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RFC1 disease, caused by biallelic repeat expansion in RFC1 , is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I–V β = −1.06, P < 0.001; lobules VI–VII β = −0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion. [ABSTRACT FROM AUTHOR]

Abstract Background Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. Results Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. Conclusions The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields.

Meiosis is a process through which diploid cells divide into haploid cells, thus promoting genetic diversity. This diversity arises from the formation of genetic crossovers (COs) that repair DNA double-strand breaks (DSBs), through homologous recombination (HR). Deficiencies in HR can lead to chromosomal abnormality resulting from chromosomal nondisjunction, and genetic disorders. Therefore, investigating the mechanisms underlying effective HR is crucial for reducing genome instability. Budding yeast serves as an ideal model for studying HR mechanisms due to its amenability to gene modifications and the ease of inducing synchronized meiosis to yield four spores. During meiosis, at the DNA level, programmed DSBs are repaired as COs or non-crossovers (NCOs) through structural alterations in the nascent D-loop, involving single-end invasions (SEIs) and double-Holliday junctions (dHJs). This repair occurs using homologous templates rather than sister templates. This protocol, using Southern blotting, allows for the analysis and monitoring of changes in DNA structures in the recombination process. One-dimensional (1D) gel electrophoresis is employed to detect DSBs, COs, and NCOs, while two-dimensional (2D) gel electrophoresis is utilized to identify joint molecules (JMs). Therefore, physical analysis is considered the most effective method for investigating the HR mechanism. Our protocol provides more comprehensive information than previous reports by introducing conditions for obtaining a greater number of cells from synchronized yeast and a method that can analyze not only meiotic/mitotic recombination but also mitotic replication. [ABSTRACT FROM AUTHOR]

Background: Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. Results: Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. Conclusions: The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields. [ABSTRACT FROM AUTHOR]

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients' blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method. [ABSTRACT FROM AUTHOR]

Telomere length (TL) limits somatic cell replication. However, the shortest among the telomeres in each nucleus, not mean TL, is thought to induce replicative senescence. Researchers have relied on Southern blotting (SB), and techniques calibrated by SB, for precise measurements of TL in epidemiological studies. However, SB provides little information on the shortest telomeres among the 92 telomeres in the nucleus of human somatic cells. Therefore, little is known about the accumulation of short telomeres with age, or whether it limits the human lifespan. To fill this knowledge void, we used the Telomere‐Shortest‐Length‐Assay (TeSLA), a method that tallies and measures single telomeres of all chromosomes. We charted the age‐dependent buildup of short telomeres (<3 kb) in human hematopoietic cells from 334 individuals (birth‐89 years) from the general population, and 18 patients with dyskeratosis congenita‐telomere biology disorders (DC/TBDs), whose hematopoietic cells have presumably reached or are close to their replicative limit. For comparison, we also measured TL with SB. We found that in hematopoietic cells, the buildup of short telomeres occurs in parallel with the shortening with age of mean TL. However, the proportion of short telomeres was lower in octogenarians from the general population than in patients with DC/TBDs. At any age, mean TL was longer and the proportion of short telomeres lower in females than in males. We conclude that though converging to the TL‐mediated replicative limit, hematopoietic cell telomeres are unlikely to reach this limit during the lifespan of most contemporary humans. [ABSTRACT FROM AUTHOR]

