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DNA Sequencing With Chain Terminating Inhibitors
Abstract
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
Supplementary resource (1)
Nucleotide Sequence
March 1995
... The phenotypic tests that were performed include Gram staining, urease, citrate, methyl red, coagulase, catalase, oxidase and sugar fermentation tests (Sanger et al. 1977;Lane, 1991). ...
... Zymo-Spin column was used to extract the DNA templates as prescribed by the manufacturer (Zymo Research Corporation, USA). The ultra-pure DNA templates were used to perform the polymerase chain reaction (PCR) as previously described (Lane, 1991) and derived amplicons subsequently sequenced with the dideoxy-chain termination method (Sanger et al. 1977). Upon confirmation of the taxonomic identity of the bacterial strains by algorithm of the United States National Center for Biotechnology Information (NCBI) GenBank, the identified bacterial strains were deposited in the GenBank database under specific accession numbers. ...
Microbial surfactants that are largely produced by bacteria and yeasts are preferentially used in relation to the synthetic surfactants. Hence there is a need to carry out investigations that will isolate efficient biosurfactant-producing microorganisms. The present study isolated potential biosurfactant-producing bacteria from soil that were polluted by spent engine oil from some automobile workshops situated at Okada, Edo State, Nigeria. The counts and isolation of hydrocarbon-utilizing bacteria (HUC) in the spent engine oil-polluted soil samples were carried out using the spread plate technique. The identification of the hydrocarbon-utilizing bacteria was carried out with standard phenotypic and 16S rRNA gene sequencing techniques. Hydrocarbon-utilizing bacterial isolates were screened for biosurfactant-producing potentials by using their culture supernatants to perform haemolytic, oil displacement and emulsification assays. The mean HUC in the engine oil-polluted soil samples ranged from 4.47±0.03 log10CFU/g to 4.84±0.05 log10CFU/g. Staphylococcus anginosus KT 1, Micrococcus luteus KT 2, Bacillus subtilis KT 3 and Staphylococcus aureus KT 4 were the bacterial strains that were isolated from the engine oil-polluted soil samples, and have been respectively deposited in the United States GenBank under accession numbers OP602363, OP602365,
... Dwa lata później udoskonalił metodę wprowadzając do reakcji tzw. terminatory, czyli trifosforany nukleozydów w formie dideoksy (ddNTP) [11]. Synteza odbywała się w 4 osobnych reakcjach, przy czym w każdej z nich oprócz tej samej matrycy i standardowych składników, w tym znakowanego radioizotopowo ( 32 P) dowolnego trifosforanu nukleozydu, znajdował się jeden rodzaj ddNTP (ddATP, ddCTP, ddGTP lub ddTTG). ...
... Początkowo możliwe było odczytanie stosunkowo krótkiej sekwencji DNA, ok. 100 nt w przypadku metody Maxama-Gilberta [10] i ok. 100-200 nt w przypadku metody Sangera [11]. Głównym ograniczeniem była rozdzielczość żelu poliakrylamidowego. ...
There is no technique that would make a greater contribution to the development of genetics, molecular biology and medicine than DNA sequencing. For many years, the method based on enzymatic DNA synthesis developed by Frederic Sanger was the gold standard in this area. At the end of the 20th century, there was a dynamic development of next-generation sequencing (NGS) technologies, which ended the era of single gene analysis and initiated the era of genome sequencing. Despite fierce competition, one NGS technology has practically completely dominated the global market. In the article, we present our own review of DNA sequencing methods, starting from the Sanger method to high-throughput second- and third-generation sequencing technologies, with particular emphasis on those that have achieved commercial success. We present their short history, principles of operation, technical possibilities, applications and limitations. In the summary, we reveal how much human genome sequencing costs at the current stage of the genomic revolution and outline the prospects for further development of genomics.
... Under Cd stress conditions, NADPH oxidase plays a role in generating reactive oxygen species (ROS), which act as signaling molecules to activate plant defense mechanisms [ 9 ]. In addition, NADPH oxidase indirectly removes toxic Cd ions by activating proton pumps and creating a proton gradient across the plasma membrane [ 10 ]. NADPH oxidase (NOX) is an important enzyme expressed by the RBOH gene [11]. ...
... After centrifugation at 14,000 rpm for 15 minutes, the ethanol was removed, and the DNA was air-dried. 10. Finally, the DNA was dissolved in 30 µl of nuclease-free water. ...
This study investigated the presence of genetic markers associated with salinity and stress tolerance in Tetradium daniellii using gel electrophoresis. Three markers (CAT, GR, RBOH) were identified out of 12 markers screened that are linked to salinity resistance and olerance to drought, cold, pathogens and cadmium stress. The identification of these markers provides insights into the genetic basis of stress tolerance in T. daniellii.
... Sequencing determines the precise order of nucleic acids in the deoxyribonucleic acid (DNA). DNA sequencing was properly introduced in 1977 with the development of Frederick Sanger's 'chain termination' technique (28). Although previous methods for DNA sequencing existed, they were time-consuming and highly expensive (29). ...
... Modern Sanger sequencing uses capillary-based electrophoresis and automated DNA sequencing machines (29). While Sanger's method was once considered the gold standard for DNA sequencing, it had significant drawbacks, primarily being expensive and time-consuming, especially considering the limited number of sequences in a single experiment (800-1000 base pairs) (28,(30)(31)(32). In 2005, a revolutionary technology called 'next generation sequencing', also referred to as 'second generation sequencing', was introduced. ...
Brain tumors and genomics have a long-standing history given that glioblastoma was the first cancer studied by the cancer genome atlas. The numerous and continuous advances through the decades in sequencing technologies have aided in the advanced molecular characterization of brain tumors for diagnosis, prognosis, and treatment. Since the implementation of molecular biomarkers by the WHO CNS in 2016, the genomics of brain tumors has been integrated into diagnostic criteria. Long-read sequencing, also known as third generation sequencing, is an emerging technique that allows for the sequencing of longer DNA segments leading to improved detection of structural variants and epigenetics. These capabilities are opening a way for better characterization of brain tumors. Here, we present a comprehensive summary of the state of the art of third-generation sequencing in the application for brain tumor diagnosis, prognosis, and treatment. We discuss the advantages and potential new implementations of long-read sequencing into clinical paradigms for neuro-oncology patients.
... The four DDNTPs (dideoxynucleotide phosphates such as ddATP, ddTTP, ddCTP, and ddGTP) are tagged with different fluorochrome dyes to facilitate laser beam detection. Each fluorescentlabeled terminated segment of DNA is recorded, and the DNA sequence is determined based on this information (Sanger, Nicklen, and Coulson 1977). ...
... The Maxam Gilbert and Sanger methods made DNA sequencing possible [1], [2]. However, these approaches were slow and expensive. ...
This review focuses on the study of biogeographic ancestry using the Accurate Ancestry Identification Panel. Autosomal markers may provide little information about the nature of an individual's admixture due to ongoing human recombination and migration. Biogeographic ancestry assessment (BGA) is a term used to describe ancestry through DNA testing. This is usually accomplished by testing specific regions of DNA called ancestry information markers (AIMs). AIMs are chosen because they expose significantly different frequencies between different populations in different parts of the world. The panels of these AIMs can be assessed using next-generation sequencing (NGS) to predict the geographical origins of a person of interest's ancestors, usually in terms of continent of origin, and sometimes by smaller geographic regions. The use of ancestry informative markers (AIM) to identify genomic ancestry can be useful for a variety of studies in evolutionary genetics, biomedical research, and forensic analyses. However, there remains a major challenge in determining AIMs for populations with complex and highly mixed ancestry.
... From a workflow perspective, both CE and massively parallel sequencing (MPS) follow a similar process as STR typing outlined in Figure 8.7 (additional processing variations are required for CE-based SNP typing). Traditional Sanger sequencing by CE (Sanger et al., 1977) reads all amplified DNA fragments in one reaction and thus is a low throughput, low resolving system. Additionally, due to its chemistry (i.e., dideoxy cycle sequencing), the method is not quantitative, limiting its use for mixture deconvolution. ...
Gender-based violence against women (GBVAW), specifically physical and sexual violence against women and girls, is a critical human rights crisis in the Southern African region, including severe cases of femicide and conflict-related sexual violence (CRSV). Over the past decades, efforts to prevent GBVAW and improve case outcomes have included the enactment of specific legislation, awareness campaigns, stakeholder engagements, victim support schemes, education, policing support and tougher consequences for offenders. However, research into the key challenges in the fight against GBVAW in the region has consistently identified a lack of reporting of cases, partly influenced by poor confidence in the justice system, and inadequate forensic science capacity to assist the investigation, prosecution, and conviction of offenders. Currently, only 4 out of the 16 member states of the SADC operate a national forensic DNA database, which is a critical intelligence tool in solving cold cases by identifying unknown offenders and linking multiple crimes to identify serial offenders. Other forensic intelligence systems, such as fingerprint and facial image databases and ballistics databases are less developed in the region. In 2023, the UNODC Regional Office for Southern Africa commissioned the handbook on forensic evidence processing in GBVAW cases for criminal justice practitioners in the SADC region. The aim of the handbook is to serve as a resource to inform capacity building on the collection and handling of forensic evidence, management of the forensic investigation of GBVAW cases, training on the interpretation, evaluation, and communication of forensic evidence, and the development of appropriate laws and policies to establish relevant intelligence databases and facilitate international cooperation.
... Billington et al. cloned and sequenced the plo gene encoding the PLO toxin, which is located in the open reading frame (ORF) of the 1605 bp gene of T. pyogenes [13]. A common ribosome binding site, two promoter sequences, and three co-repeating sequences were found upstream of the plo gene, with a transcription termination region located downstream of the gene [14]. In addition, Rudnick et al. found that the plo gene, along with the aforementioned sequence and ORF (orf121), encodes an unknown functional protein of 13.4 KDa, forming a 2.7 kb gene island characterized by a decrease in G+C content (50.2%) [15,16]. ...
