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Neutralization-sensitive merozoite surface antigens of Babesia bovis encoded by members of a polymorphic gene family.

Monospecific antibodies against native and recombinant versions of the major merozoite surface antigen (MSA-1) of Babesia bovis neutralize the infectivity of merozoites from Texas and Mexico strains in vitro. Sequence analysis shows that MSA-1 and a related, co-expressed 44 kDa merozoite surface protein (MSA-2) are encoded by members of a multigene family previously designated BabR. BabR genes, originally described in Australia strains of B. bovis, are notable because their marked polymorphism is apparently mediated by chromosomal rearrangements, but protein products of BabR genes have not previously been identified. The 3' terminal 173 nucleotides of the MSA-1 gene, including 60 nucleotides of untranslated sequence, are highly similar to the 3' terminal sequences of BabR 0.8 (84% identity) and MSA-2 (94% identity). Alignment of the predicted protein sequences demonstrates significant overall homology between MSA-1 and MSA-2, and between both proteins and the amino terminal BabR sequence. MSA-1 nucleic acid probes also hybridize weakly to genomic DNA from the Australia 'L' strain, even though this strain does not express merozoite surface epitopes cross-reactive with MSA-1 or MSA-2. Hybridization of these same probes to genomic DNA from the cloned Mexico strain reveals a pattern of bands compatible with two copies each of MSA-1 and MSA-2. Proteins encoded by this B. bovis gene family have been designated variable merozoite surface antigens (VMSA). The extent and mechanism of VMSA polymorphism among strains will be important when evaluating the role these surface proteins have in the host-parasite interaction, including immunity to blood stages.

Amino Acid Sequence↗

Accurate prediction of the functional significance of single nucleotide polymorphisms and mutations in the ABCA1 gene.

The human genome contains an estimated 100,000 to 300,000 DNA variants that alter an amino acid in an encoded protein. However, our ability to predict which of these variants are functionally significant is limited. We used a bioinformatics approach to define the functional significance of genetic variation in the ABCA1 gene, a cholesterol transporter crucial for the metabolism of high density lipoprotein cholesterol. To predict the functional consequence of each coding single nucleotide polymorphism and mutation in this gene, we calculated a substitution position-specific evolutionary conservation score for each variant, which considers site-specific variation among evolutionarily related proteins. To test the bioinformatics predictions experimentally, we evaluated the biochemical consequence of these sequence variants by examining the ability of cell lines stably transfected with the ABCA1 alleles to elicit cholesterol efflux. Our bioinformatics approach correctly predicted the functional impact of greater than 94% of the naturally occurring variants we assessed. The bioinformatics predictions were significantly correlated with the degree of functional impairment of ABCA1 mutations (r2 = 0.62, p = 0.0008). These results have allowed us to define the impact of genetic variation on ABCA1 function and to suggest that the in silico evolutionary approach we used may be a useful tool in general for predicting the effects of DNA variation on gene function. In addition, our data suggest that considering patterns of positive selection, along with patterns of negative selection such as evolutionary conservation, may improve our ability to predict the functional effects of amino acid variation.

ATP Binding Cassette Transporter 1↗

Genetic variability, haplotypes, and htSNPs for exons 1 at the human UGT1A locus.

UDP-Glucuronosyltransferases (UGTs) are a superfamily of enzymes responsible for glucuronidation of xenobiotics and endobiotics. Genetic polymorphisms have been identified in the promoter and exonic regions of several UGT genes. The UGT1As on chromosome 2q37 have unique exons 1 but share the remainder of their coding sequence. We screened exon 1 of each of the nine functional UGT1As in Asians (n=46) and Caucasians (n=92) with the aim of determining linkage disequilibrium (LD) and haplotypes across the entire locus in both populations. For polymorphisms in UGT 1A3, 1A4, 1A5, 1A7, and 1A8, we observed significant differences in the allele frequency between the two populations. The haplotype block structure across the UGT1A locus was constructed using all 83 polymorphisms and showed four and five haplotype blocks in Caucasians and Asians, respectively. There was long-distance LD between UGT pairs: 1A8 and 1A10; 1A1 and 1A3; 1A1 and 1A6; 1A6 and 1A7; and 1A7 and 1A9. Whereas both ethnic groups shared some haplotype-tagging SNPs (htSNPs), Caucasians and Asians also had unique htSNPs. This was partly due to the fact that rare variants (<5% allele frequency) were included in our analyses. Haplotypes with frequencies >5% represented only 60% of Caucasian and 65% of Asian UGT1A haplotypes. Differences in haplotype distribution patterns suggest individual and ethnic differences in glucuronidation capacity.

