{"id":604,"date":"2021-11-12T09:41:12","date_gmt":"2021-11-12T14:41:12","guid":{"rendered":"https:\/\/etiennefd.com\/dgm\/?p=604"},"modified":"2021-11-12T09:41:12","modified_gmt":"2021-11-12T14:41:12","slug":"platypus-paper-rewritten","status":"publish","type":"post","link":"https:\/\/etiennefd.com\/dgm\/platypus-paper-rewritten\/","title":{"rendered":"Platypus Paper, Rewritten"},"content":{"rendered":"<p>This is a completely rewritten version of a biology paper titled, <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;<\/strong> It is meant to be a demonstration and a proof of concept for <a href=\"https:\/\/etiennefd.com\/dgm\/the-journal-of-actually-well-written-science\/\">JAWWS<\/a>, my idea of a science journal that focusses on readability.<\/p>\n<p>You can read the original version <a href=\"https:\/\/academic.oup.com\/mbe\/article\/29\/10\/3205\/1030857\">on the <em>Molecular Biology and Evolution<\/em> journal&#8217;s website<\/a>, or <a href=\"https:\/\/etiennefd.com\/dgm\/an-annotated-reading-of-a-paper-about-platypuses\/\">here on this blog, with annotations by me<\/a>.<\/p>\n<p>Why did I choose to rewrite this paper? I wish I had a more principled answer, but the truth is that I simply went to <a href=\"https:\/\/www.researchhub.com\/\">ResearchHub<\/a>, a website where scientists share papers between themselves and upvote the most interesting one, went to the Evolutionary Biology section (because that used to be my field), and picked the first paper that was open-access and seemed fit for my purposes. In other words, a paper that seemed like it could be improved a lot because it seemed more difficult to understand than was warranted.<\/p>\n<p>Only later did I realize it was a paper from 2012, so not that recent. I don&#8217;t think that matters too much for now since it&#8217;s just a proof of concept. Nor does the topic, i.e. molecular evolution in the vertebrate immune system. The actual journal will need to pick papers in a more principled way, of course.<\/p>\n<p>What did my rewrite entail? The best way to know is to read (not necessarily closely) the original and rewritten versions. But here&#8217;s a sample of my &#8220;interventions&#8221;:<\/p>\n<ul>\n<li>I put almost all citations in collapsible footnotes.<\/li>\n<li>I cut up most long paragraphs, including the abstract.<\/li>\n<li>I added many context sentences, including at the beginning of sections, to give a better sense of why we&#8217;re reading this. One example is the first sentence of the introduction: &#8220;How did the immune system of jawed vertebrates evolve?&#8221;<\/li>\n<li>I reworked some of the paper&#8217;s structure. One major change: I put the major contribution of the study, that is, the new evolutionary model, in its own section after the introduction. This way, it is not buried deep in the discussion; readers can start with it and dig into the rest only if they want more details. I also reordered the methods so that they would match the ordering in the Results.<\/li>\n<li>I added several subheadings to the sections that didn&#8217;t already have subsections (Introduction, and Results and Discussion)<\/li>\n<li>I tried my best to avoid abbreviations. One difficulty is that some of them are probably very recognizable by people who\u00a0 know immunology, and not by me. So I left some in, while trying to make sure they don&#8217;t hamper readability. The major example is &#8220;TCR&#8221;, which means T-cell receptor and was used\u00a0<em>a lot<\/em>. It&#8217;s still used in my version, but far less often.<\/li>\n<li>I removed some jargon words. E.g. &#8220;proximal&#8221; became &#8220;closest&#8221;.<\/li>\n<li>I formatted some information in point form, such as the T-cell lineages or the protocols in the Methods.<\/li>\n<li>I added some text formatting to guide the reader. For instance, bold font for groups of animals in the introduction, and color in the text to match colored elements in figures.<\/li>\n<li>I changed one figure by reorganizing its parts to make it clearer (fig. 2, which used to be fig. 5). A lot of additional clarity could potentially be gained from editing the figures, but that&#8217;s a lot of work, so I didn&#8217;t press it further.<\/li>\n<li>I fixed a number of typos and grammatical errors. Mistakes like this are not a big problem, but there were enough that I assume little editing work was done on this paper.<\/li>\n<\/ul>\n<p>On footnotes: I&#8217;m using two different kinds of collapsible footnotes. Those in the usual style of the blog, like this one,<sup class=\"modern-footnotes-footnote \" data-mfn=\"1\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-1\">1<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-1\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"1\">Here you would usually read a citation in short form such as &#8220;Rast et al. 1997.&#8221; Head to <a href=\"https:\/\/academic.oup.com\/mbe\/article\/29\/10\/3205\/1030857#77835882\">the original paper webpage<\/a> to see the reference list in full.<\/span> contain the citations included in the original paper. \u00a0Footnotes with brackets like this<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_1');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_1');\" ><sup id=\"footnote_plugin_tooltip_604_1_1\" class=\"footnote_plugin_tooltip_text\">[1]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_1\" class=\"footnote_tooltip\">This is an example comment.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_1').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_1', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> are for comments on the rewriting process and are also shown at the bottom of the paper. I suggest you don&#8217;t click on the former, unless you want to see a reference, and hover onto the latter to read my comments.<\/p>\n<p>Overall, my rewrite increased the length of the abstract from 267 to 286 words, and of the rest of the paper from about 6000 to about 6400 words. I consider this acceptable.<\/p>\n<p>I will publish some more thoughts on the rewriting process later.<\/p>\n<h2>Abstract<\/h2>\n<p><strong>Goal<\/strong>: This study presents a new model for the evolution of part of the vertebrate immune system: the genes encoding the T-cell receptor (TCR) \u03b4 chains.<\/p>\n<p><strong>Background<\/strong>: T lymphocytes have to recognize specific antigen for the adaptive immune response to work in vertebrates. They perform this using a somatically diversified T-cell receptor. All jawed vertebrates use four T-cell receptor chains called \u03b1, \u03b2, \u03b3, and \u03b4, but some lineages have nonconventional receptor chains: monotremes and marsupials encode a fifth one, called TCR\u00b5. Its function is unknown, but it is somatically diversified like the conventional chains. Its origins are also unclear. It appears to be distantly related to the TCR\u03b4 chain, for which recent evidence from birds and frogs has provided new information that was not available from humans or other placental (eutherian) mammals.<\/p>\n<p><strong>Experiment<\/strong>: We analyzed the genes encoding the \u03b4 chains in the platypus. This revealed the presence of a highly divergent variable (V) gene, indistinguishable from immunoglobulin heavy chain V genes (VH), and related to V genes used in the \u00b5 chain. This gene is expressed as part of TCR\u03b4 repertoire, so it is designated VH\u03b4.<\/p>\n<p><strong>Conclusions<\/strong>: The VH\u03b4 gene is similar to what has been found in frogs and birds, but it is the first time such a gene has been found in a mammal. This provides a critical link in reconstructing the evolutionary history of TCR\u00b5. The current structure of the \u03b4 and \u00b5 genes in tetrapods suggests ancient and possibly recurring translocations of gene segments between the \u03b4 and immunoglobulin heavy genes, as well as translocations of \u03b4 genes out of the <em>TCR\u03b1\/\u03b4<\/em> locus early in mammals, creating the TCR\u00b5 locus. We present a detailed model of this evolutionary history.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_2');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_2');\" ><sup id=\"footnote_plugin_tooltip_604_1_2\" class=\"footnote_plugin_tooltip_text\">[2]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_2\" class=\"footnote_tooltip\">Major changes to the abstract: I split it in four paragraphs with section titles (this is common in some journals; it should be common in most journals). I also added a section at the beginning to&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_2');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_2').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_2', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<h2>Introduction<\/h2>\n<p>How did the immune system of jawed vertebrates evolve? In this study, we use genomic evidence from the platypus to propose a model for the evolution of a specific component of the vertebrate immune system: the receptors on the surface of T lymphocytes.<\/p>\n<p>As a reminder, T lymphocytes (or T cells) are white blood cells that play a critical role in the adaptive immune system. They can be classified into two main lineages based on the receptor they use:<sup class=\"modern-footnotes-footnote \" data-mfn=\"2\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-2\">2<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-2\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"2\">Rast et al. 1997; reviewed in Davis and Chein 2008<\/span><\/p>\n<ol>\n<li><strong>\u03b1\u03b2T cell lineage<\/strong>: The receptor is composed of a heterodimer of \u03b1 and \u03b2 chains. Most circulating human T cells are \u03b1\u03b2T cells, including familiar subsets such as CD4+ helper T cells and regulatory T cells, CD8+ cytotoxic T cells, and natural killer T (NKT) cells.<\/li>\n<li><strong>\u03b3\u03b4T cell lineage<\/strong>: The receptor is composed of \u03b3 and \u03b4 chains. The function of these cells is less well defined. They have been associated with a broad range of immune responses including tumor surveillance, innate responses to pathogens and stress, and wound healing.<sup class=\"modern-footnotes-footnote \" data-mfn=\"3\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-3\">3<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-3\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"3\">Hayday 2009<\/span> \u03b3\u03b4T cells are found primarily in epithelial tissues and form a lower percentage of circulating lymphocytes in some species.<\/li>\n<\/ol>\n<p>\u03b1\u03b2 and \u03b3\u03b4 T cells also differ in the way they interact with antigen. The receptors of \u03b1\u03b2T cells are &#8220;restricted&#8221; relative to the major histocompatibility complex (MHC), meaning that that they bind antigenic epitopes, such as peptide fragments, bound to, or \u201cpresented\u201d by, molecules encoded in the MHC. In contrast, \u03b3\u03b4T receptors have been found to bind antigens directly in the absence of MHC, as well as self-ligands that are often MHC-related molecules.<sup class=\"modern-footnotes-footnote \" data-mfn=\"4\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-4\">4<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-4\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"4\">Sciammas et al. 1994; Hayday 2009<\/span><\/p>\n<p>All gnathostomes (jawed vertebrates) have \u03b1\u03b2 and \u03b3\u03b4 T cells. As we will see below, marsupial and monotreme mammals have an additional type of T-cell receptor, denoted with the letter \u00b5. The platypus, a monotreme, further has a non-conventional receptor with \u03b4 chains, which is also present in birds and amphibians.<\/p>\n<p>Before presenting our evolutionary model, let&#8217;s review these types of T-cell receptors and their structure.<\/p>\n<h3 class=\"chapter-para\">Structure and Genes of Conventional T-Cell Receptors<\/h3>\n<p>The chains of conventional T-cell receptors are composed of two extracellular domains, both members of the immunoglobulin domain superfamily of cell surface proteins (fig. 1):<sup class=\"modern-footnotes-footnote \" data-mfn=\"5\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-5\">5<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-5\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"5\">reviewed in Davis and Chein 2008<\/span><\/p>\n<ul>\n<li>The closest domain to the cellular membrane is called C for <em> constant<\/em>.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_3');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_3');\" ><sup id=\"footnote_plugin_tooltip_604_1_3\" class=\"footnote_plugin_tooltip_text\">[3]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_3\" class=\"footnote_tooltip\">Since this abbreviation comes up a lot, I put it first, with its meaning in parentheses.