Codon Insertion Research Articles

Splice Sites Seldom Slide: Intron Evolution in Oomycetes.

We examine exon junctions near apparent amino acid insertions and deletions in alignments of orthologous protein-coding genes. In 1,917 ortholog families across nine oomycete genomes, 10–20% of introns are near an alignment gap, indicating at first sight that splice-site displacements are frequent. We designed a robust algorithmic procedure for the delineation of intron-containing homologous regions, and combined it with a parsimony-based reconstruction of intron loss, gain, and splice-site shift events on a phylogeny. The reconstruction implies that 12% of introns underwent an acceptor-site shift, and 10% underwent a donor-site shift. In order to offset gene annotation problems, we amended the procedure with the reannotation of intron boundaries using alignment evidence. The corresponding reconstruction involves much fewer intron gain and splice-site shift events. The frequency of acceptor- and donor-side shifts drops to 4% and 3%, respectively, which are not much different from what one would expect by random codon insertions and deletions. In other words, gaps near exon junctions are mostly artifacts of gene annotation rather than evidence of sliding intron boundaries. Our study underscores the importance of using well-supported gene structure annotations in comparative studies. When transcription evidence is not available, we propose a robust ancestral reconstruction procedure that corrects misannotated intron boundaries using sequence alignments. The results corroborate the view that boundary shifts and complete intron sliding are only accidental in eukaryotic genome evolution and have a negligible impact on protein diversity.

Hereditary Human Prion Diseases: an Update.

Prion diseases in humans are neurodegenerative diseases which are caused by an accumulation of abnormal, misfolded cellular prion protein known as scrapie prion protein (PrPSc). Genetic, acquired, or spontaneous (sporadic) forms are known. Pathogenic mutations in the human prion protein gene (PRNP) have been identified in 10-15% of CJD patients. These mutations may be single point mutations, STOP codon mutations, or insertions or deletions of octa-peptide repeats. Some non-coding mutations and new mutations in the PrP gene have been identified without clear evidence for their pathogenic significance. In the present review, we provide an updated overview of PRNP mutations, which have been documented in the literature until now, describe the change in the DNA, the family history, the pathogenicity, and the number of described cases, which has not been published in this complexity before. We also provide a description of each genetic prion disease type, present characteristic histopathological features, and the PrPSc isoform expression pattern of various familial/genetic prion diseases.

Abstract A102: Molecular profiling of a non-small cell lung PDX collection by whole exome sequencing and RNAseq revealed subtype specificities with therapeutic implications

Abstract Non-small cell lung cancer (NSCLC) is a heterogeneous disease comprising different histological subtypes with therapeutic implications. We have established a collection of 76 NSCLC patient-derived xenografts (PDX) comprising adenocarcinoma (39), squamous cell carcinoma (24) and large-cell carcinoma (11) models (2 mixed) for use in anti-cancer drug development and pharmacogenomics research. We analyzed the molecular diversity of our PDX collection using whole-exome sequencing, Affymetrix SNP6.0 and RNAseq, assessed their similarity to patient tumors and the impact on drug testing. Similar to patient lung cancers, genomic analyses revealed that the NSCLC PDX were heavily altered tumors with an overall mutations load ranking from 317 to 1549 per model (mean = 682 mutations). We retrieved signatures of mutational processes known to be associated with ageing, smoking and APOBEC alterations. In addition to the mutations, chromosomal instability characterized by polyploidy including focal gene amplifications (most frequently 2p11.2, 14q32.33) and deletions (e.g. recurrent deletions of 3p14.2, 9p21.3) and gene fusions were detected. While no significant differences in average mutation loads or levels of polyploidy between the histotypes were found, histotype-specific gene alterations and gene expression profiles were identified. In line with in vivo resistance to anti-EGFR treatment, alterations of the RAS/RAF pathway, PIK3CA and PTEN, as well as the MET amplification occurred more frequently in adenocarcinoma and large cell PDX explaining the resistance of 90% of models to anti-EGFR. Among adenocarcinoma and large cell PDX, we identified three models with EGFR mutations, one of which contained the codon insertion EGFRM766X associated with a strong sensitivity towards cetuximab but not erlotinib. In addition, we identified adenocarcinoma PDX models in which KIT was amplified and large cell PDX with deletions in CDKN2A/B, alterations in the RAS/RAF pathway (73%) and epigenetic regulation pathways (64%). Squamous cell PDX were less frequently altered in the RAS/RAF pathway, but frequently had altered TP53 (90%), PIK3CA and PTEN (30%) and high expression of TP63 and FGFR3. Furthermore, a highly expressed FGFR3-TACC3 fusion gene was identified in one squamous cell PDX, which is under evaluation for sensitivity towards anti-FGFR3. Regarding PDX response to standard-of-care agents, squamous cell PDX were the most sensitive to cisplatin whereas adenocarcinoma PDX were the most resistant (p = 0.003). In contrast, both groups were more sensitive to paclitaxel than large cell PDX (p = 0.03). These findings suggest distinct molecular profiles influence the response to cytotoxic agents. Molecular profiling of our NSCLC PDX collection confirmed its similarity to patient tumors and highlighted the distinct molecular specificities of each subtype, suggesting different therapeutic approaches. This extensive characterization will help improve the selection of PDX models in future drug development. Citation Format: Anne-Lise Peille, Bruno Zeitouni, Vanina Fiebig, Heinz-Herbert Fiebig, Vincent Vuaroqueaux. Molecular profiling of a non-small cell lung PDX collection by whole exome sequencing and RNAseq revealed subtype specificities with therapeutic implications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A102.

