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Studying the drought-responsive transcriptome is of high interest as it can serve as a blueprint for stress adaptation strategies. Despite extensive studies in this area, there are still many details to be uncovered, such as the importance of each gene involved in the stress response as well as the relationship between these genes and the physiochemical processes governing stress tolerance. This study was designed to address such important details and to gain insights into molecular responses of barley (Hordeum vulgare L.) to drought stress. To that, we combined RNA-seq data analysis with field and greenhouse drought experiments in a systems biology approach. RNA-sequence analysis identified a total of 665 differentially expressed genes (DEGs) belonging to diverse functional categories. A gene network was derived from the DEGs, which comprised of a total of 131 nodes and 257 edges. Gene network topology analysis highlighted two programmed cell death (PCD) modulating genes, MC1 (metacaspase 1) and TSN1 (Tudor-SN 1), as important (hub) genes in the predicted network. Based on the field trial, a drought-tolerant and a drought-susceptible barley genotype was identified from eight tested cultivars. Identified genotypes exhibited different physiochemical characteristics, including proline content, chlorophyll concentration, percentage of electrolyte leakage and malondialdehyde content as well as expression profiles of MC1 and TSN1 genes. Machine learning and correspondence analysis revealed a significant relationship between drought tolerance and measured characteristics in the context of PCD. Our study provides new insights which bridge barley drought tolerance to PCD through MC1 and TSN1 pathway.

Co-occurrence of abiotic stresses, especially drought and salinity, is a natural phenomenon in field conditions and is worse for crop production than any single stress. Nowadays, rigorous methods of meta-analysis and systems biology have made it possible to perform cross-study comparisons of single stress experiments, which can uncover main overlapping mechanisms underlying tolerance to combined stress. In this study, a meta-analysis of RNA-Seq data was conducted to obtain the overlapping gene network of drought and salinity stresses in barley (Hordeum vulgare L.), which identified Rubisco activase A (RcaA) as a hub gene in the dual-stress response. Thereafter, a greenhouse experiment was carried out using two barley genotypes with different abiotic stress tolerance and evaluated several physiochemical properties as well as the expression profile and protein activity of RcaA. Finally, machine learning analysis was applied to uncover relationships among combined stress tolerance and evaluated properties. We identified 441 genes which were differentially expressed under both drought and salinity stress. Results revealed that the photosynthesis pathway and, in particular, the RcaA gene are major components of the dual-stress responsive transcriptome. Comparative physiochemical and molecular evaluations further confirmed that enhanced photosynthesis capability, mainly through regulation of RcaA expression and activity as well as accumulation of proline content, have a significant association with combined drought and salinity stress tolerance in barley. Overall, our results clarify the importance of RcaA in combined stress tolerance and may provide new insights for future investigations.

Background Neuroblastoma (NB) is a childhood neural crest tumor. There are two groups of aggressive NBs, one with MYCN amplification, and another with 11q chromosomal deletion; these chromosomal aberrations are generally mutually exclusive. The DLG2 gene resides in the 11q-deleted region, thus makes it an interesting NB candidate tumor suppressor gene. Methods We evaluated the association of DLG2 gene expression in NB with patient outcomes, stage and MYCN status, using online microarray data combining independent NB patient data sets. Functional studies were also conducted using NB cell models and the fruit fly. Results Using the array data we concluded that higher DLG2 expression was positively correlated to patient survival. We could also see that expression of DLG2 was inversely correlated with MYCN status and tumor stage. Cell proliferation was lowered in both 11q-normal and 11q-deleted NB cells after DLG2 over expression, and increased in 11q-normal NB cells after DLG2 silencing. Higher level of DLG2 increased the percentage of cells in the G2/M phase and decreased the percentage of cells in the G1 phase. We detected increased protein levels of Cyclin A and Cyclin B in fruit fly models either over expressing dMyc or with RNAi-silenced dmDLG, indicating that both events resulted in enhanced cell cycling. Induced MYCN expression in NB cells lowered DLG2 gene expression, which was confirmed in the fly; when dMyc was over expressed, the dmDLG protein level was lowered, indicating a link between Myc over expression and low dmDLG level. Conclusion We conclude that low DLG2 expression level forces cell cycle progression, and that it predicts poor NB patient survival. The low DLG2 expression level could be caused by either MYCN-amplification or 11q-deletion. Graphical abstract

