RNA interference is an evolutionarily conserved gene-silencing pathway in which the nuclease Dicer cleaves double-stranded RNA into small interfering RNAs. The biological function of the RNAi-related pathway in vertebrate cells is not fully understood. Here, we report the generation of a conditional loss-of-function Dicer mutant in a chicken-human hybrid DT40 cell line that contains human chromosome 21. We show that loss of Dicer results in cell death with the accumulation of abnormal mitotic cells that show premature sister chromatid separation. Aberrant accumulation of transcripts from alpha-satellite sequences, which consist of human centromeric repeat DNAs, was detected in Dicer-deficient cells. Immunocytochemical analysis revealed abnormalities in the localization of two heterochromatin proteins, Rad21 cohesin protein and BubR1 checkpoint protein, but the localization of core kinetochore proteins such as centromere protein (CENP)-A and -C was normal. We conclude that Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells.
We used 2D protein gel electrophoresis and DNA microarray technologies to systematically analyze genes under glucose repression in B:acillus subtilis. In particular, we focused on genes expressed after the shift from glycolytic to gluconeogenic at the middle logarithmic phase of growth in a nutrient sporulation medium, which remained repressed by the addition of glucose. We also examined whether or not glucose repression of these genes was mediated by CcpA, the catabolite control protein of this bacterium. The wild-type and ccpA1 cells were grown with and without glucose, and their proteomes and transcriptomes were compared. 2D gel electrophoresis allowed us to identify 11 proteins, the synthesis of which was under glucose repression. Of these proteins, the synthesis of four (IolA, I, S and PckA) was under CcpA-independent control. Microarray analysis enabled us to detect 66 glucose-repressive genes, 22 of which (glmS, acoA, C, yisS, speD, gapB, pckA, yvdR, yxeF, iolA, B, C, D, E, F, G, H, I, J, R, S and yxbF ) were at least partially under CcpA-independent control. Furthermore, we found that CcpA and IolR, a repressor of the iol divergon, were involved in the glucose repression of the synthesis of inositol dehydrogenase encoded by iolG included in the above list. The CcpA-independent glucose repression of the iol genes appeared to be explained by inducer exclusion.
Two forms of human thyroid peroxidase cDNAs were isolated from a Xgtll cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTPO-1 and hTPO-2, we show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. These results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2.
Upon sulfonation, carcinogenic hydroxyarylamines such as N-hydroxy-2-acetylaminofluorene (N-OH-2AAF) can be further activated to form ultimate carcinogens in vivo. Previous studies have shown that a SULT1C1 sulfotransferase is primarily responsible for the sulfonation of N-OH-2AAF in rat liver. In the present study, two novel human sulfotransferases shown to be members of the SULT1C sulfotransferase subfamily based on sequence analysis have been cloned, expressed, and characterized. Comparisons of the deduced amino acid sequence encoded by the human SULT1C sulfotransferase cDNA 1 reveal 63.7, 61.6, and 85.1% identity to the amino acid sequences of rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. In contrast, the deduced amino acid sequence of the human SULT1C sulfotransferase 2 cDNA displays 62.9, 63.1, 63.1, and 62.5% identity to the amino acid sequences of the human SULT1C sulfotransferase 1, rat SULT1C1 sulfotransferase, mouse SULT1C1 sulfotransferase, and rabbit SULT1C sulfotransferase. Recombinant human SULT1C sulfotransferases 1 and 2, expressed in Escherichia coli and purified to near electrophoretic homogeneity, were shown to cross-react with the antiserum against the rat liver SULT1C1 sulfotransferase and exhibited sulfonating activities with N-OH-2AAF as substrate. Tissue-specific expression of these novel human SULT1C sulfotransferases were examined by employing the Northern blotting technique. The results provide a foundation for the investigation into the functional relevance of these new SULT1C sulfotransferases in different human tissues/organs.
Asf1 (anti-silencing function 1), a well conserved protein from yeast to humans, acts as a histone chaperone and is predicted to participate in a variety of chromatin-mediated cellular processes. To investigate the physiological role of vertebrate Asf1 in vivo, we generated a conditional Asf1-deficient mutant from chicken DT40 cells. Induction of Asf1 depletion resulted in the accumulation of cells in S phase with decreased DNA replication and increased mitotic aberrancy forming multipolar spindles, leading to cell death. In addition, nascent chromatin in Asf1-depleted cells showed increased nuclease sensitivity, indicating impaired nucleosome assembly during DNA replication. Complementation analyses revealed that the functional domain of Asf1 for cell viability was confined to the N-terminal core domain (amino acids 1-155) that is a binding platform for histones H3/H4, CAF-1p60, and HIRA, whereas Asf1 mutant proteins, abolishing binding abilities with both p60 and HIRA, exhibit no effect on viability. These results together indicate that the vertebrate Asf1 plays a crucial role in replication-coupled chromatin assembly, cell cycle progression, and cellular viability and provide a clue of a possible role in a CAF-1-and HIRA-independent chromatin-modulating process for cell proliferation.During S phase, newly synthesized histones H3-H4 are assembled behind the replication fork, followed by loading of the H2A-H2B dimer to complete de novo nucleosome formation on newly replicated DNA (1-3). Besides such a replication-coupled chromatin assembly process, a DNA synthesis-independent chromatin assembly process also exists to operate histone deposition during transcription and DNA repair (4). These processes are mediated by several specialized histone chaperones (1). CAF-1 (chromatin assembly factor-1), a trimeric protein complex consisting of p150, p60, and p48 subunits, is the most characterized chaperone responsible for loading of histones H3 and H4 onto replicating DNA through an interaction with proliferating cell nuclear antigen (PCNA), 2
The expression of six novel genes located in the region from abrB to spoVC of the Bacillus subtilis chromosome was analyzed, and one of the genes, yabG, had a predicted promoter sequence conserved among SigKdependent genes. Northern blot analysis revealed that yabG mRNA was first detected from 4 h after the cessation of logarithmic growth (T 4 ) in wild-type cells and in a gerE36 (GerE ؊ ) mutant but not in spoIIAC (SigF ؊ ), spoIIGAB (SigE ؊ ), spoIIIG (SigG ؊ ), and spoIVCB (SigK ؊ ) mutants. The transcription start point was determined by primer extension analysis; the ؊10 and ؊35 regions are very similar to the consensus sequences recognized by SigK-containing RNA polymerase. Inactivation of the yabG gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of yabG spores in L-alanine and in a mixture of L-asparagine, D-glucose, D-fructose, and potassium chloride was also the same as that of wild-type spores. On the other hand, the protein preparation from yabG spores included 15-, 18-, 21-, 23-, 31-, 45-, and 55-kDa polypeptides which were low in or not extracted from wild-type spores under the same conditions. We determined their N-terminal amino acid sequence and found that these polypeptides were CotT, YeeK, YxeE, CotF, YrbA (31 and 45 kDa), and SpoIVA, respectively. The fluorescence of YabG-green fluorescent protein fusion produced in sporulating cells was detectable in the forespores but not in the mother cell compartment under fluorescence microscopy. These results indicate that yabG encodes a sporulation-specific protein which is involved in coat protein composition in B. subtilis.
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