Southern Blot Research Articles

Host-induced gene silencing of the Verticillium dahliae thiamine transporter protein gene (VdThit) confers resistance to Verticillium wilt in cotton

Verticillium wilt (VW), induced by the soil-borne fungus Verticillium dahliae (Vd), poses a substantial threat to a diverse array of plant species. Employing molecular breeding technology for the development of cotton varieties with heightened resistance to VW stands out as one of the most efficacious protective measures. In this investigation, we successfully generated two stable transgenic lines of cotton (Gossypium hirsutum L.) VdThit-RNAi-1 and VdThit-RNAi-2 using host-induced gene silencing (HIGS) technology to introduce double-stranded RNA (dsRNA) targeting the thiamine transporter protein gene (VdThit). Southern blot analysis confirmed the presence of a single-copy insertion in both lines. Microscopic examination disclosed a marked reduction in the colonization and spread of Vd-mCherry in the roots of VdThit-RNAi cotton compared to wild type (WT). Correspondingly, the disease index and fungal biomass of VdThit-RNAi-1/2 exhibited a significant decrease. Real-time quantitative PCR (qRT-PCR) analysis demonstrated a substantial inhibition of VdThit expression following prolonged inoculation of VdThit-RNAi cotton. Small RNA sequencing (sRNA-Seq) analysis further revealed the generation of a substantial number of VdThit-specific siRNAs in the VdThit-RNAi transgenic lines. Additionally, the silencing of VdThit by siVdThit produced by VdThit-RNAi-1/2 resulted in the elevated expression of multiple genes involved in the thiamine biosynthesis pathway in Vd. In field conditions, VdThit-RNAi transgenic cotton exhibited significantly enhanced disease resistance and yield compared with WT. In summary, our findings underscore the efficacy of HIGS targeting VdThit in restraining the infection and spread of Vd in cotton, thereby potentially expediting the development of cotton breeding as a promising strategy.

Identification and functional characterisation of a locus for target site integration in Fusariumgraminearum

BackgroundFusarium Head Blight (FHB) is a destructive floral disease of different cereal crops. The Ascomycete fungus Fusariumgraminearum (Fg) is one of the main causal agents of FHB in wheat and barley. The role(s) in virulence of Fg genes include genetic studies that involve the transformation of the fungus with different expression cassettes. We have observed in several studies where Fg genes functions were characterised that integration of expression cassettes occurred randomly. Random insertion of a cassette may disrupt gene expression and/or protein functions and hence the overall conclusion of the study. Target site integration (TSI) is an approach that consists of identifying a chromosomal region where the cassette can be inserted. The identification of a suitable locus for TSI in Fg would avert the potential risks of ectopic integration.ResultsHere, we identified a highly conserved intergenic region on chromosome 1 suitable for TSI. We named this intergenic region TSI locus 1. We developed an efficient cloning vector system based on the Golden Gate method to clone different expression cassettes for use in combination with TSI locus 1. We present evidence that integrations in the TSI locus 1 affects neither fungal virulence nor fungal growth under different stress conditions. Integrations at the TSI locus 1 resulted in the expression of different gene fusions. In addition, the activities of Fg native promoters were not altered by integration into the TSI locus 1. We have developed a bespoke bioinformatic pipeline to analyse the existence of ectopic integrations, cassette truncations and tandem insertions of the cassette that may occurred during the transformation process. Finally, we established a protocol to study protein secretion in wheat coleoptiles using confocal microscopy and the TSI locus 1.ConclusionThe TSI locus 1 can be used in Fg and potentially other cereal infecting Fusarium species for diverse studies including promoter activity analysis, protein secretion, protein localisation studies and gene complementation. The bespoke bioinformatic pipeline developed in this work together with PCR amplification of the insert could be an alternative to Southern blotting, the gold standard technique used to identify ectopic integrations, cassette truncations and tandem insertions in fungal transformation.

Overexpression of cry1c* Enhances Resistance against to Soybean Pod Borer (Leguminivora glycinivorella) in Soybean.

