Location of a gene regulating drought-induced abscisic acid production on the long arm of chromosome 5A of wheat

The accumulation of abscisic acid (ABA) by detached and partially dehydrated wheat leaves is known to be inherited in a quantitative manner. The location of genes having a major effect on drought-induced ABA accumulation in wheat was determined using a set of single chromosome substitution lines and populations derived from a cross between a high-ABA- and a low-ABA-producing genotype. Examination of a series of chromosome substitution lines of the high-ABA genotype Ciano 67 into the low-ABA recipient Chinese Spring showed that chromosome 5A carries gene(s) that have a major influence on ABA accumulation in a drought test with detached and partially dehydrated leaves (DLT). A similar DLT was used to examine ABA accumulation in a population of F₂ plants and doubled haploid (DH) lines derived from the cross between Chinese Spring (low-ABA) and SQ1 (high-ABA) in which the F₂ population (139 plants) and DH lines (96 lines) were also mapped partially with molecular markers. Analysis of variance of ABA accumulation between and within marker allele classes in the F₂ confirmed the location of a gene(s) regulating ABA accumulation on the long arm of chromosome 5A. MAPMAKERQTL showed the most likely position for the ABA quantitative trait locus (QTL) to be between the loci Xpsr575 and Xpsr426, about 8 cM from Xpsr426. A similar trend for high ABA accumulation was found in DH lines having the SQ1 allele at marker loci in the same region of chromosome 5AL, but the QTL effect was not significant. The function of the QTL is discussed.     

Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.     

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval–pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.     

Genetic variation in the HSD17B1 gene and risk of prostate cancer

Steroid hormones are believed to play an important role in prostate carcinogenesis, but epidemiological evidence linking prostate cancer and steroid hormone genes has been inconclusive, in part due to small sample sizes or incomplete characterization of genetic variation at the locus of interest. Here we report on the results of a comprehensive study of the association between HSD17B1 and prostate cancer by the Breast and Prostate Cancer Cohort Consortium, a large collaborative study. HSD17B1 encodes 17β-hydroxysteroid dehydrogenase 1, an enzyme that converts dihydroepiandrosterone to the testosterone precursor Δ5-androsterone-3β,17β-diol and converts estrone to estradiol. The Breast and Prostate Cancer Cohort Consortium researchers systematically characterized variation in HSD17B1 by targeted resequencing and dense genotyping; selected haplotype-tagging single nucleotide polymorphisms (htSNPs) that efficiently predict common variants in U.S. and European whites, Latinos, Japanese Americans, and Native Hawaiians; and genotyped these htSNPs in 8,290 prostate cancer cases and 9,367 study-, age-, and ethnicity-matched controls. We found no evidence that HSD17B1 htSNPs (including the nonsynonymous coding SNP S312G) or htSNP haplotypes were associated with risk of prostate cancer or tumor stage in the pooled multiethnic sample or in U.S. and European whites. Analyses stratified by age, body mass index, and family history of disease found no subgroup-specific associations between these HSD17B1 htSNPs and prostate cancer. We found significant evidence of heterogeneity in associations between HSD17B1 haplotypes and prostate cancer across ethnicity: one haplotype had a significant (p < 0.002) inverse association with risk of prostate cancer in Latinos and Japanese Americans but showed no evidence of association in African Americans, Native Hawaiians, or whites. However, the smaller numbers of Latinos and Japanese Americans in this study makes these subgroup analyses less reliable. These results suggest that the germline variants in HSD17B1 characterized by these htSNPs do not substantially influence the risk of prostate cancer in U.S. and European whites.     

Short term increase in risk of breast cancer after full term pregnancy.

