We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.Transcription in eukaryotes is a tightly regulated, multistep process. General transcription factors, gene specific transcriptional activators, and several different cofactors are necessary to access specific loci in the context of eukaryotic chromatin to allow precise initiation of RNA polymerase II (Pol II) transcription. One of the most appealing questions in eukaryotic transcription is how activators transmit their signals to the general transcription machinery to stimulate transcription.Posttranslational modifications of nucleosomal histones have been correlated with the function of chromatin in transcription activation or repression (18, 34). One of the most extensively studied modifications is the acetylation of the highly conserved amino-terminal histone tails. The steady-state level of acetylation of histone proteins is accomplished by the action of histone acetyltransferases (HATs) and histone deacetylases (9, 37). Acetylation affects higher-order folding of chromatin fibers and histone-nonhistone protein interactions (31, 32). Thus, it can increase the affinity of transcription factors for nucleosomal DNA (40,61).A large number of recent studies have provided a direct molecular link between histone acetylation and transcriptional activation (reviewed in references 9 and 30). In these reports, it has been shown that several previously identified coactivators and adaptors of transcription possess i...
Pig muscle 3-phosphoglycerate kinase contains seven cysteine residuesimolecule enzyme. Two of them react with Ellman's reagent (Nbsz) in a second-order reaction [k = (1.1 Tt_ 0.1) x lo3 M-' s-l 1; the reaction of the other five thiols are limited by a first-order protein structural change [k = (2.0 -+_ 0.4) x S C ' ] in 0.1 M Tris/HCI buffer, pH 7.5 at 20 "C.Blocking the rapidly reacting thiols with Nbsr inactivated thc enzyme (these two -SH groups are not equivalent in this respect), but it does not abolish substrate-binding ability.The rapidly reacting thiol groups readily participate in intermolecular disulfide formation following their partial blocking with Nbsz. This type of aggregation of 3-phosphoglycerate kinase molecules also leads to inactivation.The order of effectivity of substrates in inhibiting the reaction of the slowly reacting thiols is very similar to the order of their protective effect against heat inactivation. Both phenomena presuinably reflect the structurestabilizing effect of substrates.3-Phosphoglycerate kinase has a highly conserved structure as indicated by the X-ray analysis of horse muscle [I, 21 and yeast [3] enzymes. Great similarities were also observed in the amino acid sequences ofthe horse muscle [4,5] and human [6] enzymes and in the catalytic properties of 3-phosphoglycerate kinases isolated from a wide variety of sources [7].The most remarkable feature of the enzyme structure is that its single polypeptide chain is organized into two domains [l -31. The nucleotide substrates and the triosephosphate substrates are bound to the C-terminal and N-terminal domains, respectively [2]. It was suggested [2,8] that a large-scale hinge-bending of the domains brings the two substrates together for catalysis. The results of different physicocheinical measurements [9-1 I] seem to be in agreement with this hypothesis. However, the contribution of each substrate to the conformational motion is not yet clear.The substantial conformational effects of MgATP and 3-phosphoglycerate are indicated by the fact that they affect the reactivity of the single thiol group of yeast 3-phosphoglycerate kinase; this residue is 3 nm away from the nucleotide binding site [12,13]. Chemical modification studies were also carried out with rabbit muscle 3-phosphoglycerate kinase, which has seven thiol groups/molecule enzyme. It was shown qualitatively that ADP decreased the reactivity of the two rapidly reacting thiols, whereas 3-phosphoglycerate lessened the reactivity of the five slowly reacting thiol groups [14].On the basis of affinity binding studies, the conformational effect of only 1,3-bisphosphoglycerate was UnambigOiisly Ahhr~wiuti~ii. Nbsl (Ellman's reagent), 5,5'-dithio-bis(2-nitrobenzoic acid).Fnzvmes. 3-Phosphoglycerate kinasc or ATP: 3-phospho-~-glycerate 1 -phosphotransferase (EC 2.7.2.3); glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). demonstrated and the function-related conformational change was attributed to this substrate [15].In the present work the conformational effect of subst...
Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.
A 13,863-base-pair (bp) putative centromeric DNA fragment has been isolated from a human genomic library by using a probe obtained from metaphase chromosomes of human colon carcinoma cells. The abundance of this DNA was estimated to be 16-32 copies per genome. Cotransfection of mouse cells with this sequence and a selectable marker gene (aminoglycoside 3'-phosphotransferase type II, APH-II) resulted in a transformed cell line carrying an additional centromere in a dicentric chromosome. This centromere was capable of binding an anti-centromere antibody. In situ hybridization demonstrated that the human DNA sequence as well as the APH-II gene and vector DNA sequences were located only in the additional centromere of the dicentric chromosome. The extra centromere separated from the dicentric chromosome, forming a stable minichromosome. This functional centromere linked to a dominant selectable marker may be a step toward the construction of an artificial mammalian chromosome.The centromere is a specialized region of the eukaryotic chromosome that is the site of kinetochore formation, a structure that allows the precise segregation of chromosomes during cell division and may play a role in the higher-order organization of eukaryotic chromosomes (1). (6). Isolated metaphase chromosomes were resuspended in 1 ml of buffer (150 mM NaCl/50 mM Tris-HCl, pH 7.5/10 mM MgCl2/5 mM 2-mercaptoethanol) at a DNA concentration of 1 mg/ml and digested with 500 units of EcoRI restriction endonuclease for 1 h. The suspension was diluted with 4 ml of IPP buffer (500 mM NaCI/10 mM Tris-HCl, pH 8.0/0.1% Nonidet P40
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