Rheum australe (Himalayan Rhubarb, Polygonaceae), an endangered medicinal and vegetable herb owes its age-old remedying properties to the bio-active phyto-constituents viz. anthraquinones, stilbenoids, chromones and dietary flavonoids. Polyketide pathway primarily involving the intricate Type III polyketide synthases (PKSs) contributes to the biosynthesis of these phyto-constituents. In the present study, we perform a homology-based approach to isolate an 1176 bp full-length cds sequence of the RaALS gene showing an equitable level of sequence similarity to related Type III PKSs at both nucleic acid and amino acid levels. In silico characterization revealed the presence of highly conserved amino acid residues found in nearly all Type III PKSs including the conserved active-site residues, signature motif and cyclization pocket residues with an exception of Ile256 and Gly258. Docking studies established major interactions between the starter acetyl-CoA and RaALS. Copy number analysis suggested slender evolution in Type III PKS in R. australe having a single copy of RaALS gene. qRT-PCR analyses revealed corroboration between the higher expression of RaALS in leaves followed by stem and root with that of the metabolite concentration. Expression studies further showed a direct increase of RaALS transcripts with the growing metabolite accretion in relation to altitude suggesting a probable involvement of specific Type III PKS in biosynthesis of the major phyto-constituents. Furthermore, abiotic stressors viz. methyl jasmonate, salicylic acid and UV light enhanced RaALS transcription hinting towards its role in defense mechanism in R. australe and highlighting the significance of RaALS as a prospective target for metabolic engineering. [ABSTRACT FROM AUTHOR]

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman. [ABSTRACT FROM AUTHOR]

A recessive Short Tandem Repeat expansion in RFC1 has been found to be associated with cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS), and to be a frequent cause of late onset ataxia and sensory neuropathy. The usual procedure for sizing these expansions is based on Southern Blotting (SB), a time-consuming and a relatively imprecise technique. In this paper, we compare SB with Optical Genome Mapping (OGM), a method for detecting Structural Variants (SVs) based on the measurement of distances between fluorescently labelled probes, for the diagnosis of RFC1 CANVAS and disease spectrum. The two methods are applied to 17 CANVAS patients’ blood samples and resulting sizes compared, showing a good agreement. Further, long-read sequencing is used for two patients to investigate the agreement of sizes with either SB or OGM. Our study concludes that OGM represents a viable alternative to SB, allowing for a simpler technique, a more precise sizing of the expansion and ability to expand analysis of SV in the entire genome as opposed to SB which is a locus specific method.

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus that is exerting a major restraint on global tomato production. In this transgenic approach, an RNA interference (RNAi)-based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction, and stimulus to biotic and abiotic disease was engineered targeting the AC1/Rep gene of the Oman strain of TYLCV-OM. The Rep-amiRNA constructs presented an effective approach in regulating the expression of the Rep gene against TYLCV as a silencing target to create transgenic Solanum lycopersicum L. plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a Southern hybridization analysis were performed to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1-transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with the TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the current study proposes that genetically transformed tomato plants expressing amiRNA could be a potential approach for engineering tolerance in plants against TYLCV infection and conceivably for the inhibition of viral diseases against different strains of whitefly-transmitted begomoviruses in Oman.

Telomeres, repeating TTAGGG sequences at the ends of eukaryotic chromosomes, increase genomic stability. Telomere shortening occurs not only during DNA replication associated with cell division but also under oxidative stress, where reactive oxygen species damage DNA. Therefore, changes in telomere length can be used to evaluate chronic cost or stress responses incurred by individuals. The phylogenetically unique Chondrichthyes are among the least-studied groups of marine vertebrates. Telomere data are limited and have only been reported in a few Chondrichthyes species. In this study, we measured telomere length and quantified oxidative stress and antioxidant power in 17 Chondrichthyes species whose telomere length has not been measured before. The presence of telomere sequences > 30 bp and lower values of oxidative stress were confirmed in most species. Average telomere length was not correlated with oxidative stress and antioxidant power in 15 species for which both measurements were available. It would be desirable in the future to elucidate the nature of telomeres in Chondrichthyes, and their direct relationship with reactive oxygen species. [ABSTRACT FROM AUTHOR]

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species. [ABSTRACT FROM AUTHOR]

Cultivated potato (Solanum tuberosum) is susceptible to many pests and pathogens, but the most important threat to potato production is, so far, the late blight disease, caused by the oomycete Phytophthora infestans. Resistance genes from the wild Solanum sp. have been used by breeders to develop late-blight-resistant cultivars. Two sets of Black differentials potato genotypes (R1, R2, ..., R11) were used to identify a new marker for resistance gene R11 of potato to late blight. RAPD polymorphic bands were isolated, cloned, and converted into SCAR primers. By amplification of genomic DNA with SCAR primers followed by enzymatic digestion with HinfI restriction enzyme, and verified by Southern blotting, a marker of R11 resistance gene of potato to Phytophthora infestans was identified. [ABSTRACT FROM AUTHOR]

