Identification of nutrient partitioning genes participating in rice grain filling by singular value decomposition (SVD) of genome expression data

Background

In order to identify rice genes involved in nutrient partitioning, microarray experiments have been done to quantify genomic scale gene expression. Genes involved in nutrient partitioning, specifically grain filling, will be used to identify other co-regulated genes, and DNA binding proteins. Proper identification of the initial set of bait genes used for further investigation is critical. Hierarchical clustering is useful for grouping genes with similar expression profiles, but decreases in utility as data complexity and systematic noise increases. Also, its rigid classification of genes is not consistent with our belief that some genes exhibit multifaceted, context dependent regulation.

Results

Singular value decomposition (SVD) of microarray data was investigated as a method to complement current techniques for gene expression pattern recognition. SVD's usefulness, in finding likely participants in grain filling, was measured by comparison with results obtained previously via clustering. 84 percent of these known grain-filling genes were re-identified after detailed SVD analysis. An additional set of 28 genes exhibited a stronger grain-filling pattern than those grain-filling genes that were unselected. They also had upstream sequence containing motifs over-represented among grain filling genes.

Conclusions

The pattern-based perspective that SVD provides complements to widely used clustering methods. The singular vectors provide information about patterns that exist in the data. Other aspects of the decomposition indicate the extent to which a gene exhibits a pattern similar to those provided by the singular vectors. Thus, once a set of interesting patterns has been identified, genes can be ranked by their relationship with said patterns.

     

Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes

The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.     

Quaternary organization of the goodpasture autoantigen,the alpha 3(IV) collagen chain. Sequestration of two cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains

Goodpasture's (GP) disease is caused by autoantibodies that target the alpha3(IV) collagen chain in the glomerular basement membrane (GBM). Goodpasture autoantibodies bind two conformational epitopes (E(A) and E(B)) located within the non-collagenous (NC1) domain of this chain, which are sequestered within the NC1 hexamer of the type IV collagen network containing the alpha3(IV), alpha4(IV), and alpha5(IV) chains. In this study, the quaternary organization of these chains and the molecular basis for the sequestration of the epitopes were investigated. This was accomplished by physicochemical and immunochemical characterization of the NC1 hexamers using chain-specific antibodies. The hexamers were found to have a molecular composition of (alpha3)(2)(alpha4)(2)(alpha5)(2) and to contain cross-linked alpha3-alpha5 heterodimers and alpha4-alpha4 homodimers. Together with association studies of individual NC1 domains, these findings indicate that the alpha3, alpha4, and alpha5 chains occur together in the same triple-helical protomer. In the GBM, this protomer dimerizes through NC1-NC1 domain interactions such that the alpha3, alpha4, and alpha5 chains of one protomer connect with the alpha5, alpha4, and alpha3 chains of the opposite protomer, respectively. The immunodominant Goodpasture autoepitope, located within the E(A) region, is sequestered within the alpha3alpha4alpha5 protomer near the triple-helical junction, at the interface between the alpha3NC1 and alpha5NC1 domains, whereas the E(B) epitope is sequestered at the interface between the alpha3NC1 and alpha4NC1 domains. The results also reveal the network distribution of the six chains of collagen IV in the renal glomerulus and provide a molecular explanation for the absence of the alpha3, alpha4, alpha5, and alpha6 chains in Alport syndrome.     

Trophic interactions and population growth rates: describing patterns and identifying mechanisms

While the concept of population growth rate has been of central importance in the development of the theory of population dynamics, few empirical studies consider the intrinsic growth rate in detail, let alone how it may vary within and between populations of the same species. In an attempt to link theory with data we take two approaches. First, we address the question ''what growth rate patterns does theory predict we should see in time-series?'' The models make a number of predictions, which in general are supported by a comparative study between time-series of harvesting data from 352 red grouse populations. Variations in growth rate between grouse populations were associated with factors that reflected the quality and availability of the main food plant of the grouse. However, while these results support predictions from theory, they provide no clear insight into the mechanisms influencing reductions in population growth rate and regulation. In the second part of the paper, we consider the results of experiments, first at the individual level and then at the population level, to identify the important mechanisms influencing changes in individual productivity and population growth rate. The parasitic nematode Trichostrongylus tenuis is found to have an important influence on productivity, and when incorporated into models with their patterns of distribution between individuals has a destabilizing effect and generates negative growth rates. The hypothesis that negative growth rates at the population level were caused by parasites was demonstrated by a replicated population level experiment. With a sound and tested model framework we then explore the interaction with other natural enemies and show that in general they tend to stabilize variations in growth rate. Interestingly, the models show selective predators that remove heavily infected individuals can release the grouse from parasite-induced regulation and allow equilibrium populations to rise. By contrast, a tick-borne virus that killed chicks simply leads to a reduction in the equilibrium. When humans take grouse they do not appear to stabilize populations and this may be because many of the infective stages are available for infection before harvesting commences. In our opinion, an understanding of growth rates and population dynamics is best achieved through a mechanistic approach that includes a sound experimental approach with the development of models. Models can be tested further to explore how the community of predators and others interact with their prey.     