Asian People↗

Transporter associated with antigen-processing-1 (TAP1) alleles in Gorilla gorilla: diversification of the locus postspeciation.

The transporter associated with antigen-processing (TAP) proteins are required for the transport of cytosolic peptides into the endoplasmic reticulum for assembly with class I major histocompatibility molecules. In the rat, allelic variants of the TAP genes impart specificity to the process of peptide transport. However, differential transport has yet to be demonstrated with the human molecules. TAP genes from humans and rodents have been studied thus far; analysis of another species more closely related to humans is necessary for a clearer understanding of the evolution of TAP genes. Three TAP1 alleles from four gorilla cell lines were characterized in this study. There is limited genetic distance at the locus, either within the gorilla (0.2%) or between the two hominoid species (0.8%). Nucleotide substitution analysis demonstrates that TAP1 and TAP2 are evolving at comparable rates under similar selection pressures. This pattern is in marked contrast to that observed for MHC class I genes of hominoids or rodents. Although there is limited evidence for trans-species evolution of the gorilla locus, the bulk of the diversification occurred after speciation as evidenced by a lack of shared amino acid polymorphism with human homologues. An evolutionary scheme predicts that the ancestral hominoid TAP1 molecule most closely resembled the human TAP1*02011.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

Haplotype structure, LD blocks, and uneven recombination within the LRP5 gene.

Patterns of linkage disequilibrium (LD) in the human genome are beginning to be characterized, with a paucity of haplotype diversity in "LD blocks," interspersed by apparent "hot spots" of recombination. Previously, we cloned and physically characterized the low-density lipoprotein-receptor-related protein 5 (LRP5) gene. Here, we have extensively analysed both LRP5 and its flanking three genes, spanning 269 kb, for single nucleotide polymorphisms (SNPs), and we present a comprehensive SNP map comprising 95 polymorphisms. Analysis revealed high levels of recombination across LRP5, including a hot-spot region from intron 1 to intron 7 of LRP5, where there are 109 recombinants/Mb (4882 meioses), in contrast to flanking regions of 14.6 recombinants/Mb. This region of high recombination could be delineated into three to four hot spots, one within a 601-bp interval. For LRP5, three haplotype blocks were identified, flanked by the hot spots. Each LD block comprised over 80% common haplotypes, concurring with a previous study of 14 genes that showed that common haplotypes account for at least 80% of all haplotypes. The identification of hot spots in between these LD blocks provides additional evidence that LD blocks are separated by areas of higher recombination.

3' Flanking Region↗

[Pharmacogenomics genomics approaches to optimizing drug therapy].

With the development of the research in human genomics, it is well known that genetic polymorphisms(mainly single nucleotide polymorphisms) of the genes encoding drug-metabolizing enzymes, transporters, receptors and other drug target proteins are relative to interindividual differences in the efficacy and toxicity of many medications. On the basis of functional genomics and molecular pharmacology, pharmacogenomics is elucidating the inherited nature of these differences in drug response mainly by means of analyzing the genetic variations of DNA and monitoring the gene expression pattern. It can not only improve disease diagnosis and predict the potential drug response but also speed up drug discovery and its development which will be instructive for clinical drug therapy ultimately.

Drug Therapy↗

Genome-wide association studies with experimental validation identify a protective role for B lymphocytes against chronic post-surgical pain.

