Thermotolerance and virulence of Aspergillus fumigatus: role of the fungal nucleolus.

The ability to thrive at 37 degrees C is characteristic of all human pathogens and has long been suspected to play a role in the pathogenesis of aspergillosis. As a thermotolerant fungus, Aspergillus fumigatus is capable of growth at temperatures that approach the upper limit for all eukaryotes, suggesting that the organism has evolved unique mechanisms of stress resistance that may be relevant to its ability to adapt to the stress of growth in the host. High temperature is a strain on many biological systems, particularly those involved in complex macromolecular assemblies such as ribosomes. This review will discuss the relationship between thermotolerance and virulence in pathogenic fungi, emphasizing the link to ribosome biogenesis in A. fumigatus. Future work in this area will help determine how rapid growth is accomplished at elevated temperature and may offer new avenues for the development of novel antifungals that disrupt thermotolerant ribosome assembly.

Identification of genes of Aspergillus fumigatus up-regulated during growth on endothelial cells.

Aspergillus fumigatus is an important opportunistic fungal pathogen that can cause acute invasive disease in neutropenic hosts. Invasive aspergillosis is being diagnosed with increasing frequency, and morbidity and mortality remain high despite prompt antifungal therapy. Because little is known about the virulence factors used by A. fumigatus, a tissue culture model was developed to mimic the interaction of the fungus with the endothelium. Differential display was used to compare gene expression in fungal cells grown on endothelial cells with that of cells grown in the absence of endothelial cell contact, and genes that were up-regulated were selected for analysis as putatively virulence-related genes. Two of these up-regulated genes were chosen for further study and were identified as genes encoding the regulatory subunit of cyclic adenosine monophosphate (cAMP)-dependent protein kinase and a member of the ras gene family, both of which are involved in cAMP-mediated signaling in fungi. This model system provides a new approach to the identification of potentially virulence-related genes induced in A. fumigatus by the interaction with host cells.

Molecular cloning of cgrA, the gene encoding the Aspergillus nidulans ortholog of Saccharomyces cerevisiae CGR1.

Saccharomyces cerevisiae CGR1 encodes a 120-amino acid protein with a predominant nucleolar localization. In this study we report the identification and cloning of the ortholog, cgrA, from Aspergillus nidulans. The cgrA gene is comprised of three exons on A. nidulans Chromosome 7. The cDNA contains a single open reading frame (ORF) that would encode a protein of 114 amino acids with 44% sequence identity to yeast Cgr1p. A plasmid expressing cgrA complemented the impaired growth phenotype of a yeast strain that can be inducibly depleted of CGR1, and a green fluorescent protein (GFP)-tagged CgrA protein had the same nucleolar localization as the corresponding yeast protein. These results identify cgrA as the A. nidulans ortholog of yeast CGR1 and suggest evolutionary conservation of nucleolar localization mechanisms used by these proteins.

Cgr1p, a novel nucleolar protein encoded by Saccharomyces cerevisiae orf YGL0292w.

Saccharomyces cerevisiae open reading frame (ORF) YGL029w (CGR1) encodes a small hydrophilic protein of unknown function. To investigate the role of this gene, we have determined the intracellular localization of the encoded product and examined the effects of Cgr1p depletion on cell growth. Tagging Cgr1p with the green fluorescent protein (GFP) or the myc epitope showed focal accumulation of the fusion protein in the yeast nucleolus, and this localization overlapped with the distribution of the nucleolar protein Nop1p. Cells depleted of CGR1 mRNA were growth impaired and hypersensitive to the translational inhibitor paromomycin, and this phenotype was complemented by episomal expression of the CGR1-GFP fusion gene. These results identify Cgr1p as a novel component of the yeast nucleolus and suggest a potential role in ribosome biogenesis.

Molecular cloning of Aspergillus fumigatus CgrA, the ortholog of a conserved fungal nucleolar protein.

