Discovery and Contribution of Nontypeable Haemophilus influenzae NTHI1441 to Human Respiratory Epithelial Cell Invasion.

Nontypeable Haemophilus influenzae (NTHi) is the primary cause of bacterially induced acute exacerbations of chronic obstructive pulmonary disease (COPD). NTHi adheres to and invades host respiratory epithelial cells as a means to persist in the lower airways of adults with COPD. Therefore, we mined the genomes of NTHi strains isolated from the airways of adults with COPD to identify novel proteins to investigate their role in adherence and invasion of human respiratory epithelial cells. An isogenic knockout mutant of the open reading frame NTHI1441 showed a 76.6% ± 5.5% reduction in invasion of human bronchial and alveolar epithelial cells at 1, 3, and 6 h postinfection. Decreased invasion of the NTHI1441 mutant was independent of either intracellular survival or adherence to cells. NTHI1441 is conserved among NTHi genomes. Results of whole-bacterial-cell enzyme-linked immunosorbent assay (ELISA) and flow cytometry experiments identified that NTHI1441 has epitopes expressed on the bacterial cell surface. Adults with COPD develop increased serum IgG against NTHI1441 after experiencing an exacerbation with NTHi. This study reveals NTHI1441 as a novel NTHi virulence factor expressed during infection of the COPD lower airways that contributes to invasion of host respiratory epithelial cells. The role in host cell invasion, conservation among strains, and expression of surface-exposed epitopes suggest that NTHI1441 is a potential target for preventative and therapeutic interventions for disease caused by NTHi.

Factor H uptake regulates intracellular C3 activation during apoptosis and decreases the inflammatory potential of nucleosomes.

Factor H (FH) binds apoptotic cells to limit the inflammatory potential of complement. Here we report that FH is actively internalized by apoptotic cells to enhance cathepsin L-mediated cleavage of endogenously expressed C3, which results in increased surface opsonization with iC3b. In addition, internalized FH forms complexes with nucleosomes, facilitates their phagocytosis by monocytes and induces an anti-inflammatory biased cytokine profile. A similar cytokine response was noted for apoptotic cells coated with FH, confirming that FH diminishes the immunogenic and inflammatory potential of autoantigens. These findings were supported by in vivo observations from CFH(-/-) MRL-lpr mice, which exhibited higher levels of circulating nucleosomes and necrotic cells than their CFH(+/+) littermates. This unconventional function of FH broadens the established view of apoptotic cell clearance and appears particularly important considering the strong associations with genetic FH alterations and diseases such as systemic lupus erythematosus and age-related macular degeneration.

Sequence of a turkey BAC clone identifies MHC Class III orthologs and supports ancient origins of immunological gene clusters.

Two genetically unlinked gene clusters currently define the turkey major histocompatibility complex (MHC). Previous studies identified turkey bacterial artificial chromosome (BAC) clones hypothesized as orthologs of the MHC-B and MHC-Y regions of the chicken. Physical mapping assigned these clones to the same microchromosome (MGA18) and sequencing of the MHC-B BAC found near synteny with a portion of the chicken B-locus. This study examines the sequence of the second MHC BAC clone that was hypothesized, based on subclone sequences, to be orthologous to the MHC-Y. Sequencing of this clone identified a class I locus and orthologs of additional genes found in the mammalian class III region. Approximately 50% of the BAC insert is comprised of sequence corresponding to the centromeric repeat, MGASat2. This turkey MHC BAC sequence is unique from sequences assigned to the MHC-Y in the chicken. Based on sequence comparisons, the class I gene appears to be a nonfunctional pseudogene. The class III genes (BAT1, BAT3, STK19, and a G4-like locus) represent the second class III gene cluster identified in the galliform genome. This cluster appears to be of ancient origin and provides insight into the evolution of the avian MHC.

A locus-wide approach to assessing variation in the avian MHC: the B-locus of the wild turkey.

