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PubMed1995

Biochemical characterization of recombinant yeast PPZ1, a protein phosphatase involved in salt tolerance.

The Saccharomyces cerevisiae gene PPZ1 codes for a 692-residues protein that shows in its carboxyl-terminal half about 60% identity with the catalytic subunit of mammalian and yeast protein phosphatase-1 and that is involved in salt homeostasis. The complete PPZ1 protein has been successfully expressed as a soluble glutathione-S-transferase fusion protein. The recombinant protein, after purification by a single affinity chromatography step, displayed phosphatase activity towards a number of substrates, including myelin basic protein, histone 2A and casein, but was ineffective in dephosphorylating glycogen phosphorylase. It was also active towards p-nitrophenylphosphate. The activity was severalfold increased by the presence of Mn2+ ions and by limited trypsinolysis. The enzyme was inhibited by okadaic acid and microcystin-LR at concentrations comparable to what is found for type 1 protein phosphatase although it was much less sensitive to inhibitor-2. The recombinant protein was phosphorylated in vitro by cAMP-dependent protein kinase, protein kinase C and casein kinase-2. Phosphorylation affected preferentially sites located in the amino-terminal half of the protein and did not alter the activity of the phosphatase.

Base Sequence↗
PubMed2000

Genomic structure and expression of the murine Hmgi(y) gene.

Mammalian HMGI proteins belong to the high mobility group (HMG) of small non-histone nuclear proteins, and function as architectural factors to mediate structural changes in DNA. The HMGI family consists of three members: HMGI, HMGY and HMGI-C. As pseudogenes have complicated the genomic analysis of murine Hmgi(y), a mouse lambda FIX II genomic library was screened with an intron-specific probe to identify and characterize the authentic Hmgi(y) gene. The murine Hmgi(y) gene is 7.2kb long and contains four protein coding exons and two additional exons encoding part of the 5' untranslated region. Sequencing confirms that an alternative splicing site within exon 3 results in the two protein isoforms: Hmgi and Hmgy. Primer extension experiments revealed that at least three transcription start sites exist in the 5' end of the gene. It has been well established that the expression of both Hmgi-c and Hmgi(y) is readily detectable throughout embryogenesis. Unlike Hmgi-c, whose expression is restricted to embryogenesis, a Northern hybridization analysis showed low-level expression of Hmgi(y) in adult mouse tissues. Similarly, when tissues from newborn animals were examined, Hmgi(y) expression was readily detected at a level of intensity intermediate between that found in embryos and adults. Understanding the gene structure and expression pattern will provide important insights into the in-vivo function of Hmgi(y).

Amino Acid Sequence↗
PubMed2004

Implication of BRG1 and cdk9 in the STAT3-mediated activation of the p21waf1 gene.

STAT transcription factors (signal transducers and activators of transcription) are cytoplasmic proteins that induce gene activation in response to cytokine receptor stimulation. Following tyrosine phosphorylation, STAT proteins translocate into the nucleus and activate specific target genes. We have previously reported that STAT3 activates the expression of the p21waf1 gene through its association with the NcoA/SRC1a and CBP coactivators. In this study, we explore the role of BRG1, a component of the SWI/SNF chromatin-remodeling complex, and the role of cdk9, a component of the elongation factor P-TEFb, in the STAT3-mediated expression of p21waf1. We found using pull-down experiments and co-immunoprecipitation assays that both proteins associate with STAT3. Chromatin immunoprecipitation (ChIP) experiments indicate that STAT3 DNA binding results in histone H3 acetylation and BRG1 recruitment. Using Southern blot analysis, we found that the loading of BRG1 is followed by an increased accessibility of the proximal p21waf1 promoter and by the association of RNA polymerase II. As a next step, STAT3 then recruits the cdk9 kinase to phosphorylate the carboxy-terminal domain of the RNA polymerase at serine 2. Accordingly, the elongating form of the polymerase can be detected by ChIP experiments on the coding region of the gene, probably initiating mRNA synthesis. Therefore, STAT3 not only promotes the initiation of transcription but also regulates chromatin remodeling and transcription elongation through its interaction with BRG1 and cdk9.

Cell Line, Tumor↗
PubMed1987

Isolation and characterization of a cDNA clone encoding testis protamine Z1 from the dog-fish Scylliorhinus caniculus.