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_3').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_3', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> The C domain is largely invariant among T-cell clones expressing the same class of the receptor chain.<\/li>\n<li>The domain farthest from the cellular membrane is called V for\u00a0<em>variable<\/em>. It is the region that contacts antigen and MHC. Similar to antibodies, the individual clonal diversity in the V domain is generated by somatic DNA recombination.<sup class=\"modern-footnotes-footnote \" data-mfn=\"6\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-6\">6<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-6\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"6\">Tonegawa 1983<\/span> <span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_4');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_4');\" ><sup id=\"footnote_plugin_tooltip_604_1_4\" class=\"footnote_plugin_tooltip_text\">[4]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_4\" class=\"footnote_tooltip\">I didn&#8217;t change much in the figure&#8217;s caption, but it seemed pretty trivial to add color to the text to facilitate looking up what the colors mean.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_4').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_4', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/li>\n<\/ul>\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\">Fig. 1. Cartoon diagram of the T-cell receptor (TCR) forms found in different species. Oblong circles indicate immunoglobulin superfamily domains and are color coded as <span style=\"color: #0000ff;\">C domains (blue)<\/span>, <span style=\"color: #ff0000;\">conventional V domains (red)<\/span>, and <span style=\"color: #ffcc00;\">VH\u03b4 or V\u00b5 (yellow)<\/span>. The gray shaded chains represent the hypothetical partner chain for \u00b5 and \u03b4 using VH\u03b4.<\/figcaption><\/figure>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F1\">\n<div class=\"graphic-wrap\">\n<p>While C domains are usually encoded by a single, intact exon, V domains are assembled somatically from germ-line segments in developing T cells. These segments are genes called V (again for <em>variable<\/em>), D (for <em>diversity<\/em>), and J (for <em>joining<\/em>). The assembly process depends on the enzymes encoded by two genes, the recombination activating genes (<em>RAG<\/em>)-1 and <em>RAG<\/em>-2.<sup class=\"modern-footnotes-footnote \" data-mfn=\"7\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-7\">7<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-7\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"7\">Yancopoulos et al. 1986; Schatz et al. 1989<\/span><\/p>\n<p>The various T-cell receptor chains differ in how their V domains are assembled. \u03b2 and \u03b4 chains are assembled from all three types of gene segments, whereas \u03b1 and \u03b3 chains use only V and J. The different combinations of two or three segments, selected from a large repertoire of germ-line gene segments, along with variation at the junctions due to the addition and deletion of nucleotides during recombination, contribute to a vast diversity of T-cell receptors. It is this diversity that creates the individual antigen specificity of T-cell clones.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_5');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_5');\" ><sup id=\"footnote_plugin_tooltip_604_1_5\" class=\"footnote_plugin_tooltip_text\">[5]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_5\" class=\"footnote_tooltip\">This is an example of two sentences taken verbatim from the original paper. Not all of it was poorly written!<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_5').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_5', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<p>These genes are highly conserved among species in both their genomic sequence and their organization.<sup class=\"modern-footnotes-footnote \" data-mfn=\"8\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-8\">8<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-8\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"8\">Rast et al. 1997; Parra et al. 2008, 2012; Chen et al. 2009<\/span> In all tetrapods examined, the \u03b2 and \u03b3 chains are each encoded at multiple separate loci, whereas the genes encoding the \u03b1 and \u03b4 chains are nested at a single locus, called the <em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em> locus.<sup class=\"modern-footnotes-footnote \" data-mfn=\"9\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-9\">9<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-9\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"9\">Chien et al. 1987; Satyanarayana et al. 1988; reviewed in Davis and Chein 2008<\/span> The V domains of \u03b1 and \u03b4 chains can use a common pool of V gene segments, but distinct D, J, and C genes.<\/p>\n<p>The recombination of V, J and optionally D genes, referred to as <em>V(D)J recombination<\/em>, and mediated by RAG, is also known to generate the diversity of antibodies produced by another type of lymphocyte, the B cells.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_6');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_6');\" ><sup id=\"footnote_plugin_tooltip_604_1_6\" class=\"footnote_plugin_tooltip_text\">[6]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_6\" class=\"footnote_tooltip\">It took me forever to rewrite this part. The original sentence was, &#8220;Diversity in antibodies produced by B cells is also generated by RAG-mediated V(D)J recombination and the TCR and Ig genes&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_6');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_6').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_6', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><sup class=\"modern-footnotes-footnote \" data-mfn=\"10\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-10\">10<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-10\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"10\">Flajnik and Kasahara 2010; Litman et al. 2010<\/span><\/p>\n<h3>Non-Conventional Receptors Across Vertebrates<\/h3>\n<p class=\"fig-orig original-slide\">T-cell receptor and immunoglobulin genes clearly share a common origin in the jawed vertebrates.<sup class=\"modern-footnotes-footnote \" data-mfn=\"11\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-11\">11<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-11\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"11\">Flajnik and Kasahara 2010; Litman et al. 2010<\/span> Usually, the V, D, J, and C coding regions are readily distinguishable from immunoglobulin, at least for conventional T-cell receptors, owing to divergence over the past 400 million years.<\/p>\n<p class=\"fig-orig original-slide\">Recently, however, the discovery of non-conventional isoforms of the \u03b4 chain has blurred the boundary between them. These non-conventional forms use V genes that appear indistinguishable from the immunoglobulin heavy chain V.<sup class=\"modern-footnotes-footnote \" data-mfn=\"12\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-12\">12<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-12\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"12\">Parra et al. 2010, 2012<\/span> Such V genes have been named VH\u03b4.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_7');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_7');\" ><sup id=\"footnote_plugin_tooltip_604_1_7\" class=\"footnote_plugin_tooltip_text\">[7]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_7\" class=\"footnote_tooltip\">The original combined this sentence and the next, even though they&#8217;re about quite distinct ideas: the name of the genes, and the species where they&#8217;re found.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_7').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_7', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<p>VH\u03b4 genes have been found in both <strong>amphibians and birds<\/strong> (see the rightmost part of fig. 1).<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_8');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_8');\" ><sup id=\"footnote_plugin_tooltip_604_1_8\" class=\"footnote_plugin_tooltip_text\">[8]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_8\" class=\"footnote_tooltip\">Why not indicate the part of the figure that is relevant? Whenever you can, provide reader guidance!<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_8').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_8', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> In the frog <em>Xenopus tropicalis<\/em>, as well as in a passerine bird, the zebra finch <em>Taeniopygia guttata<\/em>, the VH\u03b4 genes coexist with the conventional V\u03b1 and V\u03b4 genes at the <em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em><em>\u00a0<\/em>locus.<sup class=\"modern-footnotes-footnote \" data-mfn=\"13\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-13\">13<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-13\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"13\">Parra et al. 2010, 2012<\/span><\/p>\n<p>In <strong>galliform birds<\/strong>, such as the chicken <em>Gallus gallus<\/em>, they are instead located at a second <em style=\"letter-spacing: normal;\">TCR\u03b4 <\/em>locus that is unlinked to the conventional<em> TCR\u03b1\/\u03b4.<\/em><sup class=\"modern-footnotes-footnote \" data-mfn=\"14\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-14\">14<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-14\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"14\">Parra et al. 2012<\/span> 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 \u03b4 chains. This is true as well for frogs where the <em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em>\u00a0and\u00a0<em style=\"letter-spacing: normal;\">IgH<\/em> (immunoglobulin heavy chain) loci are tightly linked.<sup class=\"modern-footnotes-footnote \" data-mfn=\"15\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-15\">15<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-15\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"15\">Parra et al. 2010<\/span><\/p>\n<p>In <strong>mammals<\/strong>, a <em>TCR\u03b1\/\u03b4<\/em> locus has been characterized in several eutherian species and\u00a0at least one marsupial, the opossum <em>Monodelphis domestica<\/em>. VH\u03b4 genes have not been found in mammals to date.<sup class=\"modern-footnotes-footnote \" data-mfn=\"16\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-16\">16<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-16\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"16\">Satyanarayana et al.1988; Wang et al. 1994; Parra et al. 2008<\/span> However, <strong>marsupials<\/strong> do have an additional locus, unlinked to <span id=\"jumplink-mss128-B31\" class=\"xrefLink\"><em>TCR\u03b1\/\u03b4<\/em><\/span>, that uses antibody-related V genes. This fifth chain is called <span id=\"jumplink-mss128-B31\" class=\"xrefLink\">\u00b5, and the receptor that uses it is referred to as TCR\u00b5. The \u00b5 chain is related to the \u03b4 chain, but it diverges from it in both sequence and structure.<sup class=\"modern-footnotes-footnote \" data-mfn=\"17\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-17\">17<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-17\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"17\">Parra et al. 2007, 2008<\/span> It has also been found in a monotreme, t<\/span>he <strong>platypus<\/strong>.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_9');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_9');\" ><sup id=\"footnote_plugin_tooltip_604_1_9\" class=\"footnote_plugin_tooltip_text\">[9]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_9\" class=\"footnote_tooltip\">The authors like to use &#8220;duckbill platypus,&#8221; but there&#8217;s only one species of platypus, so I took that word out.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_9').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_9', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><span id=\"jumplink-mss128-B31\" class=\"xrefLink\">\u00a0The platypus and marsupial TCR\u00b5 genes are clearly orthologous, which is consistent with the idea that the \u00b5 chain is ancient in mammals, but has been lost in the eutherians.<sup class=\"modern-footnotes-footnote \" data-mfn=\"18\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-18\">18<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-18\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"18\">Parra et al. 2008; Wang et al. 2011<\/span><\/span><\/p>\n<\/div>\n<\/div>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-B31\" class=\"xrefLink\">TCR\u00b5 chains use their own unique set of V genes, called V\u00b5.