Restriction Enzyme Research Articles

DNA Barcoding of Pomegranate (Punica granatum L.) Cultivars in Duhok Province- Kurdistan Region/ Iraq Using 18S–28S rRNA and ITS Region

Pomegranate is a tree species with a significant plant diversity, therefore molecular methods are necessary to define and verify approaches to recognize rapidly and correctly. This research looks at a genetic strategy for identifying pomegranate varieties in Duhok KRG. The approach is based on application of ITS region, PCR-RFLP, sequencing and SNP identification. For this study, 14 pomegranate accessions were taken from various regions, namely the Center of Duhok, Amedi, Akre, Zaxo, the South of Duhok, and Sulav. The PCR product of the 18S–28S rDNA intergenic spacer was 854bp, and the sequence analysis revealed a 99.94 percent similarity with other accession numbers in NCBI, demonstrating the use of the 18S–28S rDNA intergenic spacer for identifying and barcoding pomegranate cultivars. The PCR product of the ITS region was 700bp. This result was then employed for PCR-RFLP using two restriction enzymes namely RsaI GT/AC and HaeIII GG/CC which helped grouping as well as genetic similarities. This study Further involved sequencing examined genes were compared using the NCBI BLASTn tool and clustalo (Version 1.2.4) to determine gene location and SNP. According to this study, the result of PCR-RFLP revealed poor association between pomegranate physical morphology and genetic features, while SNP identification was identified between studied cultivars. Moreover, this result showed possible DNA barcoding of pomegranate cultivars under the study.

Restriction site associated DNA sequencing for tumour mutation burden estimation and mutation signature analysis.

Genome-wide measures of genetic disruption such as tumour mutation burden (TMB) and mutation signatures are emerging as useful biomarkers to stratify patients for treatment. Clinicians commonly use cancer gene panels for tumour mutation burden estimation, and whole genome sequencing is the gold standard for mutation signature analysis. However, the accuracy and cost associated with these assays limits their utility at scale. WGS data from 560 breast cancer patients was used for in silico library simulations to evaluate the accuracy of an FDA approved cancer gene panel as well as restriction enzyme associated DNA sequencing (RADseq) libraries for TMB estimation and mutation signature analysis. We also transfected a mouse mammary cell line with APOBEC enzymes and sequenced resulting clones to evaluate the efficacy of RADseq in an experimental setting. RADseq had improved accuracy of TMB estimation and derivation of mutation profiles when compared to the FDA approved cancer panel. Using simulated immune checkpoint blockade (ICB) trials, we show that inaccurate TMB estimation leads to a reduction in power for deriving an optimal TMB cutoff to stratify patients for immune checkpoint blockade treatment. Additionally, prioritisation of APOBEC hypermutated tumours in these trials optimises TMB cutoff determination for breast cancer. The utility of RADseq in an experimental setting was also demonstrated, based on characterisation of an APOBEC mutation signature in an APOBEC3A transfected mouse cell line. In conclusion, our work demonstrates that RADseq has the potential to be used as a cost-effective, accurate solution for TMB estimation and mutation signature analysis by both clinicians and basic researchers.

A simple method to genetically differentiate invasive F1Typha hybrids (T. × glauca) and advanced-generation/backcrossed hybrids from parent species (T. Latifolia and T. angustifolia) in eastern Canada and northeastern USA

A widespread Typha (cattail) hybrid zone in southeastern Canada and northeastern USA comprises T. latifolia, T. angustifolia, and their hybrid T. × glauca. In this region the hybrid is considered invasive because of its detrimental impacts on wetlands and biodiversity. Researchers and managers are increasingly interested in tracking and controlling the spread of the invasive hybrid, but this can be hampered by challenges in differentiating taxa: while the parent species and F1 hybrids can often be identified from morphological characteristics, advanced-generation and backcrossed hybrids can be common, and these have obscured morphological distinctions among taxa. Here we present five species-specific genetic markers that differentiate T. latifolia, T. angustifolia, F1 hybrids, and advanced-generation/backcrossed hybrids. Unlike the pre-existing species-specific microsatellite markers, these markers require only PCR or PCR followed by a restriction enzyme digest, and can be visualized on an agarose gel. As a result, they require less specialized equipment than existing species-specific markers, and should therefore be useful for a larger group of practitioners. Future use of these markers could include monitoring range expansions, assessing the outcomes of restoration programs, and avoiding the unnecessary elimination of native Typha.

