The Landscape of the DNA Transposons in the Genome of the Horezu_LaPeri Strain of Drosophila melanogaster.

Natural transposons (NTs) represent mobile DNA sequences found in both prokaryotic and eukaryotic genomes. Drosophila melanogaster (the fruit fly) is a eukaryotic model organism with NTs standing for about 20% of its genome and has contributed significantly to the understanding of various aspects of transposon biology. Our study describes an accurate approach designed to map class II transposons (DNA transposons) in the genome of the Horezu_LaPeri fruit fly strain, consecutive to Oxford Nanopore Technology sequencing. A whole genome bioinformatics analysis was conducted using Genome ARTIST_v2, LoRTE and RepeatMasker tools to identify DNA transposons insertions. Then, a gene ontology enrichment analysis was performed in order to evaluate the potential adaptive role of some DNA transposons insertions. Herein, we describe DNA transposon insertions specific for the Horezu_LaPeri genome and a predictive functional analysis of some insertional alleles. The PCR validation of P-element insertions specific for this fruit fly strain, along with a putative consensus sequence for the KP element, is also reported. Overall, the genome of the Horezu_LaPeri strain contains several insertions of DNA transposons associated with genes known to be involved in adaptive processes. For some of these genes, insertional alleles obtained via mobilization of the artificial transposons were previously reported. This is a very alluring aspect, as it suggests that insertional mutagenesis experiments conducting adaptive predictions for laboratory strains may be confirmed by mirroring insertions which are expected to be found at least in some natural fruit fly strains.

Somaclonal Variation-Advantage or Disadvantage in Micropropagation of the Medicinal Plants.

Cell and tissue plant cultures are used either to save vulnerable species from extinction or to multiply valuable genotypes, or both, and are widely applied for economically important plant species. For medicinal plants, the use of in vitro technologies for the production of secondary metabolites and pathogen-free plants has been greatly developed. Two opposite aspects characterize the in vitro micropropagation of medicinal plants: maintaining genetic fidelity for the perpetuation and preservation of elites, and the identification and exploitation of somaclonal variations associated with new, useful traits. A balance between what is advantageous and what is undesirable is necessary, and this implies the identification of somaclonal variability at all levels, from the phenotypic to molecular ones. This review addresses the somaclonal variation arising from the in vitro multiplication of medicinal plants from three perspectives: cytogenetics, genetics, and epigenetics. The possible causes of the appearance of somaclones, the methods for their identification, and the extent to which they are desirable are presented comparatively for different plant species with therapeutic properties. The emphasis is on the subtle changes at the genetic and epigenetic level, as it results from the application of methods based on DNA markers.

ONT-Based Alternative Assemblies Impact on the Annotations of Unique versus Repetitive Features in the Genome of a Romanian Strain of Drosophila melanogaster.

To date, different strategies of whole-genome sequencing (WGS) have been developed in order to understand the genome structure and functions. However, the analysis of genomic sequences obtained from natural populations is challenging and the biological interpretation of sequencing data remains the main issue. The MinION device developed by Oxford Nanopore Technologies (ONT) is able to generate long reads with minimal costs and time requirements. These valuable assets qualify it as a suitable method for performing WGS, especially in small laboratories. The long reads resulted using this sequencing approach can cover large structural variants and repetitive sequences commonly present in the genomes of eukaryotes. Using MinION, we performed two WGS assessments of a Romanian local strain of Drosophila melanogaster, referred to as Horezu_LaPeri (Horezu). In total, 1,317,857 reads with a size of 8.9 gigabytes (Gb) were generated. Canu and Flye de novo assembly tools were employed to obtain four distinct assemblies with both unfiltered and filtered reads, achieving maximum reference genome coverages of 94.8% (Canu) and 91.4% (Flye). In order to test the quality of these assemblies, we performed a two-step evaluation. Firstly, we considered the BUSCO scores and inquired for a supplemental set of genes using BLAST. Subsequently, we appraised the total content of natural transposons (NTs) relative to the reference genome (ISO1 strain) and mapped the mdg1 retroelement as a resolution assayer. Our results reveal that filtered data provide only slightly enhanced results when considering genes identification, but the use of unfiltered data had a consistent positive impact on the global evaluation of the NTs content. Our comparative studies also revealed differences between Flye and Canu assemblies regarding the annotation of unique versus repetitive genomic features. In our hands, Flye proved to be moderately better for gene identification, while Canu clearly outperformed Flye for NTs analysis. Data concerning the NTs content were compared to those obtained with ONT for the D. melanogaster ISO1 strain, revealing that our strategy conducted to better results. Additionally, the parameters of our ONT reads and assemblies are similar to those reported for ONT experiments performed on various model organisms, revealing that our assembly data are appropriate for a proficient annotation of the Horezu genome.

