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Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes-granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples.
Newcastle disease and Avian Influenza are considered to be the most dangerous fowl diseases which may cause huge economic losses. Newcastle disease is caused by the enveloped, and single-stranded RNA virus (NDV, APMV-1; belonging to Paramyxoviridae family), which can be further divided into sixteen different genotypes grouped into five pathotypes according to their pathogenicity. It has been reported that low pathogenic virus can greatly increase its pathogenicity even during a single passage. Additionally, due to the widespread use of live vaccines, a mixture of two or more different viruses in one sample can be detected. Hence, there is a great need for establishment of fast, inexpensive, sensitive, and relatively simple diagnostic method for multistrain and quasispecies detection of NDV infection. In this paper we describe a diagnostic method based on RT-PCR followed by a modified version of single-stranded conformational polymorphism analysis using short DNA fragments of gene encoding viral F protein. The method allows for rapid diagnosis of genetic variant emerging from previously stable population which may prevent the spread of the pathogenic viral variant.
The potential association between the K121Q (A/C, rs1044498) polymorphism in the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1) gene and risk of diabetic kidney disease (DKD) has been investigated. Nevertheless, the effect of this variant on DKD risk is still under debate, and conflicting results have been reported. To this date, no meta-analysis has evaluated the association of the K121Q polymorphism with DKD. This paper describes the first meta-analysis conducted to evaluate whether the ENPP1K121Q polymorphism is associated with DKD. A literature search was conducted to identify all case-control or cross-sectional studies that evaluated associations between the ENPP1K121Q polymorphism and DKD. Pooled odds ratios (OR) and 95% confidence intervals (95% CI) were calculated for allele contrast, additive, dominant and recessive inheritance models. Seven studies were eligible for inclusion in the meta-analysis, providing data on 3571 type 1 or type 2 diabetic patients (1606 cases with DKD and 1965 diabetic controls without this complication). No significant heterogeneity was observed among the studies included in the meta-analysis when assuming different inheritance models (I² < 50% or P > 0.10 for the entire sample and after stratification by ethnicity). Meta-analysis results revealed significant associations between the K121Q polymorphism and risk of DKD in Asians and Europeans when assuming the different inheritance models analyzed. The most powerful association was observed for the additive model (OR = 1.74, 95% CI 1.27-2.38 for the total sample). In conclusion, the present meta-analysis detected a significant association between the ENPP1K121Q polymorphism and increased susceptibility of DKD in European and Asian populations.
Reactive oxygen species (ROS)-mediated damage has been hypothesized to play a role in the development and poor outcome of schizophrenia, as well as the development of neuroleptic-induced abnormal involuntary movements. Recently, the functional polymorphism (Ala-9Val) in the manganese superoxide dismutase (MnSOD) gene (part of the antioxidant defense mechanism) was found to be associated with schizophrenia in a Turkish population. This study was aimed at replicating this finding in a Xhosa population. In addition, the role of Ala-9Val in abnormal involuntary movement and tardive dyskinesia development in the Xhosa population was also investigated. The schizophrenic patient group (n=286) and a healthy control group (n=243) were genotyped for the Ala-9Val polymorphism using heteroduplex-single stranded conformational polymorphism (HEX-SSCP) analysis. No significant difference in genotype or allele frequency could be observed between the schizophrenia and control group (P=0.294 and P=0.528 respectively). In addition no association could be found between the polymorphism and symptom severity (SANS and SAPS). The Xhosa schizophrenia patient group with abnormal involuntary movements (n=54) and a subgroup with tardive dyskinesia (n=30) was found to significantly differ in Ala-9Val genotype frequency (P=0.008 and P=0.011 respectively) compared to the Xhosa schizophrenia patient group without abnormal involuntary movements (n=204). However, no significant difference was found for the allele frequencies (P=0.955 and P=0.161). Further, using ANCOVA no association was found between AIMS score and genotype in the group with abnormal involuntary movements (P=0.1234). However, in the patient group with tardive dyskinesia an association was observed between genotype and AIMS score (P=0.0365). These results do not support a major role of the MnSOD Ala-9Val polymorphism in the development of schizophrenia or symptom severity in the Xhosa population. Yet it seems to be involved in the development of abnormal involuntary movements and tardive dyskinesia and may even modulate the severity of tardive dyskinesia.