Gel image analyses are often difficult to reproduce, as the most commonly used software, the ImageJ Gels plugin, does not automatically record any steps in the analysis process. This protocol provides detailed steps for image analysis using IOCBIO Gel software with western blot as an example; however, the protocol is applicable to all images obtained by electrophoresis, such as Southern blotting, northern blotting, and isoelectric focusing. IOCBIO Gel allows multiple sample analyses, linking the original image to all the operations performed on it, which can be stored in a central database or on a PC, ensuring ease of access and the possibility to perform corrections at each analysis stage. In addition, IOCBIO Gel is lightweight, with only minimal computer requirements. Key features • Free and open-source software for analyzing gel images. • Reproducibility. • Can be used with images obtained by electrophoresis, such as western blotting, Southern blotting, isoelectric focusing, and more., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY license.)

Background: Conventional diagnosis of fragile X syndrome (FXS) is based on a combination of fragment analysis (FA) and Southern blotting (SB); however, this diagnostic approach is time- and labor-intensive and has pitfalls such as the possibility of missing large number alleles. Triplet repeat primed PCR (TP-PCR) is a current alternative used to overcome these limitations. We evaluated the diagnostic usefulness of TP-PCR compared with the conventional diagnostic protocol consisting of FA and/or SB in terms of allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers. Methods: From November 2013 to March 2018, 458 patients (326 males, 132 females) were simultaneously examined using FA and/or SB and TP-PCR by detecting CGG repeat numbers in FMR1 gene and diagnosed as per American College of Medical Genetics guidelines. Results: The TP-PCR results showed high concordance with the FA and/or SB results for all three aspects (allele categorization, repeat number correlation, and zygosity concordance in female genetic carriers). TP-PCR detected CGG expansions =200 in all full mutation (FM) allele cases in male patients, as well as both the normal allele (NL) and FM allele in female carriers. In premutation (PM) allele carriers, the TP-PCR results were consistent with the FA and/or SB results. In terms of zygosity concordance in female genetic carriers, 12 NL cases detected by TP-PCR showed a merged peak consisting of two close heterozygous peaks; however, this issue was resolved using a 10-fold dilution. Conclusions: TP-PCR may serve as a reliable alternative method for FXS diagnosis. [ABSTRACT FROM AUTHOR]

DNA methylation of three cultivars, each of the fruit tree species pear, plum and apple, was analyzed by the methylation-sensitive amplified polymorphism (MSAP) marker. All three fruit tree cultivars were found to contain apparently lower levels of methylation at the 5′-CCGG sites than all other plant species, such as rice and wheat, studied by the same method. Sequencing of the representative loci isolated from the MSAP profiles indicated that both protein-coding genes and transposable elements (TEs) were involved in low methylation. Gel blotting using isolated MSAP fragments and fragment mixtures representing two major types of TEs (copia- and gypsy-like) as hybridization probes confirmed the unexpected low DNA methylation levels at the 5′-CCGG sites in these three fruit tree genomes. Our results suggest that the three asexually propagated perennial fruit trees may indeed contain unusual lower levels of DNA methylation, especially in TEs at the 5′-CCGG sites. Additionally, our results may also suggest that the often used MSAP marker, which targets only one kind of specific methylation-sensitive sites recognized by a pair of isoschizomers (e.g., 5′-CCGG by HpaII/MspI), is not always representative of other cytosine sites (e.g., CHH) or CG sites other than those of 5′-CCGGs in some plant species.

