Identification of a small, very acidic constitutive nucleolar protein (NO29) as a member of the nucleoplasmin family

We report the discovery and molecular characterization of a small and very acidic nucleolar protein of an SDS/PAGE mobility corresponding to Mr 29,000 (NO29). The cDNA-deduced sequence of the Xenopus laevis protein defines a polypeptide of a calculated molecular mass of 20,121 and a pI of 3.75, with an extended acidic region near its C terminus, and is related to the major nucleolar protein, NO38, and the histone-binding protein, nucleoplasmin. This member of the nucleoplasmin family of proteins was immunolocalized to nucleoli in Xenopus oocytes and diverse somatic cells. Protein NO29 is associated with nuclear particles from Xenopus oocytes, partly complexed with protein NO38, and occurs in preribosomes but not in mature ribosomes. The location and the enormously high content of negatively charged amino acids lead to the hypothesis that NO29 might be involved in the nuclear and nucleolar accumulation of ribosomal proteins and the coordinated assembly of pre-ribosomal particles.

Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein

The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-à-brac tramtrack broad complex/pox viruses and zinc fingers). Using a yeast two-hybrid screen, we show here that the LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. Furthermore, DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Finally, overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation.

Mitochondrial DNA rearrangements of Podospora anserina are under the control of the nuclear gene grisea

Podospora anserina is a filamentous fungus with a limited life span. Life span is controlled by nuclear and extranuclear genetic traits. Herein we report the nature of four alterations in the nuclear gene grisea that lead to an altered morphology, a defect in the formation of female gametangia, and an increased life span. Three sequence changes are located in the 5′ upstream region of the grisea ORF. One mutation is a G → A transition at the 5′ splice site of the single intron of the gene, leading to a RNA splicing defect. This loss-of-function affects the amplification of the first intron of the mitochondrial cytochrome c oxidase subunit I gene (COI) and the specific mitochondrial DNA rearrangements that occur during senescence of wild-type strains. Our results indicate that the nuclear gene grisea is part of a molecular machinery involved in the control of mitochondrial DNA reorganizations. These DNA instabilities accelerate but are not a prerequisite for the aging of P. anserina cultures.

The unique hetero-oligomeric nature of the subunits in the catalytic cooperativity of the yeast Cct chaperonin complex

The structural and functional organization of the Cct complex was addressed by genetic analyses of subunit interactions and catalytic cooperativity among five of the eight different essential subunits, Cct1p–Cct8p, in the yeast Saccharomyces cerevisiae. The cct1–1, cct2–3, and cct3–1 alleles, containing mutations at the conserved putative ATP-binding motif, GDGTT, are cold-sensitive, whereas single and multiple replacements of the corresponding motif in Cct6p are well tolerated by the cell. We demonstrated herein that cct6–3 (L19S), but not the parolog cct1–5 (R26I), specifically suppresses the cct1–1, cct2–3, and cct3–1 alleles, and that this suppression can be modulated by mutations in a putative phosphorylation motif, RXS, and the putative ATP-binding pocket of Cct6p. Our results suggest that the Cct ring is comprised of a single hetero-oligomer containing eight subunits of differential functional hierarchy, in which catalytic cooperativity of ATP-binding/hydrolysis takes place in a sequential manner different from the concerted cooperativity proposed for GroEL.

Selective maintenance of allozyme differences among sympatric host races of the apple maggot fly

Whether phytophagous insects can speciate in sympatry when they shift and adapt to new host plants is a controversial question. One essential requirement for sympatric speciation is that disruptive selection outweighs gene flow between insect populations using different host plants. Empirical support for host-related selection (i.e., fitness trade-offs) is scant, however. Here, we test for host-dependent selection acting on apple (Malus pumila)- and hawthorn (Crataegus spp.)-infesting races of Rhagoletis pomonella (Diptera: Tephritidae). In particular, we examine whether the earlier fruiting phenology of apple trees favors pupae in deeper states of diapause (or with slower metabolisms/development rates) in the apple fly race. By experimentally lengthening the time period preceding winter, we exposed hawthorn race pupae to environmental conditions typically faced by apple flies. This exposure induced a significant genetic response at six allozyme loci in surviving hawthorn fly adults toward allele frequencies found in the apple race. The sensitivity of hawthorn fly pupae to extended periods of warm weather therefore selects against hawthorn flies that infest apples and helps to maintain the genetic integrity of the apple race by counteracting gene flow from sympatric hawthorn populations. Our findings confirm that postzygotic reproductive isolation can evolve as a pleiotropic consequence of host-associated adaptation, a central tenet of nonallopatric speciation. They also suggest that one reason for the paucity of reported fitness trade-offs is a failure to consider adequately costs associated with coordinating an insect’s life cycle with the phenology of its host plant.

