Use Of DNA Fragments Research Articles

Chloroplast genomes and ribosomal DNA provide insights into divergence and morphological evolution of alpine Tongoloa

AbstractTongoloa is a herbaceous genus of East Asia Clade (Apiaceae) distributed in the alpine regions. The use of DNA fragments has not provided a well‐resolved evolutionary history. For this research, we primarily collected samples from the type localities of Tongoloa and closely related taxa in the Hengduan Mountains. The chloroplast (cp) genomes and nuclear ribosomal (nr) DNA repeats of 27 taxa were assembled using genome skimming sequencing reads. We analyzed the characteristics of the Tongoloa cp genome, and found a remarkable expansion of the Inverted Repeats. Three genes (ndhC, ndhJ, and petG) related to photosynthesis appear to have undergone significant selective pressure. Through high‐resolution phylogenetic analysis, the cpDNA provided compelling evidence supporting the inclusion of Sinolimprichtia as an early taxon within Tongoloa. However, the nrDNA suggested that Tongoloa and Sinolimprichtia belong to distinct branches. Morphological analysis showed that Tongoloa has broadly oval fruits with a cordate base, whereas the fruits of Sinolimprichtia are long‐obovate with an obtuse base. The specific fruit morphology of Sinolimprichtia was found to be nested within Tongoloa in the cpDNA phylogenetic tree. Ancient introgression and chloroplast capture provide the most plausible explanation for the significant conflict between the nrDNA and cpDNA phylogenies. Our study highlights the potential impact of the complex evolutionary history of Tongoloa on the challenges encountered in previous taxonomic treatments.

Can the Prediction of Intrauterine Insemination Results by Used Aniline Blue Stain (ABS) and Sperm Chromatin Dispersion (SCD) Levels?

Introduction: This study aimed to perform routine seminal fluid analysis, sperm DNA fragmentation, and sperm function tests at the chromatin maturation level and evaluate pregnancy in the patients passing intrauterine insemination before starting Intrauterine Insemination (IUI) method. Materials and Methods: In this prospective study, 111 couples who underwent Intrauterine Insemination (IUI) in unexplained infertility patients were admitted to Al-Farah IVF and assisted reproductive center in Baghdad, Iraq between November 2020 and February 2021 were evaluated. Semen fluid analysis was performed based on (WHO 4th) guiding rules. In addition, Sperm Chromatin Dispersion (halo test) and sperm maturation were performed with Aniline Blue Stain (ABS). Results: Sperm Chromatin Dispersion (SCD) groups were compared in terms of pregnancy outcome; the positive pregnancy rate was found to be above in the normal SCD groups (p = 0.0005). In addition, Aniline Blue Stain (ABS) groups were compared in the terms of pregnancy outcome; the positive pregnancy rate was found to be higher in the normal ABS group (p = 0.017). Conclusion: Our study showed that the use of DNA fragmentation (SCD) and sperm maturation tests (ABS) together with routine semen analysis in intrauterine insemination cases will make a significant contribution to the prediction of Intrauterine Insemination (IUI) increased results. So, these results indicate a defect in the effect of DNA fragmentation on the outcome of intrauterine insemination.

P-999: Sperm DNA integrity evaluation using ThinPrep® cytologic preparations

There has been increasing interest in the use of DNA fragmentation as a marker of male infertility. Recently, it has been recognized that the use of sperm with a high percentage of DNA damage results in a lower pregnancy rate with IVF and a lower incidence of blastocyst development after ICSI. Increased DNA damage has also been linked to recurrent miscarriages. Sperm DNA integrity is usually evaluated by either flow cytometry (DNA Integrity Assay) or a slide based technique [TUNEL (terminal deoxynucleotidyl transferase - mediated deoxyuridine triphosphate nick end labeling)].

Conserved cysteine-rich domain of paramyxovirus simian virus 5 V protein plays an important role in blocking apoptosis.

