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At Least One Class I Gene in Restriction Fragment Pattern-Y (Rfp-Y), the Second MHC Gene Cluster in the Chicken, Is Transcribed, Polymorphic, and Shows Divergent Specialization in Antigen Binding Region
Marielle Afanassieff, Ronald M. Goto, Jennifer Ha, Mark A. Sherman, Lingwen Zhong, Charles Auffray, Françoise Coudert, Rima Zoorob and Marcia M. Miller
J Immunol March 1, 2001, 166 (5) 3324-3333; DOI: https://doi.org/10.4049/jimmunol.166.5.3324
Marielle Afanassieff
Department of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010; Institut National de la Recherche Agronomique, Station de Pathologie Aviaire et Parasitologie, Nouzilly, France;
Ronald M. Goto
Department of Molecular Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010;
Jennifer Ha
Mark A. Sherman
Department of Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010; and
Lingwen Zhong
Charles Auffray
Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Génétique Moléculaire et Biologie du Développement, Villejuif, France
Françoise Coudert
Institut National de la Recherche Agronomique, Station de Pathologie Aviaire et Parasitologie, Nouzilly, France;
Rima Zoorob
Marcia M. Miller
MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the classical B system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA hybridization. Because small numbers of MHC class I and class II genes are present in both B and Rfp-Y, the two clusters might be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to classical class Ia genes (73% with chicken B class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV, is widely transcribed and polymorphic. Mature YFV protein associated with β2m arrives on the surface of chicken B (RP9) lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized function.
MHC class I genes are often described as being either classical (class Ia) or nonclassical (class Ib). Class Ia genes are ubiquitously expressed encoding the polymorphic transplantation Ags that are the basis of rapid graft rejection. They include human HLA-A, -B, and -C; murine H2-K, -D, and -L; two loci in the chicken B system; and additional loci in a number of other species. Class Ia molecules present peptide-Ag to CTL. At the cell surface class Ia molecules are tripartite, composed of the polymorphic heavy chain, bound peptide, and β2-microglobulin (β2m).5 By contrast, the MHC class Ib genes, such as human HLA-E, -F, -G, H, I, and J; murine H2-Q, -T, and -M; mammalian CD1, Hfe, and Xenopus NCI genes, are generally not polymorphic in the manner of class Ia; but, there are exceptions, such as Q2 (1). Nonclassical molecules generally have weak influences in graft rejection. Some class Ib loci lie within or near the MHC, and a portion of these are phylogenetically close to class Ia loci. Other, usually older class Ib loci are located in paralogous regions on entirely different chromosomes. Many class Ib molecules are restricted in expression to particular tissues. Some class Ib molecules have critical roles in regulating NK cell activity (2), and trafficking of these to the cell surface can be dependent upon the binding in the Ag binding region (ABR) of peptide derived from signal sequences of other class I molecules (3). Still others, such as FcRn (4) and Hfe (5), function in the delivery of particular molecules across cellular boundaries via molecular interactions independent of the ABR. Although divergent to various degrees in primary sequences, the class Ib and class Ia heavy chain molecules share many elements of tertiary structure in common. Many, but not all, class Ib molecules bind β2m.
A characteristic often useful for distinguishing between class Ia and class Ib molecules is a set of 8 aa present in the α1 and α2 domains of the class I heavy chain (6, 7). These amino acids in the ABR are effectively invariant in all class Ia molecules and contrast strikingly with the many polymorphic residues in the region. These residues are essential in sequence-independent anchoring of peptide-Ag. Substitutions are present at one or more of these eight positions in nearly all class Ib molecules, although a few exceptions to this are found among H2-Q and H2-T loci. In some class Ib molecules, particular substitutions are associated with anchoring special forms of Ag (8). For example, N-formylated peptides (9) are bound by the mouse-nonclassical H2-M3 molecules in an ABR made more hydrophobic by the presence of phenylalanine and leucine at two of the eight critical positions. Glycolipids and lipoglycans are presented by transporter associated with Ag processing (TAP)-independent CD1 molecules (10, 11). In the ABR of CD1 there are multiple, generally hydrophobic substitutions occurring at the eight residues. Other substitutions are present in other class Ib molecules and are associated with other modifications of the ABR, such as closing of the groove in FcRn (12).
Restriction fragment pattern-Y (Rfp-Y) and B are two genetically independent clusters of MHC class I and class IIβ genes in the chicken that map to a single microchromosome (chromosome 16) (13, 14, 15, 16). Also mapping to chromosome 16 is the single nucleolar organizer region found within the chicken genome (17) and a single nonpolymorphic classical class IIα locus (18). A chromosomal region supporting highly frequent meiotic recombination, perhaps associated with the nucleolar organizer region, separates B and Rfp-Y such that the two clusters are genetically unlinked even though they are located on the same microchromosome (15, 16). This arrangement is quite different from the arrangement of class I loci in the mouse into H-2K and H-2D where the loci remain linked despite physical separation. Rfp-Y was detected initially when two sets of polymorphic restriction fragments revealed by B system class I and class II probes were found to assort independently of one another in families of fully pedigreed animals (13). Rfp-Y was later found to correspond to the cosmid clusters II/IV and III in the molecular map (19) of chicken MHC genes (14, 15).
At least two class Iα heavy chain genes (YFV and YFVI), three class IIβ genes (YLβIII, YLβIV, and YLβV) (20), a c-type lectin gene (21), and two additional genes (13.1 and 17.8) of unknown identity map to Rfp-Y. The classical B system is a compact gene region that determines rapid allograft rejection. A large portion of the B cluster has been sequenced and found to contain 19 genes within a 92-kb region, virtually all of which have counterparts in the human MHC (22). Included among these are two polymorphic class I heavy chain and two polymorphic class IIβ loci (19, 23, 24, 25).
Because the small number of chicken class Iα heavy chain and class IIβ chain genes are essentially equally divided between B and Rfp-Y, the two clusters might originate from duplication of an entire chromosomal segment providing duplicate sets of loci with similar functions. Alternatively each may perform specialized, complementary functions as is becoming apparent for mammalian classical and nonclassical regions (8). For example, the Rfp-Y loci might in some instances provide molecules supplementing the less than comprehensive Ag presentation that is so characteristic of the B system (26). To begin to define the basis of the organization of chicken MHC genes into two genetically independent clusters, we subcloned and fully sequenced the YFV and YFVI loci located in the Rfp-Y cosmid cluster map. We analyzed the sequences of these loci with respect to those of known class Ia and class Ib loci and evaluated their polymorphism. We extensively analyzed gene transcription and the capacity of YFV cDNA to produce mature Rfp-Y class I molecules upon transfection.
Clone cβ10 was isolated from a cosmid library made from line CB (B*12, Yw*7.1) (27). (We use the w* notation with all Rfp-Y haplotypes to indicate that assignments are subject to further refinement.) For transcription analysis 12- and 19-day-old line CB embryos were provided by Pierrick Thoraval from stock maintained at Institut National de la Recherche Agronomique (Nouzilly, France). One-year-old male birds from line C were provided by Larry Bacon from stock maintained at the US Department of Agriculture Avian Disease and Oncology Laboratory (ADOL) (East Lansing, MI). Lines C and CB both originate from the Reaseheath line RH-C. Lines CB and C are inbred and homozygous for the same B and Rfp-Y haplotypes. Other Rfp-Y haplotypes analyzed include Yw*1.3, Yw*2.1, Yw*3.1, and Yw*6.1 from Northern Illinois University (13); Yw*4.2 and Yw*5.3 from University of New Hampshire (R.L. Taylor, Jr. and M.M., unpublished data); and Yw*7.2, Yw*8.1, and Yw*9.1, provided by Larry Bacon (28).
Subcloning and sequencing of YFV and YFVI
The 3.55- and 4.8-kb BglII fragments of cβ10 (27) containing the YFVw*7.1 and YFVIw*7.1 genes, respectively, were subcloned into the BamHI site in Bluescript II KS (Stratagene, La Jolla, CA) to provide pYFVw*7.1 and pYFVIw*7.1. Sequences of the YFVw*7.1 and YFVIw*7.1 genes were obtained through the successive application of two techniques. First, the sequences of exons 2, 3, 4, and 5, and introns 2, 3, and 4 of each gene were defined by sequencing of products obtained by PCR with primers designed from the B-FIV*12 gene sequence (29). Two hundred nanograms of cosmid cβ10 clone DNA, 10 ng of pYFVw*7.1 and pYFVIw*7.1 plasmid DNA were used as templates. PCR amplifications consisted of 35 cycles of 95°C for 1 min, 60°C for 45 s, and 72°C for 45 s using Taq DNA polymerase buffer with 400 nM of each primer, 200 μM of each dNTP (Pharmacia, Piscataway, NJ), and 1 U of Taq DNA polymerase (PE Biosystems, Foster City, CA). A fraction of each reaction product was cloned using the TA cloning vector (Invitrogen, San Diego, CA), and the nucleotide sequence of the insert was fully determined by dideoxy chain termination with a Prism Ready Reaction Dye Terminator Cycle Sequencing Kit and a 370A DNA Sequencer (PE Biosystems). To identify any errors due to misincorporation by Taq polymerase, three to six independent PCR were conducted for each primer set and 4–10 clones per PCR were fully sequenced. The sequences upstream of exon 2 and downstream of exon 5 were obtained by direct sequencing of the pYFVw*7.1 and pYFVIw*7.1 plasmids with annealing primers designed from previously determined sequences.
Isolation of additional YFV clones
A YF-specific clone, 163/164f, was generated by PCR from YFVw*3.2 DNA and corresponds to exons for Cy1, Cy2, Cy3, and portions of surrounding introns of YFV (see Fig. 1⇓). Clone 163/164f was used in turn to isolate a full-length cDNA clone, c36f, from a cDNA library made from the small intestine of a UCD line 330 young adult bird. An additional clone, cos2, was also obtained by 163/164f screening from a SuperCos I cosmid library (Stratagene) produced from a bird (wb3078) heterozygous for two additional Rfp-Y haplotypes designated Yw*1.1 and Yw*5.1. Cos2 was determined to originate from Yw*5.1 by restriction fragment pattern. A YFV subclone, pcr75171–3, was derived from cos2 and sequenced from the clone margins.
Nucleotide sequences of the YFVw*7.1 and YFVIw*7.1 loci are aligned with the sequence of chicken classical class I locus BFIV*12. Exon sequences are presented as codons, and introns as uninterrupted sequence. Intron boundary sequences and the ATG start site are in bold print and underlined. The sequence of the hexanucleotide repeat region in YFVIw*7.1, the gene regions in YFVw*7.1 amplified with the YFα1-5′ and YFα1-3′ primer pair, the gene region corresponding to the YF gene-specific probe 163/164f, and the polyadenylation signal sequence are labeled and in bold print. Identity is marked by a dot, and a gap is shown as a dash. The GenBank accession numbers are AF218783 (YFVw*7.1) and AF218784 (YFVIw*7.1).
RNA purification