BACKGROUND: Chronic post-surgical pain (CPSP) significantly impacts patients' recovery and quality of life. Although environmental risk factors are well-established, genetic risk remains less understood. METHODS: A meta-analysis of genome-wide association studies followed by partitioned heritability was performed on 1350 individuals across five surgery types: hysterectomy, mastectomy, abdominal, hernia, and knee. In subsequent animal studies, withdrawal thresholds to evoked mechanical stimulation were measured in Rag1 null mutant and wild-type mice after plantar incision and laparotomy. Cell sorting by flow cytometry tracked recruitment of immune cell types. RESULTS: We discovered 77 genome-wide significant single-nucleotide polymorphism (SNP) hits, distributed among 24 loci and 244 genes. Meta-analysis of all cohorts estimated a SNP-based narrow-sense heritability for CPSP at &#x223c;39%, indicating a substantial genetic contribution. Partitioned heritability analysis across a wide variety of tissues revealed enrichment of heritability in immune system-related genes, particularly those associated with B and T cells. Rag1 null mutant mice lacking both T and B cells exhibited exacerbated and prolonged allodynia up to 42 days after surgery, which was rescued by B-cell transfer. Recruitment patterns of B cells but not T cells differed significantly during the first 7 days after injury in the footpad, lymph nodes, and dorsal root ganglia. CONCLUSIONS: These findings suggest a key protective role for the adaptive immune system in the development of chronic post-surgical pain.

Animals↗

High-throughput mutation detection method to scan BRCA1 and BRCA2 based on heteroduplex analysis by capillary array electrophoresis.

BACKGROUND: Scanning for mutations in BRCA1 and BRCA2 in a large number of samples is hampered by the large sizes of these genes and the scattering of mutations throughout their coding sequences. Automated capillary electrophoresis has been shown to be a powerful system to detect mutations by either single-strand conformation polymorphism or heteroduplex analysis (HA). METHODS: We investigated the adaptation of gel-based HA of BRCA1 and BRCA2 to a fluorescent multicapillary platform to increase the throughput of this technique. We combined multiplex PCR, three different fluorescent labels, and HA in a 16-capillary DNA sequencer and tested 57 DNA sequence variants (11 insertions/deletions and 46 single-nucleotide changes) of BRCA1 and BRCA2. RESULTS: We detected all 57 DNA changes in a blinded assay, and 2 additional single-nucleotide substitutions (1186 A>G of BRCA1 and 3624 A>G of BRCA2), previously unresolved by conformation-sensitive gel electrophoresis. Furthermore, different DNA changes in the same PCR fragment could be distinguished by their peak patterns. CONCLUSIONS: Capillary-based HA is a fast, efficient, and sensitive method that considerably reduces the amount of "hands-on" time for each sample. By this approach, the entire coding regions of BRCA1 and BRCA2 from two breast cancer patients can be scanned in a single run of 90 min.

Autoanalysis↗

Polymorphic forms of expressed bovine interferon-tau genes: relative transcript abundance during early placental development, promoter sequences of genes and biological activity of protein products.

Multiple interferon (IFN)-tau genes exist in cattle, but it has remained unclear how many are expressed, the extent of their variation, and whether different genes exhibit similar patterns of expression and code for proteins with similar biological activities. A total of 118 complementary DNA (cDNA) were bi-directionally sequenced from reverse-transcribed bovine (bo) conceptus RNA over the period from blastocyst formation until day 25 of pregnancy. Fourteen different cDNAs, encoding eight different IFN-tau, were confirmed unique. All showed high sequence conservation (>98% nucleotide identity; >96% amino acid identity). The cDNA fell into three, recently evolved, phylogenetic groups (tau1, 2, and 3). Mean concentrations of IFN-tau messenger RNA were greater at day 17 and day 19 than at day 14 and day 25, with different genes showing comparable expression patterns, although there appeared to be a major bias in expression of two genes (for boIFN-tau1c and tau3a) in blastocysts. Genes representing members of the three boIFN-tau groups were cloned. Their promoter regions were conserved over regions considered important for transcriptional activation. Recombinant protein generated in Escherichia coli from representative genes in the three groups had similar but not identical antiviral activities. In summary, many IFN-tau genes, which are probably under similar transcriptional control, are expressed in bovine trophoblast during the peri-implantation period of development.

