Restriction Enzyme Digestion Of DNA Research Articles

X-linked chronic granulomatous disease secondary to skewed X-chromosome inactivation in female patients.

Chronic granulomatous disease (CGD) is a heterogeneous primary immunodeficiency. X-linked (XL) CGD caused by gene defects of CYBB is the most prevalent type of CGD. We aim to understand the clinical and molecule features of XL-CGD secondary to skewed X-chromosome inactivation (XCI) in female. We retrospectively reviewed the medical records of a female patient diagnosed with XL-CGD. Flow cytometry was used to detect the respiratory burst function. After restriction enzyme digestion of DNA, XCI was calculated by detecting fluorescent PCR products with capillary electrophoresis. The previously published female XL-CGD cases secondary to skewed XCI was summarized. Clinical data were available for 15 female subjects. The median age of diagnosis was 16 years. Consistent with XL-CGD in males, infection was the most frequent manifestation in the female patients. Catalase-positive pathogens including Serratia marcescens and Staphylococcus aureus infections were the most common pathogens. Autoimmune/autoinflammation manifestations were observed in five patients. Dihydrorhodamine (DHR) assay showed that median %DHR+ values were 6.5% and the values varying with age were observed in 2 patients. All patients had a skewing XCI and there was no consistency between the daughter and carrier mother. Anti-infective treatment was effective in majority and there was no mortality reported in XL-CGD female patients to date. XL-CGD should not be neglected in female patients manifested as CGD phenotype and it is necessary to make periodic clinical evaluation of CGD female carriers as the neutrophil oxidative function may decline with aging and increase the risk for infection.

FRI031 A Novel Strategy To Detect Leptin Receptor (Lepr, fa) Mutation In Obesity And Diabetic Animal Model (Zucker Rats)

Abstract Disclosure: X. Xu: None. X. Hu: None. G. Ma: None. T. Wang: None. J. Wang: None. G. Chen: None. L. Zhao: None. J. Chen: None. Globally, diabetes is among the leading causes of premature morbidity and mortality. Early intervention is crucial to achieve optimal outcomes and minimize the burden of diabetes. Animal models are indispensable tools to investigate etiology and explore novel strategies for diabetes treatment and prevention. Zucker strain and its derivatives, Zucker Fatty (ZF) and Zucker Diabetic Fatty (ZDF) rats, are one of the most used rodent models to study diabetes and diabetes-associated metabolic disorders. Homozygous Zucker rats with leptin receptor gene mutation (Lepr, fa-/-) spontaneously develop obesity, hypercholesterolemia, and hyperlipidemia. The wildtype (fa+/+) and heterozygous (fa+/-) littermates phenotypically are indistinguishable. Furthermore, early stages of diabetes are asymptomatic, and the obese phenotype is not appreciable in homogenous (fa-/-) Zucker rats until week 4 of age. Therefore, a rapid, cost-effective, and reliable genotyping method is essential to differentiate homozygous (fa-/-) rats from either wildtype (fa+/+) or heterozygotes (fa+/-) in early interventional studies. We reported a tetra-primer Amplification-Refractory Mutation System (ARMS)—PCR approach to detect leptin receptor fa mutation using genome DNA collected from ear and tail tissues of Zucker rats. Three different types of amplicon patterns are easily identified by agarose gel electrophoresis. Wildtype (fa+/+) genotype displays a 401 bp and a 201 bp amplicon band; heterozygous (fa+/-) consists of a 401 bp, a 263 bp, and a 201 bp amplicon band on the gel; homozygous (fa-/-) comes up with two bands of 401 bp and 263 bp. The genotypes identified by our tetra-primer ARMS—PCR method are further confirmed by sequence analysis. This method requires neither DNA purification nor restriction enzyme digestion, which is suitable for research exploring gene-environment interaction, as well as implementing appropriate intervention approaches in the research areas of diabetes and metabolic diseases. Presentation: Friday, June 16, 2023

Brief restriction endonuclease digestion of genomic DNA improves PCR amplifiability and reproducibility of SSR loci: Augmenting exploration of hidden genetic variability in an endemic carp of South-East Asia