The atypical codon usage of the plant psbA gene may be the remnant of an ancestral bias

The psbA gene of the chloroplast genome has a codon usage that is unusual for plant chloroplast genes. In the present study the evolutionary status of this codon usage is tested by reconstructing putative ancestral psbA sequences to determine the pattern of change in codon bias during angiosperm divergence. It is shown that the codon biases of the ancestral genes are much stronger than all extant flowering plant psbA genes. This is related to previous work that demonstrated a significant increase in synonymous substitution in psbA relative to other chloroplast genes. It is suggested, based on the two lines of evidence, that the codon bias of this gene currently is not being maintained by selection. Rather, the atypical codon bias simply may be a remnant of an ancestral codon bias that now is being degraded by the mutation bias of the chloroplast genome, in other words, that the psbA gene is not at equilibrium. A model for the evolution of selective pressure on the codon usage of plant chloroplast genes is discussed.

Copolymer 1 induces T cells of the T helper type 2 that crossreact with myelin basic protein and suppress experimental autoimmune encephalomyelitis

The synthetic amino acid copolymer copolymer 1 (Cop 1) suppresses experimental autoimmune encephalomyelitis (EAE) and is beneficial in multiple sclerosis. To further understand Cop 1 suppressive activity, we studied the cytokine secretion profile of various Cop 1-induced T cell lines and clones. Unlike T cell lines induced by myelin basic protein (MBP), which secreted either T cell helper type 1 (Th1) or both Th1 and Th2 cytokines, the T cell lines/clones induced by Cop 1 showed a progressively polarized development toward the Th2 pathway, until they completely lost the ability to secrete Th1 cytokines. Our findings indicate that the polarization of the Cop 1-induced lines did not result from the immunization vehicle or the in vitro growing conditions, but rather from the tendency of Cop 1 to preferentially induce a Th2 response. The response of all of the Cop 1 specific lines/clones, which were originated in the (SJL/J×BALB/c)F1 hybrids, was restricted to the BALB/c parental haplotype. Even though the Cop 1-induced T cells had not been exposed to the autoantigen MBP, they crossreacted with MBP by secretion of interleukin (IL)-4, IL-6, and IL-10. Administration of these T cells in vivo resulted in suppression of EAE induced by whole mouse spinal cord homogenate, in which several autoantigens may be involved. Secretion of anti-inflammatory cytokines by Cop 1-induced suppressor cells, in response to either Cop 1 or MBP, may explain the therapeutic effect of Cop 1 in EAE and in multiple sclerosis.

Characterization of the CHD family of proteins

The murine gene CHD1 (MmCHD1) was previously isolated in a search for proteins that bound a DNA promoter element. The presence of chromo (chromatin organization modifier) domains and an SNF2-related helicase/ATPase domain led to speculation that this gene regulated chromatin structure or gene transcription. This study describes the cloning and characterization of three novel human genes related to MmCHD1. Examination of sequence databases produced several more related genes, most of which were not known to be similar to MmCHD1, yielding a total of 12 highly conserved CHD genes from organisms as diverse as yeast and mammals. The major region of sequence variation is in the C-terminal part of the protein, a region with DNA-binding activity in MmCHD1. Targeted deletion of ScCHD1, the sole Saccharomyces cerevesiae CHD gene, was performed with deletion strains being less sensitive than wild type to the cytotoxic effect of 6-azauracil. This finding suggested that enhanced transcriptional arrest at RNA polymerase II pause sites due to 6-azauracil-induced nucleotide pool depletion was reduced in the deletion strain and that ScCHD1 inhibited transcription. This observation, along with the known roles of other proteins with chromo or SNF2-related helicase/ATPase domains, suggests that alteration of gene expression by CHD genes might occur by modifications of chromatin structure, with altered access of the transcriptional apparatus to its chromosomal DNA template.

Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH−/− mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH−/− mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH−/− mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.

Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites

Immune responses of the malaria vector mosquito Anopheles gambiae were monitored systematically by the induced expression of five RNA markers after infection challenge. One newly isolated marker encodes a homologue of the moth Gram-negative bacteria-binding protein (GNBP), and another corresponds to a serine protease-like molecule. Additional previously described markers that respond to immune challenge encode the antimicrobial peptide defensin, a putative galactose lectin, and a putative serine protease. Specificity of the immune responses was indicated by differing temporal patterns of induction of specific markers in bacteria-challenged larvae and adults, and by variations in the effectiveness of different microorganisms and their components for marker induction in an immune-responsive cell line. The markers exhibit spatially distinct patterns of expression in the adult female mosquito. Two of them are highly expressed in different regions of the midgut, one in the anterior and the other in the posterior midgut. Marker induction indicates a significant role of the midgut in insect innate immunity. Immune responses to the penetration of the midgut epithelium by a malaria parasite occur both within the midgut itself and elsewhere in the body, suggesting an immune-related signaling process.

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-β superfamily

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor β (TGF-β) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1β, tumor necrosis factor α (TNF-α), interleukin 2, and macrophage colony-stimulating factor but not interferon γ, or lipopolysaccharide (LPS). Its expression is also increased by TGF-β. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-α production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-β may serve to limit the later phases of macrophage activation.

Targeted disruption of the mouse gene encoding steroidogenic acute regulatory protein provides insights into congenital lipoid adrenal hyperplasia

An essential component of regulated steroidogenesis is the translocation of cholesterol from the cytoplasm to the inner mitochondrial membrane where the cholesterol side-chain cleavage enzyme carries out the first committed step in steroidogenesis. Recent studies showed that a 30-kDa mitochondrial phosphoprotein, designated steroidogenic acute regulatory protein (StAR), is essential for this translocation. To allow us to explore the roles of StAR in a system amenable to experimental manipulation and to develop an animal model for the human disorder lipoid congenital adrenal hyperplasia (lipoid CAH), we used targeted gene disruption to produce StAR knockout mice. These StAR knockout mice were indistinguishable initially from wild-type littermates, except that males and females had female external genitalia. After birth, they failed to grow normally and died from adrenocortical insufficiency. Hormone assays confirmed severe defects in adrenal steroids—with loss of negative feedback regulation at hypothalamic–pituitary levels—whereas hormones constituting the gonadal axis did not differ significantly from levels in wild-type littermates. Histologically, the adrenal cortex of StAR knockout mice contained florid lipid deposits, with lesser deposits in the steroidogenic compartment of the testis and none in the ovary. The sex-specific differences in gonadal involvement support a two-stage model of the pathogenesis of StAR deficiency, with trophic hormone stimulation inducing progressive accumulation of lipids within the steroidogenic cells and ultimately causing their death. These StAR knockout mice provide a useful model system in which to determine the mechanisms of StAR’s essential roles in adrenocortical and gonadal steroidogenesis.

Identification of the prooxidant site of human ceruloplasmin: A model for oxidative damage by copper bound to protein surfaces