Development of Cleaved Amplified Polymorphic Sequence Marker for Cap Color Identification in Pleurotus cornucopiae

Cap color is one of the most crucial commercial traits for oyster mushrooms, and dark-colored varieties are highly favored by consumers, yet they are relatively scarce on the market. There is an urgent need for the selection and breeding of dark oyster mushrooms. Previous studies identified PcTYR, a key gene that controls the cap color of Pleurotus cornucopiae, and four SNPs were identified based on a genome-level sequence comparison of the black and white gene pools for extreme traits in the segregating populations. In this study, we verified whether these SNPs were color-specific sites via specific primer design, PCR amplification, and enzyme digestion of the entire isolated population strains and developed CAPS/dCAPS markers for the early visual identification of cap color to assist material screening in cap color breeding. One CAPS marker, TYR-CAPS-3-2, was developed for identifying the cap color of oyster mushrooms. After digestion with the restriction endonuclease Mse I, the marker generated polymorphic bands that accurately and visually distinguished dark-colored (non-white) and white strains from the cap color-segregating population. Consequently, the application of this marker during the early growth stage of oyster mushrooms can facilitate molecular-marker-assisted selection, expediting the breeding process for dark-colored varieties.

Toll-like Receptor 9 Gene in the Development of Type 2 Diabetes Mellitus in the Saudi Arabian Population.

Diabetes mellitus is a complex disease with a wide range of manifestations. Diabetes, notably type 2 diabetes mellitus (T2DM), is becoming more common in Saudi Arabia as a result of obesity and an aging population. T2DM is classified as a noncommunicable disease, and its incidence in the Saudi population continues to grow as a consequence of socioeconomic changes. Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response in diabetes mellitus. Previous studies have documented the relationship between different SNPs in the TLR9 gene in different forms of diabetes. As a result, the purpose of this study was to investigate the relationship between rs187084, rs352140, and rs5743836 SNPs in the TLR9 gene among T2DM patients in the Saudi population. This was a case-control study that included 100 T2DM cases and 100 control subjects. The three SNPs were identified in the study population (n = 200) using polymerase chain reaction (PCR), restriction enzymes for rs352140, and Sanger sequencing for rs187084 and rs5783836. Next, statistical analyses were performed using various software to determine the association between the SNPs and T2DM. rs187084 and rs5743836 were associated with an increased risk of T2DM development. rs187084 and rs5743836 allelic frequencies were associated with a 3.2 times increased risk of T2DM development (p < 0.05). DBP was associated with T2DM (p = 0.02). rs187084 was associated with TC and HDLc; rs352140 was associated with DBP, HbA1c, and HDLc; rs5743836 was associated with waist (p < 0.05). The CGT haplotype was strongly associated with T2DM (p < 0.003). Gene-gene interaction, graphical presentation, and dendrogram showed the strong association with T2DM patients (p < 0.05). This study concluded that rs187084 and rs5743836 were strongly associated with T2DM in Saudi Arabian patients. This study provides further evidence that SNPs in the TLR9 gene play a significant role in T2DM development in a Saudi community.

Heritability of Body Weight, Growth Rate Indices and Their Association with Insulin-Like Growth Factor-2 Gene Polymorphism in Purebred Nigerian Indigenous Chickens and their Crosses with Marshal Chickens

This research was conducted to estimate heritability of body weight and growth rate indices and also to determine their association with insulin-like growth factor-2 genes (IGF-2) polymorphism in purebred Nigerian indigenous chickens and their crosses with Marshal chickens. A total of 300 progenies were generated from the mating of three genotypes of F5 generation of Nigerian indigenous chickens (Normal feather (N), Frizzle feather (F), Naked neck (NN)) and Marshal (M) chickens. Blood samples were collected at twenty weeks for DNA extraction and IGF-2 gene amplification. IGF-2 gene was digested by restriction enzyme (Pst1) and genotyped using restriction fragment length polymorphism method. Growth rate indices (absolute (AGR) and relative growth rates (RGR)) were estimated and associated with IGF-2 gene polymorphism. The results show that IGF-2 allelic variants had no effect on the body weight (BW), AGR and RGR while chicken’s genotype effect was significant (p&lt;0.01) on BW and AGR. Two crossbred genotypes (NxM,1524.23±137.20 and 1894.92±122.81), (FxM, 1445.19±177.24 and 1840.88±177.86) had better performance for AGR and BW, respectively, compared to the purebred of Nigerian indigenous chickens. The heritability values ranged from low (.06±.021) to high (.77±.24).

