Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

Gene therapy restores the transcriptional program of hematopoietic stem cells in Fanconi anemia.

Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-ß and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

Shorter telomere length is associated with COVID-19 hospitalization and with persistence of radiographic lung abnormalities.

BACKGROUND: Age and comorbidity are the main determinants of COVID-19 outcome. Shorter leukocyte telomere length (TL), a hallmark of biological aging, has been associated with worse COVID-19 outcomes. We sought to determine TL in patients with severe COVID-19 requiring hospitalization to analyze whether clinical outcomes and post-COVID-19 manifestations are associated with shorter TL. RESULTS: We analyzed 251 patients with PCR-confirmed COVID-19, hospitalized in the first months of the pandemics. We determined TL in PBL at admission by quantitative-PCR (qPCR) analysis in patients. A healthy cohort from the same area with a similar age range (n = 169) was used to calculate TL Z-scores. After hospital discharge, 144 COVID-19 survivors were followed-up for persistent COVID-19 manifestations. A second TL determination was performed in a smaller group of 63 patients 1 year later and compared with baseline TL. Hospitalized COVID-19 patients had a decreased baseline age-adjusted TL Z-score compared to the reference group. No differences in Z-scores were observed in patients with different COVID-19 outcomes, classified as WHO ordinal scores. In 144 patients, followed for a median of 8 months, post-COVID manifestations were not associated to differences in TL. Persistence of lung radiographic abnormalities was associated with shorter baseline TL. In patients with a second TL determination, further telomere shortening (TS) was observed in 35% and telomere lengthening in 49%. Patients with further TS had suffered a more severe disease. CONCLUSION: Shorter TL is associated with COVID-19 hospitalization but not with hospital clinical outcomes nor with persistent post-COVID-19 manifestations. Delayed resolution of radiographic lung abnormalities was also associated with shorter TL.

Mutations in XLF/NHEJ1/Cernunnos gene results in downregulation of telomerase genes expression and telomere shortening.

NHEJ1-patients develop severe progressive lymphocytopenia and premature aging of hematopoietic stem cells (HSCs) at a young age. Here we show a patient with a homozygous-NHEJ1 mutation identified by whole exome-sequencing that developed severe pancytopenia and bone marrow aplasia correlating with the presence of short telomeres. The mutation resulted in a truncated protein. In an attempt to identify the mechanism behind the short telomere phenotype found in the NHEJ1-patient we downregulated NHEJ1 expression in 293T and CD34+cells. This downregulation resulted in reduced telomerase activity and decreased expression of several telomerase/shelterin genes. Interestingly, cell lines derived from two other NHEJ1-deficient patients with different mutations also showed increased p21 expression, inhibition in expression of several telomerase complex genes and shortened telomeres. Decrease in expression of telomerase/shelterin genes did not occur when we inhibited expression of other NHEJ genes mutated in SCID patients: DNA-PK, Artemis or LigaseIV. Because premature aging of HSCs is observed only in NHEJ1 patients, we propose that is the result of senescence induced by decreased expression of telomerase/shelterin genes that lead to an inhibition of telomerase activity. Previous reports failed to find this connection because of the use of patient´s cells immortalized by TERT expression or recombined telomeres by ALT pathway. In summary, defective regulation of telomere biology together with defective V(D)J recombination can negatively impact on the evolution of the disease in these patients. Identification of telomere shortening is important since it may open new therapeutic interventions for these patients by treatments aimed to recover the expression of telomerase genes.

Role of Dusp6 Phosphatase as a Tumor Suppressor in Non-Small Cell Lung Cancer.

DUSP6/MKP3 is a dual-specific phosphatase that regulates extracellular regulated kinase ERK1/2 and ERK5 activity, with an increasingly recognized role as tumor suppressor. In silico studies from Gene expression Omnibus (GEO) and Cancer Genome atlas (TCGA) databases reveal poor prognosis in those Non-small cell lung cancer (NSCLC) patients with low expression levels of DUSP6. In agreement with these data, here we show that DUSP6 plays a major role in the regulation of cell migration, motility and tumor growth. We have found upregulation in the expression of several genes involved in epithelial to mesenchymal transition (EMT) in NSCLC-DUSP6 depleted cells. Data obtained in RNA-seq studies carried out in DUSP6 depleted cells identified EGFR, TGF-ß and WNT signaling pathways and several genes such as VAV3, RUNXR2, LEF1, FGFR2 whose expression is upregulated in these cells and therefore affecting cellular functions such as integrin mediated cell adhesion, focal adhesion and motility. Furthermore, EGF signaling pathway is activated via ERK5 and not ERK1/2 and TGF-ß via SMAD2/3 in DUSP6 depleted cells. In summary DUSP6 is a tumor suppressor in NSCLC and re-establishment of its expression may be a potential strategy to revert poor outcome in NSCLC patients.

