Sequencing Of Genomic Regions Research Articles

NF-κB and STAT1 control CXCL1 and CXCL2 gene transcription

Diabetes mellitus results from immune cell invasion into pancreatic islets of Langerhans, eventually leading to selective destruction of the insulin-producing β-cells. How this process is initiated is not well understood. In this study, we investigated the regulation of the CXCL1 and CXCL2 genes, which encode proteins that promote migration of CXCR2(+) cells, such as neutrophils, toward secreting tissue. Herein, we found that IL-1β markedly enhanced the expression of the CXCL1 and CXCL2 genes in rat islets and β-cell lines, which resulted in increased secretion of each of these proteins. CXCL1 and CXCL2 also stimulated the expression of specific integrin proteins on the surface of human neutrophils. Mutation of a consensus NF-κB genomic sequence present in both gene promoters reduced the ability of IL-1β to promote transcription. In addition, IL-1β induced binding of the p65 and p50 subunits of NF-κB to these consensus κB regulatory elements as well as to additional κB sites located near the core promoter regions of each gene. Additionally, serine-phosphorylated STAT1 bound to the promoters of the CXCL1 and CXCL2 genes. We further found that IL-1β induced specific posttranslational modifications to histone H3 in a time frame congruent with transcription factor binding and transcript accumulation. We conclude that IL-1β-mediated regulation of the CXCL1 and CXCL2 genes in pancreatic β-cells requires stimulus-induced changes in histone chemical modifications, recruitment of the NF-κB and STAT1 transcription factors to genomic regulatory sequences within the proximal gene promoters, and increases in phosphorylated forms of RNA polymerase II.

Quantifying single nucleotide variant detection sensitivity in exome sequencing

BackgroundThe targeted capture and sequencing of genomic regions has rapidly demonstrated its utility in genetic studies. Inherent in this technology is considerable heterogeneity of target coverage and this is expected to systematically impact our sensitivity to detect genuine polymorphisms. To fully interpret the polymorphisms identified in a genetic study it is often essential to both detect polymorphisms and to understand where and with what probability real polymorphisms may have been missed.ResultsUsing down-sampling of 30 deeply sequenced exomes and a set of gold-standard single nucleotide variant (SNV) genotype calls for each sample, we developed an empirical model relating the read depth at a polymorphic site to the probability of calling the correct genotype at that site. We find that measured sensitivity in SNV detection is substantially worse than that predicted from the naive expectation of sampling from a binomial. This calibrated model allows us to produce single nucleotide resolution SNV sensitivity estimates which can be merged to give summary sensitivity measures for any arbitrary partition of the target sequences (nucleotide, exon, gene, pathway, exome). These metrics are directly comparable between platforms and can be combined between samples to give “power estimates” for an entire study. We estimate a local read depth of 13X is required to detect the alleles and genotype of a heterozygous SNV 95% of the time, but only 3X for a homozygous SNV. At a mean on-target read depth of 20X, commonly used for rare disease exome sequencing studies, we predict 5–15% of heterozygous and 1–4% of homozygous SNVs in the targeted regions will be missed.ConclusionsNon-reference alleles in the heterozygote state have a high chance of being missed when commonly applied read coverage thresholds are used despite the widely held assumption that there is good polymorphism detection at these coverage levels. Such alleles are likely to be of functional importance in population based studies of rare diseases, somatic mutations in cancer and explaining the “missing heritability” of quantitative traits.

A PCR-RFLP method for typing human papillomavirus type 16 variants

Infection with some types of human papillomavirus (HPV) is required for cervical cancer development, being HPV type 16 (HPV 16) the most common type in premalignant and malignant cervical lesions. DNA sequencing has revealed the existence of intratypic variants of HPV 16 whose genotyping is clinically useful for distinguishing between persistent and recurrent infections. From the epidemiological perspective, the frequency of diverse HPV 16 variants in several populations could correlate with the presence of precursor high-risk lesions in different anatomical locations. Currently, the "gold standard" method for identifying HPV 16 variants involves the sequencing of genomic regions to identify characteristic polymorphic sites. Although some other methods have been described, they require specialized or high-cost equipment. In this study, a robust and low cost procedure is described for HPV 16 variant typing, based on the long control region of the virus.