Studies of major histocompatibility complex (MHC) diversity in non-model vertebrates typically focus on structure and sequence variation in the antigen-presenting loci: the highly variable and polymorphic class I and class IIB genes. Although these studies provide estimates of the number of genes and alleles/locus, they often overlook variation in functionally related and co-inherited genes important in the immune response. This study utilizes the sequence of the MHC B-locus derived from a commercial turkey to investigate MHC variation in wild birds. Sequences were obtained for nine interspersed MHC amplicons (non-class I/II) from each of 40 birds representing 3 subspecies of wild turkey (Meleagris gallopavo). Analysis of aligned sequences identified 238 single-nucleotide variants approximately one-third of which had minor allele frequencies >0.2 in the sampled birds. PHASE analysis identified 70 prospective MHC haplotypes in the wild turkeys, whereas a combined analysis with commercial birds identified almost 100 haplotypes in the species. Denaturing gradient gel electrophoresis (DGGE) of the class IIB loci was used to test the efficacy of single-nucleotide polymorphism (SNP) haplotyping to capture locus-wide variation. Diversity in SNP haplotypes and haplotype sharing among individuals was directly reflected in the DGGE patterns. Utilization of a reference haplotype to sequence interspersed regions of the MHC has significant advantages over other methods of surveying diversity while identifying high-frequency SNPs for genotyping. SNP haplotyping provides a means to identify both divergent haplotypes and homozygous individuals for assessment of immunological variation in wild and domestic populations.

Recombination is suppressed over a large region of the rainbow trout Y chromosome.

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus (SEX), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy-163 marker, which maps close to SEX, was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.

Genome-wide genetic diversity of 'Nici', the DNA source for the CHORI-260 turkey BAC library and candidate for whole genome sequencing.

Vertebrate whole genome sequence assembly can benefit from a priori knowledge of variability in the target genome, with researchers often selecting highly inbred individuals for sequencing. However, for most species highly inbred research lines are lacking, requiring the use of an outbred individual(s). Here we examined the source DNA [Nicholas inbred (Nici)] of the CHORI-260 turkey bacterial artificial chromosome (BAC) library through analysis of microsatellites and BAC sequences. Heterozygosity of Nici was compared with that of individuals from several breeder lines. Seventy-eight microsatellites were screened for polymorphism in a total of 43 birds, identifying an average individual heterozygosity of 0.39, with Nici at 0.35. Additional loci (total of 147) were examined on a subset of individuals to obtain better genome coverage. The mean heterozygosity for this subset was 0.33 with Nici at 0.31. Examination of approximately 200 kb of genome sequence identified SNPs in the order of one per 200 bp in Nici. These data suggest that the heterozygosity of Nici is comparable to other birds of selected breeder lines and that whole genome sequencing would result in an abundant resource of genome-wide polymorphisms.

Simple sequence repeats for genetic studies of alpaca.

Trace sequences from the 2X alpaca genome sequencing effort were examined to identify simple sequence repeats (microsatellites) for genetic studies. A total of 6,685 repeat-containing sequences were downloaded from GenBank, processed, and assembled into contigs representing an estimated 4,278 distinct sequences. This sequence set contained 2,290 sequences of length > 100 nucleotides that contained microsatellites of length > or = 14 dinucleotide or 10 trinucleotide repeats with purity equal to 100%. An additional 13 sequences contained a GC microsatellite of length > or = 12 repeats (purity = 100%) were also obtained. Primer pairs for amplification of 1,516 putative loci are presented. Amplification of genomic DNA from alpaca and llama by PCR was demonstrated for 14 primer sets including one from each of the microsatellite repeat types. Comparative chromosomal location for the alpaca markers was predicted in the bovine genome by BLAT searches against assembly 4.0 of the bovine whole genome sequence. A total of 634 markers (41.8%) returned BLAT hits with score > 100 and Identity > 85%, with the majority assignable to unique locations. We show that microsatellites are abundant and easily identified within the alpaca genome sequence. These markers will provide a valuable resource for further genetic studies of the alpaca and related species.

An integrated and comparative genetic map of the turkey genome.