Soybean [Glycine max (L.) Merr.], an essential staple food and oil crop worldwide, boasts abundant vegetable proteins and fats beneficial for both human and animal consumption. However, the soybean pod borer (Leguminivora glycinivorella) (SPB) stands as the most destructive soybean insect pest in northeast China and other northeastern Asian regions, leading to significant annual losses in soybean yield and economic burden. Therefore, this study aims to investigate the introduction of a previously tested codon-optimized cry1c gene, cry1c*, into the soybean genome and assess its effect on the SPB infestation by generating and characterizing stable transgenic soybeans overexpressing cry1c*. The transgenic soybean lines that constitutively overexpressed cry1c* exhibited a significant reduction in the percentage of damaged seeds, reaching as low as 5% in plants under field conditions. Additionally, feeding transgenic leaves to the larvae of S. exigua, S. litura, and M. separta resulted in inhibited larval growth, decreased larval body weight, and lower survival rates compared to larvae fed on wild-type leaves. These findings showed that the transgenic lines maintained their resistance to SPB and other lepidopteran pests, especially the transgenic line KC1. Southern blotting and genome-wide resequencing analysis revealed that T-DNA integration occurred as a single copy between loci 50,868,122 and 50,868,123 of chromosome 10 in the transgenic line KC1. Therefore, the transgenic line KC1, overexpressing high levels of cry1c* in leaves and seeds, holds strong potential for commercial use in the integrated management of SPB and other lepidopteran pests.

Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197µg/100mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

T-DNA characterization of genetically modified 3-R-gene late blight-resistant potato events with a novel procedure utilizing the Samplix Xdrop® enrichment technology.

Before the commercialization of genetically modified crops, the events carrying the novel DNA must be thoroughly evaluated for agronomic, nutritional, and molecular characteristics. Over the years, polymerase chain reaction-based methods, Southern blot, and short-read sequencing techniques have been utilized for collecting molecular characterization data. Multiple genomic applications are necessary to determine the insert location, flanking sequence analysis, characterization of the inserted DNA, and determination of any interruption of native genes. These techniques are time-consuming and labor-intensive, making it difficult to characterize multiple events. Current advances in sequencing technologies are enabling whole-genomic sequencing of modified crops to obtain full molecular characterization. However, in polyploids, such as the tetraploid potato, it is a challenge to obtain whole-genomic sequencing coverage that meets the regulatory approval of the genetic modification. Here we describe an alternative to labor-intensive applications with a novel procedure using Samplix Xdrop® enrichment technology and next-generation Nanopore sequencing technology to more efficiently characterize the T-DNA insertions of four genetically modified potato events developed by the Feed the Future Global Biotech Potato Partnership: DIA_MSU_UB015, DIA_MSU_UB255, GRA_MSU_UG234, and GRA_MSU_UG265 (derived from regionally important varieties Diamant and Granola). Using the Xdrop® /Nanopore technique, we obtained a very high sequence read coverage within the T-DNA and junction regions. In three of the four events, we were able to use the data to confirm single T-DNA insertions, identify insert locations, identify flanking sequences, and characterize the inserted T-DNA. We further used the characterization data to identify native gene interruption and confirm the stability of the T-DNA across clonal cycles. These results demonstrate the functionality of using the Xdrop® /Nanopore technique for T-DNA characterization. This research will contribute to meeting regulatory safety and regulatory approval requirements for commercialization with small shareholder farmers in target countries within our partnership.

Linear DNA-driven recombination in mammalian mitochondria.

Mitochondrial DNA (mtDNA) recombination in animals has remained enigmatic due to its uniparental inheritance and subsequent homoplasmic state, which excludes the biological need for genetic recombination, as well as limits tools to study it. However, molecular recombination is an important genome maintenance mechanism for all organisms, most notably being required for double-strand break repair. To demonstrate the existence of mtDNA recombination, we took advantage of a cell model with two different types of mitochondrial genomes and impaired its ability to degrade broken mtDNA. The resulting excess of linear DNA fragments caused increased formation of cruciform mtDNA, appearance of heterodimeric mtDNA complexes and recombinant mtDNA genomes, detectable by Southern blot and by long range PacBio® HiFi sequencing approach. Besides utilizing different electrophoretic methods, we also directly observed molecular complexes between different mtDNA haplotypes and recombination intermediates using transmission electron microscopy. We propose that the known copy-choice recombination by mitochondrial replisome could be sufficient for the needs of the small genome, thus removing the requirement for a specialized mitochondrial recombinase. The error-proneness of this system is likely to contribute to the formation of pathological mtDNA rearrangements.

