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The tuberculous granuloma is a compact aggregate of dormant bacteria encapsulated by host macrophages. It is commonly regarded as a product of the host defense designed to isolate infectious mycobacteria. This work demonstrates that exposure of macrophages to the Mtb heat-shock protein Acr leads to overproduction of the chemokine CXCL16, allowing the mycobacterium to exploit the innate immune response. This induction of chemokine expression is hypothesized to occur through activation of ADAM proteases, providing an immunomodulatory role for Mtb Acr in the formation of the granuloma.

A hallmark of the research experience is encountering experimental difficulty and working through that challenge to reach success. The ability to overcome scientific challenges is essential to being a successful scientist, but replicating these challenges can be difficult in a teaching setting. The Genomics Education Partnership (GEP) is a consortium of faculty who engage their students in a genomics Course-based Undergraduate Research Experience (CURE). Students participate in genome annotation, generating gene models using multiple lines of experimental evidence. Our observations suggested that the students’ learning experience in this research is continuous and recursive, frequently beginning with frustration but eventually leading to success. In order to explore our “formative frustration” hypothesis, we gathered data about this research experience from faculty via a survey, and from students both via a general survey and a small set of focus groups administered at the end of the GEP CURE. All three datasets contained comments mentioning frustration and struggle, as well as learning and better understanding of the scientific process. Bioinformatics projects are particularly well suited to the process of iteration and refinement because iterations can be performed quickly and are inexpensive in both time and money. Based on these findings, we suggest that a dynamic of what we have deemed “formative frustration” is an important aspect for a successful CURE.

The discordance between genome size and the complexity of eukaryotes can partly be attributed to differences in repeat density. The Muller F element (∼5.2 Mb) is the smallest chromosome in Drosophila melanogaster, but it is substantially larger (>18.7 Mb) in D. ananassae. To identify the major contributors to the expansion of the F element and to assess their impact, we improved the genome sequence and annotated the genes in a 1.4-Mb region of the D. ananassae F element, and a 1.7-Mb region from the D element for comparison. We find that transposons (particularly LTR and LINE retrotransposons) are major contributors to this expansion (78.6%), while Wolbachia sequences integrated into the D. ananassae genome are minor contributors (0.02%). Both D. melanogaster and D. ananassae F-element genes exhibit distinct characteristics compared to D-element genes (e.g., larger coding spans, larger introns, more coding exons, and lower codon bias), but these differences are exaggerated in D. ananassae. Compared to D. melanogaster, the codon bias observed in D. ananassae F-element genes can primarily be attributed to mutational biases instead of selection. The 5′ ends of F-element genes in both species are enriched in dimethylation of lysine 4 on histone 3 (H3K4me2), while the coding spans are enriched in H3K9me2. Despite differences in repeat density and gene characteristics, D. ananassae F-element genes show a similar range of expression levels compared to genes in euchromatic domains. This study improves our understanding of how transposons can affect genome size and how genes can function within highly repetitive domains.

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu.

Bioinformatics, a discipline that combines aspects of biology, statistics, and computer science, is increasingly important for biological research. However, bioinformatics instruction is rarely integrated into life sciences curricula at the undergraduate level. To understand why, the Network for Integrating Bioinformatics into Life Sciences Education (NIBLSE, “nibbles”) recently undertook an extensive survey of life sciences faculty in the United States. The survey responses to open-ended questions about barriers to integration were subjected to keyword analysis. The barrier most frequently reported by the ~1,260 respondents was lack of faculty training. Faculty at associate’s-granting institutions report the least training in bioinformatics and the least integration of bioinformatics into their teaching. Faculty from underrepresented minority groups (URMs) in STEM reported training barriers at a higher rate than others, although the number of URM respondents was small. Interestingly, the cohort of faculty with the most recently awarded PhD degrees reported the most training but were teaching bioinformatics at a lower rate than faculty who earned their degrees in previous decades. Other barriers reported included lack of student interest in bioinformatics; lack of student preparation in mathematics, statistics, and computer science; already overly full curricula; and limited access to resources, including hardware, software, and vetted teaching materials. The results of the survey, the largest to date on bioinformatics education, will guide efforts to further integrate bioinformatics instruction into undergraduate life sciences education.

