Cellular Sequences Research Articles

In Vivo Analysis of Erythroid Protein 4.1 Pre-mRNA Splicing Mechanisms: Use of Antisense Morpholinos to Assay Function of Deep Intron Regulatory Elements

AbstractAbstract 815Erythroid stage-specific alternative splicing plays an essential role in the expression of protein 4.1R isoforms that interact with other skeletal proteins to strengthen the membrane. In late erythroblasts, 4.1R mRNA is processed from pre-mRNA that initiates transcription at alternative first exon 1A (E1A) and splices exclusively to the more distal of two alternative 3‘ splice sites at exon 2 (E2dis), ~100kb downstream. This splicing event is important because it is required to generate the shorter N-terminal domain characteristic of 80kDa isoforms of 4.1R protein in red cells. We have reported that E1A splicing to E2dis requires two nested intrasplicing events mediated by an essential deep intron element originally annotated as exon 1B. However, these studies employed small minigenes transfected into cultured cells, an artificial system that may not correctly reflect in vivo mechanisms. Here we used an antisense RNA strategy to explore splicing of endogenous full length 4.1R pre-mRNA in tissues of live mice and in primary erythroblasts. Chemically modified oligonucleotides known as vivo-morpholinos (vMOs), introduced via tail vein injection and internalized into selected organs, can base pair with complementary cellular RNA sequences and block function of candidate regulatory motifs. Importantly, two independent vMOs directed against the 4.1R intraexon regulatory element both substantially abrogated intrasplicing in several mouse tissues, robustly switching E1A splicing from E2dis to the proximal 3‘ splice site in E2 (E2prox). This switch results in inclusion of start codon AUG1 in mature 4.1R mRNA and synthesis of larger isoforms of 4.1R protein. These results were highly sequence-specific, since negative control vMOs directed against other genes did not alter E1A splicing to E2dis. Interestingly, we have recently used vMOs to confirm the existence of a similar deep intron element required for analogous E1A-E2dis splicing in the paralogous 4.1B gene. Together these findings strongly support the in vivo physiological function of deep intron elements in the control of intrasplicing in both 4.1R and 4.1B pre-mRNAs.To test whether the 4.1R intrasplicing mechanism is also active in erythroid cells, we incubated mouse splenic erythroblasts isolated from FVA-treated animals with morpholinos directed against the intraexon. Two independent morpholinos against its 5' splice site and branch point both induced a concentration-dependent switch in E1A splicing from E2dis to E2prox. Control morpholinos had no effect on E1A splicing. Because the splicing switch results in inclusion of alternative translation initiation codon AUG1, it was predicted to induce synthesis of larger isoforms of 4.1R including the N-terminal headpiece known to influence 4.1R binding affinities for other skeletal proteins. Western blot analysis of erythroblast proteins confirmed a switch to expression of larger 4.1R protein isoforms that are not present in normal late stage erythroblasts. Intrasplicing is mediated by deep intron elements, and is essential for accurate physiological splicing of natural 4.1R pre-mRNA in erythroid and other cells. Antisense morpholinos represent a new tool for alternative splicing studies in vivo or in cultured erythroblasts. Disclosures:No relevant conflicts of interest to declare.

Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome

Immunological detection of viral large T antigen identifies a subset of Merkel cell carcinoma tumors with higher viral abundance and better clinical outcome

Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma

To investigate whether integration of HPV DNA in cervical carcinoma is responsible for structural alterations of the host genome at the insertion site, a series of 34 primary cervical carcinomas and eight cervical cancer-derived cell lines were analysed. DNA copy number profiles were assessed using the Affymetrix GeneChip Human Mapping 250K Sty array. HPV 16, 18 or 45 integration sites were determined using the DIPS-PCR technique. The genome status at integration sites was classified as follows: no change, amplification, transition normal/gain, normal/loss or gain/LOH. A single HPV integration site was found in 34 cases; two sites were found in seven cases; and three sites in one case (51 sites). Comparison between integration sites and DNA copy number profiles showed that the genome status was altered at 17/51 (33%) integration sites, corresponding to 16/42 cases (38%). Alterations detected were amplification in nine cases, transition normal/loss in four cases, normal/gain in three cases, and gain/LOH in one case. A highly significant association was found between genomic rearrangement and integration of HPV DNA (p < 10(-10)). Activation of the replication origin located in viral integrated sequences in a cell line derived from one of the primary cervical carcinomas induced an increase of the amplification level of both viral and cellular DNA sequences flanking the integration locus. This mechanism may be implicated in the triggering of genome amplification at the HPV integration site in cervical carcinoma. Structural alterations of the host genome are frequently observed at the integration site of HPV DNA in cervical cancer and may act in oncogenesis.

