Current methods of gene prediction,their strengths and weaknesses

While the genomes of many organisms have been sequenced over the last few years, transforming such raw sequence data into knowledge remains a hard task. A great number of prediction programs have been developed that try to address one part of this problem, which consists of locating the genes along a genome. This paper reviews the existing approaches to predicting genes in eukaryotic genomes and underlines their intrinsic advantages and limitations. The main mathematical models and computational algorithms adopted are also briefly described and the resulting software classified according to both the method and the type of evidence used. Finally, the several difficulties and pitfalls encountered by the programs are detailed, showing that improvements are needed and that new directions must be considered.     

Variability of X chromosome inactivation: effect on levels of TIMP1 RNA and role of DNA methylation

X chromosome inactivation results in dosage equivalency for X-linked gene expression between males and females. However, some X-linked genes show variable X inactivation, being expressed from the inactive X in some females but subject to inactivation in other women. The human tissue inhibitor of metalloproteinases-1 ( TIMP1) gene falls into this category. As TIMP1 and its target metalloproteinases are involved in many biological processes, women with elevated TIMP1 expression may exhibit different disease susceptibilities. To address the potential impact of variable X inactivation, we analyzed TIMP1 expression levels by using an RNase protection assay. The substantial variation of TIMP1 expression observed in cells with monoallelic TIMP1 expression precluded analysis of the contribution of the inactive X to total TIMP1 RNA levels in females, so we examined expression in rodent/human somatic cell hybrids. TIMP1 expression levels varied more widely in hybrids retaining an inactive X than in those with an active X chromosome, suggesting variable retention of the epigenetic silencing mechanisms associated with X inactivation. Therefore, we investigated the contribution of methylation at the promoter to expression level variation and found that methylation of the TIMP1 promoter correlated with instability and low level expression, whereas stable TIMP1expression from the inactive X equivalent to that seen from the active X chromosome was observed when the promoter was unmethylated. Since all female cell lines examined showed methylation of the TIMP1 promoter, the contribution of expression from the inactive X appears minimal. However, as women age, they may accumulate cells stably expressing TIMP1 from the inactive X, with a resulting increase of TIMP1, which may explain some sex differences in various late-onset disorders.     

The nesprins are giant actin-binding proteins,orthologous to Drosophila melanogaster muscle protein MSP-300

Nesprin-1 and nesprin-2 (also known as Syne-1 and Syne-2,) are large ( approximately 3300-residue) vertebrate proteins associated with emerin and lamin A at the nuclear envelope of muscle cells and other cell types. We show that the previously described nesprins are short isoforms of giant proteins comprising an actin-binding amino-terminus connected to a carboxy-terminal klarsicht-related transmembrane domain by a massive ( approximately 6000-8000 amino acid) spectrin-like rod domain, making full-length nesprin-1, at one megadalton, the largest non-titin protein hitherto described in humans. We find that MSP-300, a 7000-residue Drosophila melanogaster protein whose disruption results in defects of muscle development, corresponds to the N-terminal two-thirds of the Drosophila nesprin ortholog. A nesprin-like protein is also encoded by the nematode genome. Moreover, we demonstrate that the larger isoforms of nesprin-1, like MSP-300, are localized to the sarcomeric Z-line of both skeletal and cardiac muscle. The recognition that a characteristic muscle-specific mutant phenotype in the fly results from a disruption of its nesprin ortholog reinforces the candidacy of the human proteins for involvement in genetic diseases of skeletal and cardiac muscle.     

The effects of centrally administered apelin-13 on food intake, water intake and pituitary hormone release in rats

Apelin is the recently identified endogenous ligand for the G-protein-coupled receptor, APJ. Preproapelin and APJ mRNA are found in hypothalamic regions known to be important in the regulation of food and water intake, and pituitary hormone release. The effects of intracerebroventricular (ICV) administration of pyroglutamylated apelin-13 on food and water intake and pituitary hormone release in rats were investigated. Apelin-13 had little effect on food intake, but dose-dependently increased drinking behaviour and water intake at 1 h. Apelin-13 (10 nmol) increased water intake by up to sixfold compared to saline. Compared to saline control, apelin-13 (10 nmol) significantly increased plasma ACTH and corticosterone and decreased plasma prolactin, LH and FSH at 30 min. In vitro, apelin-13 stimulated the release of CRH and AVP from hypothalamic explants, but had no effect on NPY release. These results suggest that apelin may play an important role in the hypothalamic regulation of water intake and endocrine axes.     