<sup class=\"modern-footnotes-footnote \" data-mfn=\"19\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-19\">19<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-19\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"19\">Parra et al. 2007; Wang et al. 2011<\/span> So far, no evidence has been found of V(D)J recombination between V\u00b5 genes and genes from other immunoglobulin or T-cell receptor loci.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_10');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_10');\" ><sup id=\"footnote_plugin_tooltip_604_1_10\" class=\"footnote_plugin_tooltip_text\">[10]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_10\" class=\"footnote_tooltip\">Another horrible sentence from the original, recorded for posterity: &#8220;Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCR\u00b5 genes has not been found.&#8221; That&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_10');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_10').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_10', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script>\u00a0 Neither have TCR\u00b5 homologues been found in non-mammals.<sup class=\"modern-footnotes-footnote \" data-mfn=\"20\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-20\">20<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-20\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"20\">Parra et al. 2008<\/span><\/span><\/p>\n<p>The structure of TCR\u00b5 chains is atypical. They contain three, rather than two, extra-cellular domains from the immunoglobulin superfamily;<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_11');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_11');\" ><sup id=\"footnote_plugin_tooltip_604_1_11\" class=\"footnote_plugin_tooltip_text\">[11]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_11\" class=\"footnote_tooltip\">The abbreviation IgSF was used in the paper, with no explanation. I assume the people who would read this paper tend to know what that means, but still.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_11').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_11', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> this is due to an extra N-terminal V domain (see fig. 1).<sup class=\"modern-footnotes-footnote \" data-mfn=\"21\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-21\">21<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-21\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"21\">Parra et al. 2007; Wang et al. 2011<\/span> Both V domains are encoded by a unique set of V\u00b5 genes and are more related to immunoglobulin heavy chain V than to conventional T-cell receptor V domains. The N-terminal one is diverse and encoded by genes that undergo somatic V(D)J recombination, while the second V domain (referred to as &#8220;supporting&#8221;) has little or no diversity.<\/p>\n<p>The supporting V domain differs between marsupials and monotremes. In marsupials, it is encoded by a germ-line joined, or pre-assembled, V exon that is invariant.<sup class=\"modern-footnotes-footnote \" data-mfn=\"22\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-22\">22<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-22\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"22\">Parra et al. 2007<\/span> In the platypus, it is encoded by gene segments requiring somatic DNA recombination, but with limited diversity due in part to the lack of D segments.<sup class=\"modern-footnotes-footnote \" data-mfn=\"23\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-23\">23<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-23\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"23\">Wang et al. 2011<\/span><\/p>\n<p><strong>Sharks and other cartilaginous fish<\/strong> also have a T-cell receptor chain that is structurally similar to TCR\u00b5 (see middle part of fig. 1).<sup class=\"modern-footnotes-footnote \" data-mfn=\"24\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-24\">24<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-24\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"24\">Criscitiello et al. 2006; Flajnik et al. 2011<\/span> The resulting receptor is called NAR-TCR. Like the receptor of marsupials and monotremes, it contains three extracellular domains, but its N-terminal V domain is related to chains used by IgNAR (immunoglobulin new antigen receptor) antibodies, a type of antibody found only in sharks.<sup class=\"modern-footnotes-footnote \" data-mfn=\"25\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-25\">25<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-25\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"25\">Greenberg et al. 1995<\/span> In both the TCR\u00b5 of marsupials and monotremes and the NAR-TCR of cartilaginous fishes, the current working model is that the N-terminal V domain is unpaired and acts as a single, antigen binding domain. This would be analogous to <span id=\"jumplink-mss128-B10\" class=\"xrefLink\"><span id=\"jumplink-mss128-B12\" class=\"xrefLink\"> the V domains of light-chainless antibodies found in sharks and camelids.<sup class=\"modern-footnotes-footnote \" data-mfn=\"26\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-26\">26<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-26\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"26\">Flajnik et al. 2011;\u00a0Wang et al. 2011<\/span><\/span><\/span><\/p>\n<p>How did the \u00b5 chain arise? Phylogenetic analyses support an origin after the avian\u2013mammalian split.<sup class=\"modern-footnotes-footnote \" data-mfn=\"27\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-27\">27<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-27\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"27\">Parra et al. 2007; Wang et al. 2011<\/span> Previously, we hypothesized that it originated as a recombination between ancestral immunoglobulin heavy and TCR\u03b4-like loci,<sup class=\"modern-footnotes-footnote \" data-mfn=\"28\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-28\">28<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-28\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"28\">Parra et al. 2008<\/span> but this hypothesis is problematic for several reasons. One challenge is the apparent genomic stability and ancient conserved synteny (order of genes on the chromosome) in the region surrounding the <em>TCR\u03b1\/\u03b4<\/em> locus; this region has appeared to remain stable over at least the past 350 million years of tetrapod evolution<span id=\"jumplink-mss128-B28\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"29\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-29\">29<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-29\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"29\">Parra et al. 2008,\u00a02010<\/span><\/span><\/p>\n<p>As a result, we need a new model for the evolution of TCR\u00b5 and the <em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4 <\/em>locus. Here we present the best current model, supported by an analysis of the platypus genome\u2014the first to examine a monotreme <span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><em>TCR\u03b1\/\u03b4<\/em> locus in detail\u2014<\/span>as described in the methods and results sections below.<\/p>\n<h2>The Model<\/h2>\n<p>Our model can be summarized in six stages (fig. 2).<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_12');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_12');\" ><sup id=\"footnote_plugin_tooltip_604_1_12\" class=\"footnote_plugin_tooltip_text\">[12]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_12\" class=\"footnote_tooltip\">Major change from me here. This section was moved here from the discussion, because it is the core and most interesting part of the paper. It is now its own first-level section alongside&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_12');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_12').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_12', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<figure id=\"attachment_533\" aria-describedby=\"caption-attachment-533\" style=\"width: 580px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"wp-image-533 size-large\" src=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5-1024x807.png\" alt=\"\" width=\"580\" height=\"457\" srcset=\"https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5-1024x807.png 1024w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5-300x236.png 300w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5-768x605.png 768w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5-1200x946.png 1200w, https:\/\/etiennefd.com\/dgm\/wp-content\/uploads\/sites\/2\/2021\/10\/platypus-fig5.png 1259w\" sizes=\"auto, (max-width: 580px) 100vw, 580px\" \/><figcaption id=\"caption-attachment-533\" class=\"wp-caption-text\">Fig. 2. A model of the stages of evolution of the <em>TCR\u03b1\/\u03b4<\/em> loci in tetrapods and the origins of <em>TCR\u00b5<\/em> in mammals. Refer to the text for detailed explanation of stages A-F.<\/figcaption><\/figure>\n<ol class=\"order\" style=\"list-style-type: upper-alpha;\">\n<li>\n<p class=\"chapter-para\"><strong>Duplication of the <span style=\"color: #f0a860;\">D\u03b4<\/span>\u2013<span style=\"color: #339966;\">J\u03b4<\/span>\u2013<span style=\"color: #0000ff;\">C\u03b4<\/span> cluster.<\/strong> This occurred early in the evolution of tetrapods, or earlier. The duplication resulted in two copies of the <span style=\"color: #0000ff;\">C <\/span>gene of the \u03b4 chain, each with its own set of <span style=\"color: #f0a860;\">D<\/span> and <span style=\"color: #339966;\">J<\/span> segments.<\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><strong>Insertion of <span style=\"color: #ffcc00;\">VH<\/span>.<\/strong> Recall that VH refers to the variable chain of immunoglobulin heavy (IgH). One or more genes were translocated from the <em>IgH<\/em> locus and inserted into the <em>TCR\u03b1\/\u03b4<\/em> locus, most likely to a location between the existing <span style=\"color: #ff0000;\">V\u03b1\/\u03b4<\/span> genes and the 5\u2032-proximal <span style=\"color: #f0a860;\">D\u03b4<\/span>\u2013<span style=\"color: #339966;\">J\u03b4<\/span>\u2013<span style=\"color: #0000ff;\">C\u03b4<\/span> cluster. This is the configuration found today in the zebra finch genome.<sup class=\"modern-footnotes-footnote \" data-mfn=\"30\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-30\">30<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-30\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"30\">Parra et al. 2012<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><strong>Inversion of the <span style=\"color: #ffcc00;\">VH\u03b4<\/span><em>\u2013<\/em><span style=\"color: #f0a860;\">D\u03b4<\/span>\u2013<span style=\"color: #339966;\">J\u03b4<\/span>\u2013<\/strong><span style=\"color: #0000ff;\"><strong>C\u03b4 <\/strong><span style=\"color: #000000;\"><strong>cluster in amphibians.<\/strong> This cluster of genes was translocated and inverted, and the number of <span style=\"color: #ffcc00;\">VH\u03b4<\/span> genes increased. The frog\u00a0<em>X. tropicalis<\/em> currently has the greatest number of <span style=\"color: #ffcc00;\">VH\u03b4 <\/span>genes, where they make up the majority of V genes available in the germ-line for T-cell receptor \u03b4 chains.<sup class=\"modern-footnotes-footnote \" data-mfn=\"31\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-31\">31<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-31\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"31\">Parra et al. 2010<\/span><\/span><\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><strong>Translocation of the <span style=\"color: #ffcc00;\">VH\u03b4<\/span><em>\u2013<\/em><span style=\"color: #f0a860;\">D\u03b4<\/span>\u2013<span style=\"color: #339966;\">J\u03b4<\/span>\u2013<span style=\"color: #0000ff;\">C\u03b4 <span style=\"color: #000000;\">cluster<\/span><\/span> to another site in galliforms.<\/strong> In chickens and turkeys, the same cluster that was inverted in amphibians instead moved out of the <em>TCR\u03b1\/\u03b4<\/em> locus and is now found on another chromosome. <span id=\"jumplink-mss128-F5\" class=\"xrefLink\">There are no <span style=\"color: #ff0000;\">V\u03b1<\/span> or <span style=\"color: #ff0000;\">V\u03b4<\/span> genes at this second <em>TCR\u03b4<\/em> locus in chickens, and only a single <span style=\"color: #0000ff;\">C\u03b4<\/span> gene remains at the conventional <em>TCR\u03b1\/\u03b4<\/em> locus.