Karolina Tandre

Conifer homologues to genes that control floral development in angiosperms

A set of MADS-box genes in flowering plants encode transcription factors that control both flower meristem formation and organ identity in the developing flower. In this report we present the first documentation of the presence of MADS-box genes in a non-flowering seed plant, and indeed from a plant bearing truly unisexual reproductive axes. A MADS-box-specific screening of a cDNA library from immature female strobili of the conifer Norway spruce, Picea abies (L.) Karst, resulted in cDNA clones that correspond to three different deficiens-agamous-like (dal) genes, dall, dal2 and dal3. In addition to the MADS box, the spruce genes contain a second sequence element conserved among angiosperm genes, the K box, which is located downstream to the MADS box. A phylogenetic analysis of the nucleotide sequences confirms common ancestry of the gene superfamily. dall is related to agl2, agl4 and agl6 from Arabidopsis thaliana, all genes with unknown functions, and is expressed in vegetative as well as reproductive shoots on the adult spruce tree. dal2 is sister to angiosperm genes that control the identity of sexual organs, and is expressed only in the developing male and female strobili. dal3 is related to the vegetatively expressed tomato gene tm3 and is transcribed in both vegetative and reproductive shoots. These results strongly suggest that the functional and structural complexity within the MADS-box superfamily of reproduction-control genes is an ancestral property of seed plants and not a novelty in the angiosperm lineage.

A cdc2 homologue and closely related processed retropseudogenes from Norway spruce.

The p34cdc2 protein kinase is a key component in the regulation of the eukaryotic cell cycle and has been conserved during evolution. We have isolated cDNA clones corresponding to a cdc2 gene (cdc2Pa) from the conifer Norway spruce, Picea abies (L.) Karst. The deduced amino acid sequence is 85–90% identical to p34cdc2 homologues from other plants, contains eleven subdomains characteristic for the protein kinase family, and three sequence motifs specific for the cdc2 protein kinases. A partial genomic clone of cdc2Pa reveals two introns at positions identical to intron positions in Arabidopsis thaliana cdc2a. A Southern blot analysis shows that cdc2Pa is a single-copy gene belonging to a family of about 10 related genes. Partial genomic sequences of six of the genes in this family (86–92% identical to cdc2Pa) show distinct features of processed retropseudogenes. These lack introns and contain deletions, insertions and/or non-silent point mutations. This result is consistent with the hypothesis that processed retropseudogenes in plants may be common among genes expressed in the apical meristem, that is, in cells which have the potential to take part in the formation of reproductive organs. Although cdc2Pa transcripts were abundant in the epicotyl and thus likely in the apical meristem, we observed no strict coupling of expression to cell division in embryos and seedlings.

Characterization of functional methylomes by next-generation capture sequencing identifies novel disease-associated variants.

The scaling of observable properties of galaxy clusters with mass evolves with time. Assessing the role of the evolution is crucial to study the formation and evolution of massive halos and to avoid biases in the calibration. We present a general method to infer the mass and the redshift dependence, and the time-evolving intrinsic scatter of the mass–observable relations. The procedure self-calibrates the redshift dependent completeness function of the sample. The intrinsic scatter in the mass estimates used to calibrate the relation is considered too. We apply the method to the scaling of mass M∆ versus line of sight galaxy velocity dispersion σv, optical richness, X-ray luminosity, LX, and Sunyaev-Zel’dovich signal. Masses were calibrated with weak lensing measurements. The measured relations are in good agreement with time and mass dependencies predicted in the self-similar scenario of structure formation. The lone exception is the LX-M∆ relation whose time evolution is negative in agreement with formation scenarios with additional radiative cooling and uniform preheating at high redshift. The intrinsic scatter in the σv-M∆ relation is notably small, of order of 14 per cent. Robust predictions on the observed properties of the galaxy clusters in the CLASH sample are provided as cases of study. Catalogs and scripts are publicly available at http://pico.bo.astro.it/~sereno/CoMaLit/.Most genome-wide methylation studies (EWAS) of multifactorial disease traits use targeted arrays or enrichment methodologies preferentially covering CpG-dense regions, to characterize sufficiently large samples. To overcome this limitation, we present here a new customizable, cost-effective approach, methylC-capture sequencing (MCC-Seq), for sequencing functional methylomes, while simultaneously providing genetic variation information. To illustrate MCC-Seq, we use whole-genome bisulfite sequencing on adipose tissue (AT) samples and public databases to design AT-specific panels. We establish its efficiency for high-density interrogation of methylome variability by systematic comparisons with other approaches and demonstrate its applicability by identifying novel methylation variation within enhancers strongly correlated to plasma triglyceride and HDL-cholesterol, including at CD36. Our more comprehensive AT panel assesses tissue methylation and genotypes in parallel at ∼4 and ∼3 M sites, respectively. Our study demonstrates that MCC-Seq provides comparable accuracy to alternative approaches but enables more efficient cataloguing of functional and disease-relevant epigenetic and genetic variants for large-scale EWAS.