RNA was extracted from frozen tissues with RNAzol B (Tel-Test, Friendswood, TX). For 12-day-old whole embryos and 19-day-old embryo tissues RNA was purified on cesium chloride gradients.
Southern blot analysis
Samples containing 10 μg of genomic DNA were digested with restriction endonucleases, electrophoresed in 1% agarose gels, and analyzed by Southern hybridization (13). Probes included 1) a 32P-labeled oligonucleotide (TGGGGCTGGGGCTGGGGCT) designed from the exon 1 of YFVIw*7.1; 2) a 0.9-kb SacI fragment of pYFVIw*7.1 corresponding to exons 1 to 2; and 3) 163/164f.
Transcript analysis and single-stranded conformational polymorphism (SSCP) assays
Transcription analysis by RT-PCR was performed as follows: 1) first-strand cDNA was synthesized for 15 min at 37°C using 1 μg of total RNA, 40 nM primer, 200 μM dNTPs, 20 U of AMV transcriptase (Life Sciences, St. Petersburg, FL) in reverse transcriptase buffer, and a single oligonucleotide reverse primer RTBYFα2 (CCTCGAGGATGTCACAGCC) corresponding to a site in exon 3 identical in the BF and YF genes; 2) cDNA was tested for purity with PCR performed using primer pairs that span the α1 exon/intron/α2 exon boundary so that any product originating from genomic DNA can be recognized by product length; RTBYFα with BFIVα1-5′ (GGGCAGCCGTGGTTCGTGACT) and with YFα1-5′ (GTGGACGACAAAATCTTCGGTA). Products of the PCR (35 cycles at 95°C for 1 min, at 60°C for 45 s, and at 72°C for 45 s) were analyzed for fragment length on agarose gels; 3) cDNA free of genomic DNA was used as template for PCR performed with primers specific for the two YF loci consisting of YFα1-5′ and YFα1-3′ (TTTGTTGTAGCGTTCCGGCAGCC). For BFI the primer pair was BFIα1-5′ (GGGCTGCCGTGGTTCGTGGAC) and BFIα1-3′ (GTGTTCAAGCTCACTTCCACAC). For BFIV the primer pair was BFIVα1-5′ and BFIVα1-3′ (ATGCCCAGGTTCTCGCGGTCAA); and 4) presence and absence of the BF and YF transcripts were scored on the presence or absence of amplicon in agarose gels. To distinguish the locus of origin for YF amplicons obtained with YFα1-5′ and YFα1-3′ primers were analyzed by SSCP (30). For this, 1–3 μl of the PCR products were denatured in formamide at 80°C for 5 min and electrophoresed for 105 min at 200 V in 10% polyacrylamide, 0.5% TBE (44.5 mM Tris-borate, 44.5 mM boric acid, 1 mM EDTA) gels in a Miniprotean II apparatus (Bio-Rad, Richmond, CA). The gels were fixed and stained with a Silver Stain Plus Kit (Bio-Rad) and dried in gel wrap (BioDesign, New York, NY). The resulting patterns were scored in comparison with those provided by PCR in which line C DNA, cβ10, pYFVw*7.1, and pYFVIw*7.1 served as template.
Transfection, immunoprecipitation, and immunoblotting
A FLAG epitope tag sequence was incorporated into the YFV cDNA clone c36f for tagging mature protein at the N-terminal end. The modified clone was transferred into the replication-competent RCASBP(A) vector (31), the viral plasmid was transfected into avian DF1 cells (32), and packaged virus in the DF1 culture supernatant was used to infect avian RP9 cells (33). Extracts were made of intact RP9 cells expressing FLAG-tagged c36F YFV molecule and control cells (uninfected RP9 cells, RP9 cells infected with RCASBP(A) containing FLAGc36f in the nonsense orientation, and RP9 cells expressing FLAGBFIV21; Ref. 34), electrophoresed, blotted, and developed using M2 anti-FLAG mAb and ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ). Proteins were also immunoprecipitated with M2 anti-FLAG mAb (Sigma, St. Louis, MO) or with anti-chicken β2m mAb (35), generously provided by Jim Kaufman (Institute for Animal Health, Compton, U.K.). The immunoprecipitates were electrophoresed and blotted as above.
Sequences were assembled with PC Gene and DNAStar. Similarity indices were determined with Wilbur-Lipman (DNA) and Lipman-Pearson (protein) algorithms. Deduced amino acid sequences were aligned using a mutation matrix, together with visual inspection of the modeled structures of YFVw*7.1 and B-FIV*12 and with Megalign (DNAStar). Pileup (GCG) and PaupSearch (maximum parsimony) were used to construct gene trees and to assign bootstrap values.
Molecular modeling
A homology model of the YFVw*7.1 structure was built using Insight II (Molecular Simulations, San Diego, CA) software. File 2clr.ent (HLA-A*0201) was chosen from the protein database as the template. HLA-A*0201 was one of several class Ia molecules giving essentially equal scores in FASTA alignments with YFVw*7.1 sequence. None of the class Ib molecules scored well when aligned with YFV. Amino acid insertions and deletions were readily placed between secondary structure elements at positions minimizing disruption of the overall fold by searching a high-resolution subset of the Brookhaven database for loops having the required length and similar context. Wherever more than one loop was present, the loop of the highest sequence homology was chosen. Once all coordinates were assigned and several conflicting side chains repositioned, the models were energy minimized with DISCOVER within Insight II using the consistent valence force field and default parameters. Similar steps were followed in modeling the avian β2m chain on the structure of human β2m.
YFVw*7.1 and YFVIw*7.1 are class I-like loci, but only YFVw*7.1 is intact
BglII subclones containing YFVw*7.1 and YFVIw*7.1 were prepared from the cosmid cβ10 (14, 27) and fully sequenced. The two loci are oriented with 3′ ends opposed and are separated by 11.5 kb. The sequences of both loci are presented in Fig. 1⇑ aligned with the sequence of the BFIV*12 (29), an allele at the classical class I locus that is most strongly expressed in the chicken and with which the Rfp-Y class I genes are highly similar. The exon/intron junctions for the YFVw*7.1 and YFVIw*7.1 were deduced based on the sequence of BFIV*12 and confirmed by sequencing a YFV cDNA (c36f) clone. The intron and exon organization in all three chicken genes is typical of class I genes. Eight exons are present, and their size is generally conserved with variations in the Rfp-Y genes confined to one or two codon differences from BFIV*12. Introns are generally small compared with mammalian class I genes with intron length varying between Rfp-Y and B system genes by 1–29 nucleotides. The Rfp-Y class I genes are C+G rich as has been noted for the B system class I loci (36).
The two YF loci are highly similar (93%) in nucleotide sequence (Table I⇓) except for a large repeat sequence (48 copies of the hexanucleotide GGGCTG) that disrupts exon 1 of YFVIw*7.1 (Fig. 1⇑). This insertion and the absence of a polyadenylation signal sequence downstream of the stop codon indicate that YFVI w*7.1 is most likely unexpressed. As noted below, no transcripts from the YFVI locus were found in any organs examined in RT-PCR assays. To determine whether the hexanucleotide repeat is present in other YFVI alleles in other Rfp-Y haplotypes two probes were prepared for Southern hybridizations. When an oligonucleotide containing three hexanucleotide repeats and a subclone of exon 1 from YFVIw*7.1 were used to probe DNA representing seven additional Rfp-Y haplotypes, only one was found to hybridize (data not shown), indicating that the repeat is not commonly present in other Rfp-Y haplotypes.
Table I.
Sequence similarity indices among YFw*7.1 loci and classical class I genes
There is substantial sequence similarity between the sequence of YFVw*7.1 and other MHC class I genes in extracellular domains (Table I⇑). Overall the nucleotide/predicted amino acid sequence similarities for YFVw*7.1 with BFIV*12 and HLA-A2 are 73%/60% and 49%/38%, respectively, and the similarity is generally uniform over the exons corresponding to extracellular domains. However, YFVw*7.1 and BFIV*12 are relatively less similar to each other in exon 2/α1 domain sequences (66%/49%) than they are in other extracellular exon/domain sequences suggesting that the α1 domains of YFV and BFIV molecules have divergent functional constraints. The exons corresponding to transmembrane and cytoplasmic domains of YFVw*7.1, BFIV*12, and HLA-A2 are mostly dissimilar suggesting further specialization associated with the Rfp-Y locus. As with other class Ib molecules, YFV-encoded molecules lack the phosphorylation motif found in mouse and human MHC class Ia molecules (37).
The predicted, mature product of the YFVw*7.1 gene is a 332-residue protein that has a single potential n-glycosylation site at the same residue found in many class Ia molecules (noted by • in Fig. 2⇓A). The amino acids critical for folding of class I molecules are generally conserved in the YFVw*7.1 amino acid sequence. The four cysteine residues that form the basis of the highly conserved class I disulfide loops (C98C101-C161C164, C199C203-C255C259) are present (the superscript denotes the position in HLA-A2). All 18 invariant residues (noted by I in Fig. 2⇓A) known to form various contacts within and between class I domains that are strictly conserved in the sequences of class I molecules (38) are present in YFVw*7.1. Molecular modeling of YFVw*7.1 provides a structure highly similar to that of HLA-A2 (Fig. 2⇓B). The structural integrity of the β strands and the α helices forming the ABR is mostly conserved in the YFV protein, even though the YFV ABR is three residues shorter than that of HLA-A2. The positions of the "missing" residues are easily assigned to the margins of the β strand and α helical regions and most likely do not disrupt domain folding. As reflected in the model, a proline substitution at P51E53 in YFVw*7.1 is likely to disrupt the H1 helical region typical of the α1 domain of classical class I molecules. In addition, the presence of a contiguous pair of flexible glycine residues G67A69 G68H70 in YFVw*7.1 indicate that the long H2 helical region of the α1 domain may be broken into two shorter helices. Also, the natural break in the α2 domain α helix is further accentuated in YFV by the insertion of a glycine between positions 150 and 151 (HLA-A2 numbering). In summary, it is likely that YFVw*7.1 will have a structure overall highly similar to HLA-A2 with the ABR displaying a degree of specialization associated with the locus, a feature commonly encountered in comparisons between class Ia and class Ib molecules.
Comparison of the predicted amino acid sequence of YFVw*7.1 with the sequences of human (HLA-A2) and chicken (BFIV*12) classical class I molecules. A, Structure-based alignment of the deduced amino acid sequences of three YFV alleles with chicken class Ia (BFIV*12) and human class Ia (HLA-A2) sequences. Polymorphic residues in the YFV sequences are noted in red with the position underlined. B, Stereoview of the model for the three extracellular domains of YFVw*7.1 and chicken β2m (both in red) modeled after the crystal structures of HLA-A2 and human β2m (both in black). YFVw*7.1 apparently lacks the H1 helical region (noted by arrow) found in the α1 domain of HLA-A2 and other class Ia molecules.
YFVw*7.1 encodes a distinctive class Ib molecule
In class Ia molecules eight highly conserved residues define the "left" (Y7, Y59, Y159, and Y171) and "right" (Y84, T143, K146, and W147) pockets of the ABR and secure peptide Ag by bonding with main chain atoms. In YFV two of the left-pocket tyrosine residues are replaced by H57Y59 and E156Y159. Further substitutions occur in the right pocket. Position 84 in YFV is polymorphic occupied by R, Q, and C in different Rfp-Y alleles. A further, albeit conserved substitution, R143K146, is also present in the right pocket. These left and right pocket substitutions make it highly unlikely that YFV class I molecules present Ag in the manner of class Ia molecules. Hence the YFV locus fails to meet a major criterion for inclusion in the class Ia category and, therefore, should be considered a class Ib locus.
