Rearrangements of 3-aryl-substituted cyclopropenyl anions and the gas-phase acidity of 3-(4-methylphenyl)cyclopropene.

3-(4-Methylphenyl)-3-trimethylsilylcyclopropene and 3-(4-trifluoromethylphenyl)-3-trimethylsilylcyclopropene react with fluoride ion in the gas phase to afford 6-substituted 3-indenyl anions via a spontaneous rearrangement of their corresponding cyclopropenyl anions. These isomerizations led us to reinvestigate the reported gas-phase generation of 1,2,3-triphenylcyclopropenyl anion, and contrary to the previous study, a similar rearrangement to 1,2-diphenyl-1-indenyl anion is observed. Despite the instability of 3-aryl-3-cyclopropenyl anions, we were able to measure the acidity of 3-(4-methylphenyl)cyclopropene at the allylic position (delta H(o)acid = 398.6 +/- 1.4 kcal/mol) by the DePuy kinetic method. Ab initio calculations on the structures and energies of mono- and triaryl-substituted cyclopropenyl anions also are presented.

Cloning and characterization of human histone deacetylase 8.

To date, seven different human histone deacetylases (HDACs) have been identified, which fall into two distinct classes. We have isolated and characterized a cDNA encoding a novel human HDAC, which we name HDAC8. HDAC8 shows a high degree of sequence similarity to HDAC1 and HDAC2 and thus belongs to the class I of HDACs. HDAC8 is expressed in a variety of tissues. Human cells overexpressing HDAC8 localize the protein in sub-nuclear compartments whereas HDAC1 shows an even nuclear distribution. In addition, the HDAC8 gene is localized on the X chromosome at position q13, which is close to the XIST gene and chromosomal breakpoints associated with preleukemia.

Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription.

CpG methylation in vertebrates correlates with alterations in chromatin structure and gene silencing. Differences in DNA-methylation status are associated with imprinting phenomena and carcinogenesis. In Xenopus laevis oocytes, DNA methylation dominantly silences transcription through the assembly of a repressive nucleosomal array. Methylated DNA assembled into chromatin binds the transcriptional repressor MeCP2 which cofractionates with Sin3 and histone deacetylase. Silencing conferred by MeCP2 and methylated DNA can be relieved by inhibition of histone deacetylase, facilitating the remodelling of chromatin and transcriptional activation. These results establish a direct causal relationship between DNA methylation-dependent transcriptional silencing and the modification of chromatin.

Vector methylation inhibits transcription from the SV40 early promoter.

Methylation of a plasmid containing the SV40 promoter linked to the chloramphenicol acetyl transferase (CAT) gene, with either murine DNA methylase or methylase SssI results in inhibition of the expression of the reporter gene after transfection into cultured cells. Methylation of the plasmid with the methylases HhaI and HpaII has no effect on the expression of this gene. Protein-DNA interactions in the SV40 promoter are not affected by the presence of methylcytosine suggesting that inactivation results from the formation of an inactive chromatin structure that is dependent on the high CG content of the plasmid.

The first pre-rRNA-processing event occurs in a large complex: analysis by gel retardation, sedimentation, and UV cross-linking.

The first processing event that mouse pre-rRNA undergoes occurs within the external transcribed spacer and is efficiently reproduced in vitro. Analysis with nondenaturing polyacrylamide gels revealed the formation of heparin-resistant complexes of retarded electrophoretic mobility on the substrate rRNA. The specificity of these complexes was demonstrated by their elimination due to competition with processing-competent, but not with processing-incompetent, rRNAs. Furthermore, complex formation, like the processing cleavage, required only 28 nucleotides of rRNA sequence adjacent to the processing site but was stimulated by additional downstream conserved sequences. These processing complexes formed in a time-dependent manner, and once assembled, they were stable to challenge by competitor rRNA and remained on the processed rRNA. Their sedimentation coefficient was approximately 20S. UV cross-linking studies with 4-thiouridine-substituted rRNA have identified six polypeptides, 52 to 250 kilodaltons, that are specifically bound to the rRNA processing substrate.

The U3 small nucleolar ribonucleoprotein functions in the first step of preribosomal RNA processing.

The first cleavage in mammalian pre-rRNA maturation occurs near the 5' end within the 5' external transcribed spacer. Using mouse cell extracts, we show that this processing is abolished by micrococcal nuclease pretreatment. Autoantibodies that recognize the U3, U8, and U13 snRNPs (anti-fibrillarin) deplete processing activity from the extract and selectively immunoprecipitate both rRNA substrates and processing products from the reaction. Specific involvement of the U3 snRNP is demonstrated by native gel electrophoresis of the processing reaction followed by Northern blotting and by oligonucleotide-directed RNAase H abolition of processing activity. Our identification of U3 function is discussed with respect to the molecular basis of pre-rRNA recognition by the U3 snRNP, possible roles of U3 and other nucleolar snRNPs in rRNA processing, and the morphological organization of the nucleolus and the ribosomal transcription complex.

Nucleotide sequence determining the first cleavage site in the processing of mouse precursor rRNA.

The first step in the processing of 47S precursor rRNA in mouse cells is reproduced in vitro in an S-100 transcription reaction and consists of an endonucleolytic cleavage at residue +650 of the primary transcript followed by rapid degradation of the fragment upstream from residue +650. An analogous processing occurs in human rRNA. The mouse and human rRNA sequences are approximately equal to 80% conserved for 200 nucleotides on the 3' side of these processing sites, suggesting that this conserved region may be important in specifying the processing. To test this hypothesis, we constructed a systematic series of deletion mutants approaching the mouse rDNA processing region from both the 5' and 3' directions and analyzed the processing of their transcripts in vitro. The 5' boundary of the region required for processing is quite sharp and corresponds to the rRNA cleavage site at the 5' end of the conserved sequence region. The 3' boundary is more complex: The 3' deletions extending to between 250 and 130 nucleotides beyond the processing site cause about a 50% decrease in the amount of the processed RNA. A 3' deletion that extends to 109 nucleotides beyond the processing site greatly reduces the processing efficiency. Deletions to or beyond 91 nucleotides on the 3' side of the processing site virtually eliminate processing. Under altered ionic conditions, transcripts of 3' deletions extending to only 41 nucleotides beyond the processing site can still direct a low level of accurate processing. These results demonstrate that the mouse/human conserved sequence just on the 3' side of the primary rRNA processing site consists of several domains that direct and/or augment both the initial endonucleolytic cleavage and the closely coupled selective degradation of the upstream fragment that together constitute the primary rRNA processing event.

Chemical studies on polyoma and Shope papilloma viruses.

Polyoma and Shope papilloma viruses were purified and analyzed by chemical and physical methods. Disc electrophoresis of degraded virions indicated the presence, in both cases, of only one major species of polypeptide subunit. The weight of the peptide chain of polyoma virus was estimated in 8 m urea to be about 45,000 avograms, based on the sedimentation rate in a sucrose-urea gradient and the diffusion coefficient estimated from the differential migration in electrophoresis in gels of different pore size. The presence of a minor peptide of smaller size was suggested by carboxyl-terminal and sedimentation analyses. The amino acid composition of polyoma capsid protein was reported. Chemical analyses showed that polyoma virus and Shope papilloma virus contained 16 and 17.5% deoxyribonucleic acid, respectively. Light scattering by the polyoma virion showed it to have a molecular weight of 22 x 10(6) and a diameter of 54 nm.