Chronological age estimation is a challenging marker in the field of forensic medicine. The current study aimed to investigate the accuracy of signal joint T-cell receptor rearrangement excision circles (sjTRECs) quantification and telomere length measurement as methods for estimating chronological age. Telomere length was estimated in the DNA derived from the buccal cells through estimating the telomeric restriction fragment (TRF) length using TeloTTAGGG Telomere Length Assay while the sjTRECs quantification was carried out on DNA isolated from the blood samples using qPCR. The TRF length was shortened with increased age (r = −0.722, p < 0.001). The sjTRECs were also decreased with increased age (r = −0.831, p < 0.001). Stronger coefficient and lower standard error of the estimate was obtained when multiple regression analysis for age prediction based on the values of both methods was applied (r = −0.876, p < 0.001). [ABSTRACT FROM AUTHOR]

Telomeres are located at the ends of chromosomes and have specific sequences with a distinctive structure that safeguards genes. They possess capping structures that protect chromosome ends from fusion events and ensure chromosome stability. Telomeres shorten in length during each cycle of cell division. When this length reaches a certain threshold, it can lead to genomic instability, thus being implicated in various diseases, including cancer and neurodegenerative disorders. The possibility of telomeres serving as a biomarker for aging and age-related disease is being explored, and their significance is still under study. This is because post-mitotic cells, which are mature cells that do not undergo mitosis, do not experience telomere shortening due to age. Instead, other causes, for example, exposure to oxidative stress, can directly damage the telomeres, causing genomic instability. Nonetheless, a general agreement has been established that measuring telomere length offers valuable insights and forms a crucial foundation for analyzing gene expression and epigenetic data. Numerous approaches have been developed to accurately measure telomere lengths. In this review, we summarize various methods and their advantages and limitations for assessing telomere length., Competing Interests: The authors have no potential conflicts of interest to disclose. The funder played no role in the study’s design, data collection, analysis, interpretation of the data, writing of the report, or the decision to submit the report for publication., (© 2024 The Authors.)

Telomere length is measured using Southern blotting of the chromosomal terminal restriction fragments (TRFs) released by endonuclease digestion in cells from yeast to human. In the budding yeast Saccharomyces cerevisiae, XhoI or PstI is applied to cut the subtelomere Y′ element and release TRFs from the 17 subtelomeres. However, telomeres from other 15 X‐element‐only subtelomeres are omitted from analysis. Here, we report a method for measuring all 32 telomeres in S. cerevisiae using the endonuclease MmeI. Based on analyses of the endonuclease cleavage sites, we found that the TRFs generated by MmeI displayed two distinguishable bands in the sizes of ~500 and ~700 bp comprising telomeres (300 bp) and subtelomeres (200–400 bp). The modified MmeI‐restricted TRF (mTRF) method recapitulated telomere shortening and lengthening caused by deficiencies of YKu and Rif1 respectively in S. cerevisiae. Furthermore, we found that mTRF was also applicable to telomere length analysis in S. paradoxus strains. These results demonstrate a useful tool for simultaneous detection of telomeres from all chromosomal ends with both X‐element‐only and Y′‐element subtelomeres in S. cerevisiae species. [ABSTRACT FROM AUTHOR]

Subtelomere Y′ elements get amplified by homologous recombination in sustaining the survival and division of the budding yeast Saccharomyces cerevisiae. However, current method for measurement of the subtelomere structures uses Southern blotting with labelled specific probes, which is laborious and time‐consuming. By multiple sequence alignment analysis of all 19 subtelomere Y′ elements across the 13 chromosomes of the sequenced S288C strain deposited in the yeast genome SGD database, we identified 12 consensus and relative longer fragments and 14 pairs of unique primers for real‐time quantitative PCR analysis. With a PAC2 or ACT1 located near the centromere of chromosome V and VI as internal controls, these primers were applied to real‐time quantitative PCR analysis, so the relative Y′ element intensity normalised to that of wild type (WT) cells was calculated for subtelomere Y′ element copy numbers across all different chromosomes using the formula: 2^[−((CTmutant Y′ − CTmutant control) − (CTWT Y′ − CTWT control))]. This novel quantitative subtelomere amplification assay across chromosomes by real‐time PCR proves to be a much simpler and more sensitive way than the traditional Southern blotting method to analyse the Y′ element recombination events in survivors derived from telomerase deficiency or recruitment failure. [ABSTRACT FROM AUTHOR]

