The gene for autosomal dominant polycystic kidney disease lies in a 750-kb CpG-rich region.

PKD1, the locus most commonly affected by mutations that produce autosomal dominant polycystic kidney disease (ADPKD), has previously been localized to chromosome 16p13.3. Since no cytogenetic abnormalities have been found in association with ADPKD, flanking genetic markers have been required to define an interval--the PKD1 region--that contains the PKD1 gene. In this report we demonstrate, through the construction of a long-range restriction map that links the flanking genetic markers GGG1 (D16S84) and 26.6PROX (D16S125), that the PKD1 gene lies within an extremely CpG-rich 750-kb segment of chromosome 16p13.3. Approximately 90% of this region has been cloned in three extensive cosmid/bacteriophage contigs. The cloned DNA is a valuable resource for identifying new closer flanking genetic markers and for isolating candidate genes from the region.     

The pericentriolar material in Chinese hamster ovary cells nucleates microtubule formation

The structure and function of the centrosomes from Chinese hamster ovary (CHO) cells were investigated by electron microscopy of negatively stained wholemount preparations of cell lysates. Cells were trypsinized from culture dishes, lysed with Triton X-100, sedimented onto ionized, carbon-coated grids, and negatively stained with phosphotungstate. The centrosomes from both interphase and dividing cells consisted of pairs of centrioles, a fibrous pericentriolar material, and a group of virus-like particles which were characteristic of the CHO cells and which served as markers for the pericentriolar material. Interphase centrosomes anchored up to two dozen microtubules when cells were lysed under conditions which preserved native microtubules. When Colcemid-blocked mitotic cells, initially devoid of microtubules, were allowed to recover for 10 min, microtubules formed at the pericentriolar material, but not at the centrioles. When lysates of Colcemid-blocked cells were incubated in vitro with micotubule protein purified from porcine brain tissue, up to 250 microtubules assembled at the centrosomes, similar to the number of microtubules that would normally form at the centrosome during cell division. A few microtubules could also be assembled in vitro onto the ends of isolated centrioles from which the pericentriolar material had been removed, forming characteristic axoneme- like bundles. In addition, microtubules; were assembled onto fragments of densely staining, fibrous material which was tentatively identified as periocentriolar material by its association of CHO can initiate and anchor microtubules both in vivo and in vitro.     

The nucleotide sequence of the replication origin beta of the plasmid R6K

We h ave identified by molecular cloning a region of 283 base pairs of the HindIII 2 fragment of R6K which corresponds to the region of the replication origin beta. This 283 base-pair DNA fragment, when present contiguously with the structural gene for the replication initiation protein of R6K, encoded in the HindIII 9-15 and part of HindIII 2 restriction fragments, will support the replication of a plasmid chimera containing the pBR322 replicon in a pol Ats host at the nonpermissive temperature. The nucleotide sequence of the region of replication origin beta has been determined. The nucleotide sequence has some homology with the ori gamma region of R6K; it has a 15-base-pair homology with the replication origin of Escherichia coli.     

Landscape and organismal factors affecting sagebrush‐seedling transplant survival after megafire restoration

Larger and more frequent disturbances are motivating efforts to accelerate recovery of foundational perennial species by focusing efforts into establishing island patches to sustain keystone species and facilitate recovery of the surrounding plant community. Evaluating the variability in abiotic and biotic factors that contribute to differences in survival and establishment can provide useful insight into the relative importance of these factors. In the western United States, severe degradation of the sagebrush steppe has motivated substantial efforts to restore native perennial cover, but success has been mixed. In this study, we evaluated survival of more than 3,000 sagebrush seedlings transplanted on 12 patches totaling 650 ha within a 113,000 ha burn area, and related the survival to organismal and subtaxonomic traits, and to landscape variables. Big sagebrush has high intraspecific diversity attributed to subspecies and cytotypes identifiable through ultraviolet (UV)‐induced fluorescence, length:width of leaves, or genome size (ploidy). Of these organismal traits, survival was related only to UV fluorescence, and then only so when landscape variables were excluded from analyses. The most significant landscape variable affecting survival was soil taxonomic subgroup, with much lower survival where buried restrictive layers reduce deep water infiltration. Survival also decreased with greater slope steepness, exotic annual grass cover, and burn severity. Survival was optimal where perennial bunchgrasses comprised 8–14% of total cover. These soil, topographic, and community condition factors revealed through monitoring of landscape‐level treatments can be used to explain the success of plantings and to strategically plan future restoration projects.     

