Restriction Enzymes MspI Research Articles

DNA damage and ALAD polymorphism in high blood lead (Pb) levels of pregnant women attending a tertiary care teaching hospital

Pregnant women are particularly vulnerable to lead toxicity due to increased absorption and decreased elimination of lead from their bodies. The δ-aminolevulinic acid dehydratase (ALAD) gene plays a crucial role in lead metabolism, and its polymorphisms have been implicated in modifying the susceptibility to lead toxicity. A cross-sectional study was conducted involving 90 pregnant women and blood samples were collected to measure blood lead levels (BLL) and assessed DNA damage using the comet assay. ALAD polymorphisms were genotyped using PCR-RFLP analysis with MspI restriction enzyme. Statistical analysis, including chi-square tests, logistic regression, and correlation analysis, was performed to determine associations between ALAD polymorphisms, BLL, and DNA damage. From 90 pregnant women the participants, 16 had high BLL (≥5μg/dL), while the remaining 74 had normal levels (<5μg/dL). The ALAD 1-2 genotype was found to be significantly associated with high BLL (p<0.001). Pregnant women with the ALAD 1-2 genotype exhibited higher levels of DNA damage compared to those with other genotypes (p<0.001). Furthermore, a positive correlation was observed between the transfer of lead concentration from mother to infant and DNA damage severity (r=0.511, p<0.001). The combination of comet assay and polymorphism analysis offers a comprehensive approach to understanding the impact of lead exposure during pregnancy. These findings underscore the urgent need for effective regulatory measures to reduce lead exposure in the environment and mitigate its adverse effects of lead on maternal and child health.

Sparid species discrimination by COIBar-RFLP in commercial products

Processed seafood products are subject to fraudulent species substitution practices that pose problems not only economically but also for human health due to the risk of allergies. DNA-based molecular techniques offer an undoubted contribution to unraveling commercial fraud in processed seafood products, and several investigations have been conducted to develop specific and rapid assays for the identification of fish species. In this context, we hypothesize that the Cytochrome Oxidase I (COI) Barcode-Restriction Fragment Length Polymorphisms (COIBar- RFLP) strategy may be a useful molecular tool to quickly assess the authenticity of seafood products because (i) it takes advantage of the specific discriminatory power of the COI gene as a barcode and of the robust Restriction Fragment Length Polymorphism methodology and (ii) the interspecific variation in the digestion pattern obtained using the restriction enzymes allows bypassing the gene sequencing step. The Sparidae family includes species of high commercial value and many sparid species are difficult to recognize considering only their morphological features; thus, misidentifications are frequent. The aim of this work was to identify sparid species in processed products using the COIBar-RFLP strategy with the MspI restriction enzyme which yielded differential digestion patterns and unveiled two cases of species substitution. The proposed methodology could be used in food control laboratories to combat the widespread habit of fraudulent species substitution in the fishing industry.

Three Previously Unseen Genotypes Detected in IGFBP - 3 Gene in Buffalo Breed

Anatolian water buffalo breed is Turkey’s sole water buffalo breed, and their numbers steadily increased with the national “Water Buffalo Breeding by Breeders Project”. This study aimed to investigate of the gene region polymorphisms (Intron-2, Exon-2/Intron-3, Exon-3) of the meat-yield-related Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) gene in Anatolian water buffaloes by TaqI, HaeIII, and MspI restriction endonucleases. The phenol/chloroform method was used for DNA isolation from 151 blood samples, and extracted DNAs were amplified by touchdown PCR using specific primers. Amplified PCR products were digested with restriction endonucleases (REs) and separated in 3% agarose gel electrophoresis (AGE), then genotypes were determined. Results revealed two genotypes [AA (98.68%) and AC (1.32%)] and two alleles [A (0.99) and C (0.01)] for the Exon-2 to Intron-3 region from HaeIII digestion. TaqI digestion of the Intron-2 region revealed three genotypes [AA (7.94%), AB (3.97%), and BB (88.10%)] and two alleles [A (0.10) and B (0.90)]. MspI digestion of the Exon-3 region revealed only the AA genotype and A allele, thus revealing monomorphism. Overall, HaeIII digestion revealed insignificant polymorphism (P&amp;gt;0.05), and TaqI digestion revealed significant polymorphism (P&amp;lt;0.001) for their respective regions. Gene polymorphisms of these regions were investigated for the first time in Anatolian water buffaloes. Additionally, three novel genotypes for the IGFBP-3 gene (one from HaeIII and two from TaqI) were determined for the first time. The novel B allele from TaqI digestion was observed to have a substantial frequency.