Differential amplification of the subtelomeric satellite DNA JcSAT1 in the genus Jatropha L. (Euphorbiaceae).

Satellite DNAs (satDNAs) are highly repetitive sequences that occur in virtually all eukaryotic genomes and can undergo rapid copy number and nucleotide sequence variation among relatives. After chromosomal mapping of the satDNA JcSAT1, it was found a large accumulation at subtelomeres of Jatropha curcas (subgenus Curcas), but an absence of these monomers in J. integerrima (subgenus Jatropha). This fact suggests a dynamic scenario for this satellite repeat in Jatropha genomes. Here, we used a multitasking approach (sequence analysis, DNA blotting and chromosomal mapping) to investigate the molecular organization and chromosomal abundance and distribution of JcSAT1 in a broader group of species from the subgenus Jatropha (J. gossypiifolia, J. mollissima, J. podagrica, and J. multifida) in addition to J. curcas, with the aiming of understanding the evolution of this satDNA. Based on the analysis of BAC clone sequences of J. curcas, a large array (~ 30kb) of 80 homogeneous monomers of JcSAT1 was identified in BAC 23J11. The monomer size was conserved (~ 358bp) and contained a telomeric motif at the 5' end. PCR amplification coupled with a Southern blot revealed the presence of JcSAT1-like sequences in all species examined. However, a large set of genome copies was identified only in J. curcas, where a ladder-like pattern with multimers of different sizes was observed. In situ hybridization of BAC 23J11 confirmed the subtelomeric pattern for J. curcas, but showed no signals on chromosomes of species from the subgenus Jatropha. Our data indicate that JcSAT1 is a highly homogeneous satDNA that originated from a region near the telomeres and spread throughout the chromosomal subtermini, possibly due to frequent ectopic recombination between these regions. The abundance of JcSAT1 in the genome of J. curcas suggests that an amplification event occurred either at the base of the subgenus Curcas or at least in this species, although the repeat is shared by all species of the genus studied so far.

ZNF148 inhibits HBV replication by downregulating RXRα transcription

BackgroundProgressive hepatitis B virus (HBV) infection can result in cirrhosis, hepatocellular cancer, and chronic hepatitis. While antiviral drugs that are now on the market are efficient in controlling HBV infection, finding a functional cure is still quite difficult. Identifying host factors involved in regulating the HBV life cycle will contribute to the development of new antiviral strategies. Zinc finger proteins have a significant function in HBV replication, according to earlier studies. Zinc finger protein 148 (ZNF148), a zinc finger transcription factor, regulates the expression of various genes by specifically binding to GC-rich sequences within promoter regions. The function of ZNF148 in HBV replication was investigated in this study.MethodsHepG2-Na+/taurocholate cotransporting polypeptide (HepG2-NTCP) cells and Huh7 cells were used to evaluate the function of ZNF148 in vitro. Northern blotting and real-time PCR were used to quantify the amount of viral RNA. Southern blotting and real-time PCR were used to quantify the amount of viral DNA. Viral protein levels were elevated, according to the Western blot results. Dual-luciferase reporter assays were used to examine the transcriptional activity of viral promoters. ZNF148’s impact on HBV in vivo was investigated using an established rcccDNA mouse model.ResultsZNF148 overexpression significantly decreased the levels of HBV RNAs and HBV core DNA in HBV-infected HepG2-NTCP cells and Huh7 cells expressing prcccDNA. Silencing ZNF148 exhibited the opposite effects in both cell lines. Furthermore, ZNF148 inhibited the activity of HBV ENII/Cp and the transcriptional activity of cccDNA. Mechanistic studies revealed that ZNF148 attenuated retinoid X receptor alpha (RXRα) expression by binding to the RXRα promoter sequence. RXRα binding site mutation or RXRα overexpression abolished the suppressive effect of ZNF148 on HBV replication. The inhibitory effect of ZNF148 was also observed in the rcccDNA mouse model.ConclusionsZNF148 inhibited HBV replication by downregulating RXRα transcription. Our findings reveal that ZNF148 may be a new target for anti-HBV strategies.

Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

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.

Transcribed enhancer sequences are required for maize p1 paramutation.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (red pericarp and red cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr' state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr'. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr' epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr' sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation.

