak formation to the beginning of the tail bud stage (stages 2 to 10), has shown different patterns of expression during primitive streak elongation and regression. The CdxA protein is initially detected at the posterior marginal zone and the expression moves rostrally into the primitive streak during mid-streak stages. As the primitive streak elongates, the CdxA stripe of expression moves anteriorly. By definitive streak stages, the CdxA stripe of expression delineates a position along the anterior-posterior axis in the primitive streak. CdxA, like its Drosophila homologue cad, is expressed during gastrulation in a stripe localized to the posterior region of the embryo. These observations suggest that CdxA as a homebox gene may be part of a regulatory network coupled to axial determination during gastrulation in the early chick embryo.

Animals↗
PubMed1992

Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.

Animals↗
PubMed1991

CHox E, a chicken homeogene of the H2.0 type exhibits dorso-ventral restriction in the proliferating region of the spinal cord.

CHox E is a novel chicken homeogene that belongs to the H2.0 family of homeodomains. Its homeobox sequence is interrupted by an intron between amino acids 44 and 45. Expression of CHox E during embryogenesis is localized to the central nervous system. The anterior boundary of CHox E expression can initially be localized to rhombomere number 1, later in development this boundary reaches up to the rhombencephalic isthmus. CHox E expression in the spinal cord localizes dorso-ventrally to the dorsal half of the basal plate. CHox E expression is always restricted to the proliferating region, the ventricular zone. As the ventricular zone becomes restricted laterally, so does the CHox E expressing region. Once this region of the ventricular zone ceases to exist, CHox E specific transcripts become undetectable. The site and time of CHox E expression suggest a very early function in the differentiation of the cells derived from that region of the ventricular zone.

Amino Acid Sequence↗
PubMed1991

A chicken caudal homologue, CHox-cad, is expressed in the epiblast with posterior localization and in the early endodermal lineage.

CHox-cad is a chicken homeobox gene whose homeodomain is homologous to the Drosophila caudal and the murine Cdx1 genes. Based on sequence analysis of a 2.5 kb CHox-cad cDNA clone, we deduced that the primary translation product consists of 248 amino acids. Comparison between the cDNA and genomic clones revealed the presence of an intron within the CHox-cad homeodomain between amino acids 44 and 45. The onset of CHox-cad transcription correlates temporarily with the beginning of gastrulation. During primitive streak stages CHox-cad exhibits a caudally localized pattern of expression restricted to the epiblast and the primitive streak. At these stages, CHox-cad transcripts can also be detected in the definitive endoderm cells. Later in embryogenesis CHox-cad is expressed in the epithelial lining of the embryonic gut and yolk sac. After four days of chicken development, no CHox-cad transcripts could be detected. The early CHox-cad posterior expression in the germ layer undergoing gastrulation and its continuous expression in the early endodermal lineage raise the possibility of CHox-cad involvement in the establishment of the definitive endoderm.

Amino Acid Sequence↗
PubMed1990

Effect of CpG methylation on gene expression in transfected plant protoplasts.

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.

Blotting, Southern↗