Methylase-assisted subcloning for high throughput BioBrick assembly

The BioBrick standard makes possible iterated pairwise assembly of cloned parts without any depletion of unique restriction sites. Every part that conforms to the standard is compatible with every other part, thereby fostering a worldwide user community. The assembly methods, however, are labor intensive or inefficient compared to some newer ones so the standard may be falling out of favor. An easier way to assemble BioBricks is described herein. Plasmids encoding BioBrick parts are purified from Escherichia coli cells that express a foreign site-specific DNA methyltransferase, so that each is subsequently protected in vitro from the activity of a particular restriction endonuclease. Each plasmid is double-digested and all resulting restriction fragments are ligated together without gel purification. The ligation products are subsequently double-digested with another pair of restriction endonucleases so only the desired insert-recipient vector construct retains the capacity to transform E. coli. This 4R/2M BioBrick assembly protocol is more efficient and accurate than established workflows including 3A assembly. It is also much easier than gel purification to miniaturize, automate and perform more assembly reactions in parallel. As such, it should streamline DNA assembly for the existing community of BioBrick users, and possibly encourage others to join.

FEATURES OF DIAGNOSIS OF NECROBACTERIOSIS OF COWS BY PCR-RFLP

Molecular genetic markers can detect polymorphism at the DNA level. This feature determines the possibility of their widespread use in genetics and breeding. Alleles of the BoLA-DRB3 gene (exon 2) can act as such markers if a statically significant association between the disease and the allele is established. The presence of such DNA markers in the genotype of animals makes it possible to judge the likelihood of disease in postnatal ontogenesis immediately after the birth of a heifer, based on which we can conclude about the conditions of further use of the animal in the main herd. According to the results of studying the polymorphism of the BoLA-DRB3 gene in cows of the Ukrainian black and white dairy breed resistant and susceptible to necrobacteriosis, four "informative" alleles were revealed. Two of them *03 and *22 are associated with resistance, and the other two - *16 and *23 with susceptibility to necrobacteriosis. The presence of these alleles in the genotype of the animal is determined by testing performed by PCR-RFLP. The method is time consuming, labor intensive and costly. To simplify it, the following technique is proposed. Restriction fragments of alleles *03, *16, *22 and *23 for endocluases RsaI, XhoII and HaeIII have the following DNA patterns: bbb, jbd, mba and nba. Due to the peculiarity of the restriction fragments, which is that endonuclease XhoII reveals in these alleles only one pattern b with length of 284 bp, the process of determining informative alleles can be simplified. Isolation of DNA from blood samples and amplification of a fragment of the BoLA-DRB3.2 gene with a size of 284 bp is carried out according to the established technique. Next, the restriction of the fragment by endonuclease XhoII and sampling having a pattern b. Selected samples are treated with RsaI endonuclease and only those with patterns b, j, m and n remain. The next step is to restrict the selected samples with HaeIII endonuclease and select heifers with bbb (*03) and nba (*23) genotypes. After the first restriction, blood samples without pattern b are eliminated from the experimental sample; after the second – two alleles with patterns RsaI + XhoII jb (*16) and mb (*22) are unambiguously determined, after the third – genotypes bbb and nba, which correspond to alleles *03 and *23. In total, only 75% of blood samples are typed, which reduces the material consumption, time and cost of work to identify heifers genetically susceptible (resistant) to necrobacteriosis.

Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus

ABSTRACTMagnaporthe oryzae is a filamentous ascomycete fungus that causes devastating diseases of crops that include rice and wheat, and a variety of turf, forage and wild grasses. Strains from ryegrasses possess highly stable chromosome ends that undergo frequent rearrangements during vegetative growth in culture and in planta. Instability is associated with the presence of two related retrotransposons (Magnaporthe oryzaeTelomeric Retrotransposons - MoTeRs) inserted within the telomere repeat tracts. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, restriction mapping, and sequencing of both parental and novel telomeric restriction fragments, along with MinION sequencing of DNA from three single-spore cultures, allowed us to document the molecular alterations for 109 newly-formed telomeres. Rearrangement events included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by “plain” telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; expansion and contraction of subtelomeric tandem repeats; MoTeR truncations; duplication and terminalization of internal sequences; and breakage at long, interstitial telomeres generated during MoTeR insertion. Together, our data show that when MoTeRs invade the telomeres, they can dramatically perturb the integrity of chromosome ends, leading to the generation of unprotected DNA termini whose repair has the potential to generate chromosome alterations that extend well into the genome interior.

Estimating DNA-DNA interaction frequency from Hi-C data at restriction-fragment resolution

Hi-C is a popular technique to map three-dimensional chromosome conformation. In principle, Hi-C’s resolution is only limited by the size of restriction fragments. However, insufficient sequencing depth forces researchers to artificially reduce the resolution of Hi-C matrices at a loss of biological interpretability. We present the Hi-C Interaction Frequency Inference (HIFI) algorithms that accurately estimate restriction-fragment resolution Hi-C matrices by exploiting dependencies between neighboring fragments. Cross-validation experiments and comparisons to 5C data and known regulatory interactions demonstrate HIFI’s superiority to existing approaches. In addition, HIFI’s restriction-fragment resolution reveals a new role for active regulatory regions in structuring topologically associating domains.Availability: https://github.com/BlanchetteLab/HIFI

The Dispersal of Rodent-Borne Strains of Aphanoascus Keratinophilus and Chrysosporium Tropicum by Pellets of Predatory Birds

