POPULARITY
3 episodes with Nucleotide
6 episodes with Nucleotide
2 episodes with Nucleotide
2 episodes with Nucleotide
2 episodes with Nucleotide
2 episodes with Nucleotide
2 episodes with Nucleotide
7 episodes with Nucleotide
2 episodes with Nucleotide
Folic acid plays 3 essential cellular roles, including synthesis of purines, thymidine, and metabolism of several amino acids. Nucleotide synthesis and nucleic acid synthesis are completely dependent on folic acid availability from the diet.
In this episode, we review the high-yield topic of Nucleotide Catabolism/Salvage from the Biochemistry section.Follow Medbullets on social media:Facebook: www.facebook.com/medbulletsInstagram: www.instagram.com/medbulletsofficialTwitter: www.twitter.com/medbullets
Op verzoek van luisteraar Mirjam een complete aflevering over de bevindingen van wetenschappers met betrekking tot het bodemmonster van asteroïde Bennu. Een handvol gruis die de ruimtesonde Osiris Rex naar de aarde bracht na een bezoek aan Bennu, bevat tal van organische verbindingen.Bright carbonate veins on asteroid (101955) Bennu: Implications for aqueous alteration history:https://pubmed.ncbi.nlm.nih.gov/33033155/An evaporite sequence from ancient brine recorded in Bennu samples:https://www.nature.com/articles/s41586-024-08495-6Traces of ancient brine discovered on the asteroid Bennu contain minerals crucial to life:https://www.eurekalert.org/news-releases/1071694Nucleotide base:https://en.m.wikipedia.org/wiki/Nucleotide_base101955 Bennu:https://en.wikipedia.org/wiki/101955_BennuYORP effect:https://en.wikipedia.org/wiki/YORP_effectMurchison meteoriet:https://en.wikipedia.org/wiki/Murchison_meteoriteDe Zimmerman en Space podcast is gelicenseerd onder een Creative Commons CC0 1.0 licentie.http://creativecommons.org/publicdomain/zero/1.0
References Guerra, DJ. 2024 Nucleotide Graduate lectures Hunter-Garcia 1970. "Attics of my Life" Grateful Dead [American Beauty, lp.] https://open.spotify.com/track/6WeLHc9eLjVJqOKhd97vaK?si=d93bcc38ba7d4bac --- Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support
Dr Mariko Bennett and Dr Laura Adang discuss the precarious balance between a protective and a destructive immune response, as is seen in inborn errors in nucleotide metabolism. Our discussion focuses on the most common of these disorders: Aicardi Goutières syndrome (AGS). Sadly, despite the many gains in understanding about AGS, there remain many gaps in our understanding of this condition. Nucleotide metabolism, leukodystrophies, and CNS pathology Francesco Gavazzi, et al https://doi.org/10.1002/jimd.12721
Episode 9 of the DNA Papers discusses a set of papers by the first scientist who made a sustained effort into uncovering the secret behind specificity of nucleic acids. The principle author, Erwin Chargaff, a European-American biochemist from Columbia University in New York, determined that the relative rations of the four nucleotide bases—A, T, G and C—were not present in all DNA in equal amounts as widely assumed, but rather, that they varied in proportion from one to another, with the amount of the A and G bases being equal to the T and C bases respectively. Furthermore, he also demonstrated that the ratio of these amounts was specific and consistent for a given species. He first laid out his vision for determining the role of nucleic acids in 1947, and over the next decade or so, proceeded to probe the finer details of DNA chemistry with the then state-of-the art innovations in techniques such as chromatography and UV spectroscopy. Papers discussed include: Chargaff, Erwin. “On the Nucleoproteins and Nucleic Acids of Microorganisms.” Cold Spring Harbor Symposia on Quantitative Biology 12 (January 1, 1947): 28–34. https://doi.org/10.1101/SQB.1947.012.01.006. Vischer, Ernst, and Erwin Chargaff. “The Separation and Quantitative Estimation of Purines and Pyrimidines in Minute Amounts.” Journal of Biological Chemistry 176 (1948): 703–14. Chargaff, Erwin. “Chemical Specificity of Nucleic Acids and Mechanism of Their Enzymatic Degradation.” Experientia 6, no. 6 (June 15, 1950): 201–9. Joining us to illuminate the role of Chargaff and his experiments in the history of DNA are: Pnina Abir-Am, Brandeis University Kersten Hall, University of Leeds Hans-Jörg Rheinberger, Max Planck Institute for the History of Science View more at https://www.chstm.org/video/144 Recorded August 9, 2023.
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.03.539131v1?rss=1 Authors: McCormick, L. A., Cleary, J. M., Hancock, W. O., Rice, L. M. Abstract: GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The "self-acting" (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the "interface-acting" (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with self-acting nucleotide plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and "poisoning" plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC
References J of Computational Chemistry 2011 Volume32, Issue10: Pages 2077-2083 --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message
In this episode, we review the high-yield topic of De Novo Nucleotide Synthesis from the Biochemistry section. Follow Medbullets on social media: Facebook: www.facebook.com/medbullets Instagram: www.instagram.com/medbulletsofficial Twitter: www.twitter.com/medbulletsIn this episode --- Send in a voice message: https://anchor.fm/medbulletsstep1/message
References Dr. Guerra's lecture notes Mol Genet Metab Rep. 2021 Mar; 26: 100709. --- Send in a voice message: https://anchor.fm/dr-daniel-j-guerra/message Support this podcast: https://anchor.fm/dr-daniel-j-guerra/support
In this episode, we review the high-yield topic of Nucleotide Catabolism/Salvage from the Biochemistry section. Follow Medbullets on social media: Facebook: www.facebook.com/medbullets Instagram: www.instagram.com/medbulletsofficial Twitter: www.twitter.com/medbullets --- Send in a voice message: https://anchor.fm/medbulletsstep1/message
This week we discuss how published nucleotide sequences are not always correct or to be trusted with Yasunori Park and Professor Jennifer A Byrne (@JAByrneSci), a research Assistant and PI at the University of Sydney (@Sydney_uni). We delve into the details of their nifty new text mining tool (Blast and Seek) which highlights papers with incorrect nucleotide sequences. We also discuss the most common mistakes found, the impact of these mistakes, and what we can do as researchers to prevent errors from occurring. We also talk about prepints and Australian Research Council's recent decision to ban preprints in their grant applications as well as some insight into our very own PhD's. Read the full preprint https://www.biorxiv.org/content/10.1101/2021.07.29.453321v1?fbclid=IwAR2LibD1DD8q6b5df3vMzgFXpmmA6q1E_UarbyaB0dq55fOaY8mmFvzbe14 Other links Open letter to ARC https://asapbio.org/arc This episode was produced by Emma Wilson and edited by John D Howard. If you enjoyed this show then hit that subscribe button and leave a review. If you love what we are trying to do then support us on Patreon https://www.patreon.com/preprintsinmotion where tiers start at as little as £1 a month! For the latest podcast news and updates follow us on Twitter @MotionPod or visit our website; www.preprintsinmotion.com. Produced by JEmJ Productions (find us on Twitter: Jonny @JACoates, Emma @ELWilson92, John @JohnDHoward8) and generously supported by ASAPbio (https://asapbio.org | @asapbio_).