Molecular identification and antifungal susceptibility profiles of etiologic agents of oral candidiasis among HIV-positive patients: A multicenter study.

Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is a serious risk factor for oral candidiasis (OC). In this regard, the present study aimed to investigate the frequency of Candida species collected from the oropharyngeal cavity of HIV-positive patients and the sensitivity of these isolates to antifungal drugs. Oral samples were collected from 169 HIV-positive patients. In addition to culture-based methods, a molecular assay via the polymerase chain reaction-restriction fragment length polymorphism method was applied to identify isolates using the MspI restriction enzyme. The disk diffusion method determined the susceptibility of isolated yeasts to common antifungal drugs according to the CLSI M44-A2 protocol. In total, 81 participants (47.92%) were positive for OC, and Candida albicans was the most prevalent yeast (53.98%). The median age of patients was 36 years old (IQR=10.5; 17-59), and it was found that women are 27% more susceptible to HIV-associated OC (OR=1.268; 95% CI: 0.685-2.348). Patients who received antifungal therapy had a 97.3% reduced chance for OC (OR: 0.027; 95% CI: 0.008-0.091; P-value: 0.000). Antifungal therapy reduced the risk of OC by 97.3% (OR=0.027; 95% CI=0.008-0.091; P=0.000), and antiretroviral therapy decreased the chance of OC 4.42 times (OR=4.423; 95% CI=1.697-11.528; P=0.002). The resistance rates for antifungals, namely fluconazole, ketoconazole, itraconazole, amphotericin B, and nystatin were 15.93%, 8.85%, 7.96%, 5.31%, and 4.42%, respectively. Although several decades have passed since the emergence of HIV/AIDS, little information is available about fungal colonization and infections in this population. Further investigations are suggested using novel and reference molecular identification methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequencing, respectively. In addition, more reliable methods for antifungal susceptibility testing are recommended.

A highly sensitive signal-on biosensor based on restriction enzyme-mediated molecular switch for detection of TET1

Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (CmCGG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H2O2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening.

Methylation Profile of 18S rDNA Gene in Brain and Muscle of Tambaqui Exposed to Parasiticide Trichlorfon

Until recently, there were no reports on the methylation profile of Amazonian fish specimens employing restriction enzymes. The 18S rDNA methylation status of samples of captive tambaqui (Colossoma macropomum) exposed to the antiparasitic Trichlorfon was examined in the current study. The method performed makes use of the CCGG site's sensitivity to the presence of methylation in palindrome cytosines for the restriction enzymes HpaII and MspI. The cleavage occurs by the detection or non-detection of the methyl radical in the inner (C5mCGG) or outer (5mCCGG) cytosine, which makes it possible to compare the DNA methylation profile. When compared to the specimens examined as the control group, the results obtained revealed an increase in methylated fragments of the 18S rDNA gene at LC concentrations of 30% and 50%.

