Genomic signatures of selection associated with benzimidazole drug treatments in Haemonchus contortus field populations

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how best to detect them. This study addresses these knowledge gaps by using field populations of Haemonchus contortus with well-defined benzimidazole treatment histories, leveraging a recently completed chromosomal-scale reference genome assembly. We generated a panel of 49,393 genomic markers to genotype 20 individual adult worms from each of four H. contortus populations: two from closed sheep flocks with an approximate 20 year history of frequent benzimidazole treatment, and two populations with a history of little or no treatment. Sampling occurred in the same geographical region to limit genetic differentiation and maximise the detection sensitivity. A clear signature of selection was detected on chromosome I, centred on the isotype-1 β-tubulin gene. Two additional, but weaker, signatures of selection were detected; one near the middle of chromosome I spanning 3.75 Mbp and 259 annotated genes, and one on chromosome II spanning a region of 3.3 Mbp and 206 annotated genes, including the isotype-2 β-tubulin locus. We also assessed how sensitivity was impacted by sequencing depth, worm number, and pooled versus individual worm sequence data. This study provides the first known direct genome-wide evidence for any parasitic nematode, that the isotype-1 β-tubulin gene is quantitatively the single most important benzimidazole resistance locus. It also identified two additional genomic regions that likely contain benzimidazole resistance loci of secondary importance. This study provides an experimental framework to maximise the power of genome-wide approaches to detect signatures of selection driven by anthelmintic drug treatments in field populations of parasitic nematodes.     

Epichloe canadensis, a new interspecific epichloid hybrid symbiotic with Canada wildrye (Elymus canadensis)

Many Epichlo? endophytes found in cool-season grasses are interspecific hybrids possessing much or all of the genomes of two or three progenitors. Here we characterize Epichlo? canadensis sp. nov., a hybrid species inhabiting the grass species Elymus canadensis native to North America. Three distinct morphotypes were identified that were separated into two groups by molecular phylogenetic analysis. Sequence analysis of the translation elongation factor 1-α (tefA) and β-tubulin (tubB) genes revealed two copies in all isolates examined. Phylogenetic analyses indicated that allele 1 of each gene was derived from Epichlo? amarillans and allele 2 from Epichlo? elymi. This is the first documentation of an interspecific hybrid endophyte derived from parents of strictly North American origins. Alkaloid gene profiling using primers specific to genes in the peramine, loline, indole-diterpene and ergot alkaloid pathways may indicate chemotypic variation in the ergot alkaloid and loline pathways between the assigned morphotypes. All isolates have the gene enabling the production of peramine but lack genes in the indole-diterpene biosynthesis pathway. Morphology and phylogenetic evidence support the designation of isolates from El. canadensis as a new interspecific hybrid species.     

Tubulins in Aspergillus nidulans

The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.     

Isolation of the β-tubulin gene from yeast and demonstration of its essential function in vivo

A DNA fragment from yeast (Saccharomyces cerevisiae) was identified by its homology to a chicken β-tubulin cDNA and cloned. The fragment was shown to be unique in the yeast genome and to contain the gene for yeast β-tubulin, since it can complement a benomyl-resistant conditional-lethal mutation. A smaller subfragment, when used to direct integration of a plasmid to the benomyl resistance locus in a diploid cell, disrupted one of the β-tubulin genes and concomitantly created a recessive lethal mutation, indicating that the single β-tubulin gene of yeast has an essential function. Determination of the nucleotide sequence reveals extensive amino acid sequence homology (more than 70%) between yeast and chicken brain β-tubulins.     

Benzimidazole resistance allele haplotype diversity in United Kingdom isolates of Teladorsagia circumcincta supports a hypothesis of multiple origins of resistance by recurrent mutation

Polymorphisms in the isotype I β-tubulin gene are important genetic determinants of benzimidazole (BZ) resistance in a number of parasitic nematode species including Teladorsagia circumcincta, a major gastrointestinal nematode of sheep. This study investigates the genetic diversity at this locus in a BZ-resistant isolate of T. circumcincta (MTci5) derived from a sheep farm in the United Kingdom (UK) that was open to animal, and therefore parasite, migration. Pyrosequencing was used to determine the frequency of single nucleotide polymorphisms (SNPs) known to be associated with BZ resistance. This was followed by a combination of single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing to sample allelic diversity in a 276 bp fragment immediately surrounding the isotype I β-tubulin F200Y mutation. The genetic diversity at this locus was extremely high, with seven different haplotypes found to contain the resistant F200Y polymorphism in this single resistant isolate. Genotyping by SSCP interfaced with pyrosequencing demonstrated that the P200^Y mutation is also present on multiple haplotypes in two other BZ-resistant T. circumcincta isolates from the UK. This contrasts with much lower levels of haplotype diversity in BZ-resistant alleles present in T. circumcincta isolates from French goat farms that are closed to parasite migration. Taken together with our knowledge of T. circumcincta population genetic structure, these results are most consistent with multiple independent origins of resistance and mixing of alleles due to the large amount of livestock movement in the UK.     

