A reference database for tumor-related genes co-expressed with interleukin-8 using genome-scale in silico analysis

Background

The EST database provides a rich resource for gene discovery and in silico expression analysis. We report a novel computational approach to identify co-expressed genes using EST database, and its application to IL-8.

Results

IL-8 is represented in 53 dbEST cDNA libraries. We calculated the frequency of occurrence of all the genes represented in these cDNA libraries, and ranked the candidates based on a Z-score. Additional analysis suggests that most IL-8 related genes are differentially expressed between non-tumor and tumor tissues. To focus on IL-8's function in tumor tissues, we further analyzed and ranked the genes in 16 IL-8 related tumor libraries.

Conclusions

This method generated a reference database for genes co-expressed with IL-8 and could facilitate further characterization of functional association among genes.     

Expressed Sequence Tags from a Root-Hair-Enriched Medicago truncatula cDNA Library

The root hair is a specialized cell type involved in water and nutrient uptake in plants. In legumes the root hair is also the primary site of recognition and infection by symbiotic nitrogen-fixing Rhizobium bacteria. We have studied the root hairs of Medicago truncatula, which is emerging as an increasingly important model legume for studies of symbiotic nodulation. However, only 27 genes from M. truncatula were represented in GenBank/EMBL as of October, 1997. We report here the construction of a root-hair-enriched cDNA library and single-pass sequencing of randomly selected clones. Expressed sequence tags (899 total, 603 of which have homology to known genes) were generated and made available on the Internet. We believe that the database and the associated DNA materials will provide a useful resource to the community of scientists studying the biology of roots, root tips, root hairs, and nodulation.     

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Background

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.

Results

In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.

Conclusion

The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.     

EST-based gene discovery in pig: virtual expression patterns and comparative mapping to human

A molecular understanding of porcine reproduction is of biological interest and economic importance. Our Midwest Consortium has produced cDNA libraries containing the majority of genes expressed in major female reproductive tissues, and we have deposited into public databases 21,499 expressed sequence tag (EST) gene sequences from the 3 end of clones from these libraries. These sequences represent 10,574 different genes, based on sequence comparison among these data, and comparison with existing porcine ESTs and genes indicate as many as 4652 of these EST clusters are novel. In silico analysis identified sequences that are expressed in specific pig tissues or organs and confirmed the broad expression in pig for many genes ubiquitously expressed in human tissues. Furthermore, we have developed computer software to identify sequence similarity of these pig genes with their human counterparts, and to extract the mapping information of these human homologues from genome databases. We demonstrate the utility of this software for comparative mapping by localizing 61 genes on the porcine physical map for Chromosomes (Chrs) 5, 10, and 14. The following Accession numbers were assigned to our deposited sequences: BF701840 – BF704551, BF708383, BF708386 – BF713604, BG322266 – BG322271, BI398567 – BI405235, BQ597354 – BQ605166.     

Establishment of a high throughput EST sequencing system using poly(A) tail-removed cDNA libraries and determination of 36 000 bovine ESTs

We determined 36 310 bovine expressed sequence tag (EST) sequences using 10 different cDNA libraries. For massive EST sequencing, we devised a new system with two major features. First, we constructed cDNA libraries in which the poly(A) tails were removed using nested deletion at the 3′-ends. This permitted high quality reading of sequences from the 3′-end of the cDNA, which is otherwise difficult to do. Second, we increased throughput by sequencing directly on templates generated by colony PCR. Using this system, we determined 600 cDNA sequences per day. The read-out length was >450 bases in >90% of the sequences. Furthermore, we established a data management system for analyses, storage and manipulation of the sequence data. Finally, 16 358 non-redundant ESTs were derived from ~6900 independent genes. These data will facilitate construction of a precise comparative map across mammalian species and isolate the functional genes that govern economic traits. This system is applicable to other organisms, including livestock, for which EST data are limited.     

Update of MAGEST: Maboya Gene Expression patterns and Sequence Tags

MAGEST is a database for maternal gene expression information for an ascidian, Halocynthia roretzi. The ascidian has become an animal model in developmental biological research because it shows a simple developmental process, and belongs to one of the chordate groups. Various data are deposited into the MAGEST database, e.g. the 3′- and 5′-tag sequences from the fertilized egg cDNA library, the results of similarity searches against GenBank and the expression data from whole mount in situ hybridization. Over the last 2 years, the data retrieval systems have been improved in several aspects, and the tag sequence entries have increased to over 20 000 clones. Additionally, we constructed a database, translated MAGEST, for the amino acid fragment sequences predicted from the EST data sets. Using this information comprehensively, we should obtain new information on gene functions. The MAGEST database is accessible via the Internet at http://www.genome.ad.jp/magest/.     