Do we fall into disease as a result of our DNA? Or, do we have the ability to shift things to avoid it? Understanding how and what our genes are, the complexity in each, and how this points to creation. RESOURCES 21-page guide to achieve weight loss on your keto diet FREE guide on how to start keto Keto question? Need help? Send me a message Trent's website Trent's Instagram Complete a DNA test (code KDP for $50 off) PARTNERS LMNT electrolyte powder $5 sample pack - and get 8 packets! KetoBars a low-carb bar without the fibers, code KDP20 for 20% off
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.12.380378v1?rss=1 Authors: Colquhoun, R. M., Hall, M. B., Lima, L., Roberts, L. W., Malone, K. M., Hunt, M., Letcher, B., Hawkey, J., George, S., Pankhurst, L., Iqbal, Z. Abstract: Bacterial genomes follow a U-shaped frequency distribution whereby most genomic loci are either rare (accessory) or common (core) - the alignable fraction of two genomes from a single species might be only 50%. Standard tools therefore analyse mutations only in the core genome, ignoring accessory mutations. We present a novel pan-genome graph structure and algorithms implemented in the software pandora, which approximates a sequenced genome as a recombinant of reference genomes, detects novel variation and then pan-genotypes multiple samples. Constructing a reference graph from 578 E. coli genomes, we analyse a diverse set of 20 E. coli isolates. We show, for rare variants, pandora recovers at least 13k more SNPs than single-reference based tools, achieving equal or better error rates with Nanopore as with Illumina data, and providing a stable framework for analysing diverse samples without reference bias. This is a significant step towards comprehensive analyses of bacterial genetic variation. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.08.369488v1?rss=1 Authors: Goniotaki, D., Tamagnini, F., Biasetti, L., Rumpf, S. L., Fennell, K., Pollack, S., Ukwesa, S., Sun, H., Serpell, L., Noble, W., Staras, K., Hanger, D. Abstract: Neurodegenerative tauopathies are characterized by deposition in the brain of highly phosphorylated and truncated forms of tau, but how these impact on cellular processes remains unknown. Here, we show that hyperpolarization-induced membrane voltage 'sag', which is dependent on hyperpolarization-activated inward-rectifying (Ih) current and hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels, is increased in the Tau35 mouse model of human tauopathy. Expression of Tau35, corresponding to a fragment comprising the carboxy-terminal half of tau first identified in human tauopathy brain, reduces dendritic branching in mouse brain and in cultured hippocampal neurons, and decreases synaptic density. Neuronal expression of Tau35 results in increased tau phosphorylation and significant disruption to synaptic ultrastructure, including marked and progressive reductions in synaptic vesicle counts and vesicle cluster density. Ultrastructural analysis reveals that the positioning of synaptic vesicles is perturbed by Tau35, causing vesicles to accumulate at sites adjacent to the active zone and at the lateral edges of the cluster. Structural changes induced by Tau35 is correlated with functional abnormalities in network activity, including increased width, reduced frequency and slower rate of rise of spontaneous excitatory postsynaptic currents. Collectively, these changes are consistent with a model in which disease-associated tau species disrupt network connectivity and signaling, which may underpin the catastrophic synaptic dysfunction observed during the development and progression of human tauopathy. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.30.362251v1?rss=1 Authors: Kamble, P., Hall, K., Chandak, M., Tang, Q., Caglayan, M. Abstract: DNA ligase I (LIG1) completes base excision repair (BER) pathway at the last nick sealing step following DNA polymerase (pol) {beta} gap filling DNA synthesis. We previously reported that pol {beta} 8-oxo-2'-deoxyribonucleoside 5'-triphosphate (8-oxodGTP) insertion confounds LIG1 leading to the formation of ligation failure products with 5'-adenylate (AMP) block. Here, we report the mutagenic ligation of pol {beta} 8-oxodGTP insertion products and an inefficient substrate-product channeling from pol {beta} Watson-Crick like dG:T mismatch insertion to DNA ligation by LIG1 mutant with perturbed fidelity (E346A/E592A) in vitro. Moreover, our results revealed that the substrate discrimination of LIG1 for the nicked repair intermediates with preinserted 3'-8-oxodG or mismatches is governed by the mutations at both E346 and E592 residues. Finally, we found that Aprataxin (APTX) and Flap Endonuclease 1 (FEN1), as compensatory DNA-end processing enzymes, can remove 5'-AMP block from the abortive ligation products with 3'-8-oxodG or all possible 12 non-canonical base pairs. These findings contribute to understand the role of LIG1 as an important determinant of faithful BER, and how a multi-protein complex (LIG1, pol {beta}, APTX and FEN1) can coordinate to hinder the formation of mutagenic repair intermediates with damaged or mismatched ends at the downstream steps of the BER pathway. Copy rights belong to original authors. Visit the link for more info
Dr. Becky Andrews, ND, private and Peoples Rx practitioner, discusses SIBO (small intestine bacterial overgrowth) and SNP (single nucleotide polymorphism) and how the effects they can have on your health, how to deal with them, and much more! @cultivatewellnesspodcast Always brought to you by Peoples Rx, Austin's Favorite Pharmacy, now with new online shopping options!