A clone containing a 445-bp cDNA insert was isolated from a cDNA library synthesized from dog-fish testes mRNA. The nucleotide sequence was determined and corresponded to a 50-amino-acid protein. The known five-amino-acid N-terminal sequence corresponded exactly to our deduced amino acid sequence. After in vitro transcription of this cDNA using SP6 RNA polymerase, the translated polypeptide comigrated with the Z1 scylliorhinine marker. Analysis of the cDNA 3' flanking region of our Scylliorhinus protamine Z1 revealed an inverted repeat sequence, an ACAA motif and a CAGGAAAGA box known as regulatory signals for transcription termination in histone genes. In addition, sequences homologous to the simian virus (SV 40) and polyoma virus core enhancer elements were identified in the 5' and 3' flanking regions.

Amino Acid Sequence↗
PubMed1991

Characterization of a gene encoding a basic protein of the spermatid nucleus, TNP2, and its close linkage to the protamine genes in the bull.

During elongation and condensation of the spermatid nucleus, histones are replaced by spermatid-specific transition proteins (TNP). TNP1 is well characterized at the cDNA and at the genomic level and was found to be highly conserved during mammalian evolution (similarity between 83 to 98%). We here describe for the first time the nucleotide sequence and organization of the gene for TNP2. The gene was isolated from a bull cosmid library and was found to contain a single intron of 910 bp. The coding sequence consists of 390 bp and has a similarity of about 70% to that of the TNP2 cDNAs of mouse and rat. At the basis of amino-acid sequences, the bull TNP2 is 14 and 15 amino acids longer than that of mouse and rat, respectively, and the similarity is only 45% between bull and mouse and 42% between bull and rat. However, the evolutionary divergence has not occurred at the cost of basic amino acids which are of functional importance in DNA-protein interaction in the condensing spermatid nucleus. The TNP2 gene is closely linked to the protamine genes in the bull genome.

Aging↗
PubMed1994

The role of VL gene structural determinants in the fine specificity of anti-DNA antibodies.

To investigate the structural contribution of the light chain of anti-DNA antibodies to fine specificity, the VKappa genes of two monoclonal anti-DNA antibodies, termed H241 and H102, were cloned and sequenced. H102 and H241 are independently derived from MRL-lpr/lpr mice and differ in their fine specificity: H241 binds dsDNA and normal glomeruli in vitro and deposits in the kidney in vivo, whereas H102 binds only ssDNA and does not deposit in the kidney. Both are encoded by nearly identical VH genes but different N and D regions. Our previous results have demonstrated that the VH gene for H102 and H241 encodes eight other anti-DNA antibodies that also differed in fine specificity. This suggested that the gene product encoded by the VH 102/241 gene, may have intrinsic affinity for DNA, but is unlikely to determine fine specificity or nephritogenicity. In the present study we examined whether the VKappa gene might account for the difference in nephritogenicity. The complete nucleotide and deduced amino acid sequence of VK 102 and VK241 revealed that they are very dissimilar to each other (< 60% homology). VK 241 defined a new member of the VKappa gene family and was moderately homologous to two other VK genes encoding anti-DNA antibodies and to one VK gene encoding an anti-histone antibody all from lupus strains of mice. In addition, sequence diversity in the VK CDR1 region and position 96 of the CDR3 region was observed that may be of significance in determining fine specificity. VK 102 was highly homologous to two other VKappa genes, VKs17.2 and VK C8.5, both encoding anti-DNA antibodies and members of the VK20 gene family. It was striking that all three members of the VK 20 gene family code for DNA reactivity. This suggests that certain VKappa genes may also be used to repeatedly code for anti-DNA reactivity.

Amino Acid Sequence↗
PubMed2002

[Position of the satellite DNA relative to heterochromatin in the interphase nuclei in cultured cells].

It is considered that centromeric (CEN) regions play the leading role in the formation of chromocentres predominantly consisting of satellite DNA. Cloned mouse and human satellite DNA sequences from CEN and periGEN regions were used in order to trace their positions relative to chromocentres. Methods of fluorescent in situ hybridization (FISH) and immunoFISH with antibodies against CENP-B, known is a marker of prekinetochore, were employed. Peripheral position of the signals was observed at the chromocentres, but a combined signal of GEN and periGEN satellite DNAs never covered the whole brightly DAPI stained regions. A reasonable amount of the chromocentre body is not a satellite DNA. We suppose that the matrix-associated regions (MAR) of structural genes and, probably, the heavy methylated coding sequences of the genes, which are not expressed in the given cell type, are included in the chromocentres in addition to satellite DNAs.

