Gene fusion/fission is a major contributor to evolution of multi-domain bacterial proteins

Most proteins comprise one or several domains. New domain architectures can be created by combining previously existing domains. The elementary events that create new domain architectures may be categorized into three classes, namely domain(s) insertion or deletion (indel), exchange and repetition. Using 'DomainTeam', a tool dedicated to the search for microsyntenies of domains, we quantified the relative contribution of these events. This tool allowed us to collect homologous bacterial genes encoding proteins that have obviously evolved by modular assembly of domains. We show that indels are the most frequent elementary events and that they occur in most cases at either the N- or C-terminus of the proteins. As revealed by the genomic neighbourhood/context of the corresponding genes, we show that a substantial number of these terminal indels are the consequence of gene fusions/fissions. We provide evidence showing that the contribution of gene fusion/fission to the evolution of multi-domain bacterial proteins is lower-bounded by 27% and upper-bounded by 64%. We conclude that gene fusion/fission is a major contributor to the evolution of multi-domain bacterial proteins.     

Cell enlargement: One possible mechanism underlying cellular senescence

We previously demonstrated an inverse relationship between the G1 volume of human diploid fibroblast-like (HDFL) cells obtained from foreskin tissue and clonal replicative potential. On the basis of these results, we suggested that one process underlying in vitro senescence is a progressive increase in the mean cell volume of successive progeny within clonal lineages. We now report that the size of HDFL cells, as well as of chick embryo fibroblasts, can be increased in the virtual absence of cell division by culturing at low density and at low serum concentration (0.1-1.0%). Consequent to an increase in cell size, the replicative potential of the cells is reduced to the level of later-passage cells of similar size. By clonal analysis, the populations of enlarged cells contain up to three times as many nondividing cells as do controls. In the enlarged populations, the proportion of cells producing attenuated clones (four or fewer progeny) increases by about 30%, whereas the proportion of cells yielding >32 cells declines by a similar percentage. These observations lead us to propose that replicative potential may be limited by cell size, which in turn may be regulated by a kinetic relationship between cellular growth and cell division cycles.     

Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.

Homocysteine thiolactone, a cyclic thioester, is synthesized by certain aminoacyl-tRNA synthetases in editing or proofreading reactions that prevent translational incorporation of homocysteine into proteins. Although homocysteine thiolactone is expected to acylate amino groups in proteins, virtually nothing is known regarding reactivity of the thiolactone. Here it is shown that reactions of the thiolactone with protein lysine residues were robust under physiological conditions. In human serum incubated with homocysteine thiolactone, protein homocysteinylation was a major reaction that could be observed with as little as 10 nM thiolactone. Individual proteins were homocysteinylated at rates proportional to their lysine contents. Homocysteinylation led to protein damage, manifested as multimerization and precipitation of extensively modified proteins. Model enzymes, such as methionyl-tRNA synthetase and trypsin, were inactivated by homocysteinylation. Metabolic conversion of homocysteine to the thiolactone, protein homocysteinylation, and resulting protein damage may underlie involvement of Hcy in the pathology of vascular disease.-Jakubowski, H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.     

Gene transfer is a major factor in bacterial evolution

Lateral gene transfer in four strains of Salmonella enterica has been assessed using genomic subtraction. Strain LT2 (subspecies I serovar Typhimurium) chromosomal DNA was used as target and subtracted by three subspecies I strains of serovars Typhimurium (S21), Muenchen (S71), Typhi (M229), and a subspecies V strain (M321). Data from probing random cosmids of LT2 DNA with preparations of the residual LT2 DNA after subtraction were used to estimate the amounts of LT2 DNA not able to hybridize to strains S21, S71, M229, and M321 to be in the range of 84-106, 191-355, 305-629, and 778-1,286 kb, respectively. Several lines of evidence indicate that most of this DNA is from genes not present in strain M321 and not from genes that have diverged in sequence. The amounts correlate with the divergence of the four strains as revealed by multilocus enzyme electrophoresis and sequence variation of housekeeping genes. Sequence of 39 of the fragments from the M321 subtracted residual LT2 DNA revealed only six inserts of known gene function with evidence of both gain and loss of genes during the development of S. enterica clones. Sixteen of the 39 segments have 45% or lower G+C content, below the species average, but over half are within the normal range for the species. We conclude that even within a species, clones may differ by up to 20% of chromosomal DNA, indicating a major role for lateral transfer, and that on the basis of G+C content, a significant proportion of the DNA is from distantly related species.      