How Cells Communicate in the Bone Remodelling Process

In the adult human skeleton, approximately 5 to 10% of the existing bone is replaced every year by bone remodelling. The remodelling process, which continues throughout adult life, provides for the calcium homeostatic system and is essential for resorptive removal of old bone, the removal and repair of micro-damage, and for the adaptation to mechanical stress[1,2]. The cellular sequence is initiated with signals that lead to osteoclast development and bone resorption (Fig. 1). How those signals are initiated is uncertain. In the case of micro-damage, this is proposed to lead to apoptosis of osteocytes that transmit signals to surface cells to promote production of receptor activator of NFκB ligand (RANKL) and hence osteoclast production[3]. Remodelling is essential for the maintenance of skeletal material and structural strength, with bone being continuously resorbed and reformed at about 1~2 million microscopic remodelling foci per adult skeleton. This sequence of events is initiated asynchronously throughout the skeleton, at sites that are geographically and chronologically separated from each other. Both bone resorption and bone formation occur at the same place in these “basic multicellular units” (BMUs), so that there is no change in the shape of the bone[4]. Within each of these BMUs, focal resorption is carried out by haemopoietically-derived osteoclasts and takes about 3 weeks per site, whereas the refilling of lost bone by osteoblasts, derived from bone marrow stromal cells and circulating precursors, takes about 3~4 months. In addition to remodelling, bone modelling on its periosteal surface is characterised by bone formation without prior bone resorption. This process, so vigorous during growth, establishes the adult size and shape of bone. At the completion of linear growth with closure of the epiphyses, periosteal apposition continues but markedly less so[5]. Tight regulation of these processes is essential for the achievement and maintenance of skeletal strength. Modelling and remodelling during growth achieves peak bone strength, and continued remodelling during adulthood maintains the mechanical integrity of the skeleton. Circulating hormones contribute, but the key influences are locally generated cytokines that are the signals mediating information transfer among osteoblasts, osteoclasts, immune cells and constituents of the bone matrix. How Cells Communicate in the Bone Remodelling Process

Application of computational approaches to study signalling networks of nuclear and Tyrosine kinase receptors

BackgroundNuclear receptors (NRs) and Receptor tyrosine kinases (RTKs) are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention.ResultsWe constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs.We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved.Availability: NR-RTK database is accessible at http://www.bioinfo-cbs.org/NR-RTK/ConclusionsWe infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs.ReviewersThis article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein), Dr. Arcady Mushegian and Dr. Anthony Almudevar.

Gene looping between the viral LTRs is a hallmark of transcriptionally active HIV-1 and Mo-MLV proviral DNA

By exploiting the Chromosome Conformation Capture (3C) technique, we have previously shown that the HIV-1 provirus forms a transcription-dependent gene loop structure between the 5' LTR promoter and 3' LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocked 5' to 3' LTR juxtaposition, indicating that this structure is maintained during transcription [1]. The formation of such high order chromatin structure, if aberrantly generated between a retroviral LTR sequence and the promoter of a neighboring gene, has the potential to activate transcription of the cellular gene. This would provide a molecular explanation to the insertional mutagenesis events observed in several gene therapy clinical trials entailing the use of retroviral vectors, leading to the activation of cellular protoconcogenes by means of long-range chromosomal interactions. Indeed, despite the vast amount of data on the insertion sites of retroviral vectors in preclinical and clinical studies, the molecular mechanisms connecting retrovirus integration to aberrant cellular gene activation are still almost completely obscure. We first wanted to test whether LTR-mediated gene looping is also a characteristics of Mo-MLV-based retroviral vectors. In this respect, compared to HIV-1, Mo-MLV has a much weaker poly(A) signal, having thus higher potential to mobilize cellular sequences due to the frequent 3' poly(A) read-through. We produced and characterized a number of HeLa clones harboring single integrations of a Mo-MLV vector carrying a selectable gene. Using the 3C technique, we observed that, in all the analyzed clones, a gene loop was present juxtaposing the 5' and 3' LTRs of the vector. Thus, gene looping is not a prerogative of HIV-1 but a characteristic also extended to oncoretroviruses. We are currently in the process of analyzing, in our clones, the levels of expression of the cellular genes neighboring the proviral integration sites within regions spanning 50 kb upstream or downstream of the insertions and correlating the eventual changes with the formation of aberrant loops. In addition, 3C experiments are also performed in a series of HaCaT clones in which up-regulation of genes surrounding retroviral integration has already been demonstrated (in collaboration with F. Mavilio, Milan, Italy) and will later be extended to cell clones from patients with SCID-X1 who were enrolled in one of the recent gene therapy clinical trials (with A. Thrasher, London, UK).

