Orphan nuclear receptor LRH-1 is required to maintain Oct4 expression at the epiblast stage of embryonic development

Oct4 plays an essential role in maintaining the inner cell mass and pluripotence of embryonic stem (ES) cells. The expression of Oct4 is regulated by the proximal enhancer and promoter in the epiblast and by the distal enhancer and promoter at all other stages in the pluripotent cell lineage. Here we report that the orphan nuclear receptor LRH-1, which is expressed in undifferentiated ES cells, can bind to SF-1 response elements in the proximal promoter and proximal enhancer of the Oct4 gene and activate Oct4 reporter gene expression. LRH-1 is colocalized with Oct4 in the inner cell mass and the epiblast of embryos at early developmental stages. Disruption of the LRH-1 gene results in loss of Oct4 expression at the epiblast stage and early embryonic death. Using LRH-1(-/-) ES cells, we also show that LRH-1 is required to maintain Oct4 expression at early differentiation time points. In vitro and in vivo results show that LRH-1 plays an essential role in the maintenance of Oct4 expression in ES cells at the epiblast stage of embryonic development, thereby maintaining pluripotence at this crucial developmental stage prior to segregation of the primordial germ cell lineage at gastrulation.     

Molecular cloning and characterization of AAAS-V2, a novel splice variant of human AAAS

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Much initial molecular analysis supported that Triple-A syndrome was caused by mutations in AAAS, a WD-repeat protein gene. Here we report cloning and characterization of a novel splice variant of human AAAS, which we named AAAS-v2, which is located on the human chromosome 12p13. The cDNA is 1703bp, encoding a 513-amino acid polypeptide, which contains three WD40 domains, one less than the original which we called AAAS-v1 (Gen Bank: NM_015665.3). RT-PCR analysis in our work revealed that AAAS-v2 and AAAS-v1 were ubiquitously detected in human multiple tissue cDNA (MTC) panels (CLONTECH).The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY237818.Xin Li: These two authors contributed equally to this paper.Chaoneng Ji: These two authors contributed equally to this paper.     

Simultaneous Monitoring of Phosphine and of Phosphorus Species in Taihu Lake Sediments and Phosphine Emission from Lake Sediments

Phosphine (PH₃) was monitored in the Taihu Lake in China by a GC/NPD method, coupled with cryo-trapping enrichment technology. Results showed that PH₃ was universally detected in sediments, lake water and atmosphere of the Taihu Lake area. Total phosphorus (TP_s) and fractions of different phosphorus species in lake sediments were separately measured as dissolved phosphate (DP), phosphorus bound to aluminum (Al-P), iron (Fe-P) and calcium (Ca-P), occluded phosphorus (OP), and organic phosphorus (Org-P) by sequential chemical extraction. High PH₃ levels were correlated with high TP_s values in sediments and with eutrophication at different sites. In addition, a positive linear correlation equation was obtained between the concentrations of PH₃ in lake sediments and of the phosphorus fractions. The resulting multiple linear regression equation is PH₃ = −165 + 63.3 DP + 0.736 Al-P + 2.33 Ca-P + 2.29 Org-P. The flux of PH₃ across the sediment–water interface was estimated from sediment core incubation in May and October 2002. The annual average sediment–water flux of PH₃ was estimated at ca. 0.0138±0.005 pg dm⁻² h⁻¹, the average yearly emission value of PH₃ from Taihu Lake sediments to water was calculated to be 28.3±10.2 g year⁻¹, which causes a water PH₃ concentration of up to 0.178±0.064 pmol dm⁻³. The real importance of PH₃ could be higher, because PH₃ could be consumed in the oxic sediment–water boundary layer and in the water column. Spatial and temporal distributions of total phosphorus (TP_w) and chlorophyll a (Chl-a) in the water column of Taihu Lake were measured over the study period. Higher water PH₃ has also been found where the TP_w content was high. Similarly, high Chl-a was consistent with higher water PH₃. Positive relationships between PH₃ and TP_w (average R² = 0.47±0.26) and Chl-a (average R² = 0.23±0.31) were observed in Taihu Lake water.     