Wheat is one of the most important cereals that supply needs of the human food in all countries. Fusarium head blight (FHB), caused primarily by Fusarium graminearum is one of the most destructive diseases of wheat that produces the mycotoxin deoxynivalenol (DON), a protein synthesis inhibitor which is harmful to human and livestock. Chemical modification in DON structure could reduce DON accumulation in the grain. AYT1 gene, encoding an acetyltransferase enzyme plays a major role in reducing the toxicity of DON. In this study, we used molecular techniques such as PCR, RT-PCR and southern blotting to confirm the insertion and proper expression of this transgene in the second generation of transgenic wheat plants. Obtained results showed the presence of Ayt1 gene in most of the evaluated transformed plants, and transgene expression was confirmed by RT-PCR. Moreover, southern blot analysis for evaluation of transgene copy number showed more than one copy in some of the transgenic lines.

Cultivation of human melanocytes requires several growth factors for cell proliferation. For example, basic fibroblast growth factor (bFGF) is an essential growth agent for melanocyte proliferation in vitro and has been proposed to be an autocrine growth factor in human melanoma cells. Studies using either anti-bFGF antibodies or antisense oligonucleotides partially inhibited the proliferation of human melanoma cells. However, one group was unable to detect bFGF RNA transcripts in human melanoma cells using a human complementary DNA probe. These contradictory results prompted us to investigate the bFGF gene expression in human primary melanocytes and metastatic melanoma cells using Southern, Northern, and Western blot analyses. No gross rearrangements in the bFGF gene were detected in the genomic DNA. Although high levels of bFGF RNA transcripts were detected in melanocytes, no bFGF protein was detected using Western blot analysis. In contrast, melanoma cells expressed much lower levels of bFGF RNA transcripts, and cells from three of four cell strains synthesized the multiple isoforms of bFGF protein. In one of the melanoma cell strains, no bFGF protein was detected using Western blot analysis. Although three of four melanoma cell strains expressed bFGF protein, this molecule does not appear to function as an autocrine growth factor, and expression of the bFGF protein was not a consistent alteration in all melanoma cell strains.

In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM1) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM2) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken b-actin promoter-driven firefly improved luciferase cDNA (β-act/luc+) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (β- act/luc+) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice.

Tomato yellow leaf curl virus (TYLCV) is a global spreading begomovirus exerting a major restraint on world tomato production. In this transgenic approach, an RNA interference (RNAi) based construct consisting of sequences of an artificial microRNA (amiRNA), a group of small RNA molecules necessary for plant cell development, signal transduction and stimulus to biotic and a biotic disease was engineered targeting AC1/Rep gene of Oman strain of TYLCV-OM. The Rep-amiRNA constructs presentan effective approach in regulating the expression of Rep gene against TYLCV as a silencing target to create transgenic tomato plant tolerance against TYLCV infection. Molecular diagnosis by PCR followed by a southern hybridization analysis was achieved to confirm the effectiveness of agrobacterium-mediated transformation in T0/T1 transformed plants. A substantial decrease in virus replication was observed when T1 transgenic tomato plants were challenged with TYLCV-OM infectious construct. Although natural resistance options against TYLCV infection are not accessible, the outcomes of this study advised that transformed plants expressing amiRNA could be an essential approach for engineering tolerant plants against TYLCV infection and conceivably for the inhibition of viral disease against different strains of whitefly transmitted begomoviruses in Oman. [ABSTRACT FROM AUTHOR]

We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars ('CO(Ra)-14', 'PR-202', 'Try-1' and 'Paiyur-2') of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar 'CO(Ra)-14'. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for 'CO(Ra)-14'. PCR, gus staining and Southern blot analyses were performed in T and T generations to confirm the gene integration. Six events from T had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars. [ABSTRACT FROM AUTHOR]