Virus recovery and full-length sequence analysis of atypical bovine pestivirus Th/04_KhonKaen

Phylogenetic analysis of recently identified “atypical” bovine pestiviruses, performed based on different gene regions, has revealed unclear relationships with other established species, therefore, their phylogenetic position could not be determined so far. In this study, the atypical pestivirus Th/04_KhonKaen was recovered from serum of a naturally infected calf and the complete genome sequence was determined and analysed, as means to define its position. The viral genome is 12,337 nucleotides (nt) long, and comprises a 5′-UTR of 383 nt, a 3′-UTR of 254 nt and an open reading frame of 11,700 nt, without duplication of viral sequences or insertions of cellular sequences. The phylogenetic analyses of the full-length sequence, performed by Neighbor-joining, Maximum likelihood, and the Bayesian approach, unanimously placed Th/04_KhonKaen in a single lineage, distinct from the established pestivirus species, and close to bovine viral diarrhea virus types 1 and 2. Furthermore, Th/04_KhonKaen and two previously reported atypical pestiviruses D32/00_‘HoBi’ and CH-KaHo/cont formed a well-supported monophyletic clade in trees based on the complete N pro and E2 gene regions. The finding provides conclusive classification of the Th/04_KhonKaen virus and confirms the standing of the “atypical” bovine pestiviruses as a novel pestivirus species.

A redescription of the marine hypotrichous ciliate, Nothoholosticha fasciola (Kahl, 1932) nov. gen., nov. comb. (Ciliophora: Urostylida) with brief notes on its cellular reorganization and SS rRNA gene sequence

The morphology and infraciliature of the marine hypotrichous ciliate Nothoholosticha fasciola (Kahl, 1932) nov. gen., nov. comb., isolated from mariculture waters near Qingdao, China, are redescribed based on live and protargol-impregnated specimens. Features reported for the first time include the possession of more than 50 macronuclear nodules and details of the infraciliature, i.e. 50–60 adoral membranelles, shortened paroral and endoral membranes, six frontal, one buccal and two to seven transverse cirri, ca. 40 pairs of midventral cirri, ca. 60–120 left and 70–120 right marginal cirri, three dorsal kineties, caudal and frontoterminal cirri absent. In addition, brief details of two stages of cellular reorganization in N. fasciola are supplied and comparisons with some related urostylids based on SS rRNA gene sequence data are reported. The new genus Nothoholosticha is established based primarily on the absence of frontoterminal cirri, which distinctly separates it from similar urostylid genera. Anteholosticha longissima is transferred to Nothoholosticha as N. longissima (Dragesco and Dragesco-Kernéis, 1986) nov. comb. and Holosticha antarctica is transferred to Pseudokeronopsis as P. antarctica (Wilbert and Song, 2008) nov. comb.

Histological changes occurring after endoluminal ablation with two diode lasers (940 and 1319 nm) from acute changes to 4 months

Endovenous laser ablation of the saphenous vein was studied from a histologic analysis to establish changes that occur from time of injury to 4 months when the vein is difficult to visualize by ultrasound. Twenty-four patients were examined after treatment with either a 1319 nm diode laser (Sciton, Palo Alto, CA) or a 940 nm diode (Dornier, Kennasaw, GA) 12 patients were randomly assigned to the 940 nm group and 12 patients to the 1319 nm group. Histologic evaluations were only done once per patient. All patients had symptomatic saphenous insufficiency with varicosities. All were in CEAP class 3 or 4. Sections of treated veins were submitted for evaluation after staining with hematoxylin-eosin. The evaluations were done acutely, at 1 and 4 months. Ultrasound findings were also evaluated and compared at the same intervals. Acutely, all examined veins revealed loss of intima. In the 1319 nm group numerous vacuoles were present in the subintimal layer. On gross exam at 1 month, both groups showed vein wall thickening, intraluminal thrombus and inflammatory changes. Histologic evaluation showed thrombus was present with many fibroblasts and inflammatory cells. At 4 months, collagen was the predominant histologic finding. However, the changes were less in regards to the injury response with the 1,319 nm group as manifested by less collagen deposition at 4 months. This study demonstrates the cellular sequence that occurs after endovenous ablation. Fibroblast infiltration is a result of the injury response which leads to negative modeling of the thrombus and eventual collagen deposition. Replacement of the thrombus with collagen is necessary for eventual long-term success.