A genome scan with AFLP markers to detect fearfulness-related QTLs in Japanese quail

A quantitative trait loci (QTL) study was undertaken to identify genome regions involved in the control of fearfulness in Japanese quail (Coturnix japonica). An F2 cross was made between two quail lines divergently selected over 29 generations on duration of tonic immobility (DTI), a catatonic-like state of reduced responsiveness to a stressful stimulation. A total of 1065 animals were measured for the logarithm of DTI (LOGTI), the number of inductions (NI) necessary to induce the immobility reaction, open-field behaviour including locomotor activity (MOVE), latency before first movement (LAT), number of jumps (JUMP), dejections (DEJ) and shouts (SHOUT), corticosterone level after a contention stress (LOGCORT) and body weight at 2 weeks of age (BW2). A total of 310 animals were included in a genome scan using selective genotyping with 248 AFLP markers. A total of 21 suggestive or genome-wide significant QTL were observed. Two highly significant QTL were identified on linkage group 1 (GL1), one for LOGTI and one for NI. In the vicinity of the QTL for LOGTI, a nearly significant QTL for SHOUT and a suggestive QTL for LAT were also identified. On GL3, genome-wide significant QTL were observed for JUMP and DEJ as well as suggestive QTL for LOGTI, MOVE, SHOUT and LAT. A significant QTL for BW2 was observed on GL2 and a nearly significant one on GL1. These results may be useful in the understanding of fearfulness in quail and related species provided that fearfulness has the same genetic basis.     

Evolution of Plant-Like Crystalline Storage Polysaccharide in the Protozoan Parasite Toxoplasma gondii Argues for a Red Alga Ancestry

Single-celled apicomplexan parasites are known to cause major diseases in humans and animals including malaria, toxoplasmosis, and coccidiosis. The presence of apicoplasts with the remnant of a plastid-like DNA argues that these parasites evolved from photosynthetic ancestors possibly related to the dinoflagellates. Toxoplasma gondii displays amylopectin-like polymers within the cytoplasm of the dormant brain cysts. Here we report a detailed structural and comparative analysis of the Toxoplasma gondii, green alga Chlamydomonas reinhardtii, and dinoflagellate Crypthecodinium cohnii storage polysaccharides. We show Toxoplasma gondii amylopectin to be similar to the semicrystalline floridean starch accumulated by red algae. Unlike green plants or algae, the nuclear DNA sequences as well as biochemical and phylogenetic analysis argue that the Toxoplasma gondii amylopectin pathway has evolved from a totally different UDP-glucose-based metabolism similar to that of the floridean starch accumulating red alga Cyanidioschyzon merolae and, to a lesser extent, to those of glycogen storing animals or fungi. In both red algae and apicomplexan parasites, isoamylase and glucan–water dikinase sequences are proposed to explain the appearance of semicrystalline starch-like polymers. Our results have built a case for the separate evolution of semicrystalline storage polysaccharides upon acquisition of photosynthesis in eukaryotes.This article contains online-only supplementary material.Reviewing Editor:Dr. Patrick Keeling     

A triple mutation in the second transmembrane domain of mouse dopamine transporter markedly decreases sensitivity to cocaine and methylphenidate

Previously, we reported that Phe105 in transmembrane domain 2 of the mouse dopamine transporter (DAT) is crucial for high-affinity cocaine binding. In the current study, we investigated whether other residues surrounding Phe105 also affect the potency of cocaine inhibition. After three rounds of sequential random mutagenesis at these residues, we found a triple mutant (L104V, F105C and A109V) of mouse DAT that retained over 50% uptake activity and was 69-fold less sensitive to cocaine inhibition when compared with the wild-type mouse DAT. The triple mutation also resulted in a 47-fold decrease in sensitivity to methylphenidate inhibition, suggesting that the binding sites for cocaine and methylphenidate may overlap. In contrast, the inhibition of dopamine uptake by amphetamine or methamphetamine was not significantly changed by the mutations, suggesting that the binding sites for the amphetamines differ from those for cocaine and methylphenidate. Such functional but cocaine-insensitive DAT mutants can be used to generate a knock-in mouse line to study the role of DAT in cocaine addiction.     