Comprehensive analysis of 66 complete molluscum contagiosum virus (MOCV) genomes: characterization and functional annotation of 47 novel complete MOCV genomes, including the first genome of MOCV genotype 3, and a proposal for harmonized MOCV genotyping indexing.

Four molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants (MOCV1p, MOCV1va, MOCV1vb, and MOCV1vc) were partially characterized using restriction enzyme profiling in the early 1980s/1990s. However, complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways of MOCV genotypes and subtype variants with unavailable sequences remain unclear, and also whether all MOCV genotypes/subtype variants can be reliably detected and appropriately categorized using available PCR-based protocols. We de novo fully characterized and functionally annotated 47 complete MOCV genomes, including two putative non-MOCV1/2 isolates, expanding the number of fully characterized MOCV genomes to 66. To ascertain the placement of any putative novel MOCV sequence into the restriction profiling typing scheme, we developed an original framework for extracting complete MOCV genome sequence-based restriction profiles and matching them with reference restriction profiles. We confirmed that two putative non-MOCV1/2 isolates represent the first complete genomes of MOCV3. Comprehensive phylogenomic, recombination, and restriction enzyme recognition site analysis of all 66 currently available MOCV genomes showed that they can be agglomerated into six phylogenetic subgroups (PG1-6), corresponding to the subtype variants from the pioneering studies. PG5 was a novel subtype variant of MOCV2, but no PGs corresponded to the subtype variants MOCV1vb or MOCV4. We showed that the phylogenetic subgroups may have diverged from the prototype MOCV genotype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events that spawned PG5 and the yet undetected subtype variant MOCV1vb.IMPORTANCEFour molluscum contagiosum virus (MOCV) genotypes (MOCV1-4) and four subtype variants were partially characterized using restriction enzyme profiling in the 1980s/1990s, but complete genome sequences of only MOCV1 and MOCV2 are available. The evolutionary pathways whereby genotypes/subtype variants with unavailable sequences emerged and whether all MOCVs can be detected using current diagnostic approaches remain unclear. We fully characterized 47 novel complete MOCV genomes, including the first complete MOCV3 genome, expanding the number of fully characterized genomes to 66. For reliably classifying the novel non-MOCV1/2 genomes, we developed and validated a framework for matching sequence-derived restriction maps with those defining MOCV subtypes in pioneering studies. Six phylogenetic subgroups (PG1-6) were identified, PG5 representing a novel MOCV2 subtype. The phylogenetic subgroups diverged from the prototype lineages following large-scale recombination events and hinted at partial sequence content of MOCV4 and direction of recombinant transfer in the events spawning PG5 and yet undetected MOCV1vb variant.

First Report of aac(6')-Ib and aac(6')-Ib-cr Variant Genes Associated with Mutations in gyrA Encoded Fluoroquinolone Resistance in Avian Campylobacter coli Strains Collected in Tunisia.

The aac(6')-Ib gene is the most widespread gene encoding aminoglycoside-modifying enzyme and conferring resistance to tobramycin, streptomycin and kanamycin. The variant aac(6')-Ib-cr gene confers resistance to both aminoglycosides and fluoroquinolones (FQ). A total of 132 Campylobacter isolates, including 91 C. jejuni and 41 C. coli, were selected from broiler hens isolates. The aac(6')-Ib gene was amplified using PCR and was subsequently digested with the BtsCI restriction enzyme to identify aac(6')-Ib-cr. Among these isolates, 31 out of 41 C. coli (75.6%) and 1 (0.98%) C. jejuni were positive for the aac(6')-Ib gene, which was identified as the aac(6')-Ib-cr variant in 10 (32.25%) C. coli isolates. This variant was correlated with mutations in gyrA (Thr-86-Ile), as well as resistance to FQs. This study is the first report in Tunisia on Campylobacter coli strains harboring both the aac(6')-Ib and aac(6')-Ib-cr variants. These genes were present in Campylobacter isolates exhibiting resistance to multiple antibiotics, which restricts the range of available treatments.

