ABSTRACT
As alterations in retinoblastoma (RB)/E2F pathway are commonly found in human cancers, the molecular mechanism underlying cell cycle deregulation caused by the mutations in the RB/E2F pathway needs to be investigated extensively. Compared with good understanding of RB/E2F functions in G1-S cell cycle progression, it is not fully understood how an abrogated RB pathway affects the G2-M phase of the cell cycle. Here, we report that disruption of RB accelerated G2-M progression in the presence of DNA damage by elevating the expression of a set of mitotic regulatory genes. We generated RB(+)- and (-)-matched cells using short hairpin RNA. In the RB(-) cells, the G2/M checkpoint mediated by a DNA-damaging agent was over-ridden. With microarray analysis, we found that the expression of key G2-M regulatory genes was upregulated in RB(-) cells. In particular, we demonstrated that the proto-oncogene ECT2 was directly regulated by E2Fs. Furthermore, suppression of ECT2 expression by small interfering RNA in RB(-) cells resulted in cytokinesis arrest, suggesting that RB(-) cells lack the regulation of E2F-mediated cytokinesis. These results indicate that aberrant ECT2 expression, observed in various human tumors, could be the direct result of RB/E2F pathway deficiency, thereby contributing to cell division in cancers.
Subject(s)
Cell Division , DNA Damage , G2 Phase , Proto-Oncogene Proteins/metabolism , Retinoblastoma Protein/antagonists & inhibitors , Cell Line , Cell Proliferation , E2F Transcription Factors/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Promoter Regions, Genetic , Proto-Oncogene Mas , Proto-Oncogene Proteins/genetics , RNA InterferenceABSTRACT
A new histamine receptor, HH4R, was cloned from human leukocyte cDNA. The deduced amino acid sequence showed about 40% identity to that of the human histamine H3 receptor, HH3R. HH4R-expressing cells responded to histamine, inhibiting forskolin-induced cAMP accumulation. An H3 agonist, N-alpha-methylhistamine (NAMHA), bound specifically to HH4R, while another H3 agonist, R(-)-alpha-methylhistamine (RAMHA), and the H3 antagonist, thioperamide, competed with this binding. RAMHA, NAMHA, and imetit inhibited forskolin-induced cAMP accumulation in HH4R-expressing cells. However, the binding affinities and agonistic activities of H3 agonists to HH4R were weaker than those to HH3R. Low expression of HH4R was detected in a wide variety of peripheral tissues by RT-PCR; however, in contrast with HH3R, expression was not detected in the brain. These observations indicate that the clone is a distinct histamine receptor from HH3R, and thus is named HH4R.
Subject(s)
Histamine Agonists/pharmacology , Histamine Antagonists/pharmacology , Receptors, G-Protein-Coupled , Receptors, Histamine/physiology , Amino Acid Sequence , Binding, Competitive , Cell Line , Cimetidine/pharmacology , Cloning, Molecular , Colforsin/pharmacology , Cyclic AMP/metabolism , Histamine Agonists/pharmacokinetics , Humans , Leukocytes/physiology , Methylhistamines/pharmacokinetics , Methylhistamines/pharmacology , Molecular Sequence Data , Pyrilamine/pharmacology , Receptors, Histamine/chemistry , Receptors, Histamine/genetics , Receptors, Histamine H3/chemistry , Receptors, Histamine H3/genetics , Receptors, Histamine H4 , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Stereoisomerism , TransfectionABSTRACT
A new subfamily member of thyrotropin releasing hormone (TRH) receptor gene, TRHR2, was isolated from rat brain cDNAs. The deduced amino acid sequence of TRHR2 is 51 % identical to that of rat TRH receptor gene which was reported previously. Northern blot analysis with TRHR2 probe revealed brain-specific expression of a 9.5 kb mRNA. In a binding experiment using the TRHR2-expressing COS cells, specific binding of TRH to TRHR2 was observed with Kd value of 9 nM which was equivalent to the Kd value (= 13 nM) of TRH binding to the TRH receptor previously reported. The active metabolite of TRH, histidyl-proline diketopiperazine, or cyclo(His-Pro), showed no specific binding activity. These results suggest that TRHR2 is a novel subtype of TRH receptor.
Subject(s)
Membrane Proteins/genetics , Receptors, G-Protein-Coupled , Receptors, Thyrotropin-Releasing Hormone/genetics , Amino Acid Sequence , Animals , Base Sequence , COS Cells , Cloning, Molecular , DNA, Complementary , GTP-Binding Proteins/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , Rats , Sequence Homology, Amino AcidABSTRACT
To understand the functions of rice homologues of the Arabidopsis flowering-time gene CONSTANS (CO) and salt-tolerance gene STO, we performed a similarity search of the single-run sequence data of cDNA clones accumulated by the Rice Genome Research Program, and isolated seven rice cDNA clones (S3574, C60910, S12569, R2931, R1479, R1577, and E10707) coding for proteins containing one of two zinc-finger-like motifs. Comparison of the deduced amino acid sequences between these cDNAs and the CO gene revealed significant similarities (46%-61%) in the region of zinc-finger motifs. A domain having a high content of basic amino acids at the C-terminus of the CO protein was found in the corresponding region of proteins predicted by from cDNAs S3574, C60910, and S12569. Two amino acid sequences, "CCADEAAL" and "FCV(L)EDRA," which were present inside each zinc-finger in the Arabidposis regulatory protein STO, were also found in each of the two zinc-finger regions of proteins predicted from cDNAs R2931, R1479, R1577, and E10707. Using restriction fragment length polymorphism (RFLP) linkage analysis, we determined the chromosomal location of the seven cDNA clones. The position of R2931 on the RFLP linkage map was closely linked to Hd-3, one of the putative quantitative trait loci (QTL) controlling heading date in rice.
