Identification of Y-chromosome scaffolds of the Queensland fruit fly reveals a duplicated gyf gene paralogue common to many Bactrocera pest species.

Bactrocera tryoni (Queensland fruit fly) are polyphagous horticultural pests of eastern Australia. Heterogametic males contain a sex-determining Y-chromosome thought to be gene poor and repetitive. Here, we report 39 Y-chromosome scaffolds (~700 kb) from B. tryoni identified using genotype-by-sequencing data and whole-genome resequencing. Male diagnostic PCR assays validated eight Y-scaffolds, and one (Btry4096) contained a novel gene with five exons that encode a predicted 575 amino acid protein. The Y-gene, referred to as typo-gyf, is a truncated Y-chromosome paralogue of X-chromosome gene gyf (1773 aa). The Y-chromosome contained ~41 copies of typo-gyf, and expression occurred in male flies and embryos. Analysis of 13 tephritid transcriptomes confirmed typo-gyf expression in six additional Bactrocera species, including Bactrocera latifrons, Bactrocera dorsalis and Bactrocera zonata. Molecular dating estimated typo-gyf evolved within the past 8.02 million years (95% highest posterior density 10.56-5.52 million years), after the split with Bactrocera oleae. Phylogenetic analysis also highlighted complex evolutionary histories among several Bactrocera species, as discordant nuclear (116 genes) and mitochondrial (13 genes) topologies were observed. B. tryoni Y-sequences may provide useful sites for future transgene insertions, and typo-gyf could act as a Y-chromosome diagnostic marker for many Bactrocera species, although its function is unknown.

Structure and expression of sulfatase and sulfatase modifying factor genes in the diamondback moth, Plutella xylostella.

The diamondback moth, Plutella xylostella (L.), uses sulfatases (SULF) to counteract the glucosinolate-myrosinase defensive system that cruciferous plants have evolved to deter insect feeding. Sulfatase activity is regulated by post-translational modification of a cysteine residue by sulfatase modifying factor 1 (SUMF1). We identified 12 SULF genes (PxylSulfs) and two SUMF1 genes (PxylSumf1s) in the P. xylostella genome. Phylogenetic analysis of SULFs and SUMFs from P. xylostella, Bombyx mori, Manduca sexta, Heliconius melpomene, Danaus plexippus, Drosophila melanogaster, Tetranychus urticae and Homo sapiens showed that the SULFs were clustered into five groups, and the SUMFs could be divided into two groups. Profiling of the expression of PxylSulfs and PxylSumfs by RNA-seq and by quantitative real-time polymerase chain reaction showed that two glucosinolate sulfatase genes (GSS), PxylSulf2 and PxylSulf3, were primarily expressed in the midgut of 3rd- and 4th-instar larvae. Moreover, expression of sulfatases PxylSulf2, PxylSulf3 and PxylSulf4 were correlated with expression of the sulfatases modifying factor PxylSumf1a. The findings from this study provide new insights into the structure and expression of SUMF1 and PxylSulf genes that are considered to be key factors for the evolutionary success of P. xylostella as a specialist herbivore of cruciferous plants.

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor.

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins' mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD50 concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.

Genome-wide patterns of divergence and gene flow across a butterfly radiation.

The Heliconius butterflies are a diverse recent radiation comprising multiple levels of divergence with ongoing gene flow between species. The recently sequenced genome of Heliconius melpomene allowed us to investigate the genomic evolution of this group using dense RAD marker sequencing. Phylogenetic analysis of 54 individuals robustly supported reciprocal monophyly of H. melpomene and Heliconius cydno and refuted previous phylogenetic hypotheses that H. melpomene may be paraphylectic with respect to H. cydno. Heliconius timareta also formed a monophyletic clade closely related but distinct from H. cydno with Heliconius heurippa falling within this clade. We find evidence for genetic admixture between sympatric populations of the sister clades H. melpomene and H. cydno/timareta, particularly between H. cydno and H. melpomene from Central America and between H. timareta and H. melpomene from the eastern slopes of the Andes. Between races, divergence is primarily explained by isolation by distance and there is no detectable genetic population structure between parapatric races, suggesting that hybrid zones between races are not zones of secondary contact. Our results also support previous findings that colour pattern loci are shared between populations and species with similar colour pattern elements. Furthermore, this pattern is almost unique to these genomic regions, with only a very small number of other loci showing significant similarity between populations and species with similar colour patterns.

The HER2 I655V polymorphism and risk of breast cancer in women < age 40 years.