Majority of the microsatellite primers developed through painstaking, resource intensive research often fail to amplify due to secondary structures in the repeat rich target. In the present work, we describe the use of restriction endonuclease digestion of genomic DNA prior to PCR to counter this issue. The methodology, Restriction Endonuclease Digestion of genomic DNA to improve PCR Amplifiability and Repeatability of SSR loci (REDDAR-SSR), was tested on 40 species-specific Simple Sequence Repeat (SSR) loci in Osteobrama belangeri (F: Cyprinidae). Undigested and HpaII digested pooled genomic DNA samples were used as templates. Two primer concentrations (10 pM and 25 pM) and two annealing temperatures (56 °C uniformly and individual primer-specific annealing temperatures) were tested on both template types in sets of three, repeated thrice (360 reactions/condition). The digested templates exhibited significantly higher (P < 0.05) amplifiability (93.9 % against 24.16 % of undigested counterpart) as well as repeatability (94.47 % against 21.56 % of undigested counterpart). Primer concentration had no significant effect on outcomes (P > 0.05). The number of successfully amplified loci in our method was 38 compared to 18 by the conventional protocol. All 38 loci could also be reliably amplified in individual gDNA using the developed protocol out of which 9 loci were observed to be polymorphic. Notably, 4 of the polymorohic loci could be amplified only through REDDAR-SSR. Our protocol can potentially reduce expenses; resources & time vis-a-vis enhance the reach of SSR markers for genotyping species of commercial or conservational significance.

Complete sequence of a new bipartite begomovirus infecting Sida sp. in Northeastern Brazil.

In Brazil, non-cultivated plants, especially weeds, are infected with a diversity of begomoviruses and often show striking golden mosaic symptoms. In the present study, leaves showing these symptoms were collected from Sida sp. plants in Guadalupe, Piaui State, Northeastern Brazil, in 2015 and 2016. PCR tests with degenerate primers revealed the presence of begomovirus DNA-A and DNA-B components. Restriction enzyme digestion of rollingcircle-amplified DNA revealed fragments totaling ~5.2 kb, indicating infection by a bipartite begomovirus. The DNA-A and DNA-B components have a genome organization typical of New World (NW) bipartite begomoviruses and a common region of 220 nucleotides (nt) with 96% identity, indicating these are cognate components. Comparisons performed with the DNA-A sequence revealed the highest nt sequence identity (84%) with that of sida angular mosaic virus (SiAMV), whereas those performed with the DNA-B sequence revealed highest identity (77%) with that of sida chlorotic vein virus (SiCVV). In phylogenetic analyses, the DNA-A sequence was placed in a strongly supported clade with SiAMV and SiCVV from Piaui, whereas the DNA-B sequence was placed in a clade with SiCVV and corchorus mottle virus. Based on the current ICTV criteria for the demarcation of begomovirus species (<91% nt sequence identity for the DNA-A component), this is a member of a new species for which the name "Sida yellow golden mosaic virus" is proposed.

Adapterama III: Quadruple-indexed, double/triple-enzyme RADseq libraries (2RAD/3RAD).

Molecular ecologists frequently use genome reduction strategies that rely upon restriction enzyme digestion of genomic DNA to sample consistent portions of the genome from many individuals (e.g., RADseq, GBS). However, researchers often find the existing methods expensive to initiate and/or difficult to implement consistently, especially because it is difficult to multiplex sufficient numbers of samples to fill entire sequencing lanes. Here, we introduce a low-cost and highly robust approach for the construction of dual-digest RADseq libraries that build on adapters and primers designed in Adapterama I. Major features of our method include: (1) minimizing the number of processing steps; (2) focusing on a single strand of sample DNA for library construction, allowing the use of a non-phosphorylated adapter on one end; (3) ligating adapters in the presence of active restriction enzymes, thereby reducing chimeras; (4) including an optional third restriction enzyme to cut apart adapter-dimers formed by the phosphorylated adapter, thus increasing the efficiency of adapter ligation to sample DNA, which is particularly effective when only low quantity/quality DNA samples are available; (5) interchangeable adapter designs; (6) incorporating variable-length internal indexes within the adapters to increase the scope of sample indexing, facilitate pooling, and increase sequence diversity; (7) maintaining compatibility with universal dual-indexed primers and thus, Illumina sequencing reagents and libraries; and, (8) easy modification for the identification of PCR duplicates. We present eight adapter designs that work with 72 restriction enzyme combinations. We demonstrate the efficiency of our approach by comparing it with existing methods, and we validate its utility through the discovery of many variable loci in a variety of non-model organisms. Our 2RAD/3RAD method is easy to perform, has low startup costs, has increased utility with low-concentration input DNA, and produces libraries that can be highly-multiplexed and pooled with other Illumina libraries.