Understanding how plants respond to salinity stress is essential for developing tolerant genotypes, to keep human food secure since it is threaten by climate changes and increasing population worldwide. Barley (Hordeum vulgare) is a crop that possesses various salinity tolerance mechanisms that remain to be explored. In this study, data from an RNA-Seq experiment in barley was analyzed to identify changes in genome activities as well as differentially expressed genes (DEGs) in response to salinity stress. A gene network was predicted among identified DEGs and was subjected to network topology analysis, which resulted in the prediction of a hub gene, namely low expression of osmotically responsive gene 2 (LOS2). LOS2 and its two hierarchical downstream genes, salt-tolerant zinc finger (ZAT10) and ascorbate peroxidase 1 (APX1), were used in a genome-wide association (GWA) survey to confirm their importance. A field experiment was conducted to recognize susceptible and tolerant genotypes among 10 different barley genotypes based on the principle component analysis (PCA) of stress-related indices. In a separate salinity experiment, two of the genotypes were assessed to assign their physiological and biochemical responses as well as to identify expression profiles of LOS2, ZAT10, and APX1. From the results, the activity of the barley genome was significantly altered toward response to stress. In total, 5692 DEGs were identified and the gene network derived from these genes contained 131 nodes and 257 edges. The identified genotypes clearly showed the difference in water status, osmolyte accumulation, cell membrane damages, and ion homeostasis as well as in expression profiles for studied genes during salinity stress. Our results suggest that LOS2 along with the ZAT10 and APX1 genes may serve as an important part of barley salinity stress tolerance pathways. To our knowledge, this is the first report on the role(s) of LOS2 in barley salinity stress tolerance in a gene network system.

Cis regulatory elements (CREs), located within promoter regions, play a significant role in the blueprint for transcriptional regulation of genes. There is a growing interest to study the combinatorial nature of CREs including presence or absence of CREs, the number of occurrences of each CRE, as well as of their order and location relative to their target genes. Comparative promoter analysis has been shown to be a reliable strategy to test the significance of each component of promoter architecture. However, it remains unclear what level of difference in the number of occurrences of each CRE is of statistical significance in order to explain different expression patterns of two genes. In this study, we present a novel statistical approach for pairwise comparison of promoters of Arabidopsis genes in the context of number of occurrences of each CRE within the promoters. First, using the sample of 1000 Arabidopsis promoters, the results of the goodness of fit test and non-parametric analysis revealed that the number of occurrences of CREs in a promoter sequence is Poisson distributed. As a promoter sequence contained functional and non-functional CREs, we addressed the issue of the statistical distribution of functional CREs by analyzing the ChIP-seq datasets. The results showed that the number of occurrences of functional CREs over the genomic regions was determined as being Poisson distributed. In accordance with the obtained distribution of CREs occurrences, we suggested the Audic and Claverie (AC) test to compare two promoters based on the number of occurrences for the CREs. Superiority of the AC test over Chi-square (2×2) and Fisher’s exact tests was also shown, as the AC test was able to detect a higher number of significant CREs. The two case studies on the Arabidopsis genes were performed in order to biologically verify the pairwise test for promoter comparison. Consequently, a number of CREs with significantly different occurrences was identified between the promoters. The results of the pairwise comparative analysis together with the expression data for the studied genes revealed the biological significance of the identified CREs.