Differential gene expression during germination and after the induction of adventitious bud formation in Norway spruce embryos

A pulse treatment of embryos of Norway spruce with cytokinin suppresses germinative development and induces the coordinate formation of adventitious buds from subepidermal cell layers. To analyse the patterns of gene expression associated with germination and the alterations induced by the bud induction treatment, we have isolated cDNA clones corresponding to genes that are differentially expressed in cytokinin-treated and untreatedin vitro germinating embryos. One category of 14 clones hybridized to transcripts that were abundant specifically during germination. The expression of 8 of these genes was reduced by the bud induction treatment. Four clones, including one identified as a histone H2A gene, recognized transcripts that showed an increased abundance in bud-induced versusin vitro germinating embryos. A second category of 13 clones hybridized to transcripts that increased in abundance during post-germinative development of the seedling. Among these a subset of 8 clones, including an α-tubulin clone, corresponds to genes suppressed by the bud induction treatment, whereas 5 clones, including a gene with sequence similarity to polyubiquitin, were unaffected by the treatment. One clone hybridized to a message abundant in the seed, during early germination as well as in the vegetative bud, and showed 60% partial sequence identity to a barley (1→3)-β-glucanase gene. Genes expressed exclusively in bud-induced orin vitro germinating embryos were not found.The results show that a major difference in gene expression between treated and untreated embryos is related to the shift from extensive cell proliferation to elongation and differentiation that occurs at the transition from germination to post-germinative development, and which is suppressed in the bud-induced embryos.

The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.

Transcription factors encoded by different members of the MADS‐box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen‐ and seed‐bearing organs, involves the B‐ and C‐organ‐identity MADS‐box genes. B‐ and C‐type gene orthologs with activities specifically in developing pollen‐ and seed‐bearing organs are also present in the distantly related gymnosperms: the conifers and the gneto‐phytes. We now report on the characterization of DAL10, a novel MADS‐box gene from the conifer Norway spruce, which unlike the B‐ and C‐type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B‐ and C‐type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B‐ and C‐type genes in the establishment of reproductive identity of developing cones.

cDNA sequence and expression of an intron-containing histone H2A gene from Norway spruce, Picea abies

We have isolated a cDNA clone corresponding to a histone H2A gene from Norway spruce, Picea abies (L.) Karst. The clone was isolated on the basis of the preferential expression of the corresponding gene during germination. The identification of the clone was based on the high degree of nucleotide sequence identity (60–65%) to a range of eukaryotic histone H2A genes and the presence of a 9 amino acids long sequence identical to the conserved ‘H2A box’ in the deduced amino acid sequence. Like other plant histone genes, the spruce histone H2A gene encodes a polyadenylated transcript. Further, the spruce gene contains an intervening sequence of 891 bp in the coding region. The presence of introns is typical of a distinct class of replication-independent histone genes in other eukaryotes. However, the sequence of the spruce gene and its high expression in mitotically active tissues such as the apical meristem, strongly suggests that it belongs to the class of replication-dependent histone genes. This is the first documentation of an intervening sequence in this class of histone genes and the finding implies that introns were present in the ancestral histone H2A gene before the divergence of the two classes of histone genes.

