Citrus is the main fruit crop in the world and, as such, an important commodity. For this reason, the sequencing in large scale of expressed sequence tags (ESTs) from citrus organs was performed as an approach to fill some of the gaps in knowledge concerning the genetic and molecular factors involved in several citrus diseases and fruit devel- opment. cDNAlibraries were constructed using tissues from different plant organs, such as leaf, flower, fruit, bark, seed and root, from twelve citrus species (Citrus sinensis, C. limonia, C. reticulata, C. aurantium, C. limettiodes, C. aurantifolia, C. sinensis x C. reticulata, C. sunki, C. latifolia, C. reshni, Citrumelo swingle, Fortunella margarita, Poncirus trifoliata). These plants were submit- ted to diverse situations of biotic stresses caused either by bacteria (Xylella fastidiosa), viruses (Citrus leprosis virus, Citrus tristeza virus) or Phytophthora parasitica, or abiotic stress caused by environmental factors, such as drought. Therefore, Brazilian citrus genome EST database (CitEST) covers a wide diversity of gene sequence information for the study of components of citrus defense response path- ways to pathogen, wounding and other abiotic stresses, which have often been used as marker resistance onset. In addition to providing an efficient method for gene discov- ery, the ESTs data set can also provide information about gene expression. However, the challenge is to extract bio- logical knowledge from large amounts of gene expression data deposited in databases.
To improve the probability of getting a maximum number of different ESTs, researchers have been using nor- malized and/or subtracted cDNAlibraries that bring the frequency of each clone in a cDNA library within a narrow range (Soares and Bonaldo 2000). However, normalization and/or subtraction procedures are in general laborious and have the tendency of increasing the proportion of small in- sert clones. In the SUCEST project we have implemented an efficient procedure to generate conventional cDNA li- braries to generate large scale ESTs from sugarcane. This paper describes the construction of these libraries, repre- senting all major organs, harvested at different develop- mental stages and used to generate one of the largest plant EST collections.
The analysis of the CitEST database, which was gen- erated through sequencing of ESTs from cDNAlibraries prepared from different tissues and species of Citrus (Cit- rus sinensis, Citrus reticulata, Citrus limonia, Citrus aurantium and Citrus aurantifolia), suggests the existence of at least 18 Hsp70 genes in the Citrus genome (Table 1). As the libraries displayed great variation in size and tissue representation among the species, the generated data were from a collective and global analysis. While this strategy produced statistically meaningful data, it did not rule out the possibility that the highly conserved isoforms were allelic variations of the same gene present in distinct Citrus species. Based on sequence comparison (Figure 1), predic- tion of subcellular localization (Table 1) and the presence of characteristic domains of the different HSP70 subgroups from other plant species (Sung et al., 2001a), the Citrus Hsp70-encoded proteins were grouped into five distinct classes or subfamilies. The four major subfamilies are rep- resented by eight cytosolic Hsp/Hsc70, one ER-resident BiP, four mitochondrial mtHsp70 and three csHsp70 from the chloroplast. The fifth subgroup is represented by two Hsp110/SSE-related high molecular weight Hsp70s (Table 1). Of the 18 probable genes in Citrus, only four represent full-length sequence contigs, Hsc70-5, mtHsp70-1, csHsp70-1 and Hsp70-1 (Table 1), and in the BiP-1 se- quence, a few amino acid residues are missing at the N-ter- minal region, as compared to the Lycopersicon esculentum BiP deduced protein sequence (Genbank protein accession number, gi:1346172).
cDNAlibraries were constructed from 1-2 µg poly(A)+ RNA using the SuperScript Plasmid System kit (Invitrogen), according to manufacturer’s protocol. Frac- tions containing cDNA larger than 500 bp were ligated into the SalI-NotI site of pSPORT1 vector (Invitrogen). Clones were sequenced from the 5’ends using the Big Dye Termi- nator Cycle Sequencing Ready Reaction kit (Applied Bio- systems) in conjunction with primer T7 (5’-TAATACGA CTCATATAGGG-3’).
In light of this context, we created a soybean database connecting public soybean data (like ESTs and genomic se- quences) and project data (like SuperSAGE tags, microRNAs and subtractive cDNAlibraries). This data- base offers search tools for users, including keyword searches, statistical comparisons, automatic annotation (us- ing some protein databases such as NR, Uniref, KEGG and Pfam), gene ontology classification and gene expression profiles under several conditions. Moreover, searches by sequence homology are possible using the local BLAST. All data are stored in a Fedora Linux machine, running the MySQL database server. The web interfaces (http://www.lge.ibi.unicamp.br/soybean) are based on a combination of CGI scripts using Perl language (including BioPerl module) and the Apache Web Server. As soon as the private data are published, the database will be freely available.