Triticum monococcum has recently drawn the attention of biologists to discover and utilize novel genes and alleles. To explore the molecular features of the genetic network governing floral transition in shoot apical meristem (SAM) of spring growth habit T. monococcum, two expressed sequence tag (EST) libraries containing 3,031 ESTs from vegetative SAM (VS) and 2,647 ESTs from early reproductive SAM (RS) were analyzed. Assembly of ESTs resulted in 2,303 unigenes for VS library (368 contigs and 1,935 singletons) and 1,890 unigenes (337 contigs and 1,553 singletons) for RS library. The 67.05 % of VS unigenes and 66.30 % of RS unigenes showed significant similarity with genes of known, putative and or unknown function, whereas the remaining 32.95 % of the VS unigenes and 33.7 % of RS unigenes displayed no significant match with the public protein database. The 1,064 and 866 unigenes of VS and RS libraries were assigned to functional categories using Pageman ontology tool. Further analysis revealed that the switch from VS to RS caused significant changes in the abundance of unigenes assigned to some functional categories. A total of 37 genes were identified which were significantly differentially expressed between vegetative and reproductive stages of T. monococcum SAM. Investigation of the differentially expressed genes revealed the importance of the genes involved in energy metabolism, ubiquitin/26S proteasome system, polyamines biosynthesis and signaling of reactive oxygen species in SAM differentiation towards floral transition in T. monococcum.

The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.

Motivation:Cold acclimation involves a number of different cellular processes that together increase the freezing tolerance of an organism. The DREB1/CBFs are transcription factors (TFs) that are prominent in the regulation of cold responses in Arabidopsis thaliana, rice and many other crops. We investigated if the expression of DREB1/CBFs and co-expressed genes relies on combinatorial control by several TFs. Our results support this notion and indicate that methods for studying the regulation of complex cellular processes should include identification of combinations of motifs, in addition to searching for individual overrepresented binding sites. Contact: angelica.lindlof@his.se Supplementary information: Supplementary data are available at Bioinformatics online.

DNA-analysis with flanking and intragenic markers gave confusing results in 7 out of 74 (9.5%) Finnish families with Duchenne or Becker muscular dystrophy. In five families a sister or maternal aunt of the patient had elevated serum creatine kinase (CK) activity, although DNA-analysis indicated a low risk for carriership. In one family the two affected brothers had different pERT87 alleles. In one family the intragenic deletion found in a patient was not present in his mother, who was an obligatory carrier. Deletions were detected with cDNA probes in the probands in five of the families, but the controversy regarding carriership still remained. It is necessary to combine all available data from pedigree analysis, CK measurements, and DNA studies whenever carrier studies are performed, but it appears that major problems in counselling and prenatal diagnosis will still remain in a proportion of the families.

Introduction Low temperature is one of the major environmental factors that adversely affect plant growth and yield. Many cereal crops from tropical regions, such as rice, are chilling sensitive and, therefore, are affected already at CC BY 4.0

Cancer is a broad term for a wide spectrum of diseases and which involves the alteration in expression levels of several hundreds of genes. As such, the study of the disease from a systems biology point of view becomes rational, as the properties of a system as a whole may be very different from the properties of its individual components. However, understanding a network at the systems level not only requires knowledge about the components of the network, but also the interactions between them. Here, a systems biology view of the rat PHD finger protein 5A (Phf5a) gene was attempted; a gene previously identified as aberrantly expressed in estrogen dependent endometrial adenocarcinoma tumors from both rat and human. Phf5 ais a highly conserved cysteine rich (C4HC3) zinc finger and such proteins predominantly have a role in chromatin mediated transcriptional regulation. Moreover, PHF5A is a component of the macromolecular complex spliceosome that takes part in pre-mRNA splicing and spliceosome component coding genes have previously been shown to be implicated in various cancer types and suggested to potentially be novel antitumor drugs. To derive a systems biology view, in this study, a weighted gene network was inferred from a list of genes having correlated expression profiles to Phf5a as nodes, and common transcription factors and microRNAs regulating these genes together with annotation about biological process ontology term(s) and pathway(s) as edge weights. In the inferred network a higher weight indicates more annotation shared between two genes and, hence, the network facilitates the identification of closely interacting genes with Phf5a. The results show that highly weighted edges connect Phf5a to other spliceosome components, but also to genes involved in the metabolism of proteins, proteasome and DNA replication, repair and recombination. The results also link Phf5a to the Myc/Rb/E2F pathway, one of the central pathways associated with cancer. The proposed method for inferring a weighted gene network can easily be applied to other genes and diseases. CC BYCopyright: © 2014 Vallabhu R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Low temperature is a key factor that limits growth and productivity of many important agronomical crops worldwide. Rice (Oryza sativa L.) is negatively affected already at temperatures below +10°C and is therefore denoted as chilling sensitive. However, chilling tolerant rice cultivars exist and can be commercially cultivated at altitudes up to 3,050 meters with temperatures reaching as low as +4°C. In this work, the global transcriptional response to cold stress (+4°C) was studied in the Nepalese highland variety Jumli Marshi (spp. japonica) and 4,636 genes were identified as significantly differentially expressed within 24 hours of cold stress. Comparison with previously published microarray data from one chilling tolerant and two sensitive rice cultivars identified 182 genes differentially expressed (DE) upon cold stress in all four rice cultivars and 511 genes DE only in the chilling tolerant rice. Promoter analysis of the 182 genes suggests a complex cross-talk between ABRE and CBF regulons. Promoter analysis of the 511 genes identified over-represented ABRE motifs but not DRE motifs, suggesting a role for ABA signaling in cold tolerance. Moreover, 2,101 genes were DE in Jumli Marshi alone. By chromosomal localization analysis, 473 of these cold responsive genes were located within 13 different QTLs previously identified as cold associated. CC BY 4.0