Trueperella pyogenes is an important opportunistic pathogenic bacterium widely distributed in the environment. Pyolysin (PLO) is a primary virulence factor of T. pyogenes and capable of lysing many different cells. PLO is a member of the cholesterol-dependent cytolysin (CDC) family of which the primary structure only presents a low level of homology with other members from 31% to 45%. By deeply studying PLO, we can understand the overall pathogenic mechanism of CDC family proteins. This study established a mouse muscle tissue model infected with recombinant PLO (rPLO) and its single-point mutations, rPLO N139K and rPLO F240A, and explored its mechanism of causing inflammatory damage. The inflammatory injury abilities of rPLO N139K and rPLO F240A are significantly reduced compared to rPLO. This study elaborated on the inflammatory mechanism of PLO by examining its unit point mutations in detail. Our data also provide a theoretical basis and practical significance for future research on toxins and bacteria.
... The PCR product, once purified, underwent Sanger sequencing [16] at Biokart in Bangalore, with sequencing carried out from both ends. Subsequently, the forward and reverse sequences were aligned and merged to produce a consensus sequence using Sequencher 5.3 software from Gene Codes Corporation in Ann Arbor, Michigan, USA. ...
... 4-Gel Electrophoresis: The amplification of 11 sample specimens' products was confirmed by using 1.5% agarose gel electrophoresis in a 1 X TBE buffer and then stained to visualize with 4 % ethidium bromide in UV light at 320 nm, the molecular weight of the specific target bands is 710 bp. 5-Sequencing of PCR products and phylogenetic analysis: Using the partial sequence and the Sanger Sequencing Technique (Sanger et al., 1977), the forward primer LCO 1490 was applied, for sequencing, Macrogene Company, Korea, received the PCR of the mitochondrial Cytochrome Oxidase subunit I (COI) gene product to validate the identification of the 11 specimens, the phylogenetic tree was prepared using a Mega X program. ...
The first molecular research on Iraqi centipede fauna is presented in this article. Between October 2022 and May 2023, during various climatic circumstances, centipedes were collected from several locations in four provinces of Iraq. Three families, represented by four genera, underwent molecular identification, and five species were found. From the order Scolopendromorpha family Scolopendridae, two species were recorded, Scolopendra morsitans Linnaeus, 1758, and S. cingulata Latreille, 1829, Cormocephalus sp.; while from the order Lithobiomorpha, family Lithobiidae, one species was recorded for first time in Iraq; Lithobius crassipes L. Koch, 1862 from the order Geophilomorpha family Himantariidae, one species Bothriogaster Signata Kessler, 1874. DNA was extracted from the specimens, the mtDNA fragment from the Cytochrome C Oxidase Subunit I (COI) gene was amplified by using the PCR technique with appropriate primers, and subsequently, the Basic Local Alignment Search Tool (BLAST) tool, which is accessible at the NCBI, was used. Additionally, a phylogenetic tree was built, and a distant comparison was shown.
... The amplified product was separated by electrophoresis on 1.5% agarose gel, and the target band was recovered and purified using the Wizard SV Gel and Clean-Up kit (Promega Co., USA). The purified product was sequenced using the Sanger method [19]. The taxonomic positions of strain YSY-4.3 were identified by comparing its nucleotide sequence with the nucleotide sequences of 16S rRNA genes in the Genbank/DDBJ/EMBL databases. ...
This work aimed to isolate and characterize a novel chitin-degrading bacterium from Yok Don National Park, Vietnam, for crop production studies. Among the chitinolytic isolates, strain YSY-4.3 was selected, which grew rapidly and produced a large halo around the colony. 16S rDNA analysis indicated that the strain is a novel species in the genus paenibacillus, and an in vitro evaluation showed that the strain produced phytohormones (IAA, GA3, and zeatin), biofilms, and siderophores; possessed cellulase; and exerted antifungal activity. The whole genome of the strain was 5,628,400 bp with 49.3% GC content, 5056 coding sequences, 48 tRNA, and 1 rRNA. It shared the highest values of digital DNA-DNA hybridization (67.4%) and average nucleotide identity (89.54%) with those of Paenibacillus woosongensis B2_4 (CP126084.1), suggesting a novel species. Of the coding sequences, 4287 proteins were identified by COG, and 2561 were assigned by KEGG. The genome contained at least 51 genes involved in plant growth and resistance to heavy-metal toxicity and 359 carbohydrate-active enzymes. The chitinolytic system of the strain was composed of 15 enzymes, among them, PsChiC, which contained a GH18 catalytic domain and a GH5 catalytic domain, had not been previously reported. In addition, the genome possessed 15 gene clusters encoding antimicrobial metabolites, 10 of which are possible novel clusters. This study expands knowledge regarding novel chitinolytic bacteria from Yok Don National Park and provides a valuable gene resource for future studies.
... The samples were initially confirmed as positive for A/H5 by three RT-qPCR techniques (VetMAX Gold AIV, Thermo Fisher; Lee et al., 2015; NVSL/APHIS/USDA, 2022) followed by Sanger sequencing (Sanger et al., 1977). The partial sequencing of the HA gene (segment 4) was used to confirm the presence of a polybasic motif at the cleavage site, (PLREKRKKR/GLF or PLREKRRKR/GLF) to assess the pathogenicity profile of the identified viruses (Slomka et al., 2007). ...
... To ensure adequate concentrations and purity of the PCR products, quantification of the PCR products was performed using Nanodrop (UveVis spectrophotometer Q5000/USA) [16]. According to the enzymatic chain terminator technique created by [17], PCR products with target bands in all investigated does (15 healthy and 15 mastitic) were sent for DNA sequencing in forward and reverse directions using an ABI 3730XL DNA sequencer (Applied Biosystem, USA). ...
... The widely used first generation sequencing is developed by Fredrick Sanger and co-workers, and is also known as Sanger sequencing. 24 It is considered as the "gold standard" for validating DNA sequences because of the 99.99% base accuracy. ...
Background
Molecular diagnostic technology is the foundation of precision medicine, which has the advantages of good specificity, high sensitivity, strong targeting, rapiddiagnosis, etc. It has a wide range of applications in the field of pediatrics. However, molecular diagnostic technology is characterized by complicated experimental operation, high difficulty in data analysis and interpretation, in consistent standards among laboratories, and difficult standardization of technical processes. Enhancing the application value of molecular diagnostic technology in pediatrics and promoting the high‐quality development of the discipline requires careful consideration by relevant management and professionals.
Methods
This study firstly outlines the development history of molecular diagnostic technology. Then, it analyzes the application of molecular diagnostic technology in the field of pediatrics. Finally, it explores the countermeasures for the management of molecular diagnostic laboratories.
Results
This study highlights the importance of molecular diagnostic technology in providing information and decision‐making basis for disease prevention, prediction,diagnosis, treatment and regression. It has a wide range of applications in the molecular diagnosis of pediatric hereditary diseases, malignant tumors and infectious diseases. In addition, the countermeasures for the management of molecular diagnostic laboratories are proposed from the five aspects of laboratory, personnel team construction, standardized management,multidisciplinary cross‐discipline, research and translation, safety managementand ethical supervision, and management upgrading and modernization.
Conclusions
Molecular diagnostic technology, as the basis of precision medicine, has become one of the important frontier fields in the development of contemporary pediatric medicine. Enhanced laboratory capacity in molecular diagnostic techniques can improve outcomes in the prevention, prediction, diagnosis, treatment, prognosis and research of pediatricdiseases, and lay the groundwork for child healthcare.
... Haemophilus influenzae was the first gram-negative bacterium sequenced in 1995 [15]. The DNA sequencing technique was developed by Frederick Sanger in 1977 and won the Nobel Prize [16]. Sequencing techniques have revolutionized and facilitated molecular biology by sequencing modest amounts of DNA in a single reaction. ...
Microbial forensics is a new discipline of science that analyzes evidence related to biological crime through the uniqueness and abundance of microorganisms and their toxins. Microorganisms remain alive longer than any other trace of biological evidence, such as DNA, fingerprints, and fibers, because of the protective cell membrane or capsules. Microbiological research has opened up various possibilities for forensic investigations of microbial flora. Current molecular technologies, including DNA sequencing, whole-genome sequencing, metagenomics, DNA fingerprinting, and molecular phylogeny, provide valid results for forensic investigations. Recent advancements in genome sequencing technologies, genetic data generation, and bioinformatic tools have significantly improved microbial sampling methods and forensic analyses. In this review, we discuss the applications of microbial genomic tools and technologies in forensic investigations, including human identification, geolocation, and causes of death.
... Cada producto de la PCR se purificó con la enzima Exonucleasa I (Thermo Scientific, US). La secuenciación la realizó la empresa Macrogen Inc. (Korea del Sur) en productos de PCR a una concentración de 50 ng µL -1 (Sanger, 1977). Se utilizó BioEdit Sequence Alignment Editor Versión 7.0.5.3 (Hall, 1999) para alinear y editar las secuencias. ...
