GDNF-deprived sympathetic neurons die via a novel nonmitochondrial pathway

The mitochondrial death pathway is triggered in cultured sympathetic neurons by deprivation of nerve growth factor (NGF), but the death mechanisms activated by deprivation of other neurotrophic factors are poorly studied. We compared sympathetic neurons deprived of NGF to those deprived of glial cell line-derived neurotrophic factor (GDNF). In contrast to NGF-deprived neurons, GDNF-deprived neurons did not die via the mitochondrial pathway. Indeed, cytochrome c was not released to the cytosol; Bax and caspase-9 and -3 were not involved; overexpressed Bcl-xL did not block the death; and the mitochondrial ultrastructure was not changed. Similarly to NGF-deprived neurons, the death induced by GDNF removal is associated with increased autophagy and requires multiple lineage kinases, c-Jun and caspase-2 and -7. Serine 73 of c-Jun was phosphorylated in both NGF- and GDNF-deprived neurons, whereas serine 63 was phosphorylated only in NGF-deprived neurons. In many NGF-deprived neurons, the ultrastructure of the mitochondria was changed. Thus, a novel nonmitochondrial caspase-dependent death pathway is activated in GDNF-deprived sympathetic neurons.     

Linkage and comparative mapping of the locus controlling susceptibility towards E. COLI F4ab/ac diarrhoea in pigs

In 1995, Edfors-Lilja and coworkers mapped the locus for the E. COLI K88ab (F4ab) and K88ac (F4ac) intestinal receptor to pig chromosome 13 (SSC13). Using the same family material we have refined the map position to a region between the microsatellite markers Sw207 and Sw225. Primers from these markers were used to screen a pig BAC library and the positive clones were used for fluorescent in situ hybridization (FISH) analysis. The results of the FISH analysis helped to propose a candidate gene region in the SSC13q41-->q44 interval. Shotgun sequencing of the FISH-mapped BAC clones revealed that the candidate region contains an evolutionary breakpoint between human and pig. In order to further characterise the rearrangements between SSC13 and human chromosome 3 (HSA3), detailed gene mapping of SSC13 was carried out. Based on this mapping data we have constructed a detailed comparative map between SSC13 and HSA3. Two candidate regions on human chromosome 3 have been identified that are likely to harbour the human homologue of the gene responsible for susceptibility towards E. COLI F4ab/ac diarrhoea in pigs.     

An ordered BAC contig map of the equine major histocompatibility complex

A physical map of ordered bacterial artificial chromosome (BAC) clones was constructed to determine the genetic organization of the horse major histocompatibility complex. Human, cattle, pig, mouse, and rat MHC gene sequences were compared to identify highly conserved regions which served as source templates for the design of overgo primers. Thirty-five overgo probes were designed from 24 genes and used for hybridization screening of the equine USDA CHORI 241 BAC library. Two hundred thirty-eight BAC clones were assembled into two contigs spanning the horse MHC region. The first contig contains the MHC class II region and was reduced to a minimum tiling path of nine BAC clones that span approximately 800 kb and contain at least 20 genes. A minimum tiling path of a second contig containing the class III/I region is comprised of 14 BAC clones that span approximately 1.6 Mb and contain at least 34 genes. Fluorescence in situ hybridization (FISH) using representative clones from each of the three regions of the MHC localized the contigs onto ECA20q21 and oriented the regions relative to one another and the centromere. Dual-colored FISH revealed that the class I region is proximal to the centromere, the class II region is distal, and the class III region is located between class I and II. These data indicate that the equine MHC is a single gene-dense region similar in structure and organization to the human MHC and is not disrupted as in ruminants and pigs.     

Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: ecological evidence for mixotrophy in Pyroleae

Several forest understorey achlorophyllous plants, termed mycoheterotrophs (MHs), obtain C from their mycorrhizal fungi. The latter in turn form ectomycorrhizas with trees, the ultimate C source of the entire system. A similar nutritional strategy occurs in some green forest orchids, phylogenetically close to MH species, that gain their C via a combination of MH and photosynthesis (mixotrophy). In orchid evolution, mixotrophy evolved in shaded habitats and preceded MH nutrition. By generalizing and applying this to Ericaceae, we hypothesized that green forest species phylogenetically close to MHs are mixotrophic. Using stable C isotope analysis with fungi, autotrophic, mixotrophic and MH plants as comparisons, we found the first quantitative evidence for substantial fungi-mediated mixotrophy in the Pyroleae, common ericaceous shrubs from boreal forests close to the MH Monotropoideae. Orthilia secunda, Pyrola chlorantha, Pyrola rotundifolia and Chimaphila umbellata acquired between 10.3 and 67.5% of their C from fungi. High N and ¹⁵N contents also suggest that Pyroleae nutrition partly rely on fungi. Examination of root fungal internal transcribed spacer sequences at one site revealed that 39 species of mostly endophytic or ectomycorrhizal fungi, including abundant Tricholoma spp., were associated with O. secunda, P. chlorantha and C. umbellata. These fungi, particularly ectomycorrhizal associates, could thus link mixotrophic Pyroleae spp. to surrounding trees, allowing the C flows deduced from isotopic evidence. These data suggest that we need to reconsider ecological roles of understorey plants, which could influence the dynamics and composition of forest communities.     

High-resolution gene maps of horse chromosomes 14 and 21: additional insights into evolution and rearrangements of HSA5 homologs in mammals

High-resolution physically ordered gene maps for equine homologs of human chromosome 5 (HSA5), viz., horse chromosomes 14 and 21 (ECA14 and ECA21), were generated by adding 179 new loci (131 gene-specific and 48 microsatellites) to the existing maps of the two chromosomes. The loci were mapped primarily by genotyping on a 5000-rad horse x hamster radiation hybrid panel, of which 28 were mapped by fluorescence in situ hybridization. The approximately fivefold increase in the number of mapped markers on the two chromosomes improves the average resolution of the map to 1 marker/0.9 Mb. The improved resolution is vital for rapid chromosomal localization of traits of interest on these chromosomes and for facilitating candidate gene searches. The comparative gene mapping data on ECA14 and ECA21 finely align the chromosomes to sequence/gene maps of a range of evolutionarily distantly related species. It also demonstrates that compared to ECA14, the ECA21 segment corresponding to HSA5 is a more conserved region because of preserved gene order in a larger number of and more diverse species. Further, comparison of ECA14 and the distal three-quarters region of ECA21 with corresponding chromosomal segments in 50 species belonging to 11 mammalian orders provides a broad overview of the evolution of these segments in individual orders from the putative ancestral chromosomal configuration. Of particular interest is the identification and precise demarcation of equid/Perissodactyl-specific features that for the first time clearly distinguish the origins of ECA14 and ECA21 from similar-looking status in the Cetartiodactyls.     

Mitogenetic structure of brown bears (Ursus arctos L.) in northeastern Europe and a new time frame for the formation of European brown bear lineages

We estimated the phylogenetic relationships of brown bear maternal haplotypes from countries of northeastern Europe (Estonia, Finland and European Russia), using sequences of mitochondrial DNA (mtDNA) control region of 231 bears. Twenty-five mtDNA haplotypes were identified. The brown bear population in northeastern Europe can be divided into three haplogroups: one with bears from all three countries, one with bears from Finland and Russia, and the third composed almost exclusively of bears from European Russia. Four haplotypes from Finland and European Russia matched exactly with haplotypes from Slovakia, suggesting the significance of the current territory of Slovakia in ancient demographic processes of brown bears. Based on the results of this study and those from the recent literature, we hypothesize that the West Carpathian Mountains have served either as one of the northernmost refuge areas or as an important movement corridor for brown bears of the Eastern lineage towards northern Europe during or after the last ice age. Bayesian analyses were performed to investigate the temporal framework of brown bear lineages in Europe. The molecular clock was calibrated using Beringian brown bear sequences derived from radiocarbon-dated ancient samples, and the estimated mutation rate was 29.8% (13.3%-47.6%) per million years. The whole European population and Western and Eastern lineages formed about 175,000, 70,000 and 25,000 years before present, respectively. Our approach to estimating the time frame of brown bear evolution demonstrates the importance of using an appropriate mutation rate, and this has implications for other studies of Pleistocene populations.     