Prevention of the disturbance of protein synthesis is a possible mechanism of the interferon protective activity.

Interferon and interferon inducer (Newcastle disease virus), were shown to enhance the cell resistance against the damaging effects of streptomycin. The preliminary intravenous injection of NDV to mice resulted in a diminishing of both qualitative and quantitative changes in activity of the enzyme myeloperoxydase caused by streptomycin in leucocytes from peritoneal exudate. Protective effect was conferred also by immediate pretreatment of mouse leucocytes with homologous interferon. The data presented suggest that protective action of interferon involves the control of normal protein synthesis in the cell.     

Gene conversion as a secondary mechanism of short interspersed element (SINE) evolution.

The Alu repetitive family of short interspersed elements (SINEs) in primates can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. The older subfamilies are generally very abundant, while the younger subfamilies have fewer copies. Some of the youngest Alu elements are absent in the orthologous loci of nonhuman primates, indicative of recent retroposition events, the primary mode of SINE evolution. PCR analysis of one young Alu subfamily (Sb2) member found in the low-density lipoprotein receptor gene apparently revealed the presence of this element in the green monkey, orangutan, gorilla, and chimpanzee genomes, as well as the human genome. However, sequence analysis of these genomes revealed a highly mutated, older, primate-specific Alu element was present at this position in the nonhuman primates. Comparison of the flanking DNA sequences upstream of this Alu insertion corresponded to evolution expected for standard primate phylogeny, but comparison of the Alu repeat sequences revealed that the human element departed from this phylogeny. The change in the human sequence apparently occurred by a gene conversion event only within the Alu element itself, converting it from one of the oldest to one of the youngest Alu subfamilies. Although gene conversions of Alu elements are clearly very rare, this finding shows that such events can occur and contribute to specific cases of SINE subfamily evolution.     

Interactions between different generation HIV-1 fusion inhibitors and the putative mechanism underlying the synergistic anti-HIV-1 effect resulting from their combination

We previously reported that the combinatorial use of T20 and T1144, the first and next generations of HIV fusion inhibitors, containing different functional domains resulted in synergistic anti-HIV-1 effect, but this effect diminished when T20 and T1144 were covalently linked together. To elucidate the mechanism underlying this synergistic anti-HIV-1 effect, we studied the interactions between T20 and T1144 either in a mixture state or in a covalently linked state. T20 alone in solution was largely featureless, while T1144 alone was in α-helical trimeric conformation. When mixed in solution, T20 and T1144 showed a loose and transient interaction, with a moderate 10% α-helical content increase, but this interaction was greatly enhanced in the linked state, and T20 and T1144 showed ～100% α-helical content. These results suggested that the loose and transient interaction between T20 and T1144 may destabilize the T1144 trimer, which makes its otherwise shielded binding sites more accessible to N-terminal heptad repeat (NHR) and increases its associating rate, thus increasing its anti-HIV-1 potency against the temporarily exposed target in NHR and causing the synergistic anti-HIV-1 effect. However, the strong interaction between T20 and T1144 in the covalently linked state may shield their NHR-binding sites, resulting in reduction of the synergistic effect.     

