{"id":462,"date":"2021-10-07T19:44:20","date_gmt":"2021-10-07T23:44:20","guid":{"rendered":"https:\/\/etiennefd.com\/dgm\/?p=462"},"modified":"2021-10-24T14:51:03","modified_gmt":"2021-10-24T18:51:03","slug":"an-annotated-reading-of-a-paper-about-platypuses","status":"publish","type":"post","link":"https:\/\/etiennefd.com\/dgm\/an-annotated-reading-of-a-paper-about-platypuses\/","title":{"rendered":"An Annotated Reading of a Paper about Platypuses"},"content":{"rendered":"<p><span style=\"font-family: Roboto;\">Here I present a paper I chose to rewrite as a demonstration for the JAWWS project. The original text and figures are reproduced below,<sup class=\"modern-footnotes-footnote \" data-mfn=\"1\" data-mfn-post-scope=\"000000004549207b000000005d9de537_462\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004549207b000000005d9de537_462-1\">1<\/a><\/sup><span id=\"mfn-content-000000004549207b000000005d9de537_462-1\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"1\">the paper has a Creative Commons non-commercial license<\/span> interspersed with my comments in the following format:<\/span><\/p>\n<blockquote><p><span style=\"font-family: Roboto; color: #0000ff;\">hello I am a blue comment in a quote-block<\/span><\/p><\/blockquote>\n<p><span style=\"font-family: Roboto;\">Feel free to just read the comments. Annotating the paper was a first step in the process. Next I will focus on the rewriting per se. Should be fun!<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">I didn&#8217;t have a particularly strict selection procedure \u2014 I went on <a href=\"https:\/\/www.researchhub.com\/\">ResearchHub<\/a>, in the evolutionary biology section (since that used to be my field), and picked one that seemed appropriate. A cursory skimming showed it had plenty of abbreviations and long paragraphs, which suggested there was a lot of room for improvement.<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">Also, it&#8217;s about platypuses. Or platypi. Platypodes. Whatever.<\/span><\/p>\n<p><img loading=\"lazy\" decoding=\"async\" class=\"alignnone size-large wp-image-497\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-1024x515.jpg\" alt=\"\" width=\"580\" height=\"292\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-1024x515.jpg 1024w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-300x151.jpg 300w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-768x386.jpg 768w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-1536x773.jpg 1536w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-2048x1030.jpg 2048w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-1200x604.jpg 1200w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/Platypus_Ornithorhynchus_anatinus._First_Description_1799-1980x996.jpg 1980w\" sizes=\"auto, (max-width: 580px) 100vw, 580px\" \/><\/p>\n<p><span style=\"font-family: Roboto;\">Here are the metadata:<\/span><\/p>\n<ul>\n<li><span style=\"font-family: Roboto;\"><strong>Title<\/strong>: &#8220;A Model for the Evolution of the Mammalian T-cell Receptor \u03b1\/\u03b4 and \u03bc Loci Based on Evidence from the Duckbill Platypus&#8221;<\/span><\/li>\n<li><span style=\"font-family: Roboto;\"><strong>Authors<\/strong>: Zuly E. Parra, Mette Lillie, Robert D. Miller<\/span><\/li>\n<li><span style=\"font-family: Roboto;\"><strong>Journal<\/strong>: Molecular Biology and Evolution<\/span><\/li>\n<li><span style=\"font-family: Roboto;\"><a href=\"https:\/\/academic.oup.com\/mbe\/article\/29\/10\/3205\/1030857\"><strong>Link<\/strong> to original version<\/a><\/span><\/li>\n<li><span style=\"font-family: Roboto;\"><strong>Word count<\/strong>: 5,800 words.<\/span><\/li>\n<\/ul>\n<p><span style=\"font-family: Roboto;\">A disclaimer: some of the comments below will be harsh. Again, I don&#8217;t mean to attack the authors, who did their job as well as they could, and in fact succeeded at it \u2014 after all, they managed to publish their work!<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">With that, let&#8217;s pretend we&#8217;re semi-aquatic platypuses and dive in.<\/span><\/p>\n<h2><span style=\"font-family: Roboto;\">A Model for the Evolution of the Mammalian T-cell Receptor \u03b1\/\u03b4 and \u03bc Loci Based on Evidence from the Duckbill Platypus<\/span><\/h2>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Comments: Okay, this paper is going to be about T cells (I vaguely remember this being about immunity?), platypuses, and evolution. Sounds good.<\/span><\/p><\/blockquote>\n<h2><span style=\"font-size: 48px; font-weight: bold; letter-spacing: -0.0415625em; font-family: Roboto;\">Abstract<\/span><\/h2>\n<p><span style=\"font-family: Roboto;\">The specific recognition of antigen by T cells is critical to the generation of adaptive immune responses in vertebrates. T cells recognize antigen using a somatically diversified T-cell receptor (TCR). All jawed vertebrates use four TCR chains called \u03b1, \u03b2, \u03b3, and \u03b4, which are expressed as either a \u03b1\u03b2 or \u03b3\u03b4 heterodimer. Nonplacental mammals (monotremes and marsupials) are unusual in that their genomes encode a fifth TCR chain, called TCR\u00b5, whose function is not known but is also somatically diversified like the conventional chains. The origins of TCR\u00b5 are also unclear, although it appears distantly related to TCR\u03b4. Recent analysis of avian and amphibian genomes has provided insight into a model for understanding the evolution of the TCR\u03b4 genes in tetrapods that was not evident from humans, mice, or other commonly studied placental (eutherian) mammals. An analysis of the genes encoding the TCR\u03b4 chains in the duckbill platypus revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy (IgH) chain V genes (VH) and related to V genes used in TCR\u00b5. They are expressed as part of TCR\u03b4 repertoire (VH\u03b4) and similar to what has been found in frogs and birds. This, however, is the first time a VH\u03b4 has been found in a mammal and provides a critical link in reconstructing the evolutionary history of TCR\u00b5. The current structure of TCR\u03b4 and TCR\u00b5 genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the IgH and TCR\u03b4 genes, as well as translocations of TCR\u03b4 genes out of the TCR\u03b1\/\u03b4 locus early in mammals, creating the TCR\u00b5 locus.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Comments: That&#8217;s a pretty dense abstract. There&#8217;s a lot of acronyms in there, which I find distracting. Also, it&#8217;s not immediately obvious why we should be interested in this paper. It seems to be this: studying platypuses uncovered new information about how T cells evolved. But that info is buried in the fourth sentence and beyond.<\/span><\/p><\/blockquote>\n<h2><span style=\"font-family: Roboto;\">Introduction<\/span><\/h2>\n<p><span style=\"font-family: Roboto;\">T lymphocytes are critical to the adaptive immune system of all jawed vertebrates and can be classified into two main lineages based on the T-cell receptor (TCR) they use (<span id=\"jumplink-mss128-B29\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B29\">Rast et al. 1997<\/a>; reviewed in\u00a0<span id=\"jumplink-mss128-B9\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B9\">Davis and Chein 2008<\/a>). The majority of circulating human T cells are the \u03b1\u03b2T cell lineage which use a TCR composed of a heterodimer of \u03b1 and \u03b2 TCR chains. \u03b1\u03b2T cells include the familiar T cell subsets such as CD4+ helper T cells and regulatory T cells, CD8+ cytotoxic T cells, and natural killer T (NKT) cells. T cells that are found primarily in epithelial tissues and a lower percentage of circulating lymphocytes in some species express a TCR composed of \u03b3 and \u03b4 TCR chains. The function of these \u03b3\u03b4 T cells is less well defined and they have been associated with a broad range of immune responses including tumor surveillance, innate responses to pathogens and stress, and wound healing (<span id=\"jumplink-mss128-B16\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B16\">Hayday 2009<\/a>). \u03b1\u03b2 and \u03b3\u03b4 T cells also differ in the way they interact with antigen. \u03b1\u03b2TCR are major histocompatibility complex (MHC) \u201crestricted\u201d in that they bind antigenic epitopes, such as peptide fragments, bound to, or \u201cpresented\u201d by, molecules encoded in the MHC. In contrast, \u03b3\u03b4TCR have been found to bind antigens directly in the absence of MHC, as well as self-ligands that are often MHC-related molecules (<span id=\"jumplink-mss128-B34\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B34\">Sciammas et al. 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B16\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B16\">Hayday 2009<\/a>).<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">I can hardly think of a less exciting introduction. I&#8217;m expecting talk of platypuses, of puzzling questions about evolution or the immune system \u2014 and all I get is a boring lecture on T cells. Make no mistake: all of this information is important. We need to know a T cell is, what&#8217;s a T-cell receptor, and that there exist at least two kinds (\u03b1\u03b2 and \u03b3\u03b4).<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">But this information shouldn&#8217;t be put first. And it could definitely be split up into more paragraphs.