BACKGROUND: The mountain pine beetle, Dendroctonus ponderosae Hopkins, is the most serious insect pest of western North American pine forests. A recent outbreak destroyed more than 15 million hectares of pine forests, with major environmental effects on forest health, and economic effects on the forest industry. The outbreak has in part been driven by climate change, and will contribute to increased carbon emissions through decaying forests. RESULTS: We developed a genome sequence resource for the mountain pine beetle to better understand the unique aspects of this insect's biology. A draft de novo genome sequence was assembled from paired-end, short-read sequences from an individual field-collected male pupa, and scaffolded using mate-paired, short-read genomic sequences from pooled field-collected pupae, paired-end short-insert whole-transcriptome shotgun sequencing reads of mRNA from adult beetle tissues, and paired-end Sanger EST sequences from various life stages. We describe the cytochrome P450, glutathione S-transferase, and plant cell wall-degrading enzyme gene families important to the survival of the mountain pine beetle in its harsh and nutrient-poor host environment, and examine genome-wide single-nucleotide polymorphism variation. A horizontally transferred bacterial sucrose-6-phosphate hydrolase was evident in the genome, and its tissue-specific transcription suggests a functional role for this beetle. CONCLUSIONS: Despite Coleoptera being the largest insect order with over 400,000 described species, including many agricultural and forest pest species, this is only the second genome sequence reported in Coleoptera, and will provide an important resource for the Curculionoidea and other insects.
Bark beetles (Coleoptera: Curculionidae: Scolytinae) are major insect pests of many woody plants around the world. The mountain pine beetle (MPB), Dendroctonus ponderosae Hopkins, is a significant historical pest of western North American pine forests. It is currently devastating pine forests in western North America--particularly in British Columbia, Canada--and is beginning to expand its host range eastward into the Canadian boreal forest, which extends to the Atlantic coast of North America. Limited genomic resources are available for this and other bark beetle pests, restricting the use of genomics-based information to help monitor, predict, and manage the spread of these insects. To overcome these limitations, we generated comprehensive transcriptome resources from fourteen full-length enriched cDNA libraries through paired-end Sanger sequencing of 100,000 cDNA clones, and single-end Roche 454 pyrosequencing of three of these cDNA libraries. Hybrid de novo assembly of the 3.4 million sequences resulted in 20,571 isotigs in 14,410 isogroups and 246,848 singletons. In addition, over 2300 non-redundant full-length cDNA clones putatively containing complete open reading frames, including 47 cytochrome P450s, were sequenced fully to high quality. This first large-scale genomics resource for bark beetles provides the relevant sequence information for gene discovery; functional and population genomics; comparative analyses; and for future efforts to annotate the MPB genome. These resources permit the study of this beetle at the molecular level and will inform research in other Dendroctonus spp. and more generally in the Curculionidae and other Coleoptera.
Missense mutations in exon 5 of the LPL gene are the most common reported cause of LPL deficiency. Exon 5 is also the region with the strongest homology to pancreatic and hepatic lipase, and is conserved in LPL from different species. Mutant LPL proteins from post-heparin plasma from patients homozygous for missense mutations at amino acid positions 176, 188, 194, 205, and 207, and from COS cells transiently transfected with the corresponding cDNAs were quantified and characterized, in an attempt to determine which aspect of enzyme function was affected by each specific mutation. All but one of the mutant proteins were present, mainly as partially denatured LPL monomer, rendering further detailed assessment of their catalytic activity, affinity to heparin, and binding to lipoprotein particles difficult. However, the fresh unstable Gly(188)-->Glu LPL and the stable Ile(194)-->Thr LPL, although in native conformation, did not express lipase activity. It is proposed that many of the exon 5 mutant proteins are unable to achieve or maintain native dimer conformation, and that the Ile(194)-->Thr substitution interferes with access of lipid substrate to the catalytic pocket. These results stress the importance of conformational evaluation of mutant LPL. Absence of catalytic activity does not necessarily imply that the substituted amino acid plays a specific direct role in catalysis.
Title: A novel Glu421Lys substitution in the lipoprotein lipase gene in pregnancy-induced hypertriglyceridemic pancreatitis Henderson H, Leisegang F, Hassan F, Hayden M, Marais D Ref: Clinica Chimica Acta, 269:1, 1998 : PubMed
Severe hypertriglyceridemia is an uncommon pathological finding in pregnant women if there is no prior history of hyperlipidemia. A partial reduction in lipoprotein lipase (LPL) activity due to a mutation in the LPL gene, is often an associating factor. Here we report a novel LPL gene mutation (Glu421Lys), in a previously healthy primigravid woman who died from hypertriglyceridemia-induced pancreatitis during the last trimester of pregnancy. The patient was heterozygous for this mutation which a charge inversion in the C-terminal domain of LPL resulting in a moderate reduction in catalytic activity, both in vivo and in vitro. These data support the role of partial LPL deficiency in the pathogenesis of severe gestational hypertriglyceridemia.