An integrated genetic linkage map was developed for the turkey (Meleagris gallopavo) that combines the genetic markers from the three previous mapping efforts. The UMN integrated map includes 613 loci arranged into 41 linkage groups. An additional 105 markers are tentatively placed within linkage groups based on two-point LOD scores and 19 markers remain unlinked. A total of 210 previously unmapped markers has been added to the UMN turkey genetic map. Markers from each of the 20 linkage groups identified in the Roslin map and the 22 linkage groups of the Nte map are incorporated into the new integrated map. Overall map distance contained within the 41 linkage groups is 3,365 cM (sex-averaged) with the largest linkage group (94 loci) measuring 533.1 cM. Average marker interval for the map was 7.86 cM. Sequences of markers included in the new map were compared to the chicken genome sequence by 'BLASTN'. Significant similarity scores were obtained for 95.6% of the turkey sequences encompassing an estimated 91% of the chicken genome. A physical map of the chicken genome based on positions of the turkey sequences was built and 36 of the 41 turkey linkage groups were aligned with the physical map, five linkage groups remain unassigned. Given the close similarities between the turkey and chicken genomes, the chicken genome sequence could serve as a scaffold for a genome sequencing effort in the turkey.

Characterization of the turkey MHC chromosome through genetic and physical mapping.

Previous studies in the chicken have identified a single microchromosome (GGA16) containing the ribosomal DNA (rDNA) and two genetically unlinked MHC regions, MHC-B and MHC-Y. Chicken DNA sequence from these loci was used to develop PCR primers for amplification of homologous fragments from the turkey (Meleagris gallopavo). PCR products were sequenced and overgo probes were designed to screen the CHORI 260 turkey BAC library. BAC clones corresponding to the turkey rDNA, MHC-B and MHC-Y were identified. BAC end and subclone sequencing confirmed identity and homology of the turkey BAC clones to the respective chicken loci. Based on subclone sequences, single-nucleotide polymorphisms (SNPs) segregating within the UMN/NTBF mapping population were identified and genotyped. Analysis of SNP genotypes found the B and Y to be genetically unlinked in the turkey. Silver staining of metaphase chromosomes identified a single pair of microchromosomes with nucleolar organizer regions (NORs). Physical locations of the rDNA and MHC loci were determined by fluorescence in situ hybridization (FISH) of the BAC clones to metaphase chromosomes. FISH clearly positioned the rDNA distal to the Y locus on the q-arm of the MHC chromosome and the MHC-B on the p-arm. An internal telomere array on the MHC chromosome separates the B and Y loci.

Genomic analysis of genetic markers associated with inherited cardiomyopathy (round heart disease) in the turkey (Meleagris gallopavo).

In turkeys, spontaneous cardiomyopathy or round heart (RH) disease is characterised by dilated ventricles and cardiac muscle hypertrophy. Although the aetiology of RH is still unknown, the disease can have a significant economic impact on turkey producers. In an initial attempt to identify genomic regions associated with RH, we utilised the chicken genome sequence to target short DNA sequences (sequence-characterised amplified regions, SCARs) identified in previous studies that had significant differences in frequency distribution between RH+ and RH- turkeys. SCARs were comparatively aligned with the chicken whole-genome sequence to identify flanking regions for primer design. Primers from 32 alignments were tested and target sequences were successfully amplified for 30 loci (94%). Comparative re-sequencing identified putative SNPs in 20 of the 30 loci (67%). Genetically informative SNPs at 16 loci were genotyped in the UMN/NTBF turkey mapping population. As a result of this study, 34 markers were placed on the turkey/chicken comparative map and 15 markers were added to the turkey genetic linkage map. The position of these markers relative to cardiac-related genes is presented. In addition, analysis of genotypes at 109 microsatellite loci presumed to flank the SCAR sequences in the turkey genome identified four significant associations with RH.

Assignment of linkage groups to turkey chromosome 1 (MGA1).

Previous genetic mapping identified three linkage groups (M1, M18 and M26) in the turkey corresponding to chicken chromosome 1 (GGA1). This is inconsistent with previously described chromosomal differences between these species. FISH analysis of BAC clones corresponding to microsatellite markers from each of the three turkey linkage groups, assigned all three linkage groups to a single chromosome (MGA1).

Single nucleotide polymorphisms for integrative mapping in the Turkey (Meleagris gallopavo).