Rapid and Reliable Protein-Free HBV DNA Extraction and Sensitive Branched DNA Southern Blot Assay.

HBV covalently closed circular DNA (cccDNA) plays an important role in the persistence of hepatitis B virus (HBV) infection by serving as the template for transcription of viral RNAs. To cure HBV infection, it is expected that cccDNA needs either to be eliminated or silenced. Hence, precise cccDNA quantification is essential. Sample preparation is crucial to specifically detect cccDNA. Southern blot is regarded as the "gold standard" for specific cccDNA detection but lacks sensitivity. Here, we describe a rapid and reliable modified kit-based, HBV protein-free DNA extraction method as well as a novel enhanced sensitivity Southern blot that uses branched DNA technology to detect HBV DNA in cell culture and liver tissue samples. It is useful for both HBV molecular biology and antiviral research.

ST11 KPC-2-Producing Klebsiella pneumoniae Isolated from Patient with Acute Myelocytic Leukemia.

The emergence of the ST11-CRKP (ST11-CRKP) strain is expected to become a serious public health problem in China. As one of the most serious complications in patients with acute myeloid lymphoma, infections can cause systemic infection and life-threatening sepsis, seriously affecting the morbidity, mortality, and quality of life of patients. Thus, ST11-CRKP infections in patients with acute myeloid lymphoma are worthy of our attention. To investigate the occurrence and genetic characteristics of the ST11-CRKP from a patient with acute myeloid lymphoma. Species identification was determined by MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was conducted by VITEK 2 system with AST-N335 panel. Whole-genome sequencing was performed on the Illumina NovaSeq 6000 platform. Phylogenetic analyses were performed using Snippy based on the core-genome SNPs. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot and Whole-genome analysis indicated blaKPC-2 genes were located on plasmids with a conserved genetic environment. Moreover, the eight ST11-CRKP strains carry a variety of antimicrobial resistance genes (ARGs) and virulence factors. The ability of biofilm formation of eight strains was verified by a crystal violet assay. Core genome single-nucleotide polymorphism (cgSNP) analysis suggesting a possible bacterial translocation event. We performed a comprehensive analysis of ST11-CRKP strains from a patient with acute myelocytic leukemia. Our study emphasized the need for continuous surveillance of ST11-CRKP in the clinic especially in the immunocompromised population.

Characterization of an NDM-1-Producing Citrobacter koseri Isolate from China.

The continuous rise in carbapenemase-producing Enterobacteriaceae infections is a major public health concern. However, there is limited information available on New Delhi metallo-β-lactamase-1 (NDM-1) producing Citrobacter koseri. In this study, we isolated a blaNDM-1-carrying C. koseri from a stool sample of an inpatient. Our aim was to investigate the phenotypic and genomic features of this clinically derived carbapenem-resistant C. koseri isolate and to characterize the transmission pattern of the IncFII/IncN plasmid that carries the blaNDM-1 gene. S1-PFGE, Southern blot and conjugation assay confirmed the presence of blaNDM-1 gene in a conjugative plasmid. C. koseri L2395 and transconjugant L2395-EC600 strains showed similar resistance spectrum. Whole-genome analysis revealed that pL2395_NDM is an IncFII/IncN plasmid with a length of 67,839 bp. Moreover, blaNDM-1 gene was found encoded in the ISKpn19-blaNDM-1-ble-tnpF-dsbD-cutA-ISKpn19 cassette array. Phylogenetic analysis revealed that strain L2395 was close to an IMP-4-bearing C. koseri from Australia. Ongoing surveillance will be essential to control and prevent the spread of carbapenem-resistant Citrobacter spp. in the future.

A Chromogenic In Situ Hybridization (CISH) Assay for Detection of HBV RNA, DNA, and cccDNA in Liver Tissue.

Hepatitis B virus (HBV) developed highly intricates mechanisms exploiting host resources for its multiplication within a constrained genetic coding capacity. With the aid of a series of classical analytical methods such as ultrafiltration, and Southern and Northern blots, a general framework of HBV life cycle has been established. However, this picture still lacks many key histological contexts which involves pathophysiological changes of hepatocytes, non-parenchymal cells, infiltrated leukocytes, and associated extracellular matrix. Here, we describe a CISH protocol modified from the ViewRNA assay that allows direct visualization of HBV RNA, DNA, and cccDNA in liver tissue of chronic hepatitis B patients. By coupling it with immunohistochemistry and other histological stains, much richer information regarding the HBV-induced pathological changes can be harvested.