Primary Structural Variation in Anaplasma marginale Msp2 Efficiently Generates Immune Escape Variants.

Antigenic variation allows microbial pathogens to evade immune clearance and establish persistent infection. Anaplasma marginale utilizes gene conversion of a repertoire of silent msp2 alleles into a single active expression site to encode unique Msp2 variants. As the genomic complement of msp2 alleles alone is insufficient to generate the number of variants required for persistence, A. marginale uses segmental gene conversion, in which oligonucleotide segments from multiple alleles are recombined into the expression site to generate a novel msp2 mosaic not represented elsewhere in the genome. Whether these segmental changes are sufficient to evade a broad antibody response is unknown. We addressed this question by identifying Msp2 variants that differed in primary structure within the immunogenic hypervariable region microdomains and tested whether they represented true antigenic variants. The minimal primary structural difference between variants was a single amino acid resulting from a codon insertion, and overall, the amino acid identity among paired microdomains ranged from 18 to 92%. Collectively, 89% of the expressed structural variants were also antigenic variants across all biological replicates, independent of a specific host major histocompatibility complex haplotype. Biological relevance is supported by the following: (i) all structural variants were expressed during infection of a natural host, (ii) the structural variation observed in the microdomains corresponded to the mean length of variants generated by segmental gene conversion, and (iii) antigenic variants were identified using a broad antibody response that developed during infection of a natural host. The findings demonstrate that segmental gene conversion efficiently generates Msp2 antigenic variants.

Large tandem chromosome expansions facilitate niche adaptation during persistent infection with drug-resistant Staphylococcus aureus.

We used genomics to study the evolution of meticillin-resistant Staphylococcus aureus (MRSA) during a complex, protracted clinical infection. Preparing closed MRSA genomes from days 0 and 115 allowed us to precisely reconstruct all genetic changes that occurred. Twenty-three MRSA blood cultures were also obtained during treatment, yielding 44 colony morphotypes that varied in size, haemolysis and antibiotic susceptibility. A subset of 15 isolates was sequenced and shown to harbour a total of 37 sequence polymorphisms. Eighty per cent of all mutations occurred from day 45 onwards, which coincided with the appearance of discrete chromosome expansions, and concluded in the day 115 isolate with a 98 kb tandem DNA duplication. In all heterogeneous vancomycin-intermediate Staphylococcus aureus isolates, the chromosomal amplification spanned at least a 20 kb region that notably included mprF, a gene involved in resistance to antimicrobial peptides, and parC, an essential DNA replication gene with an unusual V463 codon insertion. Restoration of the chromosome after serial passage under non-selective growth was accompanied by increased susceptibility to antimicrobial peptide killing and reduced vancomycin resistance, two signature phenotypes that help explain the clinical persistence of this strain. Elevated expression of the V463 parC was deleterious to the cell and reduced colony size, but did not alter ciprofloxacin susceptibility. In this study, we identified large DNA expansions as a clinically relevant mechanism of S. aureus resistance and persistence, demonstrating the extent to which bacterial chromosomes remodel in the face of antibiotic and host immune pressures.