Greater sage‐grouse respond positively to intensive post‐fire restoration treatments

Habitat loss is the most prevalent threat to biodiversity in North America. One of the most threatened landscapes in the United States is the sagebrush (Artemisia spp.) ecosystem, much of which has been fragmented or converted to non‐native grasslands via the cheatgrass‐fire cycle. Like many sagebrush obligates, greater sage‐grouse (Centrocercus urophasianus) depend upon sagebrush for food and cover and are affected by changes to this ecosystem. We investigated habitat selection by 28 male greater sage‐grouse during each of 3 years after a 113,000‐ha wildfire in a sagebrush steppe ecosystem in Idaho and Oregon. During the study period, seeding and herbicide treatments were applied for habitat restoration. We evaluated sage‐grouse responses to vegetation and post‐fire restoration treatments. Throughout the 3 years post‐fire, sage‐grouse avoided areas with high exotic annual grass cover but selected strongly for recovering sagebrush and moderately strongly for perennial grasses. By the third year post‐fire, they preferred high‐density sagebrush, especially in winter when sagebrush is the primary component of the sage‐grouse diet. Sage‐grouse preferred forb habitat immediately post‐fire, especially in summer, but this selection preference was less strong in later years. They also selected areas that were intensively treated with herbicide and seeded with sagebrush, grasses, and forbs, although these responses varied with time since treatment. Wildfire can have severe consequences for sagebrush‐obligate species due to loss of large sagebrush plants used for food and for protection from predators and thermal extremes. Our results show that management efforts, including herbicide application and seeding of plants, directed at controlling exotic annual grasses after a wildfire can positively affect habitat selection by sage‐grouse.     

Bilineal disease and trans-heterozygotes in autosomal dominant polycystic kidney disease

In searching for a putative third gene for autosomal dominant polycystic kidney disease (ADPKD), we studied the genetic inheritance of a large family (NFL10) previously excluded from linkage to both the PKD1 locus and the PKD2 locus. We screened 48 members of the NFL10 pedigree, by ultrasonography, and genotyped them, with informative markers, at both the PKD1 locus and the PKD2 locus. Twenty-eight of 48 individuals assessed were affected with ADPKD. Inspection of the haplotypes of these individuals suggested the possibility of bilineal disease from independently segregating PKD1 and PKD2 mutations. Using single-stranded conformational analysis, we screened for and found a PKD2 mutation (i.e., 2152delA; L736X) in 12 affected pedigree members. Additionally, when the disease status of these individuals was coded as "unknown" in linkage analysis, we also found, with markers at the PKD1 locus, significant LOD scores (i.e., >3.0). These findings strongly support the presence of a PKD1 mutation in 15 other affected pedigree members, who lack the PKD2 mutation. Two additional affected individuals had trans-heterozygous mutations involving both genes, and they had renal disease that was more severe than that in affected individuals who had either mutation alone. This is the first documentation of bilineal disease in ADPKD. In humans, trans-heterozygous mutations involving both PKD1 and PKD2 are not necessarily embryonically lethal. However, the disease associated with the presence of both mutations appears to be more severe than the disease associated with either mutation alone. The presence of bilineal disease as a confounder needs to be considered seriously in the search for the elusive PKD3 locus.     