Epigenetics for crop adaptation to climate change

Epigenetics for crop adaptation to climate change

P402 Comparison of PCR-RFLP with 21-plex PCR and rDNA: sequencing for identification of clinical yeast isolates

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesNon-albicans Candida species and other rare yeasts have emerged as major opportunistic pathogens in fungal infections. Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assay, which is time-consuming and prone to errors. The aim of the present study was to evaluate PCR-RFLP as a routinely used identification technique for the most clinically important Candida species in Iran and make a comparison with a novel multiplex PCR, called 21-plex PCR.MethodsA total of 173 yeast isolates from clinical sources were selected and identified with sequence analysis of the D1/D2 domains of rDNA (LSU rDNA) sequencing as the gold standard method.ResultsThe results were compared with those obtained by PCR-RFLP using MspI restriction enzyme and the 21-plex PCR. PCR-RFLP correctly identified 93.4% of common pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and P. kudriavsevii (=C. krusei)) and was able to identify 45.5% of isolates of the uncommon yeast species compared to the D1/D2 rDNA sequencing. Compared with PCR RFLP, all common Candida species and 72.7% of uncommon yeast species were correctly identified by the 21-plex PCR.ConclusionThe application of the 21-plex PCR assay as a non-sequence-based molecular method for the identification of common and rare yeasts can reduce turnaround time and costs for the identification of clinically important yeasts and can be applied in resource-limited settings.

P497 Candida DNA extraction from positive blood culture bottles

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMBackgroundMolecular technique of detecting candida from blood is a quick method to identify candidemia. A major limitation of the molecular method is the difficulty associated with breaking fungal cell wall, the DNA extraction step still requires more than half of a working day.The successful extraction of DNA involves effective disruption of cells, denaturation of protein, and nucleoprotein complexes, and inactivation of nucleases such as DNase. The extracted DNA should have low contamination of proteins.ObjectivesTo perform DNA extraction using phenol-chloroform method.2. To speciate candida using PCR-RFLP.Methods Candida DNA is isolated using phenol-chloroform method. The PCR products of different candida species obtained directly from blood samples were subjected to restriction enzyme (MspI).ResultsDNA was extracted from 50 samples, out of which 40 samples were from known positive blood culture bottles and the remaining 10 were known negative for candida. Of the 40 known positive blood culture bottles, 34 produced bands of various molecular weights and all 10 negative cultures did not show bands. Representative amplicon is shown in the gel picture (Figure 1).Out of 40 candida-positive blood culture samples, 7 species have been identified using PCR- RFLP method.ConclusionThe turn-around time can be highly reduced by extracting DNA from positive blood culture bottles and then performing PCR and RFLP for speciation compared with the conventional method.

Early detection of abnormality in the micropropagated Lakatan banana plants using methylation-sensitive ISSR

Somaclonal variations in banana tissue culture is not uncommon. In our tissue culture laboratory, the tissue-cultured Lakatan variety exhibited abnormalities (i.e., malformed bunches/ fingers, stunted growth or sterility) that affect the fruit yield. Using a methylation-sensitive inter-simple sequence repeats (ISSR) approach, the genomic DNA digested with methylation-sensitive restriction enzyme MspI of 274 tissue-cultured Lakatan plants were screened. The ISSR profile of the abnormal samples were identical but different from the normal samples by the absence of 997 bp band in the abnormal samples. The sensitivity of the marker employed in this study is estimated to be 93.4%. We suggest that this abnormality in the tissue-cultured Lakatan is a result of epigenetic changes which could have been induced in the banana tissue culture process. Our study also showed that this abnormality can be transmitted to the next generation through asexual propagation. The methylation-sensitive ISSR method adopted in this study is a promising tool for early detection of such abnormality in the micropropagated Lakatan banana plants.

Assessment of the association of the MOGAT1 and MOGAT3 gene with growth traits in different growth stages in Holstein calves.