In this week's 7th episode of Season 2 we recap the 8th Week of Distance Learning but 1st Week of Hybrid Teaching that was in AP/DC Biology focusing on the Monomers of Nucleic Acids the Nucleotide. In our 2nd segment, with a Quick approaching Exam over Chapters 4 and 5 I review a usually forgotten Concept...Protein Folding. In our Final Segment, I introduce a New Segment to the Podcast...Mr. V Recommends...where I recommend Music, Movies, Activities, Other Podcast, and Anything I think is the Cool Beans for you to Check Out. Remember to subscribe, like, and please comment on the podcast on your podcast listening platform. You can also e-mail me at ovelas@neisd.net with any comments or feedback. You can also follow me on twitter at OscarVelasquez@APBiologyMrV. Students can always contact me and communicate with me via Google Classroom. If you have questions or feedback you would like Mr. V to answer please e-mail me the questions or send them on Google Classroom, Instagram, or Twitter. Also follow the Instagram Page for the podcast "Evolving with Mr. V" and also now subscribe to the Wakelet Course Site Giving all the AP Biology Content by Weeks. Remember Wash Your Hands Well, Cough & Sneeze into your Elbow, Avoid Large Gatherings, Practice Social Distancing, Wear Your Mask, and Stay Safe!!! Also Study for Your Exam!!! I want to thank you for listening...I am your Host Mr. Oscar Velasquez "Master of the Biological Arts". Have a Great Week and May the Force be With Us...Always...For that is the Way. Also don't forget Rate this Podcast...GIMME Feedback!!! And Check out the Wakelet Course Page!!! Lastly...Big Shout Out to Free Music Achieve, SoundBible, and Zapsplat for the music and sound effects in the podcast. https://www.instagram.com/evolvingwithmrv/?hl=en
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.08.330985v1?rss=1 Authors: Seiler, E., Mehringer, S., Darvish, M., Turc, E., Reinert, K. Abstract: We present Raptor, a tool for approximately searching many queries in large collections of nucleotide sequences. In comparison with similar tools like Mantis and COBS, Raptor is 12 - 144 times faster and uses up to 30 times less memory. Raptor uses winnowing minimizers to define a set of representative k-mers, an extension of the Interleaved Bloom Filters (IBF) as a set membership data structure, and probabilistic thresholding for minimizers. Our approach allows compression and a partitioning of the IBF to enable the effective use of secondary memory. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.01.322164v1?rss=1 Authors: Karasikov, M., Mustafa, H., Danciu, D., Zimmermann, M., Barber, C., Ratsch, G., Kahles, A. Abstract: The amount of biological sequencing data available in public repositories is growing exponentially, forming an invaluable biomedical research resource. Yet, making all this sequencing data searchable and easily accessible to life science and data science researchers is an unsolved problem. We present MetaGraph, a versatile framework for the scalable analysis of extensive sequence repositories. MetaGraph efficiently indexes vast collections of sequences to enable fast search and comprehensive analysis. A wide range of underlying data structures offer different practically relevant trade-offs between the space taken by an index and its query performance. Achieving compression ratios of up to 1,000-fold over the already compressed raw input data, MetaGraph indexes can represent the content of large sequencing archives in the working memory of a single compute server. We demonstrate our framework's scalability by indexing over 1.4 million whole genome sequencing (WGS) records from NCBI's Sequence Read Archive, representing a total input of more than three petabases. MetaGraph provides a flexible methodological framework allowing for index construction to be scaled from consumer laptops to distribution onto a cloud compute cluster for processing terabases to petabases of input data. Notably, processing of data sets ranging from 1 TB of raw WGS reads to 20 TB of human RNA-sequencing data results in indexes whose memory footprints are small enough to host on standard desktop workstations. Besides demonstrating the utility of MetaGraph indexes on key applications, such as experiment discovery, sequence alignment, error correction, and differential assembly, we make a wide range of indexes available as a community resource, including indexes of over 450,000 microbial WGS records, more than 110,000 fungi WGS records, and more than 40,000 whole metagenome sequencing records. A subset of these indexes is made available online for interactive queries. All indexes will be available for download and in the cloud. In total, indexes comprising more than 1 million sequencing records are available for download. As an example of our indexes' integrative analysis capabilities, we introduce the concept of differential assembly, which allows for the extraction of sequences present in a foreground set of samples but absent in a given background set. We apply this technique to differentially assemble contigs to identify pathogenic agents transfected via human kidney transplants. In a second example, we indexed more than 20,000 human RNA-Seq records from the TCGA and GTEx cohorts and use them to extract transcriptome features that are hard to characterize using a classical linear reference. We discovered over 200 trans-splicing events in GTEx and found broad evidence for tissue-specific non-A-to-I RNA-editing in GTEx and TCGA. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.25.314252v1?rss=1 Authors: Feng, Z., Clemente, J., Wong, B., Schadt, E. Abstract: Cellular genetic heterogeneity is common in many biological conditions including cancer, microbiome, co-infection of multiple pathogens. Detecting and phasing minor variants, which is to determine whether multiple variants are from the same haplotype, play an instrumental role in deciphering cellular genetic heterogeneity, but are still difficult because of technological limitations. Recently, long-read sequencing technologies, including those by Pacific Biosciences and Oxford Nanopore, have provided an unprecedented opportunity to tackle these challenges. However, high error rates make it difficult to take full advantage of these technologies. To fill this gap, we introduce iGDA, an open-source tool that can accurately detect and phase minor single-nucleotide variants (SNVs), whose frequencies are as low as 0.2%, from raw long-read sequencing data. We also demonstrated that iGDA can accurately reconstruct haplotypes in closely-related strains of the same species (divergence [≥] 0.011%) from long-read metagenomic data. Our approach, therefore, presents a significant advance towards the complete deciphering of cellular genetic heterogeneity. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.25.310078v1?rss=1 Authors: Taiwo, I. A., Adeleye, N., Anwoju, F. O., Adeyinka, A., Uzoma, I. C., Bankole, T. T. Abstract: Background: Coronaviruses are a group of viruses that belong to the Family Coronaviridae, Genus Betacoronavirus. In December 2019, a new coronavirus disease (COVID-19) characterized by severe respiratory symptoms was discovered. The causative pathogen was a novel coronavirus known as 2019-nCoV and later as SARS-CoV-2. Within two months of its discovery, COVID-19 became a pandemic causing widespread morbidity and mortality. Methodology: Whole genome sequence data of SARS-CoV-2 isolated from Nigerian COVID-19 cases were retrieved by downloading from GISAID database. A total of 18 sequences that satisfied quality assurance (length > 29700 nts and number of unknown bases denoted as N < 5%) were used for the study. Multiple sequence alignment (MSA) was done in MAFFT (Version 7.471) while SNP calling was implemented in DnaSP (Version 6.12.03) respectively and then visualized in Jalview (Version 2.11.1.0). Phylogenetic analysis was with MEGA X software. Results: Nigerian SARS-CoV-2 had 99.9% genomic similarity with four large conserved genomic regions. A total of 66 SNPs were identified out of which 31 were informative. Nucleotide diversity assessment gave Pi = 0.00048 and average SNP frequency of 2.22 SNPs per 1000 nts. Non-coding genomic regions particularly 5 UTR and 3 UTR had a SNP density of 3.77 and 35.4 respectively. The region with the highest SNP density was ORF10 with a frequency of 8.55 SNPs/1000 nts). Majority (72.2%) of viruses in Nigeria are of L lineage with preponderance of D614G mutation which accounted for 11 (61.1%) out of the 18 viral sequences. Nigeria SARS-CoV-2 revealed 3 major clades namely Oyo, Ekiti and Osun on a maximum likelihood phylogenetic tree. Conclusion and Recommendation: Nigerian SARS-CoV-2 reveals high mutation rate together with preponderance of L lineage and D614G mutants. Implication of these mutations for SARS-CoV-2 virulence and the need for more aggressive testing and treatment of COVID-19 in Nigeria is discussed. Additionally, attempt to produce testing kits for COVID-19 in Nigeria should consider the conserved regions identified in this study. Strict adherence to COVID-19 preventive measure is recommended in view of Nigerian SARS-CoV-2 phylogenetic clustering pattern, which suggests intensive community transmission possibly rooted in communal culture characteristic of many ethnicities in Nigeria. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.293290v1?rss=1 Authors: Feher, K. M., Kolbanovskiy, A., Durandin, A., Shim, Y., Min, J. H., Lee, Y. C., Shafirovich, V., Mu, H., Broyde, S., Geacintov, N. E. Abstract: The Nucleotide Excision Repair (NER) mechanism removes a wide spectrum of structurally different lesions that critically depend on the binding of the DNA damage sensing NER factor XPC-RAD23B (XPC) to the lesions. The bulky mutagenic benzo[a]pyrene diol epoxide metabolite-derived cis- and trans-B[a]P-dG lesions (G*) adopt base-displaced intercalative (cis) or minor groove (trans) conformations in fully paired DNA duplexes with the canonical C opposite G* (G*:C duplexes). While XPC has a high affinity for binding to these DNA lesions in fully complementary double-stranded DNA, we show here that deleting only the C in the complementary strand opposite the lesion G* embedded in 50-mer duplexes, fully abrogates XPC binding. Accurate values of XPC dissociation constants (KD) were determined by employing an excess of unmodified DNA as a competitor; this approach eliminated the binding and accumulation of multiple XPC molecules to the same DNA duplexes, a phenomenon that prevented the accurate estimation of XPC binding affinities in previous studies. Surprisingly, a detailed comparison of XPC dissociation constants KD of unmodified and lesion-containing G*:Del complexes, showed that the KD values were ~ 2.5 - 3.6 times greater in the case of G*:Del than in the unmodified G:Del and fully base-paired G:C duplexes. The origins of this unexpected XPC lesion avoidance effect is attributed to the intercalation of the bulky, planar B[a]P aromatic ring system between adjacent DNA bases that thermodynamically stabilize the G*:Del duplexes. The strong lesion-base stacking interactions associated with the absence of the partner base, prevent the DNA structural distortions needed for the binding of the BHD2 and BHD3 {beta}-hairpins of XPC to the deletion duplexes, thus accounting for the loss of XPC binding and the known NER-resistance of G*:Del duplexes. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.27.267153v1?rss=1 Authors: Gombolay, A., Storici, F. Abstract: Ribose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze raw ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides, and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 hr of computing time for most datasets and about 30 min of hands-on time. Copy rights belong to original authors. Visit the link for more info
Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.30.228726v1?rss=1 Authors: Cloete, R., Shahbaaz, M., Grobbelaar, M., Sampson, S. L., Christoffels, A. Abstract: Nicotinamide-nucleotide adenylyl transferase (Rv2421c) was selected as a potential drug target, because it has been shown, in vitro , to be essential for Mycobacterium tuberculosis growth. It is conserved between mycobacterium species, is up-regulated during dormancy, has a known 3D crystal structure and has no known human homologs. A model of Rv2421c in complex with nicotinic acid adenine dinucleotide and magnesium ion was constructed and subject to virtual ligand screening against the Prestwick Chemical Library and the ZINC database, which yielded 155 potential hit molecules. 3D-QSAR studies of the 155 drug molecules indicated five compounds with similar inhibitory efficiencies compared to known inhibitors of Rv2421c. Molecular docking validation and molecular dynamics simulation analysis of the top five compounds indicated that the identified inhibitor molecules bind to Rv2421c with comparable efficiency as the substrate DND. Subsequent in vitro testing of the five compounds identified Novobiocin sodium salt with activity against Mycobacterium tuberculosis at 50 M, 25M and weakly at 10M concentrations. Although, Novobiocin salt targets Mycobacterium tuberculosis DNA gyrase B our studies suggest that it has the potential to be repurposed to inhibit Rv2421c. Subsequent in silico structural analysis of known Novobiocin sodium salt derivatives against Rv2421c suggest promising alternatives for the treatment of Mycobacterium tuberculosis . Copy rights belong to original authors. Visit the link for more info
Your lymphatic system functions as a sort of circulatory system for your immune system, balancing fluids and fighting viruses. This is why it is so important to know about certain nutrients that can help support it, according to Chief Scientific Director and Pharmacist Jerry Hickey, Ph. Take advantage of an exclusive podcast offer today by visiting www.invitehealth.com/podcast or by clicking here. For more information on the products or studies mentioned in this episode, click here.