Restriction enzyme digestion of host DNA enhances universal detection of parasitic pathogens in blood via targeted amplicon deep sequencing

BackgroundTargeted amplicon deep sequencing (TADS) of the 16S rRNA gene is commonly used to explore and characterize bacterial microbiomes. Meanwhile, attempts to apply TADS to the detection and characterization of entire parasitic communities have been hampered since conserved regions of many conserved parasite genes, such as the 18S rRNA gene, are also conserved in their eukaryotic hosts. As a result, targeted amplification of 18S rRNA from clinical samples using universal primers frequently results in competitive priming and preferential amplification of host DNA. Here, we describe a novel method that employs a single pair of universal primers to capture all blood-borne parasites while reducing host 18S rRNA template and enhancing the amplification of parasite 18S rRNA for TADS. This was achieved using restriction enzymes to digest the 18S rRNA gene at cut sites present only in the host sequence prior to PCR amplification.ResultsThis method was validated against 16 species of blood-borne helminths and protozoa. Enzyme digestion prior to PCR enrichment and Illumina amplicon deep sequencing led to a substantial reduction in human reads and a corresponding 5- to 10-fold increase in parasite reads relative to undigested samples. This method allowed for discrimination of all common parasitic agents found in human blood, even in cases of multi-parasite infection, and markedly reduced the limit of detection in digested versus undigested samples.ConclusionsThe results herein provide a novel methodology for the reduction of host DNA prior to TADS and establish the validity of a next-generation sequencing-based platform for universal parasite detection.

Morphological, genetic and biological characterisation of a novel alphabaculovirus isolated from Cryptophlebia peltastica (Lepidoptera: Tortricidae)

Cryptophlebia peltastica is an agricultural pest of litchis and macadamias in South Africa with phytosanitary status for certain markets. Current control methods rely on chemical, cultural and classical biological control. However, a microbial control option has not been developed. An Alphabaculovirus from C. peltastica was recovered from a laboratory reared colony and morphologically characterised by transmission electron microscopy (TEM). Analysis of occlusion bodies indicated a single NPV (SNPV) varying in size from 421 to 1263 nm. PCR amplification and sequencing of the polh gene region using universal primers followed by BLAST analysis revealed a 93% similarity to a partial polh gene sequence from Epinotia granitalis NPV. Further genetic characterisation involving single restriction endonuclease (REN) digestion of genomic DNA was carried out to generate profiles for comparison against other baculovirus species and potential new isolates of the same virus. The complete genome of the virus was sequenced, assembled and analysed for a more comprehensive genetic analysis. The genome was 115728 base pairs (bp) in length with a GC content of 37.2%. A total of 126 open reading frames (ORFs) were identified with minimal overlap and no preference in orientation. Bioassays were used to determine the virulence of the NPV against C. peltastica. The NPV was virulent against C. peltastica with an LC50 value of 6.46 × 103 OBs/ml and an LC90 value of 2.46 × 105 OBs/ml, and time mortality ranging between 76.32 h and 93.49 h. This is the first study to describe the isolation and genetic characterisation of a novel SNPV from C. peltastica, which has potential for development into a biopesticide for the control of this pest in South Africa.

Fetal and Placental DNA Stimulation of TLR9: A Mechanism Possibly Contributing to the Pro-inflammatory Events During Parturition.