Genome ARTIST_v2-An Autonomous Bioinformatics Tool for Annotation of Natural Transposons in Sequenced Genomes.

The annotation of transposable elements (transposons) is a very dynamic field of genomics and various tools assigned to support this bioinformatics endeavor have been developed and described. Genome ARTIST v1.19 (GA_v1.19) software was conceived for mapping artificial transposons mobilized during insertional mutagenesis projects, but the new functions of GA_v2 qualify it as a tool for the mapping and annotation of natural transposons (NTs) in long reads, contigs and assembled genomes. The tabular export of mapping and annotation data for high-throughput data analysis, the generation of a list of flanking sequences around the coordinates of insertion or around the target site duplications and the computing of a consensus sequence for the flanking sequences are all key assets of GA_v2. Additionally, we developed a set of scripts that enable the user to annotate NTs, to harness annotations offered by FlyBase for Drosophila melanogaster genome, to convert sequence files from .fasta to .raw, and to extract junction query sequences essential for NTs mapping. Herein, we present the applicability of GA_v2 for a preliminary annotation of P-element and hobo class II NTs and copia retrotransposon in the genome of D. melanogaster strain Horezu_LaPeri (Horezu), Romania, which was sequenced with Nanopore technology in our laboratory. We used contigs assembled with Flye tool and a Q10 quality filter of the reads. Our results suggest that GA_v2 is a reliable autonomous tool able to perform mapping and annotation of NTs in genomes sequenced by long sequencing technology. GA_v2 is open-source software compatible with Linux, Mac OS and Windows and is available at GitHub repository and dedicated website.

A Fatty Acid Fraction Purified From Sea Buckthorn Seed Oil Has Regenerative Properties on Normal Skin Cells.

In recent years, natural product's research gained momentum, fueled by technological advancement and open availability of research data. To date, sea buckthorn (Hippophae rhamnoides L. [Elaeagnaceae]) plant parts, especially berries, are well characterized and repeatedly tested for antioxidant activity and regenerative properties, in various cell types and tissues. However, fatty acids (FA) have been less investigated in term of biological effects, although, they are important bioactive components of the sea buckthorn fruit and oil. The aim of our work was to determine whether sea buckthorn seed oil is a suitable source of FA with regenerative properties on normal skin cells. Using high-performance liquid chromatography (HPLC) and liquid chromatography - mass spectrometry (LC-MS), we purified and characterized four fractions enriched in saturated (palmitic) and non-saturated (linoleic, alfa-linolenic, oleic) FA, which were tested for cytotoxicity, cytokine and growth factor production, and regenerative effect on normal keratinocytes and skin fibroblasts. Evidence is presented that the palmitic acid enriched fraction was a suitable sea buckthorn seed oil derived product with cell proliferation properties on both skin cell types.

New intergeneric orchid hybrid found in Romania × Pseudorhiza nieschalkii (Senghas) P.F.Hunt nothosubsp. siculorum H.Kertész & N.Anghelescu, 2020.

We describe the first reported intergeneric, which naturally occurs between two subspecies belonging to different genera, Dactylorhiza fuchsii subsp. sooana (genus Dactylorhiza) and Pseudorchis albida subsp. tricuspis (genus Pseudorchis), as × Pseudorhiza nieschalkii (Senghas) P.F.Hunt nothosubsp. siculorum H.Kertész & N.Anghelescu, 2020. The hybrid was found and digitally photographed for the first time by Hajnalka Kertész in June, 2020, within Terra Siculorum, in one of the Natura 2000 protected areas, known as Harghita Madaraș, ROSCI00090. Following detailed morphometric analysis using 67 characters and molecular karyological analyses, we identified this unique specimen as an intergeneric hybrid, new to science. The hybrid, an F1 generation plant, most likely representing a single intergeneric pollination event, is phenotypically intermediate between its parental species in most of the characters scored, but it significantly closely resembles Pseudorchis albida subsp. tricuspis parent. Since several individuals of the parental species occurred in near proximity, within 1-10 meters distance, we suggest that the production of this hybrid required a minimum travel distance of ca 1-10 meters, by the pollinators and frequent exchange of pollen between the parental species was very likely. The parental species and the hybrid, which display a considerable synchronicity in their flowering time, overlap in the pollinator community, sharing various species of Hymenopterans and Dipterans, very abundant in the heathland. This Terra Siculorum hybrid is thus best described as a rarely occurring intergeneric hybrid that shows strong Pseudorchis albida subsp. tricuspis parental dominance in inheritance patterns.

PCNA function in the activation and strand direction of MutLα endonuclease in mismatch repair.