Solution-based targeted genomic enrichment for precious DNA samples

BackgroundSolution-based targeted genomic enrichment (TGE) protocols permit selective sequencing of genomic regions of interest on a massively parallel scale. These protocols could be improved by: 1) modifying or eliminating time consuming steps; 2) increasing yield to reduce input DNA and excessive PCR cycling; and 3) enhancing reproducible.ResultsWe developed a solution-based TGE method for downstream Illumina sequencing in a non-automated workflow, adding standard Illumina barcode indexes during the post-hybridization amplification to allow for sample pooling prior to sequencing. The method utilizes Agilent SureSelect baits, primers and hybridization reagents for the capture, off-the-shelf reagents for the library preparation steps, and adaptor oligonucleotides for Illumina paired-end sequencing purchased directly from an oligonucleotide manufacturing company.ConclusionsThis solution-based TGE method for Illumina sequencing is optimized for small- or medium-sized laboratories and addresses the weaknesses of standard protocols by reducing the amount of input DNA required, increasing capture yield, optimizing efficiency, and improving reproducibility.

Assessment of Gastroenteric Viruses Frequency in a Children's Day Care Center in Rio De Janeiro, Brazil: A Fifteen Year Study (1994–2008)

This 15-year study aimed to determine the role of the main viruses responsible for acute infantile gastroenteritis cases in a day care center in the city of Rio de Janeiro, Brazil. From 1994 to 2008, 539 fecal samples were obtained from 23 outbreaks as well as sporadic cases that occurred in this period. The detection of Rotavirus group A (RVA), norovirus (NoV) and astrovirus (AstV) was investigated both by classical and molecular methods of viral detection. RVA was detected by enzymatic immune assay and/or polyacrylamide gel electrophoresis and genotyped by using semi-nested multiplex PCR. NoV and AstV were subsequently tested by real time PCR in all RVA-negative samples and genotyped throughout genome sequencing. Three protocols for molecular characterization of NoV nucleotide sequencing were performed with the partial nucleotide sequencing of genomic regions known as region B (polymerase gen), C and D (capsid gen).Viruses were identified in 47.7% (257/539) of the cases, and the detection rates of RVA, NoV and AstV in16.1% (87/539), 33.4% (151/452), and 6.3% (19/301), respectively. Most gastroenteritis cases were reported in autumn and winter, although NoV presented a broader monthly distribution. Viruses' detection rates were significantly higher among children aged less than 24 months old, although NoV cases were detected in all age groups. RVA genotypes as G1P[8], G9P[8], G2P[4], G3P[8] and G1+G3P[8] and RVA was no longer detected after 2005. NoV characterization revealed genotypes variability circulating in the period as GI.2, GI.3, GI.8 GII.2, GII.3, GII.4, GII.4 variants 2001 and 2006b, GII.6, GII.7, GII.12 and GII.17. AstV genotypes 1, 2, 4 and 5 were also characterized. Those data demonstrate the impact of NoV infection in cases of infantile gastroenteritis, surpassing RVA infection responsible for high morbidity rate in children under five years old.

Chicks and single-nucleotide polymorphisms: an entrée into identifying genes conferring disease resistance in chicken

Marek’s disease (MD) is one of the most serious chronic infectious disease threats to the poultry industry worldwide. Selecting for increased genetic resistance to MD is a control strategy that can augment current vaccinal control measures. Although our previous efforts integrating various genomic screens successfully identified three resistance genes, the main limitation was mapping precision, which hindered our ability to identify and further evaluate high-confidence candidate genes. Towards identifying the remaining genes of this complex trait, we incorporated three additional approaches made substantially more powerful through next-generation sequencing and that exploit the growing importance of expression variation. First, we screened for allele-specific expression (ASE) in response to Marek’s disease virus (MDV) infection, which, when allelic imbalance was identified, is sufficient to indicate a cis-acting element for a specific gene. Second, sequencing of genomic regions enriched by chromatin immunoprecipitation (ChIP) combined with transcript profiling identified motifs bound and genes directly regulated by MDV Meq, a bZIP transcription factor and the viral oncogene. Finally, analysis of genomic sequences from two experimental lines divergently selected for MD genetic resistance allowed inference about regions under selection as well as potential causative polymorphisms. These new combined approaches have resulted in a large number of high-confidence genes conferring MD resistance reflecting the multigenic basis of this trait, which expands our biological knowledge and provides corresponding single-nucleotide polymorhpisms (SNPs) that can be directly evaluated for their genetic contribution towards disease resistance.