Introducción. La identificación y detección de microorganismos a partir de técnicas moleculares se ha convertido en un insumo de gran ayuda para el diagnóstico de enfermedades, y de microorganismos presentes en los cultivos. Organismos patogénicos, no patogénicos, controladores biológicos y microorganismos utilizados como competidores, antagonistas o mutualistas pueden aislarse de cultivos agrícolas, ornamentales y forestales. Objetivo. Identificar a nivel taxonómico mediante técnicas moleculares, bacterias y levaduras patogénicas y no patogénicas en cultivos agrícolas, ornamentales y forestales de Costa Rica, y conservar el material en un banco de muestras de ADN. Materiales y métodos. Entre los años 2009 y 2018 el Laboratorio de Técnicas Moleculares del Centro de Investigación en Protección de Cultivos de la Universidad de Costa Rica recibió un total de 181 aislamientos de bacterias y levaduras para detección por medio de PCR tiempo final y tiempo real; e identificación por medio de la secuenciación de regiones específicas. Resultados. Del total de muestras el 94,2 % se analizó por medio de secuenciación y el 5,8 % por PCR. Por medio de detección por PCR en el cultivo de arroz se identificaron bacterias como Burkholderia spp., Acidovorax avenae y Pseudomonas fuscovaginae. Por medio de la secuenciación de la región parcial de 16S se identificaron 172 muestras de especies de bacterias, y cinco muestras de especies de levaduras con la región ITS del ARN ribosomal 18S. Se identificaron microorganismos aislados a partir de dieciocho especies de plantas agrícolas, ornamentales y forestales; entre estos Pseudomonas, Bacillus y Enterobacter y en el caso de levaduras Candida, Pichia y Wickerthamomyces. Conclusión. Esta investigación permitió identificar a nivel taxonómico bacterias y levaduras provenientes de cultivos de Costa Rica. Además, se desarrolla un insumo de consulta y la posibilidad de utilizar a futuro los microorganismos que se encuentran conservados dentro de un banco de muestras de ADN.
... Sequence alignment was performed using MUSCLE alignment software v. 3.8.31, aligning with the contemporary practice of utilizing advanced bioinformatics tools for precise sequence alignment [28]. The construction of the phylogenetic tree employed the Maximum Likelihood Algorithm, a methodological choice influenced by recent advancements in phylogenetic analysis and its preference for accurate tree reconstruction [29]. ...
The genus Trichoderma holds economic significance due to its widespread distribution and diverse applications, including biological control, enzyme production, and various biotechnological uses. The accurate identification of Trichoderma species is crucial given their close association with human activities. Despite previous efforts in classification, a comprehensive analysis combining morphological and molecular approaches is necessary. This study focuses on the isolation of four Trichoderma species from industrial wastewater in Pakistan, expanding on the known diversity in the region; isolation involved collecting samples from industrial wastewater effluents at specific sites in Punjab, Pakistan. Trichoderma strains were cultured and purified on solid media, with subsequent biomass production for bisorptional activity. Morphological characterization included colony features and microscopic examinations. DNA extraction, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer (ITS) region were conducted for molecular analysis. Phylogenetic analysis was performed using the Maximum Likelihood Algorithm. The study identified three Trichoderma species, viz. T. citrinoviride, T. erinaceum, and T. longibrachiatum. Each species was characterized morphologically and supported by molecular–phylogenetic analysis. Illustrations of microscopic features and a phylogenetic tree based on the ITS-nrDNA region were recorded. T. citrinoviride and T. longibrachiatum, isolated from steel mill and tanneries wastewater, respectively, were differentiated based on morphological characteristics such as phialides and conidia. The combination of morphological and molecular techniques enhances the accuracy of species identification. The study highlights the significance of Trichoderma in industrial wastewater environments and underscores the need for continued research in this area. Future research should focus on exploring the ecological roles and potential applications of the newly identified Trichoderma species. Additionally, further investigations into the biotechnological potential of these species, including enzyme production and bioremediation capabilities, would contribute to their practical applications.
... One sample from each facility and the positive control were purified with ExoSAP-I (Thermo Scientific, USA), and sequencing was performed using the Sanger method through chain termination by dideoxynucleotides. 53 The sequencing result was compared by the Basic Local Alignment Search Tool (BLAST w ; https://blast.ncbi.nlm.nih.gov) with matrix protein gene sequences available on GenBank. For phylogenetic analysis, the study sequences were compared with sequences from Brazil and other countries previously deposited in GenBank, belonging to Parrot bornavirus 2 and 4. The sequences were aligned with matrix protein gene sequences available on GenBank by Clus-talW in BioEdit 7.2 software w . ...
... To isolate DNA, a method using cetyltriethylammonium bromide was used [59]. DNA sequencing will be performed using the Sanger method using the BigDye™ Terminator v3.1 Cycle Sequencing Kit [60]. The obtained data on the primary structure of the DNA fragments under study will be analyzed through the NCBI genetic information database using the BLAST (Basic Local Alignment Search Tool) program [61]. ...
This article studies the morphological parameters of vegetative and generative organs of different age groups of Crataegus ambigua from four populations in Western Karatau (Mangistau region, Kazakhstan). The study examined four populations Pop 1 Sultan Epe, Pop 2 Karakozaiym, Pop 3 Emdikorgan, and Pop 4 Samal located in various gorges of western Karatau. Some phylogenetic inference methods were applied using six genetic markers atpF-atpH, ITS, matK, psbK-psbI, rbcL, and trnH-psbA. We also used a statistical analysis of the vegetative and generative organs of plants for three age groups of plants (virgin, young generative, and adult generative). According to the age structure, Samal populations have a high concentration of young generative plants - 42.3% and adult generative plants - 30.9%. Morphological analysis showed the significance of the parameters of the generative organs and separated the Samal population into a separate group according to the main PCoA coordinates. The results of the floristic analysis showed that the populations in the Samal have a high concentration of species diversity. The comparative dendrogram of UPGMA shows the informativeness of the use of genetic markers and the psbK-psbI region can be used to judge the difference between the fourth Samal population and the other three.
... The landscape of DNA sequencing technology and its uses has significantly changed over the past three decades, igniting the genomic age, which is characterized by an abundance of genetic data. The invention of the Sanger technique, also known as the chain termination method, as described by Sanger et al. (1977), marked a turning point. The Sanger technique served as a base upon which other advancements were built, despite its early throughput and accuracy limitations. ...
Biotechnology is one of the emerging fields that can add new and better application in a wide range of sectors like health care, service sector, agriculture, and processing industry to name some. This book will provide an excellent opportunity to focus on recent developments in the frontier areas of Biotechnology and establish new collaborations in these areas. The book will highlight multidisciplinary perspectives to interested biotechnologists, microbiologists, pharmaceutical experts, bioprocess engineers, agronomists, medical professionals, sustainability researchers and academicians. This technical publication will provide a platform for potential knowledge exhibition on recent trends, theories and practices in the field of Biotechnology. Aim of the research articles are invited in the following areas of interest.
... In 1977, a new method for DNA sequencing, commonly called Sanger sequencing method, was reported and has been widely used until now [1]. Although the Sanger sequencing method exhibited a high accuracy (> 99.99%) and could produce long reads (> 600 bp), the demand for large amounts of data for genomic research triggered the emergence of new sequencing methods. ...
DNA sequencing is the most critical technique to explore and study genomic data of human beings and surrounding environments. The history of DNA sequencing has rapidly grown since the first-generation DNA sequencing in 1977. Until now, emerging high-throughput sequencing technology has recently revolutionized omics areas, including genomics, transcriptomics, metagenomics, and metabolomics for the development of modern molecular biology. Besides the short-read sequencing method (Illumina platforms), the long-read sequencing technologies (including Pacific Biosciences and Oxford Nanopore Technologies) have been developed with the advantages of larger read sizes, portable devices, and diverse preparation kits. Consequently, the number of biological databases and study-related nanopore sequencing technologies has grown fast. This review paper summarized the principle and fundament of nanopore sequencing regarding their advanced applications in pathogenic diagnostics of different hosts. The most recent application of nanopore sequencing in the diagnostics of infectious diseases is highlighted and discussed to provide insights into the novelty of nanopore sequencing technology and its future perspectives.
... The buffers were prepared according to existing guidelines [57]. To isolate DNA, a method using cetyltrimethylammonium bromide was used [58]. DNA sequencing was performed using the Sanger method using a BigDye™ Terminator v3.1 Cycle Sequencing Kit [59]. ...
This article studies the morphological parameters of vegetative and generative organs of different age groups of Crataegus ambigua from four populations in Western Karatau (Mangistau region, Kazakhstan). In this study, we examined four populations: Sultan Epe, Karakozaiym, Emdi-korgan, and Samal, all located in various gorges of Western Karatau. Several phylogenetic inference methods were applied, using six genetic markers to reconstruct the evolutionary relationships between these populations: atpF-atpH, internal transcribed spacer (ITS), matK, psbK-psbI, rbcL, and trnH-psbA. We also used a statistical analysis of plants' vegetative and generative organs for three age groups (virgin, young, and adult generative). According to the age structure, Samal has a high concentration of young generative plants (42.3%) and adult generative plants (30.9%). Morphological analysis showed the significance of the parameters of the generative organs and separated the Samal population into a separate group according to the primary principal component analysis (PCoA) coordinates. The results of the floristic analysis showed that the Samal populations have a high concentration of species diversity. Comparative dendrograms using UPGMA (unweighted pair group method with arithmetic mean) showed that information gleaned from genetic markers and the psbK-psbI region can be used to determine the difference between the fourth Samal population and the other three.
... A final extension step was conducted at 72 • C for 10 min. Amplicons were purified with a QIAquick PCR purification kit (Qiagen) according to the manufacturer's instructions and sequenced via Sanger sequencing [76] at the Molecular Biology Service Unit (MBSU) of the University of Alberta, Edmonton, Canada. ...