MECP2, a gene associated with Rett syndrome in humans, shows conserved coding regions, independent Alu insertions, and a novel transcript across primate evolution.

BackgroundThe methyl-CpG Binding Protein two gene (MECP2) encodes a multifunctional protein comprising two isoforms involved in nuclear organization and regulation of splicing and mRNA template activity. This gene is normally expressed in all tissues, with a higher expression level in the brain during neuronal maturation. Loss of MECP2 function is the primary cause of Rett syndrome (RTT) in humans, a dominant, X-linked disorder dramatically affecting neural and motor development.ResultsWe investigated the molecular evolution of MECP2 in several primate taxa including 36 species in 16 genera of neotropical (platyrrhine) primates. The coding region of the MECP2_e2 isoform showed a high level of evolutionary conservation among humans and other primates, with amino acid substitutions in 14 codons and one in-frame insertion of a single serine codon, between codons 357 and 358, in Ateles paniscus. Most substitutions occurred in noncritical regions of MECP2 and the majority of the algorithms used for analyzing selection did not provide evidence of positive selection. Conversely, we found 48 sites under negative selection in different regions, 23 of which were consistently found by three different algorithms. Similar to an inverted Alu insert found previously in a lesser ape at a parallel location, one Alu insertion of approximately 300 bp in Cebus and Sapajus was found in intron 3. Phylogenetic reconstruction of the intron 3 data provided a topology that was coincident with the consensus arrangement of the primate taxa. RNAseq data in the neotropical primate Callimico goeldii revealed a novel transcript consisting of a noncontinuous region of the human-homologous intron 2 in this species; this transcript accounted for two putative polypeptides.ConclusionsDespite the remarkable evolutionary conservation of MECP2, one in-frame codon insertion was observed in A. paniscus, and one region of intron 3 was affected by a trans-specific Alu retrotransposition in two neotropical primate genera. Moreover, identification of novel MECP2 transcripts in Callimico suggests that part of a homologous human intronic region might be expressed, and that the potential open reading frame in this region might be a subject of interest in RTT patients who carry an apparently normal MECP2 sequence.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0240-x) contains supplementary material, which is available to authorized users.

Roles of Epstein-Barr virus BGLF3.5 gene and two upstream open reading frames in lytic viral replication in HEK293 cells

The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, but a small percentage of infected cells switch from the latent state to the lytic cycle, leading to potent viral DNA replication and progeny viruses production. We here focused on a lytic gene BGLF3.5, and first established BGLF3.5 mutants by marker cassette insertion. Unexpectedly, this insertion mutant failed to produce BGLF4 protein and thus progeny production was severely inhibited. Then we carefully made two point mutant viruses (stop codon insertion or frame-shift mutation) and found that BGLF3.5 is not essential for EBV lytic replication processes, such as viral gene expression, DNA replication, or progeny production in the HEK293 cells although its homolog in murine gammaherpesvirus 68 (MHV-68) was reported to be essential. In addition, we examined the roles of two short, upstream open reading frames within the 5′UTR of BGLF3.5 gene in translation of BGLF4.

A glycine insertion in the estrogen-related receptor (ERR) is associated with enhanced expression of three cytochrome P450 genes in transgenic Drosophila melanogaster.

Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4’-dichlorodiphenyltrichloroethane (DDT) resistant strains the glucocorticoid receptor-like (GR-like) potential transcription factor binding motifs (TFBMs) have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR) in D. melanogaster is an estrogen-related receptor (ERR) gene, which has two predicted alternative splice isoforms (ERRa and ERRb). Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G) codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G). Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera.

Ribosomes slide on lysine-encoding homopolymeric A stretches.