While course-based research in genomics can generate both knowledge gains and a greater appreciation for how science is done, a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. Nonetheless, this is a very cost-effective way to reach larger numbers of students., There is widespread agreement that science, technology, engineering, and mathematics programs should provide undergraduates with research experience. Practical issues and limited resources, however, make this a challenge. We have developed a bioinformatics project that provides a course-based research experience for students at a diverse group of schools and offers the opportunity to tailor this experience to local curriculum and institution-specific student needs. We assessed both attitude and knowledge gains, looking for insights into how students respond given this wide range of curricular and institutional variables. While different approaches all appear to result in learning gains, we find that a significant investment of course time is required to enable students to show gains commensurate to a summer research experience. An alumni survey revealed that time spent on a research project is also a significant factor in the value former students assign to the experience one or more years later. We conclude: 1) implementation of a bioinformatics project within the biology curriculum provides a mechanism for successfully engaging large numbers of students in undergraduate research; 2) benefits to students are achievable at a wide variety of academic institutions; and 3) successful implementation of course-based research experiences requires significant investment of instructional time for students to gain full benefit.

Bioinformatics, a discipline that combines aspects of biology, statistics, mathematics, and computer science, is becoming increasingly important for biological research. However, bioinformatics instruction is not yet generally integrated into undergraduate life sciences curricula. To understand why we studied how bioinformatics is being included in biology education in the US by conducting a nationwide survey of faculty at two- and four-year institutions. The survey asked several open-ended questions that probed barriers to integration, the answers to which were analyzed using a mixed-methods approach. The barrier most frequently reported by the 1,260 respondents was lack of faculty expertise/training, but other deterrents-lack of student interest, overly-full curricula, and lack of student preparation-were also common. Interestingly, the barriers faculty face depended strongly on whether they are members of an underrepresented group and on the Carnegie Classification of their home institution. We were surprised to discover that the cohort of faculty who were awarded their terminal degree most recently reported the most preparation in bioinformatics but teach it at the lowest rate.

The widespread use of nanoparticles (NPs) raises concern over their potential toxicological effects in humans and ecosystems. Here we used transcriptome sequencing (RNA-seq) to evaluate the effects of exposure to four different metal-based NPs, nano-Ag (nAg), nano-TiO 2 (nTiO 2 ), nano-ZnO (nZnO), and CdTe/CdS quantum dots (QDs), in the eukaryotic green alga Chlamydomonas reinhardtii . The transcriptome was characterized before and after exposure to each NP type. Specific toxicological effects were inferred from the functions of genes whose transcripts either increased or decreased. Data analysis resulted in important differences and also similarities among the NPs. Elevated levels of transcripts of several marker genes for stress were observed, suggesting that only nZnO caused nonspecific global stress to the cells under environmentally relevant conditions. Genes with photosynthesis-related functions were decreased drastically during exposure to nTiO 2 and slightly during exposures to the other NP types. This pattern suggests either toxicological effects in the chloroplast or effects that mimic a transition from low to high light. nAg exposure dramatically elevated the levels of transcripts encoding known or predicted components of the cell wall and the flagella, suggesting that it damages structures exposed to the external milieu. Exposures to nTiO 2 , nZnO, and QDs elevated the levels of transcripts encoding subunits of the proteasome, suggesting proteasome inhibition, a phenomenon believed to underlie the development and progression of several major diseases, including Alzheimer's disease, and used in chemotherapy against multiple myeloma.

The era of ‘big data' is also the era of abundant data, creating new opportunities for student–scientist research partnerships. By coordinating undergraduate efforts, the Genomics Education Partnership produces high-quality annotated data sets and analyses that could not be generated otherwise, leading to scientific publications while providing many students with research experience.