Delimiting a rice wide-compatibility gene <Emphasis Type="Italic">S</Emphasis><Stack><Subscript>5</Subscript><Superscript><Emphasis Type="Italic">n</Emphasis></Superscript></Stack> to a 50 kb region

Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S ₅ ⁿ gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S ₅ ⁿ gene. In the first-pass mapping, the S ₅ ⁿ gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S ₅ region. In the fine mapping, the S ₅ region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S ₅ ⁿ gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S ₅ region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S ₅ ⁿ gene. Qing Ji and Jufei Lu have contributed equally to this paper.     

Identification of SCAR markers linked to Rca2 anthracnose resistance gene and their assessment in strawberry germplasm

Bulked segregant analysis combined with AFLPs was used to identify molecular markers linked to the Rca2 gene conferring resistance to Colletotrichum acutatum pathogenicity group 2 which causes anthracnose in the octoploid strawberry Fragaria × ananassa. DNA bulks originating from a cross between the resistant cultivar ‘Capitola’ and the susceptible cultivar ‘Pajaro’ were screened with 110 EcoRI/MseI AFLP combinations. Four AFLP markers were found linked in coupling phase to Rca2 with recombination percentages between 0% and 17.7%. Among the four markers linked to the resistance gene, two were converted into SCAR markers (STS-Rca2_417 and STS-Rca2_240) and screened in a large segregating population including 179 genotypes. The Rca2 resistance gene was estimated to be 0.6 cM from STS-Rca2_417 and 2.8 cM from STS-Rca2_240. The presence/absence of the two SCAR markers was further studied in 43 cultivars of F. × ananassa, including 14 susceptible, 28 resistant, and one intermediate genotype. Results showed that 81.4% and 62.8% of the resistant/susceptible genotypes were correctly predicted by using STS-Rca2_417 and STS-Rca2_240, respectively. The 14 susceptible genotypes showed no amplification for either SCARs. These developed SCARs constitute new tools for indirect selection criteria of anthracnose resistance genotypes in strawberry breeding programs.     

Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system

Removal of a selectable marker gene from genetically modified (GM) crops alleviates the risk of its release into the environment and hastens the public acceptance of GM crops. Here we report the production of marker-free transgenic rice by using a chemically regulated, Cre/loxP-mediated site-specific DNA recombination in a single transformation. Among 86 independent transgenic lines, ten were found to be marker-free in the T₀ generation and an additional 17 lines segregated marker-free transgenic plants in the T₁ generation. Molecular and genetic analyses indicated that the DNA recombination and excision in transgenic rice were precise and the marker-free recombinant T-DNA was stable and heritable.The first two authors contributed equally to the work     

The comparison of the confidence regions in phylogeny

In this paper, several different procedures for constructing confidence regions for the true evolutionary tree are evaluated both in terms of coverage and size without considering model misspecification. The regions are constructed on the basis of tests of hypothesis using six existing tests: Shimodaira Hasegawa (SH), SOWH, star form of SOWH (SSOWH), approximately unbiased (AU), likelihood weight (LW), generalized least squares, plus two new tests proposed in this paper: single distribution nonparametric bootstrap (SDNB) and single distribution parametric bootstrap (SDPB). The procedures are evaluated on simulated trees both with small and large number of taxa. Overall, the SH, SSOWH, AU, and LW tests led to regions with higher coverage than the nominal level at the price of including large numbers of trees. Under the specified model, the SOWH test gives accurate coverage and relatively small regions. The SDNB and SDPB tests led to the small regions with occasional undercoverage. These two procedures have a substantial computational advantage over the SOWH test. Finally, the cutoff levels for the SDNB test are shown to be more variable than those for the SDPB test.