DUSP1 and SOX2 expression determine squamous cell carcinoma of the salivary gland progression.

Salivary gland squamous cell carcinomas (SG-SCCs) constitute a rare type of head and neck cancer which is linked to poor prognosis. Due to their low frequency, the molecular mechanisms responsible for their aggressiveness are poorly understood. In this work we studied the role of the phosphatase DUSP1, a negative regulator of MAPK activity, in controlling SG-SCC progression. We generated DUSP1 KO clones in A253 human cells. These clones showed a reduced ability to grow in 2D, self-renew in ECM matrices and to form tumors in immunodeficient mice. This was caused by an overactivation of the stress and apoptosis kinase JNK1/2 in DUSP1-/+ clones. Interestingly, RNAseq analysis revealed that the expression of SOX2, a well-known self-renewal gene was decreased at the mRNA and protein levels in DUSP1-/+ cells. Unexpectedly, CRISPR-KO of SOX2 did not recapitulate DUSP1-/+ phenotype, and SOX2-null cells had an enhanced ability to self-renew and to form tumors in mice. Gene expression analysis demonstrated that SOX2-null cells have a decreased squamous differentiation profile -losing TP63 expression- and an increased migratory phenotype, with an enhanced epithelial to mesenchymal transition signature. In summary, our data indicates that DUSP1 and SOX2 have opposite functions in SG-SCC, being DUSP1 necessary for tumor growth and SOX2 dispensable showing a tumor suppressor function. Our data suggest that the combined expression of SOX2 and DUSP1 could be a useful biomarker to predict progression in patients with SG-SCCs.

Mef2A, a homologue of animal Mef2 transcription factors, regulates cell differentiation in Dictyostelium discoideum.

BACKGROUND: Transcription factors from the MADS-box family play a relevant role in cell differentiation and development and include the animal SRF (serum response factor) and MEF2 (myocyte enhancer factor 2) proteins. The social amoeba Dictyostelium discoideum contains four genes coding for MADS-box transcription factors, two of these genes code for proteins that are more similar to SRF, and the other two code for proteins that are more similar to MEF2 animal factors. RESULTS: The biological function of one of the two genes that codes for MEF2-related proteins, a gene known as mef2A, is described in this article. This gene is expressed under the transcriptional control of two alternative promoters in growing cells, and its expression is induced during development in prespore cells. Mutant strains where the mef2A gene has been partially deleted were generated to study its biological function. The mutant strains showed reduced growth when feeding on bacteria and were able to develop and form fruiting bodies, but spore production was significantly reduced. A study of developmental markers showed that prespore cells differentiation was impaired in the mutant strains. When mutant and wild-type cells were set to develop in chimeras, mutant spores were underrepresented in the fruiting bodies. The mutant cells were also unable to form spores in vitro. In addition, mutant cells also showed a poor contribution to the formation of the tip-organizer and the upper region of slugs and culminant structures. In agreement with these observations, a comparison of the genes transcribed by mutant and wild-type strains during development indicated that prestalk gene expression was enhanced, while prespore gene expression decreased in the mef2A- strain. CONCLUSIONS: Our data shows that mef2A plays a role in cell differentiation in D. discoideum and modulates the expression of prespore and prestalk genes.

The dual-specificity protein phosphatase MkpB, homologous to mammalian MKP phosphatases, is required for D. discoideum post-aggregative development and cisplatin response.