The TWiM holobionts pay tribute to Stuart Levy, and reveal the remarkably diverse array of cyclic nucleotides synthesized by bacteria that likely mediate interactions with animal and plant hosts. Become a patron of TWiM. Links for this episode: Stuart Levy, Antibiotic Resistance Researcher, Dies Resistance fighter (The Scientist) Stuart Levy on TWiM 16 Stuart Levy on Meet the Scientist episode 17 Segregation of R factors (Nature) Infectious drug resistance (Sci Am) Spread of antibiotic resistance chickens to humans (Nature) Costs of antimicrobial resistance (Clin Inf Dis) Bacteria synthesize diverse nucleotide signals (Nature) Music used on TWiM is composed and performed by Ronald Jenkees and used with permission. Send your microbiology questions and comments to twim@microbe.tv
Dr Kara Fitzgerald joins Nathan Rose to discuss the epigenome, the effect of methylation on gene expression and looking at interventions outside the use of supplemental methyl donors to modify our epigenome for a healthy life.
Dr Kara Fitzgerald joins Nathan Rose to discuss the epigenome, the effect of methylation on gene expression and looking at interventions outside the use of supplemental methyl donors to modify our epigenome for a healthy life.
Sabatini focuses on a lysosomal membrane protein that his lab had found to interact with mTORC1 and to sense arginine levels inside the lysosome. In some cell types, the amino acids needed to build new proteins are not taken up as free amino acids but instead come from the breakdown of proteins in the lysosome. This led the lab to ask which arginine-rich proteins are being degraded in the lysosome, which led to the realization that ribosomal proteins are amongst the most arginine-rich proteins in mammalian cells. After many more experiments, they showed that mTORC1 regulates a balance between the biogenesis of ribosomes, and the breakdown of ribosomes (known as ribophagy), dependent on the nutritional state of the cell. Ribophagy seems to be particularly important for supplying the cell with nucleosides during nutrient starvation.
Featuring: Randalicious, Jiklim, Nucleotide, Dead Kantus, D E C O, BbtargetOriginal air date: March 18, 2018 Join the Hexis Discord - https://discord.gg/Hexis Hexis Podcast Site - https://sites.google.com/view/hexispodcasts Hexis Twitter Account - https://twitter.com/Hexis Hexis Merch Store - https://www.teepublic.com/user/randalicious Hexis Clan Thread - http://services.runescape.com/m=forum/c=J8jI1t0NuaI/forums.ws?320,321,251,66018560 Podcasts streamed at - https://www.twitch.tv/Randalicious
Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Guest: Michael Libman The TWiP-scholars solve the case of the Housewife from Kolkata, discuss mutations in the IL17 gene associated with cerebral malaria, and hear a case presentation from guest Michael Libman. Links for this episode: IL-17 mutations and risk of cerebral malaria (Inf and Imm) Echinococcosis (CDC) Echinococcus life cycle (pdf) Letters read on TWiP 103 Case study for TWiP 103 This week's case concerns a 42 yo male, refugee in Canada, from DRC, former Zaire, where there is unending civil war. Upper middle class, professor of French at university. Had been imprisoned, tortured, lived in jungle for a few years, reached refugee camp in Tanzania, moved to Canada. Came to health care system 15 months after arrived. Was sent to psych, unstable emotionally, delusions, hallucinations, depression, post traumatic issues. Was under psych care for ~1 yr, did not improve, became worse. Sent to hospital. History: talked about having minor injury, hurt lower back, pain there bothering him. Some anemia (normochromic), basic hem/chem/urine/liver nothing remarkable. Physical exam, nothing remarkable. HIV negative. Some evidence for chronic inflammatory condition: sed rate 60 (elevated), had diffuse increase in IgG, IgM. Developed some low level autoantibodies; anti-nuclear, p-anka, anti-neutrophil cytoplasmic antibodies. Slightly elevated fever for a few days, then few days or week with no fever. No eosinophilia. Radiology: on CT did have some mediastinal, aortic, axillae, lymphadenopathy. Prob screened in Africa for malaria and treated; prob also got ivermectin. Also got head MRI: not completely normal, classic nonspecific midbrain abnormality. Diffuse mild edema. Weight loss remarkable. No visual problems. Send your diagnosis to twip@microbe.tv Send your questions and comments to twip@microbe.tv
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When I was in grad school studying sports nutrition most people talked about the importance of post-workout carbs and protein in terms of actual physical muscle recovery from exercise. However, we also learned that the immune system is compromised after exercise for a brief period – basically while the body gets its bearings back. Are there any supplements we would take to prevent this or bounce back faster? We review a possible option. John always reviews the full-text of every article, but you may view the free abstract by clicking here: http://journals.lww.com/nsca-jscr/Abstract/publishahead/The_Physiological_Effects_of_Nucleotide.96819.aspx .