Amino Acid Sequence↗

Synergic effect of polymorphisms in ERCC6 5' flanking region and complement factor H on age-related macular degeneration predisposition.

This study investigates age-related macular degeneration (AMD) genetic risk factors through identification of a functional single-nucleotide polymorphism (SNP) and its disease association. We chose ERCC6 because of its roles in the aging process, DNA repair, and ocular degeneration from the gene disruption. Bioinformatics indicated a putative binding-element alteration on the sequence containing C-6530>G SNP in the 5' flanking region of ERCC6 from Sp1 on the C allele to SP1, GATA-1, and OCT-1 on the G allele. Electrophoretic mobility shift assays displayed distinctive C and G allele-binding patterns to nuclear proteins. Luciferase expression was higher in the vector construct containing the G allele than that containing the C allele. A cohort of 460 advanced AMD cases and 269 age-matched controls was examined along with pathologically diagnosed 57 AMD and 18 age-matched non-AMD archived cases. ERCC6 C-6530>G was associated with AMD susceptibility, both independently and through interaction with an SNP (rs380390) in the complement factor H (CFH) intron reported to be highly associated with AMD. A disease odds ratio of 23 was conferred by homozygozity for risk alleles at both ERCC6 and CFH compared with homozygozity for nonrisk alleles. Enhanced ERCC6 expression was observed in lymphocytes from healthy donors bearing ERCC6 C-6530>G alleles. Intense immunostaining of ERCC6 was also found in AMD eyes from ERCC6 C-6530>G carriers. The strong AMD predisposition conferred by the ERCC6 and CFH SNPs may result from biological epistasis, because ERCC6 functions in universal transcription as a component of RNA pol I transcription complex.

5' Flanking Region↗

Polymorphism at codons 114, 116, 145, and 163 muddle the typing of HLA-B*1304.

Genetic exchanges often muddle the typing of HLA class I molecules, this is also the case for HLA-B*1304. Serologic and molecular DNA class I typing report a B15/B55 type for cell 847, whereas DNA sequencing finds B*5501/B*1304. HLA-B*1304 differs by no more than four amino acids from other HLA-B13 molecules, a comparative analysis of the B13 and B15 families was therefore performed to determine why serologic and molecular DNA approaches report a B15 type for B*1304. Comparisons demonstrate that limited differences individuate the B15 and B13 molecules such that the genetic recombination of codons 145 and 163 in the class I heavy chain's alpha 2 alpha helix prompt B*1304 to exhibit a B15X21 pattern of serologic cross-reactivity. Molecular DNA class I typing approaches are also swayed by genetic recombinations to type B*1304 as a B15 molecule: B15-like nucleotide sequences encoding residues 114, 116, and 145, lead B*1304 to exhibit a B15 PCR amplification pattern. Thus, genetic exchanges encoding key amino acids in the class I heavy chain lead molecular and serologic typing approaches to categorize HLA-B*1304 as a member of the B15 family.

Alleles↗

HLA class II typing by digestion of PCR-amplified DNA with allele-specific restriction endonucleases will fail to unequivocally identify the genotypes of many homozygous and heterozygous individuals.

Recently, a new technique for HLA class II genotyping has been introduced, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, claimed to be a practical alternative to conventional serological and cellular HLA class II typing (1-3). The PCR-RFLP technique is ingenious, relatively rapid and does not require hybridization with sequence-specific oligonucleotide probes. However, analysis of whether homozygous and heterozygous combinations of PCR-RFLP patterns for the various investigated HLA class II loci are unique or not unfortunately shows that only 19% of DRB homozygous and heterozygous combinations are unique. The figures for the DQA1, DQB and DPB loci are 56%, 29% and 65%, respectively. As not all nucleotide sequences analyzed in the above-mentioned studies (1-3) gave rise to unique PCR-RFLPs and as more sequences now are known, for the DRB1, DRB3, DRB5 and DPB1 loci (4), the frequencies of unique PCR-RFLP patterns for the different HLA class II loci will be reduced even further. Thus, the present analysis demonstrates that the PCR-RFLP technique, performed as described in references 1-3, is not yet ready to be used for routine HLA class II genotyping. The resolution of the PCR-RFLP method can be improved by various modifications. However, the role of a modified PCR-RFLP technique in HLA class II typing still remains to be shown.