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.

Evolutionary genomics of animal personality

Research on animal personality can be approached from both a phenotypic and a genetic perspective. While using a phenotypic approach one can measure present selection on personality traits and their combinations. However, this approach cannot reconstruct the historical trajectory that was taken by evolution. Therefore, it is essential for our understanding of the causes and consequences of personality diversity to link phenotypic variation in personality traits with polymorphisms in genomic regions that code for this trait variation. Identifying genes or genome regions that underlie personality traits will open exciting possibilities to study natural selection at the molecular level, gene-gene and gene-environment interactions, pleiotropic effects and how gene expression shapes personality phenotypes. In this paper, we will discuss how genome information revealed by already established approaches and some more recent techniques such as high-throughput sequencing of genomic regions in a large number of individuals can be used to infer micro-evolutionary processes, historical selection and finally the maintenance of personality trait variation. We will do this by reviewing recent advances in molecular genetics of animal personality, but will also use advanced human personality studies as case studies of how molecular information may be used in animal personality research in the near future.

Molecular Phylogeny and Functional Genomics of β-Galactoside α2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes

Sialyltransferases are key enzymes in the biosynthesis of sialoglycoconjugates that catalyze the transfer of sialic residue from its activated form to an oligosaccharidic acceptor. β-Galactoside α2,6-sialyltransferases ST6Gal I and ST6Gal II are the two unique members of the ST6Gal family described in higher vertebrates. The availability of genome sequences enabled the identification of more distantly related invertebrates' st6gal gene sequences and allowed us to propose a scenario of their evolution. Using a phylogenomic approach, we present further evidence of an accelerated evolution of the st6gal1 genes both in their genomic regulatory sequences and in their coding sequence in reptiles, birds, and mammals known as amniotes, whereas st6gal2 genes conserve an ancestral profile of expression throughout vertebrate evolution.

Methylation Status of HPRT1 Promoter in HPRT Deficiency with Normal Coding Region

Lesch-Nyhan syndrome is an X-linked recessive inborn error of metabolism due to a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity (OMIM 300322). Partial deficiency of HPRT (OMIM 300323) is characterized by the effects of excess uric acid synthesis and a continuum spectrum of neurological manifestations, without the manifestations of full-blown Lesch-Nyhan syndrome. Both diseases have been associated with mutations in the HPRT1 gene. We have described one Lesch-Nyhan patient and four partial HPRT deficient patients with a normal HPRT1 coding region. These patients showed markedly decreased HPRT mRNA expression, but no mutation in their genomic regulatory sequences from HPRT1 gene. In this study, we analyzed the promoter region methylation status of the HPRT1 gene in these five HPRT deficient patients. Methods: DNA was bisulphite modified and a 620 bp fragment including 320 bp 5′ to start codon was amplified and sequenced. The methylation status of 35 CpG island 5′ to start codon and 28 CpG island 3′ to start codon were investigated in male controls, female controls, patients, and the patient's mothers. Primer pairs were designed for methylated-specific and unmethylated-specific amplification and PCR was performed employing DNA bisulphite treated as template. Results: No alterations in the methylation pattern of the HPRT1 promoter were found in the five HPRT deficient patients. Conclusions: The promoter region methylation status of these five HPRT deficient patients was similar to that of normal subjects. Thus, some other genetic alteration must explain a reduced enzyme activity with a normal gene coding region.

