Cryptic transcripts from a ubiquitous plasmid origin of replication confound tests for cis-regulatory function.

A vast amount of research on the regulation of gene expression has relied on plasmid reporter assays. In this study, we show that plasmids widely used for this purpose constitutively produce substantial amounts of RNA from a TATA-containing cryptic promoter within the origin of replication. Readthrough of these RNAs into the intended transcriptional unit potently stimulated reporter activity when the inserted test sequence contained a 3' splice site (ss). We show that two human sequences, originally reported to be internal ribosome entry sites and later to instead be promoters, mimic both types of element in dicistronic reporter assays by causing these cryptic readthrough transcripts to splice in patterns that allow efficient translation of the downstream cistron. Introduction of test sequences containing 3' ss into monocistronic luciferase reporter vectors widely used in the study of transcriptional regulation also created the false appearance of promoter function via the same mechanism. Across a large number of variants of these plasmids, we found a very highly significant correlation between reporter activity and levels of such spliced readthrough transcripts. Computational estimation of the frequency of cryptic 3' ss in genomic sequences suggests that misattribution of cis-regulatory function may be a common occurrence

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance

Background: Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology. Results: Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0–3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7. Conclusions: The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates

PCR biases distort bacterial and archaeal community structure in pyrosequencing datasets

As 16S rRNA gene targeted massively parallel sequencing has become a common tool for microbial diversity investigations, numerous advances have been made to minimize the influence of sequencing and chimeric PCR artifacts through rigorous quality control measures. However, there has been little effort towards understanding the effect of multi-template PCR biases on microbial community structure. In this study, we used three bacterial and three archaeal mock communities consisting of, respectively, 33 bacterial and 24 archaeal 16S rRNA gene sequences combined in different proportions to compare the influences of (1) sequencing depth, (2) sequencing artifacts (sequencing errors and chimeric PCR artifacts), and (3) biases in multi-template PCR, towards the interpretation of community structure in pyrosequencing datasets. We also assessed the influence of each of these three variables on α- and β-diversity metrics that rely on the number of OTUs alone (richness) and those that include both membership and the relative abundance of detected OTUs (diversity). As part of this study, we redesigned bacterial and archaeal primer sets that target the V3–V5 region of the 16S rRNA gene, along with multiplexing barcodes, to permit simultaneous sequencing of PCR products from the two domains. We conclude that the benefits of deeper sequencing efforts extend beyond greater OTU detection and result in higher precision in β-diversity analyses by reducing the variability between replicate libraries, despite the presence of more sequencing artifacts. Additionally, spurious OTUs resulting from sequencing errors have a significant impact on richness or shared-richness based α- and β-diversity metrics, whereas metrics that utilize community structure (including both richness and relative abundance of OTUs) are minimally affected by spurious OTUs. However, the greatest obstacle towards accurately evaluating community structure are the errors in estimated mean relative abundance of each detected OTU due to biases associated with multi-template PCR reactions

Physical mapping integrated with syntenic analysis to characterize the gene space of the long arm of wheat chromosome 1A

Background: Bread wheat (Triticum aestivum L.) is one of the most important crops worldwide and its production faces pressing challenges, the solution of which demands genome information. However, the large, highly repetitive hexaploid wheat genome has been considered intractable to standard sequencing approaches. Therefore the International Wheat Genome Sequencing Consortium (IWGSC) proposes to map and sequence the genome on a chromosome-by-chromosome basis. Methodology/Principal Findings: We have constructed a physical map of the long arm of bread wheat chromosome 1A using chromosome-specific BAC libraries by High Information Content Fingerprinting (HICF). Two alternative methods (FPC and LTC) were used to assemble the fingerprints into a high-resolution physical map of the chromosome arm. A total of 365 molecular markers were added to the map, in addition to 1122 putative unique transcripts that were identified by microarray hybridization. The final map consists of 1180 FPC based or 583 LTC based contigs. Conclusions/Significance: The physical map presented here marks an important step forward in mapping of hexaploid bread wheat. The map is orders of magnitude more detailed than previously available maps of this chromosome, and the assignment of over a thousand putative expressed gene sequences to specific map locations will greatly assist future functional studies. This map will be an essential tool for future sequencing of and positional cloning within chromosome 1A

Immunoglobulin Gamma Subclasses and Corresponding Fc Receptors in Rhesus Macaques: Genetic Characterization and Engineering of Recombinant Molecules