While there is evidence for a relationship between cell-free fetal DNA (cffDNA) and parturition, questions remain regarding whether cffDNA could trigger a pro-inflammatory response on the pathway to parturition. We hypothesized that placental and/or fetal DNA stimulates toll-like receptor 9 (TLR9) leading to secretion of pro-inflammatory cytokines by macrophage cells. Four in vitro DNA stimulation studies were performed using RAW 264.7 mouse peritoneal macrophage cells incubated in media containing the following DNA particles: an oligodeoxynucleotide (ODN2395), intact genomic DNA (from mouse placentas, fetuses and adult liver), mouse DNA complexed with DOTAP (a cationic liposome forming compound), and telomere-depleted mouse DNA. Interleukin 6 (IL6) secretion was measured in the media by enzyme-linked immunosorbent assay; and the cell pellet was homogenized for protein content (picograms IL6/mg protein). Robust IL6 secretion was observed in response to ODN2395 (a CpG-rich TLR9 agonist), mouse DNA-DOTAP complexes, and telomere-depleted mouse DNA in concentrations of 5 to 15 μg/mL. In contrast, ODN A151 (containing telomere sequence motifs), intact genomic mouse DNA, and restriction enzyme-digested DNA had no effect on IL6 secretion. The IL6 response was significantly inhibited by chloroquine (10 μg/mL), thereby confirming the important role for TLR9 in the response by macrophage cells. DNA derived from mouse placentas and fetuses, and depleted of telomeric sequences, stimulates a robust pro-inflammatory response by macrophage cells, thereby supporting the hypothesis that cffDNA is able to stimulate an innate immune response that could trigger the onset of parturition. These findings are of clinical importance, as we search for effective treatment/prevention of preterm parturition.

Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

Identification and molecular cloning of cysteine protease gene of Trypanosoma Evansi Isolated from Camel

A molecular study was carried out to isolate cysteine protease gene of Trypanosoma evansi using PCR. The desired amplicons of cysteine protease gene from the genomic DNA of T. evansi were successfully amplified by PCR using gene specific primers at annealing temperature of 55°C. Amplified PCR product was identified on the basis of its size in agarose gel electrophoresis as 1533 bp. For cloning the purified DNA fragment was ligated to the pGEM-T Easy vector and the ligated mixture was transformed into Escherichia coli JM109 strains. The cells containing recombinant plasmid were identified on the basis of white/blue colony selection on LB agar containing X-Gal, IPTG and ampicillin. Screening of recombinants was done by restriction enzyme digestion of plasmid DNA using EcoRI and confirmed on the basis of gene size, i. e. 1533 bp for cysteine protease gene. Colony PCR was done for quick screening of plasmid inserts directly from E. coli colonies in the presence of insert specific primers.

Multidrug resistant Pseudomonas aeruginosa survey in a stream receiving effluents from ineffective wastewater hospital plants.

BackgroundMulti-drug resistant forms of Pseudomonas aeruginosa (MDRPA) are a major source of nosocomial infections and when discharged into streams and rivers from hospital wastewater treatment plants (HWWTP) they are known to be able to persist for extended periods. In the city of Manaus (Western Brazilian Amazon), the effluent of three HWWTPs feed into the urban Mindu stream which crosses the city from its rainforest source before draining into the Rio Negro. The stream is routinely used by Manaus residents for bathing and cleaning (of clothes as well as domestic utensils) and, during periods of flooding, can contaminate wells used for drinking water.Results16S rRNA metagenomic sequence analysis of 293 cloned PCR fragments, detected an abundance of Pseudomonas aeruginosa (P. aeruginosa) at the stream’s Rio Negro drainage site, but failed to detect it at the stream’s source. An array of antimicrobial resistance profiles and resistance to all 14 tested antimicrobials was detected among P. aeruginosa cultures prepared from wastewater samples taken from water entering and being discharged from a Manaus HWWTP. Just one P. aeruginosa antimicrobial resistance profile, however, was detected from cultures made from Mindu stream isolates. Comparisons made between P. aeruginosa isolates’ genomic DNA restriction enzyme digest fingerprints, failed to determine if any of the P. aeruginosa found in the Mindu stream were of HWWTP origin, but suggested that Mindu stream P. aeruginosa are from diverse origins. Culturing experiments also showed that P. aeruginosa biofilm formation and the extent of biofilm formation produced were both significantly higher in multi drug resistant forms of P. aeruginosa.ConclusionsOur results show that a diverse range of MDRPA are being discharged in an urban stream from a HWWTP in Manaus and that P. aeruginosa strains with ampicillin and amikacin can persist well within it.