Evidence of CD8 + T-Cell-Mediated Selective Pressure on Human Immunodeficiency Virus Type 1 nef in HLA-B*57 + Elite Suppressors

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8(+) T cells play a key role in suppressing viral replication. We have previously shown that while CD8(+) T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4(+) T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57(+) ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4(+) T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8(+) T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.

Genome-Wide Analysis of the “Cut-and-Paste” Transposons of Grapevine

BackgroundThe grapevine is a widely cultivated crop and a high number of different varieties have been selected since its domestication in the Neolithic period. Although sexual crossing has been a major driver of grapevine evolution, its vegetative propagation enhanced the impact of somatic mutations and has been important for grapevine diversity. Transposable elements are known to be major contributors to genome variability and, in particular, to somatic mutations. Thus, transposable elements have probably played a major role in grapevine domestication and evolution. The recent publication of the complete grapevine genome opens the possibility for an in deep analysis of its transposon content.Principal FindingsWe present here a detailed analysis of the “cut-and-paste” class II transposons present in the genome of grapevine. We characterized 1160 potentially complete grapevine transposons as well as 2086 defective copies. We report on the structure of each element, their potentiality to encode a functional transposase, and the existence of matching ESTs that could suggest their transcription.ConclusionsOur results show that these elements have transduplicated and amplified cellular sequences and some of them have been domesticated and probably fulfill cellular functions. In addition, we provide evidences that the mobility of these elements has contributed to the genomic variability of this species.

MCLCA4 ER processing and secretion requires luminal sorting motifs

Ca(+)-activated Cl(-) channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90- and 40-kDa fragments, and the NH(2)- and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4.

The Majority of Viral-Cellular Fusion Transcripts in Cervical Carcinomas Cotranscribe Cellular Sequences of Known or Predicted Genes

Integration of human papillomavirus (HPV) DNA into the host genome is a frequent event in cervical carcinogenesis and is reported to occur at randomly selected chromosomal sites. However, as the databases are being up-dated continuously, the knowledge based on sequenced viral integration sites also expands. In this study, viral-cellular fusion transcripts of a preselected group of 74 cervical carcinoma or cervical intraepithelial neoplasia grade 3 (CIN3) biopsies harboring integrated HPV16, HPV18, HPV31, HPV33, or HPV45 DNA were amplified by 3'-rapid amplification of cDNA ends PCR and sequenced. Consistent with previous reports, integration sites were found to be distributed throughout the genome. However, 23% (17 of 74) of the integration sites were located within the cytogenetic bands 4q13.3, 8q24.21, 13q22.1, and 17q21, in clusters ranging from 86 to 900 kb. Of note is that clusters 8q24.21 and 13q22.1 are within 1.5 Mbp of an adjacent fragile site whereas clusters 4q13.3 and 17q21 are >15 Mbp distant to any known fragile sites. It is tempting to speculate that as yet unknown fragile sites may be identified on the basis of HPV integration hotspots. No correlation between HPV type and specific integration loci was found. Of 74 fusion transcripts, 28 contained cellular sequences, which were homologous to known genes, and 40 samples contained sequences of predicted genes. In 33 fusion transcripts, both viral and cellular sequences were in sense orientation, indicating that the gene itself or upstream sequences were affected by integration. These data suggest that the influence of HPV integration on host gene expression may not be a rare effect and should encourage more detailed analyses.