Here, we review the results of Southern blotting analyses of the FMR1 gene performed in our reference laboratory in Taiwan over a 15-year period. In total, 725 high-risk women with a family history of fragile X syndrome ( FXS) or idiopathic intellectual disability, 3911 low-risk pregnant women without such family history, and prenatal diagnosis data for 32 foetuses from 24 carrier mothers were included. Only 2 carriers were in the low-risk group, which indicated a prevalence of 1 of 1955 women (95% confidence interval: 1/7156-1/539). A total of 100 carriers were found to be in the high-risk group, thus revealing a significantly higher frequency than the low-risk group (100/725 vs 2/3911, P<0.0001). Eight of the 14 foetuses that inherited the maternal mutant allele were verified to have a full mutation, with the smallest maternal pre-mutation allele carrying 56 CGG repeats. The overall findings confirmed that the carrier prevalence among low-risk women in Taiwan is significantly lower than that reported in western countries. Therefore, the most important step for preventing FXS in Taiwan would be to focus on high-risk women by promoting general awareness of this disease and spreading knowledge regarding the benefits of carrier screening and prenatal testing. [ABSTRACT FROM AUTHOR]

Caldicellulosiruptor bescii is the most thermophilic cellulose degrader known and is of great interest because of its ability to degrade nonpretreated plant biomass. For biotechnological applications, an efficient genetic system is required to engineer it to convert plant biomass into desired products. To date, two different genetically tractable lineages of C. bescii strains have been generated. The first (JWCB005) is based on a random deletion within the pyrimidine biosynthesis genes pyrFA, and the second (MACB1018) is based on the targeted deletion of pyrE, making use of a kanamycin resistance marker. Importantly, an active insertion element, ISCbe4, was discovered in C. bescii when it disrupted the gene for lactate dehydrogenase (ldh) in strain JWCB018, constructed in the JWCB005 background. Additional instances of ISCbe4 movement in other strains of this lineage are presented herein. These observations raise concerns about the genetic stability of such strains and their use as metabolic engineering platforms. In order to investigate genome stability in engineered strains of C. bescii from the two lineages, genome sequencing and Southern blot analyses were performed. The evidence presented shows a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 elements within the genome of JWCB005, leading to massive genome rearrangements in its daughter strain, JWCB018. Such dramatic effects were not evident in the newer MACB1018 lineage, indicating that JWCB005 and its daughter strains are not suitable for metabolic engineering purposes in C. bescii. Furthermore, a facile approach for assessing genomic stability in C. bescii has been established. IMPORTANCE Caldicellulosiruptor bescii is a cellulolytic extremely thermophilic bacterium of great interest for metabolic engineering efforts geared toward lignocellulosic biofuel and bio-based chemical production. Genetic technology in C. bescii has led to the development of two uracil auxotrophic genetic background strains for metabolic engineering. We show that strains derived from the genetic background containing a random deletion in uracil biosynthesis genes (pyrFA) have a dramatic increase in the number of single nucleotide polymorphisms, insertions/deletions, and ISCbe4 insertion elements in their genomes compared to the wild type. At least one daughter strain of this lineage also contains large-scale genome rearrangements that are flanked by these ISCbe4 elements. In contrast, strains developed from the second background strain developed using a targeted deletion strategy of the uracil biosynthetic gene pyrE have a stable genome structure, making them preferable for future metabolic engineering studies. [ABSTRACT FROM AUTHOR]

Calamus guruba Buch.-Ham., a perennial, dioecious rattan species of the family Arecaceae has recently emerged as an important source of tribal economy owing to its high-quality flexible canes for furniture manufacturing and cottage industries. The dioecious nature together with disproportionate distribution of male and female sex organs has been the major constraint in the development of an effective breeding programme for the plant. Early identification of the male and female genotypes at the seedling stage is a pre-requisite to ensure genetic improvement of C. guruba. In the present study, we used 30 inter simple sequence repeat (ISSR) markers to develop a sequence-tagged site (STS) linked to male sex in C. guruba. Molecular analysis of bulked DNA pooled from male and female genotypes resulted in the isolation of a putative male-specific marker CgMSM. Partial sequencing of CgMSM resulted in an STS marker CgMY1 which could successfully amplify a 597-bp fragment in male but not in the female plants. DNA gel blot analysis confirmed it as a single-copy locus in the male genome of C. guruba. Further validation of CgMY1 in the natural population of C. guruba resulted in precise detection of 9 males and 6 females from 15 individuals with unknown sex. Therefore, the STS marker CgMY1 could be used as a proficient tool for early sex differentiation and form the basis of a sustainable breeding programme for genetic improvement of C. guruba. [ABSTRACT FROM AUTHOR]