Although bioinformatics is becoming increasingly central to research in the life sciences, bioinformatics skills and knowledge are not well integrated into undergraduate biology education. This curricular gap prevents biology students from harnessing the full potential of their education, limiting their career opportunities and slowing research innovation. To advance the integration of bioinformatics into life sciences education, a framework of core bioinformatics competencies is needed. To that end, we here report the results of a survey of biology faculty in the United States about teaching bioinformatics to undergraduate life scientists. Responses were received from 1,260 faculty representing institutions in all fifty states with a combined capacity to educate hundreds of thousands of students every year. Results indicate strong, widespread agreement that bioinformatics knowledge and skills are critical for undergraduate life scientists as well as considerable agreement about which skills are necessary. Perceptions of the importance of some skills varied with the respondent's degree of training, time since degree earned, and/or the Carnegie Classification of the respondent's institution. To assess which skills are currently being taught, we analyzed syllabi of courses with bioinformatics content submitted by survey respondents. Finally, we used the survey results, the analysis of the syllabi, and our collective research and teaching expertise to develop a set of bioinformatics core competencies for undergraduate biology students. These core competencies are intended to serve as a guide for institutions as they work to integrate bioinformatics into their life sciences curricula.

Genomics is not only essential for students to understand biology but also provides unprecedented opportunities for undergraduate research. The goal of the Genomics Education Partnership (GEP), a collaboration between a growing number of colleges and universities around the country and the Department of Biology and Genome Center of Washington University in St. Louis, is to provide such research opportunities. Using a versatile curriculum that has been adapted to many different class settings, GEP undergraduates undertake projects to bring draft-quality genomic sequence up to high quality and/or participate in the annotation of these sequences. GEP undergraduates have improved more than 2 million bases of draft genomic sequence from several species of Drosophila and have produced hundreds of gene models using evidence-based manual annotation. Students appreciate their ability to make a contribution to ongoing research, and report increased independence and a more active learning approach after participation in GEP projects. They show knowledge gains on pre- and postcourse quizzes about genes and genomes and in bioinformatic analysis. Participating faculty also report professional gains, increased access to genomics-related technology, and an overall positive experience. We have found that using a genomics research project as the core of a laboratory course is rewarding for both faculty and students.

The conformational flexibility exhibited by protein kinases poses an enormous challenge to the design of cancer therapeutics. Additionally the high degree of structural conservation within the kinase superfamily often leads to inhibitors that exhibit little selectivity and substantial cross reactivity. This work investigates the conformational changes that accompany the binding of Gleevec, or imatinib mesylate, to the tyrosine kinases c-Kit and c-Abl. Our analysis is that this fit is driven, at least in part, by the need to exclude water from solvent-exposed backbone hydrogen bonds. Both experimental and molecular modeling studies of the active state inhibitor of the tyrosine kinase c-Abl indicate that solvent exclusion also plays a role in this system.

Clustering and assembly of expressed sequence tags (ESTs) constitute the basis for most genomewide descriptions of a transcriptome. This approach is limited by the decline in sequence quality toward the end of each EST, impacting both sequence clustering and assembly. Here, we exploit the available draft genome sequence of the unicellular green alga Chlamydomonas reinhardtii to guide clustering and to correct errors in the ESTs. We have grouped all available EST and cDNA sequences into 12 063 ACEGs (assembly of contiguous ESTs based on genome) and generated 15 857 contigs of average length 934 nt. We predict that roughly 3000 of our contigs represent full-length transcripts. Compared to previous assemblies, ACEGs show extended contig length, increased accuracy and a reduction in redundancy. Because our assembly protocol also uses ESTs with no corresponding genomic sequences, it provides sequence information for genes interrupted by sequence gaps. Detailed analysis of randomly sampled ACEGs reveals several hundred putative cases of alternative splicing, many overlapping transcription units and new genes not identified by gene prediction algorithms. Our protocol, although developed for and tailored to the C. reinhardtii dataset, can be exploited by any eukaryotic genome project for which both a draft genome sequence and ESTs are available.