The species composition of keratinophilic fungi in 153 pellets of nine species of predatory birds was analysed. Based on morphological criteria, a total of 439 strains of non-dermatophytic fungi of the Chrysosporium group were isolated and identified. Dermatophytes were not detected. The collection was verified using molecular methods, such as PCR-RFLP (restriction fragments length polymorphism) and two potentially pathogenic species, Aphanoascus keratinophilus and Chrysosporium tropicum, were detected. Pellet colonisation by these fungi ranged between 37.5% and 91.7% depending on the bird species. As the analysis of undigested remains found in the pellets showed, rodents, mostly Microtus, which constituted from 57% to 100% of the birds’ diet, were a chief source of A. keratinophilus and Ch. tropicum strains. It was demonstrated that the survival and dispersal of A. keratinophilus strains was supported by higher pellet moisture while those of Ch. tropicum strains by drying, which was conditioned by the site where pellets were dropped and deposited by individual species of predatory birds. Based on the results, circulation routes of both opportunistic pathogens using pellets of predatory birds as carriers are proposed.

Heterozygosity for Loss-of-Function Mutation in SERPINE1 (PAI-1 Gene) Linked with Longer Absolute Telomere Length

Introduction: Plasminogen activator inhibitor-1 (PAI-1) is an important component of the senescence-associated secretome that contributes directly to cellular senescence. Telomeres, the nucleotide repeat structures located at the ends of chromosomes, shorten progressively with each round of cellular replication and telomere attrition is associated with cellular senescence. In murine models, genetic or pharmacologic inhibition of PAI-1 results in preservation of telomere length. Identification of a unique Amish kindred that harbors a loss-of-function mutation in SERPINE1 (c.699_700dupTA), which encodes PAI-1, provides a novel opportunity to assess the effects of lifelong PAI-1 deficiency on leukocyte telomere length (LTL) in humans. Methods: We conducted an observational study in the Old Order Amish, a founder population who originally settled in Berne, Indiana characterized by a homogeneous diet and lifestyle, and included participants aged 18 years and older. All study participants were genotyped for the c.699_700dupTA frameshift mutation in SERPINE1. For the primary analysis, we excluded participants who were homozygous for the null SERPINE1 mutation (n=7) due to their young age (18-34 years old) and small sample size. Relative LTL was quantified from peripheral blood using Southern blot of the terminal restriction fragments. We tested the association of SERPINE1 mutation status with LTL, after adjustment for age, sex, and relatedness in SOLAR. Results: A total of 170 participants were enrolled with mean age 46±19 years. Forty-three participants were identified as carriers of the null SERPINE1 mutation with an overall minor (null) allele frequency of 16% in the study population. SERPINE1 carriers had a significantly greater LTL compared with noncarriers, after adjustment for age, sex, and family structure (p=0.039; FIGURE). Every 1-year increase in age of study participant was associated with a 30 base pair decrease in LTL (p<0.0001). Conclusions: Heterozygosity for the null SERPINE1 gene, encoding a senescence-associated protein, PAI-1, is associated with longer LTL in a rare loss-of-function cohort. In addition, LTL is strongly and inversely correlated with chronologic age and supports the hypothesis that PAI-1 may contribute to cellular and organismal aging as reflected by LTL. Figure Mean telomere restriction fragments (kilobase pairs) as a function of age and genotype Figure. Mean telomere restriction fragments (kilobase pairs) as a function of age and genotype Disclosures Shapiro: CSL Behring: Other: Clinical research protocols; Kedrion Biopharma: Consultancy; Daiichi Sankyo: Other: Clinical research protocols; OPKO: Other: Clinical research protocols; National Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; PTC Therapeutics: Other: Clinical research protocols; Novo Nordisk Hemophilia Foundation: Membership on an entity's Board of Directors or advisory committees; Baxter/Baxalta/Shire: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Biogen: Membership on an entity's Board of Directors or advisory committees, Other: Clinical research protocols; Genentech: Membership on an entity's Board of Directors or advisory committees; American Thrombosis and Hemostasis Network: Other: Medical Director; ProMetic Life Sciences: Consultancy; Bayer healthcare Pharmaceuticals: Other: International network on pediatric hemophilia; Novartis: Other: Clinical research protocols; Selexys: Other: Clinical research protocols; Octopharma: Other: Clinical research protocols.

Identification of Candida species Isolated From Vulvovaginal Candidiasis Patients by Chromgen agar and PCR-RFLP Method

This study focuses on diagnosis of Candida species causing Vulvovaginal Candidiasis using phenotype and genotype analyzing methods, and frequencies of candida species also using Vulvovaginal Candidiasis patients. 130 samples (100 from patients and 30 from non infected women) were collected and cultured on biological media. Identifying the yeasts, initially some phenotypic experiments were carried out such as germ tube, from motion of pseudohyphae and clamydospores in CMA+TW80 medium, API20 candida and CHROMagar Candida. Genomic DNA of all species were extracted and analyzed with PCR and subsequent Polymerase Chain Reaction - Restriction Fragments Length Polymorphism (PCR-RFLP) methods. Frequency of C. albicans, C. krusei, C. tropicalis , C. parapsilosis and C. glabrata were 46.4%, 31%, 18%, 7.2%, and 1.8%, respectively.The ITS1-ITS4 region was amplified and the Restriction enzyme Msp1 digests this region and was used to identify of candida species .Electrophoretically ribosomal DNA of C. albicans, C. krusei, C. tropicalis and C. glabrata produced two bands whereas the C. parapsilosis gave one band.