Phytosulfokine-α Promotes Root Growth by Repressing Expression of <i>Pectin Methylesterase Inhibitor</i> (<i>PMEI</i>) Genes in <i>Medicago truncatula</i>

Phytosulfokine-α (PSK-α), a sulfated pentapeptide with the sequence YIYTQ, is encoded by a small precursor gene family in Arabidopsis. PSK-α regulates multiple growth and developmental processes as a novel peptide hormone. Despite its importance, functions of PSK-α in M. truncatula growth remains unknown. In this study, we identified five genes to encode PSK-α precursors in M. truncatula. All of these precursors possess conserved PSK-α signature motif. Expression pattern analysis of these MtPSK genes revealed that each gene was expressed in a tissue-specific or ubiquitous pattern and three of them were remarkably expressed in root. Treatment of M. truncatula seedlings with synthetic PSK- α peptide significantly promoted root elongation. In addition, expression analysis of downstream genes by RNA-seq and qRT-PCR assays suggested that PSK-α signaling might regulate cell wall structure via PMEI-PME module to promote root cell growth. Taken together, our results shed light on the mechanism by which PSK-α promotes root growth in M. truncatula, providing a new resource for improvement of root growth in agriculture.     

Specificity of the ModA11, ModA12 and ModD1 epigenetic regulator N6-adenine DNA methyltransferases of Neisseria meningitidis

Phase variation (random ON/OFF switching) of gene expression is a common feature of host-adapted pathogenic bacteria. Phase variably expressed N⁶-adenine DNA methyltransferases (Mod) alter global methylation patterns resulting in changes in gene expression. These systems constitute phase variable regulons called phasevarions. Neisseria meningitidis phasevarions regulate genes including virulence factors and vaccine candidates, and alter phenotypes including antibiotic resistance. The target site recognized by these Type III N⁶-adenine DNA methyltransferases is not known. Single molecule, real-time (SMRT) methylome analysis was used to identify the recognition site for three key N. meningitidis methyltransferases: ModA11 (exemplified by M.NmeMC58I) (5′-CGY^m6AG-3′), ModA12 (exemplified by M.Nme77I, M.Nme18I and M.Nme579II) (5′-AC^m6ACC-3′) and ModD1 (exemplified by M.Nme579I) (5′-CC^m6AGC-3′). Restriction inhibition assays and mutagenesis confirmed the SMRT methylome analysis. The ModA11 site is complex and atypical and is dependent on the type of pyrimidine at the central position, in combination with the bases flanking the core recognition sequence 5′-CGY^m6AG-3′. The observed efficiency of methylation in the modA11 strain (MC58) genome ranged from 4.6% at 5′-GCGC^m6AGG-3′ sites, to 100% at 5′-ACGT^m6AGG-3′ sites. Analysis of the distribution of modified sites in the respective genomes shows many cases of association with intergenic regions of genes with altered expression due to phasevarion switching.     

Genome-wide identification and expression analysis of the GRAS family proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The GRAS gene family performs a variety of functions in plant growth and development processes, and they also play essential roles in plant response to environmental stresses. Medicago truncatula is a diploid plant with a small genome used as a model organism. Despite the vital role of GRAS genes in plant growth regulation, few studies on these genes in M. truncatula have been conducted to date. Using the M. truncatula reference genome data, we identified 68 MtGRAS genes, which were classified into 16 groups by phylogenetic analysis, located on eight chromosomes. The structure analysis indicated that MtGRAS genes retained a relatively constant exon–intron composition during the evolution of the M. truncatula genome. Most of the closely related members in the phylogenetic tree had similar motif compositions. Different motifs distributed in different groups of the MtGRAS genes were the sources of their functional divergence. Twenty-eight MtGRAS genes were expressed in six tissues, namely root, bud, blade, seedpod, nodule, and flower tissues, suggesting their putative function in many aspects of plant growth and development. Nine MtGRAS genes were upregulated under cold, freezing, drought, ABA, and salt stress treatments, indicating that they play vital roles in the response to abiotic stress in M. truncatula. Our study provides valuable information that can be utilized to improve the quality and agronomic benefits of M. truncatula and other plants.     

NotI clones in the analysis of the human genome

NotI linking clones contain sequences flanking NotI recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of NotI clones in genome research, high density grids with 50 000 NotI linking clones originating from six representative NotI linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of NotI sites in the human genome. A total of 3437 sequences flanking NotI sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity ≤ 90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of NotI sites with the first exon and suggest that the so-called 3′ ESTs can actually be generated from 5′-ends of genes that contain NotI sites in their first exon.