Protein output from synonymous codons is thought to be equivalent if appropriate tRNAs are sufficiently abundant. Here we show that mRNAs encoding iterated lysine codons, AAA or AAG, differentially impact protein synthesis: insertion of iterated AAA codons into an ORF diminishes protein expression more than insertion of synonymous AAG codons. Kinetic studies in E. coli reveal that differential protein production results from pausing on consecutive AAA-lysines followed by ribosome sliding on homopolymeric A sequence. Translation in a cell-free expression system demonstrates that diminished output from AAA-codon-containing reporters results from premature translation termination on out of frame stop codons following ribosome sliding. In eukaryotes, these premature termination events target the mRNAs for Nonsense-Mediated-Decay (NMD). The finding that ribosomes slide on homopolymeric A sequences explains bioinformatic analyses indicating that consecutive AAA codons are under-represented in gene-coding sequences. Ribosome 'sliding' represents an unexpected type of ribosome movement possible during translation.

Plasmid-Encoded Pgp3 Is a Major Virulence Factor for Chlamydia muridarum To Induce Hydrosalpinx in Mice

Hydrosalpinx induction in mice by Chlamydia muridarum infection, a model that has been used to study C. trachomatis pathogenesis in women, is known to depend on the cryptic plasmid that encodes eight genes designated pgp1 to pgp8. To identify the plasmid-encoded pathogenic determinants, we evaluated C. muridarum transformants deficient in the plasmid-borne gene pgp3, -4, or -7 for induction of hydrosalpinx. C. muridarum transformants with an in-frame deletion of either pgp3 or -4 but not -7 failed to induce hydrosalpinx. The deletion mutant phenotype was reproduced by using transformants with premature termination codon insertions in the corresponding pgp genes (to minimize polar effects inherent in the deletion mutants). Pgp4 is known to regulate pgp3 expression, while lack of Pgp3 does not significantly affect Pgp4 function. Thus, we conclude that Pgp3 is an effector virulence factor and that lack of Pgp3 may be responsible for the attenuation in C. muridarum pathogenicity described above. This attenuated pathogenicity was further correlated with a rapid decrease in chlamydial survival in the lower genital tract and reduced ascension to the upper genital tract in mice infected with C. muridarum deficient in Pgp3 but not Pgp7. The Pgp3-deficient C. muridarum organisms were also less invasive when delivered directly to the oviduct on day 7 after inoculation. These observations demonstrate that plasmid-encoded Pgp3 is required for C. muridarum survival in the mouse genital tract and represents a major virulence factor in C. muridarum pathogenesis in mice.

A single codon insertion in PICALM is associated with development of familial subvalvular aortic stenosis in Newfoundland dogs.

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species.

Mechanism of the dependence of hepatitis B virus genotype G on co‐infection with other genotypes for viral replication

Hepatitis B virus (HBV) is classified into several genotypes. Genotype G (HBV/G) is characterised by worldwide dispersion, low intragenotypic diversity and a peculiar sequence of the precore and core region (stop codon and 36-nucleotide insertion). As a rule, HBV/G is detected in co-infection with another genotype, most frequently HBV/A2. In a previous in vivo study, viral replication of HBV/G was significantly enhanced by co-infection with HBV/A2. However, the mechanism by which co-infection with HBV/A2 enhances HBV/G replication is not fully understood. In this study, we employed 1.24-fold HBV/A2 clones that selectively expressed each viral protein and revealed that the core protein expressing construct significantly enhanced the replication of HBV/G in Huh7 cells. The introduction of the HBV/A2 core promoter or core protein or both genomic regions into the HBV/G genome showed that both the core promoter and core protein are required for efficient HBV/G replication. The effect of genotype on the interaction between foreign core protein and HBV/G showed that HBV/A2 was the strongest enhancer of HBV/G replication. Furthermore, Western blot analysis of Dane particles isolated from cultures of Huh7 cells co-transfected by HBV/G and a cytomegalovirus (CMV) promoter-driven HBV/A2 core protein expression construct indicated that HBV/G employed HBV/A2 core protein during particle assembly. In conclusion, HBV/G could take advantage of core proteins from other genotypes during co-infection to replicate efficiently and to effectively package HBV DNA into virions.

