cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Dissociation of Oct-1 from the Nuclear Peripheral Structure Induces the Cellular Aging-associated Collagenase Gene Expression

The cellular aging-associated transcriptional repressor that we previously named as Orpheus was identical to Oct-1, a member of the POU domain family. Oct-1 represses the collagenase gene, one of the cellular aging-associated genes, by interacting with an AT-rich cis-element in the upstream of the gene in preimmortalized cells at earlier population-doubling levels and in immortalized cells. In these stages of cells, considerable fractions of the Oct-1 protein were prominently localized in the nuclear periphery and colocalized with lamin B. During the cellular aging process, however, this subspecies of Oct-1 disappeared from the nuclear periphery. The cells lacking the nuclear peripheral Oct-1 protein exhibited strong collagenase expression and carried typical senescent morphologies. Concomitantly, the binding activity and the amount of nuclear Oct-1 protein were reduced in the aging process and resumed after immortalization. However, the whole cellular amounts of Oct-1 protein were not significantly changed during either process. Thus, the cellular aging-associated genes including the collagenase gene seemed to be derepressed by the dissociation of Oct-1 protein from the nuclear peripheral structure. Oct-1 may form a transcriptional repressive apparatus by anchoring nuclear matrix attachment regions onto the nuclear lamina in the nuclear periphery.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

The dhp1+ gene, encoding a putative nuclear 5′→3′ exoribonuclease, is required for proper chromosome segregation in fission yeast

The Schizosaccharomyces pombe dhp1+ gene is an ortholog of the Saccharomyces cerevisiae RAT1 gene, which encodes a nuclear 5′→3′ exoribonuclease, and is essential for cell viability. To clarify the cellular functions of the nuclear 5′→3′ exoribonuclease, we isolated and characterized a temperature-sensitive mutant of dhp1 (dhp1-1 mutant). The dhp1-1 mutant showed nuclear accumulation of poly(A)+ RNA at the restrictive temperature, as was already reported for the rat1 mutant. Interestingly, the dhp1-1 mutant exhibited aberrant chromosome segregation at the restrictive temperature. The dhp1-1 cells frequently contained condensed chromosomes, most of whose sister chromatids failed to separate during mitosis despite normal mitotic spindle elongation. Finally, chromosomes were displaced or unequally segregated. As similar mitotic defects were also observed in Dhp1p-depleted cells, we concluded that dhp1+ is required for proper chromosome segregation as well as for poly(A)+ RNA metabolism in fission yeast. Furthermore, we isolated a multicopy suppressor of the dhp1-1 mutant, referred to as din1+. We found that the gene product of dhp1-1 was unstable at high temperatures, but that reduced levels of Dhp1-1p could be suppressed by overexpressing Din1p at the restrictive temperature. Thus, Din1p may physically interact with Dhp1p and stabilize Dhp1p and/or restore its activity.

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.

Novel Nuclear Signaling Pathway Mediates Activation of Fibroblast Growth Factor-2 Gene by Type 1 and Type 2 Angiotensin II Receptors

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1′,3′-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

In vivo detection of gene expression in liver by 31P nuclear magnetic resonance spectroscopy employing creatine kinase as a marker gene

In vivo assessment of gene expression is desirable to obtain information on the extent and duration of transduction of tissue after gene delivery. We have developed an in vivo, potentially noninvasive, method for detecting virally mediated gene transfer to the liver. The method employs an adenoviral vector carrying the gene for the brain isozyme of murine creatine kinase (CK-B), an ATP-buffering enzyme expressed mainly in muscle and brain but absent from liver, kidney, and pancreas. Gene expression was monitored by 31P magnetic resonance spectroscopy (MRS) using the product of the CK enzymatic reaction, phosphocreatine, as an indicator of transfection. The vector was administered into nude mice by tail vein injection, and exogenous creatine was administered in the drinking water and by i.p. injection of 2% creatine solution before 31P MRS examination, which was performed on surgically exposed livers. A phosphocreatine resonance was detected in livers of mice injected with the vector and was absent from livers of control animals. CK expression was confirmed in the injected animals by Western blot analysis, enzymatic assays, and immunofluorescence measurements. We conclude that the syngeneic enzyme CK can be used as a marker gene for in vivo monitoring of gene expression after virally mediated gene transfer to the liver.

Targeted mutation of the murine arylhydrocarbon receptor nuclear translocator 2 (Arnt2) gene reveals partial redundancy with Arnt

The ubiquitously expressed basic helix–loop–helix (bHLH)-PAS protein ARNT (arylhydrocarbon receptor nuclear transporter) forms transcriptionally active heterodimers with a variety of other bHLH-PAS proteins, including HIF-1α (hypoxia-inducible factor-1α) and AHR (arylhydrocarbon receptor). These complexes regulate gene expression in response to hypoxia and xenobiotics, respectively, and mutation of the murine Arnt locus results in embryonic death by day 10.5 associated with placental, vascular, and hematopoietic defects. The closely related protein ARNT2 is highly expressed in the central nervous system and kidney and also forms complexes with HIF-1α and AHR. To assess unique roles for ARNT2 in development, and reveal potential functional overlap with ARNT, we generated a targeted null mutation of the murine Arnt2 locus. Arnt2−/− embryos die perinatally and exhibit impaired hypothalamic development, phenotypes previously observed for a targeted mutation in the murine bHLH-PAS gene Sim1 (Single-minded 1), and consistent with the recent proposal that ARNT2 and SIM1 form an essential heterodimer in vivo [Michaud, J. L., DeRossi, C., May, N. R., Holdener, B. C. & Fan, C. (2000) Mech. Dev. 90, 253–261]. In addition, cultured Arnt2−/− neurons display decreased hypoxic induction of HIF-1 target genes, demonstrating formally that ARNT2/HIF-1α complexes regulate oxygen-responsive genes. Finally, a strong genetic interaction between Arnt and Arnt2 mutations was observed, indicating that either gene can fulfill essential functions in a dose-dependent manner before embryonic day 8.5. These results demonstrate that Arnt and Arnt2 have both unique and overlapping essential functions in embryonic development.