The members of the monoacylglycerol acyltransferase (MOGAT) family are essential candidate genes that influence economic traits associated with triglyceride synthesis, dietary fat absorption, and storage in livestock. In addition, the MOGAT gene family may also play an essential function in human polygenic diseases, like type 2 diabetes and obesity. The present study was conducted on Holstein calves to find the association between MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A and growth traits. The polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method was performed for genotyping the MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A genes' locus using the TaqI, MspI, and BsuRI restriction enzyme. The allele frequency of A and G of the MOGAT1 locus was 0.79 and 0.21, respectively, while the genotype frequency was 0.65, 0.28, and 0.07 for AA, AG, and GG, respectively. While the allele and genotype frequencies of the MOGAT3/g.A229G locus were 00.57(), 0.43(), 0.35(), 0.45(), and 0.20(), the allele and genotype frequencies of the MOGAT3/g.G1627A locus were 0.49(), 0.51(), 0.25(), 0.49(), and 0.26(). Chi-square analysis showed that MOGAT3/g.G1627A distribution was at the Hardy–Weinberg disequilibrium ( 0.05), and MOGAT1 and MOGAT3/g.A229G distribution was at the Hardy–Weinberg equilibrium ( 0.05). In total, two statistical methods (general linear model (GLM) and PROC MIXED) were used to identify an association between gene locus and growth traits. An association analysis showed a statistically significant difference between the MOGAT1 and body weight, body length, and chest circumference, MOGAT3/g.A229G with average daily gain (ADG) and withers height, and MOGAT3/g.G1627A with body weight and body length ( 0.05). The results confirmed that the MOGAT1, MOGAT3/g.A229G, and MOGAT3/g.G1627A locus are strong candidate genes that could be considered molecular markers for growth traits in cattle breeding.

Hydroxymethylation-Specific Ligation-Mediated Single Quantum Dot-Based Nanosensors for Sensitive Detection of 5-Hydroxymethylcytosine in Cancer Cells.

5-Hydroxymethylcytosine (5hmC) modification is a key epigenetic regulator of cellular processes in mammalian cells, and its misregulation may lead to various diseases. Herein, we develop a hydroxymethylation-specific ligation-mediated single quantum dot (QD)-based fluorescence resonance energy transfer (FRET) nanosensor for sensitive quantification of 5hmC modification in cancer cells. We design a Cy5-modified signal probe and a biotinylated capture probe for the recognition of specific 5hmC-containing genes. 5hmC in target DNA can be selectively converted by T4 β-glucosyltransferase to produce a glycosyl-modified 5hmC, which cannot be cleaved by methylation-insensitive restriction enzyme MspI. The glycosylated 5hmC DNA may act as a template to ligate a signal probe and a capture probe, initiating hydroxymethylation-specific ligation to generate large amounts of biotin-/Cy5-modified single-stranded DNAs (ssDNAs). The assembly of biotin-/Cy5-modified ssDNAs onto a single QD through streptavidin-biotin interaction results in FRET and consequently the generation of a Cy5 signal. The nanosensor is very simple without the need for bisulfite treatment, radioactive reagents, and 5hmC-specific antibodies. Owing to excellent specificity and high amplification efficiency of hydroxymethylation-specific ligation and near-zero background of a single QD-based FRET, this nanosensor can quantify 5hmC DNA with a limit of detection of 33.61 aM and a wider linear range of 7 orders of magnitude, and it may discriminate the single-nucleotide difference among 5hmC, 5-methylcytosine, and unmodified cytosine. Moreover, this nanosensor can distinguish as low as a 0.001% 5hmC DNA in complex mixtures, and it can monitor the cellular 5hmC level and discriminate cancer cells from normal cells, holding great potential in biomedical research and clinical diagnostics.

Development of PCR-RFLP Technique for Identify Several Members of Fusarium incarnatum-equiseti Species Complex and Fusarium fujikuroi Species Complex.