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The conventional TCR chains are composed of two extracellular domains that are both members of the immunoglobulin (Ig) domain super-family (reviewed in\u00a0<span id=\"jumplink-mss128-B9\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B9\">Davis and Chein 2008<\/a>) (<span id=\"jumplink-mss128-F1\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F1\">fig. 1<\/a>). The membrane proximal domain is the constant (C) domain, which is largely invariant amongst T-cell clones expressing the same class of TCR chain, and is usually encoded by a single, intact exon. The membrane distal domain is called the variable (V) domain and is the region of the TCR that contacts antigen and MHC. Similar to antibodies, the individual clonal diversity in the TCR V domains is generated by somatic DNA recombination (<span id=\"jumplink-mss128-B36\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B36\">Tonegawa 1983<\/a>). The exons encoding TCR V domains are assembled somatically from germ-line gene segments, called the V, diversity (D), and joining (J) genes, in developing T cells, a process dependent upon the enzymes encoded by the recombination activating genes (<em>RAG<\/em>)-1 and\u00a0<em>RAG<\/em>-2 (<span id=\"jumplink-mss128-B43\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B43\">Yancopoulos et al. 1986<\/a>;\u00a0<span id=\"jumplink-mss128-B32\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B32\">Schatz et al. 1989<\/a>). The exons encoding the V domains of TCR \u03b2 and \u03b4 chains are assembled from all three types of gene segments, whereas the \u03b1 and \u03b3 chains use only V and J. The different combinations of V, D, and J or V and J, selected from a large repertoire of germ-line gene segments, along with variation at the junctions due to addition and deletion of nucleotides during recombination, contribute to a vast TCR diversity. It is this diversity that creates the individual antigen specificity of T-cell clones.<\/span><\/p>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F1\">\n<div class=\"label fig-label\"><span style=\"font-family: Roboto;\">Fig. 1.<\/span><\/div>\n<div>\n<figure id=\"attachment_528\" aria-describedby=\"caption-attachment-528\" style=\"width: 520px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-528 size-full\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f1.jpg\" alt=\"\" width=\"520\" height=\"371\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f1.jpg 520w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f1-300x214.jpg 300w\" sizes=\"auto, (max-width: 520px) 100vw, 520px\" \/><figcaption id=\"caption-attachment-528\" class=\"wp-caption-text\">Cartoon diagram of the TCR forms found in different species. Oblong circles indicate Ig super-family domains and are color coded as C domains (blue), conventional TCR V domains (red), and VH\u03b4 or V\u00b5 (yellow). The gray shaded chains represent the hypothetical partner chain for TCR\u00b5 and TCR\u03b4 using VH\u03b4.<\/figcaption><\/figure>\n<\/div>\n<div class=\"graphic-wrap\"><\/div>\n<\/div>\n<div class=\"fig fig-modal reveal-modal\">\n<div class=\"graphic-wrap\">\n<blockquote>\n<div class=\"fig-orig original-slide\"><span style=\"font-family: Roboto; letter-spacing: normal; color: #0000ff;\">The figure helps, but again, why are we reading this? This paper seems to follow the common pattern in which the introduction gradually &#8220;zooms into&#8221; the main point. This is not a good pattern, because it doesn&#8217;t tell us the reason for this information. Sure, we suspect it&#8217;s relevant to understand what comes next, but without any mystery to anchor this to, it&#8217;s hard to be really engaged.<\/span><\/div>\n<\/blockquote>\n<p class=\"fig-orig original-slide\"><span style=\"font-family: Roboto;\"><span style=\"letter-spacing: normal;\">The TCR genes are highly conserved among species in both genomic sequence and organization (<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B29\">Rast et al. 1997<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B25\">Parra et al. 2008<\/a><span style=\"letter-spacing: normal;\">,\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B27\">2012<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B5\">Chen et al. 2009<\/a><span style=\"letter-spacing: normal;\">). In all tetrapods examined, the TCR\u03b2 and \u03b3 chains are each encoded at separate loci, whereas the genes encoding the \u03b1 and \u03b4 chains are nested at a single locus (<\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">) (<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B6\">Chien et al. 1987<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B31\">Satyanarayana et al. 1988<\/a><span style=\"letter-spacing: normal;\">; reviewed in\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B9\">Davis and Chein 2008<\/a><span style=\"letter-spacing: normal;\">). The V domains of TCR\u03b1 and TCR\u03b4 chains can use a common pool of V gene segments, but distinct D, J, and C genes.<\/span><\/span><\/p>\n<p class=\"fig-orig original-slide\"><span style=\"font-family: Roboto;\"><span style=\"letter-spacing: normal;\">Diversity in antibodies produced by B cells is also generated by RAG-mediated V(D)J recombination and the TCR and Ig genes clearly share a common origin in the jawed-vertebrates (<\/span><span id=\"jumplink-mss128-B11\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B11\">Flajnik and Kasahara 2010<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><span id=\"jumplink-mss128-B21\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B21\">Litman et al. 2010<\/a><span style=\"letter-spacing: normal;\">). However, the V, D, J, and C coding regions in TCR have diverged sufficiently over the past &gt;400 million years (MY) from Ig genes that they are readily distinguishable, at least for the conventional TCR. Recently, the boundary between TCR and Ig genes has been blurred with the discovery of non-conventional TCR\u03b4 isoforms that have been found that use V genes that appear indistinguishable from Ig heavy chain V (VH) (<\/span><span id=\"jumplink-mss128-B28\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B28\">Parra et al. 2010<\/a><span style=\"letter-spacing: normal;\">,\u00a0<\/span><span id=\"jumplink-mss128-B27\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B27\">2012<\/a><span style=\"letter-spacing: normal;\">). Such V genes have been designated as VH\u03b4 and have been found in both amphibians and birds (<\/span><span id=\"jumplink-mss128-F1\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-reveal link-table xref-fig\" style=\"letter-spacing: normal;\" data-open=\"mss128-F1\">fig. 1<\/a><span style=\"letter-spacing: normal;\">). In the frog\u00a0<\/span><em style=\"letter-spacing: normal;\">Xenopus tropicalis,<\/em><span style=\"letter-spacing: normal;\">\u00a0and a passerine bird, the zebra finch\u00a0<\/span><em style=\"letter-spacing: normal;\">Taeniopygia guttata<\/em><span style=\"letter-spacing: normal;\">\u00a0the VH\u03b4 are located within the\u00a0<\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0loci where they co-exist with conventional V\u03b1 and V\u03b4 genes (<\/span><span id=\"jumplink-mss128-B28\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B28\">Parra et al. 2010<\/a><span style=\"letter-spacing: normal;\">,\u00a0<\/span><span id=\"jumplink-mss128-B27\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B27\">2012<\/a><span style=\"letter-spacing: normal;\">). In galliform birds, such as the chicken\u00a0<\/span><em style=\"letter-spacing: normal;\">Gallus gallus<\/em><span style=\"letter-spacing: normal;\">, VH\u03b4 are present but located at a second\u00a0<\/span><em style=\"letter-spacing: normal;\">TCR\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0locus that is unlinked to the conventional\u00a0<\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0(<\/span><span id=\"jumplink-mss128-B27\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B27\">Parra et al. 2012<\/a><span style=\"letter-spacing: normal;\">). VH\u03b4 are the only type of V gene segment present at the second locus and, although closely related to antibody VH genes, the VH\u03b4 appear to be used exclusively in TCR\u03b4 chains. This is true as well for frogs where the\u00a0<\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0and\u00a0<\/span><em style=\"letter-spacing: normal;\">IgH<\/em><span style=\"letter-spacing: normal;\">\u00a0loci are tightly linked (<\/span><span id=\"jumplink-mss128-B28\" class=\"xrefLink\" style=\"letter-spacing: normal;\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B28\">Parra et al. 2010<\/a><span style=\"letter-spacing: normal;\">).<\/span><\/span><\/p>\n<\/div>\n<\/div>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Okay&#8230; different species have slightly different genes&#8230; Cool.<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">Also, &#8220;MY&#8221; for million years, really? Do we really need that, especially when there are already about five abbreviations per sentence?<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci have been characterized in several eutherian mammal species and at least one marsupial, the opossum\u00a0<em>Monodelphis domestica<\/em>, and VH\u03b4 genes have not been found to date (<span id=\"jumplink-mss128-B31\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B31\">Satyanarayana et al.1988<\/a>;\u00a0<span id=\"jumplink-mss128-B39\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B39\">Wang et al. 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). However, marsupials do have an additional TCR locus, unlinked to\u00a0<em>TCR\u03b1\/\u03b4<\/em>, that uses antibody-related V genes. This fifth TCR chain is called TCR\u00b5 and is related to TCR\u03b4, although it is highly divergent in sequence and structure (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>,\u00a0<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">2008<\/a>). A TCR\u00b5 has also been found in the duckbill platypus and is clearly orthologous to the marsupial genes, consistent with this TCR chain being ancient in mammals, although it has been lost in the eutherians (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). TCR\u00b5 chains use their own unique set of V genes (V\u00b5) (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCR\u00b5 genes has not been found. So far, TCR\u00b5 homologues have not been found in non-mammals (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>).