<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><sup class=\"modern-footnotes-footnote \" data-mfn=\"32\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-32\">32<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-32\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"32\">Parra et al. 2012<\/span><\/span><\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><\/span><\/span><strong>Translocation of the <span style=\"color: #ffcc00;\">VH\u03b4<\/span><em>\u2013<\/em><span style=\"color: #f0a860;\">D\u03b4<\/span>\u2013<span style=\"color: #339966;\">J\u03b4<\/span>\u2013<span style=\"color: #0000ff;\">C\u03b4 <span style=\"color: #000000;\">cluster<\/span><\/span> to another site (TCR\u00b5) in mammals. <\/strong>A similar process to step D in galliforms happened in a common ancestor of mammals, giving rise to TCR\u00b5. Internal duplications of the <span style=\"color: #ffcc00;\">VH<\/span>, <span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span style=\"color: #f0a860;\">D<\/span><\/span>, and <span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span style=\"color: #339966;\">J<\/span><\/span> genes gave rise to the current <span id=\"jumplink-mss128-F5\" class=\"xrefLink\">[(<span style=\"color: #ffcc00;\">V<\/span>\u2013<span style=\"color: #f0a860;\">D<\/span>\u2013<span style=\"color: #339966;\">J<\/span>) \u2212 (<span style=\"color: #ffcc00;\">V<\/span>\u2013<span style=\"color: #f0a860;\">D<\/span>\u2013<span style=\"color: #339966;\">J<\/span>) \u2212 <span style=\"color: #0000ff;\">C<\/span>] organization that can encode chains with double V domains.<sup class=\"modern-footnotes-footnote \" data-mfn=\"33\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-33\">33<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-33\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"33\">Parra et al. 2007, Wang et al. 2011<\/span><\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><\/span><strong>Further changes in the three mammalian lineages.\u00a0<\/strong><\/p>\n<ul class=\"order\">\n<li>\n<p class=\"chapter-para\">In the platypus, <span id=\"jumplink-mss128-F5\" class=\"xrefLink\">the second <span style=\"color: #ffcc00;\">V<\/span>\u2013<span style=\"color: #f0a860;\">D<\/span>\u2013<span style=\"color: #339966;\">J<\/span> cluster, which encodes the supporting (non-terminal) V chain, lost its <span style=\"color: #f0a860;\">D<\/span> segments and generates V domains with short complementarity-determining region-3 (CDR3) encoded by direct V to J recombination.<sup class=\"modern-footnotes-footnote \" data-mfn=\"34\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-34\">34<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-34\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"34\">Wang et al. 2011<\/span> <\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F5\" class=\"xrefLink\">Meanwhile, in therians (marsupials and placentals), the <\/span><span style=\"color: #0000ff;\"><span style=\"color: #000000;\"><span style=\"color: #ffcc00;\">VH\u03b4<\/span><\/span><\/span> gene disappeared from the <em>TCR\u03b1\/\u03b4<\/em> locus (not shown in fig. 2).<sup class=\"modern-footnotes-footnote \" data-mfn=\"35\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-35\">35<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-35\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"35\">Parra et al. 2008<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\">Then, in placentals, the <em>TCR\u00b5 <\/em>locus was also lost.<sup class=\"modern-footnotes-footnote \" data-mfn=\"36\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-36\">36<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-36\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"36\">Parra et al. 2008<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\">The marsupials kept TCR\u00b5, but the second set of <span style=\"color: #ffcc00;\">V<\/span> and <span style=\"color: #339966;\">J<\/span> segments (which encode the supporting V domain) was replaced with a germ-line joined V gene (fused <span style=\"color: #ffcc00;\">yellow<\/span>&#8211;<span style=\"color: #339966;\">green<\/span> segment in fig. 2), probably due to germ-line V(D)J recombination and retro-transposition.<sup class=\"modern-footnotes-footnote \" data-mfn=\"37\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-37\">37<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-37\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"37\">Parra et al. 2007, 2008<\/span><\/p>\n<\/li>\n<li>\n<p class=\"chapter-para\">In both monotremes and marsupials, t<span id=\"jumplink-mss128-F5\" class=\"xrefLink\">he whole cluster from <span style=\"color: #0000ff;\"><span style=\"color: #000000;\"><span style=\"color: #ffcc00;\">VH<\/span><\/span><\/span>\u00a0to <span style=\"color: #0000ff;\">C<\/span> appears to have undergone additional tandem duplication as it exists in multiple copies in the opossum and probably in the platypus.<sup class=\"modern-footnotes-footnote \" data-mfn=\"38\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-38\">38<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-38\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"38\">Parra et al. 2007, 2008; Wang et al. 2011<\/span><\/span><\/p>\n<\/li>\n<\/ul>\n<\/li>\n<\/ol>\n<p>The rest of the paper explains the analyses that gave us with the evidence to build this model. Additional discussion of the model is provided in the last section.<\/p>\n<h2 id=\"77835853\" class=\"section-title js-splitscreen-section-title\">Materials and Methods<\/h2>\n<p>There are three parts to the analyses and experiments that allowed us to gather evidence and build our evolutionary model. First, find the <em>TCR\u03b1\/\u03b4<\/em> locus in platypus genome data. Second, perform phylogenetic analyses with the relevant genes. Third, confirm from a live specimen that the platypus expresses VH\u03b4.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_13');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_13');\" ><sup id=\"footnote_plugin_tooltip_604_1_13\" class=\"footnote_plugin_tooltip_text\">[13]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_13\" class=\"footnote_tooltip\">This new paragraph is important! It gives context to the experiments below and it guides the reader for the entire section. Also notice this is a case of an enumeration without point form. I like&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_13');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_13').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_13', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<h3 id=\"77835854\" class=\"section-title js-splitscreen-section-title\">1) Identification and Annotation of the Platypus <em>TCR\u03b1\/\u03b4<\/em> Locus<\/h3>\n<p>We analyzed the genome of the platypus, <em>Ornithorhynchus anatinus<\/em>, using the 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>). We used two genome alignment tools: whole-genome BLAST from 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 BLAST\/BLAT from Ensembl (<a class=\"link link-uri openInAnotherWindow\" href=\"http:\/\/www.ensembl.org\/\" target=\"_blank\" rel=\"noopener\">www.ensembl.org<\/a>).<\/p>\n<p>We located the V and J gene segments by looking for similarity with the corresponding segments of other species, and by identifying flanking conserved recombination signal sequences. (RSS). We annotated V segments in the 5\u2032 to 3\u2032 direction as either V\u03b1 or V\u03b4, followed by the family number and the gene segment number if there were more than one in the family. For example, V\u03b115.7 is the seventh V\u03b1 gene in family 15.<\/p>\n<p>As for the D segments, we identified them from cDNA clones using VH\u03b4, using complementarity-determining region-3 (CDR3) sequences that represent the V-D-J junctions.<\/p>\n<p>We labeled the platypus T-cell receptor gene segments 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>). We provide the location for the TCR\u03b1\/\u03b4 genes of the platypus genome version 5.0.1 in <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>, available online.<\/span><\/p>\n<h3 id=\"77835859\" class=\"section-title js-splitscreen-section-title\">2) Phylogenetic Analyses<\/h3>\n<p>We used BioEdit<sup class=\"modern-footnotes-footnote \" data-mfn=\"39\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-39\">39<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-39\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"39\">Hall 1999<\/span> as well as the <span id=\"jumplink-mss128-B14\" class=\"xrefLink\">accessory application ClustalX<\/span><sup class=\"modern-footnotes-footnote \" data-mfn=\"40\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-40\">40<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-40\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"40\">Thompson et al. 1997<\/span> to align the nucleotide sequences of the V genes regions, from the framework region FR1 to FR3, including the complementarity-determining regions CDR1 and CDR2. We established the codon position of the alignments using amino acid sequences.<sup class=\"modern-footnotes-footnote \" data-mfn=\"41\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-41\">41<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-41\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"41\">Hall 1999<\/span> When necessary, we corrected the alignments through visual inspection. We then analyzed them with MEGA Software.<sup class=\"modern-footnotes-footnote \" data-mfn=\"42\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-42\">42<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-42\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"42\">Kumar et al. 2004<\/span><\/p>\n<p>We generated phylogenetic trees using two methods: Neighbor Joining (NJ) with uncorrected nucleotide differences (p-distance), and Minimum Evolution distances.<\/p>\n<p>We evaluated support for the generated trees using bootstrap values from 1000 replicates.\u00a0<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> contains the GenBank accession numbers for the sequences used in tree construction.<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_14');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_14');\" ><sup id=\"footnote_plugin_tooltip_604_1_14\" class=\"footnote_plugin_tooltip_text\">[14]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_14\" class=\"footnote_tooltip\">In the original paper, this section comes after the Confirmation of Expression section below, but in the results section, the phylogenetic results are discussed first. I don&#8217;t know if there was&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_14');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_14').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_14', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script><\/p>\n<h3 id=\"77835856\" class=\"section-title js-splitscreen-section-title\">3) Confirmation of Expression of Platypus VH\u03b4<\/h3>\n<p>As described with more detail in the Results and Discussion section below, the annotation step allowed us to find an atypical VH\u03b4 gene in the platypus genome. To confirm that it was not an artifact of the genome assembly process, we looked at the expression of this gene in a live specimen, a male platypus from the Upper Barnard River in New South Wales, Australia.\u00a0The platypus was collected under the same permits as in Warren et al. 2008.<\/p>\n<p>We performed reverse transcription PCR (RT-PCR) on the RNA from the spleen of this New South Wales specimen. As a second point of comparison, we <span id=\"jumplink-mss128-B41\" class=\"xrefLink\">also used a previously described platypus spleen cDNA library that was constructed from RNA extracted from a Tasmanian animal<span id=\"jumplink-mss128-B38\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"43\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-43\">43<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-43\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"43\">Vernersson et al. 2002<\/span><\/span><\/span> The protocols and products used at every step are as follows:<\/p>\n<ul>\n<li><strong>cDNA synthesis<\/strong>: <span id=\"jumplink-mss128-B41\" class=\"xrefLink\">Invitrogen Superscript III-first strand synthesis kit, using the manufacturer\u2019s recommended protocol<sup class=\"modern-footnotes-footnote \" data-mfn=\"44\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-44\">44<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-44\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"44\">Invitrogen, Carlsbad, CA, USA<\/span><\/span><\/li>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><strong>PCR amplification<\/strong>: we used the QIAGEN HotStar HiFidelity Polymerase Kit<sup class=\"modern-footnotes-footnote \" data-mfn=\"45\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-45\">45<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-45\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"45\">BD Biosciences, CLONTECH Laboratories, Palo Alto, CA, USA<\/span> in total volume of 20 \u00b5l containing:<\/span>\n<ul>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">1\u00d7 Hotstar Hifi PCR Buffer (containing 0.3 mM dNTPs)<\/span><\/li>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">1\u00b5M of primers: we identified these from the platypus genome assembly step.