In the present study, a protocol for Agrobacterium tumefaciens -mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were co-cultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with β-glucuronidase ( uidA ) containing intron as the reporter gene and hygromycin phosphotransferase ( hpt ) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 20 mg/l hygromycin + 200 mg/l cefotaxime (PTSM 1 ) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 μM) and Indole-3-acetic acid IAA (1.14 μM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM 2 ) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 μM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa . Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites. [ABSTRACT FROM AUTHOR]

Histomonas meleagridis, a protozoan parasite that can infect gallinaceous birds, affects mainly the liver and caeca of infected birds. As a consequence of the recent ban of chemotherapeuticals in Europe and the USA, histomonosis gained somewhat more attention due to its re-emergence and the fact that there is no effective treatment available. Therefore, special attention is now also given towards the diagnosis and the control of the disease. In the actual study we report the development of highly specific and sensitive real-time PCR methods for detection and quantification of the parasite, based on two protein coding genes, Fe-hydrogenase ( FeHYD ) and rpb1 . Both genes seem to be in a single copy in H. meleagridis as shown by southern blotting and absolute quantification using real-time PCRs on samples containing a known amount of the parasite. The real-time PCR assays based on FeHYD and rpb1 genes were found to be an efficient method for the quantification and detection of H. meleagridis in in vitro grown cultures, tissues of infected birds and in faecal samples. Both real-time PCRs were able to detect up to a single cell in in vitro cultures of H. meleagridis and in fecal samples spiked with H. meleagridis . Finally, qPCR assays were shown to be highly specific for H. meleagridis as samples containing either of the two H. meleagridis genotypes were positive, whereas samples containing other protozoa such as Tetratrichomonas gallinarum, Trichomonas gallinae, Simplicimonas sp., Tritrichomonas sp., Parahistomonas wenrichi , Dientamoebidae sp. and Blastocystis sp. were all negative. [ABSTRACT FROM AUTHOR]

Many pronuclear stage eggs were used to generate transgenic mice (Tg) by microinjection. In this study, we used in vitro fertilized mouse eggs, followed by ultrarapid freezing to establish a simple procedure for production of Tg mice. We produced in vitro fertilized mouse eggs and cryopreserved them by ultrarapid freezing method. A total of 139 cryopreserved-thawed pronuclear eggs, of which 101 (72.6%) were survived following microinjection of chicken ϐ-actin promoter-driven firefly improved luciferase cDNA (β-act/luc+) and were transferred into 5 recipients. All recipients became pregnant and gave birth to a total of 15 (14.8%) pups. As a control, same DNA construction (β- act/luc+) was also injected into 450 in vitro fertilized eggs, of which 338 (75.1%) were survived and then were transferred into 14 recipients. Eleven (78%) mice became pregnant and littered a total of 54 (19.1%) pups. Southern blotting analysis of Tg mice indicated that one (1/15, 6.6%) and three (3/54, 5.5%) transgenic mice were production from cryopreserved and in vitro fertilized eggs, respectively. All Tg mice produced from both eggs showed the expression of improved luciferase gene. These results indicated that efficiency of produced of Tg mice from cryopreserved eggs was comparable to that from in vitro fertilized eggs. Furthermore, it is suggested that microinjection of transgene into in vitro fertilized eggs cryopreserved by ultrarapid freezing is an easy and conveniently method for production of Tg mice. [ABSTRACT FROM AUTHOR]