Family-based association study for bipolar affective disorder

In this study we aimed to evaluate 21 candidate loci for bipolar affective disorder (BPAD) identified earlier in a large genome-wide association study. We evaluated 74 pedigrees with BPAD, with a total of 411 individuals, including 96 patients who fulfilled clinical criteria for BPAD according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition classification. Family-based association analysis was performed using the UNPHASED software. We identified a single nucleotide polymorphism (rs9834970) localized on chromosome 3p22.3, showing statistically significant association with BPAD after the Bonferroni correction for multiple comparisons (P(corrected)=0.0025) with an odds ratio=2.64 (95% confidence interval: 1.30-5.35). Single nucleotide polymorphism rs9834970 is located in an intergenic region and is not known to be associated to regulatory genomic sequences.

Isolation of putative FOXO1 genomic elements using an improved in vitro technique to isolate genomic regulatory sequences

The regulation of gene expression drives many biological processes and alterations in normal regulation are integral in the development of the diseased state. Therefore, the ability to screen genomic DNA for direct targets of DNA binding proteins (DNA-BP) would provide valuable information about the mechanisms underlying these processes. At present chromatin immunoprecipitation (ChIP) and its variants are the primary methods for identifying regulatory elements. However, some DNA-BPs, such as the winged-helix transcription factor FOXO1, are difficult to ChIP thereby detracting from the use of this technique as a nonbiased screen to isolate regulatory sequences. In this report we use an improved in vitro method to Pull Out Regulatory Elements (PORE), which uses purified protein with a stable genomic library to isolate regulatory elements directly bound by a DNA-BP, to identify putative FOXO1 genomic regulatory sequences. We first validate this technique using two known DNA-BP (FOXO1 and Pax3) by demonstrating their ability to bind and amplify identified promoter elements when present in a genomic DNA context or when present in the context of our stable genomic library. Subsequent use of this technique with FOXO1 isolated regulatory elements associated with several genes known to be regulated in a FOXO1-dependent manner and multiple genes whose biological functions are consistent with the known biological functions of FOXO1 proving that the in vitro PORE is a valuable and easy to use alternative to ChIP for the isolation of genomic regulatory elements.

Detection of non-polio enteroviruses in Hungary 2000–2008 and molecular epidemiology of enterovirus 71, coxsackievirus A16, and echovirus 30

Human enteroviruses are associated with various clinical syndromes from minor febrile illness to severe, potentially fatal conditions like aseptic meningitis, paralysis, myocarditis, and neonatal enteroviral sepsis. Between June 2000 and August 2008 echovirus (E) type 2, 4, 6, 7, 9, 11, 13, 25, 30, coxsackievirus (CV) -A16, -A19, -B5, and enterovirus 71 (EV71) were reported in Hungary. In this study, 29 previously enterovirus positive samples from 28 patients diagnosed with hand, foot and mouth disease, meningitis and encephalitis, were molecularly typed. The genetic relationships of identified serotypes CV-A16, EV71, and E30 were assessed by direct sequencing of genomic region encoding the capsid protein VP1. The sequences were compared to each other and sequences from other geographical regions possessed in Genbank. The phylogenetic analysis of CV-A16 revealed that the viruses were mostly of Far-Eastern or Asia-Pacific origin. Typing of EV71 showed that one virus from 2000 belonged to genotype C1 and five viruses observed in 2004 and 2005 were identified as genotype C4. The 11 echovirus 30 strains showed homology with those of neighbor European countries. The molecular examination of E30 revealed that three separate lineages circulated in 2000, 2001, and 2004-2006 in Hungary.

A protocol describing the principles of cis-regulatory analysis in the sea urchin

cis-Regulatory analysis (CRA) is the precise identification of the cis-acting genomic sequences regulating gene transcription. As such, CRA provides essential mechanistic insight into key biological processes such as development. The first phase of this protocol involves identification of a large (approximately 100 kb) clone of genomic material surrounding the gene of interest and use of this clone to establish a reliable and unambiguous reporter assay. In the second phase, phylogenetic footprinting is used to identify candidate regulatory modules; these genomic sequences are then recursively tested for reporter activity. In the final phase, potential transcription factor binding sites are identified and disrupted in reporter constructs for individual testing. The strengths of this method reflect the use of large clones containing all relevant genomic regulatory sequences to establish a reporter assay with high fidelity. Given these foundational elements, predicted or suspected regulatory inputs can be rigorously tested and novel regulatory inputs identified. Although the expected time line varies greatly with the depth of information required, results may be obtained in as little as 4-6 months, but more detailed analyses will require several years to complete.