Growth inhibition of human cholangiocarcinoma by sorafenib‐based mono‐ and combination treatment

Background: Treatment options of advanced cholangiocarcinoma (CC) are unsatisfactory and new therapeutic approaches are mandatory. The Ras/Raf/MAPK‐pathway is one of the most significant cellular signaling sequences in the development and maintenance of cancer. Thus inhibition of this key signaling pathway appears to be a promising approach for novel CC‐treatment. Here we investigated the antineoplastic effects of sorafenib (Sor), a novel multi‐kinase inhibitor which potently suppresses wild‐type‐ and mutated Raf‐isoforms. Sor has already shown antineoplastic efficacy in some cancer types but its suitability for treatment of CC has not been investigated so far.Results: Sor dose‐dependently blocked growth‐factor‐induced MAPK‐activity and inhibited the proliferation CC cells in a time and dose‐dependent manner. At least two mechanisms accounted for the effects observed: G1/G0‐ arrest of the cell cycle and apoptosis. G1/G0‐arrest was associated with upregulation of the cdk‐inhibitor p27 and downregulation of cyclin D1. Combining Sor with doxorubicin, the HDAC inhibitor MS‐275 or IGF‐1R‐inhibition resulted in (over‐) addititve antiproliferative effects. Conclusion: Our study demonstrates that the growth of human CC cells can be potently suppressed by Sor alone or in certain combination therapies and may provide a promising rationale for future in vivo evaluations and clinical trials.

DNA sequencing: bench to bedside and beyond.

Fifteen years elapsed between the discovery of the double helix (1953) and the first DNA sequencing (1968). Modern DNA sequencing began in 1977, with development of the chemical method of Maxam and Gilbert and the dideoxy method of Sanger, Nicklen and Coulson, and with the first complete DNA sequence (phage ϕX174), which demonstrated that sequence could give profound insights into genetic organization. Incremental improvements allowed sequencing of molecules >200 kb (human cytomegalovirus) leading to an avalanche of data that demanded computational analysis and spawned the field of bioinformatics. The US Human Genome Project spurred sequencing activity. By 1992 the first ‘sequencing factory’ was established, and others soon followed. The first complete cellular genome sequences, from bacteria, appeared in 1995 and other eubacterial, archaebacterial and eukaryotic genomes were soon sequenced. Competition between the public Human Genome Project and Celera Genomics produced working drafts of the human genome sequence, published in 2001, but refinement and analysis of the human genome sequence will continue for the foreseeable future. New ‘massively parallel’ sequencing methods are greatly increasing sequencing capacity, but further innovations are needed to achieve the ‘thousand dollar genome’ that many feel is prerequisite to personalized genomic medicine. These advances will also allow new approaches to a variety of problems in biology, evolution and the environment.

Novel nickel resistance genes from the rhizosphere metagenome of plants adapted to acid mine drainage.

Metal resistance determinants have traditionally been found in cultivated bacteria. To search for genes involved in nickel resistance, we analyzed the bacterial community of the rhizosphere of Erica andevalensis, an endemic heather which grows at the banks of the Tinto River, a naturally metal-enriched and extremely acidic environment in southwestern Spain. 16S rRNA gene sequence analysis of rhizosphere DNA revealed the presence of members of five phylogenetic groups of Bacteria and the two main groups of Archaea mostly associated with sites impacted by acid mine drainage (AMD). The diversity observed and the presence of heavy metals in the rhizosphere led us to construct and screen five different metagenomic libraries hosted in Escherichia coli for searching novel nickel resistance determinants. A total of 13 positive clones were detected and analyzed. Insights about their possible mechanisms of resistance were obtained from cellular nickel content and sequence similarities. Two clones encoded putative ABC transporter components, and a novel mechanism of metal efflux is suggested. In addition, a nickel hyperaccumulation mechanism is proposed for a clone encoding a serine O-acetyltransferase. Five clones encoded proteins similar to well-characterized proteins but not previously reported to be related to nickel resistance, and the remaining six clones encoded hypothetical or conserved hypothetical proteins of uncertain functions. This is the first report documenting nickel resistance genes recovered from the metagenome of an AMD environment.

Feasibility of Detecting Human Immunodeficiency Virus Type 1 Drug Resistance in DNA Extracted from Whole Blood or Dried Blood Spots