Root rot disease poses a significant threat to canola (Brassica napus), underscoring the need for a comprehensive understanding of its causal agents for more effective disease mitigation. The composition and diversity of fungal pathogens associated with root rot of canola in Alberta, Canada, were evaluated from plant tissue samples collected in 2021 and 2022. The study revealed Fusarium spp. as the predominant pathogens found in almost all surveyed fields. Fusarium avenaceum, F. redolens, and F. solani were among the most frequently recovered species. Greenhouse trials confirmed their pathogenicity, with F. avenaceum and F. sporotrichioides found to be particularly aggressive. Additionally, F. sporotrichioides and F. commune were identified for the first time as canola root rot pathogens. Inoculation with isolates of most species resulted in significant reductions in seedling emergence, plant height, and shoot and root dry weights. Analysis of translation elongation factor 1-α (TEF-1α) and internal transcribed spacer (ITS) sequences confirmed the identity of the Fusarium spp., while concatenating the ITS and TEF-1α sequences enabled improved species differentiation. Geographic and year effects did not influence fungal diversity or aggressiveness, as determined by principal component analysis. This study emphasized the high diversity and impact of Fusarium spp. in causing canola root rot.
... In cases where instructors are not familiar with Sanger dideoxy chain termination nucleotide sequencing, it may be advisable to review the method in order to better explain the introductory figure in the introductory slides (Supporting Files S3, S4). Historical and review information may be found in Sanger et al. (6) and Shendure et al. (14). ...
... DNA products were subjected to Polymerase chain reaction (PCR) and agarose gel electrophoresis Followed by Purification of DNA fragments from the gel. DNA fragments were then subjected for DNA sequencing according to Sanger et al. (1977) and Tabor & Richardson (1995). Sample analyses were made by OpenGene software Version 3.1 from Visible Genetics, Canada at The Regional Center for Mycology and Biotechnology. ...
... This technique involves incorporating chain-terminating dideoxynucleotides during DNA replication, resulting in fragments of varying lengths that can be separated by capillary electrophoresis and read to determine the DNA sequence. Despite being labor-intensive and time-consuming, Sanger sequencing is highly accurate and remains a gold standard for smaller-scale sequencing projects and validation of nextgeneration sequencing (NGS) results [1,2]. ...
Using Sri Lanka as a case study, the paper explores how DNA analysis has transformed genetic research, particularly in low and middle-income countries (LMICs). It discusses various DNA analyzing techniques, from traditional methods like Sanger sequencing to advanced techniques such as next-generation sequencing (NGS) and CRISPR-Cas9, highlighting their applications in disease research, population genetics, and forensic science. Sri Lanka's advancements in genetic research, including DNA sequencing, typing, and recent developments in X-chromosome-based DNA typing, are emphasized. The paper also examines challenges and opportunities in LMICs regarding genetic research and underscores the importance of DNA analysis in advancing personalized medicine and understanding genetic diversity. Additionally, it discusses Sri Lanka's efforts in education and training in molecular biology. It explores the country's rich genetic diversity 55 and demographic history, focusing on ethnic studies and historical interactions among different population groups. Overall, the paper highlights the significance of DNA analysis in genetic research and its potential implications for LMICs. Sri Lanka is a notable example of progress in the field.
... Amplicon sequencing was performed by the Sanger method (Sanger et al., 1977) using the ABI PRISM 3730 DNA Analyzer (Applied Biosystems) at the Human Genome and Stem Cell Research Center, Institute of Biosciences, University of São Paulo (USP), São Paulo, SP, Brazil. The electropherograms were subjected to a quality screening test using the Phred-Phrap software (v. ...
Although bats (Mammalia: Chiroptera) act as natural reservoirs for many zoonotic pathogens around the world, few studies have investigated the occurrence of Anaplasmataceae agents in bats, especially vampire bats. The family Anaplasmataceae (order Rickettsiales) encompasses obligate intracellular bacteria of the genera Anaplasma, Ehrlichia, Neorickettsia, Neoehrlichia, Wolbachia, and Allocryptoplasma. The present study aimed to investigate, using molecular techniques, the presence of species of Anaplasma, Ehrlichia, and Neorickettsia in vampire bats sampled in northern Brazil. Between 2017 and 2019, spleen samples were collected from vampire bats belonging to two species, Desmodus rotundus (n = 228) from the states of Pará (n = 207), Amazonas (n = 1), Roraima (n = 18) and Amapá (n = 3), and Diaemus youngii (n = 1) from Pará. Positivity rates of 5.2% (12/229), 3% (7/229), and 10.9% (25/229) were found in PCR assays for Anaplasma spp. (16S rRNA gene), Ehrlichia spp. (dsb gene) and Neorickettsia spp. (16S rRNA gene), respectively. The present study revealed, for the first time, the occurrence of Anaplasma spp. and different genotypes of Ehrlichia spp. in vampire bats from Brazil. While phylogenetic analyses based on the dsb and ftsZ genes of Ehrlichia and 16S rRNA of Anaplasma spp. revealed phylogenetic proximity of the genotypes detected in vampire bats with Anaplasmataceae agents associated with domestic ruminants, phylogenetic inferences based on the gltA and groEL genes evidenced the occurrence of genotypes apparently exclusive to bats. Neorickettsia sp. phylogenetically associated with N. risticii was also detected in vampire bats sampled in northern Brazil.
... Sanger's idea was to sequence the DNA strand by chain termination. Consequently, in this case, the DNA fragments were converted into chains by DNA polymerases and by the incorporation of nucleotides [3]. Maxam and Gilbert provided a process, during which the sequences of DNA fragments were determined using the combination of radiolabeling, chemical cleaving, and gel electrophoresis of nucleotides, and autoradiography served as the detection method [4]. ...
The large-scale heterogeneity of genetic diseases necessitated the deeper examination of nucleotide sequence alterations enhancing the discovery of new targeted drug attack points. The appearance of new sequencing techniques was essential to get more interpretable genomic data. In contrast to the previous short-reads, longer lengths can provide a better insight into the potential health threatening genetic abnormalities. Long-reads offer more accurate variant identification and genome assembly methods, indicating advances in nucleotide deflect-related studies. In this review, we introduce the historical background of sequencing technologies and show their benefits and limits, as well. Furthermore, we highlight the differences between short- and long-read approaches, including their unique advances and difficulties in methodologies and evaluation. Additionally, we provide a detailed description of the corresponding bioinformatics and the current applications.
... Seven Bartonella-positive amplicons of the expected size for each assay were purified using the Promega Wizard® PCR and Gel Clean-Up (Promega®) and sequenced in both directions using the same PCR primers (forward and reverse) by Sanger sequencing (Sanger et al., 1977). Partial nucleotide sequences obtained herein of the ITS (accession nos. ...
Background: Bartonellosis, caused by bacteria of the genus Bartonella, is a zoonotic
disease with several mammalian reservoir hosts. In Somalia, a country heavily reli-
ant on livestock, zoonotic diseases pose significant public health and economic chal-
lenges. To the best of our knowledge, no study has been performed aiming to verify
the occurrence of Bartonella spp. in Somalia. This study investigated the occurrence
and molecular characterization of Bartonella in dromedary (Camelus dromedarius,
Linnaeus, 1758), cattle, sheep, and goats from Somalia.
Materials and Methods: 530 blood samples were collected from various animals
(155 dromedary, 199 goat, 131 cattle, and 45 sheep) in Benadir and Lower Shabelle
regions. DNA was extracted for molecular analysis, and a qPCR assay targeting the
NADH dehydrogenase gamma subunit (nuoG) gene was used for Bartonella screening.
Positive samples were also subjected to PCR assays targeting seven molecular mark-
ers including: nuoG, citrate synthase gene (gltA), RNA polymerase beta-subunit gene
(rpoB), riboflavin synthase gene (ribC), 60 kDa heat-shock protein gene (groEL), cell
division protein gene (ftsZ), and pap31 and qPCR targeting the 16-23S rRNA internal
transcribed spacer (ITS) followed by Sanger sequencing, BLASTn and phylogenetic
analysis.
Results: Out of 530 tested animals, 5.1% were positive for Bartonella spp. by the
nuoG qPCR assay. Goats showed the highest Bartonella occurrence (17/199, 8.5%),
followed by sheep (6/44, 6.8%), cattle (4/131, 3.1%), and dromedary (1/155, 1.9%).
Goats, sheep, and cattle had higher odds of infection compared to dromedary.
Among nuoG qPCR- positive samples, 11.1%, 14.8%, 11.1%, and 25.9% were posi -
tive in PCR assays based on nuoG, gltA, and pap31 genes, and in the qPCR based on
the ITS region, respectively. On the other hand, nuoG qPCR- positive samples were
negative in the PCR assays targeting the ribC, rpoB, ftsZ, and groEL genes. While
Bartonella bovis sequences were detected in cattle ( nuoG and ITS) and goats ( gltA), Bartonella henselae ITS sequences were detected in dromedary, goat, and sheep.
Phylogenetic analysis placed gltA Bartonella sequence from a goat in the same clade
of B. bovis.
Conclusion: The present study showed, for the first time, molecular evidence of
Bartonella spp. in dromedary and ruminants from Somalia and B. henselae in sheep and
goats globally. These findings contribute valuable insights into Bartonella spp. occur-
rence in Somali livestock, highlighting the need for comprehensive surveillance and
control measures under the One Health approach.
... The final extension was carried out at 72 °C for 7 min. The Sanger sequencing method was used to sequence PCR products [34]. The obtained sequences were analyzed through the Basic Local Alignment Search Tool (BLAST) for molecular identification, and further, the sequence data was processed for multiple sequence alignment using ClustalW tool. ...