The substitutions that are found in the predicted YFV molecules at four of the eight subclass-defining residues in the ABR are extremely rare among class Ib molecules. The E156Y159 substitution is unique. Substitution of Y159 is generally rare with substitutions of phenylalanine (H2-M9, H2-Q5k, H2-Mb-1, DLA79, FcRn), glycine (H2-T10), aspartic acid (H2-T9, H2-T22), tryptophan (Mr1), alanine (MICA), and leucine (hCD1c and mCD1) occurring in a limited number of class Ib molecules. The H57Y59 substitution is shared so far only with Mr1 and some Xenopus class Ib loci. The Y59 is conserved at most class Ib loci with other substitutions such as phenylalanine (H2-M9) and leucine (mCD1) only rarely occurring. The substitution of three different amino acids at tyrosine 84 (R/Q/C82Y84) in different alleles at any class I locus is unprecedented. Generally alternatives to tyrosine at this position are very rare with isoleucine and glutamic acid found at mouse H2-Mb-1 and CD1. Finally, the R143K146 substitution occurs occasionally in class Ib molecules (H2-M3, FcRn) and is common among chicken class Ia (BFIV) alleles. Thus no other known class I locus is closely similar to YFV in the substitutions at these four positions suggesting that YFV defines a new type of class Ib locus.
YFVw*7.1 is a recently derived class Ib locus
The α3 domain sequences of the Rfp-Y class Ib loci were aligned using PileUp (Genetics Computer Group, Madison, WI) with the corresponding sequences from class Ia and class Ib molecules from several vertebrate species. The alignment was analyzed with phylogenetic analysis using parsimony to generate the gene tree and bootstrap values presented in Fig. 3⇓ (class Ib are underlined). The two Rfp-Y class Ib α3 sequences are closely similar to those of chicken and quail class Ia molecules (see large bracket). This group forms a clade restricted to gallinaceous birds indicating that the Rfp-Y loci may be relatively young genes sharing recent ancestors with class Ia genes in gallinaceous birds. Similar relationships occur in other taxa between class Ib loci and class Ia loci as can be seen in the other bracketed regions of the tree where Xenopus, humans, and mice also form clades in which class Ib and class Ia share recent ancestry. Most of the class Ib molecules within these clades are known to require TAP for processing of specialized Ag indicating that these relatively recently derived class Ib share Ag processing pathways with class Ia (8). These molecules contrast with more distantly derived class Ib molecules known to be TAP independent in the presentation of specialized Ag.
Gene tree showing the relationship of YFVw*7.1 and YFVIw*7.1 to selected vertebrate class Ia and class Ib (underlined) genes in the α3 domain. Note that the appearance of YFVw*7.1 and YFVIw*7.1 predates the separation of chicken and Japanese quail (larger bracket). These genes share a relationship with class Ia genes similar to the relationship among class Ia and subsets of class Ib molecules in Xenopus, human, and mouse (smaller brackets).
YF class Ib genes display both haplotypic and allelic variation
Because polymorphism or the lack thereof is another characteristic that is often used to separate class Ia from class Ib loci, we examined Rfp-Y class I genes for evidence of haplotypic and allelic polymorphism. We first examined Rfp-Y class I haplotypic variability using a Rfp-Y class I specific probe, 163/164f, in Southern blots. Nine different TaqI restriction fragment patterns were obtained from nine previously defined Rfp-Y haplotypes (Fig. 4⇓A). Surprisingly, the number of restriction fragments varies among the haplotypes from only two in Yw*1.3 and Yw*7.2 to at least 10 in Yw*4.2 and Yw*6.1. Similar differences were found in PstI and BglI restriction fragment patterns (data not shown). It is likely that the number of class I loci varies among Rfp-Y haplotypes. Similar variation in gene number occurs in the class Ib region in H-2 haplotypes (39).
Evidence for genetic polymorphism associated with Rfp-Y class I genes. A, Southern blot analysis. Restriction fragment polymorphism of YF class Ib loci in nine different Rfp-Y haplotypes revealed by a YF-specific probe, 163/164f, in TaqI-digested DNA. B, Stereoview of α-carbon backbone of the α1 and α2 domains of the YFVw*7.1 model structure. Residues that are polymorphic among the three Rfp-Y sequences in Fig. 2⇑A are marked by black circles with the amino acids variants presented in single letter code.
Predicted amino acid sequences for YFVw*7.1, YFVw*3.2, and YFVw*5.1 are aligned in Fig. 2⇑A. It is likely that these three sequences all originate from the YFV locus. YFVw*7.1 and YFVw*3.2 are identical in cytoplasmic domains and differ by only one amino acid in the transmembrane domain (TM). In contrast, YFVw*3.2 differs from YFVIw*7.1 by 15 of 34 aa over the same regions making it highly unlikely that YFVw*3.2 originates from the YFVI locus. The third clone, YFVw*5.1, was obtained by PCR using primers designed for YFV specificity and differs from YFVw*7.1 by only a single amino acid in transmembrane domain sequence.
The three clones display considerable sequence variability (Fig. 2⇑, A and B). The α1 domain variability among the three sequences (21%, 18 of 87 aa) is nearly as great as the variability that is present among BFIV alleles (27%, 24 of 88 aa in 11 alleles) (40). This variability is almost entirely confined to the helical region of the α1 domain and to the floor of the ABR with a small remainder of variation present in loop regions (Fig. 2⇑B). Amino acid replacements are most often nonconservative. For example, charged residues are interchanged with neutral, nonpolar (70R/L, 59D/A, 67D/G, 85K/I, 87K/G) or with neutral, polar residues (37D/N, 55Q/R, 66Q/R, 73D/C, 82R/Q/C). In other instances neutral, polar residues are interchanged with neutral, nonpolar residues (32N/I, 35T/I, 60T/A, 78W/G, 75N/F/L, 77N/G). Conservative substitutions (47V/A and 71D/E) in these regions are more rare.
The α2 domain is less variable (10%, 9 of 92 aa). Substitutions are mostly confined to two β sheet strands in the floor of the ABR, as they are in the classical BFIV alleles (40). The residues are often hydrophobic, and substitutions are often conservative. One nonpolar residue often substitutes for another (92L/M, 95 M/I, 96I/F, 120L/I), or charged residues are interchanged (117R/K). In other instances the substitutions are nonconservative (91T/M, 94 M/R, 119F/H/Y, and 178R/T). Similar to the chicken class Ia molecules, some variability is also present in the α3 domain (Fig. 2⇑A). Whether this reflects functional specialization among the YFV alleles is not yet understood.
Nonsynonymous-to-synonymous substitution ratios vary across α helical and β sheet regions of the α1 and α2 domains of the three YFV sequences (data not shown). Briefly, the α helical portion of the α1 domain has a high ratio compared with the β sheet region suggesting that, as in class Ia loci (40, 41), this region may be under selection for interactions with diverse Ag or variability in a counterreceptor. In contrast, in the α2 domain the values for the nonsynonymous-to-synonymous substitution ratios are reversed. The YFV α2 α helical region has an extremely low ratio indicating that diversification of this region of the molecule is restricted, as apparently it is in BFIV alleles (40). So although YFV molecules are nonclassical and unlikely to bind typical peptide Ag, they are polymorphic with the distribution of sequence variability among alleles not unlike the classical class Ia molecules of the chicken.
YFV is transcriptionally active in many organs and can be expressed as a transduced gene
To determine whether the YFVw*7.1 and YFVIw*7.1 are transcriptionally active and to learn whether transcription is confined to particular organs as often is found for class Ib genes, we performed RT-PCR SSCP assays using a YF gene-specific primer set. With the exception of small regions immediately upstream of the start site, the regulatory and promoter sequence elements (see GenBank sequences) of YFVw*7.1 (and YFVIw*7.1) genes are similar to those of class Ia genes suggesting that YF could be generally active in many tissues. RNA free of genomic DNA was obtained from embryos and from a number of organs of young adult line C birds (Fig. 5⇓ and Table II⇓). The YF-specific primers provided a means of specifically detecting transcripts from the Rfp-Y class I loci, and SSCP provided a means of distinguishing between YFVw*7.1 and YFVIw*7.1 transcripts. RT-PCR assays for BFI and BFIV served as positive controls. YFVw*7.1 transcripts were detected in all organs tested, except for three (brain, heart, and pancreas). No evidence was found for transcriptional activity of YFVIw*7.1 locus. Results of the full analysis are summarized in Table II⇓ and are consistent with a limited number of RNase protection assays demonstrating the presence of YFVw*7.1 transcripts in several tissues (42). We conclude that YFVw*7.1 is constitutively transcriptionally active in many organs much like MHC class Ia genes. It remains to be determined whether YFV transcription is inherent in all or many of the tissues in these organs or whether transcription is limited to a particular cellular subset with perhaps only limited quantities of YFV reaching the surface of these cells.
Illustration of the RT-PCR SSCP assays used for scoring YF gene expression in organs from line C birds. The SSCP step was used to distinguish between transcripts originating from YFVw*7.1 and YFVIw*7.1 loci. Controls include SSCP patterns for PCR products originating from line C DNA, cosmid cβ10, and plasmids separately containing the YFVw*7.1 and YFVIw*7.1 genes. Results of the full analysis are presented in Table II⇓.
Table II.
Distribution of Rfp-Y and B class I transcripts determined using RT-PCR
To determine whether mature protein can be produced from expression of YFV cDNA, the clone c36f was FLAG-epitope tagged, inserted into the RCASBP(A) retroviral vector, and expressed in the chicken B cell lymphoma line RP9. FLAG-tagged protein was found by flow cytometry to be at the surface of cells expressing FLAGc36f inserted into the vector in the sense orientation but not in the nonsense orientation (data not shown). In immunoprecipitations with the anti-FLAG mAb M2, RP9 cells expressing FLAGc36f and the positive control FLAGBFIV*21 were both found to produce FLAG-tagged protein with masses (∼48 kDa) typical of class I molecules (Fig. 6⇓A). The FLAGc36f encoded protein could also be immunoprecipitated with an anti-chicken β2m mAb (Fig. 6⇓B) providing evidence for association between YFV protein and β2m. Hence protein similar to typical class I molecules in molecular mass and in β2m association can be obtained by expressing the YFV cDNA c36f as a transgene.
Immunoblot and immunoprecipitation of FLAG-tagged YFV molecules. A, Immunoblot was developed with anti-FLAG M2 mAb. Lysates are from RP9 cells expressing FLAGBFIV*21 (positive control), FLAGc36f in anti-sense (−) and sense (+) orientations, and uninfected cells. B, Proteins were immunoprecipitated with anti-FLAG M2 or anti-chicken β2m mAb from lysates of RP9 cells expressing FLAGc36f in anti-sense (−) and sense (+) orientations. Immunoblot was developed with anti-FLAG M2 mAb. The Ig H and L chains are derived from the precipitating mAbs. Approximate mass of the FLAG-tagged products is noted with arrowheads.