Linear Dichroism Measurements for the Study of Protein-DNA Interactions.

Linear dichroism (LD) is a differential polarized light absorption spectroscopy used for studying filamentous molecules such as DNA and protein filaments. In this study, we review the applications of LD for the analysis of DNA-protein interactions. LD signals can be measured in a solution by aligning the sample using flow-induced shear force or a strong electric field. The signal generated is related to the local orientation of chromophores, such as DNA bases, relative to the filament axis. LD can thus assess the tilt and roll of DNA bases and distinguish intercalating from groove-binding ligands. The intensity of the LD signal depends upon the degree of macroscopic orientation. Therefore, DNA shortening and bending can be detected by a decrease in LD signal intensity. As examples of LD applications, we present a kinetic study of DNA digestion by restriction enzymes and structural analyses of homologous recombination intermediates, i.e., RecA and Rad51 recombinase complexes with single-stranded DNA. LD shows that the DNA bases in these complexes are preferentially oriented perpendicular to the filament axis only in the presence of activators, suggesting the importance of organized base orientation for the reaction. LD measurements detect DNA bending by the CRP transcription activator protein, as well as by the UvrB DNA repair protein. LD can thus provide information about the structures of protein-DNA complexes under various conditions and in real time.

Cationic and anionic detergent buffers in sequence yield high-quality genomic DNA from diverse plant species

Because of the heterogeneity among seedlings of outbreeding species, the use of seedling tissues as a source of DNA is unsuitable for the genomic characterization of elite germplasms. High-quality DNA, free of RNA, proteins, polysaccharides, secondary metabolites, and shearing, is mandatory for downstream molecular biology applications, especially for next-generation genome sequencing and pangenome analysis aiming to capture the complete genetic diversity within a species. The study aimed to accomplish an efficient protocol for the extraction of high-quality DNA suitable for diverse plant species/tissues. We describe a reliable, and consistent protocol suitable for the extraction of DNA from 42 difficult-to-extract plant species belonging to 33 angiosperm (monocot and dicot) families, including tissues such as seeds, roots, endosperm, and flower/fruit tissues. The protocol was first optimized for the outbreeding recalcitrant trees viz., Prosopis cineraria, Conocarpus erectus, and Phoenix dactylifera, which are rich in proteins, polysaccharides, and secondary metabolites, and the quality of the extracted DNA was confirmed by downstream applications. Nine procedures were attempted to extract high-quality, impurities-free DNA from these three plant species. Extraction of the ethanol-precipitated DNA from cetyltrimethylammonium bromide (CTAB) protocol using sodium dodecyl sulfate (SDS) buffer, i.e., the extraction using a cationic (CTAB) detergent followed by an anionic (SDS) detergent was the key for high yield and high purity (1.75–1.85 against A260/280 and an A260/230 ratio of >2) DNA. A vice versa extraction procedure, i.e., SDS buffer followed by CTAB buffer, and also CTAB buffer followed by CTAB, did not yield good-quality DNA. PCR (using different primers) and restriction endonuclease digestion of the DNA extracted from these three plants validated the protocol. The accomplishment of the genome of P. cineraria using the DNA extracted using the modified protocol confirmed its applicability to genomic studies. The optimized protocol successful in extracting high-quality DNA from diverse plant species/tissues extends its applicability and is useful for accomplishing genome sequences of elite germplasm of recalcitrant plant species with quality reads.

Pseudorabies gD protein protects mice and piglets against lethal doses of pseudorabies virus.