<sup class=\"modern-footnotes-footnote \" data-mfn=\"46\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-46\">46<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-46\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"46\">Warren et al. 2008<\/span> We targeted T-cell receptor \u03b4 transcripts with two primers, one for VH\u03b4 and one for C\u03b4:<\/span>\n<ul>\n<li style=\"list-style-type: none;\">\n<ul>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">5\u2032-GTACCGCCAACCACCAGGGAAAG-3\u2032 for VH\u03b4 <\/span><\/li>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">5\u2032-CAGTTCACTGCTCCATCGCTTTCA-3\u2032 for C\u03b4<\/span><\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<\/li>\n<li><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">1.25U Hotstar Hifidelity DNA polymerase<\/span><\/li>\n<\/ul>\n<\/li>\n<li><strong>PCR product cloning<\/strong>: TopoTA cloning\u00ae kit <sup class=\"modern-footnotes-footnote \" data-mfn=\"47\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-47\">47<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-47\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"47\">Invitrogen<\/span><\/li>\n<li><strong>Sequencing<\/strong>: BigDye terminator cycle sequencing kit version 3<sup class=\"modern-footnotes-footnote \" data-mfn=\"48\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-48\">48<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-48\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"48\">Applied Biosystems, Foster City, CA, USA<\/span> according to the manufacturer recommendations.<\/li>\n<li><strong>Analysis of sequencing reactions<\/strong>: ABI Prism 3100 DNA automated sequences.<sup class=\"modern-footnotes-footnote \" data-mfn=\"49\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-49\">49<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-49\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"49\">PerkinElmer Life and Analytical Sciences, Wellesley, MA, USA<\/span><\/li>\n<li><strong>Chromatogram analysis<\/strong>: Sequencher 4.9 software<sup class=\"modern-footnotes-footnote \" data-mfn=\"50\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-50\">50<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-50\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"50\">Gene Codes Corporation, Ann Arbor, MI, USA<\/span><\/li>\n<\/ul>\n<p class=\"chapter-para\">We archived the sequence on GenBank under the accession numbers JQ664690\u2013JQ664710.<\/p>\n<h2 id=\"77835861\" class=\"section-title js-splitscreen-section-title\">Results and Discussion<\/h2>\n<h3>Results of the <em>TCR\u03b1\/\u03b4<\/em> Locus Identification in the Platypus<\/h3>\n<p>Here are the results of our analysis of the platypus genome from part 1 of the Materials and Methods section, which allowed us to identify the <em>TCR\u03b1\/\u03b4<\/em> locus and annotate its V, D, J and C gene segments, as well as the exons. Refer to fig. 3 below for the annotation map.<\/p>\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\">Fig. 3. Annotated map of the platypus TCR\u03b1\/\u03b4 locus, showing the locations of the <span style=\"color: #ff0000;\">V\u03b1 and V\u03b4 (red)<\/span>, <span style=\"color: #ffcc00;\">VH\u03b4 (yellow)<\/span>, <span style=\"color: #ff6600;\">D\u03b4 (orange)<\/span>, <span style=\"color: #008000;\">J\u03b1 and J\u03b4 (green)<\/span>, <span style=\"color: #0000ff;\">C\u03b4 (dark blue)<\/span>, and <span style=\"color: #00ccff;\">C\u03b1 (light blue)<\/span>. Conserved syntenic genes are in gray. The scaffold and contig numbers are indicated.<\/figcaption><\/figure>\n<p>Most of the locus is present on a single scaffold. The remainder is on a shorter contig. On either sides of the locus, we find the genes <em>SALL2<\/em>, <em>DAD1<\/em>, and several olfactory receptor genes (<em>OR<\/em>). All of these genes share conserved synteny with the <span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><em>TCR\u03b1\/\u03b4<\/em> locus in amphibians, birds, and mammals.<sup class=\"modern-footnotes-footnote \" data-mfn=\"51\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-51\">51<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-51\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"51\">Parra et al. 2008, 2010, 2012<\/span><\/span><\/p>\n<p>The platypus locus has many typical features common to\u00a0<em>TCR\u03b1\/\u03b4<\/em> loci in other tetrapods.<sup class=\"modern-footnotes-footnote \" data-mfn=\"52\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-52\">52<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-52\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"52\">Satyanarayana et al. 1988; Wang et al. 1994; Parra et al. 2008, 2010, 2012<\/span> <span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><span id=\"jumplink-mss128-B31\" class=\"xrefLink\"><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">Two C region genes are present: a C\u03b1 (<span style=\"color: #00ccff;\">light blue<\/span> in fig. 3) at the 3\u2032 end of the locus, and a C\u03b4 (<span style=\"color: #0000ff;\">dark blue<\/span>) oriented 5\u2032 of the J\u03b1 genes. These J\u03b1 genes occur in a large number (32) of fragments (in <span style=\"color: #008000;\">green<\/span>) located between C\u03b4 and C\u03b1. A large array of J\u03b1 genes like this is believed to facilitate secondary V\u03b1 to J\u03b1 rearrangements in developing \u03b1\u03b2T cells if the primary rearrangements are nonproductive or need replacement.<sup class=\"modern-footnotes-footnote \" data-mfn=\"53\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-53\">53<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-53\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"53\">Hawwari and Krangel 2007<\/span> <\/span><\/span><\/span><\/span><\/span><\/span><\/span><span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><span id=\"jumplink-mss128-B31\" class=\"xrefLink\"><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><span id=\"jumplink-mss128-B15\" class=\"xrefLink\">Primary TCR\u03b1 V\u2013J rearrangements 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 (the last <span style=\"color: #ff0000;\">red<\/span> segment in fig. 3) in reverse transcriptional orientation between the platypus C\u03b4 gene and the J\u03b1 array that is conserved in mammalian <em>TCR\u03b1\/\u03b4<\/em> both in location and orientation.<sup class=\"modern-footnotes-footnote \" data-mfn=\"54\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-54\">54<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-54\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"54\">Parra et al. 2008<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<div class=\"fig fig-modal reveal-modal\">\n<p>There are 99 conventional T-cell receptor V gene segments in the platypus <em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em> locus (<span style=\"color: #ff0000;\">red<\/span> in fig. 3). The vast majority, 89, share nucleotide identity with V\u03b1 in other species; the other 10 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 (the family and segment numbers are annotated in fig. 3). This is a typical level of complexity for mammalian V\u03b1 and V\u03b4 genes.<sup class=\"modern-footnotes-footnote \" data-mfn=\"55\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-55\">55<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-55\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"55\">Giudicelli et al. 2005;\u00a0Parra et al. 2008<\/span><\/p>\n<p>Also present were two D\u03b4 (<span style=\"color: #ff6600;\">orange<\/span>) and seven J\u03b4 (<span style=\"color: #008000;\">green<\/span>) gene segments oriented upstream of the C\u03b4. All gene segments were flanked by canonical recombination signal sequences (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 T-cell receptor D gene segments in other species.<sup class=\"modern-footnotes-footnote \" data-mfn=\"56\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-56\">56<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-56\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"56\">Carroll et al. 1993;\u00a0Parra et al. 2007,\u00a02010<\/span> In summary, the overall content and organization of the platypus\u00a0<em style=\"letter-spacing: normal;\">TCR\u03b1\/\u03b4<\/em> locus appeared fairly generic, with one exception.<\/p>\n<p>This atypical feature of the platypus locus is an additional V gene that shares greater identity to antibody VH genes than to T-cell receptor V genes. <span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">Among V genes, this segment is the closest to the D and J genes (see the <span style=\"color: #ffcc00;\">yellow<\/span> segment in fig. 3). We tentatively designated it as VH\u03b4.\u00a0<\/span><\/span><\/p>\n<\/div>\n<h3>VH<span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">\u03b4 Phylogenetics<\/span><\/span><\/h3>\n<p><span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">VH\u03b4 genes are, by definition, V genes that are indistinguishable from immunoglobulin heavy V (Ig VH) genes, but used in encoding T-cell receptor \u03b4 chains. Recall from the introduction<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_15');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_15');\" ><sup id=\"footnote_plugin_tooltip_604_1_15\" class=\"footnote_plugin_tooltip_text\">[15]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_15\" class=\"footnote_tooltip\">Yes, you are allowed to make links between the sections of your paper like this!<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_15').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_15', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> that they have previously been found only in the genomes of birds and frogs<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"57\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-57\">57<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-57\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"57\">Parra et al. 2008, 2010, 2012<\/span><\/span><\/span><\/span><\/span><\/p>\n<p>To put the platypus <span id=\"jumplink-mss128-F2\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">VH\u03b4 gene in context, let us examine the phylogeny of<\/span><\/span> VH genes. In mammals and other tetrapods, VH genes have been shown to cluster into three ancient clans (shown in fig. 4). Individual species differ in the presence of one or more of these clans in their germ-line immunoglobulin heavy locus<span id=\"jumplink-mss128-B37\" class=\"xrefLink\"><span id=\"jumplink-mss128-B24\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"58\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-58\">58<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-58\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"58\">Tutter and Riblet 1989; Ota and Nei 1994<\/span> For example, humans, mice, echidnas, and frogs have VH genes from all three clans<span id=\"jumplink-mss128-B33\" class=\"xrefLink\"><span id=\"jumplink-mss128-B2\" class=\"xrefLink\">,<sup class=\"modern-footnotes-footnote \" data-mfn=\"59\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-59\">59<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-59\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"59\">Schwager et al. 1989; Ota and Nei 1994; Belov and Hellman 2003<\/span> whereas rabbits, opossums, and chickens have only a single clan<span id=\"jumplink-mss128-B22\" class=\"xrefLink\"><span id=\"jumplink-mss128-B3\" class=\"xrefLink\"><span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><span id=\"jumplink-mss128-B1\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"60\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-60\">60<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-60\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"60\">McCormack et al. 1991;\u00a0Butler 1997;\u00a0Johansson et al. 2002;\u00a0Baker et al. 2005<\/span> <\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\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\">Fig. 4. Phylogenetic tree of mammalian VH genes, including the platypus VH\u03b4 and monotreme V\u00b5. The three major VH clans are bracketed. A box indicates the platypus VH\u03b4, and bolding indicates the clade containing platypus VH\u03b4 along with platypus and echidna V\u00b5 within 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. The numbers following the platypus and echidna V\u00b5 labels are clone numbers. The tree shown here was generated using the Minimum Evolution method; the Neighbor Joining method yielded a similar topology.