Single-nucleotide polymorphisms (SNPs) are considered to be useful polymorphic markers for genetic studies of polygenic traits. Single-stranded conformational polymorphism (SSCP) analysis has been widely applied to detect SNPs, including point mutations in cancer and congenital diseases. In this study, we describe an application of the fluorescent labeling of PCR fragments using a fluorescent-adapted primer for SSCP analysis as a novel method.
Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis was used to investigate variation in the ovine KAP6-3 gene (KRTAP6-3) in 383 Merino × Southdown-cross lambs from four sire-lines, and to determine whether this variation affects wool traits. Five PCR-SSCP banding patterns, representing five different nucleotide sequences, were detected, including four previously identified (named A, B, C, and F) variants and one newly identified (named G) variant. A new non-synonymous single nucleotide polymorphism (SNP) and a 45-bp deletion were detected in variant G. Of the three common genotypes (AA, AB, and AG) identified in these sheep, wool from sheep that were AG, on average, had a lower mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), and prickle factor (PF) than wool from AA sheep, whereas wool from AB sheep, on average, had a higher MFD, FDSD, and PF than wool from AA sheep. This suggests that variation in ovine KRTAP6-3 affect MFD, FDSD, and PF, and that this gene may have potential for use as a gene-maker for improving fibre diameter-associated wool traits.
The fatty acid binding protein 4 (FABP4) plays an important role in the regulation of lipid metabolism in mammals. In this study, two regions of ovine FABP4 spanning exon 2-intron 2 and exon 3-intron 3 were investigated in four hundred and twenty lambs derived from seven sires that were previously typed as having heterozygous genotypes in both these regions of the gene. These regions have been shown to be variable, with three SNPs plus one indel and four SNPs respectively constituting five and four allele variants in the two regions. Across these regions, fourteen haplotypes have been identified. The lambs were typed using a Polymerase Chain Reaction Single-Stranded Conformational Polymorphism (PCR-SSCP) method to identify the haplotypes inherited from the sires. Between three and four paternally-derived haplotypes were identified in the progeny of six of the seven sires, suggesting that meiotic recombination occurs within ovine FABP4. A number of sequence motifs associated with recombination "hotspots" were detected in the two regions of the gene that were analyzed and these may facilitate the recombination.
Phosphodiesterases (PDE) are key modulators of signal transduction and are involved in inflammatory cell activation, memory and cognition. There is a two-fold decrease in the expression of phosphodiesterase 8A (PDE8A) in the temporal cortex of major depressive disorder (MDD) patients. Here, we studied PDE8A mRNA-editing profile in two architectonically distinct neocortical regions in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicide decedents. By using capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), a previously validated technique to identify A-to-I RNA modifications, we report the full editing profile of PDE8A in the brain, including identification of two novel editing sites. Editing of PDE8A mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Furthermore, we report significant intra-regional differences between non-psychiatric control individuals and depressed suicide decedents, which could discriminate the two populations. Taken together, our results (i) highlight the importance of immune/inflammatory markers in major depressive disorder and suicide and (ii) establish a direct relationship between A-to-I RNA modifications of peripheral markers and A-to-I RNA editing-related modifications in brain. This work provides the first immune response-related brain marker for suicide and could pave the way for the identification of a blood-based biomarker that predicts suicidal behavior.
Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe-NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H-, K-, and N-ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3' half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR-single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG-to-CTG transition at codon 199, and the other had an ATC-to-att transition at codon 229 and two nonsense C-to-T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe-NTA.
