Analysis of the phase behavior of the aqueous poly(ethylene glycol)-Ficoll system

The PEG-Ficoll polymer phase system is one that has been overlooked in the past for biotechnology applications because of the stability of its emulsions. However, new applications, such as emulsion coating of cells, are appearing that rely on this very property. Ficoll is highly polydisperse and multimodal with three distinct Ficoll peaks in gel permeation chromatography. As a result, the transition between one-phase and two-phase systems is blurred and the binodials obtained through turbidometric titration and tie-line analysis differ significantly. Moreover, since the three Ficoll peaks partition differently, tie-line analysis cannot be described by a simple model of the aqueous two-phase system. A simple modification to the model allowed for excellent fit, and this modification may prove well-suited for the many practical cases where aqueous two-phase systems fail to display parallel tie-lines as implicitly assumed in the simpler model.

Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types

Multicellular tumor spheroids (MCTS) are used as organotypic models of normal and solid tumor tissue. Traditional techniques for generating MCTS, such as growth on nonadherent surfaces, in suspension, or on scaffolds, have a number of drawbacks, including the need for manual selection to achieve a homogeneous population and the use of nonphysiological matrix compounds. In this study we describe a mild method for the generation of MCTS, in which individual spheroids form in hanging drops suspended from a microtiter plate. The method has been successfully applied to a broad range of cell lines and shows nearly 100% efficiency (i.e., one spheroid per drop). Using the hepatoma cell line, HepG2, the hanging drop method generated well-rounded MCTS with a narrow size distribution (coefficient of variation [CV] 10% to 15%, compared with 40% to 60% for growth on nonadherent surfaces). Structural analysis of HepG2 and a mammary gland adenocarcinoma cell line, MCF-7, composed spheroids, revealed highly organized, three-dimensional, tissue-like structures with an extensive extracellular matrix. The hanging drop method represents an attractive alternative for MCTS production, because it is mild, can be applied to a wide variety of cell lines, and can produce spheroids of a homogeneous size without the need for sieving or manual selection. The method has applications for basic studies of physiology and metabolism, tumor biology, toxicology, cellular organization, and the development of bioartificial tissue. (C) 2003 Wiley Periodicals, Inc.

Growth, viral production and metabolism of a Helicoverpa zea cell line in serum-free culture

Insect cell cultures have been extensively utilised for means of production for heterologous proteins and biopesticides. Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five(TM)) cell lines have been widely used for the production of recombinant proteins, thus metabolism of these cell lines have been investigated thoroughly over recent years. The Helicoverpa zea cell line has potential use for the production of a biopesticide, specifically the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV). The growth, virus production, nutrient consumption and waste production of this cell line was investigated under serum-free culture conditions, using SF900II and a low cost medium prototype (LCM). The cell growth ( growth rates and population doubling time) was comparable in SF900II and LCM, however, lower biomass and cell specific virus yields were obtained in LCM. H. zea cells showed a preference for asparagine over glutamine, similar to the High Five(TM) cells. Ammonia was accumulated to significantly high levels (16 mM) in SF900II, which is an asparagine and glutamine rich medium. However, given the absence of asparagine and glutamine in the medium ( LCM), H. zea cells adapted and grew well in the absence of these substrates and no accumulation of ammonia was observed. The adverse effect of ammonia on H. zea cells is unknown since good production of biologically active HaSNPV was achieved in the presence of high ammonia levels. H. zea cells showed a preference for maltose even given an abundance supply of free glucose. Accumulation of lactate was observed in H. zea cell cultures.

A dynamic mathematical model for sequential leach bed anaerobic digestion of organic fraction of municipal solid waste

A mathematical model that describes the operation of a sequential leach bed process for anaerobic digestion of organic fraction of municipal solid waste (MSW) is developed and validated. This model assumes that ultimate mineralisation of the organic component of the waste occurs in three steps, namely solubilisation of particulate matter, fermentation to volatile organic acids (modelled as acetic acid) along with liberation of carbon dioxide and hydrogen, and methanogenesis from acetate and hydrogen. The model incorporates the ionic equilibrium equations arising due to dissolution of carbon dioxide, generation of alkalinity from breakdown of solids and dissociation of acetic acid. Rather than a charge balance, a mass balance on the hydronium and hydroxide ions is used to calculate pH. The flow of liquid through the bed is modelled as occurring through two zones-a permeable zone with high flushing rates and the other more stagnant. Some of the kinetic parameters for the biological processes were obtained from batch MSW digestion experiments. The parameters for flow model were obtained from residence time distribution studies conducted using tritium as a tracer. The model was validated using data from leach bed digestion experiments in which a leachate volume equal to 10% of the fresh waste bed volume was sequenced. The model was then tested, without altering any kinetic or flow parameters, by varying volume of leachate that is sequenced between the beds. Simulations for sequencing/recirculating 5 and 30% of the bed volume are presented and compared with experimental results. (C) 2002 Elsevier Science B.V. All rights reserved.

Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis

Aim: The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract. Methods and Results: Pinch samples of the terminal ileum, proximal and distal colon were taken from a healthy 35-year-old, and a 68-year-old subject with mild diverticulosis. The 16S rDNA genes were amplified using a low number of PCR cycles, cloned, and sequenced. In total, 361 sequences were obtained comprising 70 operational taxonomic units (OTU), with a calculated coverage of 82.6%. Twenty-three per cent of OTU were common to the terminal ileum, proximal colon and distal colon, but 14% OTU were only found in the terminal ileum, and 43% were only associated with the proximal or distal colon. The most frequently represented clones were from the Clostridium group XIVa (24.7%), and the Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides ) cluster (27.7%). Conclusion: Comparison of 16S rDNA clone libraries of the hindgut across mammalian species confirms that the distribution of phylogenetic groups is similar irrespective of the host species. Lesser site-related differences within groups or clusters of organisms, are probable. Significance and Impact: This study provides further evidence of the distribution of the bacteria on the mucosal surfaces of the human hindgut. Data contribute to the benchmarking of the microbial composition of the human digestive tract.

Phosphoregulators: Protein kinases and protein phosphatases of mouse

With the completion of the human and mouse genome sequences, the task now turns to identifying their encoded transcripts and assigning gene function. In this study, we have undertaken a computational approach to identify and classify all of the protein kinases and phosphatases present in the mouse gene complement. A nonredundant set of these sequences was produced by mining Ensembl gene predictions and publicly available cDNA sequences with a panel of InterPro domains. This approach identified 561 candidate protein kinases and 162 candidate protein phosphatases. This cohort was then analyzed using TribeMCL protein sequence similarity clustering followed by CLUSTALV alignment and hierarchical tree generation. This approach allowed us to (1) distinguish between true members of the protein kinase and phosphatase families and enzymes of related biochemistry, (2) determine the structure of the families, and (3) suggest functions for previously uncharacterized members. The classifications obtained by this approach were in good agreement with previous schemes and allowed us to demonstrate domain associations with a number of clusters. Finally, we comment on the complementary nature of cDNA and genome-based gene detection and the impact of the FANTOM2 transcriptome project.

Exploration of the cell-cycle genes found within the RIKEN FANTOM2 data set

The cell cycle is one of the most fundamental processes within a cell. Phase-dependent expression and cell-cycle checkpoints require a high level of control. A large number of genes with varying functions and modes of action are responsible for this biology. In a targeted exploration of the FANTOM2-Variable Protein Set, a number of mouse homologs to known cell-cycle regulators as well as novel members of cell-cycle families were identified. Focusing on two prototype cell-cycle families, the cyclins and the NIMA-related kinases (NEKs), we believe we have identified all of the mouse members of these families, 24 cyclins and 10 NEKs, and mapped them to ENSEMBL transcripts. To attempt to globally identify all potential cell cycle-related genes within mouse, the MGI (Mouse Genome Database) assignments for the RIKEN Representative Set (RPS) and the results from two homology-based queries were merged. We identified 1415 genes with possible cell-cycle roles, and 1758 potential paralogs. We comment on the genes identified in this screen and evaluate the merits of each approach.

Characterizing embryonic gene expression patterns in the mouse using nonredundant sequence-based selection

This article investigates the expression patterns of 160 genes that are expressed during early mouse development. The cDNAs were isolated from 7.5 d postcoitum (dpc) encloderm, a region that comprises visceral encloderm (VE), definitive encloderm, and the node-tissues that are required for the initial steps of axial specification and tissue patterning in the mouse. To avoid examining the same gene more than once, and to exclude potentially ubiquitously expressed housekeeping genes, cDNA sequence was derived from 1978 clones of the Endoderm library. These yielded 1440 distinct cDNAs, of which 123 proved to be novel in the mouse. In situ hybridization analysis was carried out on 160 of the cDNAs, and of these, 29 (18%) proved to have restricted expression patterns.

Systematic expression profiling of the mouse transcriptome using RIKEN cDNA mircoarrays

The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of 'molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as unknown EST, and 1969 (58%) clones of 3388 clones that were categorized as unclassifiable were also shown to be reproducibly expressed.

Targeting a complex transcriptome: The construction of the mouse full-length cDNA encyclopedia

We report the construction of the mouse full-length cDNA encyclopedia, the most extensive view of a complex transcriptome, on the basis of preparing and sequencing 246 libraries. Before cloning, cDNAs were enriched in full-length by Cap-Trapper, and in most cases, aggressively subtracted/normalized. We have produced 1,442,236 successful 3'-end sequences clustered into 171,144 groups, from which 60,770 clones were fully sequenced cDNAs annotated in the FANTOM-2 annotation. We have also produced 547,149 5' end reads, which clustered into 124,258 groups. Altogether, these cDNAs were further grouped in 70,000 transcriptional units (TU), which represent the best coverage of a transcriptome so far. By monitoring the extent of normalization/subtraction, we define the tentative equivalent coverage (TEC), which was estimated to be equivalent to >12,000,000 ESTs derived from standard libraries. High coverage explains discrepancies between the very large. numbers of clusters (and TUs) of this project, which also include non-protein-coding RNAs, and the lower gene number estimation of genome annotations. Altogether, S'-end clusters identify regions that are potential promoters for 8637 known genes and S'-end clusters suggest the presence of almost 63,000 transcriptional starting points. An estimate of the frequency of polyadenylation signals suggests that at least half of the singletons in the EST set represent real mRNAs. Clones accounting for about half of the predicted TUs await further sequencing. The continued high-discovery rate suggests that the task of transcriptome discovery is not yet complete.

