Accuracy improvement for identifying translation initiation sites in microbial genomes

MOTIVATION: At present the computational gene identification methods in microbial genomes have a high prediction accuracy of verified translation termination site (3' end), but a much lower accuracy of the translation initiation site (TIS, 5' end). The latter is important to the analysis and the understanding of the putative protein of a gene and the regulatory machinery of the translation. Improving the accuracy of prediction of TIS is one of the remaining open problems. RESULTS: In this paper, we develop a four-component statistical model to describe the TIS of prokaryotic genes. The model incorporates several features with biological meanings, including the correlation between translation termination site and TIS of genes, the sequence content around the start codon; the sequence content of the consensus signal related to ribosomal binding sites (RBSs), and the correlation between TIS and the upstream consensus signal. An entirely non-supervised training system is constructed, which takes as input a set of annotated coding open reading frames (ORFs) by any gene finder, and gives as output a set of organism-specific parameters (without any prior knowledge or empirical constants and formulas). The novel algorithm is tested on a set of reliable datasets of genes from Escherichia coli and Bacillus subtillis. MED-Start may correctly predict 95.4% of the start sites of 195 experimentally confirmed E.coli genes, 96.6% of 58 reliable B.subtillis genes. Moreover, the test results indicate that the algorithm gives higher accuracy for more reliable datasets, and is robust to the variation of gene length. MED-Start may be used as a postprocessor for a gene finder. After processing by our program, the improvement of gene start prediction of gene finder system is remarkable, e.g. the accuracy of TIS predicted by MED 1.0 increases from 61.7 to 91.5% for 854 E.coli verified genes, while that by GLIMMER 2.02 increases from 63.2 to 92.0% for the same dataset. These results show that our algorithm is one of the most accurate methods to identify TIS of prokaryotic genomes. AVAILABILITY: The program MED-Start can be accessed through the website of CTB at Peking University: http://ctb.pku.edu.cn/main/SheGroup/MED_Start.htm.     

Using amino acid patterns to accurately predict translation initiation sites

The translation initiation site (TIS) prediction problem is about how to correctly identify TIS in mRNA, cDNA, or other types of genomic sequences. High prediction accuracy can be helpful in a better understanding of protein coding from nucleotide sequences. This is an important step in genomic analysis to determine protein coding from nucleotide sequences. In this paper, we present an in silico method to predict translation initiation sites in vertebrate cDNA or mRNA sequences. This method consists of three sequential steps as follows. In the first step, candidate features are generated using k-gram amino acid patterns. In the second step, a small number of top-ranked features are selected by an entropy-based algorithm. In the third step, a classification model is built to recognize true TISs by applying support vector machines or ensembles of decision trees to the selected features. We have tested our method on several independent data sets, including two public ones and our own extracted sequences. The experimental results achieved are better than those reported previously using the same data sets. Our high accuracy not only demonstrates the feasibility of our method, but also indicates that there might be "amino acid" patterns around TIS in cDNA and mRNA sequences.     

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.     

A computational study of RNA binding and specificity in the tandem zinc finger domain of TIS11d

TIS11d is a member of the CCCH-type family of tandem zinc finger (TZF) proteins; the TZF domain of TIS11d (residues 151–220) is sufficient to bind and destabilize its target mRNAs with high specificity. In this study, the TZF domain of TIS11d is simulated in an aqueous environment in both the free and RNA-bound states. Multiple nanosecond timescale molecular dynamics trajectories of TIS11d wild-type and E157R/E195K mutant with different RNA sequences were performed to investigate the molecular basis for RNA binding specificities of this TZF domain. A variety of measures of the protein structure, fluctuations, and dynamics were used to analyze the trajectories. The results of this study support the following conclusions: (1) the structure of the two fingers is maintained in the free state but a global reorientation occurs to yield a more compact structure; (2) mutation of the glutamate residues at positions 157 and 195 to arginine and lysine, respectively, affects the RNA recognition by this TIS11d mutant in agreement with the findings of Pagano et al. (J Biol Chem 2007; 282:8883–8894); and (3) we predict that the E157R/E195K mutant will present a more relaxed RNA binding specificity relative to wild-type TIS11d based on the more favorable nonsequence-specific Coulomb interaction of the two positively charged residues at positions 157 and 195 with the RNA backbone, which compensates for a partial loss of the stacking interaction of aromatic side chains with the RNA bases.     