Animals↗
PubMed2006

Protein interactions within the Set1 complex and their roles in the regulation of histone 3 lysine 4 methylation.

Set1 is the catalytic subunit and the central component of the evolutionarily conserved Set1 complex (Set1C) that methylates histone 3 lysine 4 (H3K4). Here we have determined protein/protein interactions within the complex and related the substructure to function. The loss of individual Set1C subunits differentially affects Set1 stability, complex integrity, global H3K4 methylation, and distribution of H3K4 methylation along active genes. The complex requires Set1, Swd1, and Swd3 for integrity, and Set1 amount is greatly reduced in the absence of the Swd1-Swd3 heterodimer. Bre2 and Sdc1 also form a heteromeric subunit, which requires the SET domain for interaction with the complex, and Sdc1 strongly interacts with itself. Inactivation of either Bre2 or Sdc1 has very similar effects. Neither is required for complex integrity, and their removal results in an increase of H3K4 mono- and dimethylation and a severe decrease of trimethylation at the 5' end of active coding regions but a decrease of H3K4 dimethylation at the 3' end of coding regions. Cells lacking Spp1 have a reduced amount of Set1 and retain a fraction of trimethylated H3K4, whereas cells lacking Shg1 show slightly elevated levels of both di- and trimethylation. Set1C associates with both serine 5- and serine 2-phosphorylated forms of polymerase II, indicating that the association persists to the 3' end of transcribed genes. Taken together, our results suggest that Set1C subunits stimulate Set1 catalytic activity all along active genes.

DNA-Binding Proteins↗
PubMed2003

HP1 binding to native chromatin in vitro is determined by the hinge region and not by the chromodomain.

We have isolated the complete coding sequences for two Xenopus laevis isoforms of heterochromatin protein 1, corresponding to HP1alpha and HP1gamma. The sequence of xHP1alpha shows considerable divergence from its mammalian homologues, whereas xHP1gamma is highly conserved. Functionally, xHP1alpha behaves identically to human HP1alpha. We observe unexpected differences between the two HP1 variants in binding native soluble chromatin, which seem to correlate with their distinct nuclear distributions in vivo. A surprising finding is that the characteristic interaction of HP1 chromodomains with histone H3 at methylated lysine 9 is not detected in preformed chromatin due to its inaccessibility. Instead, we localize a strong chromatin-binding activity to the short hinge region between the chromodomain and the chromoshadow domain of xHP1alpha but not xHP1gamma. This novel chromatin-binding activity has a non-specific DNA-binding component in addition to a linker histone-dependent preference for an altered chromatin structure with a likely heterochromatin organization.

Amino Acid Sequence↗
PubMed2004

Predicting gene expression from sequence.

We describe a systematic genome-wide approach for learning the complex combinatorial code underlying gene expression. Our probabilistic approach identifies local DNA-sequence elements and the positional and combinatorial constraints that determine their context-dependent role in transcriptional regulation. The inferred regulatory rules correctly predict expression patterns for 73% of genes in Saccharomyces cerevisiae, utilizing microarray expression data and sequences in the 800 bp upstream of genes. Application to Caenorhabditis elegans identifies predictive regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes. Successful prediction requires diverse and complex rules utilizing AND, OR, and NOT logic, with significant constraints on motif strength, orientation, and relative position. This system generates a large number of mechanistic hypotheses for focused experimental validation, and establishes a predictive dynamical framework for understanding cellular behavior from genomic sequence.

Animals↗
PubMed1986

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Amino Acid Sequence↗
PubMed1991

Cloning and sequencing of cDNA encoding rat GTP cyclohydrolase I. The first enzyme of the tetrahydrobiopterin biosynthetic pathway.

