Pattern of Internal Domain Duplications in the Chicken Protein ENSGALP00000020382, with 66 Repeating Nebulin Domains (Pfam)

<div><p>(A) The intensity of the squares is related to alignment scores, and the numbers on both axes indicate the domains in N-to-C terminal orientation. As there were gaps in the repeat sequence (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020114#pcbi-0020114-g001" target="_blank">Figure 1</a>), these were introduced as domains at positions 6, 18, 25, and 32.</p><p>(B) ACV calculated from the alignment scores in (A) with the average similarity to domains at distance 1, 2, 3, etc. The ACV are normalized around zero, hence the dotted line at zero is the mean score between all domains in the protein. The ACV was calculated before introducing the gaps as domains (dashed line) and after (solid line). When the regions with no domain assignments were regarded as domains, the pattern of seven repeating units became much clearer, indicating that the gaps are also domains.</p></div

Hierarchical Clustering of the ACVs from Each Protein

<div><p>(A) Dendrogram of the 20 clusters. Each cluster is indicated by a cluster number followed by the number of proteins in the cluster.</p><p>(B) The average ACV for each cluster with red color for values below the average and green for values above.</p><p>(C) Distribution of the ten largest domain families, as well as nebulin, in the different clusters. The expected number of proteins from a domain family in each cluster was calculated using random shuffling, and Z-scores for overrepresentation (green) and underrepresentation (red) in the cluster were calculated. The numbers after the domain family names is the number of repeats of the family.</p></div

Overview of the Methodology

<div><p>(A) In a protein with five domains, a unit of three N-terminal domains has been duplicated in tandem.</p><p>(B) To identify this evolutionary event, alignment of all domain pairs in the protein is performed.</p><p>(C) The alignment scores between the domains displayed in a matrix with increasing color intensity for higher scores. The diagonal shows alignment scores for each domain to itself, while square 1,2 gives the score between the first and the second domain. A pattern where domain pairs 3–6, 4–7, and 5–8 have the highest alignment scores can be seen.</p><p>(D) From the alignment scores, an ACV is calculated as the mean alignment score at each distance normalized around zero. The distance between the domains is defined as one for neighbouring domains, while domain pairs with one domain between them have distance two, etc. In this example a peak at distance three can be seen. Hence, we assume that this protein has evolved through the duplication of three domains.</p></div

Pattern of Internal Domain Duplications in Two Human Proteins, ENSP00000319007 and ENSP00000303696, both with C2H2 Zinc Finger Repeats

<div><p>(A) ENSP00000319007.</p><p>(B) ENSP00000303696.</p><p>The intensity of the squares reflects the alignment score with darker color for higher scores. The numbers at each axis indicate the domains in N-to-C terminal orientation within the repeat. In these two examples, patterns of duplication of six domains (A) and two domains (B) can be seen.</p></div

ACVs for Proteins with Repeats of Eight Different Domain Families

<p>Solid line shows ACVs for proteins with repeats of eight different domain families. In the bottom right diagram, the ACV for all proteins with repeats is displayed. The ACV for each family was normalized around zero, hence the dashed line at zero is the mean bit score between all domains in the family. The <i>p</i>-value for each datapoint was calculated from random shuffling of domains, and peaks with <i>p</i>-values below 10⁻⁵ are indicated with an asterisk. The dotted line illustrates the fraction of repeats of the domain family with each repeat length, i.e., nonrepeated proteins have length one. The number of proteins/domains that goes into each figure can be found in Materials and Methods. Data for the remaining domain families can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.0020114#pcbi-0020114-sg002" target="_blank">Figure S2</a>.</p

<i>Tcs1</i> and <i>Tcs2</i>-congenic strains show similar variation in extracellular MHC expression in thymus and spleen.

<p>(A) Thymic conventional DCs (CD103+, CD11b/c+) stained extracellularly for class I (OX18). Scatterplots show results from two different experiments with <i>Tcs1</i> and <i>Tcs2</i>-congenic strains. The variation between the strains is comparable to data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004151#pgen-1004151-g005" target="_blank">Figure 5</a> for CD68+ cells. (B–C) Splenic DCs (CD103+, CD68+, CD11b/c+) (B) and thymic DCs (C) stained extracellularly for RT1-D (OX17) and RT1-B (OX6). Histograms show representative samples from DA (solid lines) and DA.1HR10 (dashed lines).</p

CD4∶CD8 T cell ratio and extracellular MHC class I and II expression in spleens of congenic and recombinant strains.

a<p>RT1 haplotype designations are based on genotyping data (see <i>Methods</i>);</p>b<p>Strains were compared in separate experiments to DA littermate controls. The mean fluorescent intensity was then normalized to the expression of DA, which was given an arbitrary value of 100.</p>c<p>MHC-I (class Ia and Ib; OX18) extracellular expression on WBCs.</p>d<p>MHC class II expression on B cells. </p><p>Significant differences compared to DA:</p>e<p><i>P<0.001</i>;</p>f<p><i>P<0.01</i>;</p>g<p><i>P<0.05</i>.</p><p>WT, wildtype; PS, parental strain; RCS, recombinant congenic strain. Shown are mean values ±SD.</p

Regulation of class I expression by classical and inverse cim.