MutLα (MLH1-PMS2) is a latent endonuclease that is activated in a mismatch-, MutSα-, proliferating cell nuclear antigen (PCNA)-, replication factor C (RFC)-, and ATP-dependent manner, with nuclease action directed to the heteroduplex strand that contains a preexisting break. RFC depletion experiments and use of linear DNAs indicate that RFC function in endonuclease activation is limited to PCNA loading. Whereas nicked circular heteroduplex DNA is a good substrate for PCNA loading and for endonuclease activation on the incised strand, covalently closed, relaxed circular DNA is a poor substrate for both reactions. However, covalently closed supercoiled or bubble-containing relaxed heteroduplexes, which do support PCNA loading, also support MutLα activation, but in this case cleavage strand bias is largely abolished. Based on these findings we suggest that PCNA has two roles in MutLα function: The clamp is required for endonuclease activation, an effect that apparently involves interaction of the two proteins, and by virtue of its loading orientation, PCNA determines the strand direction of MutLα incision. These results also provide a potential mechanism for activation of mismatch repair on nonreplicating DNA, an effect that may have implications for the somatic phase of triplet repeat expansion.

Endonucleolytic function of MutLalpha in human mismatch repair.

Half of hereditary nonpolyposis colon cancer kindreds harbor mutations that inactivate MutLalpha (MLH1*PMS2 heterodimer). MutLalpha is required for mismatch repair, but its function in this process is unclear. We show that human MutLalpha is a latent endonuclease that is activated in a mismatch-, MutSalpha-, RFC-, PCNA-, and ATP-dependent manner. Incision of a nicked mismatch-containing DNA heteroduplex by this four-protein system is strongly biased to the nicked strand. A mismatch-containing DNA segment spanned by two strand breaks is removed by the 5'-to-3' activity of MutSalpha-activated exonuclease I. The probable endonuclease active site has been localized to a PMS2 DQHA(X)(2)E(X)(4)E motif. This motif is conserved in eukaryotic PMS2 homologs and in MutL proteins from a number of bacterial species but is lacking in MutL proteins from bacteria that rely on d(GATC) methylation for strand discrimination in mismatch repair. Therefore, the mode of excision initiation may differ in these organisms.

Human mismatch repair: reconstitution of a nick-directed bidirectional reaction.

Bidirectional mismatch repair directed by a strand break located 3' or 5' to the mispair has been reconstituted using seven purified human activities: MutSalpha, MutLalpha, EXOI, replication protein A (RPA), proliferating cell nuclear antigen (PCNA), replication factor C (RFC) and DNA polymerase delta. In addition to DNA polymerase delta, PCNA, RFC, and RPA, 5'-directed repair depends on MutSalpha and EXOI, whereas 3'-directed mismatch correction also requires MutLalpha. The repair reaction displays specificity for DNA polymerase delta, an effect that presumably reflects interactions with other repair activities. Because previous studies have suggested potential involvement of the editing function of a replicative polymerase in mismatch-provoked excision, we have evaluated possible participation of DNA polymerase delta in the excision step of repair. RFC and PCNA dramatically activate polymerase delta-mediated hydrolysis of a primer-template. Nevertheless, the contribution of the polymerase to mismatch-provoked excision is very limited, both in the purified system and in HeLa extracts, as judged by in vitro assay using nicked circular heteroplex DNAs. Thus, excision and repair in the purified system containing polymerase delta are reduced 10-fold upon omission of EXOI or by substitution of a catalytically dead form of the exonuclease. Furthermore, aphidicolin inhibits both 3'- and 5'-directed excision in HeLa nuclear extracts by only 20-30%. Although this modest inhibition could be because of nonspecific effects, it may indicate limited dependence of bidirectional excision on an aphidicolin-sensitive DNA polymerase.

A defined human system that supports bidirectional mismatch-provoked excision.

Mismatch-provoked excision directed by a strand break located 3' or 5' to the mispair has been reconstituted using purified human proteins. While MutSalpha, EXOI, and RPA are sufficient to support hydrolysis directed by a 5' strand break, 3' directed excision also requires MutLalpha, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5' to 3' hydrolysis when the nick that directs excision is located 3' to the mispair and activate 3' to 5' excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3' to 5' hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5' to 3' excision directed by a 5' strand break.

Two-hybrid analysis of the interaction between the UL52 and UL8 subunits of the herpes simplex virus type 1 helicase-primase.

Herpes simplex virus type 1 expresses a heterotrimeric helicase-primase, the subunits of which are encoded by the viral UL5, UL8 and UL52 genes. The interactions of the UL52 protein with the UL8 and UL5 proteins were analysed by using the yeast two-hybrid system. The UL52-UL5 interaction gave a specific but weak signal in the two-hybrid system. In contrast, the UL52-UL8 interaction gave a strong signal in the two-hybrid system. Deletion analysis showed that a 548 amino acid fragment of UL52 (amino acids 366-914) retains the ability to interact with UL8 and that the N-terminal 349 amino acids are dispensable for the interaction. A fragment library screen and co-immunoprecipitation experiments confirmed the deletion analysis results.