<\/figcaption><\/figure>\n<p><span id=\"jumplink-mss128-B37\" class=\"xrefLink\"><span id=\"jumplink-mss128-B24\" class=\"xrefLink\"><span id=\"jumplink-mss128-B33\" class=\"xrefLink\"><span id=\"jumplink-mss128-B2\" class=\"xrefLink\"><span id=\"jumplink-mss128-B22\" class=\"xrefLink\"><span id=\"jumplink-mss128-B3\" class=\"xrefLink\"><span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><span id=\"jumplink-mss128-B1\" class=\"xrefLink\">Our phylogenetic analyses showed that the platypus VH\u03b4 was most related to the platypus V\u00b5 genes found at the <em>TCR\u00b5<\/em> locus (see the boxed and bolded parts of fig. 4)<span id=\"jumplink-mss128-F3\" class=\"xrefLink\">. Platypus VH\u03b4, however, shares 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.<sup class=\"modern-footnotes-footnote \" data-mfn=\"61\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-61\">61<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-61\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"61\">Wang et al. 2011<\/span> This is noteworthy since VH genes in the platypus IgH locus are also from clan III and, in general, clan III is the most ubiquitous and conserved lineage of VH.<sup class=\"modern-footnotes-footnote \" data-mfn=\"62\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-62\">62<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-62\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"62\">Johansson et al. 2002; Tutter and Riblet 1989<\/span> Although clearly related to platypus VH, the VH\u03b4 gene shares only 34\u201365% nucleotide identity (average 56.9%) with the &#8220;authentic&#8221;<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_16');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_16');\" ><sup id=\"footnote_plugin_tooltip_604_1_16\" class=\"footnote_plugin_tooltip_text\">[16]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_16\" class=\"footnote_tooltip\">I&#8217;m not sure about this but the original phrase was bona fide, which I had to look up. Maybe &#8220;authentic&#8221; between quotes isn&#8217;t the best translation, but a translation is better&nbsp;&#x2026; <span class=\"footnote_tooltip_continue\"  onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_16');\">Continue reading<\/span><\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_16').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_16', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> VH used in antibody heavy chains in this species.<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<h3>Results of the Confirmation of VH\u03b4 Expression<\/h3>\n<p class=\"chapter-para\">It was necessary to rule out that the VH\u03b4 gene present in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em> locus was not an artifact of the genome assembly process. This is why we performed a &#8220;wet lab&#8221; verification step on cDNA synthesized from the splenic RNA of two platypuses, one from New South Wales and one from Tasmania (see Materials and Methods). We performed RT-PCR with primers that were specific for VH\u03b4 and C\u03b4. We were successful in amplifying the PCR products of the NSW specimen, but not for the Tasmanian one.<\/p>\n<p class=\"chapter-para\">One piece of supporting evidence for the expression of VH\u03b4 would be the demonstration that it is recombined to downstream D\u03b4 and J\u03b4 segments, and expressed with C\u03b4 in complete T-cell receptor \u03b4 transcripts. This is what we found from the twenty sequenced clones we obtained from PCR in the New South Wales platypus. Each clone contained a unique nucleotide sequence that comprised the VH\u03b4 gene recombined to the D\u03b4 and J\u03b4 gene segments (see fig. 4A). Of these 20, 11 had unique V, D, and J combinations that would therefore encode 11 different <span id=\"jumplink-mss128-F4\" class=\"xrefLink\">complementarity-determining regions-3 (CDR3; see fig. 4B). More then half of these (8 out of 11) contained evidence of using both D genes, giving a VDDJ pattern. This is a common feature of \u03b4 V domains where multiple D genes can be incorporated into the recombination due to the presence of asymmetrical RSS.<sup class=\"modern-footnotes-footnote \" data-mfn=\"63\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-63\">63<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-63\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"63\">Carroll et al. 1993<\/span><\/span><\/p>\n<figure id=\"attachment_540\" aria-describedby=\"caption-attachment-540\" style=\"width: 580px\" class=\"wp-caption alignnone\"><img loading=\"lazy\" decoding=\"async\" class=\"size-large wp-image-540\" 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\">Fig. 4. <strong>(A)<\/strong> 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. <strong>(B)<\/strong> 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<p class=\"chapter-para\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B4\" class=\"xrefLink\">The region corresponding to the junctions between the V, D, and J segments contained an additional sequence that could not be accounted for by the germ-line gene segments (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\">fig. 4B). There are two possible sources of such a sequence. One is palindromic 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\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"64\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-64\">64<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-64\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"64\">Lewis 1994<\/span> The second is non-templated nucleotides that can be added by the enzyme terminal deoxynucleotidyl transferase (TdT) during the V(D)J recombination process.<\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B20\" class=\"xrefLink\">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 immunoglobulin domains (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\">fig. 4A). Additional cysteines in the complementarity-determining region 3 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\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"65\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-65\">65<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-65\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"65\">Johansson et al. 2002<\/span> The CDR3 of T-cell receptor \u03b4 using VH\u03b4 are only slightly longer than conventional \u03b4 chains (ranging 10\u201320 residues)<span id=\"jumplink-mss128-B30\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"66\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-66\">66<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-66\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"66\">Rock et al. 1994;\u00a0Wang et al. 2011<\/span> 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\">fig. 4A).\u00a0<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<h5>The Tasmanian specimen<\/h5>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B20\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><span id=\"jumplink-mss128-B30\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">The above concerns the animal collected from New South Wales. With the Tasmanian specimen, we were unable to amplify T-cell receptor \u03b4 transcripts containing VH\u03b4 from its splenic cDNA. We did, however, successfully isolate transcripts containing conventional V\u03b1\/\u03b4 segments, which provides a positive control. <\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B20\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><span id=\"jumplink-mss128-B30\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">It is possible t<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B4\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B20\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B17\" class=\"xrefLink\"><span id=\"jumplink-mss128-B30\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">hat Tasmanian platypuses, which have been separated from the mainland population at least 14,000 years ago, either have a divergent VH\u03b4 or have deleted this single V gene altogether<span id=\"jumplink-mss128-B19\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"67\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-67\">67<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-67\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"67\">Lambeck and Chappell 2001<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<h5>Sequence variation in VH\u03b4<\/h5>\n<div class=\"fig fig-section js-fig-section\" data-id=\"mss128-F4\">\n<div class=\"graphic-wrap\">\n<p>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 (see <span id=\"jumplink-mss128-F4\" class=\"xrefLink\">fig. 4A; the sequence data are not shown here, but are available in GenBank). We have three potential explanations for this variation:<\/span><\/p>\n<ol>\n<li>Two alleles of a single <span id=\"jumplink-mss128-F4\" class=\"xrefLink\">VH\u03b4 gene<\/span><\/li>\n<li><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">Somatic mutation of expressed VH\u03b4 genes<\/span><\/span><\/li>\n<li><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\">Allelic variation in gene copy number<\/span><\/span><\/li>\n<\/ol>\n<p>The two-allele explanation makes sense given that\u00a0<span id=\"jumplink-mss128-F4\" class=\"xrefLink\">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, which was found to contain substantial heterozygosity<span id=\"jumplink-mss128-B41\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"68\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-68\">68<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-68\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"68\">Warren et al. 2008<\/span> However, the variation was too large to be fully explained by this.<\/span><\/span><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">\u00a0<\/span><\/span><\/span><\/p>\n<p><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">The second possibility, somatic mutation (i.e. mutation not occurring in germ cells), is considered controversial for T-cell receptor chains. Nonetheless, it has been invoked in sharks and postulated in salmonids to explain the variation that exceeds the apparent gene copy number in these vertebrates<span id=\"jumplink-mss128-B44\" class=\"xrefLink\"><span id=\"jumplink-mss128-B5\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"69\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-69\">69<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-69\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"69\">Yazawa et al. 2008; Chen et al. 2009<\/span> Therefore, it seems possible<span class=\"footnote_referrer\"><a role=\"button\" tabindex=\"0\" onclick=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_17');\" onkeypress=\"footnote_moveToReference_604_1('footnote_plugin_reference_604_1_17');\" ><sup id=\"footnote_plugin_tooltip_604_1_17\" class=\"footnote_plugin_tooltip_text\">[17]<\/sup><\/a><span id=\"footnote_plugin_tooltip_text_604_1_17\" class=\"footnote_tooltip\">I kind of like the original phrasing &#8220;it does not seem to be out of the realm of possibility&#8221; but that could be easily simplified, so I did.<\/span><\/span><script type=\"text\/javascript\"> jQuery('#footnote_plugin_tooltip_604_1_17').tooltip({ tip: '#footnote_plugin_tooltip_text_604_1_17', tipClass: 'footnote_tooltip', effect: 'fade', predelay: 0, fadeInSpeed: 200, delay: 400, fadeOutSpeed: 200, position: 'top center', relative: true, offset: [-7, 0], });<\/script> that somatic mutation is occurring in platypus VH\u03b4. One piece of evidence in favor of this is that the mutations appear to be localized to the V region with no variation in the C region (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\">fig. 4A). This may be due to the relatedness between VH\u03b4 and immunoglobulin VH genes where somatic hyper-mutation is well documented. Somatic mutation in immunoglobulin VH contributes to overall affinity maturation in secondary antibody responses.<span id=\"jumplink-mss128-B42\" class=\"xrefLink\"><sup class=\"modern-footnotes-footnote \" data-mfn=\"70\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-70\">70<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-70\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"70\">Wysocki et al. 1986<\/span> However, this means that the evidence is mixed: the pattern of mutation seen in the platypus is found in the complementarity-determining region 3, which would be indicative of selection for affinity maturation, but was also found in the framework regions, which does not indicate this. As further evidence against the somatic mutation explanation, there is no evidence of somatic mutation in the V regions of birds, which also have\u00a0<\/span><\/span><\/span><\/span><\/span><\/span><\/span><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B44\" class=\"xrefLink\"><span id=\"jumplink-mss128-B5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B42\" class=\"xrefLink\">only a single VH\u03b4.