An unsupervised classification scheme for improving predictions of prokaryotic TIS

Background  

Although it is not difficult for state-of-the-art gene finders to identify coding regions in prokaryotic genomes, exact prediction of the corresponding translation initiation sites (TIS) is still a challenging problem. Recently a number of post-processing tools have been proposed for improving the annotation of prokaryotic TIS. However, inherent difficulties of these approaches arise from the considerable variation of TIS characteristics across different species. Therefore prior assumptions about the properties of prokaryotic gene starts may cause suboptimal predictions for newly sequenced genomes with TIS signals differing from those of well-investigated genomes.     

Use of the ThinPrep Imaging System for internal quality control of cervical cytology

T. Heard, A. Chandra, G. Culora, S.S. Gupta, A. Herbert and M. Morgan
Use of the ThinPrep Imaging System for internal quality control of cervical cytology Objective: To audit the use of the ThinPrep Imaging System (TIS) for internal quality control (IQC) in the place of rapid review (RR), and to compare its performance with routine primary screening. Method: During 9 months, 16 462 ThinPrep slides were processed by TIS. Slides were initially reviewed using the TIS review scope, as recommended by the manufacturer: 22 fields of view were observed and, if considered abnormal, a full microscopic review was conducted using the review scope. Different biomedical scientists (BMSs), working on each procedure in rotation, performed batches of TIS‐assisted quality control and routine primary screening independently on unmarked slides. Any slides with abnormalities detected by either method were referred to a consultant pathologist or advanced BMS practitioner for a final report. TIS results were compared with both previous records of RR and routine primary screening carried out on the same slides. We used the UK terminology in which ‘dyskaryosis’ is equivalent to squamous intraepithelial lesion (SIL) and borderline to atypical (including squamous and glandular cells). Results: TIS preview detected significantly more high‐grade dyskaryosis compared with RR during the previous 4 years: 2.0–4.2 compared with 0.1–1.8 detected per 1000 slides (P = 0.0001). TIS and routine screening were equivalent in sensitivity and specificity for the final cytology result, but BMSs were significantly more likely to classify slides as dyskaryotic rather than borderline when using TIS compared with routine screening. Referrals for potentially high‐grade abnormalities detected by TIS‐assisted IQC alone found 28 biopsies of at least cervical intraepithelial neoplasia grade 2 (CIN2+), whereas 15 CIN2+ biopsies were found on routine screening but missed using TIS. There was no significant change in the rates of inadequate tests, high‐ or low‐grade cytological abnormalities, or positive predictive value for CIN2+ when TIS was in use. Conclusions: Screening with TIS was more sensitive than RR for IQC, providing a rescreening method equivalent to routine primary screening in overall accuracy.     

Modular organization of protein interaction networks

MOTIVATION: Accumulating evidence suggests that biological systems are composed of interacting, separable, functional modules. Identifying these modules is essential to understand the organization of biological systems. RESULT: In this paper, we present a framework to identify modules within biological networks. In this approach, the concept of degree is extended from the single vertex to the sub-graph, and a formal definition of module in a network is used. A new agglomerative algorithm was developed to identify modules from the network by combining the new module definition with the relative edge order generated by the Girvan-Newman (G-N) algorithm. A JAVA program, MoNet, was developed to implement the algorithm. Applying MoNet to the yeast core protein interaction network from the database of interacting proteins (DIP) identified 86 simple modules with sizes larger than three proteins. The modules obtained are significantly enriched in proteins with related biological process Gene Ontology terms. A comparison between the MoNet modules and modules defined by Radicchi et al. (2004) indicates that MoNet modules show stronger co-clustering of related genes and are more robust to ties in betweenness values. Further, the MoNet output retains the adjacent relationships between modules and allows the construction of an interaction web of modules providing insight regarding the relationships between different functional modules. Thus, MoNet provides an objective approach to understand the organization and interactions of biological processes in cellular systems. AVAILABILITY: MoNet is available upon request from the authors.     