In these experiments we have shown that the YFV locus shares many qualities with MHC class Ia genes. As summarized in the structural model in Fig. 2⇑B, the YFV molecule is likely to be structurally similar to class Ia, as are several other class Ib molecules. The unusual substitutions in the putative ABR of the YFV molecules are the strongest characteristic separating YFV from class Ia loci. Alleles at the YFV locus differ from one another by multiple changes in predicted amino acid sequence. Many of the polymorphic residues surround the ABR. Over 20 aa differences in the ABR separate the three YFV alleles in this study from each other. Such variability is on par with the differences between alleles at class Ia loci and between alleles at the one previously identified highly polymorphic class Ib locus, H2-Q2 (1). In addition, there may be many YFV alleles. SSCP analyses of the Rfp-Y class I exon 2 sequences in a variety of genetic lines provide indirect evidence for many more YFV alleles than the three presented here (M.M., unpublished data). As with class Ia loci, polymorphism and the relatively high ratios of nonsynonymous-to-synonymous substitutions over portions of the YFV ABR indicate that this region may be under selection for diversity. YFVw*7.1 and YFVIw*7.1 occupy positions in the evolutionary tree in Fig. 3⇑ that further emphasize their close relationship with class Ia genes in gallinaceous birds. The gene tree in Fig. 3⇑ indicates that the YFVw*7.1 and YFVIw*7.1 were derived relatively recently with their appearance likely predating only the separation of gallinaceous taxa (Fig. 3⇑). Perhaps YFVw*7.1 and YFVIw*7.1 are intermediates in the evolutionary tide postulated by Shawar and colleagues (37) to flow between loci encoding molecules with less selective (class Ia) and highly selective (class Ib) ABRs.
Given the close relationship the YF loci have with avian class Ia genes and their sequence polymorphism, it seems likely that YFV molecules bind peptide Ag. The unique substitutions of glutamic acid and histidine in the left pocket of the ABR may provide a means for selecting a particular form of Ag. Perhaps the charged residues form salt bridges with Ag in the ABR providing a means for selection of a particular subset of Ags. Or alternatively, perhaps the left end of the putative ABR of YFV is actually closed by interactions between these and other residues surrounding this region of the groove. In this instance, shorter forms of peptide might fill the remaining open portion of the groove through a selective interaction based on another characteristic of antigenic peptide, such as hydrophobicity of amino acid side chains. Because YFV molecules are apparently close relatives of the MHC class Ia molecules, it seems likely that peptide Ag load into the ABR through a TAP-dependent pathway. It would make sense that the YFV Ag is peptide, but this remains to be determined. How YFV molecules bind Ag and what form the Ag has will be the subject of additional experiments, as will be the consequence of YFV expression in cellular interactions with cytotoxic T and NK cells.
The YF loci have other features that define them as class Ib. Most class Ib loci have little or only weak influences in graft rejection. The structural identification of YFV as a class Ib gene is consistent with conclusions drawn by Pharr and colleagues (28) on the influence of Rfp-Y incompatibility on transplantation immunity. In experiments conducted with carefully defined genetic stock, Pharr et al. found skin graft rejections attributable to Rfp-Y incompatibilities occurred at moderate rates. They were clearly slower than B incompatibilities but significantly faster than Rfp-Y-compatible grafts. These authors suggested that one reason for the observed intermediate rate of graft rejection with Rfp-Y incompatibility might be the presence of class I-like (nonclassical) loci within the Rfp-Y gene region.
The YFV locus may share another feature with class Ib genes. There may be little YFV normally on the surface of cells despite the presence of YFV transcripts. Other investigators have found no evidence that Y-FV molecules are immunoprecipitated by anti-chicken β2m (43) and so YFV molecules may be normally less abundant at the cell surface than, for example, chicken class Ia molecules derived from the BFIV locus. It will be interesting to learn whether there are conditions under which surface expression of YFV becomes abundant. Because the Ag for YFV is likely to be atypical, it may be that it is not normally abundant and that trafficking of YFV to the cell surface is limited by Ag availability. This would be particularly interesting to explore given the evidence that in some but not all instances Rfp-Y haplotype has been found to influence resistance to virally induced tumors in chickens (44, 45, 46). If YFV is dependent on TAP molecules encoded in the B system for Ag processing, it could also be that interaction with chicken TAP affects YFV surface expression. Because chicken TAP genes are themselves polymorphic (47) and the YFV and TAP loci are unlinked, it might be that in particular combinations of TAP and YFV alleles there is either less or more YFV at the cell surface even in the presence of ample YFV Ag.
Finally, the organization of chicken class I genes into two genetic units composed of class Ia and class Ib loci is not unique. The class Ia and class Ib loci in Xenopus are also located in two independent genetic units and, just as in chickens, the two regions map to the same chromosome (48) separated by a region supporting highly frequent recombination. Considering the evolutionary relationship that exists between class Ia and class Ib genes in these two species, as illustrated in Fig. 3⇑A, it is likely that this manner of organizing class I genes has been arrived at independently in these two species. Genetic separation of the two class I subclasses could be a means by which the integrity of class Ia and class Ib loci are maintained. Perhaps the class Ia loci isolated by this arrangement evolve in concert with adjacent Ag processing loci as has been suggested by others (47, 48), whereas the class Ib loci are free to evolve in a different manner. In isolation the class Ib loci may be able to change in number, allelic variation, and ABR specificity through a variety of recombination events in a system for selective Ag presentation that evolves rapidly in response to disease challenge.
We thank Larry Bacon for providing C line birds and DNA from Cornell line N and P; Henry Hunt for providing RP9 cells expressing FLAGBFIV*21; Pierrick Thoraval for providing tissue from line CB embryos; and Jim Kaufman for providing anti-β2m mAb. Elwood Briles and Robert Taylor, Jr. generously provided blood samples from Rfp-Y-typed birds. We thank Larry Bacon, Pamela Bjorkman, Louis DuPasquier, Henry Hunt, and Iwona Stroynowski for helpful discussions.
↵1 This material is based upon work supported in part by the U.S. Department of Agriculture/National Research Initiative Competitive Grants Program (92-37204-8244), the U.S. Department of Agriculture/Foreign Agricultural Service/International Collaborative Research/Research and Scientific Exchange Division (58-3148-5-023), and by the National Science Foundation under Grants 9118199 and 9604589.
↵2 Sequences submitted to the GenBank database are Y-FVw*7.1 (AF218783) and Y-FVIw*7.1 (AF218784).
↵3 Current address: Laboratoire de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique Unité Mixte de Recherche 5665, Institut National de la Recherche Agronomique, LA 913, Ecole Normale Supérieure de Lyon, Lyon, France.
↵4 Address correspondence and reprint requests to Dr. Marcia M. Miller, Department of Molecular Biology, Beckman Research Institute of the City of Hope National Medical Center, 1450 East Duarte Road, Duarte, CA 91010-3011. E-mail address: mamiller{at}coh.org
↵5 Abbreviations used in this paper: β2m, β2-microglobulin; Rfp-Y, restriction fragment pattern-Y, ABR, Ag binding region; SSCP, single-stranded conformational polymorphism.
Received June 12, 2000.
Accepted December 11, 2000.
Copyright © 2001 by The American Association of Immunologists
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Pope Francis waves to the faithful as he delivers the traditional Urbi et Orbi Christmas Day message from the central balcony of St. Peter's Basilica at the Vatican, on Dec. 25, 2017. Angelo. FULL TEXT: Pope Francis' Christmas 2017 message. On Christmas Day, Pope Francis prays for peace and urges Pope christmas day message world to see Jesus in the faces of little children caught in the middle of war Pope John Paul II shared his fears about world hot spots and expressed his hopes in a Christmas message Saturday that peace-building efforts will bring the world a better future.
Justin Welby joins Pope Francis in using traditional Christmas Day message to make oblique criticism of Donald Trump over Jerusalem decision Pope Francis offers the traditional Christmas Day message address from the Vatican: 'Urbi et Orbi' (To the City and the World). Watch it live here. On Tuesday he gave his first Christmas Eve mass. This marks the second Christmas since his election last year.
Here is the official Vatican English-language translation of Pope Francis' Christmas Day message, which he delivered in Italian: Dear Brothers and Sisters, Happy Christmas! Pope Francis used a traditional Christmas address on Thursday to emphasize the plight of. 2014, on Page A6 of the New York edition with the headline: Francis, in Christmas Day Message.
Pope Francis' Christmas message wishes peace for troubled world. VATICAN CITY – Pope Francis has wished Christmas peace for people scarred by wars and those who. In his annual Christmas. In his annual Christmas Day address, known as "Urbi et Orbi" ("to the city and the world" in Latin), the pope touched on troubled places around the globe and the especially tragic impact.
REUTERS/Alessandro Bianchi Pope Francis delivered his Christmas day message on Sunday, offering hope for" those scarred by war and harsh conflicts that seem stronger than the yearning for peace. " Pope Francis waves as he leads the Urbi et Orbi (to the city and the world) message from the balcony overlooking St. Peter's Square at the Vatican on Christmas Day. (Alessandro Bianchi/Reuters) text of pope francis' traditional christmas day message He offered a prayer that" confrontation may be overcome on the Korean Peninsula and that mutual trust may increase in the interest of the.
Here is the official Vatican English translation of the" Urbi et Orbi" Christmas Day message read in Italian by Pope Benedict XVI Tuesday. " Urbi et Orbi" is Latin for to" The city and to the world. ROME — Pope Francis used his annual Christmas Day address on Monday to make clear his concern that serenity is sorely lacking at a time when the "winds of war" and an "outdated model of. Dec 25, 2017.
Pope Francis used his traditional Christmas Day message to call for a negotiated end to the Israeli-Palestinian conflict, including the dispute.
Pope Francis had some words of warning about the Middle East in his annual Christmas Day address. Pope Francis appeared on the balcony of St. Peter's Basilica today to deliver a Christmas Day message to Rome and to the world. Now, this pope has traditionally used his messages to focus on. Full text of pope's 'peace in Jerusalem' Christmas message.
Christmas' day blessing from the main balcony of St. Peter's Basilica at the Vatican, Monday, Dec. 25, 2017. VATICAN CITY. Pope calls for peace in world's conflict areas in Christmas Day message Francis appeals for support for all scarred by war and terrorism, as UK's Christian leaders also address tumultuous events.
Dec 25, 2017 · pope francis prays for peace, laments 'winds of war' in christmas message The first people to see the humble glory of the Savior, after Mary and Joseph, were the shepherds of Bethlehem. VATICAN CITY (AP) — Here is the official Vatican English-language translation of Pope Francis Urbi et Orbi' Christmas Day message, which he delivered in It.
Pope Francis on Christmas Day called for peace in the Holy Land and elsewhere throughout the world. " Where God is born, peace is born, " the Pope said. " And where peace is born, there is no longer.