Pseudorabies (PR) is a highly contagious viral disease caused by the pseudorabies virus (PRV), which can cause disease in a wide range of domestic and wild animals. Studies have shown that new mutant strains have emerged in pig farms in many regions and that commercial inactivated and live attenuated vaccines are becoming less effective at protecting pigs. Porcine pseudorabies glycoprotein D (gD) gene (GenBank: QEY95774.1) with hexa-His tag to the C terminus for further purification processes was cloned into the lentiviral expression plasmid pLV-CMV-eGFP by restriction enzyme, the resulting plasmid was designated as pLV-CMV-gD. HEK-293T cells with robust and stable expression of recombinant gD protein was established by infection with recombinant lentivirus vector pLV-CMV-gD. We expressed porcine pseudorabies virus gD protein using HEK-293T cells. We describe in this study that individual gD proteins produced by a mammalian cell expression system are well immunogenic and stimulate high levels of PRV-specific and neutralizing antibodies in mice and piglets. All mice and piglets survived lethal doses of PRV, significantly reducing the amount of PRV virus in piglets' lymph nodes, lungs, spleen, and other tissues. It also significantly reduced the time cycle and amount of viral excretion from piglets to the environment through the nasal and anal cavities. The results suggest that PRV gD protein is expected to be a potential candidate for the preparation of genetically engineered PR vaccines for the prevention of PRV infection and the control of PR epidemics.

Correction of a homoplasmic mitochondrial tRNA mutation in patient-derived iPSCs via a mitochondrial base editor

Pathogenic mutations in mitochondrial DNA cause severe and often lethal multi-system symptoms in primary mitochondrial defects. However, effective therapies for these defects are still lacking. Strategies such as employing mitochondrially targeted restriction enzymes or programmable nucleases to shift the ratio of heteroplasmic mutations and allotopic expression of mitochondrial protein-coding genes have limitations in treating mitochondrial homoplasmic mutations, especially in non-coding genes. Here, we conduct a proof of concept study applying a screened DdCBE pair to correct the homoplasmic m.A4300G mutation in induced pluripotent stem cells derived from a patient with hypertrophic cardiomyopathy. We achieve efficient G4300A correction with limited off-target editing, and successfully restore mitochondrial function in corrected induced pluripotent stem cell clones. Our study demonstrates the feasibility of using DdCBE to treat primary mitochondrial defects caused by homoplasmic pathogenic mitochondrial DNA mutations.

Evaluation of the polymorphism of the GDF9 and BMP15 genes and their relationship with the reproductive functions of sheep of different breeds

Aim. Polymorphism is the result of evolutionary processes and is inherited, associated with biodiversity and modified by natural selection and functions to preserve various forms of population. It is economically advantageous to find genes that can be used in breeding in such a way as to increase the fertility of animals in different natural geographical zones, since the adaptive abilities of organisms are complicated. In this regard, the purpose of this study was to study the genetic potential of sheep of different breeds bred in various ecological and geographical regions of the North Caucasus with pasture-pasture and pasture-stall conditions of sheep breeding.Material and Methods. Genotyping was undertaken of sheep of different breeds contained in various natural and climatic zones of the Republic of Dagestan and the most arid region of Stavropol Territory in the zone of risky agriculture where the climate is sharply continental. Genotyping in the loci of the genes of differential growth factor (GDF9) and bone morphogenetic protein (BMP15) was carried out by PCR-PDRF (polymorphism of the lengths of restriction fragments) by cutting DNA using endonuclease restriction and further analysis of the size of the resulting PCR fragments. The genetic structure of genes has been studied by methods of genetic and statistical analysis of the data obtained.Conclusion. The genetic structure of sheep populations of different breeds bred in the foothills of the Republic of Dagestan and the arid region of Stavropol Territory was studied for the first time. Information obtained on the role of the degree of genetic variability of sheep of different breeds provides data which can contribute to the ecological well-being of herds and the status characterising the signs of multiple birth rates is also determined. This will further improve and significantly accelerate breeding work with livestock, since an important task of further increasing the efficiency of the industry is the reproduction of the herd together with a simultaneous increase in the productivity of animals.

Inducible Expression ofthe Restriction Enzyme Uncovered Genome-Wide Distribution andDynamic Behavior ofHistones H4K16ac andH2A.Z at DNA Double-Strand Breaks inArabidopsis.