Genetic risk and transcriptional variability of amyloid precursor protein in Alzheimer's disease

It is well established that Alzheimer's disease causing mutations in APP, PSEN1 and PSEN2 lead to a relative increased production of Abeta42, thereby fostering its deposition in plaques. Recently others and we showed that amyloid precursor protein (APP) overproduction, either as a result of genomic locus duplication or altered regulatory sequences in the APP promoter region, leads to early-onset disease. Here, we have expanded our study of genetic variability in the APP promoter to a large group of well-documented Belgian patients (n = 750, mean onset age = 75.0 +/- 8.6, range = 37-96). We identified three different APP promoter mutations (-369C-->G, -534G-->A and -479C-->T) in seven patients. In patients with onset < or =70 years (n = 204), we identified one patient carrying the London APP V717I mutation while no patients carried an APP locus duplication, indicating that APP promoter mutations (n = 2) were more frequently associated with increased risk for early-onset Alzheimer's disease. The two mutations (-369C-->G and -534G-->A) increasing APP promoter activity by nearly 2-fold and mimicking an APP duplication, appeared in probands of families with multiple patients with dementia. The -479C-->T mutation that increased APP expression only mildly (1.2-fold), was observed in four patients with onset ages ranging from 62 to 79 years (mean 71.5 years), suggesting that its contribution to disease risk is more pronounced at later age due to modulating factors. In conclusion, we provided evidence that mutations in APP regulatory sequences are more frequent than APP coding mutations, and that increased APP transcriptional activity constitutes a risk factor for Alzheimer's disease with onset ages inversely correlated with levels of APP expression.

Regulation of Endogenous Melanocortin-4 Receptor Expression and Signaling by Glucocorticoids

The melanocortin-4 (MC4) receptor plays a pivotal role in regulating food intake and energy expenditure, and obesity results from mutations that interfere with the MC4 receptor pathway. We investigated the effect of glucocorticoids on endogenous MC4 receptors expressed in GT1-1 cells, an immortalized hypothalamic neuronal cell line. Dexamethasone (Dex) caused a 5- to 10-fold increase in the cAMP response to the MC4 receptor agonist, NDP-alphaMSH. The stimulatory effect of Dex reached a maximum within 24 h and was blocked by the glucocorticoid antagonist RU486. This glucocorticoid effect was specific for the MC4 receptor and not a result of up-regulation of another component of the cAMP cascade, because the response to endogenous beta-adrenergic receptor stimulation was not altered by Dex. Dex also potentiated NDP-alphaMSH-mediated ERK1/2 activation. After 12 h, Dex caused a 3- to 5-fold increase in [125I]NDP-alphaMSH binding, which was maintained for at least 48 h and prevented by RU486. Dex withdrawal caused a rapid return of MC4 receptor concentration to the basal level. Dex-mediated increases in MC4 receptor concentration resulted from a rapid but transient increase in MC4 receptor mRNA. This regulation apparently requires genomic regulatory sequences because Dex did not increase MC4 receptor expression or signaling in CHO cells expressing the MC4 receptor under the control of a cytomegalovirus promoter. We conclude that in GT1-1 hypothalamic neurons, glucocorticoids increase the amplitude of MC4 receptor signaling. This regulation may serve as a control to limit the effects of glucocorticoids on food intake.

Ultra-Sensitive Immunodetection of 5'Methyl Cytosine for DNA Methylation Analysis on Oligonucleotide Microarrays

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.

Towards the Human Cancer Genome Project: A Sequence-Ready Physical Map of a Follicular Lymphoma Genome.