Fusarium incarnatum-equiseti species complex (FIESC) contain over 40 members. The primer pair Smibo1FM/Semi1RM on the RPB2 partial gene has been reported to be able to identify Fusarium semitectum. The F. fujikuroi species complex (FFSC) contains more than 50 members. The F. verticillioides as a member of this complex can be identified by using VER1/VER2 primer pair on the CaM partial gene. In this research, the Smibo1FM/Semi1RM can amplify F. sulawesiense, F. hainanense, F. bubalinum, and F. tanahbumbuense, members of FIESC at 424 bp. The VER1/VER2 can amplify F. verticillioides, F. andiyazi, and F. pseudocircinatum, members of FFSC at 578 bp. Polymerase chain reaction-restriction fragment length polymorphism by using the combination of three restriction enzymes EcoRV, MspI, and HpyAV can differentiate each species of FIESC used. The two restriction enzymes HpaII and NspI can distinguish each species of FFSC used. The proper identification process is required for pathogen control in the field in order to reduce crop yield loss.

Evaluation of candidate genotype of GH gene associated with growth, production and reproduction traits in Dairy Cows.

Growth hormone (GH) is a major regulator of postnatal growth and metabolism in mammals and plays a critical role in growth, production and fertility in cattle. The present study was conducted in dairy cattle to find the association of g.48769565 C>T mutation with growth, production and reproduction traits in Sahiwal and Hardhenu cattle. PCR-RFLP was performed to genotype g.48769565 C>T mutation using the MspI restriction enzyme in our resource cattle population. In Hardhenu cattle, the frequencies of C and T alleles were 0.59 and 0.41, respectively, while genotypic frequencies were 0.33, 0.53 and 0.14 for CC, CT and TT respectively. The frequencies of the C and T alleles were 0.24 and 0.76, respectively, in Sahiwal cattle and it was observed that the highest frequency was for the TT genotype (0.58) and the lowest was for the CC genotype (0.06). Chi-square analysis showed that g.48769565C>T SNP loci meet with the Hardy-Weinberg equilibrium in Sahiwal and Hardhenu cattle. From the least-squares analysis, it was observed that CC genotype was significantly associated with total milk yield (TMY), 300days milk yield (300D MY), lactation length (LL), dry period (DP) and artificial insemination (AI)/conception (p<.05). We also observed a significant association (p<.05) of genotype CT with 3-month calves body weight. Cows with TT genotype revealed comparatively favourable service period (SP) and calving interval (CI) in our resource population. These observed differences in their allelic and genotypic frequencies in association with the traits underlying production and fertility can be utilized for genetic improvement in dairy cattle.

Genotyping-by-sequencing and genomic selection applications in hexaploid triticale.

Triticale, a hybrid species between wheat and rye, is one of the newest additions to the plant kingdom with a very short history of improvement. It has very limited genomic resources because of its large and complex genome. Objectives of this study were to generate dense marker data, understand genetic diversity, population structure, linkage disequilibrium (LD), and estimate accuracies of commonly used genomic selection (GS) models on forage yield of triticale. Genotyping-by-sequencing (GBS), using PstI and MspI restriction enzymes for reducing genome complexity, was performed on a triticale diversity panel (n = 289). After filtering for biallelic loci with more than 70% genome coverage, and minor allele frequency (MAF) > 0.05, de novo variant calling identified 16,378 single nucleotide polymorphism (SNP) markers. Sequences of these variants were mapped to wheat and rye reference genomes to infer their homologous groups and chromosome positions. About 45% (7430), and 58% (9500) of the de novo identified SNPs were mapped to the wheat and rye reference genomes, respectively. Interestingly, 28.9% (2151) of the 7430 SNPs were mapped to the D genome of hexaploid wheat, indicating substantial substitution of the R genome with D genome in cultivated triticale. About 27% of marker pairs were in significant LD with an average r2 > 0.18 (P < 0.05). Genome-wide LD declined rapidly to r2 < 0.1 beyond 10 kb physical distance. The three sub-genomes (A, B, and R) showed comparable LD decay patterns. Genetic diversity and population structure analyses identified five distinct clusters. Genotype grouping did not follow prior winter vs spring-type classification. However, one of the clusters was largely dominated by winter triticale. GS accuracies were estimated for forage yield using three commonly used models with different training population sizes and marker densities. GS accuracy increased with increasing training population size while gain in accuracy tended to plateau with marker densities of 2000 SNPs or more. Average GS accuracy was about 0.52, indicating the potential of using GS in triticale forage yield improvement.