Alleles↗

Effectiveness of enterobacterial repetitive intergenic consensus PCR and random amplified polymorphic DNA fingerprinting for Helicobacter pylori strain differentiation.

We compared the robustness and discriminatory power of the enterobacterial repetitive intergenic consensus (ERIC) and random amplified polymorphic DNA (RAPD) fingerprinting methods for detecting cases of mixed Helicobacter pylori infection in Peruvian shantytown residents. H. pylori isolates from 63 participants were cultured, and five single colonies and a pool of additional colonies from each participant were analyzed by ERIC-PCR and by RAPD tests with four 10-nucleotide primers (one primer per reaction). There was 94% agreement between the ERIC and RAPD profiles in classifying sets of isolates as uniform versus closely related but not identical versus probably unrelated, indicating a high kappa statistic of 0.8942. Subtle differences in related ERIC or RAPD patterns likely reflect gene transfer between strains, recombination, and/or mutation, whereas markedly different patterns reflect infection by unrelated strains. At least half of infected shantytown residents seemed to carry more than one H. pylori strain, although in 19 of 31 persons, the strains were closely related. Three RAPD tests, each with a different primer, were needed to achieve the sensitivity of one ERIC test. ERIC-PCR constitutes a resource- and time-efficient method for H. pylori strain differentiation.

Bacterial Typing Techniques↗

Integrating biogeographic and genetic approaches to investigate the history of bioluminescent colour alleles in the Jamaican click beetle, Pyrophorus plagiophthalamus.

Bioluminescent colour in the Jamaican click beetle, Pyrophorus plagiophthalamus, is an ideal system for studies moving from gene to landscape to gain a holistic understanding of the molecular, ecological, and historical bases for adaptation. Previous studies have established the genetics of bioluminescent colour variation in the beetle to the level of the nucleotide base pair in the target gene luciferase. Three different luciferase colour alleles affecting ventral light organ colour [yellow-green (vYG), yellow (vYE), and orange (vOR)] were found segregating in P. plagiophthalamus populations. These alleles differ from each other in a number of replacement mutations (14 total), the majority of which (11) have a measurable effect on colour. Phylogenetic analysis revealed a long-term adaptive trend on Jamaica towards longer wavelength bioluminescence, culminating in the most recently derived vOR allele. Here, we further investigate the historical and geographic context of adaptive colour evolution by testing a vicariance model for the origins of the extant ventral light organ polymorphism: that the vOR allele arose and differentiated in an isolated deme on the east side of Jamaica before spreading westward. Comparisons of colour phenotypes, luciferase coding sequences, the third intron of the gene, mtDNA, and microsatellite data provided evidence for past population subdivision on Jamaica and ongoing gene flow, as has been found for other island endemics. However, the pattern of differentiation supported the allopatric divergence of vYG and vYE alleles. The vOR gene appears to have arisen relatively recently from a vYE precursor and postdates the period of major biogeographic isolation. We discuss the implications of the results for discerning ecological causation in the adaptive sequence from nucleotide to landscape to population change for bioluminescent colour.

Adaptation, Physiological↗

Contribution of the LRP5 gene to normal variation in peak BMD in women.