<span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><sup class=\"modern-footnotes-footnote \" data-mfn=\"71\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-71\">71<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-71\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"71\">Parra et al. 2012<\/span> The contribution of mutation to the platypus TCR\u03b4 repertoire, if it is occurring, remains to be determined. <\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<p><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B41\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B44\" class=\"xrefLink\"><span id=\"jumplink-mss128-B5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F4\" class=\"xrefLink\"><span id=\"jumplink-mss128-B42\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">Alternatively, the sequence polymorphism may be due to VH\u03b4 gene copy number variation between individual <em>TCR\u03b1\/\u03b4<\/em> alleles.<\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/span><\/p>\n<\/div>\n<\/div>\n<p class=\"chapter-para\">Irrespective of the number of VH\u03b4 genes in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em> locus, the results clearly support T-cell receptor \u03b4 transcripts containing VH\u03b4 recombined to D\u03b4 and J\u03b4 gene segments in the <em>TCR\u03b1\/\u03b4<\/em>\u00a0locus (<span id=\"jumplink-mss128-F4\" class=\"xrefLink\">fig. 4). A VH\u03b4 gene or genes in the platypus\u00a0<em>TCR\u03b1\/\u03b4<\/em> locus in the genome assembly, therefore, does not appear to be an assembly artifact. <\/span><span id=\"jumplink-mss128-F4\" class=\"xrefLink\">Rather, it is present and functional, and contributes to the expressed T-cell receptor \u03b4 chain repertoire. The possibility that some platypus <em>TCR\u03b1\/\u03b4<\/em> loci contain more than a single VH\u03b4 does not alter the principal conclusions of this study.<\/span><\/p>\n<h3>Discussion of our Model of the Evolution of TCR\u03b1\/\u03b4 and TCR\u00b5<\/h3>\n<p>The results above make up the evidence that allowed us to construct the model shown after the introduction section (see fig. 2). Here we discuss various considerations about the model.<\/p>\n<h5>Previous hypothesis of the origin of TCR\u00b5 in mammals<\/h5>\n<p>Our previous hypothesis<sup class=\"modern-footnotes-footnote \" data-mfn=\"72\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-72\">72<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-72\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"72\">Parra et al. 2008<\/span> about the origin of T-cell receptor \u00b5 (TCR\u00b5) in mammals involved the recombination between an ancestral <em>TCR\u03b1\/\u03b4<\/em> locus and an immunoglobulin heavy (<em>IgH<\/em>) locus. The <em>IgH<\/em> locus would have contributed the V gene segments at the 5\u2032-end, while the T-cell receptor \u03b4 would have contributed the D, J, and C genes at the 3\u2032-end of the locus.<\/p>\n<p>The difficulty with this hypothesis was the clear stability of the genome region surrounding the <em>TCR\u03b1\/\u03b4<\/em>\u00a0locus. In other words, the chromosomal region containing the\u00a0<em>TCR\u03b1\/\u03b4<\/em> locus appears to have remained relatively undisrupted for at least the past 360 million years<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"73\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-73\">73<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-73\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"73\">Parra et al. 2008, 2010, 2012<\/span><\/span><\/span><\/p>\n<h5><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">VH\u03b4<\/span><\/span> in different vertebrate lineages<\/h5>\n<p>An alternative model for the origins of TCR\u00b5 emerged from the discovery, in amphibians and birds, of VH\u03b4 genes inserted into the <em>TCR\u03b1\/\u03b4<\/em> locus. This model involves the insertion of VH (fig. 2B) followed by the duplication and translocation of T-cell receptor genes (fig. 2C-E).<\/p>\n<p>The insertion in<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">\u00a0the <em>TCR\u03b1\/\u03b4<\/em> locus seems to occur without disrupting the local syntenic region, as we know from zebra finches<\/span><\/span><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">\u00a0and frogs. <\/span><\/span><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">In frogs, the <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> locus.<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><sup class=\"modern-footnotes-footnote \" data-mfn=\"74\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-74\">74<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-74\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"74\">Parra et al. 2010<\/span> <\/span><\/span><\/span><\/p>\n<p><span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\"><span id=\"jumplink-mss128-B28\" class=\"xrefLink\">But close linkage is not a requirement. The\u00a0genomes of birds and platypuses do not show such linkage, and<\/span><\/span><\/span><span class=\"xrefLink\"><span class=\"xrefLink\"><span class=\"xrefLink\"> the translocation of VH genes to the <em>TCR\u03b1\/\u03b4<\/em> locus appears to have occurred independently from frogs in these two lineages. We know this from the lack of similarity and relatedness between the VH\u03b4 genes of frogs, birds, and monotremes<span class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"75\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-75\">75<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-75\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"75\">Parra et al. 2012<\/span> As can be seen in the phylogenetic tree of fig. 4, they<\/span><\/span><\/span><\/span><span id=\"jumplink-mss128-B12\" class=\"xrefLink\"><span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">\u00a0appear derived each from different, ancient VH clans:<\/span><\/span><\/span><\/p>\n<ul>\n<li>Clan I for birds<\/li>\n<li>Clan II for frogs<\/li>\n<li>Clan III for platypuses<\/li>\n<\/ul>\n<p><span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">Therefore, we suggest that the transfer of <span class=\"xrefLink\">VH\u03b4 <\/span>occurred independently in the different lineages. Another possibility is that<\/span><\/span><span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\"> transfers of <span class=\"xrefLink\">VH\u03b4<\/span> may have occurred frequently and repeatedly in the past. Gene replacement may be the best explanation for the current content of these genes in the different tetrapod lineages.<\/span><\/span><\/p>\n<p>The new evidence of platypus VH\u03b4 from this study allows us to update the model.<\/p>\n<h5>Updating the model for mammalian <span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">TCR\u00b5<\/span><\/span><\/h5>\n<p>Let us contrast the evidence from marsupials with the evidence we have gathered from the platypus. In marsupials, there is no <span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">VH\u03b4; the V\u00b5 genes are highly divergent; and at least in the opossum, there is no conserved synteny with genes linked to TCR\u00b5. These facts provide little insight into the origins of T-cell receptor \u00b5 and its relationship to other T-cell receptor chains like \u03b4 or the conventional ones.<sup class=\"modern-footnotes-footnote \" data-mfn=\"76\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-76\">76<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-76\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"76\">Parra et al. 2008<\/span><\/span><\/span><\/p>\n<p>In the platypus genome, however, we notice a striking similarity between <span id=\"jumplink-mss128-F5\" class=\"xrefLink\"><span id=\"jumplink-mss128-F3\" class=\"xrefLink\">VH, VH\u03b4, and V\u00b5. These genes are all in clan III. In particular, the close relationship between the platypus VH\u03b4 and V\u00b5 genes lends greater support for the model presented in fig. 2E, with TCR\u00b5 having been derived from TCR\u03b4 genes.<\/span><\/span><\/p>\n<p>The similarity that we found here between the platypus VH\u03b4 and V genes in the <em>TCR\u00b5<\/em> locus is, so far, the clearest evolutionary association between the \u00b5 and \u03b4 loci in one species.<\/p>\n<h5>Evolution of chains with three extracellular domains<\/h5>\n<p class=\"chapter-para\">TCR\u00b5 is an example of a T-cell receptor form with\u00a0three extracellular domains (refer back to fig. 1). These forms have evolved at least twice in vertebrates. The first was in the ancestors of the cartilaginous fish in the form of NAR-TCR.<sup class=\"modern-footnotes-footnote \" data-mfn=\"77\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-77\">77<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-77\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"77\">Criscitiello et al. 2006<\/span> The second was in the mammals as TCR\u00b5.<sup class=\"modern-footnotes-footnote \" data-mfn=\"78\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-78\">78<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-78\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"78\">Parra et al. 2007<\/span><\/p>\n<p class=\"chapter-para\">As we discussed in the introduction, NAR-TCR uses an N-terminal V domain that is related to the V domains found in IgNAR antibodies, which are unique to cartilaginous fish<span id=\"jumplink-mss128-B12\" class=\"xrefLink\">,<sup class=\"modern-footnotes-footnote \" data-mfn=\"79\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-79\">79<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-79\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"79\">Greenberg et al. 1995; Criscitiello et al. 2006<\/span> 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.<sup class=\"modern-footnotes-footnote \" data-mfn=\"80\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-80\">80<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-80\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"80\">Parra et al. 2007;\u00a0Wang et al. 2011<\/span><\/span><\/p>\n<h5>Evolution of chains with antibody-like V domains<\/h5>\n<p class=\"chapter-para\">T-cell receptor chains that use antibody-like V domains, such as TCR\u03b4 using VH\u03b4, NAR-TCR, or TCR\u00b5 (i.e. the receptors with yellow ovals in fig. 1) are widely distributed in vertebrates. Only the bony fish and placental mammals lack them.<\/p>\n<p class=\"chapter-para\">In addition to NAR-TCR, some shark species appear to generate T-cell receptor chains using antibody V genes. This occurs via trans-locus V(D)J recombination between immunoglobulin IgM and IgW heavy chain V genes and TCR\u03b4 and TCR\u03b1 D and J genes<span id=\"jumplink-mss128-B7\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"81\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-81\">81<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-81\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"81\">Criscitiello et al. 2010<\/span> This may be possible, in part, due to the multiple clusters of immunoglobulin genes found in the cartilaginous fish. It also illustrates that there have been independent solutions to generating T-cell receptor chains with antibody V domains in different vertebrate lineages. <\/span><\/p>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-B7\" class=\"xrefLink\">In the tetrapods, the VH genes were trans-located into the T-cell receptor loci where they became part of the germ-line repertoire. By comparison, 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 T-cell receptors using antibody V genes over much of vertebrate evolutionary history.<\/span><\/p>\n<p class=\"chapter-para\">What function do the antibody V chains serve? The current working hypothesis is that they are able to bind native antigen directly. This is consistent with a selective pressure for T-cell receptor chains that may bind or recognize antigen in ways similar to antibodies in many different lineages of vertebrates.<\/p>\n<p class=\"chapter-para\">In the case of NAR-TCR and TCR\u00b5, the N-terminal V domain (the &#8220;third&#8221; one) is likely to be unpaired and bind antigen as a single domain (see fig. 1), as has been described for IgNAR and some IgG antibodies in camels (recently reviewed in <span id=\"jumplink-mss128-B10\" class=\"xrefLink\">Flajnik et al. 2011). 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 and presumably perform a structural role rather than one of antigen recognition.