Smoking has no effect on the amino acid composition of apolipoprotein B100 of LDL while directly influencing the antioxidant status

Previous studies have demonstrated increased plasma levels of oxidised low-density lipoprotein (oxLDL) in chronic smokers, which has been associated with the extent of endothelial dysfunction. In this study we examine the relationship between the amino acid composition of apolipoprotein B100 (apo B) of low-density lipoprotein (LDL), by reverse phase HPLC after precolumn derivatisation, between smokers (> or =40 cigarettes/day) and nonsmokers in relation to their plasma and LDL antioxidant status. While there was a significant difference in the levels of plasma vitamin C and alpha-tocopherol between female smokers and nonsmokers, as well as in the levels of LDL alpha-tocopherol, there was no significant difference in the amino acid composition of apo B between the two groups.     

Molecular organization of amyloid protofilament-like assembly of betabellin 15D: helical array of beta-sandwiches

Betabellin is a 32-residue peptide engineered to fold into a four-stranded antiparallel beta-sheet protein. Upon air oxidation, the betabellin peptides can fold and assemble into a disulfide-bridged homodimer, or beta-sandwich, of 64 residues. Recent biophysical and ultrastructural studies indicate that betabellin 15D (B15D) (a homodimer of HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) forms unbranched, 35-A wide assemblies that resemble the protofilaments of amyloid fibers. In the present study, we have analyzed in detail the X-ray diffraction patterns of B15D prepared from acetonitrile. The fiber diffraction analysis indicated that the B15D fibril was composed of a double helix defined by the selection rule l = n + 7m (where l is even, and n and m are any integers), and having a 199-A period and pitch, 28-A rise per unit, and 10-A radius. This helical model is equivalent to a reverse-handed, single helix with half the period and defined by the selection rule l = -3n + 7m (where l is any integer). The asymmetric unit is the single B15D beta-sandwich molecule. These results suggest that the betabellin assembly that models the protofilaments of amyloid fibers is made up of discrete subunits on a helical array. Multiple intersheet hydrogen bonds in the axial direction and intersandwich polar interactions in the lateral direction stabilize the array.     

xMLP is an early response calcium target gene in neural determination in Xenopus laevis

In vertebrates, neural induction occurs during gastrulation when ectodermal cells choose between two fates, neural and epidermal. In Xenopus, neural induction has been regarded as a default pathway as it occurs, in dorsal ectoderm, when ventralizing signals (mainly Bone Morphogenesis Proteins, BMPs, potent epidermal inducers) are inhibited by dorsalizing signals, including factors such as noggin, chordin, and follistatin. However, our previous studies demonstrated that an instructive signal triggered by the activation of L-type voltage-sensitive calcium channels, resulting in a transient increase in intracellular free calcium, appears to be a necessary and sufficient requirement to induce the competent ectoderm toward the neural pathway. Here we further explore the relationship between the Ca2+ transient signals observed and the expression of early neural genes. We have performed a subtractive approach to identify the genes which are transcribed early after the calcium signal and involved in neural determination. We have analyzed a candidate gene (xMLP) which encodes a MARCKS-like protein, a substrate for PKC. We show that this gene is activated by a calcium transient signals and induced by noggin overexpression. xMLP is expressed at the right time in presumptive neural territories. The putative role of xMLP in the process of neural induction is discussed.     

Selection of the simplest RNA that binds isoleucine

We have identified the simplest RNA binding site for isoleucine using selection-amplification (SELEX), by shrinking the size of the randomized region until affinity selection is extinguished. Such a protocol can be useful because selection does not necessarily make the simplest active motif most prominent, as is often assumed. We find an isoleucine binding site that behaves exactly as predicted for the site that requires fewest nucleotides. This UAUU motif (16 highly conserved positions; 27 total), is also the most abundant site in successful selections on short random tracts. The UAUU site, now isolated independently at least 63 times, is a small asymmetric internal loop. Conserved loop sequences include isoleucine codon and anticodon triplets, whose nucleotides are required for amino acid binding. This reproducible association between isoleucine and its coding sequences supports the idea that the genetic code is, at least in part, a stereochemical residue of the most easily isolated RNA-amino acid binding structures.     

Heterologous expression of the<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> alcohol acetyltransferase genes in<Emphasis Type="Italic"> Clostridium acetobutylicum</Emphasis> and<Emphasis Type="Italic"> Escherichia coli</Emphasis> for the production of isoamyl acetate

Esters are formed by the condensation of acids with alcohols. The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings. Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates. The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized. The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity. This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C. acetobutylicum. ATF1 and ATF2 were cloned and expressed in E. coli and ATF2 was expressed in C. acetobutylicum. Isoamyl acetate production from the substrate isoamyl alcohol in E. coli and C. acetobutylicum cultures was determined by head-space gas analysis. Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector. The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1and ATF2 in bacteria for ester production.