To determine whether there is a short term increase in the risk of breast cancer after a full term birth data from two hospital based, case-control studies in Italy were pooled. Analysis was restricted to women aged under 50 with two or more children (573 women with cancer and 570 controls). A relative risk for breast cancer of 2.66 was seen in women who had given birth during the three years preceding the interview compared with women whose last birth had occurred 10 or more years before, after adjustment for age, age at first birth, and parity. The relative risk slowly decreased for women who had last given birth three to 10 years before. Multivariate analyses confirmed the protective effect of an early age at first birth and the age dependent effect of parity on the risk of breast cancer--that is, a direct relation below age 40 and an inverse one in older women. These data provide epidemiological evidence that a full term birth is followed by a transient increase in the risk of breast cancer, which for some time contrasts with and overcomes the long term protection of pregnancy at an early age. They therefore confirm predictions from animal studies and theoretical models that pregnancy prevents the early stages of breast carcinogenesis but promotes the late stages of the process.     

Double-strand break DNA repair genotype predictive of later mortality and cancer incidence in a cohort of non-smokers

We followed-up for mortality and cancer incidence 1088 healthy non-smokers from a population-based study, who were characterized for 22 variants in 16 genes involved in DNA repair pathways. Follow-up was 100% complete. The association between polymorphism and mortality or cancer incidence was analyzed using Cox Proportional Hazard regression models. Ninety-five subjects had died in a median follow-up time of 78 months (inter-quartile range 59-93 months). None of the genotypes was clearly associated with total mortality, except variants for two Double-Strand Break DNA repair genes, XRCC3 18067 C>T (rs#861539) and XRCC2 31479 G>A (rs#3218536). Adjusted hazard ratios were 2.25 (1.32-3.83) for the XRCC3 C/T genotype and 2.04 (1.00-4.13) for the T/T genotype (reference C/C), and 2.12 (1.14-3.97) for the XRCC2 G/A genotype (reference G/G). For total cancer mortality, the adjusted hazard ratios were 3.29 (1.23-7.82) for XRCC3 C/T, 2.84 (0.81-9.90) for XRCC3 T/T and 3.17 (1.21-8.30) for XRCC2 G/A. With combinations of three or more adverse alleles, the adjusted hazard ratio for all cause mortality was 17.29 (95% C.I. 8.13-36.74), and for all incident cancers the HR was 5.28 (95% C.I. 2.17-12.85). Observations from this prospective study suggest that polymorphisms of genes involved in the repair of DNA double-strand breaks significantly influence the risk of cancer and non-cancer disease, and can influence mortality.     

Synthesis of the same two proteins prior to larval diapause and pupation in the spruce budworm,Choristoneura fumiferana

Spruce budworm larvae produce large quantities of two proteins (Choristoneura fumiferana diapause associated proteins 1 and 2, CfDAP1 and CfDAP2) that are diapause related. These proteins appeared soon after hatching and increased in abundance, reaching maximum levels by four days into the 1st instar, and they remained at high levels until three days after the termination of diapause. These two proteins were purified to homogeneity and their NH2-terminal sequences were obtained. Oligonucleotide primers designed on the basis of these NH2-terminal sequences were used in RT-PCR to isolate the cDNA fragments coding for these proteins. These PCR fragments were then used as probes to isolate the cDNAs that contained the complete coding region. The 2.5kb mRNAs coding for these proteins started to appear 24hr after hatching and large quantities of these mRNAs were detected in 1st instar and 2nd instar larvae until the 2nd instar larvae entered diapause. Low levels of these mRNAs were detected in the 2nd instar larvae that were preparing to enter diapause, in those that were in diapause as well as in those that terminated diapause. Low levels of CfDAP1 mRNA were also detected on days 1 and 2 after ecdysis to the 3rd instar. However, no CfDAP1 and CfDAP2 mRNAs could be detected during the 4th and 5th instar larval stages. The mRNAs reappeared 24hr after the 5th instar larvae molted into the 6th instar and increased to reach maximum levels by 60hr after ecdysis. The mRNA levels remained high until 156hr after ecdysis into the 6th instar (36-48hr before pupal ecdysis), after which they disappeared once again. Immunocytochemical analyses showed that CfDAP1 protein was present in 2nd and 6th instar larval fat body but not in 5th instar larval fat body. Thus, the same two genes were expressed for the first time before C. fumiferana larvae entered diapause and for a 2nd time before pupation.