to the children's medical office Dr. Med. Martin Hirte.
Welcome to the children's medical office Dr. Med. Martin Hirte.
I have a group practice with Dr. Christine Bäumler. Our practice is a private health insurance and self-paying practice; this means that we do not have a contract with the health insurance funds. Members of health insurance funds are welcome in our practice, yet they will also receive a bill in accordance with the medical fee schedule (Gebührenordnung für Ärzte, GOÄ), that are generally only covered by private health insurances or private subsidiary health insurances.
Classic Homeopathic medicine with treatments for chronic and recurring illnesses.
Consultation on allergies and their treatment (asthma, neurodermitis, hay fever, etc).
Consultation on vaccination (including alternatives to the official vaccination schedule), chiefly for parents whose children we treat in our practice.


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Santiago Roncagliolo Interviewd at Bookslut
June 30, 2011 June 29, 2011 ~ bythefirelight
Bookslut has an interview with the Peruvian author Santiago Roncagliolo. It doesn't go into some of the scandals he has been involved in (some of which I've mentioned here). It is a review worth reading to get a sense of where he is coming from and they go more into his childhood than I have come across in the Spanish press. I wonder if I would find Red April funny. Listening him talk I know he's got a sense of humor.
Red April is terrifying in parts, and gory, and Kafkaesque, in other places, but at the same time it made me laugh my socks off – and I felt peculiar about that.
[Laughing] I spent one night with a couple of African writers a few years ago. We were making political jokes and we found that had the same jokes, we just used different names. In poor countries humour is blacker than in rich countries, and more politically incorrect. I had to moderate myself when I arrived in Spain because I would say things I found really funny and everyone would look at me.
It's beautiful when someone tells me I made them laugh. I love to write humour and terror because I like very physical emotions. When you laugh it's your body moving, when you're scared, it's your skin crawling. These are powerful emotions.
For us, Red April is relatively new, but you wrote it ages ago. Is it hard to reconnect with the man you were then?
Yes. It's a date with your own past. I am surprised at what this novel did. It won the big prize in Spain, and it was a big bestseller, and keeps being reprinted. In Peruvian society it was the moment to begin to talk about the past. After I wrote about Abimael Guzman, I went to the jails to talk about the books, and it was amazing. I was talking to army people and Shining Path people and guerrilla people in the same auditorium. I would speak then they all begin to tell their own stories. It was a bit of a metaphor of what was going on in the country. Everybody had the feeling that it was time to listen to each other, even to the killers, even to the evil ones, to understand why people arrived at this place. My generation can do it, because we were children when this happened. We were innocent, we have no past. So it was like a liberation for us, and for the people participating.
Posted in Books Latin American LiteratureSantiago Roncagliolo
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elf says:
When I started reading the novel, I thought it was wickedly funny…The narrator makes these snide comments about the protagonist and about how life is led in the third world. I also felt kind of a guilty fun, but it later turns very dark and you cannot laugh after that. I think the interviewer got it just right.