Simple, experimentally tractable systems such Saccharomyces cerevisiae, Chlamydomonas reinhardtii, and Arabidopsis thaliana are powerful models for dissecting basic biological processes. The unicellular green alga C. reinhardtii is amenable to a diversity of genetic and molecular manipulations. This haploid organism grows rapidly in axenic cultures, on both solid and liquid medium, with a sexual cycle that can be precisely controlled. Vegetative diploids are readily selected through the use of complementing auxotrophic markers and are useful for analyses of deleterious recessive alleles. These genetic features have permitted the generation and characterization of a wealth of mutants with lesions in structural, metabolic and regulatory genes. Another important feature of C. reinhardtii is that it has the capacity to grow with light as a sole energy source (photoautotrophic growth) or on acetate in the dark (heterotrophically), facilitating detailed examination of genes and proteins critical for photosynthetic or respiratory function. Other important topics being studied using C. reinhardtii, many of which have direct application to elucidation of protein function in animal cells (26), include flagellum structure and assembly, cell wall biogenesis, gametogenesis, mating, phototaxis, and adaptive responses to light and nutrient environments (32, 44). Some of these studies are directly relevant to applied problems in biology, including the production of clean, solar-generated energy in the form of H2, and bioremediation of heavy metal wastes. Recent years have seen the development of a molecular toolkit for C. reinhardtii (42, 44, 66, 98, 99). Selectable markers are available for nuclear and chloroplast transformation (4, 5, 12, 13, 30, 44, 56, 82). The Arg7 (22) and Nit1 (30) genes are routinely used to rescue recessive mutant phenotypes. The bacterial ble gene (which codes for zeocin resistance [70, 112]) is an easily scored marker for nuclear transformation, and the bacterial aadA gene (which codes for spectinomycin and streptomycin resistance) is a reliable marker for chloroplast transformation (39). Nuclear transformation can be achieved by

The National Science Foundation-funded Chlamydomonas reinhardtii genome project involves (a) construction and sequencing of cDNAs isolated from cells exposed to various environmental conditions, (b) construction of a high-density cDNA microarray, (c) generation of genomic contigs that are nucleated around specific physical and genetic markers, (d) generation of a complete chloroplast genome sequence and analyses of chloroplast gene expression, and (e) the creation of a Web-based resource that allows for easy access of the information in a format that can be readily queried. Phases of the project performed by the groups at the Carnegie Institution and Duke University involve the generation of normalized cDNA libraries, sequencing of cDNAs, analysis and assembly of these sequences to generate contigs and a set of predicted unique genes, and the use of this information to construct a high-density DNA microarray. In this paper, we discuss techniques involved in obtaining cDNA end-sequence information and the ways in which this information is assembled and analyzed. Descriptions of protocols for preparing cDNA libraries, assembling cDNA sequences and annotating the sequence information are provided (the reader is directed to Web sites for more detailed descriptions of these methods). We also discuss preliminary results in which the different cDNA libraries are used to identify genes that are potentially differentially expressed.

There have been numerous calls to engage students in science as science is done. A survey of 90-plus faculty members explores barriers and incentives when developing a research-based genomics course. The results indicate that a central core supporting a national experiment can help overcome local obstacles., In their 2012 report, the President's Council of Advisors on Science and Technology advocated “replacing standard science laboratory courses with discovery-based research courses”—a challenging proposition that presents practical and pedagogical difficulties. In this paper, we describe our collective experiences working with the Genomics Education Partnership, a nationwide faculty consortium that aims to provide undergraduates with a research experience in genomics through a scheduled course (a classroom-based undergraduate research experience, or CURE). We examine the common barriers encountered in implementing a CURE, program elements of most value to faculty, ways in which a shared core support system can help, and the incentives for and rewards of establishing a CURE on our diverse campuses. While some of the barriers and rewards are specific to a research project utilizing a genomics approach, other lessons learned should be broadly applicable. We find that a central system that supports a shared investigation can mitigate some shortfalls in campus infrastructure (such as time for new curriculum development, availability of IT services) and provides collegial support for change. Our findings should be useful for designing similar supportive programs to facilitate change in the way we teach science for undergraduates.