Hosts: Vincent Racaniello, Alan Dove, and Kathy Spindler Vincent, Alan, and Kathy discuss finding viruses in outer space, varying results obtained from personal genetic testing, and depletion of CD4 cells during HIV infection by pyroptosis. Links for this episode We should hunt for viruses in space (New Scientist) I had my DNA picture taken (NY Times) CD4 cell death by pyroptosis (Nature) Abortive HIV infection in lymphoid tissue (Cell) IFI16 sensor required for HIV mediated cell death (Science) PubMed Commons Preferential CTL targeting of Gag (Cell Rep) Exonic transcription protein binding (Science) Letters read on TWiV 266 Video of this episode - view at YouTube Weekly Science Picks Kathy - 17 mundane things, mind blowing viewsAlan - SnowovelVincent - Public's views on human evolution Listener Pick of the Week Emily - Dengue pillow Send your virology questions and comments (email or mp3 file) to twiv@twiv.tv
Today's Neuroscience, Tomorrow's History - Professor Geoffrey Burnstock
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06
Thu, 20 May 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11908/ https://edoc.ub.uni-muenchen.de/11908/1/Becirovic_Elvir.pdf Becirovic, Elvir
An introduction to the processes of transcription and translation.
Transcript -- An introduction to the processes of transcription and translation.
An introduction to the processes of transcription and translation.
Transcript -- An introduction to the processes of transcription and translation.
Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 10/19
Thu, 7 May 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/10138/ https://edoc.ub.uni-muenchen.de/10138/1/Schickinger_Veronika.pdf Schickinger, Veronika
Enhanced Video PodcastAired date: 1/28/2008 12:00:00 PM Eastern Time
Enhanced Audio PodcastAired date: 1/28/2008 12:00:00 PM Eastern Time
Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06
The present thesis deals with the development of nanoparticles based on the proteinaceous macromolecule gelatin as delivery system for various nucleotide-based drugs. Since a method to produce homogenous nanoparticles was already described in principle (Coester et al. 2000), it was the first approach to characterize, optimize, and standardize this manufacturing process. The next goal was to advance these plain gelatin nanoparticles via modification of the surface towards a delivery system for nucleotide-based drugs. Subsequent to this, the newly established carrier system should be evaluated in preclinical trials. In addition to these main projects, it was also an aim of this study to investigate and influence the biodistribution of gelatin nanoparticles. Due to the multitude of independent projects, the present work is divided into five self-contained chapters. In Chapter I, fundamental research data concerning the preparation of gelatin nanoparticles is described. Thereby, the work was focused on process optimization of the existing preparation procedure. Moreover, new analytical tools to characterize gelatin and the gelatin nanoparticles are introduced. Chapter II features the data that were produced in cooperation with the Department of Pharmaceutical Biotechnology at Ludwig-Maximilians-University Munich. In this cooperation, plasmid DNA was bound onto the surface of previously modified gelatin nanoparticles by electrostatic interactions. Subsequent preparation optimization, this simple non-viral gene delivery system was investigated in vitro on murine melanoma cells. The major project of this work, the development and evaluation of gelatin nanoparticles as carrier system for immunogenic so called CpG oligonucleotides is presented in Chapter III and Chapter IV. The data has been generated in cooperation with the Department of Internal Medicine at the Ludwig-Maximilians-University Munich and initially during a 3-month research stay at the Faculty of Pharmacy at the University of Alberta in Edmonton, Canada. Chapter III features extensive in vitro investigations on the respective primary murine and human target cells such as dendritic cells and B cells, whereas in Chapter IV, the results of in vivo experiments are presented. Here, the immunogenic effects of CpG oligonucleotide-loaded nanoparticles alone and their adjuvant activity in combination with the model protein antigen ovalbumin (OVA) were explored. In the final chapter, Chapter V, first PEGylation experiments of gelatin nanoparticles are described, with special emphasis on the establishment of new analytical tools for the quality control of the PEGylation process. In the second part of this chapter, radiolabeling strategies were developed in cooperation with the Department of Nuclear Medicine (TU Munich) to enable real-time in vivo tracking of the gelatin nanoparticles via positron emission tomography (PET).
Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 02/06
Cytokinesis is the process that divides the cytoplasm of a parent cell into two. In animal cells, cytokinesis requires the formation of the central spindle and the contractile ring structures. The onset of cytokinesis is marked during anaphase with the specification of the division site, followed by cleavage furrow formation and ingression, midbody formation and abscission. The astral microtubules that originate from the centrosomes and the anti-parallel microtubules of the central spindle are proposed to determine the site of cleavage furrow formation (Bringmann and Hyman, 2005). The acto-myosin based contractile ring assembles at the division site and constricts the cytoplasm which is supported by the fusion of membrane vesicles to the ingressing plasma membrane. All these processes together result in the formation of two daughter cells. The small GTPase RhoA is one of the most upstream regulators of contractile ring assembly at the cortex. Rho proteins are activated by GEF’s (guanine nucleotide exchange factors) and one GEF that is required for cytokinesis is Ect2 (epithelial cell transforming protein2) (Tatsumoto et al., 1999). The Drosophila pebble (pbl) gene product is the founding member of the Ect2 protein family and has been shown to be required for cytokinesis (Lehner, 1992). In mammals, Ect2 was originally identified as a transforming protein in an expression cloning assay (Miki et al., 1993) and subsequently shown to be essential for cytokinesis. In this study, we have explored the temporal and spatial mechanisms that regulate Ect2 function. In agreement with previous studies, we show that Ect2 is a cell cycle regulated protein and is phosphorylated during mitosis. We identify a number of potentially interesting endogenous phosphorylation sites in Ect2, including potential Plk1 and Cdk1 sites. Although we have not been able to determine the function of these phosphorylation sites, their strong conservation among different species implies that they accomplish evolutionarily conserved roles.The identification of these phosphorylation sites sets the stage for future functional analyses. In complementary studies, we have shown that the central spindle and cell cortex localizations of Ect2 are facilitated by the BRCT and PH domains, respectively. The targeting of Ect2 to the central spindle is mediated by the MKlp1/MgcRacGAP and MKlp2/Aurora-B complexes. Of the two complexes, we show that Ect2 interacts and colocalizes only with the MKlp1/MgcRacGAP complex in telophase and propose that this interaction is mediated by a phosphorylation dependent docking mechanism that targets Ect2 to the central spindle. Interestingly, the displacement of Ect2 from the central spindle did not prevent cytokinesis, suggesting that localized GEF activity is not absolutely essential for cleavage furrow ingression and cytokinesis. In the second part of this thesis, we have explored the role of Ect2 during cytokinesis and show that, in Ect2 depleted cells, levels of RhoA and Citron kinase are diminished at the cleavage site, concomitant with the impairment of cleavage furrow formation and ingression. Additionally, overexpression of appropriate amino-terminal Ect2 fragments in cells also hinders cytokinesis. In these cells, RhoA and Citron kinase localize to the cortex and cleavage furrow ingression occurs, but, the subsequent abscission fails. Taken together, these results suggest that proper function of Ect2 is not only important for cleavage furrow ingression, but also for cell abscission. Finally, we investigate the overexpression phenotypes of different Ect2 truncation mutants. We show that abscission failure correlates with the persistence of amino-terminal Ect2 fragments at striking ring-like structures surrounding the midbody, indicating that completion of cell division requires the displacement of Ect2 from the contractile ring and its re-import into the reforming cell nucleus. Collectively, our data indicate that multiple mechanisms cooperate to regulate Ect2 in a spatial-temporal manner.