The gene encoding the high glycine/tyrosine keratin-associated protein 20-2 (KAP20-2) gene has been described in humans, but has not been identified in any livestock species. A search for similar sequences in the caprine genome using the human KAP20-2 gene (KRTAP20-2) revealed a homologous sequence on chromosome 1. Three different banding patterns representing distinct sequences (A-C) in Longdong cashmere goats were identified using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis. These sequences shared high sequence similarity with the human and mouse KRTAP20-2 sequences, suggesting that A-C are caprine variants of the human and mouse genes. Four single nucleotide polymorphisms (SNPs) were identified, and three of them were non-synonymous. KRTAP20-2 was found to be expressed in secondary hair follicles, but not in heart, liver, lung, kidney, spleen, or longissimus dorsi muscle. The presence of A was associated with increased cashmere fibre weight, while the presence of B was associated with a decrease in cashmere fibre weight and curly fibre length. Goats with genotype AA had a higher cashmere fibre weight and a higher curly fibre length than those with genotypes AB or BB. These results indicate that caprine KRTAP20-2 variation may have value as a genetic marker for improving cashmere fibre weight.
Patients successfully treated for a malignancy with cytotoxic therapy have an increased risk of developing secondary myelodysplasia (MDS) and acute myeloid leukemia (AML). We report a patient in remission from Hodgkin's disease (HD) who remains hematologically normal 4 years after combination chemotherapy, but who has biological and genetic abnormalities characteristic of myelodysplasia. X-inactivation analysis using a 5' phosphoglycerate kinase (PGK) probe demonstrates polyclonal hematopoiesis, but cytogenetic analysis reveals a clonal population with a minority of metaphases having a 7q-deletion. NRAS mutations are not detectable 1 year after treatment, but are present in two separate clones (at codons 12 and 15) analyzed by single-stranded conformational polymorphism (SSCP), followed by cloning and sequencing 4 years after treatment. The presence of an activated NRAS with the same codon 12 mutation was independently confirmed by the nude mouse tumorigenicity assay. In vitro peripheral blood granulocyte-macrophage colony-forming units (CFU-GM) have changed from normal to undetectable levels while erythroid burst forming units (BFU-E) were significantly reduced on two occasions during the period of observation. These abnormalities are characteristic of MDS. Continued clinical follow-up will determine whether these evolving genetic and biological abnormalities pre-date the onset of clinical and morphological features of MDS.
Keratin-associated proteins (KAPs) are structural components of wool and hair fibers. To date, eight high glycine/tyrosine KAP (HGT-KAP) families have been identified in humans, but only three have been identified in sheep. In this study, the putative ovine homolog of the human KAP22-1 gene (KRTAP22-1) was amplified using primers designed based on a human KRTAP22-1 sequence. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was used to screen for variation in KRTAP22-1 in 390 Merino × Southdown-cross lambs and 75 New Zealand (NZ) Romney sheep. Three PCR-SSCP banding patterns were detected and DNA sequencing revealed that the banding patterns represented three different nucleotide sequences (A-C). Two single nucleotide polymorphisms (SNPs) were identified in these sequences. Variant B was most common with a frequency of 81.3% in NZ Romney sheep, while in the Merino × Southdown-cross lambs, A was more common with a frequency of 51.8%. The presence of B was found to be associated with increased wool yield and decreased mean fiber curvature (MFC). Sheep of genotype BB or AB had a higher wool yield than those of genotype AA. These results suggest that ovine KRTAP22-1 variation may be useful when developing breeding programs based on increasing wool yield, or decreasing wool curvature.
The LIM-homeobox gene 3 (LHX3) plays an essential role in pituitary gland and nervous system development. Sequence variants (SVs) in coding and non-coding regions of LHX3 gene have an impact on LHX3 transcription and growth traits in cattle. Previously, we have identified 3 single nucleotide polymorphisms (SNPs: 1-3) in all exons and intron 2 regions of the LHX3 gene in cattle. Here, 7 novel SNPs (SNPs: 4-10) were identified by DNA sequencing and polymerase chain reaction single-stranded conformational polymorphism (PCR-SSCP) methods. In the present study, a total of 10 SNPs were assessed linkage disequilibrium (LD) in 802 cows representing four main cattle breeds from China (Nanyang, Qinchuan, Jiaxian, and Chinese Holstein). The assessment results demonstrated that 17 haplotypes and 18 diplotypes were revealed in these cattle populations. Moreover, association analysis indicated that the genotypes of SNPs 1-6 are associated with the body weight at 6, 12 and 18months of age in Nanyang cattle (P<0.01 or P<0.05), whereas no significant association was found between the 18 diplotypes and growth traits. Our results provide evidence that some SNPs in LHX3 gene may be associated with body weight at certain age, and LHX3 gene may be used as candidate gene for marker-assisted selection (MAS) in beef cattle breeding.