With the aim of identifying and sequencing mutations in follicular lymphoma genomes, we have begun a project to generate at least 24 deeply redundant sequence-ready Bacterial Artificial Clone (BAC) - based whole genome maps, each from a different individual's lymphoma. BAC-array CGH and Affymetrix whole-genome sampling assays (WGSA) will be used along with the mapping data to identify genomic amplifications and losses in the lymphomas. Results from the mapping and array studies will be used to prioritize BAC clones for sequence analysis. Because each map will span essentially the entire genome of the corresponding lymphoma, we anticipate that essentially all regions of each tumor genome will be represented in easily sequenced BAC clones. This approach facilitates targeted sequencing of genomic regions of interest, including those containing genes relevant to cancer or harboring amplifications or deletions.Our mapping strategy hinges on the successful creation of deeply redundant high quality BAC libraries from primary lymphomas and large scale high throughput restriction enzyme fingerprinting of individual BACs with a version of the technology we used to map the human, mouse, rat and other genomes. The effort is large-scale, and will result in the generation of at least 2.5 million fingerprinted BAC clones over the next three years. Using the fingerprints, we will align the BACs to the reference human genome to assess genome coverage and to identify candidate genome rearrangements. In parallel, we will assemble the fingerprints into genome maps, looking for larger-scale genome variations between the lymphoma maps and the reference genome sequence. To test the feasibility of our approach, we obtained two restriction digest fingerprints from each of 140,000 individual BAC clones. BACs were sampled from a 7-fold redundant BAC library that had been created from genomic DNA purified from a primary follicular lymphoma sample. The fingerprints are being assembled into a clone map with the intent of reconstructing the entire tumor genome. 90,377 fingerprinted clones with unambiguous single alignments to the reference sequence were automatically assembled into 15,538 contigs. Subsequent rounds of semi-automatic contig merging further reduced the number of contigs to 5,433. Only 1,241 clones remained unassembled. We anchored the tumor genome map to the reference human genome sequence by aligning the clone fingerprints to the restriction map computed from the reference sequence assembly. As a result of this, we identified a BAC that captured the canonical t(14;18) translocation characteristic of follicular lymphomas. We sequenced this BAC and confirmed that it contains the expected translocation.Almost 2.6 gigabases (~91%) of the reference genome are represented in the evolving map, with an additional 50,000 clone fingerprints awaiting incorporation into the map assembly. Among these are repeat-rich and other clones that may well harbor genome rearrangements. Additional prioritization of sequencing targets will be undertaken when map construction and analysis of genome copy number alterations are complete.

Porcine Toll-like receptor 1, 6, and 10 genes: Complete sequencing of genomic region and expression analysis

Toll-like receptors (TLRs) recognize various microbial components and play key roles in activating the innate immune system. Hence, their function is important in swine infectious diseases. We completely determined 173,804 bp of nucleotide sequence of a genomic region including porcine TLR6 and the newly identified porcine TLR homologues TLR1 and TLR10. The porcine genomic structure of these genes was highly conserved in comparison with the corresponding region in humans. Analysis of their expression in porcine tissues showed differences in expression patterns between porcine TLR10 and TLR1 or TLR6. Moreover, phylogenetic analysis of the cytoplasmic regions of TLR genes suggested that the signal transduction pathway of TLR10 was different from those of TLR1 and TLR6. We also developed six polymorphic microsatellite markers within this genomic region; these markers will be valuable for association studies between TLR genes and resistance or susceptibility to infectious diseases in swine.

Function and regulation of Alx4 in limb development: Complex genetic interactions with Gli3 and Shh

The role of the aristaless-related homeobox gene Alx4 in antero-posterior (AP-) patterning of the developing vertebrate limb has remained somewhat elusive. Polydactyly of Alx4 mutant mice is known to be accompanied by ectopic anterior expression of genes like Shh, Fgf4 and 5′Hoxd. We reported previously that polydactyly in Alx4 mutant mice requires SHH signaling, but we now show that in early Alx4 −/− limb buds the anterior ectopic expression of Fgf4 and Hoxd13, and therefore disruption of AP-patterning, occurs independently of SHH signaling. To better understand how Alx4 functions in the pathways that regulate AP-patterning, we also studied genomic regulatory sequences that are capable of directing expression of a reporter gene in a pattern corresponding to endogenous Alx4 expression in anterior limb bud mesenchyme. We observed, as expected for authentic Alx4 expression, expansion of reporter construct expression in a Shh −/− background. Total lack of reporter expression in a Gli3 −/− background confirms the existence of Gli3-dependent and -independent Alx4 expression in the limb bud. Apparently, these two modules of Alx4 expression are linked to dissimilar functions.