Durable remission with lenalidomide in a patient with early relapse of adult T-cell leukemia/lymphoma after cord blood transplantation

A 62-year-old woman with adult T-cell leukemia/lymphoma (ATL) received umbilical cord blood transplantation (CBT) in first complete remission. However, relapse of ATL was detected on day 74 post-transplantation, as evidenced by the rapid growth of lymphoma cells in peripheral blood and an increase in soluble interleukin-2 receptor (sIL2R) levels. Discontinuation of immunosuppressant therapy alone did not improve ATL findings, but treatment with lenalidomide caused lymphoma cells to disappear from the peripheral blood and sIL2R levels to return to normal. Pancytopenia was observed as a lenalidomide-associated adverse effect, but lymphocyte counts were not reduced. The patient was judged to be in complete remission based on results of Southern blot analysis and human T-cell leukemia virus 1 (HTLV-1)-infected cell analysis using flow cytometry (HAS-Flow). Flow cytometric analysis of peripheral blood and FISH analysis of X and Y chromosomes revealed that the therapeutic effect of lenalidomide was associated with an increase in the number of donor-derived peripheral natural killer cells. ATL relapse was not observed at 13 months into lenalidomide treatment. Our results suggest that lenalidomide is an effective option for the treatment of post-transplant relapsed ATL.

Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of thehepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

High-Resolution Characterization of DNA/Protein Complexes in Living Bacteria.

The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.

Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and ofits reliable detection due to its low abundance and thepresence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA andall DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.

Clinical and molecular characteristics of FMR1 microdeletion in patient with fragile X syndrome and review of the literature

BackgroundFragile X syndrome (FXS) is mainly caused by FMR1 CGG repeat expansions. Other types of mutations, particularly deletions, are also responsible for FXS phenotypes, however these mutations are often missed by routine clinical testing. Materials and methodsMolecular diagnosis in cases of suspected FXS was a combination of PCR and Southern blot. Measurement of the FMRP protein level was useful for detecting potentially deleterious impact. ResultsPCR analysis and Southern blot revealed a case with premutation and suspected deletion alleles. Sanger sequencing showed that the deletion involved 313 bp upstream of repeats and some parts of CGG repeat tract, leaving transcription start site. FMRP was detected in 5.5 % of blood lymphocytes. ConclusionAccording to our review of case reports, most patients carrying microdeletion and full mutation had typical features of FXS. To our knowledge, our case is the first to describe mosaicism of a premutation and microdeletion in the FMR1 gene. The patient was probably protected from the effects of the deletion by mosaicism with premutation allele, leading to milder phenotype. It is thus important to consider appropriate techniques for detecting FMR1 variants other than repeat expansions which cannot be detected by routine FXS diagnosis.

10221-ML-11 A CASE OF PRIMARY CENTRAL NERVOUS SYSTEM ADULT T CELL LEUKEMIA

Abstract Adult T cell leukemia (ATL) manifesting only in the central nervous system (CNS) at presentation is rare. We present a case of primary CNS ATL. 54 year old male presenting with multiple enhanced lesions in the cerebrum went under biopsy and was diagnosed as T-cell lymphoma. While systemic workup was negative, human T-cell leukemia virus type 1 (HTLV-1) antibody was found to be seropositive. Southern blotting using peripheral blood DNA revealed positivity for clonal proliferation of HTLV-1 infected cells. He was therefore clinically diagnosed as primary CNS ATL. He received chemotherapy using methotrexate, procarbazine and vincristine (MPV), and gained a complete response unconfirmed (CRu) after completion of four cycles. However, relapse occurred after the fifth cycle of MPV. Salvage treatment using etoposide, cisplatin, cytarabine and prednisone (ESHAP), whole brain radiation therapy, and lenalidomide, were delivered as second, third, and fourth line therapies, but were ineffective and disease continued to progress. He was therefore treated by best supportive care. Primary CNS ATL should be considered in the differential diagnosis of brain tumors. Southern blotting using peripheral blood might be useful for clinical diagnosis in such cases.