Inhibition of HIV-1 envelope glycoprotein-mediated cell fusion by a DL-amino acid-containing fusion peptide: possible recognition of the fusion complex

The N-terminal fusion peptide (FP) of human immunodeficiency virus-1 (HIV-1) is a potent inhibitor of cell-cell fusion, possibly because of its ability to recognize the corresponding segments inside the fusion complex within the membrane. Here we show that a fusion peptide in which the highly conserved Ile(4), Phe(8), Phe(11), and Ala(14) were replaced by their d-enantiomers (IFFA) is a potent inhibitor of cell-cell fusion. Fourier transform infrared spectroscopy confirmed that despite these drastic modifications, the peptide preserved most of its structure within the membrane. Fluorescence energy transfer studies demonstrated that the diastereomeric peptide interacted with the wild type FP, suggesting this segment as the target site for inhibition of membrane fusion. This is further supported by the similar localization of the wild type and IFFA FPs to microdomains in T cells and the preferred partitioning into ordered regions within sphingomyelin/phosphatidyl-choline/cholesterol giant vesicles. These studies provide insight into the mechanism of molecular recognition within the membrane milieu and may serve in designing novel HIV entry inhibitors.     

Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism

Transformation (i.e., genetic modification of a cell by the incorporation of exogenous DNA) is indispensable for manipulating fungi. Here, we review the transformation methods for Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris and Aspergillus species and discuss some common modifications to improve transformation efficiency. We also present a model of the mechanism underlying S. cerevisiae transformation, based on recent reports and the mechanism of transfection in mammalian systems. This model predicts that DNA attaches to the cell wall and enters the cell via endocytotic membrane invagination, although how DNA reaches the nucleus is unknown. Polyethylene glycol is indispensable for successful transformation of intact cells and the attachment of DNA and also possibly acts on the membrane to increase the transformation efficiency. Both lithium acetate and heat shock, which enhance the transformation efficiency of intact cells but not that of spheroplasts, probably help DNA to pass through the cell wall.     

miR-21 Gene expression triggered by AP-1 is sustained through a double-negative feedback mechanism

Methylation of cytosine residues in CpG dinucleotides plays an important role in epigenetic regulation of gene expression and chromatin structure/stability in higher eukaryotes. DNA methylation patterns are established and maintained at CpG dinucleotides by DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b). In mammals and many other eukaryotes, the CpG dinucleotide is underrepresented in the genome. This loss is postulated to be the result of unrepaired deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively. Two thymine glycosylases are believed to reduce the impact of 5-methylcytosine deamination. G/T mismatch-specific thymine-DNA glycosylase (Tdg) and methyl-CpG binding domain protein 4 can both excise uracil or thymine at U·G and T·G mismatches to initiate base excision repair. Here, we report the characterization of interactions between Dnmt3b and both Tdg and methyl-CpG binding domain protein 4. Our results demonstrate (1) that both Tdg and Dnmt3b are colocalized to heterochromatin and (2) reduction of T·G mismatch repair efficiency upon loss of DNA methyltransferase expression, as well as a requirement for an RNA component for correct T·G mismatch repair.     

Inhibition of microtubule assembly is a possible mechanism of action of mitoxantrone

We have found that mitoxantrone can inhibit the polymerization of brain tubulin in a dose dependent manner. MXT had relatively high affinity for tubulin but had no appreciable effect on tubulin associated guanosine-triphosphatase (GTPase) activity nor could it compete with vinblastine (VB) and colchicine (Col) for tubulin binding sites. Furthermore, MXT (0.1-10 microM) is antiproliferative to cold-treated (0 degree C) epithelial cells after only brief exposure (30 min). These results indicated that MXT is a microtubule inhibitory agent and can exert its anticellular effect through modulation of microtubule assembly.     

Molecular mechanism underlying the di-uridylation activity of Arabidopsis TUTase URT1

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5′-AAAU-3′ RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3′ extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an ‘open’ to a ‘closed’ state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.     

A possible mechanism for the influence of geomagnetic field on the evolution of life

The mechanism through which the geomagnetic field might have influenced the extinction of the species has been proposed. It has been assumed that the concentration factor for essential trace elements is dependent on the magnetic field.Supported in part by the R. A. Welch Foundation