<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">After an overview of non-mammal tetrapods (frogs, birds), we&#8217;re now talking about mammals: platypuses, marsupials, eutherians. It seems like the zooming in is coming to an end&#8230;<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">TCR\u00b5 chains are atypical in that they contain three extra-cellular IgSF domains rather than the conventional two, due to an extra N-terminal V domain (<span id=\"jumplink-mss128-F1\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F1\">fig. 1<\/a>) (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). Both V domains are encoded by a unique set of V\u00b5 genes and are more related to Ig VH than to conventional TCR V domains. The N-terminal V domain is diverse and encoded by genes that undergo somatic V(D)J recombination. The second or supporting V domain has little or no diversity. In marsupials this V domain is encoded by a germ-line joined, or pre-assembled, V exon that is invariant (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>). The second V domain in platypus is encoded by gene segments requiring somatic DNA recombination; however, only limited diversity is generated partly due to the lack of D segments (<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). A TCR chain structurally similar to TCR\u00b5 has also been described in sharks and other cartilaginous fish (<span id=\"jumplink-mss128-F1\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F1\">fig. 1<\/a>) (<span id=\"jumplink-mss128-B8\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B8\">Criscitiello et al. 2006<\/a>;\u00a0<span id=\"jumplink-mss128-B10\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B10\">Flajnik et al. 2011<\/a>). This TCR, called NAR-TCR, also contains three extracellular domains, with the N-terminal V domain being related to those used by IgNAR antibodies, a type of antibody found only in sharks (<span id=\"jumplink-mss128-B12\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B12\">Greenberg et al. 1995<\/a>). The current working model for both TCR\u00b5 and NAR-TCR is that the N-terminal V domain is unpaired and acts as a single, antigen binding domain, analogous to the V domains of light-chainless antibodies found in sharks and camelids (<span id=\"jumplink-mss128-B10\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B10\">Flajnik et al. 2011<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>).<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">I&#8217;ve tried reading this paragraph like five times and I&#8217;m still not sure what it&#8217;s trying to say. It feels like it&#8217;s mostly disjointed sentences that\u00a0<em>had<\/em> to be included so the authors can assume you know this, but since we <em>still<\/em> don&#8217;t have a vision of the larger picture, it&#8217;s really hard to pay attention.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Phylogenetic analyses support the origins of TCR\u00b5 occurring after the avian\u2013mammalian split (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). Previously, we hypothesized the origin of TCR\u00b5 being the result of a recombination between ancestral IgH and TCR\u03b4-like loci (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). This hypothesis, however, is problematic for a number of reasons. One challenge is the apparent genomic stability and ancient conserved synteny in the region surrounding the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus; this region has appeared to remain stable over at least the past 350 MY of tetrapod evolution (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>,\u00a0<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">2010<\/a>). The discovery of VH\u03b4 genes inserted into the TCR\u03b1\/\u03b4 locus of amphibians and birds has provided an alternative model for the origins of TCR\u00b5; this model involves both the insertion of VH followed by the duplication and translocation of TCR genes. Here we present the model along with supporting evidence drawn from the structure of the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus, which is also the first analysis of this complex locus in a monotreme.<\/span><\/p>\n<blockquote><p><span style=\"font-family: Roboto; color: #0000ff;\">The last sentence is the first interesting one of the entire paper. It could have come earlier. Technically we should know this from the abstract, but the abstract was pretty difficult to read too.<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">Also, this is definitely at least two paragraphs merged into one: the first about the previous hypothesis, and the second about the alternative model that is going to be presented.<\/span><\/p><\/blockquote>\n<h2 id=\"77835853\" class=\"section-title js-splitscreen-section-title\"><span style=\"font-family: Roboto;\">Materials and Methods<\/span><\/h2>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">The intro was painful, and usually materials and methods are even worse. We&#8217;ll see! \ud83d\ude42<\/span><\/p><\/blockquote>\n<h3 id=\"77835854\" class=\"section-title js-splitscreen-section-title\"><span style=\"font-family: Roboto;\">Identification and Annotation of the Platypus TCR\u03b1\/\u03b4 Locus<\/span><\/h3>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The analyses were performed using the platypus (<em>Ornithorhynchus anatinus<\/em>) genome assembly version 5.0.1 (<a class=\"link link-uri openInAnotherWindow\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/genome\/guide\/platypus\/\" target=\"_blank\" rel=\"noopener\">http:\/\/www.ncbi.nlm.nih.gov\/genome\/guide\/platypus\/<\/a>). The platypus genome was analyzed using the whole-genome BLAST available at NCBI (<a class=\"link link-uri openInAnotherWindow\" href=\"http:\/\/www.ncbi.nlm.nih.gov\/\" target=\"_blank\" rel=\"noopener\">www.ncbi.nlm.nih.gov\/<\/a>) and the BLAST\/BLAT tool from Ensembl (<a class=\"link link-uri openInAnotherWindow\" href=\"http:\/\/www.ensembl.org\/\" target=\"_blank\" rel=\"noopener\">www.ensembl.org<\/a>). The V and J segments were located by similarity to corresponding segments from other species and by identifying the flanking conserved recombination signal sequences (RSS). V gene segments were annotated 5\u2032 to 3\u2032 as V\u03b1 or V\u03b4 followed by the family number and the gene segment number if there were greater than one in the family. For example, V\u03b115.7 is the seventh V\u03b1 gene in family 15. The D segments were identified using complementarity-determining region-3 (CDR3) sequences that represent the V\u2013D\u2013J junctions, from cDNA clones using VH\u03b4. Platypus TCR gene segments were labeled according to the IMGT nomenclature (<a class=\"link link-uri openInAnotherWindow\" href=\"http:\/\/www.imgt.org\/\" target=\"_blank\" rel=\"noopener\">http:\/\/www.imgt.org\/<\/a>). The location for the TCR\u03b1\/\u03b4 genes in the platypus genome version 5.0.1 is provided in\u00a0<span class=\"content-section supplementary-material\"><a href=\"https:\/\/oup.silverchair-cdn.com\/oup\/backfile\/Content_public\/Journal\/mbe\/29\/10\/10.1093_molbev_mss128\/2\/mss128_Supplementary_Data.zip?Expires=1634352201&amp;Signature=neboZ0FNmw047UwMfYUhEy2IWIl3C7EoJTcRuEhjqgbYDkr6Hl0kC-wC7nJ8hP6rSGtMrtQdWlICuoeBVPv2NFN7~0YriPkOjyk-cYzkM~j4LmodwZlf60noDxlTUgUiv7EE~zCNMy8QU3EV4BhoP1Lw3lyeOcnbFw8U9N~9hHlzUym-4QspBMAIPfQf2VbvzdiMVm-N6051W-nXV9N8jR-pXczAQFql2Q8lA7VLt-W0kEVw-BIN3a3uWZf~ZTUrSFT1WLPYv86FRKEvkP0YEPjYUHApbkCJUvJCVfbgc9MgvhVEKWGnHkh1Q6eiU-7LVuXx7GYzL6X8m40~IguZyA__&amp;Key-Pair-Id=APKAIE5G5CRDK6RD3PGA\">supplementary table S1<\/a><\/span>,\u00a0<span class=\"content-section supplementary-material\"><a href=\"https:\/\/oup.silverchair-cdn.com\/oup\/backfile\/Content_public\/Journal\/mbe\/29\/10\/10.1093_molbev_mss128\/2\/mss128_Supplementary_Data.zip?Expires=1634352201&amp;Signature=neboZ0FNmw047UwMfYUhEy2IWIl3C7EoJTcRuEhjqgbYDkr6Hl0kC-wC7nJ8hP6rSGtMrtQdWlICuoeBVPv2NFN7~0YriPkOjyk-cYzkM~j4LmodwZlf60noDxlTUgUiv7EE~zCNMy8QU3EV4BhoP1Lw3lyeOcnbFw8U9N~9hHlzUym-4QspBMAIPfQf2VbvzdiMVm-N6051W-nXV9N8jR-pXczAQFql2Q8lA7VLt-W0kEVw-BIN3a3uWZf~ZTUrSFT1WLPYv86FRKEvkP0YEPjYUHApbkCJUvJCVfbgc9MgvhVEKWGnHkh1Q6eiU-7LVuXx7GYzL6X8m40~IguZyA__&amp;Key-Pair-Id=APKAIE5G5CRDK6RD3PGA\">Supplementary Material online<\/a><\/span>.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Actually, this isn&#8217;t <em>that<\/em> bad: it&#8217;s easier to follow than the introduction because it tells us sequential actions. They make sense together.<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">But there are a few things wrong here. First, the use of the dreaded passive voice. &#8220;The analyses were performed &#8230;&#8221; No! Tell us who performed it! Second, it&#8217;s a pretty dense paragraph and the only one in its section (Identification and Annotation &#8230;), which means there&#8217;s no benefit to bundling all these sentences together: the title already serves this purpose. Third, it lacks some sentence to tell us what the goal is. The intro was not clear enough to assume readers know what the end point of these analyses is.