Olga Zhuchenko

Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α(1A)-voltage-dependent calcium channel

A polymorphic CAG repeat was identified in the human α1A voltage-dependent calcium channel subunit. To test the hypothesis that expansion of this CAG repeat could be the cause of an inherited progressive ataxia, we genotyped a large number of unrelated controls and ataxia patients. Eight unrelated patients with late onset ataxia had alleles with larger repeat numbers (21‐27) compared to the number of repeats (4‐16) in 475 non‐ataxia individuals. Analysis of the repeat length in families of the affected individuals revealed that the expansion segregated with the phenotype in every patient. We identified six isoforms of the human α1A calcium channel subunit. The CAG repeat is within the open reading frame and is predicted to encode glutamine in three of the isoforms. We conclude that a small polyglutamine expansion in the human α1A calcium channel is most likely the cause of a newly classified autosomal dominant spinocerebellar ataxia, SCA6.

RIGUI, A PUTATIVE MAMMALIAN ORTHOLOG OF THE DROSOPHILA PERIOD GENE

The molecular components of mammalian circadian clocks are elusive. We have isolated a human gene termed RIGUI that encodes a bHLH/PAS protein 44% homologous to Drosophila period. The highly conserved mouse homolog (m-rigui) is expressed in a circadian pattern in the suprachiasmatic nucleus (SCN), the master regulator of circadian clocks in mammals. Circadian expression in the SCN continues in constant darkness, and a shift in the light/dark cycle evokes a proportional shift of m-rigui expression in the SCN. m-rigui transcripts also appear in a periodic pattern in Purkinje neurons, pars tuberalis, and retina, but with a timing of oscillation different from that seen in the SCN. Sequence homology and circadian patterns of expression suggest that RIGUI is a mammalian ortholog of the Drosophila period gene, raising the possibility that a regulator of circadian clocks is conserved.

Social amoebae trap and kill bacteria by casting DNA nets

Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans.

Microarray phenotyping in Dictyostelium reveals a regulon of chemotaxis genes

MOTIVATION Coordinate regulation of gene expression can provide information on gene function. To begin a large-scale analysis of Dictyostelium gene function, we clustered genes based on their expression in wild-type and mutant strains and analyzed their functions. RESULTS We found 17 modes of wild-type gene expression and refined them into 57 submodes considering mutant data. Annotation analyses revealed correlations between co-expression and function and an unexpected correlation between expression and function of genes involved in various aspects of chemotaxis. Co-regulation of chemotaxis genes was also found in published data from neutrophils. To test the predictive power of the analysis, we examined the phenotypes of mutations in seven co-regulated genes that had no published role in chemotaxis. Six mutants exhibited chemotaxis defects, supporting the idea that function can be inferred from co-expression. The clustering and annotation analyses provide a public resource for Dictyostelium functional genomics.

ABC Transporters in Dictyostelium discoideum Development

ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.

Lectins modulate the microbiota of social amoebae

Sticky bacteria tolerated as future food Dictyostelium discoideum amoebae consume bacteria until the supply is exhausted. Then the amoeba cells clump together into a “slug” and initiate a complex multicellular reproductive phase. Specialized cells within aggregates rid the slug of any extracellular bacteria. However, some strains of amoeba tolerate live, intracellular bacteria. Dinh et al. discovered that these carrier strains bear surface lectins that bind Klebsiella bacteria, promote cell entry, and prevent the bacteria from being immediately digested. These bacteria then provide a future food source. Moreover, the internalized bacteria transfer DNA into the amoeba nucleus, resulting in transient genetic transformation. Science, this issue p. 402 Lectins facilitate bacterial uptake by eukaryotic cells through specific receptor recognition. The social amoeba Dictyostelium discoideum maintains a microbiome during multicellular development; bacteria are carried in migrating slugs and as endosymbionts within amoebae and spores. Bacterial carriage and endosymbiosis are induced by the secreted lectin discoidin I that binds bacteria, protects them from extracellular killing, and alters their retention within amoebae. This altered handling of bacteria also occurs with bacteria coated by plant lectins and leads to DNA transfer from bacteria to amoebae. Thus, lectins alter the cellular response of D. discoideum to bacteria to establish the amoebae’s microbiome. Mammalian cells can also maintain intracellular bacteria when presented with bacteria coated with lectins, so heterologous lectins may induce endosymbiosis in animals. Our results suggest that endogenous or environmental lectins may influence microbiome homeostasis across eukaryotic phylogeny.