Tierärztliche Fakultät - Digitale Hochschulschriften der LMU - Teil 02/07
Lipoprotein Lipase (LPL) is a key enzyme in lipid transport. It catalyses the hydrolysis of the triacylglycerol component of chylomicrons and very low-density lipoproteins (VLDL), providing non-esterified fatty acids for tissue utilization. The gene encoding for LPL has already been identified in several species except the dog. Mutations of the human LPL-gene have been shown to cause partial or complete malfunction of the enzyme, resulting in accumulation of lipoproteins in the blood. This condition is called familial LPL deficiency. LPL malfunction results in hyperlipoproteinemia, recurrent acute pancreatitis, and ultimately pancreatic insufficiency. Several authors have postulated a genetic cause for pancreatitis in the Miniature Schnauzer. An idiopathic increase in serum triglyceride concentration can also be found in this breed. Based on these findings we were evaluating a possible role of the lipoprotein lipase gene in the development of pancreatitis and hyperlipidemia in the Miniature Schnauzer. First, we identified the genetic sequence of the LPL gene in the dog. We determined clones on the Trace Archive database for the canine genome project that contain the genomic sequence of a particular exon as well as its adjacent intronic regions. Based on these findings we designed primers for each exon using the software Netprimer (www.premierbiosoft.com/netprimer/index.html). Canine subjects were chosen from a pool of 170 Miniature Schnauzers from the database at the Gastrointestinal Laboratory at Texas A&M University. Based on clinical history, serum cPLI concentrations, and serum triglyceride concentrations 21 Miniature Schnauzers were chosen and were selected into a clinically normal control group (9 dogs) and an affected group (12 dogs). DNA was then collected from either white blood cells or mucosal cells of these dogs. After PCR optimization, exon 1 through 9 including the adjacent intronic regions were amplified in all dogs using MasterAmp Extra – Long PCR Kit (Epicentre, WI, USA) and were sequenced in triplicates. Differences in the nucleotide sequences were then compared among the two groups. 10 exonic SNPs and 9 intronic SNPs were identified. Upon analysis, none of these variations could be associated with the disease status. We conclude that pancreatitis associated with hyperlipidemia in the Miniature Schnauzer is not linked to mutations of the lipoprotein lipase gene or its splicing regions.
Objectives: DNA sequences are very rich in short repeats and their pattern can be altered by point mutations. We wanted to investigate the effect of single nucleotide polymorphism (SNP) on the pattern of short DNA repeats and its biological consequences. Methods: Analysis of the pattern of short DNA repeats of the Thy-1 sequence with and without SNP. Searching for DNA-binding factors in any region of significance. Results: Comparing the pattern of short repeats in the Thy-1 gene sequences of Turkish patients with ataxia telangiectasia (AT) with the `wild type' sequence from the DNA database, we identified a missing 8-bp repeat element due to an SNP in position 1271 (intron II) in AT-DNA sequences. Only the mutated sequence had the potential for the formation of a stem loop in DNA or pre-mRNA. In super-shift experiments we found that DNA oligomers covering the area of this SNP formed a complex with proteins amongst which we identified the proliferating cell nuclear antigen (PCNA) protein. Conclusion: SNPs have the potential to alter DNA or pre-mRNA conformation. Although no SNP-depeding formation of the DNA-protein complex was evident, future investigations could reveal differential molecular mechanisms of cellular regulation. Copyright (C) 2001 S. Karger AG, Basel.
Sat, 1 Oct 1994 12:00:00 +0100 https://epub.ub.uni-muenchen.de/9517/1/stief_christian_9517.pdf Jonas, Udo; Stief, Christian Georg; Forssmann, W. G.; Truss, Michael C.; Meyer, M. F.; Schulz-Knappe, P.; Taher, A.
Sat, 1 Jan 1994 12:00:00 +0100 http://epub.ub.uni-muenchen.de/3654/ http://epub.ub.uni-muenchen.de/3654/1/3654.pdf Distler, Madeleine; Biel, Martin; Flockerzi, Veit; Hofmann, Franz Distler, Madeleine; Biel, Martin; Flockerzi, Veit und Hofmann, Franz (1994): Expression of cyclic nucleotide-gated cation channels in non-sensory tissues and cells. In: Neuropharmacology, Vol. 33: pp. 1275-1282. Chemi
Cyclic nucleotide-gated cation channels are essential in visual and olfactory signal transduction. An additional member of the cGMP-gated channel family, termed CNG-3, has been cloned from bovine kidney. Its deduced amino acid sequence is 60% and 62% identical with the CNG-channel proteins from bovine rod outer segment and bovine olfactory epithelium, respectively. Northern analysis and sequences amplified by the PCR showed that the CNG-3 mRNA is present in testis, kidney, and heart. Calcium permeated the expressed channel in the presence of extracellular Mg2+ and Na+ at membrane potentials from -100 to +45 mV. It is likely that CNG-3 protein is responsible for cGMP-induced Ca2+ entry in cells other than sensory cells.
Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.
Fri, 15 Jan 1993 12:00:00 +0100 https://epub.ub.uni-muenchen.de/8828/1/kinetic_and_structural_analysis_of_the_mg2plus-binding_site_8828.pdf Wittinghofer, A.; Goody, R. S.; Borasio, Gian Domenico; Vetter, I.; Schlichting, I.; Rensland, H.; John, J.
Fetuins are among the major plasma proteins, yet their biological role has remained elusive. Here we report the molecular cloning of rat fetuin and the sequence analysis of a full-length clone, RF619 of 1456 bp with an open reading frame of 1056 bp encoding 352 amino acid residues. The coding part of RF619 was identical with the cDNA sequence of the natural inhibitor of the insulin receptor tyrosine kinase from rat (pp63) except for four substitutions and a single base insertion causing divergence of the predicted protein sequences. Partial amino acid sequences of rat plasma fetuin were in agreement with the predictions based on the RF619 cDNA. Purified rat fetuin inhibited the insulin receptor tyrosine kinase in vitro. Therefore, we conclude that RF619 and pp63 cDNA encode the same protein, i.e. authentic rat fetuin which is a functional tyrosine kinase inhibitor.
The nucleotide and complete amino acid sequence for the human ß2-glycoprotein I (ß,I) was derived by sequencing the cDNA clone pB2I- 1. In addition to the 326 amino acid residues of the mature protein this clone codes for a putative leader peptide and contains sequence representing 5' and 3' untranslated regions. When this amino acid sequence was compared with the previously published primary sequence, three major amino acid substitutions were found, two involving cysteine residues. These substitutions lead to a new alignment of the complement control protein (CCP) repeats present in fl2I and a prediction of the complete disulphide bond organization. Northern-blot analysis indicates that hepatocytes are a major site of biosynthesis for this protein. A transcription signal of about 1.5 kb was detected by using RNA from HepG2 cells
Sat, 25 Aug 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7603/1/Neupert_Walter_7603.pdf Tropschug, Maximilian; Neupert, Walter; Müller, Harald A.; Harmey, Matthew A.; Rassow, Joachim
Mon, 1 Jan 1990 12:00:00 +0100 https://epub.ub.uni-muenchen.de/8665/1/molecular_cloning_and_complete_nucleotides_sequence_of_the_attenuated_rabies_virus_sad_b19_8665.pdf Thiel, Heinz-Jürgen; Schneider, Lothar G.; Cox, James H.; Conzelmann, Karl-Klaus
Thu, 1 Jan 1987 12:00:00 +0100 https://epub.ub.uni-muenchen.de/8669/1/nucleotide_sequence_and_expression_of_two_beta-tubulin_genes_in_stylonychia_lemnae_8669.pdf Helftenbein, E.; Conzelmann, Karl-Klaus dd
Antigen HLA-B27 is a high-risk genetic factor with respect to a group of rheumatoid disorders, especially ankylosing spondylitis. A cDNA library was constructed from an autozygous B-cell line expressing HLA-B27, HLA-Cw1, and the previously cloned HLA-A2 antigen. Clones detected with an HLA probe' were isolated and sorted into homology groups by differential hybridization and restriction maps. Nucleotide sequencing allowed the unambiguous assignment of cDNAs to HL4-A, -B, and -C loci. The HLA-B27 mRNA has the structural features and the codon variability typical of an HLA class I transcript but it specifies two uncommon amino acid replacements: a cysteine in position 67 and a serine in position 131. The latter substitution may have functional consequences, because it occurs in a conserved region and at a position invariably occupied by a species-specific arginine in humans and lysine in mice. The availability of the complete sequence of HLA-B27 and of the partial sequence of HLA-Cw1 allows the recognition of locus-specific sequence markers, particularly, but not exclusively, in the transmembrane and cytoplasmic domains.
G-Proteins are membrane-bound heterotrimeric polypeptides that couple receptor signals to second messenger systems such as cAMP. Recently, point mutations at 2 codons of the highly preserved alpha-chain of Gs, the adenyl cyclase-stimulating G-protein, were found in GH-secreting pituitary tumors. These mutations resulted in constitutively activated Gs alpha and high intracellular cAMP levels. In addition, point mutations at similar codons of a different G-protein, G(i) alpha 2, were reported in adrenocortical neoplasms, suggesting a potential role of this isoform in the genesis of these tumors. We reevaluated the frequency of constitutively activating point mutations in the alpha- chain of the stimulatory (Gs alpha) and inhibitory (G(i) alpha 2) G- proteins in human adrenocortical tumors. Seven adrenocortical carcinomas, 2 human adrenocortical tumor cell lines, and 11 adrenocortical adenomas were studied. Genomic DNA was purified from either frozen tumor tissue or paraffin-embedded sections. Using specific primers and the polymerase chain reaction, DNA fragments surrounding codons 201 and 227 (Gs alpha) and 179 and 205 (G(i) alpha 2) were amplified and visualized on a 2% agarose gel. In a second asymmetric polymerase chain reaction, using nested primers, single stranded DNA was generated using 1-10 microL of the initial amplification mixture and directly sequenced using the dideoxy chain termination method of Sanger. We found no mutations at codons 201, 227 and 179, 205 of Gs alpha and G(i) alpha 2, respectively, in the tumors studied. We conclude that previously identified oncogenic point mutations in the stimulatory and inhibitory alpha-chain of G-proteins do not appear to be present at high frequency in adrenal neoplasms. Thus, the mechanism(s) of tumorigenesis in these tumors is different from that in GH-secreting adenomas and may involve oncogenic mutations of other cell constituents.
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