In this report we describe the cloning of cgrA, the Aspergillus fumigatus ortholog of the yeast nucleolar protein Cgr1p. The cgrA complementary DNA (cDNA) contains a single open reading frame that would encode a protein of 114 amino acids that has 42% sequence identity to yeast Cgrlp. Heterologous expression of a green fluorescent protein (GFP)-tagged A. fumigatus cgrA gene demonstrated that the CgrA protein could localize to the yeast nucleolus. Moreover, the cgrA cDNA complemented the growth deficiency caused by inducible depletion of intracellular Cgr1p levels in yeast. These results support an orthologous relationship between the CgrA and Cgr1 proteins, and open the way for future studies into the potential value of nucleolar proteins as antifungal targets.

Identification of a cell type-specific silencer in the first exon of the His-1 gene.

The His-1 gene is developmentally expressed in the murine choroid plexus but is silenced in the adult brain. To test the hypothesis that the gene contains cis-acting elements that contribute to this repression, we have analyzed segments of the proximal promoter for negative regulatory sequences by transient transfection analysis. The activity of the proximal promoter was moderately influenced by positively and negatively acting sequences located from -335 to -168 and -617 to -335, respectively. A strong His-1-positive regulatory element (HPRE, +18 to +29) was essential for maximal promoter activity and could also enhance the activity of the heterologous SV40 promoter in an orientation-dependent manner. The HPRE contains homology to the neuronal restrictive silencer element (NRSE) but interacted with nuclear proteins that were distinct from the NRSE-binding factor (NRSF). By contrast, a potent negative regulatory sequence (HNRE) was identified in the first exon that repressed either the His-1 or SV40 promoters by greater than 80%. This negative regulatory sequence interacted with nuclear proteins from cells that contain a silent His-1 gene but showed no interaction with nuclear proteins from cells that actively transcribe the endogenous gene. HNRE-mediated repression was orientation independent; most of this activity was mapped to a minimal 26-bp sequence. These findings suggest that the first exon of the His-1 gene contains a cell type-specific silencer that contributes to the regulation of His-1 transcription.

New insights into the function of noncoding RNA and its potential role in disease pathogenesis.

All polyadenylated RNAs expressed in mammalian tissues are assumed to be transported to the cytoplasm where they direct the synthesis of a protein product. This mainstream view of the function of polyadenylated transcripts is currently being challenged by the identification of a novel class of genes which, although they encode polyadenylated RNA, do not make a translated protein. Many of these noncoding RNAs are developmentally regulated or show highly restricted patterns of gene expression, and their functions are providing important insight into RNA-based mechanisms of gene expression, genomic imprinting, cell cycle progression, and differentiation. The purpose of this review is to discuss the current understanding of mammalian noncoding RNAs, and to highlight their potential for identifying new pathways of human disease.

Graduate education in microscopic anatomy.

One of the most striking changes to affect the direction of current biomedical research is the increasing use of transgenic or gene-targeted mice as models of gene function and human disease. The proliferation of transgenic and gene-targeting technology has contributed to a rebirth of histology as an important research tool and is driving the need for broadly trained investigators with expertise at both the molecular and organismal levels. Since the ultimate goal of graduate-student education is the training of the next generation of independent scientists, it is important that graduate training programs provide students with the background required to take advantage of the unique resources provided by these mouse models. Anatomists are well suited to provide such training by incorporating mouse anatomy, physiology, and genetics into traditional coursework in microscopic anatomy.

Sequencing of a gene encoding a member of the mitochondrial carrier family of transport proteins from Aspergillus nidulans.