The LAN blood group system: a review

Abstract LAN (Langereis) was officially recognized by the International Society of Blood Transfusion in 2012 as being the 33rd human blood group system. It consists of one single high-prevalence antigen, Lan (LAN1). The ABCB6 protein is the carrier of the Lan blood group antigen. The ABCB6gene (chromosome 2q36, 19 exons) encodes the ABCB6 polypeptide (ATP-binding cassette protein, subfamily B, member 6), known as a porphyrin transporter. The exceptional Lan– people do not express ABCB6 (Lan null phenotype), owing to several different molecular mechanisms affecting ABCB6:frameshift leading to a premature stop codon (deletion, insertion, or nonsense mutation of nucleotides); missense mutation; or intronic mutation responsible for RNA splicing defect. Despite the Lan antigen’s being reported to play a key role in erythropoiesis and detoxification of cells, Lan– people do not appear to demonstrate susceptibility to any disease or seemingly physiologic disorder. Anti-Lan has been described as having variable clinical significance, either for hemolytic transfusion reactions (none to severe) or hemolytic disease of the fetus and newborn (none to mild). Despite challenging conditions caused by the scarcity of Lan– donors worldwide, Lan– blood should ideally be given to patients with anti-Lan, especially those with a high-titer antibody. Immunohematology 2013;29:131–135.

Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport.

Zebrafish mll Gene Is Essential for Hematopoiesis

Studies implicate an important role for the mixed lineage leukemia (Mll) gene in hematopoiesis, mainly through maintaining Hox gene expression. However, the mechanisms underlying Mll-mediated hematopoiesis during embryogenesis remain largely unclear. Here, we investigate the role of mll during zebrafish embryogenesis, particularly hematopoiesis. Mll depletion caused severe defects in hematopoiesis as indicated by a lack of blood flow and mature blood cells as well as a significant reduction in expression of hematopoietic progenitor and mature blood cell markers. Furthermore, mll depletion prevented the differentiation of hematopoietic progenitors. In addition, we identified the N-terminal mini-peptide of Mll that acted as a dominant negative form to disrupt normal function of mll during embryogenesis. As expected, mll knockdown altered the expression of a subset of Hox genes. However, overexpression of these down-regulated Hox genes only partially rescued the blood deficiency, suggesting that mll may target additional genes to regulate hematopoiesis. In the mll morphants, microarray analysis revealed a dramatic up-regulation of gadd45αa. Multiple assays indicate that mll inhibited gadd45αa expression and that overexpression of gadd45αa mRNA led to a phenotype similar to the one seen in the mll morphants. Taken together, these findings demonstrate that zebrafish mll plays essential roles in hematopoiesis and that gadd45αa may serve as a potential downstream target for mediating the function of mll in hematopoiesis.

Targets for cystic fibrosis therapy: proteomic analysis and correction of mutant cystic fibrosis transmembrane conductance regulator

Proteomic analysis has proved to be an important tool for understanding the complex nature of genetic disorders, such as cystic fibrosis (CF), by defining the cellular protein environment (proteome) associated with wild-type and mutant proteins. Proteomic screens identified the proteome of CF transmembrane conductance regulator (CFTR), and provided fundamental information to studies designed for understanding the crucial components of physiological CFTR function. Simultaneously, high-throughput screens for small-molecular correctors of CFTR mutants provided promising candidates for therapy. The majority of CF cases are caused by nucleotide deletions (ΔF508 CFTR; >75%), resulting in CFTR misfolding, or insertion of premature termination codons (∼10%), leading to unstable mRNA and reduced levels of truncated dysfunctional CFTR. In this article, we review recent results of proteomic screens, developments in identifying correctors for the most frequent CFTR mutants, and comment on how integration of the knowledge gained from these studies may aid in finding a cure for CF and a number of other genetic disorders.

