Age at puberty and estrous activity of straightbred and reciprocal crossbred gilts

Age at puberty, estrous activity and growth were evaluated in 294 straightbred and reciprocal crossbred gilts maintained in confinement conditions. Trial 1 used straightbred Landrace (L), Yorkshire (Y) and reciprocal crossbred (LY, YL) gilts. Trial 2 used LY and YL gilts, and Trial 3 used L and Duroc (D) reciprocal crossbred gilts (LD, DL). Daily observations for estrous activity with a mature boar were initiated at 150 days of age and continued until gilts reached 255 days of age. Gilts not exhibiting estrus by 255 days of age were examined by laparoscopy to determine reproductive status. In Trial 1, age at puberty was greater for Y gilts compared to L and reciprocal crossbred gilts. Differences between reciprocal crosses for age at puberty were not significant in any trial. In Trial 1, the percentage of gilts exhibiting regular estrous cycles at 195 days of age was lower for Y gilts than for the other three groups of gilts; however, by 225 days of age, this difference was not significant. In all trials, reciprocal crossbred gilts did not differ with respect to percentage exhibiting regular estrous cycles or in percentage prepubertal. In Trial 1, Y gilts had a higher percentage prepubertal at 255 days of age than the reciprocal crossbred gilts. The percentage of gilts exhibiting behavioral anestrus at 255 days of age did not differ among breed groups within any trial. Based on these observations, we concluded the breed maternal effects were small or nonexistent relative to direct additive and nonadditive genetic effects in the breed combinations that were investigated.     

Low functional redundancy among mammalian browsers in regulating an encroaching shrub (Solanum campylacanthum) in African savannah

Large herbivorous mammals play an important role in structuring African savannahs and are undergoing widespread population declines and local extinctions, with the largest species being the most vulnerable. The impact of these declines on key ecological processes hinges on the degree of functional redundancy within large-herbivore assemblages, a subject that has received little study. We experimentally quantified the effects of three browser species (elephant, impala and dik-dik) on individual- and population-level attributes of Solanum campylacanthum (Solanum incanum sensu lato), an encroaching woody shrub, using semi-permeable exclosures that selectively removed different-sized herbivores. After nearly 5 years, shrub abundance was lowest where all browser species were present and increased with each successive species deletion. Different browsers ate the same plant species in different ways, thereby exerting distinct suites of direct and indirect effects on plant performance and density. Not all of these effects were negative: elephants and impala also dispersed viable seeds and indirectly reduced seed predation by rodents and insects. We integrated these diffuse positive effects with the direct negative effects of folivory using a simple population model, which reinforced the conclusion that different browsers have complementary net effects on plant populations, and further suggested that under some conditions, these net effects may even differ in direction.     

Fucci2a: A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.     

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice

Drinking in the dark (DID) is a limited access ethanol‐drinking phenotype in mice. High Drinking in the Dark (HDID‐1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4‐h period of access to 20% ethanol. A second replicate line (HDID‐2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID‐1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h² = 0.09) but the lines have shown 4–5 fold increases in BEC since S0; 80% of HDID‐1 and 60% of HDID‐2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID‐1 and 60% in HDID‐2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol .     

Metastatic tumor evolution and organoid modeling implicate TGFBR2 as a cancer driver in diffuse gastric cancer

Background

Gastric cancer is the second-leading cause of global cancer deaths, with metastatic disease representing the primary cause of mortality. To identify candidate drivers involved in oncogenesis and tumor evolution, we conduct an extensive genome sequencing analysis of metastatic progression in a diffuse gastric cancer. This involves a comparison between a primary tumor from a hereditary diffuse gastric cancer syndrome proband and its recurrence as an ovarian metastasis.

Results

Both the primary tumor and ovarian metastasis have common biallelic loss-of-function of both the CDH1 and TP53 tumor suppressors, indicating a common genetic origin. While the primary tumor exhibits amplification of the Fibroblast growth factor receptor 2 (FGFR2) gene, the metastasis notably lacks FGFR2 amplification but rather possesses unique biallelic alterations of Transforming growth factor-beta receptor 2 (TGFBR2), indicating the divergent in vivo evolution of a TGFBR2-mutant metastatic clonal population in this patient. As TGFBR2 mutations have not previously been functionally validated in gastric cancer, we modeled the metastatic potential of TGFBR2 loss in a murine three-dimensional primary gastric organoid culture. The Tgfbr2 shRNA knockdown within Cdh1^-/-; Tp53^-/- organoids generates invasion in vitro and robust metastatic tumorigenicity in vivo, confirming Tgfbr2 metastasis suppressor activity.

Conclusions

We document the metastatic differentiation and genetic heterogeneity of diffuse gastric cancer and reveal the potential metastatic role of TGFBR2 loss-of-function. In support of this study, we apply a murine primary organoid culture method capable of recapitulating in vivo metastatic gastric cancer. Overall, we describe an integrated approach to identify and functionally validate putative cancer drivers involved in metastasis.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0428-9) contains supplementary material, which is available to authorized users.     

Xeroderma pigmentosum p48 gene enhances global genomic repair and suppresses UV-induced mutagenesis

UV-damaged DNA-binding activity (UV-DDB) is deficient in some xeroderma pigmentosum group E individuals due to mutation of the p48 gene, but its role in DNA repair has been obscure. We found that UV-DDB is also deficient in cell lines and primary tissues from rodents. Transfection of p48 conferred UV-DDB to hamster cells, and enhanced removal of cyclobutane pyrimidine dimers (CPDs) from genomic DNA and from the nontranscribed strand of an expressed gene. Expression of p48 suppressed UV-induced mutations arising from the nontranscribed strand, but had no effect on cellular UV sensitivity. These results define the role of p48 in DNA repair, demonstrate the importance of CPDs in mutagenesis, and suggest how rodent models can be improved to better reflect cancer susceptibility in humans.     