Mitochondrial carrier proteins comprise a superfamily of evolutionarily conserved proteins that regulate the specific transport of essential metabolites across the mitochondrial membranes. In this report we describe the cloning and sequencing of a gene from Aspergillus nidulans, amc-1, that encodes the first reported example of a mitochondrial carrier protein in Aspergillus species. The amc-1 gene is located on chromosome 7, and is transcribed as a 1.6 kb unspliced polyadenylated RNA. The predicted translation product of the amc-1. cDNA displays three tandemly repeated domains which possess protein signature motifs that are characteristic of mitochondrial carrier proteins that localize to the inner mitochondrial membrane. amc-1 shares the greatest similarity with a Neurospora mitochondrial carrier protein that is implicated in basic amino acid transport, suggesting that the amc-1 protein may provide a related function.

Human and murine high endothelial venule cells phagocytose apoptotic leukocytes.

Apoptotic cell death occurs during normal lymphocyte development and differentiation as well as following lymphocyte exposure to endogenous corticosteroids released during stress, malnutrition, and trauma. Recognition and engulfment of these apoptotic cells is important for the clearance of dying cells before they release potent inflammatory mediators into the vasculature or tissues. Phagocytosis of apoptotic cells is accomplished in part by macrophages. We report for the first time that apoptotic lymphocytes are also phagocytosed by high endothelial venule (HEV) cells. The murine HEV cell line mHEVa rapidly phagocytosed apoptotic lymphoid and myeloid cells with the greatest rate of phagocytosis occurring at 0-6 h. To confirm HEV cell interaction with apoptotic cells, we demonstrated that apoptotic human tonsil lymphocytes were phagocytosed by human tonsil HEV cells in primary cultures. Furthermore, we examined HEV cell phagocytosis in vivo. Mice were treated with a natural corticosterone (4-pregnene-11 beta,21-diol-3,20-dione) at levels detected during stress or malnutrition (93-180 micrograms serum cortisol/dl). At 4-12 h posttreatment, apoptotic lymphocytes were present inside vacuoles of HEV cells in axillary lymph node tissue sections, as determined by transmission electron microscopy. These data suggest that, in addition to macrophages, lymph node HEV cells also play a role in the removal of apoptotic lymphocytes. Moreover, since HEV cells are specialized endothelial cells that regulate lymphocyte migration into peripheral lymphoid tissues, they may provide an important checkpoint for clearance of apoptotic lymphocytes within the vasculature, as well as limiting entrance of nonfunctional lymphocytes into the lymph node.

Expression of the putative proto-oncogene His-1 in normal and neoplastic tissues.

The His-1 gene is expressed as a 3-kb spliced and polyadenylated RNA that is believed to function in the absence of an encoded protein. The precise function of the His-1 gene is unknown, but its transcriptional activation in a series of mouse leukemias has implicated the His-1 RNA in leukemogenesis when it is abnormally expressed. To study the oncogenic potential of this gene in more detail, we have examined the normal tissue distribution of His-1 RNA during mouse embryogenesis and in various adult tissues. His-1 expression was detected at low levels in the epithelia of the adult mouse stomach, prostate, seminal vesicle, and the developing choroid plexus by in situ hybridization. All other tissues examined lacked detectable levels of hybridizing RNA, suggesting that normal His-1 gene expression is highly restricted to these epithelial sites. These transcripts were not detectable by Northern blot analysis of normal tissues but were readily identified in five mouse leukemias and in five carcinomas of the choroid plexus. These data indicate that the His-1 gene expression is highly restricted and suggest that inappropriate activation of this gene may contribute to carcinogenesis.

A novel flow cytometric method for quantifying phagocytosis of apoptotic cells.