Aromatase is an important enzyme in the local synthesis of oestrogens and its expression has been shown to be increased in breast cancer through the activation of multiple promoters. However, the mechanisms behind this are not yet fully understood. A novel candidate in this context is the transcription factor forkhead box L2 (FOXL2), which has been recognised to be co-expressed with aromatase and transcriptionally active promoter II in developing goat and chicken ovaries. We propose that FOXL2 could be involved in the increased expression of aromatase in breast cancer. We examined FOXL2 and its relation to aromatase in 132 post-menopausal breast cancer patients by immunohistochemistry. Using in silico analysis, we further searched for FOXL2 binding-elements in the aromatase gene promoters. The results demonstrate that FOXL2 is expressed in breast cancer and influences clinical outcome with improved recurrence-free survival in cases with nuclear expression. In a multivariate Cox model, nuclear FOXL2 was a significant prognostic factor in ER-positive patients treated with tamoxifen (HR=0.18, 95% confidence interval (CI)=0.04-0.81, P=0.03). Tumours expressing nuclear FOXL2 were also more likely positive for stromal and/or cytoplasmic aromatase (P=0.03 and P=0.008, respectively). In silico analyses revealed binding elements of FOXL2 in promoters I.3, II and I.7 of the aromatase gene of which promoter I.7 was most significant. In conclusion, this is the first study to report that FOXL2 is expressed in breast cancer and correlates with aromatase as well as with clinical outcome. The results further strengthen a possible binding of FOXL2 to aromatase promoter I.7. Nevertheless, whether FOXL2 is a direct activator of aromatase requires further investigation.

A ‘B’ cell line, originating from a patient with chronic myeloid leukemia and containing the Philadelphia chromosome, was established after Epstein-Barr virus transformation. The Philadelphia chromosome was the sole chromosomal abnormality in this line, designated as PhBl cell line. In DNA hybridization studies we detected rearrangements in the bcr gene and in the immunoglobulin heavy chain joining region. The phenotypes of the cells were typical of mature B cells expressing antigens CDI9, CD20, CD22, CD23, CD39, HLADR, IgM, and kappa. The expression of the 210 bcr-abl chimeric protein was detected by means of an immunoprecipitate assay.