The HER2 gene controls cellular function and has prognostic significance in breast cancer. The I655V polymorphism was associated with increased risk of breast cancer in Chinese women under the age of 45 years and in women with a first-degree family history of the disease. These associations, however, have not been confirmed in several studies of older women. We conducted a population-based case-control-family study of the I655V polymorphism using 409 Australian women with breast cancer diagnosed before the age of 40 years and 299 controls frequency matched for age. The I655V polymorphism was more common in cases (P = 0.01). A recessive model, in which homozygotes were associated with an adjusted odds ratio of 2.8 (95% confidence interval 1.3-6.2; P = 0.005), gave the best fit under parsimony. Although the biological role of the I655V polymorphism is not known, large independent studies of early onset breast cancer are warranted to attempt to replicate this finding.

Genetic analysis of benign ovarian tumors.

Ovarian cancer represents a major cause of cancer death among women and yet remarkably little is known about its etiology. The paradigm established by the colorectal carcinogenesis model would suggest that ovarian cancers are likely to arise through malignant transformation of benign ovarian tumors. However, molecular genetic data that could answer this important question is lacking. In our study, we analyzed 80 benign ovarian tumors for TP53 and K-ras mutations and for LOH on chromosomes 6, 7, 9, 11 and 17 using 56 microsatellite markers. Twenty-five percent (5/20) of non-epithelial tumors and 73% (44/60) of epithelial tumors exhibited LOH on at least 1 chromosome arm. A particularly high frequency of LOH was detected among the epithelial tumors on chromosome arms 6q (17%), 7p (17%), 7q (27%) and 11p (18%), which are also regions of frequent LOH among ovarian carcinomas. No K-ras mutations were detected in any tumor but somatic TP53 mutations were detected in 2/34 (6%) serous and 1/26 (4%) mucinous epithelial tumors. In contrast to most previous studies our data is derived from a relatively large number of microdissected tumors and is likely to represent a more accurate picture of the frequency of alterations in these tumors. We conclude that LOH is common in benign ovarian tumors, suggesting that inactivation of tumor suppressor genes are pivotal in their development. The high frequency of alterations is consistent with their being precursors to malignant disease but does not unequivocally prove this continuum. It does however provide a framework for future analysis of the molecular genetic etiology of ovarian tumorigenesis.

Methylenetetrahydrofolate reductase polymorphism and susceptibility to breast cancer.

BACKGROUND: A growing body of evidence suggests that variations in the levels of folate may contribute to the development of cancer. A functional polymorphic variant (C-->T substitution at nucleotide 677) in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene results in the conversion of an alanine to a valine and may modify the risk of breast and other cancers. METHOD: We have investigated the possible influence of this MTHFR variant on breast cancer risk in a case-control study of 233 healthy women and 335 women who had breast cancer that occurred under the age of 40 years, bilateral breast cancer or a family history of breast cancer. RESULTS: A significant excess of the valine genotypes was observed among the cases (odds ratio 1.43, 95% confidence interval 1.02-2.00). The effect was more pronounced among the cases with a breast cancer diagnosis under the age of 40 years, with an odds ratio of 1.66 (95% confidence interval 1.12-2.41). A nonsignificant excess of the valine genotypes was observed among the cases with a family history of breast cancer or bilateral breast cancer. CONCLUSIONS: The low activity C677T (valine) genotype of MTHFR may increase the risk of early onset breast cancer.

Mutation analysis of EP300 in colon, breast and ovarian carcinomas.

The putative tumour suppressor gene EP300 is located on chromosome 22q13 which is a region showing frequent loss of heterozygosity (LOH) in colon, breast and ovarian cancers. We analysed 203 human breast, colon and ovarian primary tumours and cell lines for somatic mutations in EP300. LOH across the EP300 locus was detected in 38% of colon, 36% of breast, and 49% of ovarian primary tumours but no somatic mutations in EP300 were identified in any primary tumour. Analysis of 17 colon, 11 breast, and 11 ovarian cancer cell lines identified truncating mutations in 4 colon cancer cell lines (HCT116, HT29, LIM2405 and LIM2412). We confirmed the presence of a previously reported frameshift mutation in HCT116 at codon 1699 and identified a second frameshift mutation at codon 1468. Bi-allelic inactivation of EP300 was also detected in LIM2405 that harbours an insC mutation at codon 927 as well an insA mutation at codon 1468. An insA mutation at codon 1468 was identified in HT29 and a CGA>TGA mutation at codon 86 was identified in LIM2412. Both these lines were heterozygous across the EP300 locus and western blot analysis confirmed the presence of an apparently wild-type protein. Our study has established that genetic inactivation of EP300 is rare in primary colorectal, breast and ovarian cancers. In contrast, mutations are common among colorectal cancer cell lines with 4/17 harbouring homozygous or heterozygous mutations. The rarity of EP300 mutations among these tumour types that show a high frequency of LOH across 22q13 may indicate that another gene is the target of the loss. It is possible that bi-allelic inactivation of EP300 is not necessary and that haploinsufficiency is sufficient to promote tumorigenesis. Alternatively, silencing of EP300 may be achieved by epigenetic mechanisms such as promoter methylation.