Structural and functional characterization of the upstream regulatory region of the human gene encoding prostate apoptosis response factor-4

Prostate apoptosis response factor-4 (Par-4) is critical to cell growth and apoptosis. Induction of Par-4 expression has been shown to be required for apoptosis in a diversity of cellular systems, including neurons. Neuronal populations in individuals with degenerative disorders show elevated levels of Par-4 protein in advance of cellular and functional loss. To understand the regulation of par-4 expression, we isolated and characterized 5.7 kb of the human par-4 promoter. We demonstrated that the isolated promoter was functional. Similar to the endogenous par-4 gene, par-4 expression could be induced upon apoptotic insult with thapsigargin following introduction of the promoter DNA into human A375 cells. Also, increased levels of the atypical protein kinase C, zetaPKC, was shown to negatively regulate expression from the ectopic par-4 promoter. A 550 bp sequence immediately upstream to the 5'-untranslated region of the gene was found to be responsible for par-4 promoter induction to apoptosis by thapsigargin.     

The Genome of Thermosipho africanus TCF52B: Lateral Genetic Connections to the Firmicutes and Archaea

Lateral gene transfers (LGT) (also called horizontal gene transfers) have been a major force shaping the Thermosipho africanus TCF52B genome, whose sequence we describe here. Firmicutes emerge as the principal LGT partner. Twenty-six percent of phylogenetic trees suggest LGT with this group, while 13% of the open reading frames indicate LGT with Archaea.Thermosipho africanus TCF52B was isolated from produced fluids of a high-temperature oil reservoir in the North Sea using fish waste as the only substrate (4). Phylogenetic analyses based on the 16S rRNA gene sequence and DNA-DNA hybridization placed it as a strain of Thermosipho africanus, which was first isolated from a shallow marine hydrothermal system in Djibouti, Africa (8, 21).The complete genome sequence of this strain was determined by the conventional whole-genome shotgun strategy. Genomic libraries containing 1- to 4-kb and 40-kb fragments were constructed, and sequence chromatograms were produced using a MegaBACE 1000 capillary DNA sequencer (GE Healthcare). Nucleotide skews were computed as described previously (11). Automated open reading frame (ORF) identification and annotation were performed using the annotation software Manatee made available by TIGR (23). Pseudogenes were identified by doing BLAST searches of neighboring ORFs with the same or similar annotations and by using the program Psi-phi (9, 10), and clustered regularly interspaced short palindromic repeat loci (CRISPRs) were identified using the web site http://crispr.u-psud.fr/crispr/CRISPRHomePage.php with the default parameters (6). Maximum-likelihood (ML) trees (WAG [Γ+Ι model, four categories]) were constructed from protein-coding ORFs using PHYML and the PhyloGenie package (5). Recently, several Thermotogales genomes have become available in GenBank. As these genomes had not been published yet, we did not include them in any “genome-scale” analyses (i.e., the phylogenetic analyses). We did, however, include them in the BLAST analyses of mobile Thermosipho africanus genes.The genome of Thermosipho africanus strain TCF52B is a single circular chromosome consisting of 2,016,657 bp with an average G+C content of 30.8%. Strand asymmetries, such as GC skew and tetramer skews, are pronounced and show two clear singularity points, located at roughly 8 kb and 1033 kb from the +1 site (see Fig. S1 in the supplemental material). Since these two points are diametrically opposed on the circular chromosome, dividing it into two halves with opposite compositional skews, they make good candidates for the putative origin and termination of replication. The 1,033-kb region is likely to harbor the origin, since GC skew becomes positive past this location, as in most bacterial genomes with a known origin.The genome contains 2,000 potential coding sequences, of which 1913 are putative protein-coding ORFs, 30 are putatively assigned as pseudogenes, and 57 encode RNA. A comparison to the genome of Thermotoga maritima is given in Table ​Table1.1. The Thermosipho africanus genome is about 156 kb larger than the Thermotoga maritima genome and carries 36 more ORFs. The genome contains duplicated regions comprising paralogous gene copies, CRISPRs, and mobile genetic elements, which collectively provide considerable indirect evidence for genomic instability and acquisition of exogenous genetic information.

TABLE 1.

General features of the Thermosipho africanus genome, with a comparison to Thermotoga maritima

Analysis of a diverse global Pisum sp. collection and comparison to a Chinese local P. sativum collection with microsatellite markers

Twenty-one informative microsatellite loci were used to assess and compare the genetic diversity among Pisum genotypes sourced from within and outside China. The Chinese germplasm comprised 1243 P. sativum genotypes from 28 provinces and this was compared to 774 P. sativum genotypes that represented a globally diverse germplasm collection, as well as 103 genotypes from related Pisum species. The Chinese P. sativum germplasm was found to contain genotypes genetically distinct from the global gene pool sourced outside China. The Chinese spring type genotypes were separate from the global gene pool and from the other main Chinese gene pool of winter types. The distinct Chinese spring gene pool comprised genotypes from Inner Mongolia and Sha'anxi provinces, with those from Sha'anxi showing the greatest diversity. The other main gene pool within China included both spring types from other northern provinces and winter types from central and southern China, plus some accessions from Inner Mongolia and Sha'anxi. A core collection of Chinese landraces chosen to represent molecular diversity was compared both to the wider Chinese collection and to a geographically diverse core collection of Chinese landraces. The average gene diversity and allelic richness per locus of both the micro-satellite based core and the wider collection were similar, and greater than the geographically diverse core. The genetic diversity of P. sativum within China appears to be quite different to that detected in the global gene pool, including the presence of several rare alleles, and may be a useful source of allelic variation for both major gene and quantitative traits.