原核基因翻译起始位点预测的新方法

翻译起始位点（TIS,即基因5’端）的精确定位是原核生物基因预测的一个关键问题,而基因组GC含量和翻译起始机制的多样性是影响当前TIS预测水平的重要因素．结合基因组结构的复杂信息（包括GC含量、TIS邻近序列及上游调控信号、序列编码潜能、操纵子结构等）,发展刻画翻译起始机制的数学统计模型,据此设计TIS预测的新算法MED．StartPlus．并将MED．StartPlus与同类方法RBSfinder、GS．Finder、MED-Start、TiCo和Hon-yaku等进行系统地比较和评价．测试针对两种数据集进行：当前14个已知的TIS被确认的基因数据集,以及300个物种中功能已知的基因数据集．测试结果表明,MED-StartPlus的预测精度在总体上超过同类方法．尤其是对高GC含量基因组以及具有复杂翻译起始机制的基因组,MED-StartPlus具有明显的优势．     

TICO: a tool for improving predictions of prokaryotic translation initiation sites

SUMMARY: We provide the tool 'TICO' (Translation Initiation site COrrection) for improving the results of conventional gene finders for prokaryotic genomes with regard to exact localization of the translation initiation site (TIS). At the current state TICO provides an interface for direct post processing of the predictions obtained from the widely used program GLIMMER. Our program is based on a clustering algorithm for completely unsupervised scoring of potential TIS locations. AVAILABILITY: Our tool can be freely accessed through a web interface at http://tico.gobics.de/ CONTACT: maike@gobics.de     

Engineering support vector machine kernels that recognize translation initiation sites

MOTIVATION: In order to extract protein sequences from nucleotide sequences, it is an important step to recognize points at which regions start that code for proteins. These points are called translation initiation sites (TIS). RESULTS: The task of finding TIS can be modeled as a classification problem. We demonstrate the applicability of support vector machines for this task, and show how to incorporate prior biological knowledge by engineering an appropriate kernel function. With the described techniques the recognition performance can be improved by 26% over leading existing approaches. We provide evidence that existing related methods (e.g. ESTScan) could profit from advanced TIS recognition.     

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.     

Improving secondary embryogenesis in <Emphasis Type="Italic">Quercus robur</Emphasis>: application of temporary immersion for mass propagation

The effects of the culture system used for embryo proliferation were investigated with the aim of improving multiplication rates and somatic embryo quality in two embryogenic lines of Quercus robur derived from mature trees (B-17 and Sainza). Embryo proliferation medium was defined following comparison of five different semi-solid media, and the highest multiplication rates (based on the total number of embryos and number of cotyledonary-shaped embryos) were achieved with medium supplemented with 0.44 μM benzyladenine for both lines. Embryo proliferation on semi-solid medium was compared with that obtained by a temporary immersion system (TIS), in which four cycles with immersion frequencies of 1 min every 6, 8, 12 or 24 h were tested. TIS promoted a significant increase in proliferated embryo biomass, with the growth index (GI) two and four times higher than in semi-solid medium in B-17 and Sainza genotypes, respectively. An immersion cycle of 1 min every 8 or 12 h produced approximately 700 somatic embryos (B-17) and 1,500 somatic embryos (Sainza) per RITA^® bioreactor, with significant differences in the latter genotype with respect to gelled medium. TIS had also a significant effect on somatic embryo synchronization as it enabled a higher production of cotyledonary embryos (90%), which represents increases of 14% (B-17) and 20% (Sainza) with respect to gelled medium. For germination of embryos proliferated in TIS two maturation systems were applied: (1) culture in semi-solid medium containing 6% sorbitol or (2) culture by TIS (without sorbitol) at a frequency of 1 min immersion every 48 h. Germination ability was higher after maturation on sorbitol medium and plantlet conversion occurred in 48% (B-17) and 13% (Sainza) embryos. TIS produced large numbers of well-developed cotyledonary embryos, hence reduced the cost and labor.