Genes coding for drought response element binding (DREB) proteins regulate transcription of a large number of downstream genes involved in the plant response to abiotic stresses. However the regulation of DREB genes themselves is not well understood. Using a bioinformatics approach, we identified the over-represented motifs in promoters of DREB genes of sorghum and rice as compared to all the other promoters in their genomes. Aligned orthologous promoter pairs of sorghum and rice DREBs were then used to identify co-localized motifs from among the over-represented ones, assuming that such motifs were likely to play a regulatory role. Finally the motifs over-represented in sorghum DREBs in comparison to their rice orthologs were identified. Results indicated over-representation of motifs pertaining to calcium, light, sugar and hormone signaling in the DREB promoters. The co-localized motifs in DREB promoters were mainly those involved in abscisic acid-, light- and calcium-mediated regulation. These motifs, along with others pertaining to ethylene signaling, were over-represented in sorghum DREB promoters as compared to their orthologs from rice and could possibly contribute to its drought tolerance. Besides calcium, an integration of abscisic acid, ethylene, auxin and methyl jasmonate signaling was probably involved in regulating expression of the drought response through DREB transcription factors.

Low temperature is one of the major factors that adversely affect crop yields by causing restraints on plant growth and productivity. However, most temperate plants have the ability to acclimate to cooler temperatures. Cold acclimation is a process which increases the freezing tolerance of an organism after exposure to low, non-freezing temperatures. The main trigger is a decrease in temperature levels, but light reduction has also been shown to have an important impact on acquired tolerance. Since the lowest temperatures are commonly reached during the night hours in winter time and is an annually recurring event, a favorable trait for plants is the possibility of sensing an imminent cold period. Consequently, extensive crosstalk between light- and temperature signaling pathways has been demonstrated and in this review interesting interaction points that have been previously reported in the literature are highlighted.

The detection of differentially expressed genes from EST data is of importance for the discovery of potential biological or pharmaceutical targets, especially when studying biological processes in less characterized organisms and where large-scale microarrays are not an option. We present a comparison of five different statistical methods for identifying up-regulated genes through pairwise comparison of EST sets, where one of the sets is generated from a treatment and the other one serves as a control. In addition, we specifically address situations where the sets are relatively small (~2,000– 10,000 ESTs) and may differ in size. The methods were tested on both simulated and experimentally derived data, and compared to a collection of cold stress induced genes identified by microarrays. We found that combining the method proposed by Audic and Claverie with Fisher's exact test and a method based on calculating the difference in relative frequency was the best combination for maximizing the detection of up-regulated genes. We also introduced the use of a flexible cutoff, which takes the size of the EST sets into consideration. This could be considered as an alternative to a static cutoff. Finally, the detected genes showed a low overlap with those identified by microarrays, which indicates, as in previous studies, low overall concordance between the two platforms.

Background With the advent of microarray technology, it has become feasible to identify virtually all genes in an organism that are induced by developmental or environmental changes. However, relying solely on gene expression data may be of limited value if the aim is to infer the underlying genetic networks. Development of computational methods to combine microarray data with other information sources is therefore necessary. Here we describe one such method. Results By means of our method, previously published Arabidopsis microarray data from cold acclimated plants at six different time points, promoter motif sequence data extracted from ~24,000 Arabidopsis promoters and known transcription factor binding sites were combined to construct a putative genetic regulatory interaction network. The inferred network includes both previously characterised and hitherto un-described regulatory interactions between transcription factor (TF) genes and genes that encode other TFs or other proteins. Part of the obtained transcription factor regulatory network is presented here. More detailed information is available in the additional files. Conclusion The rule-based method described here can be used to infer genetic networks by combining data from microarrays, promoter sequences and known promoter binding sites. This method should in principle be applicable to any biological system. We tested the method on the cold acclimation process in Arabidopsis and could identify a more complex putative genetic regulatory network than previously described. However, it should be noted that information on specific binding sites for individual TFs were in most cases not available. Thus, gene targets for the entire TF gene families were predicted. In addition, the networks were built solely by a bioinformatics approach and experimental verifications will be necessary for their final validation. On the other hand, since our method highlights putative novel interactions, more directed experiments could now be performed.

Background Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required. Results From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4°C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10-10) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values ≤ 10-10) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs. Conclusion The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.