When multiple genetic maps exist for a species, integration of these maps requires a set of common markers be genotyped across the individual mapping populations. In the turkey, three genetic maps based on separate mapping populations are available. In this study, SNP-based markers were developed for integrating the cDNA/RFLP-based map (1) with microsatellite markers of the second-generation turkey genome map (2). Forty-eight primer sets were designed and tested and 33 (69%) correctly amplified turkey genomic DNA by PCR. Putative SNPs were detected in 20 (61%) of the amplified gene fragments, and 10 SNP markers were subsequently genotyped by PCR/RFLP for segregation analysis. Eight SNP markers were incorporated into the turkey genetic map.

Using the chicken genome sequence in the development and mapping of genetic markers in the turkey (Meleagris gallopavo).

The efficacy of employing the chicken genome sequence in developing genetic markers and in mapping the turkey genome was studied. Eighty previously uncharacterized microsatellite markers were identified for the turkey using BLAST alignment to the chicken genome. The chicken sequence was then used to develop primers for polymerase chain reaction where the turkey sequence was either unavailable or insufficient. A total of 78 primer sets were tested for amplification and polymorphism in the turkey, and informative markers were genetically mapped. Sixty-five (83%) amplified turkey genomic DNA, and 33 (42%) were polymorphic in the University of Minnesota/Nicholas Turkey Breeding Farms mapping families. All but one marker genetically mapped to the position predicted from the chicken genome sequence. These results demonstrate the usefulness of the chicken sequence for the development of genomic resources in other avian species.

Integration of microsatellite-based genetic maps for the turkey (Meleagris gallopavo).

Integration of turkey genetic maps and their associated markers is essential to increase marker density in support of map-based genetic studies. The objectives of this study were to integrate 2 microsatellite-based turkey genetic maps--the Roslin map and the University of Minnesota (UMN) map--by genotyping markers from the Roslin study on the mapping families of the UMN study. A total of 279 markers was tested, and 240 were subsequently screened for polymorphisms in the UMN/Nicholas Turkey Breeding Farms (NTBF) mapping families. Of the 240 markers, 89 were genetically informative and were used for genotyping the F2 offspring. Significant genetic linkages (log of odds > 3.0) were found for 84 markers from the Roslin study. BLASTn comparison of marker sequences with the draft assembly of the chicken genome found 263 significant matches. The combination of genetic and in silico mapping allowed for the alignment of all linkage groups of the Roslin map with those of the UMN map. With the addition of the markers from the Roslin map, 438 markers are now genetically linked in the UMN/NTBF families, and more than 1700 turkey sequences have now been assigned to likely positions in the chicken-genome sequence.

A comparative genetic map of the turkey genome.

Genetic markers (microsatellites and SNPs) were used to create and compare maps of the turkey and chicken genomes. A physical map of the chicken genome was built by comparing sequences of turkey markers with the chicken whole-genome sequence by BLAST analysis. A genetic linkage map of the turkey genome (Meleagris gallopavo) was developed by segregation analysis of genetic markers within the University of Minnesota/Nicholas Turkey Breeding Farms (UMN/NTBF) resource population. This linkage map of the turkey genome includes 314 loci arranged into 29 linkage groups. An additional 40 markers are tentatively placed within linkage groups based on two-point LOD scores and 16 markers remain unlinked. Total map distance contained within linkage groups is 2,011 cM with the longest linkage group (47 loci) measuring 413.3 cM. Average marker interval over the 29 linkage groups was 6.4 cM. All but one turkey linkage group could be aligned with the physical map of the chicken genome. The present genetic map of the turkey provides a comparative framework for future genomic studies.

Survey of a cDNA library from the turkey (Meleagris gallopavo).

Genome characterization and analysis is an imperative step in identifying and selectively breeding for improved traits of agriculturally important species. Expressed sequence tags (ESTs) represent a transcribed portion of the genome and are an effective way to identify genes within a species. Downstream applications of EST projects include DNA microarray construction and interspecies comparisons. In this study, 694 ESTs were sequenced and analyzed from a library derived from a 24-day-old turkey embryo. The 437 unique sequences identified were divided into 76 assembled contigs and 361 singletons. The majority of significant comparative matches occurred between the turkey sequences and sequences reported from the chicken. Whole genome sequence from the chicken was used to identify potential exon-intron boundaries for selected turkey clones and intron-amplifying primers were developed for sequence analysis and single nucleotide polymorphism (SNP) discovery. Identified SNPs were genotyped for linkage analysis on two turkey reference populations. This study significantly increases the number of EST sequences available for the turkey.