The co-existence of Zebu animals with the tick Riphicephalus (Boophilus) microplus over the centuries seems to have conferred greater resistance to these animals compared to Taurine animals. Little is known so far about the genetic mechanisms involved in the genetic resistance of Zebu animals to ticks. The identification of new genes and host antigens involved in the mechanism of resistance/susceptibility to the parasite are a promising approach. In this work, we compared the relative expression of susceptible and resistant animals groups using real-time polymerase chain reaction to determine the expression level of the calcium-binding proteins translationally-controlled tumor protein 1 (TPT1) and allergen Bos d3 (S100A7), and of the calcium channel protein transient receptor potential vanilloid 6 (TRPV6). The three genes were identified in cDNAlibraries prepared from skin lesions of susceptible animals and from healthy skin of resistant animals. Skin biopsies were obtained from F 2 cattle
In the second chapter the comparison of three sequenced cDNAlibraries from divergent genetic background identified sequences that were differentially expressed among them. From 34 differentially distributed gene sequences identified, 21 could be grouped into a network of functional relevance for muscularity; this may be helpful to better understand the molecular mechanisms involved in skeletal muscle development. However, this is a complex trait that is subject to action from a large number of genes that are regulated by several transcription regulatory elements (Carvajal & Rigby, 2010). To better understand the DEGs role during muscle development, their expression levels were measured during different muscle periods. Thus it was possible to confirm genes which are known to be related with myogenic stage as ANKRD2, MYBPC1, NEB and MYL2 (Bean et al. 2008; Gautel et al., 1998 & Kurasawa et al., 1999; Bang et al. 2006 and Zhang et al. 2009 respectively) having a prenatal high expression in this study. Furthermore, novel candidates for muscle development (TPM2, TP53 and DCTN1) can be listed.
One approach used to analyzing P. brasiliensis gene expression in host-like conditions involves using cDNA Representational Difference Analysis (cDNA-RDA) Hu- bank & Schatz 1994) to identify P. brasiliensis genes in- duced during the infective process in a mouse model of infection and under conditions that mimicked the hemato- genic route of fungal dissemination (Bailão et al. 2006), as well as infection sites with inflammation (Bailão et al. 2007). Subtracted cDNAlibraries were made using driver cDNAs from seven-day-old yeast cell cultures and tester cDNAs were synthesized from fungal cell RNAs recovered from the liver of infected animals and from yeast cells after artificial infection with human blood or plasma. The subtracted cDNAlibraries and the number of transcripts generated are summarized in Fig. 1. A total of 45, 86 and 99 over-expressed sequences were obtained from the subtracted cDNAlibraries of yeast cells recov- ered from infected liver (Bailão et al. 2006) and from yeast cells incubated with human blood or plasma (Bailão et al. 2006, 2007), respectively, as shown in Fig. 1.
In order to obtain a comprehensive profile of genes expressed in the rubber tree, we constructed a cDNA library from the latex of H. brasiliensis and analysed the expressed sequence tag (EST) of these H. brasiliensis cDNAlibraries. Identification of several novel genes expressed in the rubber tree latex could provide information that would provide us with a better understanding of the molecular events involved in rubber biosynthesis and/or plant defensive mechanisms against pathogens.
USA, for Illumina cDNA library constructions and sequencing. Prior to cDNA library constructions, the RNA quality and concentration were checked with the Agilent Bioanalyzer 2100 using an Agilent RNA 6000 Nano Chip (Agilent Technologies, USA) and 0.5 m g of each cDNA library was used to prepare Illumina cDNA sequencing libraries. Poly-A mRNA was purified using the oligo-dT beads provided in the NEBNExt Poly(A) mRNA Magnetic Isolation Module (New England Biolabs (NEB), USA). Libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit (NEB) for Illumina and indexed with the NEBNext Mulitplex Oligos for Illumina (NEB). The Poly-A mRNA was chemically fragmented and primed with random oligos for first strand cDNA synthesis with a heating step of 94 uC for 5 min. First strand cDNA synthesis was performed with an incubation time of 10 min at 25uC, 50 min at 42uC, and 15 min at 70uC. Second strand cDNA synthesis was carried out with dUTPs to preserve strand orientation information. The sample was purified, end repaired and dA-tailed prior to adaptor ligation. Illumina Multiplex Adaptors were ligated, the ligation reaction was purified according to the protocol for a 500–700 bp insert, and a PCR amplification of 15 cycles was performed. The PCR product was purified and the cDNAlibraries were checked for quality and concentration with the Agilent Bioanalyzer 2100 using a High Sensitivity DNA Chip (Agilent Technologies, USA). The 10 prepared Illumina cDNAlibraries were pooled in equal molar amounts and the resulted, final pooled cDNA library was run on a Illumina MiSeq instrument using the MiSeq Reagent Kit v3 (Illumina, USA). Ten independent 26300 bp paired-end runs were carried out and clustered at a concentration of 8pM. The software packages Real Time Analysis (RTA), version 1.18.42, MiSeq Control Software (MCS), version 2.3.0, and MiSeq Reporter, version 2.3.32, were used for sequencing and to generate fastq files.