<sup class=\"modern-footnotes-footnote \" data-mfn=\"82\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-82\">82<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-82\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"82\">Parra et al. 2007;\u00a0Wang et al. 2011<\/span> <\/span><\/p>\n<p class=\"chapter-para\"><span id=\"jumplink-mss128-B10\" class=\"xrefLink\">There is no evidence of double V domains in TCR\u03b4 chains using VH\u03b4 in frogs, birds, or platypus (rightmost part of fig. 1)<span id=\"jumplink-mss128-B28\" class=\"xrefLink\"><span id=\"jumplink-mss128-B27\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"83\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-83\">83<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-83\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"83\">Parra et al. 2010, 2012<\/span> Rather, the complex containing VH\u03b4 would likely be structured similar to a conventional \u03b3\u03b4 receptors with a single V domain on each chain. It is possible that such receptors also bind antigen directly, but this remains to be determined.<\/span><\/span><\/span><\/p>\n<p class=\"chapter-para\">A compelling model for the evolution of the immunoglobulin and T-cell receptor loci has been one of internal duplication, divergence and deletion. This is the so-called birth-and-death model of evolution of immune genes and was promoted by Nei and colleagues<span id=\"jumplink-mss128-B24\" class=\"xrefLink\"><span id=\"jumplink-mss128-B23\" class=\"xrefLink\">.<sup class=\"modern-footnotes-footnote \" data-mfn=\"84\" data-mfn-post-scope=\"000000004d86c196000000005a895d32_604\"><a href=\"javascript:void(0)\"  role=\"button\" aria-pressed=\"false\" aria-describedby=\"mfn-content-000000004d86c196000000005a895d32_604-84\">84<\/a><\/sup><span id=\"mfn-content-000000004d86c196000000005a895d32_604-84\" role=\"tooltip\" class=\"modern-footnotes-footnote__note\" tabindex=\"0\" data-mfn=\"84\">Ota and Nei 1994; Nei et al. 1997<\/span> Our results do not 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 had 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><\/span><\/p>\n<div class=\"speaker-mute footnotes_reference_container\"> <div class=\"footnote_container_prepare\"><p><span role=\"button\" tabindex=\"0\" class=\"footnote_reference_container_label pointer\" onclick=\"footnote_expand_collapse_reference_container_604_1();\">Notes<\/span><span role=\"button\" tabindex=\"0\" class=\"footnote_reference_container_collapse_button\" style=\"display: none;\" onclick=\"footnote_expand_collapse_reference_container_604_1();\">[<a id=\"footnote_reference_container_collapse_button_604_1\">+<\/a>]<\/span><\/p><\/div> <div id=\"footnote_references_container_604_1\" style=\"\"><table class=\"footnotes_table footnote-reference-container\"><caption class=\"accessibility\">Notes<\/caption> <tbody> \r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_1');\"><a id=\"footnote_plugin_reference_604_1_1\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>1<\/a><\/th> <td class=\"footnote_plugin_text\">This is an example comment.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_2');\"><a id=\"footnote_plugin_reference_604_1_2\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>2<\/a><\/th> <td class=\"footnote_plugin_text\">Major changes to the abstract: I split it in four paragraphs with section titles (this is common in some journals; it should be common in most journals). I also added a section at the beginning to state the main point of the paper. Scientists have the bad habit of starting with background information before we even know why we&#8217;re supposed to care. This fixes that.<\/p>\n<p>The abstract is longer now, but not terribly so (286 vs. 267 words), so I think it&#8217;s fine; it could be shortened some more, but that would be a question of picking what to remove, which I&#8217;m less confident to do as I&#8217;m not the author.<\/p>\n<p>Note that I rewrote the abstract after rewriting the rest.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_3');\"><a id=\"footnote_plugin_reference_604_1_3\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>3<\/a><\/th> <td class=\"footnote_plugin_text\">Since this abbreviation comes up a lot, I put it first, with its meaning in parentheses.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_4');\"><a id=\"footnote_plugin_reference_604_1_4\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>4<\/a><\/th> <td class=\"footnote_plugin_text\">I didn&#8217;t change much in the figure&#8217;s caption, but it seemed pretty trivial to add color to the text to facilitate looking up what the colors mean.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_5');\"><a id=\"footnote_plugin_reference_604_1_5\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>5<\/a><\/th> <td class=\"footnote_plugin_text\">This is an example of two sentences taken verbatim from the original paper. Not all of it was poorly written!<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_6');\"><a id=\"footnote_plugin_reference_604_1_6\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>6<\/a><\/th> <td class=\"footnote_plugin_text\">It took me forever to rewrite this part. The original sentence was, &#8220;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.&#8221; Soooo many things wrong here.<\/p>\n<p>First, the weirdly formatted term &#8220;V(D)J&#8221; was not defined anywhere. I assume it means &#8220;V, J, and optionally D,&#8221; but it&#8217;s not as obvious as the authors seem to think.<\/p>\n<p>Second, why are we talking about B cells? They don&#8217;t come up anywhere else except in the very last sentence of the paper. We&#8217;ve been talking about T cells; if you&#8217;re going to switch to a different but similarly named type of cell, then you should tell the reader explicitly.<\/p>\n<p>Third, this is two different ideas linked together with an &#8220;and&#8221;. I have no clue why it was written as a single sentence, except maybe for the bad reason of having the citations refer to both ideas. They&#8217;re so different that it made sense to split them into not only distinct sentences or paragraphs, but actual sections!<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_7');\"><a id=\"footnote_plugin_reference_604_1_7\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>7<\/a><\/th> <td class=\"footnote_plugin_text\">The original combined this sentence and the next, even though they&#8217;re about quite distinct ideas: the name of the genes, and the species where they&#8217;re found.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_8');\"><a id=\"footnote_plugin_reference_604_1_8\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>8<\/a><\/th> <td class=\"footnote_plugin_text\">Why not indicate the part of the figure that is relevant? Whenever you can, provide reader guidance!<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_9');\"><a id=\"footnote_plugin_reference_604_1_9\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>9<\/a><\/th> <td class=\"footnote_plugin_text\">The authors like to use &#8220;duckbill platypus,&#8221; but there&#8217;s only one species of platypus, so I took that word out.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_10');\"><a id=\"footnote_plugin_reference_604_1_10\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>10<\/a><\/th> <td class=\"footnote_plugin_text\">Another horrible sentence from the original, recorded for posterity: &#8220;Trans-locus V(D)J recombination of V genes from other Ig and TCR loci with TCR\u00b5 genes has not been found.&#8221; That distance between the subject (recombination) and the verb (has). Ugh.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_11');\"><a id=\"footnote_plugin_reference_604_1_11\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>11<\/a><\/th> <td class=\"footnote_plugin_text\">The abbreviation IgSF was used in the paper, with no explanation. I assume the people who would read this paper tend to know what that means, but still.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_12');\"><a id=\"footnote_plugin_reference_604_1_12\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>12<\/a><\/th> <td class=\"footnote_plugin_text\">Major change from me here. This section was moved here from the discussion, because it is the core and most interesting part of the paper. It is now its own first-level section alongside Introduction, Materials and Methods, etc.<\/p>\n<p>I also simplified the contents. The six stages used to be identified with letters A-F in the figure, and 1-6 in the text. I changed that to use letters everywhere. I removed most of the figure&#8217;s caption since it repeats the text.<\/p>\n<p>There was an inconsistency in calling the same thing the D\u03b4\u2013J\u03b4\u2013C\u03b4 cluster in the figure and D\u2013J\u2013C\u03b4 cluster in the text. I fixed that. I also color-coded the elements in the text according to the figure.<\/p>\n<p>One thing I didn&#8217;t like about the original figure is that the six stages aren&#8217;t sequential. The figure presented steps A to F as if they followed one another, but steps C, D and E-F describe the evolution in different animal lineages. So I reorganized the contents and added some arrows for clarification. It also seems that the steps 5-6 in the text and E-F in the figure didn&#8217;t quite match, with some parts illustrated in step F being explained in step 5; I edited the text so that they do match. I think the figure could be improved much more, notably by splitting the complex F stage in multiple steps, but I don&#8217;t want to change it too much.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_13');\"><a id=\"footnote_plugin_reference_604_1_13\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>13<\/a><\/th> <td class=\"footnote_plugin_text\">This new paragraph is important! It gives context to the experiments below and it guides the reader for the entire section. Also notice this is a case of an enumeration without point form. I like point form, but it must not be overused.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_14');\"><a id=\"footnote_plugin_reference_604_1_14\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>14<\/a><\/th> <td class=\"footnote_plugin_text\">In the original paper, this section comes after the Confirmation of Expression section below, but in the results section, the phylogenetic results are discussed first. I don&#8217;t know if there was a reason for this (maybe they performed the phylogenetic analysis later) but it seems better to keep the same order in both sections, which is why I&#8217;m placing this part here.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_15');\"><a id=\"footnote_plugin_reference_604_1_15\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>15<\/a><\/th> <td class=\"footnote_plugin_text\">Yes, you are allowed to make links between the sections of your paper like this!<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_16');\"><a id=\"footnote_plugin_reference_604_1_16\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>16<\/a><\/th> <td class=\"footnote_plugin_text\">I&#8217;m not sure about this but the original phrase was <em>bona fide<\/em>, which I had to look up. Maybe &#8220;authentic&#8221; between quotes isn&#8217;t the best translation, but a translation is better than a Latin phrase that many people will not get.<\/td><\/tr>\r\n\r\n<tr class=\"footnotes_plugin_reference_row\"> <th scope=\"row\" class=\"footnote_plugin_index_combi pointer\"  onclick=\"footnote_moveToAnchor_604_1('footnote_plugin_tooltip_604_1_17');\"><a id=\"footnote_plugin_reference_604_1_17\" class=\"footnote_backlink\"><span class=\"footnote_index_arrow\">&#8593;<\/span>17<\/a><\/th> <td class=\"footnote_plugin_text\">I kind of like the original phrasing &#8220;it does not seem to be out of the realm of possibility&#8221; but that could be easily simplified, so I did.<\/td><\/tr>\r\n\r\n <\/tbody> <\/table> <\/div><\/div><script type=\"text\/javascript\"> function footnote_expand_reference_container_604_1() { jQuery('#footnote_references_container_604_1').show(); jQuery('#footnote_reference_container_collapse_button_604_1').text('\u2212'); } function footnote_collapse_reference_container_604_1() { jQuery('#footnote_references_container_604_1').hide(); 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It is meant to be a demonstration and a proof of concept for JAWWS, my idea of a science journal that focusses on [&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-604","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 v27.4 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>Platypus Paper, Rewritten &#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\/platypus-paper-rewritten\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Platypus Paper, Rewritten &#8212; 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