Comparison of horse Chromosome 3 with donkey and human chromosomes by cross-species painting and heterologous FISH mapping

The melanocortin 1 receptor (MC1R), mast/stem cell growth factor receptor (KIT), and platelet-derived growth factor receptor α (PDGFRA) are loci that all belong to equine linkage group 2 (LG2). Of these, KIT was fluorescent in situ hybridization (FISH) mapped to ECA3q21 with equine cDNA and heterologous porcine BAC probes, while MC1R was localized to ECA3p12 and PDGFRA to ECA3q21 with heterologous porcine BAC probes. A three-step comparison between ECA3 and donkey chromosomes was carried out. First, microdissected ECA3 painting probe was used on donkey chromosomes, which showed disruption of the equine synteny. Next, human (HSA) Chromosomes (Chrs) 16q and 4 specific paints, known to be homologous to ECA3p and 3q, respectively, were applied to detect homologous chromosomal segment(s) in donkey. Finally, four genes (MC1R, ALB, PDGFRA, KIT) and two equine microsatellite markers (SGCV18 and SGCV33) located on ECA3 were FISH mapped to donkey chromosomes. The findings refined the cross species painting homology results and added six new markers to the nascent donkey gene map. The hypothesis that Tobiano coat color in horses may be associated with a chromosomal inversion involving genes within LG2 was tested by G-banding-based cytogenetic analysis and ordering of four loci—KIT, PDGFRA, albumin (ALB), and MC1R—in Tobiano and non-tobiano (homozygous as well as heterozygous) horses. However, no difference either in banding patterns or location/relative order of the genes was observed in the three classes. The study highlights successful FISH mapping of BAC probes across evolutionarily diverged species, viz., pig and horse/donkey, and represents the first use of large-sized individual clones across distantly related farm animals. Received: 2 September 1998 / Accepted: 20 October 1998     

Fruiting body-guided molecular identification of root-tip mantle mycelia provides strong indications of ectomycorrhizal associations in two species of Sistotrema (Basidiomycota)

Fruiting body guided sequence analysis of mycorrhizal root-tip mycelia is a powerful yet relatively sparsely explored method for species-level identification of mycorrhizal fungi. It is used in this study to indicate mycorrhizal associations in the corticioid (resupinate) genus Sistotrema of the cantharelloid clade through phylogenetic analysis of the ITS and nuLSU rDNA regions of two spatiotemporally co-occurring Sistotrema fruiting bodies and ectomycorrhizal root tips. The genus Sistotrema is confirmed to be polyphyletic, and the mycorrhizal species form a strongly supported monophyletic clade together with the stipitate genus Hydnum. The remaining lineages of Sistotrema may well be saprotrophic, the nutritional mode traditionally attributed to the genus, but the phylogenetic analyses show that they should be excluded from Sistotrema. The cantharelloid clade contains several mycorrhizal genera, but no symbiotic associations have previously been demonstrated for Sistotrema.     

Genetics of Sex-linked yellow in the Syrian Hamster

Alternating patches of black and yellow pigment are a ubiquitous feature of mammalian color variation that contributes to camouflage, species recognition, and morphologic diversity. X-linked determinants of this pattern—recognized by variegation in females but not in males—have been described in the domestic cat as Orange, and in the Syrian hamster as Sex-linked yellow (Sly), but are curiously absent from other vertebrate species. Using a comparative genomic approach, we develop molecular markers and a linkage map for the euchromatic region of the Syrian hamster X chromosome that places Sly in a region homologous to the centromere-proximal region of human Xp. Comparison to analogous work carried out for Orange in domestic cats indicates, surprisingly, that the cat and hamster mutations lie in nonhomologous regions of the X chromosome. We also identify the molecular cause of recessively inherited black coat color in hamsters (historically referred to as nonagouti) as a Cys115Tyr mutation in the Agouti gene. Animals doubly mutant for Sly and nonagouti exhibit a Sly phenotype. Our results indicate that Sly represents a melanocortin pathway component that acts similarly to, but is genetically distinct from, Mc1r and that has implications for understanding both the evolutionary history and the mutational mechanisms of pigment-type switching.