Subject(s)
Arabidopsis Proteins , Chromosome Mapping , Genes, Plant , Oryza/genetics , Plant Proteins/genetics , Zinc Fingers/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary/isolation & purification , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Genetic Linkage , Molecular Sequence Data , Oryza/chemistry , Plant Proteins/chemistry , Polymorphism, Restriction Fragment Length , Quantitative Trait, Heritable , Restriction Mapping , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription, GeneticABSTRACT
The two gene clusters rps3-rpl16 and nad3-rps12 are separated from each other in the mitochondrial genome and are expressed as the individual transcription units in many plants. In rice mitochondrial DNA (mtDNA), the four genes rps3, rpl16, nad3 and rps12 are located within a region of 6 kbp. Northern-blot analysis revealed that a large transcript (6.6 kb) hybridized to both the rps3-rpl16 and the nad3-rps12 gene clusters. Using RT-PCR, we amplified a fragment of anticipated size (790 bp) from two primers that corresponded to sequences in the coding regions of rpl16 and nad3, demonstrating that at least two of the four genes, namely rpl16 and nad3, were co-transcribed. These results together indicated that all four genes, namely, rps3, rpl16, nad3 and rps12, were co-transcribed in rice mitochondria. Transcription initiation sites were determined by an in vitro capping/ribonuclease protection assay and primer extension analysis. Two initiation sites were identified in the rps3-rpl16-nad3-rps12 gene cluster: one was located upstream of rps3 and the other was located between rpl16 and nad3. This evidence indicates that the rps3-rpl16-nad3-rps12 gene cluster is transcribed from two alternative promoters.
Subject(s)
DNA, Mitochondrial/genetics , Multigene Family , Oryza/genetics , Promoter Regions, Genetic , Transcription, Genetic , Base Sequence , Blotting, Southern , Molecular Sequence Data , RNA Caps/metabolism , RNA, Messenger/genetics , Restriction Mapping , Ribosomal Proteins/geneticsABSTRACT
A part of the tRNA(Leu)(UAA) gene containing a 240-nucleotide group I intron was amplified by PCR from cyanobacterium Synechococcus PCC 6301 genomic DNA. The pre-tRNA synthesized from the cloned PCR product was efficiently self-spliced in vitro under physiological conditions. The gene encoding the tRNA(Leu)(UAA), trnL-UAA, was isolated from a Synechococcus PCC 6301 genomic library and the nucleotide sequence of a 2,167-bp portion was determined. The trnL-UAA consists of a 34-bp 5' exon, a 240-bp group I intron and a 50-bp 3' exon. In addition, three open reading frames (ORF1, ORF2 and ORF3) were found in the 5' and 3' flanking regions of trnL-UAA. The predicted protein sequence of ORF3, which is located 74-bp upstream from trnL-UAA on the opposite strand, shows 66.2% amino acid identity to that of the Synechocystis PCC 6803 gene encoding subunit L of NADH dehydrogenase (ndhL).
Subject(s)
Cyanobacteria/genetics , Genes, Bacterial/genetics , Introns/genetics , NADH Dehydrogenase/genetics , RNA, Transfer, Leu/genetics , Amino Acid Sequence , Base Sequence , Cyanobacteria/enzymology , DNA, Bacterial/genetics , Genome, Bacterial , Molecular Sequence Data , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA Splicing , Selection, GeneticABSTRACT
The nucleotide sequence of a 27,588-bp region of rice mitochondrial DNA was determined. This sequence contains putative genes that encode initiator methionine tRNA (trnfM), subunits III (nad3) and IV (nad4) of the NADH dehydrogenase complex, and ribosomal proteins S3 (rps3), S12 (rps12) and L16 (rpl16). An open reading frame that contains sequences homologous to parts of rps2 and atpA is also present. In addition to these regions, there are many short sequences with homology to fragments of mitochondrial DNAs from rice or other plants. These sequences may be remnants of multiple rearrangements of the genome and their presence seems to explain, in part, the large sizes of the mitochondrial genomes of higher plants.
Subject(s)
DNA, Mitochondrial/genetics , Genes, Plant/genetics , Oryza/genetics , Base Sequence , Gene Library , Molecular Sequence Data , NADH Dehydrogenase/genetics , Oryza/enzymology , Proton-Translocating ATPases/genetics , RNA, Transfer, Met/genetics , Ribosomal Proteins/genetics , Sequence Analysis, DNAABSTRACT
The human P68 protein is an ATP-dependent RNA helicase and thought to be involved in cell growth and division. We have isolated a Nicotiana sylvestris cDNA which encodes a p68-like protein. Northern blot analysis showed that the transcript from the gene is accumulated in N. sylvestris leaves, roots and flowers, but not in N. tabacum-cultured cells.
Subject(s)
Nicotiana/genetics , Plant Proteins/genetics , Plants, Toxic , RNA Nucleotidyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , DNA, Complementary , Humans , Molecular Sequence Data , RNA Helicases , Sequence Homology, Amino Acid , Nicotiana/enzymologyABSTRACT
Tobacco chloroplast ribosomal protein L12 was isolated as a ssDNA-cellulose-binding protein from a chloroplast soluble protein fraction. Based on the N-terminal amino acid sequence of chloroplast L12, a cDNA clone was isolated and characterized. The precursor protein deduced from the DNA sequence consists of a transient peptide of 53 amino acid residues and a mature L12 protein of 133 amino acid residues. The chloroplast L12 protein was synthesized with a reticulocyte lysate and subjected to nucleic acid-binding assays. L12 synthesized in vitro does not bind to ssDNA, dsDNA nor ribonucleotide homopolymers, but it binds to cellulose matrix.