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Using whole cosmids as probes, we have mapped 242 DMD BMD deletion breakpoints located in the major deletion hot spot of the DMD gene. Of these, 113 breakpoints were mapped more precisely to individual restriction enzyme fragments in the distal 80 kb of the 170-kb intron 44. An additional 12 breakpoints were mapped to the adjacent 10 kb of introm 45. The breakpoints are distributed over the entire region, with no significant local variation in frequency. Furthermore, deletion sizes vary and are not influenced by the positions of the breakpoints. This argues against a predominant role of one or a few specific sequences in causing frequent rearrangements. It suggests that structural characteristics or a more widespread recombinogenic sequence makes this region so susceptible to deletion. Our study revealed several RFLPs, one of which is a 300-bp insertion/deletion polymorphism. Abnormally migrating junction fragments are found in 81% of the precisely mapped deletions and are highly valuable in the diagnosis of carrier females.

Linkage analysis of five marker loci in and around the Duchenne muscular dystrophy (DMD) locus, DXS84, DXS206, DXS164, DXS270, and DXS28, was conducted with 499 families. Overall, the best multipoint distances were found to be DXS84-3.7 +/- 0.6 cM-DXS206-1.0 +/- 0.4 cM-DXS164-1.9 +/- 0.6 cM-DXS270-12.0 +/- 1.1 cM-DXS28. A comparison of this linkage map with the established physical map suggests the presence of hot spots for recombination in the DMD locus.

Summary Ten polymorphic DNA markers, including gene specific markers of loci DXS164 and DXS206, were tested for allele frequencies, degree of heterozygosity and linkage in 34 Finnish families with X-linked muscular dystrophy. With the exception of the BamHI RFLP of DXS164 subclone pERT87-15, allele frequencies and the degree of heterozygosity failed to show any significant deviation from the data published elsewhere. We document a high degree of linkage disequilibrium between several RFLPs belonging to locus DXS164. Our linkage data include one recombination between DMD and DXS164 enabling a tentative location of the mutation site distal to DXS164. The maximum lod score for linkage between the disease locus and DX164 was 7.828 at a recombination fraction of 0.02. According to our data DXS28 and DXS43 may be located further away from the disease locus than previously thought. We use only gene specific markers for genetic counselling. Excluding deletions, 97.1 % of women were heterozygous for at least one such marker. A diagnostic procedure in which useful information can be obtained in over 90 % of all diagnostic situations, using only four filters, is proposed.

A family in which the proband showed phenotypic signs of both the Turner and Down syndromes was studied cytogenetically and with restriction fragment length polymorphisms. The proband's karyotype was 46,X,+21, showing double aneuploidy without any signs of mosaicism. The single X and one chromosome 21 were of paternal origin while two chromosome 21 were of maternal origin. The nondisjunction of chromosome 21 took place in maternal meiosis II. If it is assumed that the absence of mosaicism renders postzygotic mitotic loss of the X chromosome unlikely, then the X chromosome would have been lost in maternal meiosis I or II. Recombination had occurred between the nondisjoined chromosomes 21. We conclude that double nondisjunction took place in one patient and that asynapsis was not a prerequisite for the autosomal nondisjunction.

With the aim of offering carrier detection, genetic counselling, and prenatal diagnosis to as many families with Duchenne (DMD) and Becker (BMD) muscular dystrophy as possible, we used available DNA probes to determine the usefulness of the RFLP approach. We report in detail the risks calculated using Bayesian theory and combining pedigree and creatine kinase (CK) data with information derived from the RFLP studies. To date we have analysed members of 28 DMD families (10 familial, 18 sporadic) and six BMD families (four familial, two sporadic) with the closely linked pERT probes 87-1, 87-8, and 87-15 (DXS164). In addition, key members of all families were analysed with probes D2 (DXS43), C7 (DXS28), 754 (DXS84), and L1 X 28 (DXS7). Of the 97 females at risk of being carriers (not including 26 obligate carriers), the RFLP results were compatible with carriership in 22 and not in 51. In 24 females (including 17 mothers of sporadic cases), no information regarding carriership was derived from the RFLP studies. There was no disagreement between pedigree information, clearly raised CK values, and DNA studies. Of 52 obligate or possible carriers under the age of 45, prenatal diagnosis is possible in 49. Prenatal diagnostic RFLP studies have so far been done in three women. In one sporadic DMD family and one BMD family with three affected males the probands showed a deletion involving the three pERT87 subclones used. Experience derived from these families indicates that in our society genetic counselling in X linked muscular dystrophy is received with approval or even enthusiasm in spite of the 5% error estimate that we have quoted for pERT87 derived results.