The rs2238296 polymorphism of the mitochondrial DNA polymerase gamma gene and postinfarction left ventricular remodeling in patients with heart failure

Abstract Introduction Mitochondria carry out respiratory function in cells, ensuring the operation of the electron transport chain and the production of ATP. The internal environment of mitochondria, including mitochondrial DNA (mtDNA), is susceptible to free radicals. DNA polymerase gamma mediates mtDNA replication. Mutations in the POLG1 gene cause, in particular, mtDNA depletion syndrome. This leads to mitochondrial dysfunction and reduces oxidative stress resistance, which is one of the main pathological factors in myocardial infarction (MI). Purpose To evaluate the association between rs2238296 polymorphism of the POLG1 gene and the features of left ventricular remodeling after MI in patients with chronic heart failure (CHF). Methods A total of 153 patients with CHF and history of myocardial infarction were enrolled in the study. According to echocardiography data, 56 patients (36.6%) were diagnosed with left ventricular aneurysm. Thus, the first group consisted of patients with postinfarction left ventricular aneurysm (n=56), the second group consisted of patients without left ventricular aneurysm (n=97). Genotyping of the rs2238296 polymorphism of the POLG1 gene (substitution of T for C) located in the first intron of the gene was performed using polymerase chain reaction (PCR), cleavage of PCR products with MspI restriction enzyme, and electrophoretic detection of the results. Results Groups of patients were comparable of gender (92,9% and 87,6% men, p=0.3) and age (57 [50; 60] and 55 [50; 60.5] years, p=0,8). The anamnesis of arterial hypertension (83.9% and 88.7%, p=0,3), diabetes mellitus (14.3% and 19.6%, p=0,4) and stroke (8.9% and 8.3%, p=0,9) in both group of patients was comparable too. Patients of the first group had a significantly lower left ventricular ejection fraction (43 [30; 53.5]% and 54.5 (44; 64)%, p&amp;lt;0.001). In addition, clinical severity of CHF was higher in the first group, but the differences did not reach statistical significance. Thus, NYHA functional class III-IV was diagnosed in 60.7% of patients in group 1 and in 49.5% in group 2 (p=0.2). It was found that haplotype TT of the POLG1 gene rs2238296 polymorphism occurred equally often in the study groups (29.6% and 34.7%, p=0.5). TC was found with frequency 40.7% in patients with aneurism and 52.6% in patients without aneurism (p=0.2). Haplotype CC was found in almost every third patient with left ventricular aneurysm (29.6%), and only in 12.6% of cases in the group without aneurysm (OR 2.3; 95% CI 1.1–5.3, p=0.01). The studied polymorphism is located in the intron, but it can be localized in the region associated with the regulation of gene expression, or linked to a functionally significant polymorphism in another region of the gene. Conclusion The CC genotype of the POLG1 gene rs2238296 polymorphism is associated with an increased risk of developing left ventricular aneurysm after MI in patients with CHF. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): State assignment No. AAAA-A20-120041090007-8 (10.04.2020)

Повреждение ДНК в тканях белых крыс при воздействии дыма лесных пожаров

Wildfire smoke affects the health of the population of the entire planet and each year the situation is getting worse. Fire smoke has mutagenic, carcinogenic and other long-term effects. The aim of this paper was to study the genotoxic effect of wildfire smoke on male white rats and the transgenerational effect of wildfire smoke on their offspring. Smoke exposure simulation was performed on 40 male white rats in 200-litre exposure chambers. Forest litter, branches, fallen bark, and the upper layer of soil served as a combustible substrate. The exposure was carried out for 4 hours 5 days a week in the course of 4 weeks. Offspring of both sexes were obtained from the exposed animals and intact females. We analysed DNA fragmentation and global DNA methylation in the gonadal tissue and blood cells of the exposed animals immediately after the exposure and global DNA methylation in the blood cells of the offspring upon reaching sexual maturity. The research was performed using the comet assay with modifications to study global DNA methylation with MspI and HpaII restriction enzymes. The exposed animals showed an increased level of global DNA methylation in their blood cells. In male offspring, a decrease in the level of global DNA methylation in the blood cells was revealed, compared with the controls, while females showed no differences from the controls. The established facts of changes in global DNA methylation after exposure to wildfire smoke require further in-depth research, since the mechanism of the development of this phenomenon remains rather unclear.