A full-length cDNA clone for GTP cyclohydrolase I, the first enzyme of the tetrahydrobiopterin biosynthetic pathway, was isolated and characterized. Synthetic oligonucleotides, constructed according to selected amino acid sequences of purified GTP cyclohydrolase I, were used to screen a rat liver cDNA library. Four clones were isolated, and the length of the longest cDNA insert was 1024 base pairs. The identity of the cDNA was confirmed by amino acid sequence data for eight fragments obtained by lysyl endopeptidase digestion of the purified protein. The coding region encoded a protein of 241 amino acid residues, but the NH2 terminus of the protein contained 11 additional amino acid residues not present in the purified protein. RNA blot analysis showed a single mRNA species of 1.2 kilobases in rat liver. A characteristic feature of the deduced amino acid sequence of GTP cyclohydrolase I was the presence of sequences similar to those proposed for the phosphorylation sites for casein kinase II and growth-associated histone H1 kinase. Furthermore, significant similarity was found to the highly conserved sequences of dihydrofolate reductases, which are known to be involved in the binding of the pterin group of dihydrofolate to the reductases. This region in GTP cyclohydrolase I may be assigned to the binding site of tetrahydrobiopterin, one of the inhibitors of this enzyme.

Amino Acid Sequence↗
PubMed2000

Acetylation of importin-alpha nuclear import factors by CBP/p300.

Histone acetylases were originally identified because of their ability to acetylate histone substrates [1] [2] [3]. Acetylases can also target other proteins such as transcription factors [4] [5] [6] [7]. We asked whether the acetylase CREB-binding protein (CBP) could acetylate proteins not directly involved in transcription. A large panel of proteins, involved in a variety of cellular processes, were tested as substrates for recombinant CBP. This screen identified two proteins involved in nuclear import, Rch1 (human importin-alpha) and importin-alpha7, as targets for CBP. The acetylation site within Rch1 was mapped to a single residue, Lys22. By comparing the context of Lys22 with the sequences of other known substrates of CBP and the closely related acetylase p300, we identified G/SK (in the single-letter amino acid code) as a consensus acetylation motif. Mutagenesis of the glycine, as well as the lysine, severely impaired Rch1 acetylation, supporting the view that GK is part of a recognition motif for acetylation by CBP/p300. Using an antibody raised against an acetylated Rch1 peptide, we show that Rch1 was acetylated at Lys22 in vivo and that CBP or p300 could mediate this reaction. Lys22 lies within the binding site for a second nuclear import factor, importin-beta. Acetylation of Lys22 promoted interaction with importin-beta in vitro. Collectively, these results demonstrate that acetylation is not unique to proteins involved in transcription. Acetylation may regulate a variety of biological processes, including nuclear import.

Acetylation↗
PubMed2005

MeCP2 deficiency in Rett syndrome causes epigenetic aberrations at the PWS/AS imprinting center that affects UBE3A expression.

Rett syndrome (RS) is a severe and progressive neurodevelopmental disorder caused by heterozygous mutations in the X-linked methyl CpG binding protein 2 (MeCP2) gene. MeCP2 is a nuclear protein that binds specifically to methylated DNA and functions as a general transcription repressor in the context of chromatin remodeling complexes. RS shares clinical features with those of Angelman syndrome (AS), an imprinting neurodevelopmental disorder. In AS patients, the maternally expressed copy of UBE3A that codes for the ubiquitin protein ligase 3A (E6-AP) is repressed. The similar phenotype of these two syndromes led us to hypothesize that part of the RS phenotype is due to MeCP2-associated silencing of UBE3A. Indeed, UBE3A mRNA and protein are shown here to be significantly reduced in human and mouse MECP2 deficient brains. This reduced UBE3A level was associated with biallelic production of the UBE3A antisense RNA. In addition, MeCP2 deficiency resulted in elevated histone H3 acetylation and H3(K4) methylation and reduced H3(K9) methylation at the PWS/AS imprinting center, with no effect on DNA methylation or SNRPN expression. We conclude, therefore, that MeCP2 deficiency causes epigenetic aberrations at the PWS imprinting center. These changes in histone modifications result in loss of imprinting of the UBE3A antisense gene in the brain, increase in UBE3A antisense RNA level and, consequently reduction in UBE3A production.

Angelman Syndrome↗
PubMed2006

In vivo color-coded imaging of the interaction of colon cancer cells and splenocytes in the formation of liver metastases.