IFN-α production by plasmacytoid dendritic cell associations with polymorphisms in gene loci related to autoimmune and inflammatory diseases

The type I interferon (IFN) system is persistently activated in systemic lupus erythematosus (SLE) and many other systemic autoimmune diseases. Studies have shown an association between SLE and several gene variants within the type I IFN system. We investigated whether single-nucleotide polymorphisms (SNPs) associated with SLE and other autoimmune diseases affect the IFN-α production in healthy individuals. Plasmacytoid dendritic cells (pDCs), B cells and NK cells were isolated from peripheral blood of healthy individuals and stimulated with RNA-containing immune complexes (ICs), herpes simplex virus (HSV) or the oligonucleotide ODN2216. IFN-α production by pDCs alone or in cocultures with B or NK cells was measured by an immunoassay. All donors were genotyped with the 200K ImmunoChip, and a 5 bp CGGGG length polymorphism in the IFN regulatory factor 5 gene (IRF5) was genotyped by PCR. We found associations between IFN-α production and 18-86 SNPs (P ≤ 0.001), depending on the combination of the stimulated cell types. However, only three of these associated SNPs were shared between the cell-type combinations. Several SNPs showed novel associations to the type I IFN system among all the associated SNPs, whereas some loci have been described earlier for their association with SLE. Furthermore, we found that the SLE-risk variant of the IRF5 CGGGG-indel was associated with lower IFN-α production. We conclude that the genetic variants affecting the IFN-α production highlight the intricate regulation of the type I IFN system and the importance of understanding the mechanisms behind the dysregulated type I IFN system in SLE.

AGAMOUS subfamily MADS-box genes and the evolution of seed cone morphology in Cupressaceae and Taxodiaceae

SUMMARY In this comparative developmental genetics study, we test hypotheses based on fossil and morphological data on reproductive organ morphology and evolution in conifers—specifically, the ovule‐bearing organ in Cupressaceae and Taxodiaceae. Genes homologous to the Arabidopsis gene AGAMOUS are expressed in ovuliferous scales of spruces (Picea) throughout development. Previous studies have shown that the AGAMOUS subfamily of MADS‐box genes predates the split between angiosperms and gymnosperms, and that these genes have in part conserved functions in reproductive development among seed plants, especially in the specification of identity of the ovule‐bearing organs. These data indicate that their expression in conifer families other than Pinaceae might be used as markers for organs homologous to the Pinaceae ovuliferous scale. Here we have isolated putative AGAMOUS orthologs from Cupressaceae and Taxodiaceae and analyzed their expression pattern in seed cones to test for the presence of morphological homologs of ovuliferous scales. Our results were not congruent with the hypothesis that the tooth of the Cryptomeria seed cone is homologous to the Picea ovuliferous scale. Likewise, the hypothesis that the bracts of Thujopsis and Juniperus contain fused ovuliferous scales was not supported. However, we found expression of AGAMOUS homologs in the sterile bracts of Cupressaceae seed cones at late developmental stages. This expression probably represents a novel gene function in these conifer families, since no corresponding expression has been identified in Pinaceae. Our study suggests that the evolutionary history of modern conifer cones is more diverse than previously thought.

A Novel HLA-DRB1*10:01-Restricted T Cell Epitope From Citrullinated Type II Collagen Relevant to Rheumatoid Arthritis.

Antibodies against citrullinated type II collagen (Cit‐CII) are common in the sera and synovial fluid of patients with rheumatoid arthritis (RA); however, the known T cell epitope of CII is not dependent on citrullination. The aim of this study was to identify and functionally characterize the Cit‐CII–restricted T cell epitopes that are relevant to RA.

Extended exome sequencing identifies BACH2 as a novel major risk locus for Addison's disease

Autoimmune disease is one of the leading causes of morbidity and mortality worldwide. In Addisons disease, the adrenal glands are targeted by destructive autoimmunity. Despite being the most common cause of primary adrenal failure, little is known about its aetiology.