The present study was executed to evaluate the impact of mixed microbial culture and modified electrodes on the operational capability of microbial fuel cells (MFCs) for the treatment of leather tannnery wastewater with simultaneous bioelctricity generation. The impact of nutrient addition to support microbial growth and catholyte buffer strength was also evaluated. Studies were conducted in dual-chamber MFC fed with leather tannery wastewater. The anode materials used were bare carbon felt/nickel foam (CF/NF) and polyaniline (PANI)-modified carbon felt (PANI@CF) and nickel foam (PANI@NF). The PANI@CF anode in MFC with mixed microbial culture along with nutrients addition in anolyte and catholyte buffer strength (500 mM) resulted in power density 234.67 mW/m², while in the case of PANI@NF, the recorded power density was 373.01 mW/m². Wastewater treatment efficiency of MFCs was assessed based on the %COD removal. The %COD removal in MFC operations using PANI@CF and PANI@NF anode was depicted to be 81% and 84%, respectively, which were considerably higher than those equipped with CF and NF anodes. The major bacteria identified in mixed microbial culture were closely related to Bacillus haikouensis, Rhizobium daejeonense, Pseudomonas aeruginosa, Bacillus cereus, and Cytobacillus oceanisediminis. Conslusively, the modification of MFC anode with PANI, use of mixed microbial culture along with nutrient addition and catholyte buffer strength (500 mM) significantly improved MFC performance regarding leather tannery wastewater treatment with simultaneous power generation. By improving MFC performance in real-world wastewater treatment systems, particularly those with mixed microbial communities, this study bids to contribute substantially to sustainable industrial wastewater management practices.
The bacterial mannose phosphotransferase system (Man-PTS) mediates uptake of selected monosaccharides. Simultaneously, it is a receptor for diverse bacteriocins such as subclass IIa pediocin-like bacteriocins and some subclass IId ones (garvicins ABCQ, lactococcins ABZ, BacSJ, ubericin K, and angicin). So far, no attempt has been made to categorize this ever-expanding group of bacteriocins. Here, we identified Man-PTS as a receptor for a number of novel bacteriocins and demonstrated that they all belong to a large family of Man-PTS-binding non-pediocin-like peptides. Based on amino acid sequence similarities between members of this family, we propose their classification into five groups. This classification conveniently distinguishes bacteriocins with specific structures and properties regarding their spectrum of antimicrobial activity and pattern of interaction with Man-PTS. With respect to the latter, we indicate individual amino acid residues or regions of Man-PTS and the bacteriocin responsible for their interaction. In Man-PTS these residues localize to the exterior of the transport complex, specifically the extracellular loop of the so-called Vmotif domain containing regions γ and/or γ+, and to the interior of the transport complex, specifically the interface between the Core and Vmotif domains. Finally, we propose that while the bacteriocins from separate groups display specific binding patterns to Man-PTS, the general mechanism of their interaction with the receptor is universal despite significant differences in their predicted structures, i.e., after initial docking on the bacterial cell through an interaction with the Man-PTS regions γ and/or γ+, they pull away its Core and Vmotif from one another to form a pore across the membrane.
In the dynamic field of DNA sequencing, continuous advancements in science and technology drive progress. Notably, recent developments have focused on the refinement of techniques and the introduction of innovative tools to enhance sequencing capabilities. For instance, Michael's work in 2005 showcased the integration of microfluidic separation platforms into Sanger sequencing methods, marking a significant milestone in the field. Furthermore, Jay A.'s research in 2008 contributed to the optimization of dideoxy sequencing protocols, further improving the efficiency and accuracy of DNA sequencing processes. Additionally, the pioneering work of Sanger and subsequent reports by Clyde A. Hutchinson in 2007 introduced novel techniques such as plus and minus sequencing, expanding the repertoire of available sequencing methods. Moreover, advancements have been made in the sequencing of methylated DNA and the investigation of DNA and RNA protein interactions, as evidenced by George M. Church's seminal work in 1988. This review endeavors to synthesize and present a comprehensive overview of these cutting-edge techniques and their implications for DNA sequencing methodologies.
The recent rise of ‘omics and other molecular research technologies alongside improved techniques for tissue preservation have broadened the scope of marine mammal research. Collecting biological samples from wild marine mammals is both logistically challenging and expensive. To enhance the power of marine mammal research, great effort has been made in both the field and the laboratory to ensure the scientific integrity of samples from collection through processing, supporting the long‐term use of precious samples across a broad range of studies. However, identifying the best methods of sample preservation can be challenging, especially as this technological toolkit continues to evolve and expand. Standardizing best practices could maximize the scientific value of biological samples, foster multi‐institutional collaborative efforts across fields, and improve the quality of individual studies by removing potential sources of error from the collection, handling, and preservation processes. With these aims in mind, we summarize relevant literature, share current expert knowledge, and suggest best practices for sample collection and preservation. This manuscript is intended as a reference resource for scientists interested in exploring collaborative studies and preserving samples in a suitable manner for a broad spectrum of analyses, emphasizing support for ‘omics technologies.
The microbiome plays a key role in the health of all metazoans. Whether and how the microbiome favors the adaptation processes of organisms to extreme conditions, such as those of Antarctica, which are incompatible with most metazoans, is still unknown. We investigated the microbiome of three endemic and widespread species of Antarctic polychaetes: Leitoscoloplos geminus , Aphelochaeta palmeri , and Aglaophamus trissophyllus . We report here that these invertebrates contain a stable bacterial core dominated by Meiothermus and Anoxybacillus , equipped with a versatile genetic makeup and a unique portfolio of proteins useful for coping with extremely cold conditions as revealed by pangenomic and metaproteomic analyses. The close phylosymbiosis between Meiothermus and Anoxybacillus and these Antarctic polychaetes indicates a connection with their hosts that started in the past to support holobiont adaptation to the Antarctic Ocean. The wide suite of bacterial cryoprotective proteins found in Antarctic polychaetes may be useful for the development of nature-based biotechnological applications.
Accurate and timely diagnosis of oral squamous cell carcinoma (OSCC) is crucial in preventing its progression to advanced stages with a poor prognosis. As such, the construction of sensors capable of detecting previously established disease biomarkers for the early and non-invasive diagnosis of this and many other conditions has enormous therapeutic potential. In this work, we apply synthetic biology techniques for the development of a whole-cell biosensor (WCB) that leverages the physiology of engineered bacteria in vivo to promote the expression of an observable effector upon detection of a soluble molecule. To this end, we have constructed a bacterial strain expressing a novel chimeric transcription factor (Sphnx) for the detection of N-acetylneuraminic acid (Neu5Ac), a salivary biomolecule correlated with the onset of OSCC. This WCB serves as the proof-of-concept of a platform that can eventually be applied to clinical screening panels for a multitude of oral and systemic medical conditions whose biomarkers are present in saliva.
Over the last few decades, the study of congenital heart disease (CHD) has benefited from various model systems and the development of molecular biological techniques enabling the analysis of single gene as well as global effects. In this chapter, we first describe different models including CHD patients and their families, animal models ranging from invertebrates to mammals, and various cell culture systems. Moreover, techniques to experimentally manipulate these models are discussed. Second, we introduce cardiac phenotyping technologies comprising the analysis of mouse and cell culture models, live imaging of cardiogenesis, and histological methods for fixed hearts. Finally, the most important and latest molecular biotechniques are described. These include genotyping technologies, different applications of next-generation sequencing, and the analysis of transcriptome, epigenome, proteome, and metabolome. In summary, the models and technologies presented in this chapter are essential to study the function and development of the heart and to understand the molecular pathways underlying CHD.
The current investigation was conducted to assess the antagonistic activity of Lacticaseibacillus paracasei M12, a novel strain, against the pathogenic agent Erwinia amylovora E22, which causes fire blight in fruit crops. The E. amylovora E22 strain was isolated from Golden Delicious floral colonization dynamics and specificity of Aureobasidium pullulans strains used to suppress fire blight in apple fruit. The L. paracasei M12 strain was collected from the phyllosphere of the garden cenosis in the Almaty region of Kazakhstan. Identification of both isolates relied on morphological and biochemical tests as well as PCR analysis. Results indicated that the zone of pathogen growth inhibition due to the action of the L. paracasei M12 strain was 41.5 ± 1.5 mm. The maximum inhibitory activity of this antagonist was observed on the 7th day of fermentation. Lactic acid constituted the largest percentage in the cultural broth component composition (43.55%) and exhibited inhibitory effects against E. amylovora E22. Evaluation of various concentrations of the broth in vitro demonstrated that the use of the original broth (100%) resulted in complete inhibition of pathogen growth. A parallel outcome was observed in tests conducted on immature pear fruits, where those treated with 100% of M12 culture broth exhibited complete protection against fire blight infection, while 50% and 20% concentrations showed a slight appearance of the disease. Furthermore, the incorporation of TWEEN® 80 as an adhesive agent enhanced the antagonistic activity of the L. paracasei M12 strain in vitro. Consequently, the L. paracasei strain M12 emerges as a promising candidate for the development of a novel biopesticide targeting fire blight. It was shown that the biological efficacy of L. paracasei M12 against fire blight during double treatment of Aport and Starkrimson fruit trees was 80.3% and 80.7%, respectively.
Ulva is an unutilized green seaweed that grows in the intertidal zone around the coast of Java Island, Indonesia. In this study, U. lactuca samples collected from three regions in south of Java Island (Cihara, Surade, and Tepus) were studied in terms of their chemical composition, physical properties and its bioactivity, to determine the best regions for establishing seaweed industries. The chemical characteristics differed significantly among different regions, where the seaweed from Tepus, Surade and Cihara had the highest content of protein (22.93%), carbohydrate (61.58%), and mineral (28.72%), respectively. The amino acids were dominated with L-aspartic acid and L-glutamic acid. All U. lactuca samples contained abundant pigments, such as chlorophylls and carotenoids, especially samples from Tepus. The highest content of crude ulvan was found in Surade seaweed (26.9%). Chemical and physical analyses showed the presence of S = O and C–O–S functional groups in ulvan, a sulfated polysaccharide unique to Ulva sp., with thermal degradation up to 220 °C. Crude ulvan from Surade and Tepus seaweed exhibited bioactivity to support proliferation of fibroblast cells at 100 and 1000 ppm, respectively. Based on the properties of U. lactuca, Tepus and Surade were identified as potential sites to establish aquaculture and/or processing industries.