Summary During winter-time, two groups of captive female Mountain hares were fed food of high and normal quality respectively, in order to study the impact of winter nutrition on reproduction the following summer. On average, the animals fed on high quality food increased their weight 19.2% from December to April, while the corresponding figure for the other group was 7.1%. Neither the size of the first, nor the second litter of the year differed between the two feeding regimes. In the first litter, the leverets from the females fed high quality food had significantly higher birth weights (149.5±24.4 g) than the leverets from the normally fed females (108.5±25.6 g). The second litter of the year showed no such differences. Gestation time was similar for the two groups and showed small fluctuations around a mean value of 51 days. Juvenile growth rate amounted to an average of slightly below 30 g per day during the first 2–3 months. We are most grateful to Dr Olav Hjeljord and Dr Jonathan Reynolds for valuable criticism of the manuscript. Dr Reynolds also kindly checked the language for us.

Carrier determination and prenatal diagnosis in Norrie disease (ND) has so far not been reported. We describe a kindred with 4 members affected by ND in which a deletion comprising gene locus DXS7 on the short arm of the X chromosome defined by probe L1.28 causes the disorder. This allowed us to predict via chorion villus biopsy that a male foetus of a carrier woman is unaffected.

The DNA from 68 patients with X-linked (Duchenne and Becker) muscular dystrophy belonging to 49 unrelated families was analyzed for microdeletions using 13 closely linked or gene-specific DNA-markers. Fourteen patients from eight families showed a deletion involving a least one of the markers used, giving a deletion frequency of 16%. The proportion of families with deletions was 36% in the Becker and 8% in the Duchenne form of the disease. With one exception, the extent of the deletion was different in different families. All living, affected males from the same family carried the same deletion. The extent or the localization of the deletion did not correlate with clinical features such as severity of disease or mental retardation.

Inherited mutations in the erythropoietin receptor (EPOR) causing premature termination of the receptor cytoplasmic region are associated with dominant familial erythrocytosis (FE), a benign clinical condition characterized by hypersensitivity of erythroid progenitor cells to EPO and low serum EPO (S-EPO) levels. We describe a Swedish family with dominant FE in which erythrocytosis segregates with a new truncation in the negative control domain of the EPOR. We show that cells engineered to concomitantly express the wild-type (WT) EPOR and mutant EPORs associated with FE (FE EPORs) are hypersensitive to EPO-stimulated proliferation and activation of Jak2 and Stat5. These results demonstrate that FE is caused by hyperresponsiveness of receptor-mediated signaling pathways and that this is dominant with respect to WT EPOR signaling.

Freezing tolerance in plants is obtained during a period of low nonfreezing temperatures before the winter sets on, through a biological process known as cold acclimation. Cold is one of the major stress factors that limits the growth, productivity and distribution of plants, and understanding the mechanism of cold tolerance is therefore important for crop improvement. Expressed sequence tags (EST) analysis is a powerful, economical and time-efficient way of assembling information on the transcriptome. To date, several EST sets have been generated from cold-induced cDNA libraries from several different plant species. In this study we utilize the variation in the frequency of ESTs sampled from different cold-stressed plant libraries, in order to identify genes preferentially expressed in cold in comparison to a number of control sets. The species included in the comparative study are oat (Avena sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), rice (Oryza sativa) and Arabidopsis thaliana. However, in order to get comparable gene expression estimates across multiple species and data sets, we choose to compare the expression of tentative ortholog groups (TOGs) instead of single genes, as in the normal procedure. We consider TOGs as preferentially expressed if they are detected as differentially expressed by a test statistic and up-regulated in comparison to all control sets, and/or uniquely expressed during cold stress, i.e., not present in any of the control sets. The result of this analysis revealed a diverse representation of genes in the different species. In addition, the derived TOGs mainly represent genes that are long-term highly or moderately expressed in response to cold and/or other stresses.