Due to high cost, availability of human immunodeficiency virus type 1 (HIV-1) drug resistance testing in resource-poor settings is still limited. We therefore evaluated the usefulness of viral DNA extracted from either whole blood or dried blood spots (DBS). Samples were collected from 50 patients receiving therapy and 10 therapy-naïve patients. Amplification and sequencing of RNA and DNA was performed using an in-house assay. Protease (PR) and reverse transcriptase (RT) sequences of plasma viral RNA were obtained for 96.6% and 89.7%, respectively, of the 29 patients with a detectable viral load. For cellular viral DNA, useful PR and RT sequences were obtained for 96.6% and 93.1% of the whole-blood-cell samples and for 93.1% and 93.1% of the DBS samples, respectively. For the 31 patients with an undetectable viral load, PR and RT sequences were obtained for 67.7% and 61.3% of the whole-blood-cell DNA preparations and for 54.8% and 58.1% of the DBS DNA preparations, respectively. A good correlation between RNA and DNA sequences was found; most discordances were caused by the detection of mixed amino acids. Of the RT drug-resistant mutations, 13 (38.2%) were seen in RNA only, 6 (17.6%) in DNA only, and 15 (44.1%) in both. Repeated amplification and sequencing of DNA extracts revealed a lack of reproducibility for the detection of drug resistance mutations in a number of samples, indicating a possible founder effect. In conclusion, this study shows the feasibility of genotypic drug resistance testing on whole blood cells or DBS and its possible usefulness for HIV-1 subtyping or examining the overall distribution of drug resistance in a population. For individual patients, RNA sequencing was shown to be superior to DNA sequencing, especially for patients who experienced early treatment failure. The use of DNA extracted from whole blood or DBS for the detection of archived drug resistance mutations deserves further study.

Biotransformation of isoeugenol to vanillin by a newly isolated Bacillus pumilus strain: Identification of major metabolites

A bacterial strain S-1 capable of transforming isoeugenol to vanillin was isolated. The strain was identified as Bacillus pumilus based on biochemical tests, cellular fatty acid composition, riboprint pattern and 16S rRNA gene sequence analyses. In the biotransformation of isoeugenol, vanillin was the main product. With the growing culture of B. pumilus S-1, 10g l(-1) isoeugenol was converted to 3.75g l(-1) vanillin in 150 h, with a molar yield of 40.5% that is the highest up to now. Dehydrodiisoeugenol, a dimer of isoeugenol, was separated by preparative thin layer chromatography and identified by gas chromatography-mass spectrometry. Based on the accurate masses obtained from gas chromatography-high resolution mass spectrometry, two key intermediates, isoeugenol-epoxide (IE) and isoeugenol-diol (ID), were identified by mass spectra interpretations. The biotransformation with resting cells showed that vanillin was oxidized to vanillic acid and then to protocatechuic acid before the aromatic ring was broken. These findings suggest that isoeugenol is degraded through an epoxide-diol pathway.

Functional Anthology of Intrinsic Disorder. 2. Cellular Components, Domains, Technical Terms, Developmental Processes, and Coding Sequence Diversities Correlated with Long Disordered Regions

Biologically active proteins without stable ordered structure (i.e., intrinsically disordered proteins) are attracting increased attention. Functional repertoires of ordered and disordered proteins are very different, and the ability to differentiate whether a given function is associated with intrinsic disorder or with a well-folded protein is crucial for modern protein science. However, there is a large gap between the number of proteins experimentally confirmed to be disordered and their actual number in nature. As a result, studies of functional properties of confirmed disordered proteins, while helpful in revealing the functional diversity of protein disorder, provide only a limited view. To overcome this problem, a bioinformatics approach for comprehensive study of functional roles of protein disorder was proposed in the first paper of this series (Xie, H.; Vucetic, S.; Iakoucheva, L. M.; Oldfield, C. J.; Dunker, A. K.; Obradovic, Z.; Uversky, V. N. Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions. J. Proteome Res. 2007, 5, 1882-1898). Applying this novel approach to Swiss-Prot sequences and functional keywords, we found over 238 and 302 keywords to be strongly positively or negatively correlated, respectively, with long intrinsically disordered regions. This paper describes approximately 90 Swiss-Prot keywords attributed to the cellular components, domains, technical terms, developmental processes, and coding sequence diversities possessing strong positive and negative correlation with long disordered regions.

Genomic instability of the host cell induced by the human papillomavirus replication machinery

Development of invasive cervical cancer upon infection by 'high-risk' human papillomavirus (HPV) in humans is a stepwise process in which some of the initially episomal 'high-risk' type of HPVs (HR-HPVs) integrate randomly into the host cell genome. We show that HPV replication proteins E1 and E2 are capable of inducing overamplification of the genomic locus where HPV origin has been integrated. Clonal analysis of the cells in which the replication from integrated HPV origin was induced showed excision, rearrangement and de novo integration of the HPV containing and flanking cellular sequences. These data suggest that papillomavirus replication machinery is capable of inducing genomic changes of the host cell that may facilitate the formation of the HPV-dependent cancer cell.