In plant and algal cells, inhibition of the enzyme protoporphyrinogen oxidase (Protox) by the N-phenyl heterocyclic herbicide S-23142 causes massive protoporphyrin IX accumulation, resulting in membrane deterioration and cell lethality in the light. We have identified a 40.4 kb genomic fragment encoding S-23142 resistance by using transformation to screen an indexed cosmid library made from nuclear DNA of the dominant rs-3 mutant of Chlamydomonas reinhardtii. A 10.0 kb HindIII subclone (Hind10) of this insert yields a high frequency of herbicide-resistant transformants, consistent with frequent non-homologous integration of the complete RS-3 gene. A 3.4 kb XhoI subfragment (Xho3.4) yields rare herbicide-resistant transformants, suggestive of homologous integration of a portion of the coding sequence containing the mutation. Molecular and genetic analysis of the transformants localized the rs-3 mutation conferring S-23142 resistance to the Xho3.4 fragment, which was found to contain five putative exons encoding a protein with identity to the C-terminus of the Arabidopsis Protox enzyme. A cDNA clone containing a 1698 bp ORF that encodes a 563 amino acid peptide with 51% and 53% identity to Arabidopsis and tobacco Protox I, respectively, was isolated from a wild-type C. reinhardtii library. Comparison of the wild-type cDNA sequence with the putative exon sequences present in the mutant Xho3.4 fragment revealed a G→A change at 291 in the first putative exon, resulting in a Val→Met substitution at a conserved position equivalent to Val-389 of the wild-type C. reinhardtii cDNA. A sequence comparison of genomic Hind10 fragments from C. reinhardtii rs-3 and its wild-type progenitor CC-407 showed this G→A change at the equivalent position (5751) within exon 10.

We have examined the effects of illumination, carbon source, and levels of chloroplast protein synthesis on trans-acting proteins that bind to the leaders of five representative chloroplast mRNAs. The accumulation of these five chloroplast mRNAs and the proteins they encode were measured in cells grown under identical conditions. Extracts from all cell types examined contain a minimum set of six chloroplast 5′-untranslated region (UTR)-binding proteins (81, 62, 56, 47, 38, and 15 kDa). Fractionation results suggest that multiple forms of the 81-, 62-, and 47-kDa proteins may exist. A 36-kDa protein was found in all cells except those deficient in chloroplast protein synthesis. Binding of the 81-, 47-, and 38-kDa proteins to the rps12 leader is effectively competed by the atpB or rbcL 5′-UTRs, indicating that the same proteins bind to all three leaders. In contrast, these three proteins do not bind to the nuclear-encoded α-1 tubulin leader, which bound novel proteins of 110, 70, and 43 kDa. Cis-acting sequences within the 5′-UTRs of two chloroplast mRNAs (rps7 and atpB) have been identified which are protected from digestion by RNase T1 by extracts enriched for the 81-, 47-, and 38-kDa proteins.

In order to properly assess the environmental risk of engineered nanoparticles (ENP), it is necessary to determine their fate (including dissolution, aggregation, and bioaccumulation) under representative environmental conditions. CdTe/CdS quantum dots (QD), such as those used in medical imaging, are known to release Cd(2+) due (mainly) to the dissolution of their outer shell. In this study, Chlamydomonas reinhardtii was exposed to either a soluble Cd salt or QD at similar concentrations of total Cd. Free Cd concentrations were measured using the Absence of Gradients and Nernstian Equilibrium Stripping technique. QD dissolution increased with decreasing pH and with increasing QD concentration. When exposed to QD, bioaccumulation was largely accounted for by dissolved Cd. Nonetheless, QD were shown to be taken up by the cells and to provoke unique biological effects. Whole transcriptome screening using RNA-Seq analysis showed that the free Cd and the QD had distinctly different biological effects.

Under certain conditions, a ketone having an alpha-hydrogen atom may be acylated with an ester, an acid anhydride, or an acid chloride to form a β-ketone or, when the acylating agent is a formic ester, a β-keto aldehyde. The process consists in the replacement of an alpha-hydrogen atom of the ketone by an acyl group. The reaction involves a carbon-carbon condensation. The acylation of ketones with esters has generally been effected by means of a basic reagent, such as sodium, sodium ethoxide, etc. The acylation of ketones to form β-diketones may also occur with acid anhydrides by means of an acidic reagent boron trifluoride. In this chapter the basic reagent and boron trifluoride methods of acylation are considered separately. They are compared with other methods. Keywords: acylation; ketones; β-ketones; β-aldehdyes; basic reagents; boron trifluoride; comparison of methods; experimental procedures