Rhesus macaques represent a valuable model in biomedical research and in development of vaccines and therapeutics. Due to the lack of reagents, the general properties of IgG and corresponding cellular receptors (FcγR) in this species are poorly characterized. We engineered recombinant IgGs containing each of the four rhesus macaque heavy constant region (CH) subclasses. To define FcγRs that mediate IgGs, we identified and characterized three FcγR classes, and generated recombinant cDNA constructs. cDNA IgH constructs were created by fusing – by sequence overlap extension PCRs – a gene segment encoding the murine variable heavy domain specific for the hapten NIP, an established specificity system for assessing antibody effector functions, with rhesus macaque CH fragments. The complete IgH constructs were transfected into J558L cells, a murine IgH-lost myeloma cell line expressing anti-NIP light chain. Secretion of engineered IgGs was determined by ELISAs using NIP-BSA and anti-monkey IgG-specific antibodies. Molecular cloning methods were applied to identify and clone FcγR genes, and recombinant FcγR cDNA constructs were created by the recombinant DNA method. Four engineered IgH cDNA constructs were successfully created. Recombinant IgGs, in the intact Ig form and retaining the original anti-NIP specificity, were successfully produced. Compared to those in humans, FcγRs in rhesus macaques share high homology, yet also feature a relatively high level of intra-species polymorphism and possess different N-linked glycosylation patterns. FcγR constructs and expression vectors were successfully generated. The chimeric recombinant IgGs are powerful tools for defining IgG functional properties and studying CH structure/function relationship. These molecules can also be used as immunogens for generation of antibodies capable of unequivocally detecting individual IgG subclasses. The findings on FcγRs validate rhesus macaques as a model for studying antibody responses, and underscore the need to take into account of the genetic heterogeneity. The FcγR constructs and vectors serve as a tool for further studies of IgG/FcγR interactions. We also reported here our findings from a separate study that the main female hormone, 17β-estradiol, is capable of restoring antibody responses to an influenza vaccine in a postmenopausal mouse model, suggesting that immunogenicity and efficacy of influenza vaccines should be evaluated in postmenopausal women

Over-expression of a chimeric gene of the transcriptional co-activator MBF1 fused to the EAR repressor motif causes developmental alteration in Arabidopsis and tomato

Transcriptional co-activators of the Multiprotein Bridging Factor1 (MBF1) type belong to a multigenic family that encode key components of the machinery controlling gene expression by communicating between transcription factors and the basal transcription machinery. Knocking-down the expression of one member of the family has proved difficult probably due to functional redundancy. We show here that a fusion of SlER24, an MBF1 type gene of tomato, to the Ethylene-responsive element-binding associated Amphiphilic Repression (EAR) motif is capable of slowing down significantly the expression of the GFP protein driven by a synthetic ethylene-responsive GCC-rich promoter in a single cell transient expression system. A fusion of AtMBF1c of Arabidopsis to EAR, driven by the 35S promoter, caused a reduction of the percentage of seed germination and dwarfism of the plant. Similar fusion with the SlER24 of tomato in the MicroTom cultivar induced a delay of seed germination and no obvious effect on plant growth. Besides giving information on the role of the MBF1 genes in plant development, this study demonstrates that the EAR strategy is efficient not only for regular transcription factors as demonstrated so far, but also in the case of co-activators known to not bind directly to DNA