Isolation and molecular characterisation of actin gene of Trypanosoma evansi from Indian dromedaries

The present study was carried out to isolate the actin gene of Trypanosoma evansi by reverse transcription polymerase chain reaction, clone the amplicon in a suitable bacterial plasmid vector, characterisation of gene through sequencing and molecular modeling of obtained actin protein. Total RNA of T. evansi was used to amplify the actin gene by RT-PCR, amplification was done using gene specific primers and desired amplicon was identified on the basis of size of the gene. For cloning, DNA fragment of interest was then ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains. After cloning, screening of recombinants was done by restriction enzyme digestion of plasmid DNA. After confirmation of clone, the plasmid DNA was sequenced and coding sequences of actin gene was of 1131bp. Sequence analysis revealed that actin gene of Trypanosoma evansi from Indian isolate shared 81.4–99.5% and 93.4–98.7% sequence identity at the nucleotide and amino acid level, respectively with isolates from different geographical areas of the world.

Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides.

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.

Diagnostic Performance of Plasma DNA Methylation Profiles in Lung Cancer, Pulmonary Fibrosis and COPD

Disease-specific alterations of the cell-free DNA methylation status are frequently found in serum samples and are currently considered to be suitable biomarkers.Candidate markers were identified by bisulfite conversion-based genome-wide methylation screening of lung tissue from lung cancer, fibrotic ILD, and COPD. cfDNA from 400μl serum (n=204) served to test the diagnostic performance of these markers. Following methylation-sensitive restriction enzyme digestion and enrichment of methylated DNA via targeted amplification (multiplexed MSRE enrichment), a total of 96 markers were addressed by highly parallel qPCR.Lung cancer was efficiently separated from non-cancer and controls with a sensitivity of 87.8%, (95%CI: 0.67–0.97) and specificity 90.2%, (95%CI: 0.65–0.98). Cancer was distinguished from ILD with a specificity of 88%, (95%CI: 0.57–1), and COPD from cancer with a specificity of 88% (95%CI: 0.64–0.97). Separation of ILD from COPD and controls was possible with a sensitivity of 63.1% (95%CI: 0.4–0.78) and a specificity of 70% (95%CI: 0.54–0.81). The results were confirmed using an independent sample set (n=46) by use of the four top markers discovered in the study (HOXD10, PAX9, PTPRN2, and STAG3) yielding an AUC of 0.85 (95%CI: 0.72–0.95).This technique was capable of distinguishing interrelated complex pulmonary diseases suggesting that multiplexed MSRE enrichment might be useful for simple and reliable diagnosis of diverse multifactorial disease states.

Separation of DNA by length in rotational flow: Lattice-Boltzmann-based simulations.

We use a lattice-Boltzmann based Brownian dynamics simulation to investigate the separation of different lengths of DNA through the combination of a trapping force and the microflow created by counter-rotating vortices. We can separate most long DNA molecules from shorter chains that have lengths differing by as little as 30%. The sensitivity of this technique is determined by the flow rate, size of the trapping region, and the trapping strength. We expect that this technique can be used in microfluidic devices to separate long DNA fragments that result from techniques such as restriction enzyme digests of genomic DNA.

Enhanced reduced representation bisulfite sequencing for assessment of DNA methylation at base pair resolution.