Crambe abyssinica is a dedicated oilseed crop suitable for production of industrial feedstocks. Genetic modification of crambe has progressed substantially in the last few years, but the transformation efficiency needs to be further improved. Meanwhile, developing a reliable molecular system including Southern blot and qRT-PCR analyses is desired for effectively evaluating transgenic lines and gene expression levels of both endogenous and transgenes. In this study, we have developed an efficient transformation protocol with hygromycin as the selective agent for crambe transformation. In the regeneration test, addition of hygromycin at concentration of 5 mg L-1 resulted in 18% of shoot regeneration using crambe hypocotyls as explants, while no regeneration occurred when the hygromycin concentration reached 10 mg L-1. Based on this result, the hygromycin concentration up to 10 mg L-1 was used in the subsequent transformations. The results showed that the transformation efficiency under constant low selection pressure (H3-H3) was similar to that under higher selection pressure first followed by transfer to lower selection pressure (H10-H3). The PCR, Southern blot and fatty acid composition analyses confirmed the integration of transgenes in the crambe genome. We have also optimised the Southern and qRT-PCR methods for future studies on crambe or related species. For Southern blot analysis on crambe, more than 50 µg DNA is required for a clear band. The choice of enzymes for DNA digestion was not rigid for confirmation of the T-DNA integration, while for determining the copy number of transgenes, suitable enzymes should be chosen. Increasing the enzyme concentration could improve the digestion and 20 µl enzyme was recomended for a complete digestion of up to 80 µg crambe DNA. For qRT-PCR analysis, around 20 days after flowering was observed to be the suitable sampling time for expresseion analysis of genes invovled in the seed oil biosynthesis.

Background Telomeres are repeated sequences (the hexanucleotide TTAGGG in vertebrates) located at chromosome ends of eukaryotes, protecting DNA from end joining or degradation. Telomeres become shorter with each cell cycle, but telomerase, a ribonucleoprotein complex, alleviates this attrition. The telomerase RNA component (TERC) is an essential element of telomerase, serving as a template for telomere elongation. The H/ACA domain of TERC is indispensable for telomere biogenesis. Mutations in the telomerase components allow accelerated telomere loss, resulting in various disease manifestations, including bone marrow failure. To date, this is the first detailed report of an H-box mutation in TERC that is related to human disease. Case presentation A 26-year-old man with myelodysplastic syndrome (MDS) had very short telomeres. Sequencing identified a single heterozygous mutation in the H box of the patient's TERC gene. The same mutation was also present in his father and his son, demonstrating that it was germline in origin. The telomere length in the father's blood was shorter compared to agematched healthy controls, while it was normal in the son and also in the sperm cells of the patient. In vitro experiments suggested that the mutation was responsible for the telomere shortening in the patient's leukocytes and contributed to the pathogenesis of bone marrow failure in our patient. Conclusion We analyzed a mutation (A377G) in the H box of TERC in a young MDS patient who had significantly short-for-age telomeres. As telomeres protect chromosomes from instability, it is highly plausible that this genetic lesion was responsible for the patient's hematological manifestations, including marrow failure and aneuploidy in the hematopoietic stem cell compartment. [ABSTRACT FROM AUTHOR]

DOF1 (DNA binding with one finger) plays an important role in regulating C/N metabolism in cereals. In order to validate its role in the regulation of nitrogen use efficiency (NUE) and photosynthetic efficiency in finger millet, 5′-3′ RACE PCR was performed to obtain and characterize full-length Dof1 genes of high and low grain protein finger millet genotypes. The full-length DOF1 ORFs were both 1,284 nt long and were 98.8 % similar over 427 amino acids containing the characteristic Dof domain. Comparison of both the EcDof1 protein sequences with the Dof1 of other cereals revealed high sequence similarity to the Dof1 of rice. Southern hybridization carried out using the probe developed from the region encoding the highly variable C-terminal region of EcDof1 showed the presence of four copies of the DOF1 gene in finger millet, which might explain the high NUE and photosynthetic performance of finger millet. Since the genes involved in C/N metabolism are regulated diurnally and play crucial roles in determining grain protein content during grain filling, the diurnal expression of EcDOF1 was assessed in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 % and 6.15 % respectively). It was found that EcDOF1 exhibited diurnal regulation and peak differential pattern expression with early phasing in GE3885 and late phasing in GE1437. Differential expression of DOF1 might alter the regulation of genes involved in C/N metabolism affecting grain protein composition of finger millet genotypes. [ABSTRACT FROM AUTHOR]