redpajama_set_name: RedPajamaCommonCrawl


Still unsure about the potential of digital book publishing, or, more specifically, the trajectory of the e-reader market? Sales of portable devices such as Amazon's Kindle and Barnes & Noble's Nook that are used solely for the purpose of downloading and reading e-books are outpacing the more interactive, Web-enabled tablet computers such as Apple's iPad and the Samsung Galaxy Tab. Twelve percent of adults in the U.S. owned an e-reader as of May 2011, twice the amount recorded by the same Pew Internet research project in November 2010. Eight percent of American adults owned tablets in May 2011, up from 5 percent in November 2010. There is, however, some overlap among ownership of the two devices. Three percent of U.S. adults own both an e-reader and a tablet, while 9 percent own an e-book reader but not a tablet, and 5 percent own a tablet computer but not an e-reader.


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Homburg (Frans: Hombourg; Platdiets: Homerech) is een deelgemeente van Plombières in de Belgische provincie Luik. Het plaatsje ligt in het Land van Herve en in de zogeheten Platdietse streek. Tot 1977 was Homburg een zelfstandige gemeente.

Geschiedenis
De geschiedenis van Homburg gaat mogelijk terug tot de Merovingische tijd; rond 1070 wordt de naam Humborc (volgens sommigen Hunborc) aangetroffen in manuscripten.

Tot de opheffing van het hertogdom Limburg hoorde Homburg tot de Limburgse hoogbank Montzen. Net als de rest van het hertogdom werd Homburg bij de annexatie van de Zuidelijke Nederlanden door de Franse Republiek in 1795 opgenomen in het toen gevormde departement van de Ourthe.

Bij de onafhankelijkheid van België inventariseerde geograaf Philippe Vandermaelen in dit dorp (dat toen ook Remersdaal omvatte) 151 landelijke woningen, 20 hoeves, 2 herenhuizen, 141 hutten, 2 kerken, 2 gemeentehuizen, 3 watermolens, 2 kleine kaliumfabrieken, 3 siroopfabriekjes en 2 scholen. Er waren 1657 inwoners die Duits en Waals spraken. Het inventaris omvat verder details over de natuurlijke omgeving (o.a. enkele wolven), bodems, landbouwproductie en veestapel. Ook het wegennetwerk van toen is beschreven. De omschrijving door Vandermaelen geeft een interessante inkijk in het dagelijkse leven rond 1830.

Op het grondgebied van Homburg, bij het gehucht Vogelsang, bevindt zich een groot Amerikaans oorlogskerkhof uit de Tweede Wereldoorlog. Hoewel het kerkhof zich in de deelgemeente Homburg bevindt, wordt het meestal naar het dichtbijgelegen Hendrik-Kapelle (Henri-Chapelle) genoemd.

Demografische ontwikkeling

... was een zelfstandige gemeente tot aan de gemeentelijke herindeling van 1977.

Bronnen:NIS, Opm:1831 tot en met 1970=volkstellingen; 1976 = inwoneraantal op 31 december

Geografie
Homburg ligt op een hoogte van ongeveer 240 meter, in het verlengde tussen twee heuvelplateaus in. Aan de noordzijde is dat het Plateau van Crapoel dat helemaal doorloopt tot in Gulpen in Nederland met op het Belgische deel het Beusdalbos. In het zuiden ligt er een heuvelrug richting Hendrik-Kapelle. Ook het dorp zelf ligt op een relatief hoog punt in het landschap. Verder ligt ten noordwesten van het dorp de heuvel Schaesberg.

Het dorp ligt in het stroomgebied van de Geul. Aan de westzijde van het dorp en het westen van de beide heuvelplateaus stroomt de Gulp in het Gulpdal.

Tussen Homburg en het gehucht Montzen-Station ligt het uitgestrekte rangeerterrein van Montzen.

Bezienswaardigheden
Sint-Brixiuskerk
Agnus Deikerk
Molen van Medael
Gulpermolen
Kasteel van Berlieren
Kasteel van Vieljaeren

Verkeer en vervoer
Homburg had een spoorwegstation aan de lijn Chênée - Plombières (spoorlijn 38). Tijdens de Eerste Wereldoorlog werd er een aansluiting aangelegd tussen Homburg en het nabijgelegen Montzen. Het treinverkeer op de lijn stopte in 1957.

Ten noorden van Homburg ligt de Spoorlijn 24 met in het noordwesten de Tunnel van Remersdaal onder het Beusdalbos en in het noordoosten een groot spoorwegemplacement van Station Montzen. Hier gaat veel goederenvervoer langs van en naar Aken.

Door Homburg loopt de N608.

Talen
Het dorp ligt van oudsher op de grens van het Nederlandse, het Duitse en het Franse cultuurgebied. Vroeger sprak de bevolking Platdiets, een Limburgs-Ripuarisch grensdialect. Bij de eerste talentelling in 1846 gaf 90% van de bevolking aan Nederlands te spreken, tegenover 8% Frans en 2% Duits. De kerktaal was toen echter Duits. Mede daarom werd Remersdaal, waar de kerktaal Nederlands was, in 1851 als onafhankelijke gemeente van Homburg afgescheiden. Na de Eerste Wereldoorlog zocht de bevolking om vaderlandslievende redenen nog sterker aansluiting bij het Franse taalgebied. Bij de talentelling van 1930 gaf 76% van de bevolking aan Franstalig te zijn; in 1947 was dat 82%. Bij de vastlegging van de taalgrens in 1962 werd Homburg bij het Franse taalgebied ingedeeld. Het Platdiets is tegenwoordig in deze streek zo goed als verdwenen.

Trivia
 Bij Homburg hebben twee watermolens gelegen, stroomopwaarts was dat Moulins Meyers, stroomafwaarts de Molen van Medael.

Nabijgelegen kernen
Henri-Chapelle, Plombières, Sippenaeken, Hagelstein, Aubel

Geboren
 Johan Meens (7 juli 1999), wielrenner

Externe link
 Officiële toeristische site van Homburg (gearchiveerd)

Plaats in Luik (provincie)
Blieberg

redpajama_set_name: RedPajamaWikipedia


They even give you a little pamphlet about how to care for it! Pretty cool, really.
They can't engrave the Oscars before the ceremony, of course, because then everyone who saw the statue would know who won. This is almost as fast.
Here's Leonardo DiCaprio a little later, carrying the now-engraved Oscar as he enters the famous Vanity Fair afterparty.
Maybe it's me, but it looks like the first "I" in DiCaprio is even a little shorter than the rest, to emphasize that the "C" is capitalized. Nice touch!


