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.      

Mechanism of free fatty acid re-esterification in human adipocytes in vitro

Within adipose tissue, free fatty acids liberated by lipolysis may be re-esterified into newly synthesized triacylglycerol. We hypothesized that re-esterification may occur via an extracellular route, such that free fatty acids arising from lipolysis must leave the adipocyte and be taken up again before they can be re-esterified. We simultaneously measured rates of lipolysis, acylglycerol synthesis, and free fatty acid re-esterification in human adipose tissue and isolated adipocytes in vitro, utilizing a dual-isotopic technique. We manipulated incubations to increase mixing of released free fatty acids with the incubation medium. Such manipulations should decrease the probability that released free fatty acids would be taken up and re-esterified. We found that re-esterification was decreased in isolated adipocytes compared to fragments of tissue, in shaken compared to unshaken incubations, and in low adipocyte concentrations compared to high adipocyte concentrations. Rates of acylglycerol synthesis and lipolysis were unaltered by these manipulations, indicating that changes in free fatty acid re-esterification are not secondary to effects on these processes. The results are consistent with an extracellular route for free fatty acid re-esterification. Such a mechanism suggests that adipose tissue blood flow may play an important role in the regulation of free fatty acid release from adipose tissue.     

Variants of ST8SIA1 are associated with risk of developing multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system of unknown etiology with both genetic and environmental factors playing a role in susceptibility. To date, the HLA DR15/DQ6 haplotype within the major histocompatibility complex on chromosome 6p, is the strongest genetic risk factor associated with MS susceptibility. Additional alleles of IL7 and IL2 have been identified as risk factors for MS with small effect. Here we present two independent studies supporting an allelic association of MS with polymorphisms in the ST8SIA1 gene, located on chromosome 12p12 and encoding ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1. The initial association was made in a single three-generation family where a single-nucleotide polymorphism (SNP) rs4762896, was segregating together with HLA DR15/DQ6 in MS patients. A study of 274 family trios (affected child and both unaffected parents) from Australia validated the association of ST8SIA1 in individuals with MS, showing transmission disequilibrium of the paternal alleles for three additional SNPs, namely rs704219, rs2041906, and rs1558793, with p = 0.001, p = 0.01 and p = 0.01 respectively. These findings implicate ST8SIA1 as a possible novel susceptibility gene for MS.     

Ratio-dependent parasitism with Trissolcus basalis (Wollaston) (Hymenoptera: Scelionidae) on egg rafts of Nezara viridula (Linnaeus) (Hemiptera: Pentatomidae): effect of experimental variables and compatibility of 'ratio' and 'Holling' models

Abstract Egg rafts of Nezara viridula were exposed to the parasitoid wasp Trissolcus basalis in experimental arenas to establish the relationship of the rates of attack and parasitism to various combinations of arena size, parasitoid density, host density and parasitoid-to-host ratio. Arena sizes were varied in the ratio 1:9:63, with the largest having a search area of 1.44 m². Parasitoid and host densities were varied over a 27-fold range. The parasitoid-to-host ratios used were 1:1, 3:1 and 6:1. Finding time was related to a constant factor (flight propensity), rather than to the difficulty of finding (density of hosts). Initial attack rates were therefore related only to parasitoid numbers (or density), even at the lower densities and ratios. Parasitism rates (a function of attack rate per host) were thus also strongly related to parasitoid to host ratio, regardless of densities used and arena sizes. Even reducing host density, while keeping time and parasitoid density constant, increased the parasitism rate. A ratio model for parasitism rate was therefore compatible with the data but the more explicit Holling 'disc' equation was also compatible because handling time was sufficiently large to make it sensitive to the ratio of parasitoids to hosts for the densities used. We conclude that the two models would predict different results if the density of host egg rafts was in a range below one per square metre.     

ER Stress Induced by Tunicamycin Triggers α-Synuclein Oligomerization,Dopaminergic Neurons Death and Locomotor Impairment: a New Model of Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD’s etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 μg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 μg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.     

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.     

Internalization-dependent and -independent requirements for transforming growth factor beta receptor signaling via the Smad pathway

Members of the transforming growth factor β (TGF-β) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-β signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-β receptor (TGF-βR) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-βR internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-β-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-βR complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-βR endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.     

Bovine herpesvirus-1 associated with single, trypsin-treated embryos was not infective for uterine tubal cells

It has been reported that bovine herpesvirus-1 (BHV-1) remains associated with in vitro-produced (IVP) bovine embryos after exposure to the virus and either washing or trypsin treatment. However, it is not known if the quantity of virus associated with an exposed IVP embryo is likely to infect a recipient cow after transfer. The specific objective of this study was to determine if IVP embryos that were exposed to BHV-1 would infect uterine tubal cells (UTC) in a co-culture system. In vitro-produced Day 7 embryos were exposed to BHV-1 and then washed or trypsin treated according to the IETS guidelines. These embryos were then co-cultured individually or in groups with UTC in microdrops of tissue culture medium 199 (TCM 199) supplemented with 10% equine serum. Following co-culture for 48 h, virus isolation was attempted on the embryos and the UTC from each drop. Virus was detected in washed individual embryos, groups of washed embryos, groups of trypsin-treated embryos and the UTC co-cultured with each of these treatments. However, BHV-1 was not detected in the individual, trypsin-treated embryos or the UTC co-cultured with them. It is concluded that trypsin treatment might effectively prevent infection of recipients if individual, Day 7, exposed embryos were transferred into the uterus.