The monograph "Virus, phytoplasma and bacterial diseases of the grapevine" presents the phytoplasmas, viruses and bacteria that cause diseases of the grapevine and reveals the approaches and methods for their diagnosis. Damage to the buds, leaves, shoots, roots, fronds and bunches caused the death of the plants and sometimes the destruction of entire vineyards. Maintaining the vines in good health and obtaining high and quality grape yields is impossible only by applying high agricultural techniques. It is necessary to carry out plant protection, which, in addition to protecting the vine from diseases and enemies, also observes the protection of nature from pollution with pesticides. Its implementation is possible only with a good knowledge of viral, bacterial and phytoplasma diseases and their vectors, as well as modern directions and future strategy in plant protection. Tactics in the fight against enemies should be aimed at regulating them below a certain level, since their complete destruction is practically impossible. This approach makes it possible for beneficial species of insects and mites to multiply in vineyards, which, together with climatic factors, are often able to keep enemies below the thresholds of economic harm without human intervention.
The measures against grapevine diseases is extremely prophylactic. For the implementation of the new diagnostic methods in plant protection, it is necessary that the vine plantations are under constant surveillance. Knowing the signs of diseased plants is an important condition for performing differential diagnostics, and the new methods for detecting and confirming viral, bacterial and phytoplasma diseases for improving the phytosanitary condition of the grapevine.
The monographic work is based on the author's theoretical statements and empirical research in wine-growing regions of Bulgaria and on vine planting material from wine-growing regions in France, Italy, Serbia, Germany and Austria.
The monograph "Virus, Phytoplasma and Bacterial Diseases of the Grapevine" is aimed at specialists in the study of grapevine infections, grape growers, students and a wide range of readers who are interested in the cultivation of grapevines.
Background
The Middle East and North Africa (MENA) offer optimal climatic conditions for tick reproduction and dispersal. Research on tick-borne pathogens in this region is scarce. Despite recent advances in the characterization and taxonomic explanation of various tick-borne illnesses affecting animals in Egypt, no comprehensive examination of TBP (tick-borne pathogen) statuses has been performed. Therefore, the present study aims to detect the prevalence of pathogens harbored by ticks in Egypt.
Methodology/Principal findings
A four-year PCR-based study was conducted to detect a wide range of tick-borne pathogens (TBPs) harbored by three economically important tick species in Egypt. Approximately 86.7% (902/1,040) of the investigated Hyalomma dromedarii ticks from camels were found positive with Candidatus Anaplasma camelii (18.8%), Ehrlichia ruminantium (16.5%), Rickettsia africae (12.6%), Theileria annulata (11.9%), Mycoplasma arginini (9.9%), Borrelia burgdorferi (7.7%), Spiroplasma-like endosymbiont (4.0%), Hepatozoon canis (2.4%), Coxiella burnetii (1.6%) and Leishmania infantum (1.3%). Double co-infections were recorded in 3.0% (27/902) of Hy. dromedarii ticks, triple co-infections (simultaneous infection of the tick by three pathogen species) were found in 9.6% (87/902) of Hy. dromedarii ticks, whereas multiple co-infections (simultaneous infection of the tick by ≥ four pathogen species) comprised 12% (108/902). Out of 1,435 investigated Rhipicephalus rutilus ticks collected from dogs and sheep, 816 (56.9%) ticks harbored Babesia canis vogeli (17.1%), Rickettsia conorii (16.2%), Ehrlichia canis (15.4%), H. canis (13.6%), Bo. burgdorferi (9.7%), L. infantum (8.4%), C. burnetii (7.3%) and Trypanosoma evansi (6.6%) in dogs, and 242 (16.9%) ticks harbored Theileria lestoquardi (21.6%), Theileria ovis (20.0%) and Eh. ruminantium (0.3%) in sheep. Double, triple, and multiple co-infections represented 11% (90/816), 7.6% (62/816), and 10.3% (84/816), respectively in Rh. rutilus from dogs, whereas double and triple co-infections represented 30.2% (73/242) and 2.1% (5/242), respectively in Rh. rutilus from sheep. Approximately 92.5% (1,355/1,465) of Rhipicephalus annulatus ticks of cattle carried a burden of Anaplasma marginale (21.3%), Babesia bigemina (18.2%), Babesia bovis (14.0%), Borrelia theleri (12.8%), R. africae (12.4%), Th. annulata (8.7%), Bo. burgdorferi (2.7%), and Eh. ruminantium (2.5%). Double, triple, and multiple co-infections represented 1.8% (25/1,355), 11.5% (156/1,355), and 12.9% (175/1,355), respectively. The detected pathogens’ sequences had 98.76–100% similarity to the available database with genetic divergence ranged between 0.0001 to 0.0009% to closest sequences from other African, Asian, and European countries. Phylogenetic analysis revealed close similarities between the detected pathogens and other isolates mostly from African and Asian countries.
Conclusions/Significance
Continuous PCR-detection of pathogens transmitted by ticks is necessary to overcome the consequences of these infection to the hosts. More restrictions should be applied from the Egyptian authorities on animal importations to limit the emergence and re-emergence of tick-borne pathogens in the country. This is the first in-depth investigation of TBPs in Egypt.
Historically, the most important source of both antibiotics and anticancer medications has been microorganisms. Bacillus thuringiensis (Bt) is one of the most prominent bacterial species used as a therapeutic agent targeting cancerous cells in recent worldwide investigations. This study was designed to isolate, molecularly identify, and discover novel Saudi Arabian Bt strains that selectively exhibit cytotoxic properties against MDA-MB-231, a human triple-negative breast cancer (TNBC) cell model. The bacterial strain under investigation was biochemically typed using API 20E and API CH50 and molecularly typed using 16S rDNA sequencing. Flow cytometry and immunoblotting were performed to elucidate the mechanism-of-action (MOA). Molecular typing confirmed the identity of the isolated non-hemolytic strain to be Bt and was named Bt HAU-145. Microscopic examination showed that the strain possessed a parasporal (PS) crystal protein with a spherical morphology. Data of cytotoxicity assay based on MTT revealed that Bt HAU-145 strain exhibited selective and potent cytotoxicity against MDA-MB-231, with a 50 percent inhibition (IC50) of 28 µg/ml. FACS analysis revealed that PS proteins induced both late and early apoptosis in a ROS-dependent manner. Immunoblotting assays showed increased expression of caspase-3 in response to PS treatment, paralleled by a reduction in Bcl-2 expression. This is the first study to investigate the MOA of PS proteins from the Saudi Arabian Bt strain, showing an induction of apoptosis through a ROS-dependent mechanism in TNBC cells. It is hoped that PS-based therapeutic strategies will be investigated at the preclinical scale in non-human primates prior to the clinical scale in randomized clinical trials.
This chapter overviews genetic techniques’ fundamentals and methodological features, including different approaches, analyses, and applications that have contributed to advancing health and disease. The aim is to describe laboratory methodologies and analyses employed to understand the genetic landscape of different biological contexts, from conventional techniques to cutting-edge technologies. Besides describing detailed aspects of the polymerase chain reaction (PCR) and derived types as one of the principles for many novel techniques, we also discuss microarray analysis, next-generation sequencing, and genome editing technologies such as transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems. These techniques study several phenotypes, ranging from autoimmune disorders to viral diseases. The significance of integrating diverse genetic methodologies and tools to understand host genetics comprehensively and addressing the ethical, legal, and social implications (ELSI) associated with using genetic information is highlighted. Overall, the methods, procedures, and applications in host genetic analysis provided in this chapter furnish researchers and practitioners with a roadmap for navigating the dynamic landscape of host-genome interactions.
BACKGROUND: The purpose of the work is to assess the populations of the fungi Parastagonospora nodorum and P. pseudonodorum in 2023 based on the presence of effector genes, as well as to identify alleles of Snn1/snn1, Snn3/snn3 genes that control the sensitivity or resistance of wheat to PtrTox1 and PtrTox3 toxins. MATERIALS AND METHODS: Infectious samples were collected in 2023 from spring wheat leaves. In addition, the material for the study was 2 varieties and 23 lines of spring soft wheat of local selection. Using the molecular markers Xfcp624 and Xcfd20, the presence of the Snn1 and Snn3-B1 alleles, which control sensitivity to the fungal toxins PtrTox1 and PtrTox3, was detected. RESULTS: Using molecular screening, ToxA and Tox1 genes were identified in genotypes of P. pseudonodorum isolates; Tox3 and Tox267 in P. nodorum isolates. 2 varieties of spring soft wheat and 11 hybrid lines carry a recessive allele snn1, which protects against the phytopathogen toxin PtrTox1; wheat variety Tambovchanka and 2 hybrid lines (Stb-7/15, Rl-6-22) carry the recessive allele snn3 on chromosome B1, which confers resistance to the fungal toxin PtrTox3. CONCLUSIONS: The ToxA gene was found only among monoconidial isolates of P. pseudonodorum species obtained from leaves of spring soft wheat of Lebedushka variety. As a result of molecular screening, the Tox1 gene was identified among 70 P. pseudonodorum isolates. The presence of the Tox3 and Tox267 genes was established in 30 isolates of P. nodorum species obtained from plant samples of spring durum wheat Donskaya Elegiya.The variety of Pamyati Plakhotnika and 11 hybrid lines carry one recessive allele tsn1, snn1 or snn3, protecting the plant at the genetic level from the toxins PtrToxA, PtrTox1 and PtrTox3, respectively. Variety Tambovchanka and 7 lines have protection against two toxins at once.