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

XAGE-1b Cancer/Testis Antigen Is a Potential Target for Immunotherapy in Prostate Cancer

Introduction: Gene-modified cell vaccines are now considered to be the best way to achieve immunotherapy for a variety of cancers including prostate cancer (PCa). XAGE-1b is a member of the cancer/testis antigen family which has demonstrated strong immunogenicity. We investigated whether XAGE-1b is an ideal target for PCa immunotherapy. Materials and Methods: The recombinant eukaryotic expression vector pDisplay-XAGE-1b was constructed. Then the recombinant vector was transfected into Myc-CaP cells and its immunogenicity in vitro was studied. After transfection, the Myc-CaP-XAGE-1b cells were injected into FVB mice subcutaneously. Tumor growth was periodically observed and the anti-tumor effect and mechanism in vivo were further studied. Results: The recombinant vector was correctly constructed by DNA sequencing and restriction endonuclease digestion. Myc-CaP cells were successfully transfected with XAGE-1b gene by immunofluorescence staining and Western blot. The transfected cells exhibited increased IFN-γ secretion, decreased IL-6 secretion and enhanced killing activity. Tumor grew slower in XAGE-1b-modified FVB mice than in wild-type FVB mice. High dendritic cell expression and low myeloid-derived suppressor cell expression were observed in tumor tissues expressed with XAGE-1b. Conclusions: XAGE-1b gene transfection could significantly enhance the immunogenicity of Myc-CaP cells. Therefore, XAGE-1b may be an attractive target for antigen-specific immunotherapy in PCa.

Isolation, PCR Amplification and Cloning of Heat Shock Protein Gene from Salivary Glands ofHyalomma dromedariiTicks fromCamelus dromedarius

A molecular study was carried out to isolate the heat shock protein gene of Hyalomma dromedarii ticks of camels, which play important role in their survival under harsh environmental conditions. Engorged adult H. dromedarii ticks were collected from camel herds in the Bikaner district of Rajasthan. Total genomic DNA and RNA was isolated from the salivary glands of ticks. Primers were designed for amplification of heat shock protein gene from H. dromedarii by using base sequence of Haemaphysalis longicornis. The heat shock protein gene of H. dromedarii was successfully amplified from genomic DNA and cDNA and was identified on the basis of its size in agarose gel electrophoresis as 560 bp. The amplicon of expected size was purified from the 1% low melting agarose gel. DNA fragment of interest was then ligated to the pGEM-T Easy vector and ligated mixture was transformed into Escherichia coli JM109 strains for cloning. Screening of recombinants was done by restriction enzyme digestion of plasmid DNA and by colony PCR for quick screening of plasmid insert directly from E. coli colonies in the presence of insert specific primers.

Genomic and proteomic dissection and characterization of the human sperm chromatin.

The mammalian spermatozoon has a unique chromatin structure where the majority of DNA is packaged by protamines, while a small fraction (∼8%) remains associated with nucleosomes. However, the chromatin affinity and repertoire of the additional proteins constituting the different sperm chromatin fractions have not yet been explored. To address this we have carried out a genomic and proteomic characterization of human sperm samples subjected to chromatin fractionation using either 0.65 M NaCl extraction followed by EcoRI/BamHI DNA restriction enzyme digestion, or micrococcal nuclease digestion. DNA fractions corresponding to the nucleosome-packaged DNA were sequenced, confirming an appropriate dissection of the sperm chromatin. In addition we detected and sequenced a subnucleosomal particle. Although both fractions were highly enriched at gene promoters, some sequences were found to be exclusively associated with one of those. The results of the proteomic analyses demonstrate that there are two distinct sets of sperm proteins which differ in chromatin affinity. Histone variants, transcription factors, chromatin-associated and modifying proteins involved in regulatory roles were identified as weakly attached to the sperm DNA, whereas proteins with structural roles were identified in the condensed fraction. Many factors, such as the histone lysine demethylase PHF8 identified for the first time in the human sperm cell in this study, were identified exclusively in soluble fraction. Our results provide additional support to the possibility that all of these factors may constitute additional layers of sperm epigenetic information or have structural or regulatory roles transmitted by the sperm cell to the oocyte at fertilization.