Dual-specificity protein phosphatases participate in signal transduction pathways inactivating mitogen-activated protein kinases (MAP kinases). These signaling pathways are of critical importance in the regulation of numerous biological processes, including cell proliferation, differentiation and development. The social ameba Dictyostelium discoideum harbors 14 genes coding for proteins containing regions very similar to the dual-specificity protein phosphatase domain. One of these genes, mkpB, additionally codes for a region similar to the Rhodanase domain, characteristic of animal MAP kinase-phosphatases, in its N-terminal region. Cells that over-express this gene show increased protein phosphatase activity. mkpB is expressed in D. discoideum ameba at growth but it is greatly induced at 12h of multicellular development. Although it is expressed in all the cells of developmental structures, mkpB mRNA is enriched in cells with a distribution typical of anterior-like cells. Cells that express a catalytically inactive mutant of MkpB grow and aggregate like wild-type cells but show a greatly impaired post-aggregative development. In addition, the expression of cell-type specific genes is very delayed, indicating that this protein plays an important role in cell differentiation and development. Cells expressing the MkpB catalytically inactive mutant show increased sensitivity to cisplatin, while cells over-expressing wild type MkpB, or MkpA, proteins or mutated in the MAP kinase erkB gene are more resistant to this chemotherapeutic drug, as also shown in human tumor cells.

Comparison of Colorectal Cancer Stem Cells and Oxaliplatin-Resistant Cells Unveils Functional Similarities.

Colorectal cancer is the second most common cancer in women, the third in men, and an important cause of cancer-related mortality. Recurrence and the development of chemotherapy resistance are major hindrances for patients' treatment. The presence of cancer stem cells with chemotherapy resistance able to generate proliferating tumor cells contributes to tumor recurrence and resistance. In addition, tumor cells can develop chemoresistance through adaptation mechanisms. In this article, cancer stem cells were isolated from HT29 and SW620 colorectal cancer cell lines. Oxaliplatin resistance was induced by a single drug treatment simulating the usual guidelines of patient treatment. A comparison of these two populations showed similarities since cancer stem cells presented increased oxaliplatin resistance, and resistant cells contained an increased number of cancer stem cells. Cancer stem cells isolated from resistant cells showed increased oxaliplatin resistance. Cell invasion capacity and epithelial-mesenchymal transition were increased both in cancer stem cells and oxaliplatin-resistant cells. mRNA expression analysis showed that both cell types shared a significant proportion of commonly regulated genes. In summary, the data presented indicate that colorectal cancer stem cells and oxaliplatin-resistant cells are highly related cell populations that might have interesting implications in the development of tumor recurrence and resistance to chemotherapy.

Generation of dyskeratosis congenita-like hematopoietic stem cells through the stable inhibition of DKC1.

Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.

Next-generation Sequencing in Bone Marrow Failure Syndromes and Isolated Cytopenias: Experience of the Spanish Network on Bone Marrow Failure Syndromes.

Inherited bone marrow failure syndromes (IBMFSs) are a group of congenital rare diseases characterized by bone marrow failure, congenital anomalies, high genetic heterogeneity, and predisposition to cancer. Appropriate treatment and cancer surveillance ideally depend on the identification of the mutated gene. A next-generation sequencing (NGS) panel of genes could be 1 initial genetic screening test to be carried out in a comprehensive study of IBMFSs, allowing molecular detection in affected patients. We designed 2 NGS panels of IBMFS genes: version 1 included 129 genes and version 2 involved 145 genes. The cohort included a total of 204 patients with suspected IBMFSs without molecular diagnosis. Capture-based targeted sequencing covered > 99% of the target regions of 145 genes, with more than 20 independent reads. No differences were seen between the 2 versions of the panel. The NGS tool allowed a total of 91 patients to be diagnosed, with an overall molecular diagnostic rate of 44%. Among the 167 patients with classified IBMFSs, 81 patients (48%) were diagnosed. Unclassified IBMFSs involved a total of 37 patients, of whom 9 patients (24%) were diagnosed. The preexisting diagnosis of 6 clinically classified patients (6%) was amended, implying a change of therapy for some of them. Our NGS IBMFS gene panel assay is a useful tool in the molecular diagnosis of IBMFSs and a reasonable option as the first tier genetic test in these disorders.

SrfB, a member of the Serum Response Factor family of transcription factors, regulates starvation response and early development in Dictyostelium.