Two mutations in the lipoprotein lipase (LPL) gene, a T to G transition at position -93 of the proximal promoter region and an Asp9Asn substitution in exon 2, were examined in 762 Dutch males with angiographically-diagnosed coronary artery disease (CAD) and 296 healthy normolipidemic Dutch males. The two mutations exhibited strong linkage disequilibrium (D' = 0.975). A significantly higher proportion of cases (4.86%) than controls (1.37%) carried the -93G/Asn9 allele (p = 0.008). In the combined sample of cases and controls, adjusted mean plasma total cholesterol (TC) levels were significantly higher in -93G/Asn9 carriers (6.20+/-0.13 mmol/l) than in non-carriers (5.93+/-0.03 mmol/l; p = 0.048), while mean high-density lipoprotein cholesterol (HDL-C) levels were lower in carriers (0.88+/-0.03 mmol/l) than in non-carriers (0.98+/-0.01 mmol/l; p = 0.002). There was a trend towards higher triglyceride (TG) levels in carriers (1.96+/-0.14 mmol/l) compared with non-carriers (1.73+/-0.03 mmol/l) (p = 0.08). Additionally, carrier frequencies in tertiles of TC, HDL-C, TG, and LPL activity, suggested an association of the -93G/Asn9 variant with higher TC and TG levels, and with lower HDL-C and LPL activity levels. Logistic regression revealed a significant odds ratio (OR) for the combined -93G/Asn9 genotype in CAD cases relative to controls (OR: 5.36; 95% CI: 1.57-18.24), with age, body mass index (BMI), smoking, and plasma total- and HDL-cholesterol levels included in the model. In conclusion, we show that the LPL Asp9Asn mutation is in non-random association with a T G substitution at position -93 of the proximal promoter region and that the combined -93G/Asn9 genotype predisposes to decreased HDL-C levels and an increased risk of CAD.
Lipoprotein lipase (LPL) plays a crucial role in the regulation of lipoprotein metabolism by hydrolysing the core triglycerides of circulating chylomicrons and VLDL. Human, bovine, mouse, and guinea pig complementary DNA clones have recently been isolated and the organization of the human LPL gene is now known to comprise 10 exons spanning approximately 30 kb. Here we report a similar mutation on 21 alleles from 13 unrelated affected probands with LPL deficiency of French Canadian, English, Polish, German, Dutch, and East Indian ancestry. We show that an identical missense mutation within exon 5, resulting in an amino acid substitution of glutamic acid for glycine at position 188, is responsible for LPL deficiency in 21 of 88 LPL alleles assessed. This mutation alters an Ava II restriction site in exon 5 and will allow a rapid screening test for this mutation in patients with LPL deficiency. This mutation has occurred on the same haplotype in all the unrelated affected persons suggesting a common origin. The amino acid substitution lies within the longest segment of homology for LPL in different species and results in a protein that is catalytically defective.
Title: The molecular biology of hypertriglyceridemia: characterization of mutations in patients with lipoprotein lipase deficiency. (Abstract) Devlin R, Henderson H, Monsalve V, Brunzell J, Deeb S, Hayden MR Ref: American Journal of Human Genetics, 45 (suppl.):A4, 1989 : PubMed
Lipoprotein lipase (LPL) is a crucial enzyme in the hydrolysis of triglyceride-rich lipoproteins liberating free fatty acids for storage or energy production in the cell. Human LPL deficiency is associated with massive hypertriglyceridemia and presents usually in childhood with abdominal pain, pancreatitis and occasionally xanthomata. The LPL gene is 30 kb in size and contains 10 exons. We now report the characterization of some gene mutations underlying lipoprotein lipase deficiency. Patients studied are mainly of Northern European ancestry but include individuals of Indian and Indonesian extraction. Approximately 25% of the patients of European ancestry have a 2 kb direct tandem duplication of in the LPL gene between exon 6 and an Alu element within intron 6. The common duplication event appears in persons of different ancestries in Europe suggesting that the mutation predates the spread of these populations. The second mutation found in a single family consisted of a 6 kb deletion with breakpoints in introns 2 and 5. In order to define point mutations and small DNA rearrangements we have used PCR amplification of each of the exons. Direct sequencing of these PCR products revealed another mutation in a consanguineous patient of Malay extraction. This alteration is a 5 bp insertion in exon 3, leading to an altered reading frame and premature stop codon in exon 4. Analysis of these mutations together with detailed assessment of the protein and its function will provide information on the relationship between the gene structure and the functional domains that it encodes