Rapid exchange of mammalian topoisomerase II alpha at kinetochores and chromosome arms in mitosis

A stable cell line (GT2-LPk) derived from LLC-Pk was created in which endogenous DNA topoisomerase II alpha (topoII alpha) protein was downregulated and replaced by the expression of topoII alpha fused with enhanced green fluorescent protein (EGFP-topoII alpha). The EGFP-topoII alpha faithfully mimicked the distribution of the endogenous protein in both interphase and mitosis. In early stages of mitosis, EGFP-topoII alpha accumulated at kinetochores and in axial lines extending along the chromosome arms. During anaphase, EGFP-topoII alpha diminished at kinetochores and increased in the cytoplasm with a portion accumulating into large circular foci that were mobile and appeared to fuse with the reforming nuclei. These cytoplasmic foci appearing at anaphase were coincident with precursor organelles of the reforming nucleolus called nucleolus-derived foci (NDF). Photobleaching of EGFP-topoII alpha associated with kinetochores and chromosome arms showed that the majority of the protein rapidly exchanges (t1/2 of 16 s). Catalytic activity of topoII alpha was essential for rapid dynamics, as ICRF-187, an inhibitor of topoII alpha, blocked recovery after photobleaching. Although some topoII alpha may be stably associated with chromosomes, these studies indicate that the majority undergoes rapid dynamic exchange. Rapid mobility of topoII alpha in chromosomes may be essential to resolve strain imparted during chromosome condensation and segregation.     

Complete one-stage,immediate breast reconstruction with prosthetic material in patients with large or ptotic breasts

Immediate prosthetic breast reconstruction is a relatively simple, quick procedure with no donor site morbidity. This report discusses immediate one-stage breast reconstruction using prostheses in 18 patients (19 breasts) who also required a contralateral reduction or mastopexy. In all cases, an inverted-T pattern was applied to both breasts. The mean age of the patients was 49 years (range, 32 to 62 years), and the mean size of the gel implant used was 330 ml (range, 120 to 550 ml); the implant was inserted in a total submuscular pocket in seven patients and subcutaneously in 11 patients. In two patients with multiple risk factors, the prosthesis extruded, and one patient required removal for a periprosthetic infection. In 10 patients with early stage disease (T1 or T2) with tumors more than 5 cm from the nipple-areola complex, the original areola (n = 3) or nipple-areola complex (n = 7) was retained as a full-thickness skin graft.The breast shape after submuscular prosthesis insertion is different than that of the contralateral breast after a mastopexy or reduction, and nipple-areola complex symmetry was difficult to obtain; thus, this technique was abandoned in favor of the subcutaneous position (using a modified Wise keyhole pattern with a de-epithelialized portion, which still allows two-layer closure).In the subgroup of patients with large breasts or marked ptosis, a single-stage breast reconstruction procedure can be performed with symmetrical incisions. The subcutaneous position allows for symmetrical shape and nipple-areola complex symmetry to be obtained. When the tumors are small and situated in the periphery of the breast, the nipple-areola complex may be retained as a full-thickness graft.     

Inferences about human demography based on multilocus analyses of noncoding sequences

Data from 10 unlinked autosomal noncoding regions, resequenced in 15 individuals from each of three populations, were used in a multilocus analysis to test models of human demography. Each of the 10 regions consisted of approximately 2500 bp. The multilocus analysis, based on summary statistics (average and variance of Tajima's D and Fu and Li's D*), was used to test a family of models with recent population expansion. The African sample (Hausa of Cameroon) is compatible with a constant population size model and a range of models with recent expansion. For this population sample, we estimated confidence sets that showed the limited range of parameter values compatible with growth. For an exponential growth rate as low as 1 x 10(-3)/generation, population growth is unlikely to have started prior to 50,000 years ago. For higher growth rates, the onset of growth must be more recent. On the basis of the average value of Tajima's D, our sample from an Italian population was found to be incompatible with a constant population size model or any simple expansion model. In the Chinese sample, the variance of Tajima's D was too large to be compatible with the constant population size model or any simple expansion model.     

A missense mutation (R565W) in cirhin (FLJ14728) in North American Indian childhood cirrhosis

North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology. It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension. Clinical and physiological investigations have not revealed the underlying cause of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease. We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22. Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein. Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds.     

Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein

To design anti-nucleocapsid drugs, it is useful to know the affinities the protein has for its natural substrates under physiological conditions. Dissociation equilibrium constants are reported for seven RNA stem-loops bound to the mature HIV-1 nucleocapsid protein, NCp7. The loops include SL1, SL2, SL3, and SL4 from the major packaging domain of genomic RNA. The binding assay is based on quenching the fluorescence of tryptophan-37 in the protein by G residues in the single-stranded loops. Tightly bound RNA molecules quench nearly all the fluorescence of freshly purified NCp7 in 0.2 M NaCl. In contrast, when the GGAG-tetraloop of tight-binding SL3 is replaced with UUCG or GAUA, quenching is almost nil, indicating very low affinity. Interpreting fluorescence titrations in terms of a rapidly equilibrating 1:1 complex explains nearly all of the experimental variance for the loops. Analyzed in this way, the highest affinities are for 20mer SL3 and 19mer SL2 hairpin constructs (K(d) = 28 +/- 3 and 23 +/- 2 nM, respectively). The 20mer stem-UUCG-loop and GAUA-loop constructs have <0.5% of the affinity for NCp7 relative to SL3. Affinities relative to SL3 for the other stem-loops are the following: 10% for a 16mer construct to model SL4, 30% for a 27mer model of the 9-residue apical loop of SL1, and 20% for a 23mer model of a 1 x 3 asymmetric internal loop in SL1. A 154mer construct that includes all four stem-loops binds tightly to NCp7, with the equivalent of three NCp7 molecules bound with high affinity per RNA; it is also possible that two strong sites and several weaker ones combine to give the appearance of three strong sites.