Understanding the process of speciation requires understanding how gene flow influences divergence. Recent analyses indicate that divergence can take place despite gene flow and that the sex chromosomes can exhibit different levels of gene flow than autosomes and mitochondrial DNA. Using an eight marker dataset including autosomal, z-linked, and mitochondrial loci we tested the hypothesis that blue-footed (Sula nebouxii) and Peruvian (S. variegata) boobies diverged from their common ancestor with gene flow, paying specific attention to the differences in gene flow estimates from nuclear and mitochondrial markers. We found no gene flow at mitochondrial markers, but found evidence from the combined autosomal and z-linked dataset that blue-footed and Peruvian boobies experienced asymmetrical gene flow during or after their initial divergence, predominantly from Peruvian boobies into blue-footed boobies. This gene exchange may have occurred either sporadically between periods of allopatry, or regularly throughout the divergence process. Our results add to growing evidence that diverging species can remain distinct but exchange genes.
The 5'AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that controls cellular energy homeostasis in response to environmental or nutritional stress. The PRKAG3 gene (PRKAG3) encodes the γ3 subunit of the AMPK. Variation in this gene has been found to be associated with meat quality traits in pigs. In this study, we used polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) to investigate variation in exon 3 and exons 4-6 of ovine PRKAG3. In 160 New Zealand Suffolk sheep, two variant sequences (named a and b) were identified in the exon 3 region of the gene and three variant sequences (named A, B and C) were identified in the exon 4-6 region of the gene, respectively. A total of three nucleotide substitutions were revealed and these were located in intron 4, exon 4 and intron 5, respectively. The nucleotide substitution identified in the exon 4 (g.2656 C>T) could nominally lead to an amino acid substitution of tryptophan to arginine at position 230 (R230W) in ovine PRKAG3. In comparison with the PRKAG3 amino acid sequences in other species, this R230W substitution appeared to occur only in sheep. This is the first report of genetic variation in ovine PRKAG3, and the variation found in this study could be functionally important for AMPK activity, which in turn may affect meat quality traits in sheep.
The keratin-associated proteins (KAPs) are constituents of cashmere fibers and variation in many KAP genes (KRTAPs) has been found to be associated with fiber traits. The gene encoding the high-sulphur KAP28-1 has been described in sheep, but it has not been identified in the goat genome. In this study, a 255-bp open reading frame on goat chromosome 1 was identified using a search of similar sequence to ovine KRTAP28-1, and that would if transcribed and translated encode a high sulphur KAP. Based on the analysis of polymerase chain reaction (PCR) amplicons for the goat nucleotide sequences in 385 Longdong cashmere goats in China, five unique banding patterns were detected using single-stranded conformational polymorphism (SSCP). These represented five DNA sequences (named variants A to E) and they had the highest resemblance to KRTAP28-1 sequences from sheep, suggesting A-E are variants of caprine KRTAP28-1. DNA sequencing revealed a 2 or 4-bp deletion and eleven nucleotide sequence differences, including four non-synonymous substitutions. Of the four common variants (A, B, C and D) found in these goats, the presence of variant A was associated with decreased mean fiber diameter and this effect appeared to be additive. These results indicate that caprine KRTAP28-1 variation might have value as a molecular marker for reducing cashmere mean fiber diameter.