<\/span><\/p><\/blockquote>\n<h3 id=\"77835856\" class=\"section-title js-splitscreen-section-title\"><span style=\"font-family: Roboto;\">Confirmation of Expression of Platypus VH\u03b4<\/span><\/h3>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Reverse transcription PCR (RT\u2013PCR) was performed on total splenic RNA extracted from a male platypus from the Upper Barnard River, New South Wales, Australia. This platypus was collected under the same permits as in\u00a0<span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B41\">Warren et al. (2008)<\/a>. The cDNA synthesis step was carried out using the Invitrogen Superscript III-first strand synthesis kit according to the manufacturer\u2019s recommended protocol (Invitrogen, Carlsbad, CA, USA). TCR\u03b4 transcripts containing VH\u03b4 were targeted using primers specific for the C\u03b4 and VH\u03b4 genes identified in the platypus genome assembly (<span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B41\">Warren et al. 2008<\/a>). PCR amplification was performed using the QIAGEN HotStar HiFidelity Polymerase Kit (BD Biosciences, CLONTECH Laboratories, Palo Alto, CA, USA) in total volume of 20 \u00b5l containing 1\u00d7 Hotstar Hifi PCR Buffer (containing 0.3 mM dNTPs), 1\u00b5M of primers, and 1.25U Hotstar Hifidelity DNA polymerase. The PCR primers used were 5\u2032-GTACCGCCAACCACCAGGGAAAG-3\u2032 and 5\u2032-CAGTTCACTGCTCCATCGCTTTCA-3\u2032 for the VH\u03b4 and C\u03b4, respectively. A previously described platypus spleen cDNA library constructed from RNA extracted from tissue from a Tasmanian animal was also used (<span id=\"jumplink-mss128-B38\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B38\">Vernersson et al. 2002<\/a>).<\/span><\/p>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">PCR products were cloned using TopoTA cloning\u00ae kit (Invitrogen). Sequencing was performed using the BigDye terminator cycle sequencing kit version 3 (Applied Biosystems, Foster City, CA, USA) and according to the manufacturer recommendations. Sequencing reactions were analyzed using the ABI Prism 3100 DNA automated sequences (PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA). Chromatograms were analyzed using the Sequencher 4.9 software (Gene Codes Corporation, Ann Arbor, MI, USA). Sequences have been archived on GenBank under accession numbers JQ664690\u2013JQ664710.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">This seems to be mostly a list of the machines, substances, protocols etc. that were used. Accordingly, it should be formatted as a list. It doesn&#8217;t read well as a paragraph (nor should it be expected to).<\/span><\/p><\/blockquote>\n<h3 id=\"77835859\" class=\"section-title js-splitscreen-section-title\"><span style=\"font-family: Roboto;\">Phylogenetic Analyses<\/span><\/h3>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Nucleotide sequences from FR1 to FR3 of the V genes regions, including CDR1 and CDR2, were aligned using BioEdit (<span id=\"jumplink-mss128-B14\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B14\">Hall 1999<\/a>) and the accessory application ClustalX (<span id=\"jumplink-mss128-B35\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B35\">Thompson et al. 1997<\/a>). Nucleotide alignments analyzed were based on amino acid sequence to establish codon position (<span id=\"jumplink-mss128-B14\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B14\">Hall 1999<\/a>). Alignments were corrected by visual inspection when necessary and were then analyzed using the MEGA Software (<span id=\"jumplink-mss128-B18\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B18\">Kumar et al. 2004<\/a>). Neighbor joining (NJ) with uncorrected nucleotide differences (p-distance) and minimum evolution distances methods were used. Support for the generated trees was evaluated based on bootstrap values generated by 1000 replicates. GenBank accession numbers for sequences used in the tree construction are in\u00a0<span class=\"content-section supplementary-material\"><a href=\"https:\/\/oup.silverchair-cdn.com\/oup\/backfile\/Content_public\/Journal\/mbe\/29\/10\/10.1093_molbev_mss128\/2\/mss128_Supplementary_Data.zip?Expires=1634352201&amp;Signature=neboZ0FNmw047UwMfYUhEy2IWIl3C7EoJTcRuEhjqgbYDkr6Hl0kC-wC7nJ8hP6rSGtMrtQdWlICuoeBVPv2NFN7~0YriPkOjyk-cYzkM~j4LmodwZlf60noDxlTUgUiv7EE~zCNMy8QU3EV4BhoP1Lw3lyeOcnbFw8U9N~9hHlzUym-4QspBMAIPfQf2VbvzdiMVm-N6051W-nXV9N8jR-pXczAQFql2Q8lA7VLt-W0kEVw-BIN3a3uWZf~ZTUrSFT1WLPYv86FRKEvkP0YEPjYUHApbkCJUvJCVfbgc9MgvhVEKWGnHkh1Q6eiU-7LVuXx7GYzL6X8m40~IguZyA__&amp;Key-Pair-Id=APKAIE5G5CRDK6RD3PGA\">supplementary table S2<\/a><\/span>,\u00a0<span class=\"content-section supplementary-material\"><a href=\"https:\/\/oup.silverchair-cdn.com\/oup\/backfile\/Content_public\/Journal\/mbe\/29\/10\/10.1093_molbev_mss128\/2\/mss128_Supplementary_Data.zip?Expires=1634352201&amp;Signature=neboZ0FNmw047UwMfYUhEy2IWIl3C7EoJTcRuEhjqgbYDkr6Hl0kC-wC7nJ8hP6rSGtMrtQdWlICuoeBVPv2NFN7~0YriPkOjyk-cYzkM~j4LmodwZlf60noDxlTUgUiv7EE~zCNMy8QU3EV4BhoP1Lw3lyeOcnbFw8U9N~9hHlzUym-4QspBMAIPfQf2VbvzdiMVm-N6051W-nXV9N8jR-pXczAQFql2Q8lA7VLt-W0kEVw-BIN3a3uWZf~ZTUrSFT1WLPYv86FRKEvkP0YEPjYUHApbkCJUvJCVfbgc9MgvhVEKWGnHkh1Q6eiU-7LVuXx7GYzL6X8m40~IguZyA__&amp;Key-Pair-Id=APKAIE5G5CRDK6RD3PGA\">Supplementary Material online<\/a><\/span>.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">I have a graduate degree in evolutionary biology, I&#8217;ve done plenty of phylogenetic analyses (building trees of life), and somehow I hadn&#8217;t understood yet that this is what this paper was about. Maybe that&#8217;s really obvious to practicing evolutionary biologists, but it seems to me that the kind of analysis could have been made more obvious earlier.<\/span><\/p><\/blockquote>\n<h2 id=\"77835861\" class=\"section-title js-splitscreen-section-title\"><span style=\"font-family: Roboto;\">Results and Discussion<\/span><\/h2>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Not a bad idea to merge results and discussion together IMO, as long as it doesn&#8217;t hinder comprehension.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus was identified in the current platypus genome assembly and the V, D, J, and C gene segments and exons were annotated and characterized (<span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F2\">fig. 2<\/a>). The majority of the locus was present on a single scaffold, with the remainder on a shorter contig (<span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F2\">fig. 2<\/a>). Flanking the locus were\u00a0<em>SALL2<\/em>,\u00a0<em>DAD1<\/em>\u00a0and several olfactory receptor (<em>OR<\/em>) genes, all of which share conserved synteny with the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus in amphibians, birds, and mammals (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>,\u00a0<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">2010<\/a>,\u00a0<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">2012<\/a>). The platypus locus has many typical features common to\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci in other tetrapods (<span id=\"jumplink-mss128-B31\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B31\">Satyanarayana et al. 1988<\/a>;\u00a0<span id=\"jumplink-mss128-B39\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B39\">Wang et al. 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>,\u00a0<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">2010<\/a>,\u00a0<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">2012<\/a>). Two C region genes were present: a C\u03b1 that is the most 3\u2032 coding segment in the locus, and a C\u03b4 oriented 5\u2032 of the J\u03b1 genes. There is a large number of J\u03b1 gene segments (<em>n<\/em>\u00a0= 32) located between the C\u03b4 and C\u03b1 genes. Such a large array of J\u03b1 genes are believed to facilitate secondary V\u03b1 to J\u03b1 rearrangements in developing \u03b1\u03b2T cells if the primary rearrangements are nonproductive or need replacement (<span id=\"jumplink-mss128-B15\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B15\">Hawwari and Krangel 2007<\/a>). Primary TCR\u03b1 V\u2013J rearrangments generally use J\u03b1 segments towards the 5\u2032-end of the array and can progressively use downstream J\u03b1 in subsequent rearrangements. There is also a single V\u03b4 gene in reverse transcriptional orientation between the platypus C\u03b4 gene and the J\u03b1 array that is conserved in mammalian\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0both in location and orientation (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>).<\/span><\/p>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F2\">\n<div class=\"label fig-label\"><span style=\"font-family: Roboto;\">Fig. 2.<\/span><\/div>\n<div>\n<figure id=\"attachment_538\" aria-describedby=\"caption-attachment-538\" style=\"width: 520px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-538 size-full\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f2.jpg\" alt=\"\" width=\"520\" height=\"640\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f2.jpg 520w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f2-244x300.jpg 244w\" sizes=\"auto, (max-width: 520px) 100vw, 520px\" \/><figcaption id=\"caption-attachment-538\" class=\"wp-caption-text\">Annotated map of the platypus TCR\u03b1\/\u03b4 locus showing the locations of the V\u03b1 and V\u03b4 (red), VH\u03b4 (yellow), D\u03b4 (orange), J\u03b1 and J\u03b4 (green), C\u03b4 (dark blue), and C\u03b1 (light blue). Conserved syntenic genes are in gray. The scaffold and contig numbers are indicated.<\/figcaption><\/figure>\n<\/div>\n<div class=\"graphic-wrap\"><\/div>\n<\/div>\n<div class=\"fig fig-modal reveal-modal\">\n<div class=\"graphic-wrap\">\n<blockquote>\n<div class=\"fig-orig original-slide\"><span style=\"font-family: Roboto; color: #0000ff;\"><span style=\"letter-spacing: normal;\">Oof. I had to actually add line breaks to this paragraph to parse it. It mostly says the same things as the figure, which isn&#8217;t too bad. Repeating important info in multiple formats is a good idea. The figure itself could have been clearer, though \u2014 it took me a few minutes to understand that the multiple lines in it represent contiguous segments of the chromosome (at least that&#8217;s what I think it means). I also had to look up what &#8220;synteny&#8221; means: it&#8217;s having the same order for genetic elements across species.