The role of host cells in tumor progression and metastasis is critical. Intrasplenic injection of tumor cells has long been known as an effective method of developing liver metastases in nude mice, whereas portal vein (PV) injection of tumor cells can result in rapid death of the tumor cells. Host cells were thought to play a role in these phenomena. We report here that after splenic injection of tumor cells, splenocytes cotraffic with the tumor cells to the liver and facilitate metastatic colony formation. Human colon cancer cells that express green fluorescent protein (GFP) linked to histone H2B in the nucleus and red fluorescent protein (RFP) in the cytoplasm (HCT-116-GFP-RFP) were injected in either the PV or spleen of nude mice and imaged at the subcellular level in vivo. Extensive clasmocytosis (destruction of the cytoplasm) of the cancer cells occurred within 6 hours after PV injection and essentially all the cancer cells died. In contrast, splenic injection of these tumor cells resulted in the aggressive formation of liver and distant metastasis. GFP spleen cells were found in the liver metastases that resulted from intrasplenic injection of the tumor cells in transgenic nude mice ubiquitously expressing GFP. When GFP spleen cells and the RFP cancer cells were coinjected in the PV, liver metastasis resulted that contained GFP spleen cells. These results suggest a novel tumor-host interaction that enables efficient formation of liver metastasis via intrasplenic injection.

Animals↗
PubMed2000

Mouse scrapie responsive gene 1 (Scrg1): genomic organization, physical linkage to sap30, genetic mapping on chromosome 8, and expression in neuronal primary cell cultures.

We have previously reported a transcript of a novel mouse gene (Scrg1) with increased expression in transmissible spongiform encephalopathies and the cloning of the human mRNA analogue. In this paper, we present the genomic organization of the mouse and human SCRG1 loci, which exhibit a high degree of conservation. The genes are composed of three exons; the two downstream exons contain the protein coding region. The mouse gene is expressed in brain tissue essentially as a 0.7-kb message but also as a minor 2.6-kb mRNA. We have sequenced 20 kb of DNA at the mouse Scrg1 locus and found that the longer transcript is the prolongation of the 0.7-kb mRNA to a polyadenylation site located about 2 kb further downstream. Sequencing revealed that the mouse Scrg1 gene is physically linked to Sap30, a gene that encodes a protein of the histone deacetylase complex, and genetic linkage mapping assigned the localization of Scrg1 to chromosome 8 between Ant1 and Hmg2. Northern blot analysis showed that Scrg1 is under strict developmental control in mouse embryo and is expressed by cells of neuronal origin in vitro. Comparison of the rat, mouse, and human SCRG1 proteins identified a box of 35 identical contiguous amino acids and a characteristic cysteine distribution pattern defining a new protein signature.

Amino Acid Sequence↗
PubMedDate not supplied

[Chromatin structure of ribosomal genes of Drosophila melanogaster. Random location of nucleosomes in DNA and characteristics of organization of non-transcribed spacer].

Chromatin structure of ribosomal genes from nuclei of Drosophila melanogaster embryos was studied by using micrococcal nuclease cleavage. End-directed labelling with short cloned fragments of the ribosomal repeat was carried out. It shows, that the micrococcal nuclease prefers specific sites in naked DNA, and the pattern of DNA cleavage is essentially conserved when the nuclei are digested. Only minor differences are visible. Hence, there are no specific positions of nucleosomes on the ribosomal repeat. The DNA fragments from the nuclei treated with micrococcal nuclease were electrophoresed, transferred to DBM-paper and hybridized with different probes subcloned from the ribosomal repeat. The non-transcribed spacer and the region of the beginning of transcription are hydrolysed significantly faster than the coding region or inactive ribosomal insertion. The region of NTS and the beginning of transcription do not give normal nucleosomal fragment in the range of 145-185 bp; instead they produce a heterogeneous band 200-280 bp in length even after prolonged digestion. Dinucleosomal fragments are also slightly longer and more heterogeneous than in other parts of the ribosomal repeat. Higher oligomers are similar throughout the ribosomal repeat. We suggest that a hypothetical transcription factor interacts in a way with histones and protects unusual fragments of DNA from digestion.

Animals↗
PubMed1986

Immunofractionation of chromatin regions associated with histone H1o.

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1o but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr-Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme-linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1o. Oligonucleosomes, prepared from the livers of either untreated or 3-methylcholanthrene-treated adult rats, were fractionated on the anti-H1o affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P-labeled probes. A slight enrichment in H1o was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3'-terminal region of a cytochrome P-450 gene, which is inducible by the chemical carcinogen 3-methylcholanthrene, regardless of whether isolated from control or from carcinogen-treated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1o content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non-transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1o.

Animals↗