Turkey microsatellite loci from an embryonic cDNA library.

Expressed sequence tags (EST) containing microsatellites have been used in the development of both genetic linkage maps and syntenic maps for various species and, thus, offer the advantage of being a convenient tool in comparative mapping studies. A turkey embryonic cDNA library was constructed and screened with (CA/TG)15, (GA/CT)15, (AGG)10, and (AAAC)7 probes for the development of polymorphic microsatellite markers. Sequences of 128 cDNA revealed 42 new loci containing microsatellites. BLAST nucleotide analysis demonstrated significant homology to known mammalian or avian coding regions for 15 of the turkey EST, five of which matched chicken transcripts. The remaining 27 EST represented novel sequences. Of the 42 new loci, 31 were polymorphic when tested on commercial turkey lines, including the founding individuals of a new resource population developed for genetic linkage mapping. Comparative mapping of these markers will provide new information toward the evolutionary divergence of turkey and chicken as well as other species.

Microsatellite loci for genetic mapping in the turkey (Meleagris gallopavo).

New microsatellite loci for the turkey (Meleagris gallopavo) were developed from two small insert DNA libraries. Polymorphism at these new loci was examined in domestic birds and two resource populations designed for genetic linkage mapping. The majority of loci (152 of 168) was polymorphic in domestic turkeys and informative in two mapping resource populations and thus will be useful for genetic linkage mapping.

Allelic variation and genetic linkage of avian microsatellites in a new turkey population for genetic mapping.

Efforts to build a comprehensive genetic linkage map for the turkey (Meleagris gallopavo) have focused on development of genetic markers and experimental resource families. In this study, PCR amplification was attempted for 772 microsatellite markers that had been previously developed for three avian species (chicken, quail and turkey). Allelic polymorphism at 410 markers (53.1% of total examined) was determined by genotyping ten individuals (six F1 parents and four grandparents) in a new resource population specifically developed for genetic linkage mapping. Of these 410 markers, 109 (26.6%) were polymorphic in the tested individuals, with an average of 2.3 alleles per marker. Higher levels of polymorphism were found for the turkey-specific markers (61.1%) than for the chicken (22.7%) or quail-specific markers (33.3%). To test the fidelity of the matings, demonstrate the power of these families for linkage analysis, and determine genetic linkage relationships, 86 polymorphic markers were genotyped for up to 224 birds including founder grandparents, parents and F2 progeny. Linkage relationships for many of the chicken markers elucidated in the turkey were comparable to those observed in the chicken. These data demonstrate that the new UMN/NTBF resource population will provide a solid foundation for constructing a comparative genetic map of the turkey.

Myosin light chain genes in the turkey (Meleagris gallopavo).

Myosin light chains associate with the motor protein myosin and are believed to play a role in the regulation of its actin-based ATPase activity. Myosin light chain cDNA clones from the turkey (Meleagris gallopavo) were isolated and sequenced. One sequence corresponded to an alternative transcript, the skeletal muscle essential light chain (MYL1 isoform 1) and a second to the smooth muscle isoform of myosin light chain (MYL6). The DNA and predicted amino acid sequences of both light chain genes were compared to that of the chicken. Based on the cDNA sequence, oligonucleotide primers were designed to amplify genomic DNA from six of the seven introns of the MYL1 gene. Approximately 5 kb of DNA was sequenced (introns and 3' UTR) and evaluated for the presence of single nucleotide polymorphisms (SNPs). SNPs were verified by sequencing common intron regions from multiple individuals and three polymorphisms were used to genotype pedigreed families. MYL1 is assigned to a turkey linkage group that corresponds to a region of chicken chromosome 7 (GGA7). The results of this study provide genomic reagents for comparative studies of avian muscle components and muscle biology.