Many eukaryotic cell types are capable of specific recognition and phagocytosis of apoptotic cells, and there is increasing interest in the mechanisms involved in this process. To facilitate analysis of these mechanisms, we designed a novel fluorescence-based method to quantify phagocytosis in vitro using endothelial cell engulfment of apoptotic cells as a model. The B-cell line WEHI-231 was labeled with the fluorophore 5-(&-6)-carboxytetramethyl-rhodamine-succinimidyl-ester (TAMRA) and then induced to undergo apoptosis by crosslinking cell surface immunoglobulin. An endothelial cell line was subsequently allowed to ingest these TAMRA-labeled apoptotic lymphocytes. After 24 h, nonbound lymphocytes were removed and the mono-layers were dissociated. Any nonphagocytosed lymphocytes that remained tightly bound to the endothelial cells were then indirectly immunofluorescein labeled for the pan leukocyte-specific marker CD45. Flow cytometric analysis of the cells distinguished three endothellal cell populations: 1) endothelial cells with surface bound lymphocytes (TAMRA+ CD45+); 2) endothelial cells containing phagocytosed apoptotic lymphocytes (TAMRA+ CD45-); and 3) endothelial cells that were not associated with lymphocytes. The identification of these populations was verified by confocal microscopy of sorted cells. The method described herein will facilitate detailed studies on phagocytic recognition of apoptotic cells and should have broad applications to other phagocytic cell systems.

Evolutionary conservation of putative functional domains in the human homolog of the murine His-1 gene.

The mouse His-1 gene encodes a spliced and polyadenylated RNA with no long open reading frame (ORF), making it difficult to distinguish a functional protein coding domain. To identify candidate protein coding ORFs, and other functionally significant regions, we have isolated and sequenced 8.5 kb of a human genomic DNA that is homologous to the mouse His-1 gene. Alignment of the mouse and human sequences required no extensive gapping, indicating that evolutionary constraints have maintained a requirement for colinearity in genomic organization. We have identified the mouse transcriptional start point (tsp) and shown that the sequence of the 5'-flanking region is highly conserved in the human homolog. Sequence comparisons between the mouse and human genes identified conservation of other putative functional domains in exon 3 and in each of the two introns. Southern blot analysis with probes from each of these regions detected homologs in multiple other vertebrate species. However, none of the multiple candidate ORFs in the mouse RNA were conserved in the human sequence, suggesting that the RNA is unlikely to encode a protein. These data suggest that the RNA may be the final and functional product from the mouse His-1 gene.

The Evi-1 zinc finger myeloid transforming protein binds to genomic fragments containing (GATA)n sequences.

The EVI1 gene is activated by chromosomal translocations and inversions in approximately 5% of human acute myeloid leukemia (AML) and by retroviral insertion in approximately 20% of murine myeloid leukemias. EVI1 encodes a nuclear DNA-binding protein having 10 zinc finger motifs in two noncontiguous domains consisting of an amino-terminal domain of seven fingers and a carboxyl domain containing three fingers. To evaluate the sequence specificity of Evi-1 binding and potentially identify genomic targets, whole-genome PCR was utilized to isolate multiple Sau3A fragments which specifically bind to the amino-terminal zinc finger domain. The majority of these clones represented single copy sequences and virtually all contained variable numbers of repeats of the GATA motif, the target sequence for the erythroid-specific transcription factor GATA-1. GST/Evi-1 fusion proteins containing the amino-terminal domain of zinc fingers bound the GATA motif in these clones as well as to those present in the human gamma-globin promoter, similar to the binding of purified GATA-1 protein. By obtaining corresponding large genomic clones for eight of these fragments, transcription units were found associated with two. One corresponded to the glyceraldehyde-3-phosphate dehydrogenase gene and its expression was not affected by Evi-1. The second is a novel gene whose expression is repressed in murine myeloid cell lines that express Evi-1.

Regulation of T cell receptor gamma gene expression by cytokines in murine hematopoietic cells.