Brazil is the largest citrus producer in the world, being responsible for more than 20% of its production, which is, however still low due to phytosanitary issues such as citrus blight. Citrus blight is an anomaly whose causes still have not yet been determined, therefore there are no efficient control measures to minimize the production losses with the use of resistant varieties being considered the most appropriate method. However, little is known about the genes involved in the defense response of the plants to this anomaly. Considering that many physiological alterations associated with plant stress responses are controlled at a transcriptional level, in this study we sought the identification and characterization of the gene expression products differentially expressed in the response to the citrus blight. Through the suppressive subtractive hybridization technique, expressed cDNAlibraries were built using mRNAs isolated from “Cravo” lemon tree roots (Citrus limonia L. Osbeck) under “Pera” orange (Citrus sinensis L. Osbeck) of healthy and sick plants. 129 clones were obtained by subtraction and their sequences were compared in databases. 34 of them linked to proteins associated to stress processes, while the others were similar to sequences of unknown functions or did not present similarity with sequences deposited in the databases. 3 genes were selected and their expressions were studied by RT - qPCR in real-time. Plants with citrus blight presented an increase of the expression level in two of those genes, suggesting that these can be directly involved with this anomaly.
Libraries are expensive and research libraries are particularly expensive. Even in the United States, few people can afford good access to primary scientific, medical, legal and scholarly information. Members of major universities have excellent library services. So do people who work in teaching hospitals, or for drug companies or rich law firms. Others have access to information only through the tedious, inefficient system of interlibrary lending. In less affluent countries the situation is worse; even the best universities cannot afford good libraries. Must access to scientific and professional information always be expensive, or is it possible that digital libraries might change this sad situation? The costs of a conventional research library fall into three main categories: facilities (which include buildings), library materials and staff. In digital libraries, the facilities costs are small, since digital libraries avoid the need for expensive buildings. Digital libraries require computers and networks, but these are relatively inexpensive, and the costs to users are shared with other services, such as electronic mail and word processing.
University libraries provide an important service to society, contributing to spread knowledge and cultivating new talents in the academic environment. The main objective of this article is to perform a review of the literature on efficiency assessments in the context of university libraries. The databases Web of Science and Scopus were adopted as reference to search for papers in the aforementioned context and identify the methodologies used and perspectives set out by different authors. A complementary search was also made on Google Scholar to obtain additional articles. In sum, 34 papers were found to compose the core of analyzed publications. As result, we observed that nearly 90% of the papers use Data Envelopment Analysis to assess the efficiency of university libraries and other methodologies with the same proposal were identified. Moreover, the variables used in these publications were analyzed, contributing to the mapping of main inputs and outputs that directly affect the services of university libraries. Furthermore, other characteristics were also considered, such as: temporal placement of publications and countries with the largest production of papers. Finally, based on the results of this study, further researches are suggested. Keywords: Academic libraries. Evaluation methods. Bibliographical revision.
We are interested in how intragenic recombination contributes to the evolution of proteins and how this mechanism complements and enhances the diversity generated by random mutation. Experiments have revealed that proteins are highly tolerant to recombination with homologous sequences (mutation by recombination is conservative); more surprisingly, they have also shown that homologous sequence fragments make largely additive contributions to biophysical properties such as stability. Here, we develop a random field model to describe the statistical features of the subset of protein space accessible by recombination, which we refer to as the recombinational landscape. This model shows quantitative agreement with experimental results compiled from eight libraries of proteins that were generated by recombining gene fragments from homologous proteins. The model reveals a recombinational landscape that is highly enriched in functional sequences, with properties dominated by a large-scale additive structure. It also quantifies the relative contributions of parent sequence identity, crossover locations, and protein fold to the tolerance of proteins to recombination. Intragenic recombination explores a unique subset of sequence space that promotes rapid molecular diversification and functional adaptation.