UNLABELLED: The role of the LRP5 gene in rare BMD-related traits has recently been shown. We tested whether variation in this gene might play a role in normal variation in peak BMD. Association between SNPs in LRP5 and hip and spine BMD was measured in 1301 premenopausal women. Only a small proportion of the BMD variation was attributable to LRP5 in our sample. INTRODUCTION: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene have been implicated as the cause of multiple distinct BMD-related rare Mendelian phenotypes. We sought to examine whether the LRP5 gene contributes to the observed variation in peak BMD in the normal population. MATERIALS AND METHODS: We genotyped 12 single nucleotide polymorphisms (SNPs) in LRP5 using allele-specific PCR and mass spectrometry methods. Linkage disequilibrium between the genotyped LRP5 SNPs was measured. We tested for association between these SNPs and both hip and spine BMD (adjusted for age and body weight) in 1301 healthy premenopausal women who took part in a sibling pair study aimed at identifying the genes underlying peak bone mass. Our study used both population-based (ANOVA) and family-based (quantitative transmission disequilibrium test) association methodology. RESULTS AND CONCLUSIONS: The linkage disequilibrium pattern and haplotype block structure within the LRP5 gene were consistent with that observed in other studies. Although significant evidence of association was found between LRP5 SNPs and both hip and spine BMD, only a small proportion of the total variation in these phenotypes was accounted for. The genotyped SNPs accounted for approximately 0.8% of the variation in femoral neck BMD and 1.1% of the variation in spine BMD. Results from our sample suggest that natural variation in and around LRP5 is not a major contributor to the observed variability in peak BMD at either the femoral neck or lumbar spine in white women.

Adult↗

Cloning, mapping and expression analysis of barley MADS-box genes.

Six MADS-box cDNA clones were isolated by heterologous screening from a barley inflorescence cDNA library. Based on sequence comparison to known MADS-box genes, the barley MADS-box (BM) genes were grouped into three distinct phylogenetic subclasses of the MADS-box gene family. The three MADS-box genes BM3, BM5 and BM8 share similarities with genes of the SQUAMOSA (SQUA) subgroup, while BM7 and BM9 belong to the AGAMOUS-LIKE 2 (AGL2) subgroup. BM1 resembles MADS-box genes described as solitary sequences or orphan genes. Expression analysis of the barley MADS-box genes revealed expression patterns that are not characteristic of the barley MADS-box genes of the SQUA subgroup. while expression of BM7 and BM9 was largely as expected for the AGL2 subgroup. BM1 is mainly expressed in vegetative tissues and its primary transcript undergoes alternative splicing such that the corresponding mRNAs differ by two codons. The genes BM1, BM3 and BM8 were mapped by analysis of single-nucleotide polymorphisms onto barley chromosomes 4, 2 and 7, respectively.

Alternative Splicing↗

Allelic diversity of S-RNase at the self-incompatibility locus in natural flowering cherry populations (Prunus lannesiana var. speciosa).

In the Rosaceae family, which includes Prunus, gametophytic self-incompatibility (GSI) is controlled by a single multiallelic locus (S-locus), and the S-locus product expressed in the pistils is a glycoprotein with ribonuclease activity (S-RNase). Two populations of flowering cherry (Prunus lannesiana var. speciosa), located on Hachijo Island in Japan's Izu Islands, were sampled, and S-allele diversity was surveyed based on the sequence polymorphism of S-RNase. A total of seven S-alleles were cloned and sequenced. The S-RNases of flowering cherry showed high homology to those of Prunus cultivars (P. avium and P. dulcis). In the phylogenetic tree, the S-RNases of flowering cherry and other Prunus cultivars formed a distinct group, but they did not form species-specific subgroups. The nucleotide substitution pattern in S-RNases of flowering cherry showed no excess of nonsynonymous substitutions relative to synonymous substitutions. However, the S-RNases of flowering cherry had a higher Ka/Ks ratio than those of other Prunus cultivars, and a subtle heterogeneity in the nucleotide substitution rates was observed among the Prunus species. The S-genotype of each individual was determined by Southern blotting of restriction enzyme-digested genomic DNA, using cDNA for S-RNase as a probe. A total of 22 S-alleles were identified. All individuals examined were heterozygous, as expected under GSI. The allele frequencies were, contrary to the expectation under GSI, significantly unequal. The two populations studied showed a high degree of overlap, with 18 shared alleles. However, the allele frequencies differed considerably between the two populations.

Alleles↗

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed.

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.

Animals↗