Stamcell chimärism i groddbanan? : en polymorfisk märkör undersökning

Primordial germ cells are the embryonic precursor cells of sperm and ova. They are concluded to differentiate outside the gonadal area probably in the extraembryonic mesoderm. Their invasive migration through the stromal tissues of the dorsal mesentery adherent to the extracellular matrix is a well accepted fact in mice. The aim of this study was to set up a technique based on the use of individual-specific markers to test the hypothesis on the migration of primordial germ cells in a large animal model. The hypothesis, supported by feeble proof until now, states that there would be substantial chimerism in the progeny of primordial germ cells in bovine twins originating from transmission of the cells through placental vascular anastomoses during fetal life. Our aim was pursued by microsatellite analysis of the highly polymorphic bovine MHC locus DRB3. Polymerase chain reaction and capillary electrophoresis were optimized using 21 non-chimeric animals, ten cows and eleven bulls. The DRB3 microsatellite of one bull was cloned to isolate single alleles. Whole blood-derived DNA from 11 chimeric animals, the leukocyte chimerims of which had earlier been quantified by in situ hybridisation, and 47 semen-derived DNA samples from twin borne AI bulls were analysed in the last phase of this study. Primordial germ cell chimerism did not seem to occur in a substantial quantity in twin borne bovine bulls. Extension of the analysis described here to other polymorphic repeat loci should clarify the cases were traces of possible chimerism was found by analysing the above locus.Primordiala groddceller är de embryonala föregångarna till spermier och ägg. Man har enats om att de utvecklas utanför den gonadala regionen och man tror att de differentierar sig i det extraembryonala mesodermet. Deras invasiva migration, fastbundna till det extracellulära matrixet genom det dorsala mesenteriets stromala vävnad, har blivit godkänd som fakta i en musmodell. Syftet med denna undersökning var att hitta en teknik som baserar sig på individ specifika markörer för att testa en hypotes angående de embryonala groddcellernas vandring i en stordjursmodell. Hypotesen, som fram till nu har stöttats med bara föga bevis, konstaterar att det skulle finnas substantiell chimärism bland descendenter från de primordiala groddcellerna i nötdjurstvillingar. Dessa härstammar från cellernas transmission genom vaskulära anastomoser i placenta under fostertiden. Vi analyserade ett synnerligen polymorfiskt mikrosatellitlocus, DRB3, för att se mängden av olika genotyper i spermaprov. Optimeringen av metoden som baserar sig på PCR och kapillär elektrofores utfördes med 21 icke-chimära djur; tio kor och elva tjurar. DRB3 mikrosatelliten från en tjur var klonad för att isolera enskilda allel. Till sist analyserades sperman av 47 tjurar som är födda som tvillingar. Helblod av 11 djur som tidigare hade verifierats vara leukocytchimära med in situ hybridisation analyserades också. Man kunde visa att de embryonala groddcellernas chimärism, förmodligen inte sker som ett substantiellt fenomen hos nötdjurstvillingar. En spridning av den nu använda metoden till andra polymorfiska repetition sekvens loci förklarar de tvivelaktiga fallen som hittades vid analysering av DRB3 locus.Primordiaaliset ituradan solut, niin sanotut PGC solut, ovat siittiöiden ja munasolujen sikiönkehityksen aikaisia edeltäjäsoluja. PGC solujen on päätelty erilaistuvan gonadialueen ulkopuolella, luultavasti sikiön ulkopuolella sijaitsevassa mesodermissä. Hiirellä näiden solujen invasiivinen vaellus suoliliepeen strooman läpi, soluväliaineeseen liittyneenä, on laajalti hyväksytty malli. Tämän tutkimuksen tavoite oli pystyttää yksilöspesifisiin markkereihin perustuva tekniikka, jolla kyettäisiin testaamaan hypoteesia PGC solujen vaelluksesta suureläin mallissa. Hypoteesin mukaan nauta kaksosilla esiintyisi pysyvää ituratakimerismiä PGC solujen siirtyessä sikiöstä toiseen istukan verisuonianastomoosien kautta. Tavoitteeseen pyrittiin analysoimalla erittäin polymorfista naudan MHC-kompleksin lokusta; DRB3 geenin mikrosatelliittia. Polymeraasiketjureaktion ja kapillaari geelielektroforeesin optimointiin käytettiin 21:stä ei-kimeeristä eläintä; kymmentä lehmää ja yhtätoista sonnia. Yhden sonnin DRB3 mikrosatelliitti kloonattiin yksittäisten alleelien eristämiseksi. 11:sta, aikaisemmin in situ tekniikalla leukosyyttikimeeriseksi varmistetun, eläimen kokoverestä eristetyt DNA-näytteet ja 47:n keinosiemennykseen käytetyn, kaksosena syntyneen sonnin spermasta eristetyt DNA näytteet analysoitiin DRB3 mikrosatelliittialleelien lukumäärän suhteen. PGC solu kimerismi osoittautui esiintyessään enintään marginaaliseksi ilmiöksi kaksoissonneilla. 47:n sonnin spermanäytteistä ainoastaan yhdestä löydettiin mahdollisia viitteitä neljästä DRB3 alleelista ja siten kimerismistä. Jatkotutkimuksissa pyritään täydentämään tekniikkaa käyttämällä useampia polymorfisia mikrosatelliittilokuksia analyysissä, jolloin suuremmalla todennäköisyydellä havaitaan normaaliin homo- tai heterotsygoottitilanteeseen nähden kasvanut alleelimäärä kimerismin esiintyessä

MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus. The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development

PacBio amplicon sequencing for metabarcoding of mixed DNA samples from lichen herbarium specimens.

The detection and identification of species of fungi in the environment using molecular methods heavily depends on reliable reference sequence databases. However, these databases are largely incomplete in terms of taxon coverage, and a significant effort is required from herbaria and living fungal collections for the mass-barcoding of well-identified and well-curated fungal specimens or strains. Here, a PacBio amplicon sequencing approach is applied to recent lichen herbarium specimens for the sequencing of the fungal ITS barcode, allowing a higher throughput sample processing than Sanger sequencing, which often required the use of cloning. Out of 96 multiplexed samples, a full-length ITS sequence of the target lichenised fungal species was recovered for 85 specimens. In addition, sequences obtained for co-amplified fungi gave an interesting insight into the diversity of endolichenic fungi. Challenges encountered at both the laboratory and bioinformatic stages are discussed, and cost and quality are compared with Sanger sequencing. With increasing data output and reducing sequencing cost, PacBio amplicon sequencing is seen as a promising approach for the generation of reference sequences for lichenised fungi as well as the characterisation of lichen-associated fungal communities