The objective of this study was to determine the prevalence and identification of leukotoxin gene, lktA, variant strains of Fusobacterium necrophorum in the footrot lesions of sheep. The detection of F. necrophorum was carried out by PCR targeting the lktA gene fragment and identification of lktA variant strains was done by PCR-single stranded conformational polymorphism (PCR-SSCP) and gene sequencing. Of the 450 swabs collected from footrot lesions of sheep, 117 were lktA-positive for F. necrophorum. Of the 50 swabs collected from apparently asymptomatic sheep, only one was lktA-positive for F. necrophorum. The overall prevalence of F. necrophorum in footrot affected sheep in Kashmir valley was 26%, and ranged from 20 to 34.8%, respectively. PCR-SSCP of lktA gene fragment analysis revealed three lktA variants, designated as JKS-F1/F2/F3, while two samples (1.7%) showed multiple lktA variant strains of F. necrophorum in a single footrot-affected sheep hoof. This appears to be the first report on the presence of more than one lktA variant of F. necrophorum in a footrot lesion of sheep. The JKS-F3 lktA variant was the most frequent (75.4%), followed by JKS-F2 (14.4%) and JKS-F1 (8.4%), respectively. Among the three lktA variants identified, JKS-F3 was detected in 74 (86.0%) samples from severe footrot affected sheep with a lesion score of 4. The data suggest that JKS-F3 is the predominant lktA variant of F. necrophorum and is associated with severe footrot in sheep. Hence, JKS-F3 may be a significant variant contributing to the severity and duration of the disease in sheep.
Cloned animals often suffer from loss of development to term and abnormalities, typically classified under the umbrella term of Large Offspring Syndrome (LOS). Cattle are an interesting species to study because of the relatively greater success rate of nuclear transfer in this species compared with all species cloned to date. The imprinted insulin-like growth factor receptor (IGF2R; mannose-6-phosphate) gene was chosen to investigate aspects of fetal growth and development in cloned cattle in the present study. IGF2R gene expression patterns in identical genetic clones of several age groups were assessed in day 25, day 45, and day 75 fetuses as well as spontaneously aborted fetuses, calves that died shortly after birth and healthy cloned calves using single stranded conformational polymorphism gel electrophoresis. A variable pattern of IGF2R allelic expression in major organs such as the brain, cotyledon, heart, liver, lung, spleen, kidney and intercotyledon was observed using a G/A transition in the 3'UTR of IGF2R. IGF2R gene expression was also assessed by real time RT-PCR and found to be highly variable among the clone groups. Proper IGF2R gene expression is necessary for survival to term, but is most likely not a cause of early fetal lethality or an indicator of postnatal fitness. Contrary to previous reports of the transmission of imprinting patterns from somatic donor cells to cloned animals within organs in the same cloned animal the paternal allele of IGF2R can be imprinted in one tissue while the maternal allele is imprinted in another tissue. This observation has never been reported in any species in which imprinting has been studied.
Uncoupling protein-1 gene (UCP1) plays an important role in the regulation of thermogenesis, energy expenditure, and protection against oxidative stress. In this study, six separate UCP1 regions: region-1 and region-2 (two parts of the promoter), region-3 and region-4 (two parts of intron 1), region-5 (spanning part of intron 5 and part of exon 6), and region-6 (spanning part of exon 6 and part of the 3'-UTR) from a variety of sheep breeds, were analysed using polymerase chain reaction-single-stranded conformational polymorphism (PCR-SSCP) analyses. In total, 30 single nucleotide polymorphisms (SNPs) were detected. Of these, 14 were located in the promoter, eight were found in intron 1, six were found in intron 5, and one was found in the 3'-UTR. One substitution in exon 6 (c.910A/G) would putatively result in an amino acid change (p.Thr304Ala). Twenty-eight novel SNPs and nine new haplotypes spanning region-2 to region-5 were identified. Of these nine haplotypes, five were common (B₂-A₅, C₂-A₅, C₂-C₅, A₂-A₅, and A₂-B₅) and four were rare (C₂-B₅, A₂-C₅, B₂-C₅, and B₂-B₅) in the sheep investigated. Of the five common haplotypes found in 314 New Zealand Romney sheep for which growth and carcass trait data were available, the presence of A₂-B₅ was associated with decreased hot carcass weight (HCW) and loin lean-meat yield (p = 0.006, p = 0.032, respectively), and the presence of C₂-C₅ was associated with a decreased proportion of leg lean-meat yield (p = 0.047) in the carcasses. No associations were found with growth traits. These results confirm that ovine UCP1 is a variable gene and may have value as a genetic marker for sheep breeding.
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