The acetoacetic ester condensation consist in the reaction in the presence of certain bases, of an ester having hydrogen on the alpha carbon atom with a second molecule of the same ester or with another ester to form a beta-ketoester. The bases capable of effecting such reactions include sodium alkoxides, triphenylmethylsodium, sodium amide, certain Grignard reagents. Metallic sodium effects certain condensations, the sodium, alkoxide which is formed in the reaction mixture probably serves as the active condensing agents. The classical example of the acetoacetic ester reaction is the formation of acetoacetic ester itself by the condensation of ethyl acetate by means of sodium ethoxide. Keywords: acetoacetic ester; condensation; side reactions; Dieckmann reaction; acylation; esters; acid chlorides; sodium; experimental conditions

Publisher Summary Translational control functions in diverse biological processes including developmental pattern formation, intracellular protein targeting, the control of homeostasis, and a variety of stress responses. Since the discovery of genomes in plastids, Chlamydomonas research has played a central role in the development of our understanding of the plastid translation machinery and the mechanisms that regulate translation in chloroplasts, and this chapter reviews relevant findings and models. Protein synthesis rates in the Chlamydomonas chloroplast are routinely measured using pulse labeling of intact cells with 35SO4 or [14C] acetate, or isolated chloroplasts with [35S]methionine. This part, radioisotope pulse labeling of newly synthesized proteins, is described under methodologies of protein synthesis. Genetics has been used extensively for the identification and characterization of trans-acting factors and cis-acting sequences that function in the translation of chloroplast mRNAs. Chloroplast ribosomes are introduced and updated with recent research findings. Translation requires general translation factors (GTF), many of which are GTPases activated by the ribosome. The identities and properties of the Chlamydomonas GTFs are covered in this section. Chlamydomonas responds to light in a variety of ways, including changes in chloroplast gene expression at the translational level. This section reviews distinct translational regulatory responses during dark-to-light transitions and under high light stress. Reverse genetic approaches are used to address the functions of other translation factors originally identified through biochemical approaches.

During the evolution of chloroplasts from cyanobacterial endosymbionts, many genes encoding components necessary for protein synthesis and photosynthesis have been transferred to the nucleus. Assembly of the machinery for both processes now relies on the concerted expression of genes in the nuclear and plastid genomes. Evidence accumulating in C. reinhardtii indicates that the expression and regulation of chloroplast genes probably differs in certain respects from the prokaryotic (E. coli) paradigm. Both mutational analysis of putative Shine-Dalgarno (SD) sequences, and creation of canonical SD sequences (-9 to -5) reveals that initiation is not mediated by SD-16S rRNA interactions for the majority of chloroplast-encoded mRNAs. Recent evidence in cyanobacteria, the most likely ancestors of the Chlamydomonas chloroplast, indicates that most genes lack SD sequences at the classical position. Interactions between cis-acting sequences in the 5′ untranslated regions (UTRs) and specific trans-acting nuclear gene products appear to mediate translation of chloroplast-encoded mRNAs for specific genes. Both UV crosslinking and gel mobility shift assays with several chloroplast leaders show that multiple proteins interact with these sequences. Some of these appear to be involved in gene-specific regulation, whereas others may be core proteins of a general ribonucleoprotein (RNP) complex. Inverted repeat sequences present in most chloroplast leaders predict higher order structures within the 5′ UTR of mRNAs that could serve as scaffolding for the formation of the RNP complex. One model predicts that the RNP complex directly enhances ribosome binding and translation initiation. A second model predicts that the RNP complex plays a role in making topological distinctions in the sites of synthesis of chloroplast mRNAs. A challenge in the coming years will be to determine which, if any, of these models are correct.

We present an efficient, fully automated algorithm to assemble ESTs into full-length cDNA sequences that represent the complete coding regions of a gene. Our EST clustering algorithm is neither hierarchical nor incremental, but recursive, processing each EST once. The algorithm exploits a variety of syntactic and statistical features of the ESTs. The resulting assembly shows significant improvement in computational efficiency and information extraction over a previous assembly of C. reinhardtii ESTs. The algorithm was developed using iterative and participatory design on C. reinhardtii; however, it can be used for any organism with a draft genomic sequence.