DNA double-strand breaks (DSBs) are among the most serious types of DNA damage, causing mutations and chromosomal rearrangements. In eukaryotes, DSBs are immediately repaired in coordination with chromatin remodeling for the deposition of DSB-related histone modifications and variants. To elucidate the details of DSB-dependent chromatin remodeling throughout the genome, artificial DSBs need to be reproducibly induced at various genomic loci. Recently, a comprehensive method for elucidating chromatin remodeling at multiple DSB loci via chemically induced expression of a restriction enzyme was developed in mammals. However, this DSB induction system is unsuitable for investigating chromatin remodeling during and after DSB repair, and such an approach has not been performed in plants. Here, we established a transgenic Arabidopsis plant harboring a restriction enzyme gene Sbf I driven by a heat-inducible promoter. Using this transgenic line, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) of histones H4K16ac and H2A.Z and investigated the dynamics of these histone marks around the endogenous 623 Sbf I recognition sites. We also precisely quantified DSB efficiency at all cleavage sites using the DNA resequencing data obtained by the ChIP-seq procedure. From the results, Sbf I-induced DSBs were detected at 360 loci, which induced the transient deposition of H4K16ac and H2A.Z around these regions. Interestingly, we also observed the co-localization of H4K16ac and H2A.Z at some DSB loci. Overall, DSB-dependent chromatin remodeling was found to be highly conserved between plants and animals. These findings provide new insights into chromatin remodeling that occurs in response to DSBs in Arabidopsis.

Determination of &lt;i&gt;G&lt;/i&gt;/&lt;i&gt;C&lt;/i&gt; Polymorphism at chr20:37352001 Position in Human Blood DNA Preparations by GlaI- and Bst2UI-PCR Analyses Methods

Single nucleotide polymorphism (SNP) is a change of one nucleotide by another. This change often leads to an emergence (or disappearance) of a site recognized by a certain restriction endonuclease. As a result amplification of DNA fragment using primers surrounding SNP point (containing either N1 or N2 nucleotide) followed by hydrolysis of the amplicon with this restriction enzyme will be different for three possible variants in a diploid genome (genotypes N1/N1, N1/N2 and N2/N2). This method of restriction fragments length polymorphism (RFLP) is widely used in the genetic studies. Earlier we have developed GlaI- and FatI-PCR analyses methods which allowed to carry out real-time PCR and showed it applicability for SNP determination. In the current work a new way to determine the single nucleotide polymorphism G/C by the Bst2UI-PCR analysis is proposed. GlaI- and Bst2UI-PCR analyses have been used to determine the frequency of G/C polymorphism variants at the chr20:37352001 position (according to GRCH38.p14 genomic assembly) in the blood DNA samples of 161 donors. The study included: 1) the isolation of leukocyte DNA from blood cells; 2) GlaI- and Bst2UI-PCR analyses of the DNA fragment chr20:37351957-37352083, 3) determination of cytosine and guanine at the chr20:37352001 position in the analyzed DNA preparations, and 4) comparative analysis of the obtained results. It has been shown that 68 donors (42.2%) have a heterozygous set of G/C at the chr20:37352001 position, 89 donors (55.3%) are homozygous by G, and 4 donors (2.5%) are homozygous by C. Thus, taking into account that blood cells have a diploid set of chromosomes, G to C replacement occurs in 76 out of 322 analyzed cases (23.6%). At the same time, from the results obtained it follows that the cytosine residue complementary to G at the chr20:37352001 position exists in methylated form (5-methylcytosine) in most of the DNA molecules, both in homo- and heterozygotes. The proposed method of Bst2UI-PCR analyses extends the possibilites of SNP determination using real-time PCR.

IMPRESS: Improved Multiplex Methylation Profiling Using Restriction Enzymes and smMIP Sequencing for Highly Sensitive Multi-Cancer Detection

IMPRESS: Improved Multiplex Methylation Profiling Using Restriction Enzymes and smMIP Sequencing for Highly Sensitive Multi-Cancer Detection

Exploring Bacterial Diversity: How Far Have We Reached?