啤酒酵母菌的细胞融合

啤酒多倍体酵母菌原生质体已成功地与单倍体原生质体进行融合。经细胞壁再生后,稳定的融合重组体被分离出来。这些融合体的基因分析表明,融合体中含有双亲的基因型。孢子形成良好,且每个子囊中含有四个孢子,每个孢子确实是二倍体。这样原生质体融合就提供了一个对啤酒酿造酵母进行遗传分析的方法。但是如果没有一个方便的杂交技术,这个方法将是很困难的。     

Impaired glomerulogenesis and endothelial cell migration in Pkd1-deficient renal organ cultures

The PKD1 gene is essential for a number of biological functions, and its loss-of-function causes autosomal dominant polycystic kidney disease (ADPKD). The gene is developmentally regulated and believed to play an essential role in renal development. Previous studies have shown that manipulating murine renal organ cultures with dominant-negative forms of the Pkd1 gene impaired ureteric bud (UB) branching. In the current study, we analyzed different stages of renal development in two distinct mouse models carrying either a null mutation or inactivation of the last two exons of Pkd1. Surprisingly, metanephric explants from Pkd1-deleted kidneys harvested at day E11.5 did not show defects of UB branching and elongation, estimated by cytokeratin staining on fixed tissues or by Hoxb7-GFP time-lapse imaging. However, renal explants from Pkd1-mutants isolated at day E14.5 showed impaired nephrogenesis. Notably, we observed cell migratory defects in the developing endothelial compartment. Previous studies had implicated the Pkd1 gene in controlling cell migration and collagen deposition through PI3 kinases. In line with these studies, our results show that wild-type explants treated with PI3-kinase inhibitors recapitulate the endothelial defects observed in Pkd1 mutants, whereas treatment with VEGF only partially rescued the defects. Our data are consistent with a role for the Pkd1 gene in the endothelium that may be required for proper nephrogenesis.     

Prescribed fires in Artemisia tridentata ssp. vaseyana steppe have minor and transient effects on vegetation cover and composition

Question: What is the impact of prescribed fires on the cover and composition of vegetation in Artemisia tridentata ssp. vaseyana steppe? Location: United States Department of Agriculture, Agricultural Research Service, United States Sheep Experiment Station, eastern Idaho (44°14′44′’ N, 112°12′47′’ W). Methods: Multiple prescribed fires were lit in 2002 and 2003 in an Artemisia tridentata ssp. vaseyana (mountain big sagebrush) steppe ecosystem that was relatively free of exotic plants. Measurements of cover components and plant species frequencies were taken pre‐ and for 2 to 3 years post‐fire. Results: Cover of forbs and grasses returned to pre‐fire levels after two years. Shrub cover declined from 36 to 6% in the first year post‐fire. Fire reduced the frequencies of three species, A. tridentata ssp. vaseyana, Festuca idahoensis, and Cordylanthus ramosus, of rangeland plants. Frequencies of four plant species, Hesperostipa comata, Polygonum douglasii, Chenopodium fremontii and Chenopodium leptophyllum increased, but only P. douglasii increased for more than a year. Conclusion: This study demonstrates that in an Artemisia tridentata ssp. vaseyana steppe ecosystem without significant non‐native species or anthropogenic disturbances vegetative cover and species composition of the herbaceous community are only minimally altered by fire. The herbaceous component returned to pre‐fire conditions within three years of a fire.     

Variability of Cenococcum colonization and its ecophysiological significance for young conifers at alpine-treeline

* Plants establishing in environments that are marginal for growth could be particularly sensitive to mycorrhizal associations. We investigated ectomycorrhizal colonization and its significance for young conifers growing at, or above, their normal limits for growth, in the alpine-treeline ecotone. * Colonization of seedlings (<1 yr old) and juveniles (2- to 10-yr-old) of Picea engelmannii and Abies lasiocarpa by Cenococcum geophilum was determined in a field study, and effects of Cenococcum on Picea seedling ecophysiology were investigated in a glasshouse. * Colonization by Cenococcum was c. 20-fold greater for juveniles than seedlings, and approximately 4-fold greater adjacent compared with approximately 7 m away from trees. Juveniles of Picea were more colonized at timberline than Abies, and the opposite relationship was observed in forest. Colonization enhanced seedling water potential, but not phosphorus concentrations or photosynthesis. * These landscape and age-dependent variations in colonization correspond well with known variations in conifer physiology and establishment near timberline. Facilitation of seedling establishment by older trees at alpine-treeline may include a below-ground, mycorrhizal component that complements previously reported effects of trees on the microclimate and ecophysiology of seedlings.