Ethyl β-hydroxy-β,β-diphenylpropionate product: ethyl β-hydroxy-β,β-diphenylpropionate reactant: benzophenone product: Isopropyl and t-butyl β-hydroxy-β,β-diphenylpropionate Keywords: addition, to CO; condensation, ester-ketone condensations; lithium amide, preparation of; ammonia

3-(4-Chlorophenyl)-5-(4-methoxyphenyl)isoxazole reactant: 16.96 g. (0.1000 mole) of 4-chloroacetophenone oxime product: 3-(4-chlorophenyl)-5-(4-methoxyphenyl)isoxazole Keywords: annulation, heterocyclic-[5]; condensation, miscellaneous; 4-chloroacetophenone oxime, preparation of; lithium diisopropylamide (LDA), preparation of; tetrahydrofuran

o-Methylbenzyl alcohol intermediate: o-Methylbenzyl acetate intermediate: 2-methylbenzylethyldimethylammonium bromide product: o-Methylbenzyl alcohol intermediate: 2,3-dimethylbenzyl acetate product: 2,3-dimethylbenzyl alcohol Keywords: alkylation, N-alkylation; hydrolysis, esters and lactones; replacement reactions, ammonium by oxygen; replacement reactions, miscellaneous; (2-methylbenzyl)ethyldimethylammonium bromide, preparation of

Ethyl α-isopropylacetoacetate product: ethyl α-cyclohexylacetoacetate product: ethyl α-tert-butylacetoacetate product: Ethyl α-isopropylacetoacetate Keywords: alkylation, C-alkylation; boron trifluoride

tert-Butyl acetate product: tert-butyl acetate product: tert-butyl propionate product: tert-Butyl isobutyrate product: tert-butyl isovalerate product: tert-butyl cinnamate product: tert-Butyl chloroacetate Keywords: acylation; tert-butyl alcohol; tert-butyl alcohol

β-Diketones from methyl alkyl ketones: 3-n-butyl-2,4-pentanedione reactant: 28.6 g. (0.251 mole) of 2-heptanone reactant: 51.0 g. (0.500 mole) of acetic anhydride solvent: 100-ml. portions of petroleum ether product: 3-n-butyl-2,4-pentanedione product: 3-methyl-2,4-pentanedione product: 3-ethyl-2,4-pentanedione product: 3-phenyl-2,4-pentanedione product: 3,3-dimethyl-2,4-pentanedione product: 2-acetylcyclohexanone product: 2-acetylcyclopentanone product: 3-methyl-2,4-hexanedione product: 1,3-diphenyl-2,4-pentanedione product: benzoylacetone Keywords: acylation; boron trifluoride-acetic acid complex, 1:1, preparation

2-n-Butyl-2-methylcyclohexanone reactant: 123 g. (1.10 moles) of 2-methylcyclohexanone reactant: 81.4 g. (1.10 moles) of ethyl formate intermediate: Sodio-2-formyl-6-methylcyclohexanone reactant: 28.2 g. (0.21 mole) of n-butyl bromide product: 2-n-butyl-2-methylcyclohexanone byproduct: 2-formyl-6-n-butyl-6-methylcyclohexanone product: 2,2-Dimethylcyclohexanone product: 2-benzyl-2-methylcyclohexanone Keywords: acylation; alkylation, C-alkylation; cleavage, ketonic; ethyl formate; potassium amide, preparation of; ammonia; potassium

5-Methyl-5-hexen-2-one reactant: Eastman Organic Chemicals practical grade methallyl chloride reactant: Union Carbide Chemicals Co. 2,4-pentanedione product: 5-Methyl-5-hexen-2-one product: 4-Phenyl-2-butanone product: 4-(o-Bromophenyl)-2-butanone product: 4-(m-Bromophenyl)-2-butanone product: 4-(o-Chlorophenyl)-2-butanone product: 4-(m-Chlorophenyl)-2-butanone product: 4-(p-Chlorophenyl)-2-butanone product: 4-(m-Fluorophenyl)-2-butanone product: 4-(m-Nitrophenyl)-2-butanone product: 4-(α-Naphthyl)-2-butanone product: 1-Phenyl-1,4-pentanedione product: 2-Heptanone Keywords: alkylation, C-alkylation; cleavage, ketonic

2-Methyl-4-hydroxyquinoline solvent: 200 ml. of petroleum ether product: 2-methyl-4-hydroxyquinoline Keywords: annulation, heterocyclic-[6]; cyclization, condensation