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Mariners Notebook: Kyle Lewis to IL with meniscus tear; T-Mobile Park capacity to expand
Jun 1, 2021, 1:58 PM | Updated: 3:29 pm
Mariners CF Kyle Lewis injured his right knee on this play Monday. (Getty)
BY BRENT STECKER
Editor of SeattleSports.com
The Mariners have seen their fair share of injuries already this season. So many that it seems they're now starting to repeat themselves.
Scott Servais Show: M's takeaways on Gilbert, Kelenic and France
Kyle Lewis, the Mariners' center fielder and the 2020 American League Rookie of the Year, is headed to the 10-day injured list again for a right knee injury. It's the same right knee that Lewis suffered a deep bruise on late in spring training, keeping him out of action for nearly three weeks to start the season.
Manager Scott Servais said during his weekly interview on 710 ESPN Seattle that Lewis has a "very small" meniscus tear in his right knee. Not only is it the same knee Lewis injured in March, but it's the knee he had surgically repaired in 2016 after suffering a devastating injury sliding into home plate during his first season in the minor leagues.
Servais told 710 ESPN Seattle's Shannon Drayer, Stacy Rost and Jake Heaps that Lewis suffered this most recent injury in Monday's win over the A's when he made a leap trying to catch a deep fly ball to right-center that turned into a double for Oakland's Sean Murphy.
"It was kind of a funky play yesterday where he was trying to run down that ball in the gap there in about the eighth inning and he felt it when he landed. He kind of jumped awkwardly for it," Servais said. "So we took him out of the game right away and he had that checked on this morning (and got) a very early MRI and it was determined he does have a very small meniscus tear in his right knee. I'm not exactly sure how long he's going to be out. Is it a scope-type surgery thing? I'm not sure yet. We'll find out more today."
Lewis, 25, is hitting .246 with a .333 on-base percentage, .726 OPS, five home runs and four doubles in 36 games this season. He also has two stolen bases.
The Mariners have called up outfielder Taylor Trammell from Triple-A Tacoma to replace Lewis on the roster. Trammell, 23, has excelled with the Rainiers after playing 27 games with Seattle to open the season. In MLB action this season, he has a .157/.255/.337 slash line (.593 OPS) with four home runs, three doubles and two steals. In 17 games with Tacoma, he's slashing .384/.413/.726 (1.139 OPS) with six homers, seven doubles and three steals.
Jarred Kelenic will shift from left field to center field in Lewis' absence, Servais said, with Trammell taking over in left.
Also Tuesday, the Mariners activated left-handed pitcher Marco Gonzales from the IL, as expected. Gonzales has recovered from a left forearm strain and is Tuesday's scheduled starter against Oakland in a 7:10 p.m. game (coverage begins at 6 p.m. with the pregame show on 710 ESPN Seattle). Right-handed pitcher Robert Dugger was optioned to Triple-A to make room for Gonzales.
With Tuesday's moves, the Mariners' injured list now currently includes Lewis; first baseman Evan White (hip); starting pitchers James Paxton (Tommy John surgery), Ljay Newsome (elbow) and Nick Margevicius (shoulder); relievers Erik Swanson (groin), Casey Sadler (shoulder), Drew Steckenrider and Kendall Graveman (reportedly COVID-19 protocol); and utility players Dylan Moore (calf), Sam Haggerty (shoulder) and Shed Long (on a rehab assignment for a shin injury).
T-Mobile Park increasing capacity
The Mariners will be able to have even more fans in attendance for their next homestand.
The team announced Tuesday that the state of Washington and King County have given permission to increase seating capacity at T-Mobile Park in time for when the Mariners return home for games from June 14-23.
Per a press release: "The updated operations plan allows 26,265 tickets to be sold as vaccine only with no social distancing. The remaining capacity will be sold in socially distanced pods of 1-6 seats for a total of 4,680 seats. Total ballpark capacity under the approved plan is 30,945 (out of 47,000)."
To this point, the Mariners have had a reduced capacity at T-Mobile Park with socially distanced seating of up to 9,000 and vaccinated-only sections of up to approximately 5,000.
The new capacity also comes with an updated policy for bags and outside food. You can find more details here.
Follow Brent Stecker on Twitter.
Ken Griffey Jr. shares advice for Mariners rookie Jarred Kelenic
Follow @BrentStecker
Mariners Odds & Ends: Uniform notes, Julio Rodríguez snub
Seattle Sports' Brent Stecker provides a few notes on the lighter side of baseball regarding the Mariners, including their 2023 uniforms.
Mariners Breakdown: What veteran A.J. Pollock brings to the table
The Mariners are reportedly bringing in veteran slugger A.J. Pollock on a one-year deal. We look at what he adds to Seattle's roster, including insight from Dodgers announcer Joe Davis.
Are Mariners right about free agency? AL West rivals will test that theory
The Mariners will chase Houston again in 2023, but they need to keep their eyes on two AL West rivals who have turned to free agency for help.
Mariners Offseason: 6 under the radar free agents to fill need for 2 bats
The Mariners are looking for one or two more bats, and they have spots to fill on the corners. We look at free agents who would fit the bill.
Sweep or not, Astros now know Mariners are coming for them
Sweeps don't get any closer than the Mariners' ALDS loss to the Astros, and there are signs the perennial AL West power knows it has company.
How did Mariners make it this far? Same reason they could keep going
The same old Mariners are gone, and the reason they made it to the ALDS is the same reason they will have a chance against the Astros.


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Gamescom 2019: Cyberpunk 2077's Open World Map Will Be Smaller, Denser Than The Witcher 3's
Aug 24, 2019 Aaron Hans 0 comments 846
Credit: CD Projekt Red via YouTube
In open-world gaming, developers always go with the mindset that their new project has to feature a bigger world than their previous one did, which has led to conflicting results. But, CD Projekt Red won't take the "bigger is better" approach with Cyberpunk 2077, as it'll have a smaller world map than CDPR's previous open-world title, The Witcher 3: Wild Hunt.
While some fans might find it disheartening that Cyberpunk 2077's world will be smaller than The Witcher 3's, the developers have promised a much denser, and more intricate open-world than in their previous title.
CD Projekt Red dev Richard Borzymowski talked about Cyberpunk's world in comparison to The Witcher 3's during this past week's Gamescom event, where he said: "it's basically — you take the world of The Witcher and you squeeze it right in." He also added that the game is supposed to have more of a humane crunch than in The Witcher 3, which is why the world is going to be smaller.
Also during this past week's Gamescom event, Borzymowski explained why Night City, which is where most of Cyberpunk 2077 will be set in, has to be denser than any of their other worlds: "Night City is an integral part of the setting; it's essentially a protagonist if you want to call it that, so it has to be denser."
It was also recently revealed that, for the most part, players will be able to enter and explore every building they see in Night City, which is rarely the case with any other open-world titles. Even more linear single-player experiences don't allow players to explore every part of the world that they've created, so this will likely be a welcomed addition to open-world gaming. Also, if it's done well, it could convince other devs to stop focusing on size, and start focusing on quality and detail.
CD Projekt Red is planning on showing more gameplay from Cyberpunk 2077 on August 30 at 11 AM Pacific, 2 PM Eastern. During the stream, the developers will show footage taken from their behind-closed-doors demos at both E3 2019 and this past week's Gamescom. The stream will show approximately 15 minutes of brand new gameplay footage.
As for what the stream could offer, well, CDPR has promised that it'll give players better insight into the process behind their creative decisions, information regarding the districts in Night City, and much more information on the various playstyles that you'll be able to implement once the game launches next spring.
CYBERPUNK 2077CYBERPUNK
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Konami Announces Release Date For eFootball PES 2020 Mobile Version, Gameplay Features And Changes
Destiny 2: Xur Offers Players The Chance For Yet Another Annual Pass Item On August 23rd
CD Projekt Red And Go On Board Announce The Witcher: Old World Board Game
Feb 14, 2021 Lisa Nguyen 3440
CD Projekt Red is teaming up with Go On Board for a new game in The Witcher series. The Witcher: Old World is a board game designed by Łukasz...
CyberPunk 2077 Collaborates With Kosuke Kawamura For Original Merchandise
Feb 3, 2021 Lisa Nguyen 5270
Good Smile Company and CD Projekt Red have been working together to release several pieces of CyberPunk 2077 merchandise. Previously announced were...
Cyberpunk 2077 Now Has Modding Support, Which Should Enhance Modding Possibilities
Jan 26, 2021 Jeremy Green 4640
It's pretty evident the development for Cyberpunk 2077 didn't go according to plan for CD Projekt Red. They were hoping to deliver a next-gen...