\u00a0<\/span><\/span><\/div>\n<\/blockquote>\n<div class=\"fig-orig original-slide\"><span style=\"font-family: Roboto;\"><span style=\"letter-spacing: normal;\">There <\/span><span style=\"letter-spacing: normal;\">are 99 conventional TCR V gene segments in the platypus <\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0locus, 89 of which share nucleotide identity with V\u03b1 in other species and 10 that share identity with V\u03b4 genes. The V\u03b4 genes are clustered towards the 3\u2032-end of the locus. Based on nucleotide identity shared among the platypus V genes they can be classified into 17 different V\u03b1 families and two different V\u03b4 families, based on the criteria of a V family sharing &gt;80% nucleotide identity (not shown, but annotated in\u00a0<\/span><a class=\"link link-reveal link-table xref-fig\" style=\"letter-spacing: normal;\" data-open=\"mss128-F2\">fig. 2<\/a><span style=\"letter-spacing: normal;\">). This is also a typical level of complexity for mammalian V\u03b1 and V\u03b4 genes (<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B13\">Giudicelli et al. 2005<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B25\">Parra et al. 2008<\/a><span style=\"letter-spacing: normal;\">). Also present were two D\u03b4 and seven J\u03b4 gene segments oriented upstream of the C\u03b4. All gene segments were flanked by canonical RSS, which are the recognition substrate of the RAG recombinase. The D segments were asymmetrically flanked by an RSS containing at 12 bp spacer on the 5\u2032-side and 23 bp spacer on the 3\u2032-side, as has been shown previously for TCR D gene segments in other species (<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B4\">Carroll et al. 1993<\/a><span style=\"letter-spacing: normal;\">;\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B26\">Parra et al. 2007<\/a><span style=\"letter-spacing: normal;\">,\u00a0<\/span><a class=\"link link-ref link-reveal xref-bibr\" style=\"letter-spacing: normal;\" data-open=\"mss128-B28\">2010<\/a><span style=\"letter-spacing: normal;\">). In summary, the overall content and organization of the platypus\u00a0<\/span><em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><span style=\"letter-spacing: normal;\">\u00a0locus appeared fairly generic.<\/span><\/span><\/div>\n<div><\/div>\n<\/div>\n<\/div>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">The last sentence seems to be the main takeaway. I would have put it first.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">What is atypical in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus was the presence of an additional V gene that shared greater identity to antibody VH genes than to TCR V genes (<span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F2\">figs. 2<\/a>\u00a0and\u00a0<span id=\"jumplink-mss128-F3\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F3\">3<\/a>). This V gene segment was the most proximal of the V genes to the D and J genes and was tentatively designated as VH\u03b4. VH\u03b4 are, by definition, V genes indistinguishable from Ig VH genes but used in encoding TCR\u03b4 chains and have previously been found only in the genomes of birds and frogs (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>,\u00a0<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">2010<\/a>,\u00a0<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">2012<\/a>).<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Shortish paragraph, intriguing first sentence \u2014 good job!<\/span><\/p><\/blockquote>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F3\">\n<div class=\"label fig-label\"><span style=\"font-family: Roboto;\">Fig. 3.<\/span><\/div>\n<div>\n<figure id=\"attachment_539\" aria-describedby=\"caption-attachment-539\" style=\"width: 520px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-539 size-full\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f3.jpg\" alt=\"\" width=\"520\" height=\"1625\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f3.jpg 520w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f3-96x300.jpg 96w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/m_mss128f3-328x1024.jpg 328w\" sizes=\"auto, (max-width: 520px) 100vw, 520px\" \/><figcaption id=\"caption-attachment-539\" class=\"wp-caption-text\">Phylogenetic tree of mammalian VH genes including the platypus VH\u03b4 and monotreme V\u00b5. The three major VH clans are bracketed. The platypus VH\u03b4 is boxed and the clade containing platypus VH\u03b4 along with platypus and echidna V\u00b5 is in bold and indicated by a smaller bracket in VH clan III. The three-digit numbers following the VH gene labels are the last three digits of the GenBank accession number referenced in supplementary table S2, Supplementary Material online. The numbers following the platypus and echidna V\u00b5 labels are clone numbers. The tree presented was generated using the Minimum Evolution method. Similar topology was generation using the Neighbor Joining method.<\/figcaption><\/figure>\n<\/div>\n<div class=\"graphic-wrap\"><\/div>\n<\/div>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Maybe that&#8217;s the ex-biologist speaking, but I personally really like phylogenetic trees. I find them quite illustrative. On the other hand, I, uh, didn&#8217;t remember at all what a VH gene is, so I had to go back to the introduction. There should have been a way to make it clearer, since VH genes play a big role in the results.<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">Also, not important, but there&#8217;s a big typo in the last sentence (<em>generation<\/em> should have been <em>generated<\/em>).<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">VH genes from mammals and other tetrapods have been shown to cluster into three ancient clans and individual species differ in the presence of one or more of these clans in their germ-line IgH locus (<span id=\"jumplink-mss128-B37\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B37\">Tutter and Riblet 1989<\/a>;\u00a0<span id=\"jumplink-mss128-B24\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B24\">Ota and Nei 1994<\/a>). For example, humans, mice, echidnas, and frogs have VH genes from all three clans (<span id=\"jumplink-mss128-B33\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B33\">Schwager et al. 1989<\/a>;\u00a0<span id=\"jumplink-mss128-B24\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B24\">Ota and Nei 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B2\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B2\">Belov and Hellman 2003<\/a>), whereas rabbits, opossums, and chickens have only a single clan (<span id=\"jumplink-mss128-B22\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B22\">McCormack et al. 1991<\/a>;\u00a0<span id=\"jumplink-mss128-B3\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B3\">Butler 1997<\/a>;\u00a0<span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B17\">Johansson et al. 2002<\/a>;\u00a0<span id=\"jumplink-mss128-B1\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B1\">Baker et al. 2005<\/a>). In phylogenetic analyses, the platypus VH\u03b4 was most related to the platypus V\u00b5 genes found in the TCR\u00b5 locus in this species (<span id=\"jumplink-mss128-F3\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F3\">fig. 3<\/a>). Platypus VH\u03b4, however, share only 51\u201361% nucleotide identity (average 56.6%) with the platypus V\u00b5 genes. Both the platypus V\u00b5 and VH\u03b4 clustered within clan III (<span id=\"jumplink-mss128-F3\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F3\">fig. 3<\/a>) (<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). This is noteworthy given that VH genes in the platypus IgH locus are also clan III and, in general, clan III VH are the most ubiquitous and conserved lineage of VH (<span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B17\">Johansson et al. 2002<\/a>;\u00a0<span id=\"jumplink-mss128-B37\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B37\">Tutter and Riblet 1989<\/a>). Although clearly related to platypus VH, the VH\u03b4 gene share only 34\u201365% nucleotide identity (average 56.9%) with the\u00a0<em>bona fide<\/em>\u00a0VH used in antibody heavy chains in this species.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Okay, this explains the three VH parts in the tree. It&#8217;s pretty clear.