20-Hydroxyecdysone induces the expression of one β-tubulin gene in Drosophila Kc cells

The expression of 56D and 60C β-tubulin genes has been examined in Drosophila melanogaster Kc cells in response to the insect moulting hormone, 20-hydroxyecdysone (20-OH-E). Northern blots probed with β-tubulin subclones show that the 56D β-tubulin gene encodes a 1.8 kb mRNA whose abundance is not affected by 20-OH-E. The 60C gene probe detects two mRNAs: one of 1.8 kb present in untreated and 20-OH-E-treated cells, and one of 2.6 kb present only in 20-OH-E-treated cells; using a 60C 3′-specific probe, only the 2.6 kb is revealed. Hybrid selection translation experiment demonstrates that a 20-OH-E-inducible mRNA homologous to the 60C gene encodes a β-tubulin subunit (P4); this subunit is the so-called β3-tubulin. Translation of size-fractionated mRNA shows that the 20-OH-E-induced β3-tubulin subunit is encoded, in treated cells, by the 2.6 kb mRNA.     

Identification of a 4-coumarate:CoA ligase gene family in the moss, Physcomitrella patens

Since the early evolution of land plants from primitive green algae, phenylpropanoid compounds have played an important role. In the biosynthesis of phenylpropanoids, 4-coumarate:CoA ligase (4CL; EC 6.2.1.12) has a pivotal role at the divergence point from general phenylpropanoid metabolism to several major branch pathways. Although higher plant 4CLs have been extensively studied, little information is available on the enzymes from bryophytes. In Physcomitrella patens, we have identified a 4CL gene family consisting of four members, taking advantage of the available EST sequences and a draft sequence of the P. patens genome. The encoded proteins of three of the genes display similar substrate utilization profiles with highest catalytic efficiency towards 4-coumarate. Interestingly, the efficiency with cinnamate as substrate is in the same range as with caffeate and ferulate. The deduced proteins of the four genes share sequence identities between 78% and 86%. The intron/exon structures are pair wise similar. Pp4CL2 and Pp4CL3 each consists of four exons and three introns, whereas Pp4CL1 and Pp4CL4 are characterized each by five exons and four introns. Pp4CL1, Pp4CL2 and Pp4CL3 are expressed in both gametophore and protonema tissue of P. patens, unlike Pp4CL4 whose expression could not be demonstrated under the conditions employed. Phylogenetic analysis suggests an early evolutionary divergence of Pp4CL gene family members. Using Streptomyces coelicolor cinnamate:CoA ligase (ScCCL) as an outgroup, the P. patens 4CLs are clearly separated from the spermatophyte proteins, but are intercalated between the angiosperm 4CL class I and class II. A comparison of three P. patens subspecies from diverse geographical locations shows high sequence identities for the four 4CL isoforms.     

Crystal structure of tubulin folding cofactor A from Arabidopsis thaliana and its β-tubulin binding characterization

Microtubules are composed of polymerized α/β-tubulin heterodimers. Biogenesis of assembly-competent tubulin dimers is a complex multistep process that requires sequential actions of distinct molecular chaperones and cofactors. Tubulin folding cofactor A (TFCA), which captures β-tubulin during the folding pathway, has been identified in many organisms. Here, we report the crystal structure of Arabidopsis thaliana TFC A (KIESEL, KIS), which forms a monomeric three-helix bundle. The functional binding analysis demonstrated that KIS interacts with β-tubulin in plant. Furthermore, mutagenesis studies indicated that the α-helical regions of KIS participate in β-tubulin binding. Unlike the budding yeast TFC A, the two loop regions of KIS are not required for this interaction suggesting a distinct binding mechanism of TFC A to β-tubulin in plants.

Structured summary

MINT-7968902, MINT-7968915, MINT-7968951, MINT-7968966: KIS (uniprotkb:O04350) physically interacts (MI:0915) with Tub9 (uniprotkb:P29517) by anti tag coimmunoprecipitation (MI:0007)MINT-7968928: KIS (uniprotkb:O04350) and Tub9 (uniprotkb:P29517) physically interact (MI:0915) by bimolecular fluorescence complementation (MI:0809)     

Either alpha-tubulin isogene product is sufficient for microtubule function during all stages of growth and differentiation in Aspergillus nidulans.