msg1, a novel melanocyte-specific gene, encodes a nuclear protein and is associated with pigmentation.

Messenger RNA transcripts of the highly pigmented murine melanoma B16-F1 cells were compared with those from their weakly pigmented derivative B16-F10 cells by differential display. A novel gene called msg1 (melanocyte-specific gene) was found to be expressed at high levels in B16-F1 cells but at low levels in B16-F10 cells. Expression of msg1 was undetectable in the amelanotic K1735 murine melanoma cells. The pigmented murine melanocyte cell line melan-a expressed msg1, as did pigmented primary cultures of murine and human melanocytes; however, seven amelanotic or very weakly pigmented human melanoma cell lines were negative. Transformation of murine melanocytes by transfection with v-Ha-ras or Ela was accompanied by depigmentation and led to complete loss of msg1 expression. The normal tissue distribution of msg1 mRNA transcripts in adult mice was confined to melanocytes and testis. Murine msg1 and human MSG1 genes encode a predicted protein of 27 kDa with 75% overall amino acid identity and 96% identity within the C-terminal acidic domain of 54 amino acids. This C-terminal domain was conserved with 76% amino acid identity in another protein product of a novel human gene, MRG1 (msg1-related gene), isolated from normal human melanocyte cDNA by 5'-rapid amplification of cDNA ends based on the homology to msg1. The msg1 protein was localized to the melanocyte nucleus by immunofluorescence cytochemistry. We conclude that msg1 encodes a nuclear protein, is melanocyte-specific, and appears to be lost in depigmented melanoma cells.

Transposition of DNase hypersensitive chromatin to the nuclear periphery coincides temporally with nerve growth factor-induced up-regulation of gene expression in PC12 cells.

To test the hypothesis that the nonrandom organization of the contents of interphase nuclei represents a compartmentalization of function, we examined the relative, spatial relationship of small nuclear ribonucleoproteins (snRNPs) and of DNase I hypersensitive chromatin (DHC) in rat pheochromocytoma cells. In controls, DHC and snRNPs colocalized as pan-nuclear speckles. During nerve growth factor-induced differentiation, both snRNPs and DHC migrated to the nuclear periphery with the migration of DHC preceding that of snRNPs, resulting in their transient separation. The formation of DHC shells temporally coincided with an up-regulation of neurofilament light chain mRNA. This indicates that the expression of this sequence may be associated with its spatial transposition to the nuclear periphery.

The SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex is required for RNA polymerase II and III snRNA gene transcription and interacts with the TATA box binding protein.

The RNA polymerase II and III small nuclear RNA (snRNA) promoters contain a common basal promoter element, the proximal sequence element (PSE). The PSE binds a multisubunit complex we refer to as the snRNA activating protein complex (SNAPc). At least four polypeptides are visible in purified SNAPc preparations, which migrate with apparent molecular masses of 43, 45, 50, and 190 kDa on SDS/polyacrylamide gels. In addition, purified preparations of SNAPc contain variable amounts of TATA box binding protein (TBP). An important question is whether the PSEs of RNA polymerase II and III snRNA promoters recruit the exact same SNAP complex or slightly different versions of SNAPc, differing, for example, by the presence or absence of a subunit. To address this question, we are isolating cDNAs encoding different subunits of SNAPc. We have previously isolated the cDNA encoding the 43-kDa subunit SNAP43. We now report the isolation of the cDNA that encodes the p45 polypeptide. Antibodies directed against p45 retard the mobility of the SNAPc-PSE complex in an electrophoretic mobility shift assay, indicating that p45 is indeed part of SNAPc. We therefore refer to this protein as SNAP45. SNAP45 is exceptionally proline-rich, interacts strongly with TBP, and, like SNAP43, is required for both RNA polymerase II and III transcription of snRNA genes.

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.

Nuclear/cytoplasmic localization of the von Hippel-Lindau tumor suppressor gene product is determined by cell density.

The product of the von Hippel-Lindau (VHL) tumor suppressor gene, the gene inactivated in VHL disease and in sporadic clear-cell renal carcinomas, has recently been shown to have as a functional target the transcription elongation complex, elongin (also called SIII). Here it is shown that there is a tightly regulated, cell-density-dependent transport of VHL into and/or out of the nucleus. In densely grown cells, the VHL protein is predominantly in the cytoplasm, whereas in sparse cultures, most of the protein can be detected in the nucleus. We have identified a putative nuclear localization signal in the first 60 and first 28 amino acids of the human and rat VHL protein, respectively. Sequences in the C-terminal region of the VHL protein may also be required for localization to the cytosol. These findings provide the initial indication of a novel cell density-dependent pathway that is responsible for the regulation of VHL cellular localization.