We determined the contribution of exopolysaccharide and LPS to histone resistance and in the pathogenic Gram-negative bacteria, S. flexneri and K. pneumoniae. Both of these species produce O-antigen+ LPS but only K. pneumoniae is a capsulated. We compared the MBC90 and MBC99 of wild type K. pneumoniae Kp52145 strain with the acapsulate mutant 52K10 . K. pneumoniae with or without capsule were resistant to histones (Fig. 5A), but the MBC90 of the acapsulate mutant is significantly higher than that of the wild type strain. The capsule seemed to have a limited impact on histone resistance. S. flexneri is naturally acapsulated but O-antigen+, showed a higher histone resistance than MACH1 E. coli carrying empty vectors (Fig. 5B, Fig. 3A–F). To address histone protection via Figure 1. Screening of histone resistant E. coli from pooled over-expression libraries. . The libraries consisted of a pooled of E. coli harboring plasmids carrying random fragments of E. coli DNA of 1, 2, 4 and 8 kb. The selection of the libraries consisted of elimination of histone susceptible clones (3 h), followed by a recovery step (2 h), and this repeated 3 times. After the selection, the plasmid population was extracted and analyzed by microarray. (B) During the selection process, the bacterial population was quantified by serial dilution and plating. Two controls were running in parallel, one consisting of the libraries without histone selection, and another of E. coli carrying empty vector submitted to histone killing. (C) Several clones from the different steps of the selection were isolated to identify the plasmid insert by sequencing. An insert corresponding to the genomic region from wcaI to wcaJ was enriched during the histone selection. The wcaI/J region is part of the colanic acid cluster. (D) After microarray analysis of the histone selected population, two plasmid inserts showed up belonging to the colanic acid cluster: wcaI/cpsB/cpsG/wcaJ and wzc/wcaA. The fitness for each gene was calculated by the logarithmic value of the intensity of the DNA fragment divided by the logarithmic value of the vector’s backbone and compared to the normalized intensity before selection.
The authors are grateful to Professor Norman Maclean for his helpful suggestions and Dr. Olga Francino for fruitful discussions. We also thank Rodrigo Maggioni for technical assistance with cDNA sequencing and Dr. Arati Iyengar for her careful reading of the manuscript. This research was partially supported by IFS (International Foundation for Science, Stockholm, Sweden) through a grant to L.F. Marins (Research Grant Agreement No. A/2915-1), CAPES (Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil), UAB (Universitat Autònoma de Barcelona, Spain) and FURG (Fundação Universidade Fe- deral do Rio Grande, Brazil).
During the period of 1992-2002, more than 700 colleges had been incorporated in China. At the same time, the university libraries also had been incorporated. This article is to analysis on the university libraries after colleges have been incorporated and what the university libraries ought to do under the mode of school management. Keywords : University library; Library work; Mode of school management; University merge
chlorophyll a-b binding protein, plastocyanin oxidoreductase iron–sulfur protein, and ribulose-1,5-bisphosphate carboxylase oxygenase large/small subunit. These proteins suggest the robust ability of Chlorella to restart photosynthetic function to protect heterotrophically grown cells from photodamage under sudden light stress. Likewise, the upregulation of these proteins in response to light stress has also been reported in higher plants and algae [37,38]. By contrast, the heterotrophic cells were expectedly deprived of photosynthetic performance; thus, few genes related to photosynthetic pathways were found in the reverse library. However, two special cases were found, namely, the antenna proteins of light-harvesting complexes (RYG131 and RYG034), which showed enhanced expression in response to heterotrophy. Plant and green algal light-harvesting complexes are composed of more than 20 different antenna proteins associated with photo- systems I and II . Although the function of antenna proteins in darkness-grown cells was not explored, the relatively increased expression indicates their importance in heterotrophic Chlorella. In unicellular green algae Dunaliella tertiolecta, an increase in the relative abundance of chlorophyll a/b light-harvesting complex mRNA was also found following a shift from high light to darkness and from high light to low light . LaRoche et al proposed that changes in energy balance brought about by a change in light intensity may control a regulatory factor acting to repress chlorophyll a/b binding protein mRNA expression . Further- more, a unigene that encodes glutamate dehydrogenase (GDH), was also found in the forward library. GDH is an important intermediate enzyme between carbon and nitrogen metabolism and it functions in multiple directions, depending on the environment and the stress [41,42]. In transgenic plants, the overexpression of microbial GDH reportedly confers improved tolerance to herbicides, drought, and pathogenic infections . Figure 3. Gene Ontology (GO) annotation of genes obtained from the SSH libraries. GO predictions identified several categories based on the three terms cellular component, molecular function, and biological process, and were plotted by WEGO. LI represents forward library under the light-induced treatment; HC represents the reverse library under the heterotrophic culture process.