The effects of 1-beta-D-arabinofuranosyl CTP (ara-CTP) on DNA replication were studied in an in vitro system from polyoma-infected BALB/3T3 cells. Ara-CTP concentrations of larger than or equal to 150 muM were found to block in vitro DNA synthesis completely, and concentrations of smaller than or equal to 0.3 muM had no inhibitory effect. Intermediate concentrations resulted in a concentration-dependent reduction of the in vitro synthesis rate. Long-term labeling with [alpha-32-P]ara-CTP demonstrated the incorporation of the analogue into cellular and viral DNA concomitantly with [3-H]TTP. In pulse-labeling experiments, at noninhibitory concentrations of the analogue, ara-CTP was incorporated into short DNA fragments and long growing strands to relatively the same extent as TTP. Partial venom phosphodiesterase digestion liberated the incoporated are-CTP at essentially the same rate as incorporated TTP, excluding a predominantly terminal incorporation, and after total venom phosphodiesterase digestion greater than 80% of the incorporated ara-CTP was recovered as 5'-ara-CMP. Analysis of the long-term in vitro viral DNA product made in the presence of partially inhibiting ara-CTP concentrations demonstrated that none of the steps leading to mature viral DNA were totally inhibited at the ara-CTP concentrations used. Pulse labeling of replicating viral DNA in the presence of ara-CTP revealed two consistent differences in the pattern found in control pulses: (i) predominant labeling of short chains (5S) with reduced amounts of radioactivity in the longer growing viral DNA strands (smaller than or equal to 16S), and (ii) a one-third to one-half reduction in size for short DNA chains labeled in the presence of ara-CTP. Release of the ara-CTP inhibition with excess dCTP resulted in covalent extension of these smaller short chans to approximately the size of regular short chains labeled in the absence of the inhibitor. Isolated short chains synthesized in the presence of ara-CTP exhibited a slightly lower degree of self-complementarity than regular short chains. The predominant labeling of short chains during pulses is, therefore, not a consequence of discontinuous growth on both sides of the replication fork. Similar results were obtained with ara-ATP and N-ethylmaleimide. The experiments indicate that ara-CTP acts primarily on DNA-polymerizing activities, affecting different DNA polymerases to varying degrees. The results are discussed in terms of the possible number and identity of polymerases involved in viral (and cellular) DNA replication.
The inhibition of protein synthesis by diphtheria toxin in extracts of mammalian cells results from the inactivation of elongation factor 2 (EF-2) by covalent attachment of the adenosine diphosphate ribose (ADPR) moiety of NAD⁺.
NAD⁺ + EF-2 ⇌ ADPR-EF-2 + nicotinamide + H⁺
This reaction is catalyzed by Fragment A (mol wt 24,000) or other less common fragments generated by limited proteolysis and reduction of the toxin, but not by the toxin itself (mol wt 63,000). This report describes studies of the interaction of NAD⁺ with Fragment A.
In addition to its major enzymic activity, Fragment A also catalyzes the slow hydrolysis of the nicotinamide-ribose linkage of NAD⁺.
NAD⁺ + H2O ⇌ ADPR + nicotinamide + H⁺
This activity (NAD⁺-glycohydrolase; EC 3.2.2.5) is several orders of magnitude lower than the NAD⁺:EF-2-ADPR-transferase activity and probably does not contribute to the toxicity of diphtheria toxin. However, its existence implies a direct binding of NAD⁺ to Fragment A and is of interest with regard to the mechanism of transfer of ADPR to EF-2.
Binding of NAD⁺ and related compounds to Fragment A was studied by dynamic dialysis, fluorescence quenching, and spectral absorbance techniques. The fragment contains a single binding site for NAD⁺ with a Kd of about 8 µm. Binding of NAD⁺ is rapidly reversible, and no evidence of a covalent ADPR-Fragment A intermediate was found. NAD⁺ strongly quenches the intrinsic fluorescence of the fragment (emission maximum 333 nm) and induces a broad peak of absorbance at 360 nm (ε ∼ 500). Both effects are interpreted to result from formation of a charge transfer complex between the nicotinamide moiety of NAD⁺ and at least one of the tryptophan residues of the fragment. Binding of NAD⁺ also enhances the resistance of the fragment to trypsin or chymotrypsin. No significant change in either the sedimentation coefficient (2.19 S) or the rotational relaxation time was found upon addition of NAD⁺.
The affinities of various NAD⁺ analogs and partial structures for the binding site on Fragment A closely paralleled their activities as inhibitors or substrates of ADPR transfer. These results, together with the fact that Fragment A alone hydrolyzes the same linkage which is ruptured during ADPR transfer, leave little doubt that it is this binding site which is involved in transfer of ADPR to EF-2. A model of the ADP-ribosylation of EF-2 is proposed based on these and other results.
The effects of nucleotide analogues on DNA synthesis were studied in nucleotide-permeable Escherichia coli cells. 2′,3′-Dideoxyribosylthymine 5′-triphosphate inhibited replicative DNA synthesis far more strongly than endonuclease-induced DNA repair synthesis. In φX174-infected cells this analogue caused the formation of short pieces (7 S) of φX replicative form DNA in which newly synthesized DNA was found covalently linked to preformed primer. Deoxyuridine triphosphate led to even shorter pieces (3 S) of φX replicative form DNA. Nicotinamide mononucleotide, an inhibitor of E. coli DNA ligase, promoted accumulation of short (10 S) E. coli DNA pieces.
Newly synthesized 5′-triphosphate ends were not detected in these nucleotide-permeable cells during the early stages of φX replicative form replication, nor was evidence obtained that nucleoside monophosphates are immediate precursors of DNA.
The nucleotide sequence of the coding region of gene G of φX174 and the amino acid sequence of the G-coded “spike” protein of the virion have now been completed. From the 5′ A of the initiating ATG to the 3′ end of the terminator triplet, the gene consists of 528 nucleotides and codes for a protein of 175 amino acids, molecular weight 19,053.
DNA can be sequenced by a chemical procedure that breaks a terminally labeled DNA molecule partially at each repetition of a base. The lengths of the labeled fragments then identify the positions of that base. We describe reactions that cleave DNA preferentially at guanines, at adenines, at cytosines and thymines equally, and at cytosines alone. When the products of these four reactions are resolved by size, by electrophoresis on a polyacrylamide gel, the DNA sequence can be read from the pattern of radioactive bands. The technique will permit sequencing of at least 100 bases from the point of labeling.
A DNA sequence for the genome of bacteriophage phiX174 of approximately 5,375 nucleotides has been determined using the rapid and simple `plus and minus' method. The sequence identifies many of the features responsible for the production of the proteins of the nine known genes of the organism, including initiation and termination sites for the proteins and RNAs. Two pairs of genes are coded by the same region of DNA using different reading frames.
A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.
2′,3′-Dideoxyribonucleoside triphosphates are analogs of the natural 2′-deoxyribonucleotide substrates of deoxyribonucleic acid polymerase but lack the 3′-hydroxyl group required for deoxyribonucleic acid chain growth. Attachment of a dideoxynucleotide blocks deoxyribonucleic acid synthesis and inhibits related reactions (pyrophosphorolysis, pyrophosphate exchange, and hydrolysis) which occur at the primer site of deoxyribonucleic acid polymerase from Escherichia coli. Attachment of a chain-terminating dideoxythymidylate group to deoxyribonucleic acid and to oligo-and polydeoxynucleotide chains is approximately a thousand times slower than that of deoxyribothymidylate. Hydrolysis and pyrophosphate exchange are inhibited to a similar extent. The requirement for a 3′-hydroxyl group for optimal rates of these reactions is discussed in terms of a model for deoxyribonucleic acid polymerase action. In the presence of an excess of polymerase, the extent of incorporation of dideoxythymidylate residues at the 3′ terminus of poly d(A-T) and of deoxyribonucleic acid chains is proportional to polynucleotide concentration, and thus permits a determination of available primer sites.
Phosphorylation of 3′-deoxy-3′-iodothymidine gives the corresponding 3′-deoxy-3′-iodothymidine 5′-phosphate in high yield. Activation of the phosphate group can be achieved by formation of the phosphoromorpholidate under anhydrous conditions, and subsequent condensation with tributylammonium pyrophosphate in anhydrous dimethyl sulfoxide gives 3′-deoxy-3′-iodothymidine 5′-triphosphate in modest yield. The latter reaction is complicated by simultaneous dehydrohalogenation giving the related 2′,3′-unsaturated nucleoside 5′-triphosphate and by extensive intramolecular displacement of iodide ion by phosphate giving a 3′,5′-cyclic phosphate with the 2-deoxy-β-D-threo-pentofuranosyl configuration. The same spectrum of products is obtained using 3′-deoxy-3′-iodothymidine 5′-phosphoroimidazolate prepared from the parent nucleoside and triimidazolephosphine oxide. The various products are characterized by enzymatic and spectroscopic techniques, and reduction of either the iodotriphosphate or the unsaturated triphosphate with hydrogen and palladium gives 3′-deoxythymidine 5′-triphosphate, the enzymatic properties of which are discussed in the accompanying paper. Phosphorylation of 1-(2-deoxy-β-D-threo-pentofuranosyl)thymine with diphenyl phosphorochloridate gives the crystalline 5′-diphenyl phosphate ester that can be converted with base into the same 3′,5′-cyclic phosphate obtained as a by-product during preparation of the triphosphates above. A pair of 3′,5′-cyclic phosphate triesters diastereoisomeric about their phosphorus atoms are intermediates in this cyclization reaction.
An active Neurospora-like assimilatory NADPH-nitrate reductase (EC 1.6.6.2), which can be formed in vitro by incubation of extracts of nitrate-induced Neurospora crassa mutant nit-1 with extracts of (a) certain other nonallelic nitrate reductase mutants, (b) uninduced wild type, or (c) xanthine oxidizing and liver aldehyde-oxidase systems was also formed by combination of the nit-1 extract with other acid-treated enzymes known to contain molybdenum. These molybdenum enzymes included (a) nitrogenase, or its molybdenum-iron protein, from Clostridium, Azotobacter, and soybeannodule bacteroids, (b) bovine liver sulfite oxidase, (c) respiratory formate-nitrate reductase from Escherichia coli, (d) NADH-nitrate reductase from foxtail grass (Setaria faberii), and (e) FADH(2)- and reduced methyl viologennitrate reductase preparations from certain Neurospora mutants. Several molybdenum-amino-acid complexes, as possible catalytic models of nitrogenase, were inactive (as were some previously tested 20 nonmolybdenum enzymes) in place of the acid-treated molybdenum-containing enzymes. The results imply the existence of a molybdenum-containing component shared by the known molybdenum-enzymes.
