Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Molecular evolution of a repetitive region within the per gene of Drosophila

The clock gene period (per) controls a number of biological rhythms in Drosophila. In D. melanogaster, per has a repetitive region that encodes a number of alternating threonine-glycine residues. We sequenced and compared this region from several different Drosophila species belonging to various groups within the Drosophila and Sophophora subgenera. This part of per shows a great variability in both DNA sequence and length. Furthermore, analysis of the data suggests that changes in the length of this variable region might be associated with amino acid replacements in the more conserved flanking sequences.      

Anatomical and chemical characteristics of the seed coat of <i>Medicago sativa</i> L. (alfalfa) cv. Baralfa 85 seeds and their association with seed dormancy

Seeds of alfalfa (Medicago sativa L.) can exhibit seedcoat imposed dormancy, which produces hard seeds within a seed lot. These seeds do not germinate because they do not imbibe water due to a barrier to water entry in the seed coat. The aim of this work was to analyze the anatomical and chemical characteristics of the testa of alfalfa seeds with respect to water permeability levels. The anatomy of seeds of the cv. Baralfa 85 was studied and structural substances, polyphenols, tannins and cutin present in the testa of seeds of different water permeability levels were determined. The anatomical characteristics of the seed coat and the proportions of components were found to determine the permeability level of the seed coat, an aspect that is associated with the physical seed dormancy level. Anatomically, increased thickness of the testa was associated with a lower permeability level. The difference may be attributed to the variation in cuticle thickness, length of macrosclereids and thickness of the cell wall, and presence and development of osteosclereids. From the physiological and chemical points of view, the mechanism of physical dormancy of the testa is explained by a greater amount of components that repel water and cement the cell wall, such as polyphenols, lignins, condensed tannins, pectic substances, and a lower proportion of cellulose and hemicellulose.     

The genetic architecture of fitness in a seed beetle: assessing the potential for indirect genetic benefits of female choice

Background  

Quantifying the amount of standing genetic variation in fitness represents an empirical challenge. Unfortunately, the shortage of detailed studies of the genetic architecture of fitness has hampered progress in several domains of evolutionary biology. One such area is the study of sexual selection. In particular, the evolution of adaptive female choice by indirect genetic benefits relies on the presence of genetic variation for fitness. Female choice by genetic benefits fall broadly into good genes (additive) models and compatibility (non-additive) models where the strength of selection is dictated by the genetic architecture of fitness. To characterize the genetic architecture of fitness, we employed a quantitative genetic design (the diallel cross) in a population of the seed beetle Callosobruchus maculatus, which is known to exhibit post-copulatory female choice. From reciprocal crosses of inbred lines, we assayed egg production, egg-to-adult survival, and lifetime offspring production of the outbred F1 daughters (F1 productivity).     

Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death

Inner ear sensory hair cell death is observed in the majority of hearing and balance disorders, affecting the health of more than 600 million people worldwide. While normal aging is the single greatest contributor, exposure to environmental toxins and therapeutic drugs such as aminoglycoside antibiotics and antineoplastic agents are significant contributors. Genetic variation contributes markedly to differences in normal disease progression during aging and in susceptibility to ototoxic agents. Using the lateral line system of larval zebrafish, we developed an in vivo drug toxicity interaction screen to uncover genetic modulators of antibiotic-induced hair cell death and to identify compounds that confer protection. We have identified 5 mutations that modulate aminoglycoside susceptibility. Further characterization and identification of one protective mutant, sentinel (snl), revealed a novel conserved vertebrate gene. A similar screen identified a new class of drug-like small molecules, benzothiophene carboxamides, that prevent aminoglycoside-induced hair cell death in zebrafish and in mammals. Testing for interaction with the sentinel mutation suggests that the gene and compounds may operate in different pathways. The combination of chemical screening with traditional genetic approaches is a new strategy for identifying drugs and drug targets to attenuate hearing and balance disorders.     

Alternative splicing and protein function

Background  

Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data.     