Free transition metal ions oxidize lipids and lipoproteins in vitro; however, recent evidence suggests that free metal ion-independent mechanisms are more likely in vivo. We have shown previously that human ceruloplasmin (Cp), a serum protein containing seven Cu atoms, induces low density lipoprotein oxidation in vitro and that the activity depends on the presence of a single, chelatable Cu atom. We here use biochemical and molecular approaches to determine the site responsible for Cp prooxidant activity. Experiments with the His-specific reagent diethylpyrocarbonate (DEPC) showed that one or more His residues was specifically required. Quantitative [14C]DEPC binding studies indicated the importance of a single His residue because only one was exposed upon removal of the prooxidant Cu. Plasmin digestion of [14C]DEPC-treated Cp (and N-terminal sequence analysis of the fragments) showed that the critical His was in a 17-kDa region containing four His residues in the second major sequence homology domain of Cp. A full length human Cp cDNA was modified by site-directed mutagenesis to give His-to-Ala substitutions at each of the four positions and was transfected into COS-7 cells, and low density lipoprotein oxidation was measured. The prooxidant site was localized to a region containing His426 because CpH426A almost completely lacked prooxidant activity whereas the other mutants expressed normal activity. These observations support the hypothesis that Cu bound at specific sites on protein surfaces can cause oxidative damage to macromolecules in their environment. Cp may serve as a model protein for understanding mechanisms of oxidant damage by copper-containing (or -binding) proteins such as Cu, Zn superoxide dismutase, and amyloid precursor protein.

Transfer of the HIV-1 cyclophilin-binding site to simian immunodeficiency virus from Macaca mulatta can confer both cyclosporin sensitivity and cyclosporin dependence

HIV-1 specifically incorporates the peptidyl prolyl isomerase cyclophilin A (CyPA), the cytosolic receptor for the immunosuppressant cyclosporin A (CsA). HIV-1 replication is inhibited by CsA as well as by nonimmunosuppressive CsA analogues that bind to CyPA and interfere with its virion association. In contrast, the related simian immunodeficiency virus SIVmac, which does not interact with CyPA, is resistant to these compounds. The incorporation of CyPA into HIV-1 virions is mediated by a specific interaction between the active site of the enzyme and the capsid (CA) domain of the HIV-1 Gag polyprotein. We report here that the transfer of HIV-1 CA residues 86–93, which form part of an exposed loop, to the corresponding position in SIVmac resulted in the efficient incorporation of CyPA and conferred an HIV-1-like sensitivity to a nonimmunosuppressive cyclosporin. HIV-1 CA residues 86–90 were also sufficient to transfer the ability to efficiently incorporate CyPA, provided that the length of the CyPA-binding loop was preserved. However, the resulting SIVmac mutant required the presence of cyclosporin for efficient virus replication. The results indicate that the presence or absence of a type II tight turn adjacent to the primary CyPA-binding site determines whether CyPA incorporation enhances or inhibits viral replication. By demonstrating that CyPA-binding-site residues can induce cyclosporin sensitivity in a heterologous context, this study provides direct in vivo evidence that the exposed loop between helices IV and V of HIV-1 CA not merely constitutes a docking site for CyPA but is a functional target of this cellular protein.

Decreased blood pressure response in mice deficient of the α1b-adrenergic receptor

To investigate the functional role of different α1-adrenergic receptor (α1-AR) subtypes in vivo, we have applied a gene targeting approach to create a mouse model lacking the α1b-AR (α1b−/−). Reverse transcription–PCR and ligand binding studies were combined to elucidate the expression of the α1-AR subtypes in various tissues of α1b +/+ and −/− mice. Total α1-AR sites were decreased by 98% in liver, 74% in heart, and 42% in cerebral cortex of the α1b −/− as compared with +/+ mice. Because of the large decrease of α1-AR in the heart and the loss of the α1b-AR mRNA in the aorta of the α1b−/− mice, the in vivo blood pressure and in vitro aorta contractile responses to α1-agonists were investigated in α1b +/+ and −/− mice. Our findings provide strong evidence that the α1b-AR is a mediator of the blood pressure and the aorta contractile responses induced by α1 agonists. This was demonstrated by the finding that the mean arterial blood pressure response to phenylephrine was decreased by 45% in α1b −/− as compared with +/+ mice. In addition, phenylephrine-induced contractions of aortic rings also were decreased by 25% in α1b−/− mice. The α1b-AR knockout mouse model provides a potentially useful tool to elucidate the functional specificity of different α1-AR subtypes, to better understand the effects of adrenergic drugs, and to investigate the multiple mechanisms involved in the control of blood pressure.