The filamentous fungus Aspergillus nidulans has two genes encoding alpha-tubulin, tubA and tubB, which are differentially required at distinct stages during the life cycle. The tubA gene is required during vegetative growth for mitosis and nuclear migration (B. R. Oakley, C. E. Oakley, and J. E. Rinehart, Mol. Gen. Genet. 208:135-144, 1987; P. Doshi, C. A. Bossie, J. H. Doonan, G. S. May, and N. R. Morris, Mol. Gen. Genet. 225:129-141, 1991). The tubB gene is not required for any detectable aspect of vegetative growth or asexual reproduction but is essential during sexual development prior to the first meiotic division (K. E. Kirk and N. R. Morris, Genes Dev. 5:2014-2023, 1991). In this study, we determined whether the role of each alpha-tubulin gene is to provide a specific isotype necessary for a particular microtubule function or whether either alpha-tubulin isotype, if present in sufficient quantities, can participate effectively in all types of microtubule. Strains carrying a deletion allele of tubB (tubB delta) produce no ascospores from a cross. When one copy of a plasmid containing the region upstream of the tubB gene fused to the tubA coding region was integrated into a tubB delta strain, ascosporogenesis proceeded beyond the tubB delta block and resulted in the formation of sexual spores. However, irregular numbers of spores formed in some asci during development, and the ascospores had greatly diminished viability and aberrant morphologies. These defects were nearly corrected when two additional copies of the tubA coding region were integrated into the tubB delta strain. These results indicate that the tubA alpha-tubulin isotype can form functional microtubules during sexual development in the absence of tubB protein. In a reciprocal set of experiments, we examined whether upregulation of tubB can complement the tubA4 mutation, which causes supersensitivity to benomyl during vegetative growth. When tubA4 strains integrated a plasmid containing an alcohol-inducible promoter joined to the tubB coding region and subsequently overexpressed the tubB isotype, the benomyl supersensitivity normally caused by the tubA4 allele was relieved. These results indicate that when enough tubB alpha-tubulin is supplied, strains lacking functional tubA isotype can still form microtubules which effectively carry out mitosis and nuclear migration.     

Molecular Characterization of β-tubulin gene from Pleurotus sajor-caju

A β-tubulin gene (TUB1) from the basidiomycete Pleurotus sajor-caju was sequenced. TUB1 encodes a 446-amino-acid protein. The coding region is interrupted by 9 introns, all of which had a 5'-GTRNGT…YAG-3' sequence at the boundaries. Locations of the introns in TUB1 were common between the β-tubulin genes of other basidiomycetes, but not with animals, ascomycetes, or plants. This suggests that the introns were inserted independently into the β-tubulin gene after these divisions had diverged.     

牧草盲蝽实时定量PCR内参基因的筛选

【目的】筛选出适合牧草盲蝽Lygus pratensis在不同条件下稳定表达的内参基因。【方法】选取编码牧草盲蝽α-微管蛋白、β-微管蛋白、肌动蛋白、延伸因子、琥珀酸脱氢酶复合体A、泛素、转录起始因子TFIID亚基、谷胱甘肽S-转移酶、核糖体蛋白L32及TATA盒结合蛋白的10条基因为候选内参基因,采用qRT-PCR技术测定其在牧草盲蝽不同性别成虫、成虫不同组织、不同龄期、对功夫菊酯不同抗性成虫、不同温度及不同杀虫剂分别处理后的成虫共6类样品中的表达量;采用BestKeeper, geNorm, NormFinder及RefFinder 4种计算程序对各候选基因的表达稳定性进行评价。【结果】依据获得的有效qRT-PCR数据前提,利用不同计算方法综合分析得出：在牧草盲蝽成虫不同组织中和对功夫菊酯不同抗性成虫中最稳定表达的内参基因均为RPL32;不同温度处理、不同性别成虫中、不同杀虫剂处理和不同龄期牧草盲蝽中最稳定表达的内参基因分别为UBQ, SDHA, β-tubulin和TAF。通过geNorm软件判断配对变异数确定的内参基因最佳数目,结合RefFinder对基因表达稳定性综合排序的结果可得：牧草盲蝽不同成虫组织中、不同性别成虫中、不同龄期、对功夫菊酯不同抗性成虫中、不同温度及杀虫剂分别处理的成虫等各组的最优内参基因组合分别为RPL32+TAF, SDHA+GST, TAF+GST+UBQ, RPL32+GST, UBQ+ACT+β-tubulin和β-tubulin+TAF+RPL32。【结论】本研究获得的牧草盲蝽在不同条件下表达最稳定及最适内参基因组合,都可用于后续的基因定量表达研究。     

Evaluation of ��-Tubulin as an Antigenic and Molecular Probe to Detect Giardia lamblia

The α/β-tubulin heterodimer is the basic subunit of microtubules in eukaryotes. Polyclonal antibodies specific to recombinant α-tubulin of Giardia lamblia were made, and found effective as a probe to specifically detect G. lamblia by immunofluorescence assays. Nucleotide sequences of α-tubulin genes were compared between G. lamblia WB and GS strains, prototypes of assemblage A and assemblage B, respectively. A set of primers was designed and used to amplify a portion of the α-tubulin gene from G. lamblia. PCR-RFLP analysis of this α-tubulin PCR product successfully differentiated G. lamblia into 2 distinct groups, assemblages A and B. The results indicate that α-tubulin can be used as a molecular probe to detect G. lamblia.