Murine IL-3-dependent myeloid cell lines express transcripts from non-rearranged TCR gamma genes and this expression is dependent upon IL-3. To investigate this observation in general terms we examined various IL-3 dependent cell lines for TCR gamma gene expression. We also examined various cytokines to test their potential to induce TCR gamma gene expression. All IL-3 dependent cell lines expressed TCR gamma transcripts. The IL-3 induced expression was sensitive to protein synthesis inhibitors. This demonstrated that the TCR gamma genes belong to the early growth factor response class. IL-3, IL-4, GM-CSF and Erytropoietin (EPO), but not G-CSF, induced TCR gene expression. 32D cells transfected with the IL-2 beta chain receptor became responsive to IL-2 as a growth factor and induced TCR gamma gene expression. The induction of TCR gamma gene expression by the cytokines was not correlated to their growth promoting activity. This indicated different signaling pathways.

v-raf suppresses apoptosis and promotes growth of interleukin-3-dependent myeloid cells.

Interleukin-3 (IL-3) is required for the proliferation, survival and differentiation of myeloid progenitors. In the absence of IL-3, murine myeloid 32D.3 cells accumulate in the G1 phase of the cell cycle and subsequently undergo programmed cell death, or apoptosis. Here we demonstrate that enforced expression of the v-raf oncogene suppresses apoptosis of myeloid 32D.3 cells following the withdrawal of IL-3. Surprisingly, steady state levels of Bcl-2, an oncogene known to suppress apoptosis, were not dependent upon IL-3 in 32D.3 cells and its levels were not augmented in v-raf clones. This suggests that ability of v-raf to suppress apoptosis in the absence of ligand is either Bcl-2 independent or that v-raf kinase promotes Bcl-2 function. v-raf also promoted growth of these cells in the presence of IL-3. v-raf clones proliferated at an increased rate due to a shortened G1 phase and had decreased requirements for IL-3 for growth. Therefore, transformation of myeloid cells by v-raf involves signaling pathways which promote both cell cycle progression and cell survival.

Retroviral insertions in the murine His-1 locus activate the expression of a novel RNA that lacks an extensive open reading frame.

The His-1 locus is a common site of viral insertion in murine myeloid leukemias induced by the wild mouse ecotropic retrovirus, CasBrM. In this report, we describe the cloning of a novel gene at the His-1 locus and show that His-1 expression is associated with the transformed phenotype. Northern (RNA) blot analysis identified His-1 transcripts in four transformed myeloid cell lines but in no normal tissues examined. Two of these cell lines were derived from retrovirus-induced myeloid leukemias that harbor integrated proviruses which drive His-1 gene expression by promoter insertion. The two other cell lines expressed a discrete 3-kb His-1 RNA that is derived from a novel gene consisting of three exons that span 6 kb on mouse chromosome 2. The His-1 gene is conserved as a single-copy sequence in multiple vertebrate species and is expressed as a spliced and polyadenylated RNA. A protein-coding region is not evident from analysis of the His-1 sequence because of the presence of multiple small open reading frames, none of which are greater than 219 bp. This lack of an extensive open reading frame is an unusual feature that is shared by other RNA molecules believed to function in the absence of translation.

Activation of apoptosis associated with enforced myc expression in myeloid progenitor cells is dominant to the suppression of apoptosis by interleukin-3 or erythropoietin.

The inappropriate expression of c-myc in cells deprived of growth factors has recently been implicated in the activation of programmed cell death (apoptosis). The studies described here examine the ability of interleukin-3 (IL-3) or erythropoietin (Epo) to suppress apoptosis that occurs in association with enforced myc expression during cell cycle arrest of a murine IL-3-dependent myeloid progenitor cell line, 32D. G1 arrest was observed when culturing 32D cells to high density in medium supplemented with IL-3, or at subconfluent densities in medium supplemented with Epo. Under both conditions, endogenous c-myc expression was downregulated and viability was maintained. In clones of cells in which c-myc is constitutively expressed from a retroviral vector, enforced c-myc expression was associated with the activation of apoptosis at high cell densities. Similarly, enforced c-myc expression was deleterious to cell survival when these cells were cultured in Epo, as apoptosis was evident within 6 hours. The results support the concept that inappropriate c-myc expression activates apoptosis and that neither IL-3 nor Epo can suppress this program under these conditions.