The Serum Response Factor (SRF) is an important regulator of cell proliferation and differentiation. Dictyostelium discoideum srfB gene codes for an SRF homologue and is expressed in vegetative cells and during development under the control of three alternative promoters, which show different cell-type specific patterns of expression. The two more proximal promoters directed gene transcription in prestalk AB, stalk and lower-cup cells. The generation of a strain where the srfB gene has been interrupted (srfB(-)) has shown that this gene is required for regulation of actin-cytoskeleton-related functions, such as cytokinesis and macropinocytosis. The mutant failed to develop well in suspension, but could be rescued by cAMP pulsing, suggesting a defect in cAMP signaling. srfB(-) cells showed impaired chemotaxis to cAMP and defective lateral pseudopodium inhibition. Nevertheless, srfB(-) cells aggregated on agar plates and nitrocellulose filters 2 h earlier than wild type cells, and completed development, showing an increased tendency to form slug structures. Analysis of wild type and srfB(-) strains detected significant differences in the regulation of gene expression upon starvation. Genes coding for lysosomal and ribosomal proteins, developmentally-regulated genes, and some genes coding for proteins involved in cytoskeleton regulation were deregulated during the first stages of development.

Dyskerin Mutations Present in Dyskeratosis Congenita Patients Increase Oxidative Stress and DNA Damage Signalling in Dictyostelium Discoideum.

Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease.

Structure of Dictyostelium discoideum telomeres. Analysis of possible replication mechanisms.

Telomeres are nucleo-protein structures that protect the ends of eukaryotic chromosomes. They are not completely synthesized during DNA replication and are elongated by specific mechanisms. The structure of the telomeres and the elongation mechanism have not been determined in Dictyostelium discoideum. This organism presents extrachromosomal palindromic elements containing two copies of the rDNA, also present at the end of the chromosomes. In this article the structure of the terminal region of the rDNA is shown to consist of repetitions of the A(G)n sequence where the number of Gs is variable. These repeats extend as a 3' single stranded region. The G-rich region is preceded by four tandem repetitions of two different DNA motifs. D. discoideum telomere reverse transcriptase homologous protein (TERTHP) presented RNase-sensitive enzymatic activity and was required to maintain telomere structure since terthp-mutant strains presented reorganizations of the DNA terminal regions. These modifications were different in several terthp-mutants and changed with their prolonged culture and subcloning. However, the terthp gene is not essential for D. discoideum proliferation. Telomeres could be maintained in terthp-mutant strains by homologous recombination mechanisms such as ALT (Alternative Lengthening of Telomeres) or HAATI (heterochromatin amplification-mediated and telomerase-independent). In agreement with this hypothesis, the expression of mRNAs coding for several proteins involved in homologous recombination was induced in terthp-mutant strains. Extrachromosomal rDNA could serve as substrate in these DNA homologous recombination reactions.

GSE4 peptide suppresses oxidative and telomere deficiencies in ataxia telangiectasia patient cells.

Ataxia telangiectasia (AT) is a genetic disease caused by mutations in the ATM gene but the mechanisms underlying AT are not completely understood. Key functions of the ATM protein are to sense and regulate cellular redox status and to transduce DNA double-strand break signals to downstream effectors. ATM-deficient cells show increased ROS accumulation, activation of p38 protein kinase, and increased levels of DNA damage. GSE24.2 peptide and a short derivative GSE4 peptide corresponding to an internal domain of Dyskerin have proved to induce telomerase activity, decrease oxidative stress, and protect from DNA damage in dyskeratosis congenita (DC) cells. We have found that expression of GSE24.2 and GSE4 in human AT fibroblast is able to decrease DNA damage, detected by γ-H2A.X and 53BP1 foci. However, GSE24.2/GSE4 expression does not improve double-strand break signaling and repair caused by the lack of ATM activity. In contrast, they cause a decrease in 8-oxoguanine and OGG1-derived lesions, particularly at telomeres and mitochondrial DNA, as well as in reactive oxygen species, in parallel with increased expression of SOD1. These cells also showed lower levels of IL6 and decreased p38 phosphorylation, decreased senescence and increased ability to divide for longer times. Additionally, these cells are more resistant to treatment with H202 and the radiomimetic-drug bleomycin. Finally, we found shorter telomere length (TL) in AT cells, lower levels of TERT expression, and telomerase activity that were also partially reverted by GSE4. These observations suggest that GSE4 may be considered as a new therapy for the treatment of AT that counteracts the cellular effects of high ROS levels generated in AT cells and in addition increases telomerase activity contributing to increased cell proliferation.