<p>(A) CD68+ cells were stained on the cell surface with OX18 (anti-class Ia and Ib). Histograms show representative samples from DA, DA.1H and DA.1H derived strains with different alleles of <i>RT1-A</i> and <i>Tap2</i> as stated on top. Data from all individuals are shown in scatterplot (far right); * significant compared to DA.1H (1H); ** significant compared to DA. (B) CD68+ cells stained with a class Ia specific antibody (F16-4-4). (C) T cells from animals shown in (A) stained intracellularly with OX18 (scatterplot) and F16-4-4 (histogram). (D–E) Subsets of leukocytes from DA and DA.1IR85 spleen stained extracellularly (D) and intracellularly (E) with OX18. Data are representative of 6 individuals per group. (F) Surface expression of MHC class I (OX18) on CD68+ cells from DA and DA.1U congenic strains. (G) CD68+ cells (same as in F) stained with F16-4-4. Vertical lines in scatterplots show mean values. Representative results of at least two independent experiments are shown.</p

Sequence variants and allele distribution of genes in six rat MHC haplotypes.

a<p>All genes except <i>RT1-DMa</i> and <i>Tapbp</i> are encoded within the <i>Tcs2</i> locus.</p>b<p>Size of the coding sequences (cds) in bp.</p>c<p>Total number of synonymous and nonsynonymous SNPs in the cds.</p>d<p>Total number of insertions/deletions on gene level.</p>e<p>Allele distribution between MHC haplotypes; DA (RT1^a), DA.1F (RT1^f), DA.1I (RT1ⁱ), DA.1H (RT1^h), DA.1U (RT1^u). Sequence information for RT1ⁿ was obtained from NCBI GenBank.</p>f<p>Total number of amino acid substitutions.</p>g<p>Number of alternative isoforms at the protein level.</p>h<p><i>Btnl2</i> was analyzed in only two strains.</p>I<p>Sequence information for RT1-DOb refers to the full-length transcript of this gene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004151#pgen.1004151.s001" target="_blank">Fig. S1</a> for alternative transcript).</p

Class-I modification reduces negative selection of CD8 cells.

<p>(A) The QTLs in <i>Tcs1</i> and <i>Tcs2</i> did not affect the total number of double negative cells (DN; CD4−, CD8b−). B cells (CD45RA+) were excluded from the DN gate. (B) DN cell maturation is defined by CD45RC and CD2 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004151#pgen.1004151.s010" target="_blank">Fig. S10</a>). DN cells in DA.1FR9 showed a lower frequency of early thymic precursors (ETP) compared to DA. Counter plot shows gating strategy with numbers indicating percent (%) of parent population (stated above plot). (C) Counter plot shows CD45RC−, CD2^hi DN cells from DA.1UR83, and scatter plots the frequency of TCRβ− and TCRβ+ (DN4 in mouse) cells in DA and DA.1UR83. (D) Thymi from <i>Tcs2</i>-congenic strains with TAP-B (HR10 and UR10) contained fewer double positive (DP) cells but more cells (in %) with high TCR expression. (E) TAP-B strains (HR10 and UR10) showed higher frequencies (histograms) and total numbers (scatter plots) of CD8 single positive (SP) cells with high TCR expression. Numbers (%) in histograms represent mean-values ±SD of cells with high TCR expression (gated, <i>n</i> = 5). (F) Frequencies (histograms; <i>n</i> = 5) and total numbers (scatter plot) of CD8SP cells with high TCR expression in DA.1H (RT1-A^h, TAP-B) and in strains with low levels of surface MHC class I (HR83 [RT1-A^h, TAP-A] and IR85 [RT1-Aⁿ, TAP-A]). (G) Virtually all CD4SP cells express high levels of TCR. Histogram shows expression of TCR on CD4SP thymocytes in DA (<i>n</i> = 5). Scatterplot shows total number of CD4SP cells per thymus in <i>Tcs2</i>-congenic strains.</p