Histatins In Non-Human Primates: Gene Variations and Functional Effects

Human histatins are histidine-rich, low molecular weight salivary proteins that contribute to the immune system of the oral cavity. In this work, nucleotide sequences of the HIS1 (coding for histatin 1) and HIS2 (coding for histatin 3) genes, homologous to the human ones, have been sequenced and analysed in five primates species including Great Ape, Hylobatidae and Cercopithecidae. In HIS1, the region corresponding to the putative mature peptide shows a premature stop codon in Macaca and Cercopithecus, while HIS2 a six codon insertion in the Cercopithecidae. Histatin 5, a 24-residue peptide derived from histatin 3, is the most antimicrobially active among human histatins, thus macaque and nomascus orthologues of histatin 5 were selected for chemical synthesis and functional characterization, in comparison to the human peptide. All synthesized histatins are predicted to be poorly amphipathic, depending on the charged state of His residues and assume partially a-helical conformations only in lipophilic conditions. Antimicrobial assays against Candida and Criptococcus spp. indicate somewhat different spectra of in vitro activity against the tested fungi. We have described HIS1 and HIS2 gene variations in primates and have analysed their functional effects on selected Hst5 orthologues. The human antimicrobial peptide has been proposed to represent an important lead for new generation of antimicrobial compounds for the treatment of oral mycoses, thus the information from the non-human primates histatins studied may aid strategies for drugs design.

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As(2)O(3)) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 micromol/L As(2)O(3) in vitro, and the primary APL cells were treated with 2.0 micromol/L As(2)O(3) in vitro and 0.16 mg kg(-1) d(-1) As(2)O(3) in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As(2)O(3) use, but the mutation spots were remarkably increased after As(2)O(3) treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: r (NB4-As2O3)=0.973818, and r (APL-As2O3)=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As(2)O(3) aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As(2)O(3) in APL treatment.

The worldwide holoparasitic Apodanthaceae confidently placed in the Cucurbitales by nuclear and mitochondrial gene trees

BackgroundOf the c. 450 families of flowering plants, only two are left "unplaced" in the most recent APG classification of angiosperms. One of these is the Apodanthaceae, a clade of c. 19 holoparasitic species in two or three genera occurring in North and South America, Africa, the Near East, and Australia. Because of lateral gene transfer between Apodanthaceae and their hosts it has been difficult to infer the family's true closest relatives.ResultsHere we report a phylogenetic analysis of 16 accessions representing six species of Apodanthaceae from the United States, Chile, Iran, and Australia, using the mitochondrial matR gene and the nuclear 18S gene. Data matrices include 190 matR sequences from up to 95 families in 39 orders of flowering plants and 197 18S sequences from 101 families representing the 16 orders of rosids. Analyses were performed at the nucleotide and at the amino acid level. Both gene trees agree with angiosperm phylogenies found in other studies using more genes. Apodanthaceae and the seven families of the order Cucurbitales form a clade with 100% bootstrap support from matR and 56% from 18 S. In addition, the Apodanthaceae and Cucurbitales matR gene sequences uniquely share two non-synonymous codon changes and one synonymous change, as well as a codon insertion, already found by Barkman et al. (2007).ConclusionsApodanthaceae belong in the Cucurbitales with which they share inferior ovaries, parietal placentation and a dioecious mating system, traits that are ancestral in Cucurbitales and which can now be interpreted as possible synapomorphies of an enlarged order Cucurbitales. The occurrence of Apodanthaceae in the Americas, Africa, the Near East, and Australia, and their adaptation to distantly related host species in the Fabaceae and Salicaceae suggest a long evolutionary history.

Identical Skin Toxins by Convergent Molecular Adaptation in Frogs

The Tree of Life is rife with adaptive convergences at all scales and biological levels of complexity. However, natural selection is not likely to result in the independent evolution of identical gene products. Here we report such a striking example of evolutionary convergence in the toxic skin secretions of two distantly related frog lineages. Caeruleins are important decapeptides in pharmacological and clinical research [1] and are commonly believed to represent a single evolutionary class of peptides [2-4]. Instead, our phylogenetic analyses combining transcriptome and genome data reveal that independently evolved precursor genes encode identical caeruleins in Xenopus and Litoria frogs. The former arose by duplication from the cholecystokinin (cck) gene, whereas the latter was derived from the gastrin gene. These hormone genes that are involved in many physiological processes diverged early in vertebrate evolution, after a segmental duplication during the Cambrian period. Besides implicating convergent mutations of the peptide-encoding sequence, recurrent caerulein origins entail parallel shifts of expression from the gut-brain axis to skin secretory glands. These results highlight extreme structural convergence in anciently diverged genes as an evolutionary mechanism through which recurrent adaptation is attained across large phylogenetic distances.