Development and evaluation of an automated annotation pipeline and cDNA annotation system

Manual curation has long been held to be the gold standard for functional annotation of DNA sequence. Our experience with the annotation of more than 20,000 full-length cDNA sequences revealed problems with this approach, including inaccurate and inconsistent assignment of gene names, as well as many good assignments that were difficult to reproduce using only computational methods. For the FANTOM2 annotation of more than 60,000 cDNA clones, we developed a number of methods and tools to circumvent some of these problems, including an automated annotation pipeline that provides high-quality preliminary annotation for each sequence by introducing an uninformative filter that eliminates uninformative annotations, controlled vocabularies to accurately reflect both the functional assignments and the evidence supporting them, and a highly refined, Web-based manual annotation tool that allows users to view a wide array of sequence analyses and to assign gene names and putative functions using a consistent nomenclature. The ultimate utility of our approach is reflected in the low rate of reassignment of automated assignments by manual curation. Based on these results, we propose a new standard for large-scale annotation, in which the initial automated annotations are manually investigated and then computational methods are iteratively modified and improved based on the results of manual curation.

Identification of putative noncoding RNAs among the RIKEN mouse full-length cDNA collection

With the sequencing and annotation of genomes and transcriptomes of several eukaryotes, the importance of noncoding RNA (ncRNA)-RNA molecules that are not translated to protein products-has become more evident. A subclass of ncRNA transcripts are encoded by highly regulated, multi-exon, transcriptional units, are processed like typical protein-coding mRNAs and are increasingly implicated in regulation of many cellular functions in eukaryotes. This study describes the identification of candidate functional ncRNAs from among the RIKEN mouse full-length cDNA collection, which contains 60,770 sequences, by using a systematic computational filtering approach. We initially searched for previously reported ncRNAs and found nine murine ncRNAs and homologs of several previously described nonmouse ncRNAs. Through our computational approach to filter artifact-free clones that lack protein coding potential, we extracted 4280 transcripts as the largest-candidate set. Many clones in the set had EST hits, potential CpG islands surrounding the transcription start sites, and homologies with the human genome. This implies that many candidates are indeed transcribed in a regulated manner. Our results demonstrate that ncRNAs are a major functional subclass of processed transcripts in mammals.

Systematic characterization of the zinc-finger-containing proteins in the mouse transcriptome

Zinc-finger-containing proteins can be classified into evolutionary and functionally divergent protein families that share one or more domains in which a zinc ion is tetrahedrally coordinated by cysteines and histidines. The zinc finger domain defines one of the largest protein superfamilies in mammalian genomes; 46 different conserved zinc finger domains are listed in InterPro (http://www.ebi.ac.uk/InterPro). Zinc finger proteins can bind to DNA, RNA, other proteins, or lipids as a modular domain in combination with other conserved structures. Owing to this combinatorial diversity, different members of zinc finger superfamilies contribute to many distinct cellular processes, including transcriptional regulation, mRNA stability and processing, and protein turnover. Accordingly, mutations of zinc finger genes lead to aberrations in a broad spectrum of biological processes such as development, differentiation, apoptosis, and immunological responses. This study provides the first comprehensive classification of zinc finger proteins in a mammalian transcriptome. Specific detailed analysis of the SP/Kruppel-like factors and the E3 ubiquitin-ligase RING-H2 families illustrates the importance of such an analysis for a more comprehensive functional classification of large protein families. We describe the characterization of a new family of C2H2 zinc-finger-containing proteins and a new conserved domain characteristic of this family, the identification and characterization of Sp8, a new member of the Sp family of transcriptional regulators, and the identification of five new RING-H2 proteins.

Continued discovery of transcriptional units expressed in cells of the mouse mononuclear phagocyte lineage

The current RIKEN transcript set represents a significant proportion of the mouse transcriptome but transcripts expressed in the innate and acquired immune systems are poorly represented. In the present study we have assessed the complexity of the transcriptome expressed in mouse macrophages before and after treatment with lipopolysaccharide, a global regulator of macrophage gene expression, using existing RIKEN 19K arrays. By comparison to array profiles of other cells and tissues, we identify a large set of macrophage-enriched genes, many of which have obvious functions in endocytosis and phagocytosis. In addition, a significant number of LPS-inducible genes were identified. The data suggest that macrophages are a complex source of mRNA for transcriptome studies. To assess complexity and identify additional macrophage expressed genes, cDNA libraries were created from purified populations of macrophage and dendritic cells, a functionally related cell type. Sequence analysis revealed a high incidence of novel mRNAs within these cDNA libraries. These studies provide insights into the depths of transcriptional complexity still untapped amongst products of inducible genes, and identify macrophage and dendritic cell populations as a starting point for sampling the inducible mammalian transcriptome.

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleoticle distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.