Genetic analyses of aplastic anemia and idiopathic pulmonary fibrosis patients with short telomeres, possible implication of DNA-repair genes.

BACKGROUND: Telomeres are nucleoprotein structures present at the terminal region of the chromosomes. Mutations in genes coding for proteins involved in telomere maintenance are causative of a number of disorders known as telomeropathies. The genetic origin of these diseases is heterogeneous and has not been determined for a significant proportion of patients. METHODS: This article describes the genetic characterization of a cohort of patients. Telomere length was determined by Southern blot and quantitative PCR. Nucleotide variants were analyzed either by high-resolution melting analysis and Sanger sequencing of selected exons or by massive sequencing of a panel of genes. RESULTS: Forty-seven patients with telomere length below the 10% of normal population, affected with three telomeropathies: dyskeratosis congenita (4), aplastic anemia (22) or pulmonary fibrosis (21) were analyzed. Eighteen of these patients presented known pathogenic or novel possibly pathogenic variants in the telomere-related genes TERT, TERC, RTEL1, CTC1 and ACD. In addition, the analyses of a panel of 188 genes related to haematological disorders indicated that a relevant proportion of the patients (up to 35%) presented rare variants in genes related to DNA repair or in genes coding for proteins involved in the resolution of complex DNA structures, that participate in telomere replication. Mutations in some of these genes are causative of several syndromes previously associated to telomere shortening. CONCLUSION: Novel variants in telomere, DNA repair and replication genes are described that might indicate the contribution of variants in these genes to the development of telomeropathies. Patients carrying variants in telomere-related genes presented worse evolution after diagnosis than the rest of patients analyzed.

Structural and functional studies of a family of Dictyostelium discoideum developmentally regulated, prestalk genes coding for small proteins.

BACKGROUND: The social amoeba Dictyostelium discoideum executes a multicellular development program upon starvation. This morphogenetic process requires the differential regulation of a large number of genes and is coordinated by extracellular signals. The MADS-box transcription factor SrfA is required for several stages of development, including slug migration and spore terminal differentiation. RESULTS: Subtractive hybridization allowed the isolation of a gene, sigN (SrfA-induced gene N), that was dependent on the transcription factor SrfA for expression at the slug stage of development. Homology searches detected the existence of a large family of sigN-related genes in the Dictyostelium discoideum genome. The 13 most similar genes are grouped in two regions of chromosome 2 and have been named Group1 and Group2 sigN genes. The putative encoded proteins are 87-89 amino acids long. All these genes have a similar structure, composed of a first exon containing a 13 nucleotides long open reading frame and a second exon comprising the remaining of the putative coding region. The expression of these genes is induced at10 hours of development. Analyses of their promoter regions indicate that these genes are expressed in the prestalk region of developing structures. The addition of antibodies raised against SigN Group 2 proteins induced disintegration of multi-cellular structures at the mound stage of development. CONCLUSION: A large family of genes coding for small proteins has been identified in D. discoideum. Two groups of very similar genes from this family have been shown to be specifically expressed in prestalk cells during development. Functional studies using antibodies raised against Group 2 SigN proteins indicate that these genes could play a role during multicellular development.

Investigating gene expression: in situ hybridization and reporter genes.

Coordinated cell type differentiation is essential for morphogenesis during Dictyostelium development. The specification of different cell types and the regulation of temporal and spatial patterns of expression of cell type-specific genes are important problems currently being addressed in many laboratories. Besides, determination of gene expression patterns provides significant information in the characterization of developmental mutants. Cell type-specific probes and well characterized promoters are available that allow the identification of most cell types during Dictyostelium development. Expression patterns can be studied by whole-mount in situ messenger RNA (mRNA) detection and by the use of reporter genes under the control of specific promoters. The most common in situ hybridization technique, based on nonradioactive ribo-probes that are hybridized to fixed whole-mounts prepared at different developmental stages, is described. Several reporter genes have been used to characterize gene expression patterns and to determine functional promoter elements. The lacZ gene, coding for the beta-galactosidase enzyme, is the reporter most frequently used in Dictyostelium because both temporal- and spatial-patterns of expression can be easily determined. Generally used beta-galactosidase detection methods are described.

Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene.

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.