Abstract Many methods have been developed for studying and comparing bacterial diversity. These methods suffer from a number of drawbacks. Culture-dependent methods have a drawback that only a small number of bacteria can be cultured. Although many modifications in the traditional cultivation approach have been made, such as the use of gellan instead of agar and high throughput dilution to extinction culturing, but a large fraction of microbes still remain uncultured. Culture-independent methods were developed to explore uncultured bacterial diversity but they have their own drawbacks. PCR-based methods have biases during DNA extraction and the removal of substances that may inhibit polymerase activity during PCR and digestion with restriction enzymes. “Omics” approach, i.e., metagenomics, metatranscriptomics, and metaproteomics, aim to link bacterial community structure with function. Different combinations of methods can be used to know more precisely about the bacterial diversity. To date, no known method can reveal the exact bacterial diversity of different environments. This lacuna needs to be filled and newer methods must be developed that can help in exploring the immense bacterial diversity created by nature.

Effect of Reversion Back to Cys11 on the Structure and Function of S11C Cys-free Nt.BspD6I

The three-dimensional structure of recombinant nicking endonuclease S11C Cys-free Nt.BspD6I was determined at 1.85 Å resolution. Nickase S11C Cys-free Nt.BspD6I was produced by the reversion back to Cys11 in Cys-free Nt.BspD6I using site-directed mutagenesis. An analysis of the crystal structure of nickase S11C Cys-free Nt.BspD6I demonstrated that the reversion back to Cys11 induces significant conformational changes in the recognition domain of nickase, which are accompanied by changes in its functions, such as a decrease in the affinity to DNA, the loss of the ability to undergo oligomerization, and high activity of restriction endonuclease S11C Cys-free R.BspD6I.

Lack of evidence for association between the leptin/Sau3AI gene and milk yield traits in Holstein Friesian dairy cattle.

This study aimed to investigate the effect of leptin gene polymorphism and some environmental factors on milk production traits. Blood samples from 212 Holstein Friesian dairy cattle reared on a private farm were used. The intron 2 region of the leptin gene was digested with Sau3AI restriction enzyme using the PCR-RFLP method. A and B alleles and AA, AB, and BB genotype frequencies for the Sau3AI polymorphism were determined as 0.8821 and 0.1179, and 0.764, 0.236 and 0.000, respectively. Chi-square analysis revealed that the leptin gene polymorphism followed the Hardy-Weinberg equilibrium, including the absence of animals with the BB genotype. The effect of leptin gene polymorphism on all milk production traits was insignificant. For milk production traits, direct heritability (ha2) varied between 0.03 ± 0.283 (for the dry period) and 0.50 ± 0.183 (for milk conductivity). Regarding the milking time (MT), the estimated breeding values (EBVs) of cattle with the AA genotype were higher than the AB genotype (P < 0.05). As a result of this study, in the selection program, allele or genotype could not be suggested as a marker for milk yield characteristics except for the possible exception of milking time and its relationship to mastitis incidence.

The Phylogenetic Characterization of Cockroaches (Insecta: Blattaria) in Türkiye and Determination of Their Vector Potential for Medically Important Parasites

This study was performed to investigate the phylogenetic characters of the cockroaches in XXX region for mitochondrial cytochrome c oxidase subunit I (mt-COI), mt-COII, and internal transcribed spacer-2 (ITS-2) gene regions, and to determine their mechanical transmission of medically important parasites. PCR-restriction fragment length polymorphism (RFLP) was performed using mt-COI, mt-COII, and ITS-2 DNA gene regions to identify cockroach species (n=220) collected from different regions. Differentiation of cockroach species was based on RFLP models using two restriction enzymes: AvaI and EciI. For phylogenetic analysis, mt-COI, mt-COII, and ITS-2 DNA barcode regions were amplified with standard primers. The amplicons obtained were purified and sequenced with PCR primers. According to PCR-RFLP, cockroach species were identified as Blattella germanica (n =105), Blatta orientalis (n = 86), and Periplaneta americana (n = 29). A total of 13 haplotypes were detected and Maximum likelihood (ML) analyses revealed that the sequences of all three species for the three gene regions showed monophyletic structuring. The cockroaches were examined for the presence of parasites. It was determined that of the 58 parasitic forms identified belonged to helminth species 46 (79.3%) and protozoan species and 12 (20.7%). The results showed that B. germanica (58.6%) had the highest prevalence, followed by Bl. orientalis (32.8%) and P. americana (8.6%). The study findings not only contribute to the molecular epidemiology of cockroaches but also confirm that they play an important role as mechanical vectors of protozoan and helminth parasites.