Feedback equilibrium control during human standing

Equilibrium maintenance during standing in humans was investigated with a 3-joint (ankle, knee and hip) sagittal model of body movement. The experimental paradigm consisted of sudden perturbations of humans in quiet stance by backward displacements of the support platform. Data analysis was performed using eigenvectors of motion equation. The results supported three conclusions. First, independent feedback control of movements along eigenvectors (eigenmovements) can adequately describe human postural responses to stance perturbations. This conclusion is consistent with previous observations (Alexandrov et al., 2001b) that these same eigenmovements are also independently controlled in a feed-forward manner during voluntary upper-trunk bending. Second, independent feedback control of each eigenmovement is sufficient to provide its stability. Third, the feedback loop in each eigenmovement can be modeled as a linear visco-elastic spring with delay. Visco-elastic parameters and time-delay values result from the combined contribution of passive visco-elastic mechanisms and sensory systems of different modalities     

Low density lipoprotein (LDL) receptor-related protein 1B impairs urokinase receptor regeneration on the cell surface and inhibits cell migration

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family and is closely related to LRP. It was discovered as a putative tumor suppressor and is frequently inactivated in lung cancer cells. In the present study, we used an LRP1B minireceptor (mLRP1B4), which mimics the function and trafficking of LRP1B, to explore the roles of LRP1B on the plasminogen activation system. We found that mLRP1B4 and urokinase plasminogen activator receptor (uPAR) form immunoprecipitable complexes on the cell surface in the presence of complexes of uPA and its inhibitor, plasminogen activator inhibitor type-1 (PAI-1). However, compared with cells expressing the analogous LRP minireceptor (mLRP4), cells expressing mLRP1B4 display a substantially slower rate of uPA.PAI-1 complex internalization. Expression of mLRP1B4, or an mLRP4 mutant deficient in endocytosis, leads to an accumulation of uPAR at the cell surface and increased cell-associated uPA and PAI-1 when compared with cells expressing mLRP4. In addition, we found that expression of mLRP1B or the mLRP4 endocytosis mutant impairs the regeneration of unoccupied uPAR on the cell surface and that this correlates with a diminished rate of cell migration. Taken together, these results demonstrate that LRP1B can function as a negative regulator of uPAR regeneration and cell migration.     

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.     

The PINE study: rationale and design of a randomised comparison of epidural injection of local anaesthetics and steroids versus care-as-usual to prevent postherpetic neuralgia in the elderly [ISRCTN32866390]

BACKGROUND: Postherpetic neuralgia (PHN) is by far the most common complication of herpes zoster (HZ) and one of the most intractable pain disorders. Since PHN is seen most often in the elderly, the number of patients with this disorder is expected to increase in our ageing society. PHN may last for months to years and has a high impact on the quality of life. The results of PHN treatment are rather disappointing. Epidural injection of local anaesthetics and steroids in the acute phase of HZ is a promising therapy for the prevention of PHN. Since randomised trials on the effectiveness of this intervention are lacking, the PINE (Prevention by epidural Injection of postherpetic Neuralgia in the Elderly) study was set up. The PINE study compares the effectiveness and cost-effectiveness of a single epidural injection of local anaesthetics and steroids during the acute phase of HZ with that of care-as-usual (i.e. antivirals and analgesics) in preventing PHN in elderly patients. METHODS / DESIGN: The PINE study is an open, multicenter clinical trial in which 550 elderly (age >/= 50 yr.) patients who consult their general practitioner in the acute phase of HZ (rash < 7 days) are randomised to one of the treatment groups. The primary clinical endpoint is the presence of HZ-related pain one month after the onset of the rash. Secondary endpoints include duration and severity of pain, re-interventions aiming to treat the existing pain, side effects, quality of life, and cost-effectiveness. CONCLUSION: The PINE study is aimed to quantify the (cost-) effectiveness of a single epidural injection during the acute phase of HZ on the prevention of PHN.