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">It was necessary to rule out that the VH\u03b4 gene present in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus was not an artifact of the genome assembly process. One piece of supporting evidence would be the demonstration that the VH\u03b4 is recombined to downstream D\u03b4 and J\u03b4 segments and expressed with C\u03b4 in complete TCR\u03b4 transcripts. PCR using primers specific for VH\u03b4 and C\u03b4 was performed on cDNA synthesized from splenic RNA from two different platypuses, one from New South Wales and the other from Tasmania. PCR products were successfully amplified from the NSW animal and these were cloned and sequenced. Twenty clones, each containing unique nucleotide sequence, were characterized and found to contain the VH\u03b4 recombined to the D\u03b4 and J\u03b4 gene segments (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A). Of these 20, 11 had unique V, D, and J combinations that would encode 11 different complementarity-determining regions-3 (CDR3) (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>B). More than half of the CDR3 (8 out of 11) contained evidence of using both D genes (VDDJ) (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>B). This is a common feature of TCR\u03b4 V domains where multiple D genes can be incorporated into the recombination due to the presence of asymmetrical RSS (<span id=\"jumplink-mss128-B4\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B4\">Carroll et al. 1993<\/a>). The region corresponding to the junctions between the V, D, and J segments, contained additional sequence that could not be accounted for by the germ-line gene segments (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>B). There are two possible sources of such sequence. One are palindromic (P) nucleotides that are created during V(D)J recombination when the RAG generates hairpin structures that are resolved asymmetrically during the re-ligation process (<span id=\"jumplink-mss128-B20\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B20\">Lewis 1994<\/a>). The second are non-templated (N) nucleotides that can be added by the enzyme terminal deoxynucleotidyl transferase (TdT) during the V(D)J recombination process. An unusual feature of the platypus VH\u03b4 is the presence of a second cysteine encoded near the 3\u2032-end of the gene, directly next to the cysteine predicted to form the intra-domain disulfide bond in Ig domains (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A). Additional cysteines in the CDR3 region of VH domains have been thought to provide stability to unusually long CDR3 loops, as has been described for cattle and the platypus previously (<span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B17\">Johansson et al. 2002<\/a>). The CDR3 of TCR\u03b4 using VH\u03b4 are only slightly longer than conventional TCR\u03b4 chains (ranging 10\u201320 residues) (<span id=\"jumplink-mss128-B30\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B30\">Rock et al. 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). Furthermore, the stabilization of CDR3 generally involves multiple pairs of cysteines, which were not present in the platypus VH\u03b4 clones (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A). Attempts to amplify TCR\u03b4 transcripts containing VH\u03b4 from splenic RNA obtained from the Tasmanian animal were unsuccessful. As a positive control, TCR\u03b4 transcripts containing conventional V\u03b1\/\u03b4 were successfully isolated, however. It is possible that Tasmanian platypuses, which have been separated from the mainland population at least 14,000 years either have a divergent VH\u03b4 or have deleted this single V gene altogether (<span id=\"jumplink-mss128-B19\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B19\">Lambeck and Chappell 2001<\/a>).<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">I like the thought process: &#8220;hey, our results may have been an artifact, here&#8217;s what we did to prove it wasn&#8217;t.&#8221; But why is this paragraph so<em> long<\/em>? Seems like it could have been multiple smaller ones, perhaps with a section subheading.<\/span><\/p><\/blockquote>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F4\">\n<div class=\"label fig-label\"><span style=\"font-family: Roboto;\">Fig. 4.<\/span><\/div>\n<div>\n<figure id=\"attachment_540\" aria-describedby=\"caption-attachment-540\" style=\"width: 580px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-540 size-large\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-1024x490.jpg\" alt=\"\" width=\"580\" height=\"278\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-1024x490.jpg 1024w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-300x144.jpg 300w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-768x368.jpg 768w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-1536x736.jpg 1536w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4-1200x575.jpg 1200w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f4.jpg 1800w\" sizes=\"auto, (max-width: 580px) 100vw, 580px\" \/><figcaption id=\"caption-attachment-540\" class=\"wp-caption-text\">(A) Alignment of predicted protein sequence of transcripts containing a recombined VH\u03b4 gene isolated from platypus spleen RNA. The individual clones are identified by the last three digits of their GenBank accession numbers (JQ664690\u2013JQ664710). Shown is the region from FR3 of the VH\u03b4 through the beginning of the C\u03b4 domain. The sequence in bold at the top of the alignment is the germ-line VH\u03b4 and C\u03b4 gene sequence. The double cysteines at the end of FR3 and unpaired cysteines in CDR3 are shaded, as is the canonical FGXG in FR4. (B) Nucleotide sequence of the CDR3 region of the eleven unique V(D)J recombinants using VH\u03b4 described in the text. The germ-line sequence of the 3\u2032-end of VH\u03b4, the two D\u03b4, are shown at the top. The germ-line J\u03b4 sequences are shown on the right-hand side of the alignment interspersed amongst the cDNA sequences using each. Nucleotides in the junctions between the V, D, and J segments, shown italicized, are most likely N-nucleotides added by TdT.<\/figcaption><\/figure>\n<\/div>\n<div class=\"graphic-wrap\">\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">This figure is probably good to visualize what their results actually looked like, but it also seems like a way to cram as much information in a visual and its caption as humanly possible&#8230; I&#8217;ll let it pass. It&#8217;s fine that some parts of the paper go more in depth, if they can be easily ignored, as I think is the case here.<\/span><\/p>\n<p><span style=\"color: #0000ff; font-family: Roboto;\">Small nitpick: This is two figures, and I would preferred that this fact would have been clearer. A small &#8220;(A)&#8221; and &#8220;(B)&#8221; in the paragraph doesn&#8217;t really help the reader.<\/span><\/p><\/blockquote>\n<p><span style=\"font-family: Roboto;\">Although there is only a single VH\u03b4 in the current platypus genome assembly, there was sequence variation in the region corresponding to FR1 through FR3 of the V domains (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A and sequence data not shown but available in GenBank). Some of this variation could represent two alleles of a single VH\u03b4 gene. Indeed, the RNA used in this experiment is from a wild-caught individual from the same population that was used to generate the whole-genome sequence and was found to contain substantial heterozygosity (<span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B41\">Warren et al. 2008<\/a>). There was greater variation in the transcribed sequences, however, than could be explained simply by two alleles of a single gene (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A). Two alternative explanations are the occurrence of somatic mutation of expressed VH\u03b4 genes or allelic variation in gene copy number. Somatic mutation in TCR chains is controversial. Nonetheless, it has been invoked to explain the variation in expressed TCR chains that exceeds the apparent gene copy number in sharks, and has also been postulated to occur in salmonids (<span id=\"jumplink-mss128-B44\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B44\">Yazawa et al. 2008<\/a>;\u00a0<span id=\"jumplink-mss128-B5\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B5\">Chen et al. 2009<\/a>). Therefore, it does not seem to be out of the realm of possibility that somatic mutation is occurring in platypus VH\u03b4. Indeed, the mutations appear to be localized to the V region with no variation in the C region (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>A). This may be due to its relatedness of VH\u03b4 to Ig VH genes where somatic hyper-mutation is well documented. Such somatic mutation contributes to overall affinity maturation in secondary antibody responses (<span id=\"jumplink-mss128-B42\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B42\">Wysocki et al. 1986<\/a>). The pattern of mutation seen in platypus VH\u03b4 however, is not localized to the CDR3, which would be indicative of selection for affinity maturation, but was also found in the framework regions. Furthermore, in the avian genomes where there is also only a single VH\u03b4, there was no evidence of somatic mutation in the V regions (<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">Parra et al. 2012<\/a>). The contribution of mutation to the platypus TCR\u03b4 repertoire, if it is occurring, remains to be determined. Alternatively, the sequence polymorphism may be due to VH\u03b4 gene copy number variation between individual TCR\u03b1\/\u03b4 alleles.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Not the worst paragraph, but again, doesn&#8217;t need to be a Wall of Text.<\/span><\/p><\/blockquote>\n<\/div>\n<\/div>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Irrespective of the number of VH\u03b4 genes in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus, the results clearly support TCR\u03b4 transcripts containing VH\u03b4 recombined to D\u03b4 and J\u03b4 gene segments in the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F4\">fig. 4<\/a>). A VH\u03b4 gene or genes in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus in the genome assembly, therefore, does not appear to be an assembly artifact. Rather it is present, functional and contributes to the expressed TCR\u03b4 chain repertoire. The possibility that some platypus TCR\u03b1\/\u03b4 loci contain more than a single VH\u03b4 does not alter the principal conclusions of this study.