Growth hormone gene polymorphism in Jaisalmeri and Sindhi camels

The present study was aimed to assess the genetic polymorphism of growth hormone (GH) gene in Jaisalmeri and Sindhi camels using polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique. Genomic DNA were isolated from venous blood samples collected from 93 Indian camels (Jaisalmeri- 38 and Sindhi-55 camels) and 613-base pair fragment of GH gene was amplified from each genomic DNA sample by polymerase chain reaction. The PCR-RFLP was done using restriction enzyme MspI which revealed three genotypes, viz. CC, CT and TT in both the breeds. Our result indicated that both the breeds were polymorphic in nature. The corresponding genotype frequency in Jaisalmeri and Sindhi breeds were 0.474 and 0.345 for the CC, 0.395 and 0.564 for CT, 0.131 and 0.091 for TT genotypes and the corresponding C allele frequency was 0.671 and 0.627 and T allele frequency was 0.329 and 0.373. The CT genotype was found predominant in the Sindhi breed, whereas CC genotype was predominant in Jaisalmeri breed. The frequency of C allele was higher than the T allele in both of the breeds. These camel populations were in Hardy-Weinberg Equilibrium indicating that the populations meet the HWE assumptions and genetic variations were conserved. It could be concluded that Jaisalmeri and Sindhi camels having different genetic variants of GH gene might be investigated for production a DNA marker for growth and production traits in Indian camels.

Growth Hormone Gene Polymorphism and its Relation to Quail Growth and Carcass

In this study, it was aimed to determine the polymorphism of GH (growth hormone) gene in Japanese quails and the relationships between these genes and body weight and carcass traits. 3 genotypes (AA, AB and BB), 2 (A and B) alleles were detected by cutting the GH intron 1 region with restriction enzyme MspI. As a result of variance analysis, it was determined that the hatching weights of the animals with B allele and liver weights were higher. Thus, it is seen that GH gene and PCR-RFLP technique can be used in breeding studies.

The impact of promoter single nucleotide polymorphism (-11391 G/A) on type II diabetes mellitus in Iraqi population

BackgroundType two diabetes mellitus (Type II DM) in Iraq is exceeding and exhibited a large demand on health care systems. Many studies have explained polymorphism within the promoter adiponectin gene like -11391G>A (rs17300539) is associated with (Type II DM) and this polymorphism can cause changes in body mass index (BMI), blood glucose level insulin sensitivity and lipid profile. MethodsThe current study included 400 Type II DM patients and 400 apparently healthy control subjects. Genotyping of rs17300539 polymorphism was performed by PCR–RFLP technique by using restriction enzyme (MspI) then agarose gel electrophoresis. ResultsThe analysis of the genotype distribution of rs17300539 emphasized significant differences among Type II DM group after comparison with the control subjects under the codominant and dominant models. The homozygous genotype (AA) and heterozygous (GA) genotype of rs17300539 was significantly (OR = 2.52, CI 95%:1.04–6.12, P = 0.04) (OR = 2.27, CI 95%; 1.62–3.19, P = 0.001) respectively, raised the risk of Type II DM by two folds, as compared to the wild type (GG) after adjustment for age, gender and BMI. ConclusionAdiponectin gene polymorphism of promoter gene location rs17300539 was implicated in the development of Type II DM. this polymorphism also affect the metabolic changes in diabetic patients.