Car insurance is a necessity for it helps to maintain the car and also meets the emergencies. Car insurance gives you the confidence to drive fearlessly on the roads. Now with so many car insurance companies vying for customer base in the market, it is quite perplexing to look out for the right insurance company and scheme, which fulfills your requirement and is cost effective. There is a lot of difference in the offerings of the different companies.
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We at moneyduniya.com assist you in this endeavor of car insurance. You fill a simple online form, and your specific requirements and then our expert team will work on it. We bring the best and the most competitive cost quote and benefits of all insurance companies under one roof. Comparative analysis of quotes from the leading car insurance brands will make your choice easier and accurate. Usually it is noticed that car insurances is a significant expense, and worth spending 5 minutes on if you can save upto 40%, and get a better product and service.
Never leave your keys in the vehicle.
Turn off your ignition whenever you leave your car.
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Conceal items left in your car, keep things in the trunk and never leave money or compact discs in the open.
Ask your neighbours to watch out for your vehicle and do the same for them.
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"All that Apurva wanted was to live a happily married life with the woman he loved the most, but what he didn't expect to face was a drastic and dangerous challenge on the first night of his honeymoon. He woke up the first morning and discovered his wife to be affected by something he didn't have any clue about.He was faced with two options, either leave her and save himself or stay and find a solution. What will Apurva choose to do? Will he be successful in his decision? 7 days is a romantic, psychological thriller with paranormal element in it."


Advanced Course of Mechanical and Electronic System Engineering, Advanced Course of Akashi National Colleage of Technology.
Electrical and Computer Engineering, Akashi National Colleage of Technology.
PDR(Pedestrian Dead Reckoning) requires to estimate the exact step count, step length and direction. This study aimed to improve the accuracy of the step length reasoning. The conventional PDRs require a pre-learning such as walking several hundreds meters and manually input parameters. Furthermore, the conventional PDRs don't adapt to circumstance variations such as time periods, a number of users companion. To solve these problems, we propose the step length reasoning method which is automatically learning step length from the GPS before starting PDR. As a result of evaluation, mean absolute error of step length becomes about 2.81 cm when walking alone, about 3 cm to 4 cm when using the proposed method.


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Skip to main content Books Fiction Books Nonfiction Books Ron Charles Michael Dirda Carlos Lozada
The enchanting history of Ben Franklin's glass armonica — 'the first musical instrument ever invented by an American'
By Eugenia Zukerman
Benjamin Franklin may have helped give birth to a new nation, but the glass armonica was the Founding Father's "most cherished creation," according to English professor Corey Mead. Best known as a statesman and a scientist, Franklin had a deep appreciation for the power and beauty of music. In fact, music was his passion and joy, and Mead's new book, "Angelic Music," resonates with that enchantment.
(Simon & Schuster)
But what exactly is a glass armonica, which Mead notes was "the first musical instrument ever invented by an American"? It's unknown when or where glass vessels were first used to create sound, but by the 1700s, Europeans knew that "by filling glasses with different amounts of wine and rubbing a wet finger around their rims," they could make "a cheerful wine-music."
[Listen to Dennis James play a glass armonica.]
Enter our tireless inventor. In 1757, when Franklin was sent to London to lobby for Pennsylvania's right to self-government, he enjoyed going to concerts. When he heard Edward Delaval perform on the musical glasses, he was charmed. But "in typical Franklin fashion, he saw room for improvement in the instrument's design." He experimented to improve its form and found a solution that allowed the performer to create "a much fuller sound than the musical glasses had previously allowed," Mead writes. With other innovations, his new invention "improved the musical glasses, and formed them into a compleat instrument to accompany the voice; capable of a thorough bass, and never out of tune," according to the Bristol Journal in 1762.
Mead takes us on an instructive ride from early America to London and continental Europe, where the armonica was at its most popular. Some loved it, some insisted it caused insanity, while others praised its healing powers. Great composers, including Handel, Beethoven and Mozart, wrote for it. Many gifted women played it. Marie Antoinette popularized it. Ann Ford was "the first famous performer on the musical glasses in eighteenth-century London."
With spirited charm, Mead weaves history, music, science and medicine into the story. There is a delightful chapter on the history of glass, sidetrips to Ireland and Germany, and an intriguing section about Franz Mesmer, who used the glass armonica to hypnotize — or mesmerize — his patients.
The glass armonica eventually fell out of favor, but "after lingering in obscurity for a century and a half, the instrument has in recent decades enjoyed a revival," Mead writes. Composers are writing music for it again in many different genres. Fascinating, insightful and, best of all, great fun, "Angelic Music" will certainly add to this enthusiasm.
Eugenia Zukerman is a flutist and the music director of Clarion Concerts in Columbia County, as well as the artistic director of Classics on Hudson.
Angelic Music
The Story of Benjamin Franklin's Glass Armonica
By Corey Mead
Simon & Schuster. 288 pp. $28


This key chain features a handmade polymer clay round with a handwritten F initial on it. It features a black crafted chain and a silver tone keyring.
Wonderful gift for anyone with an F in their first or last name or for you!


text:
World News | Europe
Facebook Post Featuring Nazi Easter Eggs Gets German Woman $820 Fine
The woman, 70, had tried to argue that she didn't know what the symbols were, that she was protected by freedom of speech, and that it was a joke
Illustration: An Easter basket.Credit: /AP
A court says a 70-year-old woman in eastern Germany has dropped an appeal against her conviction for posting a photo on Facebook of eggs she'd decorated with the Nazi swastika and other runes, wishing people "Happy Easter."
Girl scout goes viral after confronting neo-Nazi at Czech rally
The Star of David: More than just a symbol of the Jewish people or Nazi persecution
Israeli museum honoring Jews who fought the Nazis stands empty and awaits funding
The Dresden administrative court said Thursday that Margit H. withdrew the appeal of her conviction for displaying banned symbols and agreed to pay a 750-euro ($820) fine.
H., whose last name wasn't given in line with privacy laws, posted the picture over Easter 2016 of the basket of 10 eggs decorated with the Nazi German flag, and with the SS symbol and other runes.
The woman had tried to argue that she didn't know what the symbols were, that she was protected by freedom of speech, and that it was a joke.


And now that Obama Administration is in the process of allowing the slaughter of eagles on wind-farms they are ok with that.
You tell me yourself, right here and now that what the Obama Administration is doing with this policy that would allow the killing of eagles for decades to come is wrong, that President Obama himself is wrong on this issue, I will concede this.
Again...point to where anyone is okay with it, but the killing of less migratory birds minus the pollution factor is preferable to the killing of more migratory birds with the pollution factor. Common sense...get some.
It's a drop in the bucket compared to the amount of birds killed by cats so spare me about the birds.
How is this particular related to the bolded? Do you think renewable energy contributes more than oil, gas, and coal to campaigns?
This is not a battle between Exxon and Solyndra. This is between Solyndra and the Bird Conservancy. Which do you think contributed more to Obama 2012?
And who decide which is an acceptable number of killing?. You think 30 years worth of unrestrained killing of eagles, a species that was once on the endangered species list is preferable?.
You go on and on about the loss of wildlife and then turn around and say well, loss of wildlife is ok when we do it. And then want to talk about my "common sense"? That's what I get for trying to have a mature and intellectually honest conversation with someone on the left. Get your head out of your backside before you try and tell others about "common sense".
A president of the U.S. waves his wand and gives permission, exemptions from federal laws, to wind farm operators to have unlimited amounts of eagle kills for 30 years. There are some on here making the dumbest, lamest excuses as to why it should be acceptable.
You go on and on about the loss of wildlife and then turn around and say some loss of wildlife is ok when we do it. And then what to talk about my "common sense"? That's what I get for trying to have a mature and intellectually honest conversation with someone on the left. Get your head out of your backside before you try and tell others about "common sense".
When you are living off the grid and not using any common energy resources is the day that you can get up on a soapbox and preach your environmental purity, but until then don't point fingers at others with self-righteous indignation. It's way too hypocritical and transparent and btw...there is no common sense utilized in your thought process if you were unable to figure out the obvious.
See what I mean?....Did I say anything about me caring about birds? Spin, spin, spin. And yes, the left, in general tend to be environmentalist and pro-green, no denying that and like you said, there are exceptions, like with everything else. What point was you trying to make? I wanna "LOLZ" too, so please tell me.
Here's the LOLZ, you are lumping a group of people together and assuming they all think and believe the same thing just so you can come back and point at them for their "hypocrisy". It's about scoring points against the left and environmentalist for you.
And who decide which is an acceptable number of killing?. You think 30 years worth of unrestrained killing of eagles, a species that was once on the endangered species list is preferable?
How many eagles are being killed. I want numbers instead of just you throwing out opinions.
Who is we? Texas is the leading state with wind farms, you think that's because environmentalists or because of Bush Jr, conservative state legislatures, and Obama?
Get your head outta your backside and stop generalizing, lest you keep getting PWNED.
There is no wand waving, stop being dense. How many eagles have been killed? Do you know? Because it matters with the argument youre presenting.
Does that mean we can hunt those eagles now?
What's the alternative ? do you want more Fukushima/ I mean nuclear reactors?
If it honestly concerns you, stop using electricity, or shut up.
The is, solar ya know .
Photovoltaic solar has been available for over 100 years , What exactly are you doing ? nothing right .


text:
Pachypaederus is a genus of small beetles of the family Staphylinidae ("rove beetles").

Notes

References
 ION –  Pachypaederus
 Nomenclator Zoologicus – Pachypaederus

Staphylinidae genera
Paederinae