<\/span><\/p>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Previously, we hypothesized the origin of TCR\u00b5 in mammals involving the recombination between and ancestral\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus and an IgH locus (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). The\u00a0<em>IgH<\/em>\u00a0locus would have contributed the V gene segments at the 5\u2032-end of the locus, with the TCR\u03b4 contributing the D, J, and C genes at the 3\u2032-end of the locus. The difficulty with this hypothesis was the clear stability of the genome region surrounding the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus. In other words, the chromosomal region containing the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus appears to have remained relatively undisrupted for at least the past 360 million years (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>,\u00a0<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">2010<\/a>,\u00a0<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">2012<\/a>). The discovery of VH\u03b4 genes within the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci of frog and zebra finch is consistent with insertions occurring without apparently disrupting the local syntenic region. In frogs, the\u00a0<em>IgH<\/em>\u00a0and\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci are tightly linked, which may have facilitated the translocation of VH genes into the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus (<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">Parra et al. 2010<\/a>). However, close linkage is not a requirement since the translocation of VH genes appears to have occurred independently in birds and monotremes, due to the lack of similarity between the VH\u03b4 in frogs, birds, and monotremes (<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">Parra et al. 2012<\/a>). Indeed, it would appear is if the acquisition of VH genes into the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus occurred independently in each lineage.<\/span><\/p>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The similarity between the platypus VH\u03b4 and V genes in the TCR\u00b5 locus is, so far, the clearest evolutionary association between the TCR\u00b5 and TCR\u03b4 loci in one species. From the comparison of the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci in frogs, birds, and monotremes, a model for the evolution of TCR\u00b5 and other TCR\u03b4 forms emerges (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>), which can be summarized as follows:<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Oooh, exciting! The title promised a model, and at last we get it. Also it seems that below we get point-form stuff! I like point-form stuff. It&#8217;s often really helpful to guide the reader.<\/span><\/p><\/blockquote>\n<ol class=\"order\">\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Early in the evolution of tetrapods, or earlier, a duplication of the D\u2013J\u2013C\u03b4 cluster occurred resulting in the presence of two C\u03b4 each with its own set of D\u03b4 and J\u03b4 segments (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>A).<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Subsequently, a VH gene or genes was translocated from the\u00a0<em>IgH<\/em>\u00a0locus and inserted into the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus, most likely to a location between the existing V\u03b1\/V\u03b4 genes and the 5\u2032-proximal D\u2013J\u2013C\u03b4 cluster (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>B). This resulted in the configuration like that which currently exists in the zebra finch genome (<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">Parra et al. 2012<\/a>).<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">In the amphibian lineage there was an inversion of the region containing VH\u03b4\u2013D\u03b4\u2013J\u03b4\u2013C\u03b4 cluster and an expansion in the number of VH\u03b4 genes (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>C). Currently,\u00a0<em>X. tropicalis<\/em>\u00a0has the greatest number of VH\u03b4 genes, where they make up the majority of V genes available in the germ-line for use in TCR\u03b4 chains (<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">Parra et al. 2010<\/a>).<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">In the galliform lineage (chicken and turkey), the VH\u03b4\u2013D\u03b4\u2013J\u03b4\u2013C\u03b4 cluster was trans-located out of the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus where it currently resides on another chromosome (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>D). There are no V\u03b1 or V\u03b4 genes at the site of the second chicken TCR\u03b4 locus and only a single C\u03b4 gene remains in the conventional\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus (<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">Parra et al. 2012<\/a>).<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">Similar to galliform birds, the VH\u03b4\u2013D\u03b4\u2013J\u03b4\u2013C\u03b4 cluster was trans-located out of the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus in presumably the last common ancestor of mammals, giving rise to TCR\u00b5 (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>E). Internal duplications of the VH\u03b4\u2013D\u03b4\u2013J\u03b4 genes gave rise to the current [(V\u2013D\u2013J) \u2212 (V\u2013D\u2013J) \u2212 C] organization necessary to encode TCR chains with double V domains (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>,\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). In the platypus, the second V\u2013D\u2013J cluster, encoding the supporting V, has lost its D segments and generates V domains with short CDR3 encoded by direct V to J recombination (<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). The whole cluster appears to have undergone additional tandem duplication as it exists in multiple tandem copies in the opossum and also likely in the platypus (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>,\u00a0<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">2008<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>).<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">In the therian lineage (marsupials and placentals), the VH\u03b4 was lost from the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). In placental mammals, the\u00a0<em>TCR\u00b5<\/em>\u00a0locus was also lost (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). The marsupials retained TCR\u00b5, however the second set of V and J segments, encoding the supporting V domain in the protein chain, were replaced with a germ-line joined V gene, in a process most likely involving germ-line V(D)J recombination and retro-transposition (<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>F) (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>,\u00a0<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">2008<\/a>).<\/span><\/p>\n<\/li>\n<\/ol>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\"><strong>Yeah, this was good<\/strong>. These point-form paragraphs, combined with Fig. 5 (below) did more to help me understand the paper than anything else so far. I kind of wish the paper had just opened with this, and then proceeded to explain the reasoning behind.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">TCR forms such as TCR\u00b5, which contain three extracellular domains, have evolved at least twice in vertebrates. The first was in the ancestors of the cartilaginous fish in the form of NAR-TCR (<span id=\"jumplink-mss128-B8\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B8\">Criscitiello et al. 2006<\/a>) and the second in the mammals as TCR\u00b5 (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>). NAR-TCR uses an N-terminal V domain related to the V domains found in IgNAR antibodies, which are unique to cartilaginous fish (<span id=\"jumplink-mss128-B12\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B12\">Greenberg et al. 1995<\/a>;\u00a0<span id=\"jumplink-mss128-B8\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B8\">Criscitiello et al. 2006<\/a>), and not closely related to antibody VH domains. Therefore, it appears that NAR-TCR and TCR\u00b5 are more likely the result of convergent evolution rather than being related by direct descent (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). Similarly, the model proposed in\u00a0<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>\u00a0posits the direct transfer of VH genes from an\u00a0<em>IgH<\/em>\u00a0locus to the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus. But it should be pointed out the VH\u03b4 found in frogs, birds, and monotremes are not closely related (<span id=\"jumplink-mss128-F3\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F3\">fig. 3<\/a>); indeed, they appear derived each from different, ancient VH clans (birds, VH clan I; frogs VH clan II; platypus VH clan III). This observation would suggest that the transfer of VH\u03b4 into the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0loci occurred independently in the different lineages. Alternatively, the transfer of VH genes into the\u00a0<em>TCR\u03b1\/\u03b4<\/em>\u00a0locus may have occurred frequently and repeatedly in the past and gene replacement is the best explanation for the current content of these genes in the different tetrapod lineages. The absence of VH\u03b4 in marsupials, the highly divergent nature of V\u00b5 genes in this lineage, and the absence of conserved synteny with genes linked to TCR\u00b5 in the opossum, provide little insight into the origins of TCR\u00b5 and its relationship to TCR\u03b4 or the other conventional TCR (<span id=\"jumplink-mss128-B25\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B25\">Parra et al. 2008<\/a>). The similarity between VH, VH\u03b4, and V\u00b5 genes in the platypus genome, which are all clan III, however is striking. In particular, the close relationship between the platypus VH\u03b4 and V\u00b5 genes lends greater support for the model presented in\u00a0<span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F5\">fig. 5<\/a>E, with TCR\u00b5 having been derived from TCR\u03b4 genes.