The paramyxovirus family includes many well-known human and animal pathogens as well as emerging viruses such as Hendra virus and Nipah virus. The V protein of simian virus 5 (SV5), a prototype of the paramyxoviruses, contains a cysteine-rich C-terminal domain which is conserved among all paramyxovirus V proteins. The V protein can block both interferon (IFN) signaling by causing degradation of STAT1 and IFN production by blocking IRF-3 nuclear import. Previously, it was reported that recombinant SV5 lacking the C terminus of the V protein (rSV5VDeltaC) induces a severe cytopathic effect (CPE) in tissue culture whereas wild-type (wt) SV5 infection does not induce CPE. In this study, the nature of the CPE and the mechanism of the induction of CPE were investigated. Through the use of DNA fragmentation, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling, and propidium iodide staining assays, it was shown that rSV5VDeltaC induced apoptosis. Expression of wt V protein prevented apoptosis induced by rSV5VDeltaC, suggesting that the V protein has an antiapoptotic function. Interestingly, rSV5VDeltaC induced apoptosis in U3A cells (a STAT1-deficient cell line) and in the presence of neutralizing antibody against IFN, suggesting that the induction of apoptosis by rSV5VDeltaC was independent of IFN and IFN-signaling pathways. Apoptosis induced by rSV5VDeltaC was blocked by a general caspase inhibitor, Z-VAD-FMK, but not by specific inhibitors against caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13, suggesting that rSV5VDeltaC-induced apoptosis can occur in a caspase 12-dependent manner. Endoplasmic reticulum stress can lead to activation of caspase 12; compared to the results seen with mock and wt SV5 infection, rSV5VDeltaC infection induced ER stress, as demonstrated by increased expression levels of known ER stress indicators GRP 78, GRP 94, and GADD153. These data suggest that rSV5VDeltaC can trigger cell death by inducing ER stress.

Cis-regulatory sequences from the first intron of the rat glutamine synthetase gene are involved in hepatocyte specific expression of the enzyme.

In order to identify regulatory elements involved in the hepatocyte specific expression of the enzyme glutamine synthetase [GS (E.C. 6.3.1.2)] we analyzed the first intron of the rat GS gene. A sequence analysis detected clusters of potential transcription factor binding sites in regions that are hypersensitive for DNase I, including sites for Sp1, HNF3 and elements related to binding of members from the C/EBP family. By use of DNA fragments with putative regulatory elements, reporter genes have been constructed that were transfected into isolated hepatocytes in primary culture and into HepG2 hepatoblastoma cells. By these experiments we cold show that sequences from the first intron are able to enhance transcription specifically in hepatocytes but not in cells from the hepatoblastoma cell line. The existence of enhancer effects in the first intron of the GS gene and their restriction to hepatocytes demonstrates that aside from regulatory regions upstream of the transcription start point, there are also downstream regions involved in the specific expression of the gene. We conclude that intronic elements are involved in the pretranslational regulation of the expression of the GS as part of a complex interplay between different regions of the gene.

Isolation of chromosome-associated proteins from Drosophila melanogaster that bind a human centromeric DNA sequence.

The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is though to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.

Use of DNA probes and plasmid capture in a search for new interesting environmental genes

Adaptation to a stressed environment leads to organisms bearing DNA, encoding defense mechanisms. These mechanisms can be heavy metal resistance, catabolism of organic xenobiotics or stress reactions. Genes responsible for these mechanisms can be used for monitoring changing environments and therefore it can be important to store such bacteria in a bank. DNA-probing will be presented by the use of DNA fragments (of Alcaligenes eutrophus) coding for heavy metal resistance or xenobiotic degradation. Some strains do not grow on petri dishes and accordingly cannot be isolated from soils. In order to isolate plasmids from such strains, coding for heavy metal resistances or xenobiotic degradations, an exogenous plasmid isolation method was developed. In this method, the endogenous population is conjugated with Pseudomonas or Alcaligenes strains bearing a retrotransfer plasmid like RP4. In that way new plasmids from various sources including non-culturable strains could be obtained. With these methods, a large number of specimens adapted to stressed situations can be isolated or constructed (in the case of the exogenous plasmid isolation method). They form a source of interesting genetic material that can be used to restore polluted areas in natural areas, if necessary with the aid of genetic engineering (in vitro or in vivo techniques). Full knowledge of such bacteria and their resistance mechanisms or degradation pathways, can lead to new constructions able to attack recalcitrant mixtures of different organics and to resist heavy metals.

31] The use of DNA fragments of defined sequence for the study of DNA damage and repair

This chapter discusses the use of DNA fragments of defined sequence for the study of DNA damage and repair. A wide variety of physical and chemical agents damages the DNA. The biological effects of such agents include cell death, neoplastic transformation, and mutations. Cells are normally capable of repairing some types of DNA damage by enzymatic modification of the DNA. In most cases, the details of the repair pathways will determine the ultimate biological effect of the initial lesions. The chapter applies some of the remarkable progress that has been made in nucleic acid technology to such problems. The basic approach involves the use of a DNA fragment of defined sequence as a probe to study the damage and repair of DNA. The advantage of using such a sequence is that the precise site of damage and the subsequent fate of the modified DNA can be determined rapidly. The chapter also overviews some of the examples related to DNA DNA sequence for studies of DNA damage and repair.