The icosanucleotide having the sequence, d-G-C-T-C-C-C-T-T-A-G-C-A-T-G-G-G-A-G-A-G- has been chemically synthesized. This and the icosanucleotide described in the preceding paper (CIV) are the longest polydeoxynucleotides with specific non-repeating sequences which have been chemically synthesized to date. The present icosanucleotide covers the nucleotide sequence 31 to 50 of one of the strands in the DNA duplex representing the gene for yeast alanine transfer RNA. The nucleotide sequence and the polarity of the DNA strand, of which the icosanucleotide herein described forms a part, is the same as that of the tRNA itself (Fig. 1). The synthesis involved the following steps, using fully protected intermediates. The nucleoside d-MMTr-G1B was condensed successively with d-pCAnOAc and d-pT-OAc to give d-MMTr-G1BpCAnpT. The latter was condensed successively with the three dinueleotide blocks, d-pCAnpCAn-OAc, d-pCAnpT-OAc and d-pTpABz-OAc, to give the protected nonanucleotide, d-MMTr-G1BpCAn-pTpCAnpCAnpCAnpTpTpABz. Then followed condensations with the following trinucleotide and tetranucleotide blocks in the order that the blocks are written: d-pG1BpCAnpABz-OAc, d-pTpG1BpG1BpG1B-OiB and d-pABzpG1BpABzpG1B-OAc. All the blocks, the intermediate polydeoxynucleotides and the final product were characterized in a variety of ways, both in the protected and the unprotected form.
The possible mechanisms by which the interaction of cholera toxin with isolated fat cells leads to an enhancement in the rate of lipolysis were examined, with special emphasis on the biochemical basis of the marked lag phase which exists before the onset of the lipolytic response is evident. Cells exposed to cholera toxin can be washed and replaced with fresh medium after 8 min or after 60 min of incubation at 37° without altering the characteristic 1-hr lag phase or the subsequent course of the lipolytic response. During the lag phase lipolytic metabolites or products are not secreted and accumulated in the incubation medium. Fat cells obtained from young rats (50-100 g) are much more sensitive to cholera toxin than cells obtained from large (>200 g) animals. Increasing the concentration of cholera toxin in the medium to very high values does not appreciably shorten the lag phase of the lipolytic response. In the presence of cholera toxin the lipolytic effects of epinephrine and of glucagon are not appreciably altered during the period of the lag phase. During the course of incubation the toxin does not appear to be secreted into the medium in an immediately active form. Incubation of fat cells at 37° in the absence of toxin does not alter the subsequent course of toxin-induced lipolysis. The lag phase is very dependent on the temperature of incubation. Prolonged incubation of the cell-toxin complex at 4 or 24° does not modify the length of the lag phase upon subsequent incubation at 37°. The length of the lag phase is the same whether the cells are incubated at 37 or 48°. The lipolytic response of fat cells to cholera toxin can be effectively inhibited by insulin or by alloxan, compounds which are capable of inhibiting the activity of adenylate cyclase. The inhibition by insulin is equally effective whether it is added at the start of the incubation with toxin or whether it is added after the lag period has transpired. Alloxan, however, becomes progressively less effective when it is added after increasing periods of incubation of the toxin-cell complex. Various inhibitors of prostaglandin biosynthesis or action (indomethacin, sodium salicylate, diphloretin phosphate) and inhibitors of RNA and protein synthesis (actinomycin D, cycloheximide, puromycin) do not affect the lipolytic activity of cholera toxin. With increasing length and temperature of incubation of the cell-toxin complex the rate and extent to which the complex can spontaneously dissociate are progressively decreased. However, even with very prolonged incubation the toxin does not appear to form stable covalent bonds with membrane macromolecules. The length of the lipolytic lag phase cannot be decreased by exposing the cells for a brief period to cholera toxin in the presence of low concentrations of detergent (to enhance permeability). Despite the ability of tetanus toxin to bind to gangliosides, this bacterial toxin has no lipolytic or antilipolytic activities in fat cells, even when ganglioside-treated fat cells are used. Tetanus toxin does not compete with 125I-labeled cholera toxin for binding to membranes. It is suggested that cholera toxin initially forms an inactive toxin-ganglioside receptor complex on the cell membrane, and that this complex is transformed into a biologically active complex by a special transition which involves a major, spontaneous relocation of the complex within the two-dimensional structure of the membrane.
Colicinogenic factor E1 (Col E1) DNA isolated from Escherichia coli by phenol extraction followed by dye-buoyant centrifugation was found to consist almost exclusively of the monomer 23 s supercoiled circular DNA form. Little if any of the larger multiple forms which are characteristic of Col E1 DNA isolated from Proteus mirabilis were observed. The Col E1 DNA isolated from E. coli was shown to be similar, if not identical, to the monomer form of P. mirabilis by co-purification through alkali denaturation, neutralization and nitrocellulose filtration, and by co-sedimentation in sucrose gradients. The Col E1 DNA of E. coli also gives rise to a 17 s form with first-order kinetics during degradation with pancreatic deoxyribonuclease, as does the 23 s form isolated from P. mirabilis.A rapid technique of isolation of closed circular DNA molecules from E. coli, consisting of direct dye—buoyant centrifugation of a sheared sarkosyl lysate was developed. Using this technique, Col E1 from E. coli was again isolated as the 23 s form exclusively, while colicinogenic factors E2 and E3 were both found to be predominantly 25 s DNA forms. The sedimentation properties of the Col E2 and Col E3 DNA indicate a molecular weight of 5 × 106 for these factors.
The behavior of each of a series of proteins during chromatography on columns of Sephadex G-200 may be correlated with the Stokes radius of the protein, but does not correlate with molecular weight. Proteins with Stokes radii as high as 107 Å or molecular weights as high as 1 300 000 may be characterized by the use of such columns. The Sephadex data are used in a critique of earlier mathematical treatments of the phenomenon known as "gel filtration". With a Stokes radius measured by the chromatographic method and a sedimentation coefficient determined by density gradient centrifugation, reasonable estimates for both the molecular weight and the frictional ratio (f/f0) of a macromolecule are available. Since both of these methods are applicable to proteins present in mixtures, valuable information concerning the molecular weights and shapes of proteins may be obtained in anticipation of the achievement of high degrees of purity. The determination of the molecular weight and the f/f0 for each of several enzymes in unfractionated extracts of Salmonella typhimurium and Neurospora crassa illustrates this application.
3′-O-Tosyl-2′-deoxyadenosine (I) has been treated with sodium methoxide in dimethylformamide to provide the first reported synthesis of 2′,3′-dideoxy-2′,3′-didehydroadenosine (II). Studies have been made which support a simple E2 mechanism for the general introduction of a 2′,3′ double bond from the corresponding 3′-O-tosyl-2′-deoxyadenosine derivative under these conditions. In the presence of potassium t-butoxide in dimethyl sulfoxide further elimination occurs with 5′-S-ethyl-3′-O-tosyl-5′-thio-2′,5′- dideoxyadenosine (V) to yield 9-(5′-methyl-2′-furyl)adenine (X) as a final product. A mechanism has been proposed for the formation of X consistent with the present work. The preparation of these unsaturated adenine nucleosides has provided a new route to the synthesis of 2′,3′-dideoxyadenosine (III) and 2′,3′,5′-trideoxyadenosine (VIII) by direct hydrogenation procedures. The direct utilization of these novel unsaturated nucleoside derivatives as reaction intermediates offers a unique opportunity for future synthetic studies.
General synthetic routes have now been devised for the chemical preparation of a number of new deoxyadenosines via the requisite intermediate 3′- or 5′-alkylthionucleosides. 5′-O-Trityl-3′-O-tosyl - 2′- de- oxyadenosine (II) has been converted in good yield to 3′-O-tosyl-2′-deoxyadenosine (III) which in turn has been treated with ethanethiol to yield 6-amino-9-(3′-S-ethyl-3′-thio-2′,3′-dideoxy-β - D - threo - pentofuranosyl)- purine (IV). Raney nickel desulfurization of IV yielded 2′,3′-dideoxyadenosine (V). Selective 5′-O-tosylation of 2′-deoxyadenosine has been successfully accomplished. Treatment of 5′-O-tosyl-2′-deoxyadenosine (VII) with ethanethiol yielded 5′-S-ethyl-5-thio-2′,5′-dideoxyadenosine (VIII). The structure of VIII was verified by an unambiguous synthesis from 3′-O-acetyl-2′-deoxyadenosine (XI) via 5′-O-tosyl-3′-O-acetyl-2′-deoxyadenosine (XII). Raney nickel desulfurization of VIII gave a good yield of 2′,5′-dideoxyadenosine (IX). Similarly, 3′,5′-di-O-tosyl-2′-deoxyadenosine (XIII) yielded 2′,3′,5′-trideoxyadenosine (XVI) via the 3′,5′-diethylthio derivative. The significance of these compounds as potential inhibitors of deoxyribonucleic acid biosynthesis is discussed.
Reaction of the phosphorimidazohdate formed from a nucleotide and 1,1′-carbonyldiimidazole with inorganic pyrophosphate provides the nucleoside triphosphate in good yield. The method is convenient, generally applicable, and particularly suitable for microscale syntheses from mono- or oligonucleotides.
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