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">My comments are getting repetitive. This could have been multiple paragraphs etc. etc. It&#8217;s easy enough to find the joints where it should be carved, by the way: right before the sentences that start with &#8220;Similarly&#8221; and &#8220;Alternatively&#8221; would be a good start, since these words indicate that we&#8217;re switching to a new idea.<\/span><\/p><\/blockquote>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F5\">\n<div class=\"label fig-label\"><span style=\"font-family: Roboto;\">Fig. 5.<\/span><\/div>\n<\/div>\n<div>\n<figure id=\"attachment_541\" aria-describedby=\"caption-attachment-541\" style=\"width: 580px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-541 size-large\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f5-630x1024.jpg\" alt=\"\" width=\"580\" height=\"943\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f5-630x1024.jpg 630w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f5-184x300.jpg 184w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f5-768x1249.jpg 768w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/mss128f5.jpg 787w\" sizes=\"auto, (max-width: 580px) 100vw, 580px\" \/><figcaption id=\"caption-attachment-541\" class=\"wp-caption-text\">A model of the stages of evolution of the TCR\u03b1\/\u03b4 loci in tetrapods and the origins of TCR\u00b5 in mammals. A color key of the gene segments is presented at the bottom. (A) Depiction of the D\u03b4-J\u03b4-C\u03b4 duplication in an ancestral TCR\u03b1\/\u03b4 locus that provides a second C\u03b4 gene found in frogs and zebra finch. (B) Depiction of the insertion of a VH gene into the TCR\u03b1\/\u03b4 locus producing a current organization as it is found in zebra finch. (C) Depiction of the inversion\/translocation and VH\u03b4 gene duplication that yielded the current organization found in frogs. (D) Depiction of the translocation of a VH\u03b4\u2013D\u03b4\u2013J\u03b4\u2013C\u03b4 cluster to a location outside the TCR\u03b1\/\u03b4 locus generating a second TCR\u03b4 locus as it is currently found in chicken and turkey. (E) Depiction the translocation that took place in mammals giving rise to the TCR\u00b5 locus. (F) Loss of TCR\u00b5 in placental mammals, loss of D gene segments in cluster encoding the support V domain, retro-transpostion to form a germ-line joined V in marsupials, and duplication of TCR\u00b5 clusters in both monotremes and marsupials.<\/figcaption><\/figure>\n<\/div>\n<div class=\"graphic-wrap\"><\/div>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Super helpful figure. Although I&#8217;m generally in favor of repeating important info, I do feel that the caption could have simply referred to the 6-point model in the text. The caption as it stands doesn&#8217;t add much and looks like a Wall of Text. But that&#8217;s not a big deal.<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The presence of TCR chains that use antibody like V domains, such as TCR\u03b4 using VH\u03b4, NAR-TCR or TCR\u00b5 are widely distributed in vertebrates with only the bony fish and placental mammals missing. In addition to NAR-TCR, some shark species also appear to generate TCR chains using antibody V genes. This occurs via trans-locus V(D)J recombination between IgM and IgW heavy chain V genes and TCR\u03b4 and TCR\u03b1 D and J genes (<span id=\"jumplink-mss128-B7\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B7\">Criscitiello et al. 2010<\/a>). This may be possible, in part, due to the multiple clusters of Ig genes found in the cartilaginous fish. It also illustrates that there has been independent solutions to generating TCR chains with antibody V domains in different vertebrate lineages. In the tetrapods, the VH genes were trans-located into the TCR loci where they became part of the germ-line repertoire. Whereas in cartilaginous fish something equivalent may occur somatically during V(D)J recombination in developing T cells. Either mechanism suggests there has been selection for having TCR using antibody V genes over much of vertebrate evolutionary history.<\/span><\/p>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">The current working hypothesis for such chains is that they are able to bind native antigen directly. This is consistent with a selective pressure for TCR chains that may bind or recognize antigen in ways similar to antibodies in many different lineages of vertebrates. In the case of NAR-TCR and TCR\u00b5, the N-terminal V domain is likely to be unpaired and bind antigen as a single domain (<span id=\"jumplink-mss128-F1\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F1\">fig. 1<\/a>), as has been described for IgNAR and some IgG antibodies in camels (recently reviewed in\u00a0<span id=\"jumplink-mss128-B10\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B10\">Flajnik et al. 2011<\/a>). This model of antigen binding is consistent with the evidence that the N-terminal V domains in TCR\u00b5 are somatically diverse, while the second, supporting V domains have limited diversity with the latter presumably performing a structural role rather than one of antigen recognition (<span id=\"jumplink-mss128-B26\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B26\">Parra et al. 2007<\/a>;\u00a0<span id=\"jumplink-mss128-B40\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B40\">Wang et al. 2011<\/a>). There is no evidence of double V domains in TCR\u03b4 chains using VH\u03b4 in frogs, birds, or platypus (<span id=\"jumplink-mss128-F1\" class=\"xrefLink\"><\/span><a class=\"link link-reveal link-table xref-fig\" data-open=\"mss128-F1\">fig. 1<\/a>) (<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B28\">Parra et al. 2010<\/a>,\u00a0<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B27\">2012<\/a>). Rather, the TCR complex containing VH\u03b4 would likely be structured similar to a conventional \u03b3\u03b4TCR with a single V domain on each chain. It is possible that such receptors also bind antigen directly, however this remains to be determined.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Not much to add except that I just had a thought that subheadings would have greatly eased this section (like they did the Methods section).<\/span><\/p><\/blockquote>\n<p class=\"chapter-para\"><span style=\"font-family: Roboto;\">A compelling model for the evolution of the Ig and TCR loci has been one of internal duplication, divergence and deletion; the so-called birth-and-death model of evolution of immune genes promoted by Nei and colleagues (<span id=\"jumplink-mss128-B24\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B24\">Ota and Nei 1994<\/a>;\u00a0<span id=\"jumplink-mss128-B23\" class=\"xrefLink\"><\/span><a class=\"link link-ref link-reveal xref-bibr\" data-open=\"mss128-B23\">Nei et al. 1997<\/a>). Our results in no way contradict that the birth-and-death mode of gene evolution has played a significant role in shaping these complex loci. However, our results do support the role of horizontal transfer of gene segments between the loci that has not been previously appreciated. With this mechanism T cells may have been able to acquire the ability to recognize native, rather than processed antigen, much like B cells.<\/span><\/p>\n<blockquote><p><span style=\"color: #0000ff; font-family: Roboto;\">Pretty good conclusion, opening on new ideas and showing the significance of this work in the field.<\/span><\/p><\/blockquote>\n<hr \/>\n<p><span style=\"font-family: Roboto;\">Phew. I&#8217;m done.<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">Reading this paper took me several days, although I could have been more focussed in general. But this shows how much work is required to read papers! I had to push myself to read. Many times I caught myself skimming paragraphs without understanding anything, and I had to read again. Right now I think I would benefit from reading it all a second time, but I resist the thought, because it&#8217;s\u00a0<em>work<\/em>.<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">But I think it&#8217;s a good candidate for my rewriting project. It should be relatively easy to cut down the number of abbreviations, split long paragraphs, and add subheadings. More thorough rewriting will probably involve clarifying the main points and claims right at the start. At the most extreme (I&#8217;m not sure I&#8217;ll go there), it could be beneficial to change the entire structure: give the detailed model first, and only then explain the background and methods.<\/span><\/p>\n<p><span style=\"font-family: Roboto;\">Stay tuned!<\/span><\/p>\n","protected":false},"excerpt":{"rendered":"<p>Here I present a paper I chose to rewrite as a demonstration for the JAWWS project. The original text and figures are reproduced below, interspersed with my comments in the following format: hello I am a blue comment in a quote-block Feel free to just read the comments. Annotating the paper was a first step [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":497,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[7],"tags":[30,26,18],"class_list":["post-462","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-original-research","tag-biology","tag-science","tag-writing"],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v27.5 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>An Annotated Reading of a Paper about Platypuses &#8212; Dark Gray Matters<\/title>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/etiennefd.com\/dgm\/an-annotated-reading-of-a-paper-about-platypuses\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"An Annotated Reading of a Paper about Platypuses &#8212; Dark Gray Matters\" \/>\n<meta property=\"og:description\" content=\"Here I present a paper I chose to rewrite as a demonstration for the JAWWS project. 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