α,β-Diphenylpropionic acid reactant: 14.2 g. (0.104 mole) of phenylacetic acid reactant: 13.2 g. (0.104 mole) of benzyl chloride product: α,β-diphenylpropionic acid solvent: 60 ml. of petroleum ether Keywords: alkylation, C-alkylation; ammonia

Phenylation with diphenyliodonium chloride: 1-phenyl-2,4-pentanedione reactant: 40.0 g. (0.400 mole) of 2,4-pentanedione product: 1-phenyl-2,4-pentanedione byproduct: iodobenzene product: 1-phenyl-1,3-butanedione product: 2,4-heptanedione product: 2,4-nonanedione product: 2,4-tridecanedione product: 3,5-heptanedione product: 1-(4-chlorophenyl)-2,4-pentanedione product: 4-(4-methylphenyl)-1-phenyl-1,3-butanedione product: 1-(4-methylphenyl)-2,4-nonanedione Keywords: arylation; 2,4-pentanedione; sodium amide, preparation of; ammonia

Ethyl β-phenyl-β-hydroxypropionate product: Ethyl α,α-dimethyl-β-phenyl-β-hydroxypropionate product: Ethyl β-phenyl-β-hydroxypropionate Keywords: Grignard and related reactions; benzaldehyde; ethyl bromoacetate; zinc; ethyl bromoacetate, lachrymator

α-(N,N-dimethylamino)phenylacetonitrile product: α-(N,N-dimethylamino)phenylacetonitrile Keywords: addition, to CO; replacement reactions, miscellaneous; hydrogen cyanide

Esterification of hindered alcohols: tert-butyl p-toluate reactant: 5.42 g. (0.0351 mole) of p-toluoyl chloride product: tert-butyl p-toluate product: tert-butyl phenylacetate product: triethylcarbinol ester of p-toluic acid product: 2,2-diphenylethanol ester of benzoic acid product: C6H5CH2CO2C(CH3)3 product: C6H5CO2CH2CH(C6H5)2 product: C6H5CO2C(CH3)3 product: C6H5CO2CCH3(C2H5)2 product: C6H5CO2C(C2H5)3 product: C6H5CO2CH2(t-C4H9) product: (CH3)3CCO2C(CH3)3 product: (CH3)3CCO2C(C2H5) product: (i-C3H7)2CHCO2C(C2H5)3 product: C6H5CHCHCO2C(CH3)3 product: (tC4H9)CH2CO2C(C2H5)3 product: C6H5CH2CH2CO2C(CH3)3 product: (tC4H9)2CHCO2C(C2H5)3 Keywords: esterification, unsubstituted monobasic acids; tert-butyl alcohol; diethyl ether; lithium tert-butoxide, preparation of; p-toluoyl chloride, preparation of

1-(p-Methoxyphenyl)-5-phenyl-1,3,5-pentanetrione product: 1-(p-methoxyphenyl)-5-phenyl-1,3,5-pentanetrione reactant: benzoylacetone was obtained from Eastman Kodak (Eastman grade) product: 1-phenyl-1,3,5-hexanetrione product: 1,7-diphenyl-1,3,5,7-heptanetetraone product: 1,5-diphenyl-1,3,5-pentanetrione product: 1,5-di(p-methoxyphenyl)-1,3,5-pentanetrione Keywords: acylation; 1,2-dimethoxyethane, purification of; sodium hydride, handling of; sodium hydride, handling

α,α,β-Triphenylpropionitrile reactant: 30.5 g. (0.24 mole) of benzyl chloride product: α,α,β-triphenylpropionitrile Keywords: alkylation, C-alkylation; potassium amide, preparation of; ammonia; potassium

N,N-Dimethylaminomethylferrocene methiodide reactant: 43.2 g. (0.422 mole) of bis(dimethylamino)-methane intermediate: dimethylaminomethylferrocene product: N,N-dimethylaminomethylferrocene methiodide Keywords: alkylation, N-alkylation; amination, Mannich reaction; enamine or imine formation; bis(dimethylamino)methane, lachrymator

Ethyl 2,4-diphenylbutanoate product: ethyl 2,4-diphenylbutanoate product: ethyl 2-methyl-2-phenylpropanoate Keywords: alkylation, C-alkylation; ammonia