Genetic polymorphisms of gene conversion within the duplicated human α-globin loci

Summary Of the 645 Japanese subjects studied, we have identified 10 individuals heterozygous for a chromosome with the triplicated -globin loci. The frequency of the triple -loci was 0.008 in this population, while that of the single -locus, i.e., -thalassemia 2 gene, might be lower than 0.0008. Analysis of haplotypes using particular RsaI site polymorphism in the -globin gene complex strongly suggests that the triple loci may have had multiple origins in this population.     

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.     

Transgenic resistance to Mirafiori lettuce virus in lettuce carrying inverted repeats of the viral coat protein gene

Mirafiori lettuce virus (MiLV), a plant RNA virus belonging to the genus Ophiovirus, is considered to be a causal agent of lettuce big-vein disease. In this study, inverted repeats of a fragment of the coat protein (CP) gene of MiLV in a binary vector pBI121 were transferred via Agrobacterium tumefaciens-mediated transformation into lettuce (Lactuca sativa L.) in order to generate MiLV-resistant lettuce. Forty T₁ lines were analyzed for resistance to MiLV by detecting MiLV in leaves, and two lines (lines 408 and 495) were selected as resistant to MiLV. Both lines were susceptible to Lettuce big-vein associated virus (LBVaV), and line 495 showed higher resistance to MiLV than line 408. Further analysis indicated that line 495 showed resistance to big-vein symptoms expression. Small interfering RNA (siRNA) molecules derived from the transgene were detected in plants of line 495. MiLV was detected in roots but not in leaves of line 495 plants after MiLV inoculation, suggesting that resistance to MiLV is less effective in roots than in leaves.     

Genetic basis for established and novel host plant use in a herbivorous ladybird beetle, Epilachna vigintioctomaculata

Genetic trade-offs in host plant use are thought to promote the evolution of host specificity. Experiments on a range of herbivorous insects, however, have found negative genetic correlation in host plant use in only a limited number of species. To account for the general lack of negative genetic correlation, recent hypotheses advocate that different stages in evolution of host use must be distinguished: initial performance on a novel host in comparison with the established host, and performance on both hosts after the insect population has interacted with both hosts for a long time. The hypotheses suggest that genetic correlation may not necessarily be negative at the initial stage. The present study examines growth performance on both the established and a novel host in a herbivorous ladybird beetle, Epilachna vigintioctomaculata Motschulsky (Coccinellidae, Epilachninae). The results show that traits of growth performance across hosts were positively or neutrally correlated, but there was no evidence of a negative genetic correlation. In addition, significant genetic variance of growth performance on each host was detected, suggesting that E. vigintioctomaculata can potentially respond to selection for increased performance on both plant species. These results and similar results from experiments on other herbivores suggest that host expansion may not be constrained genetically, at least at the initial stage of host range evolution.     

Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

DEAD-box RNA helicase subunits of the Drosha complex are required for processing of rRNA and a subset of microRNAs

MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.     

HJURP is involved in the expansion of centromeric chromatin

The CENP-A–specific chaperone HJURP mediates CENP-A deposition at centromeres. The N-terminal region of HJURP is responsible for binding to soluble CENP-A. However, it is unclear whether other regions of HJURP have additional functions for centromere formation and maintenance. In this study, we generated chicken DT40 knockout cell lines and gene replacement constructs for HJURP to assess the additional functions of HJURP in vivo. Our analysis revealed that the middle region of HJURP associates with the Mis18 complex protein M18BP1/KNL2 and that the HJURP-M18BP1 association is required for HJURP function. In addition, on the basis of the analysis of artificial centromeres induced by ectopic HJURP localization, we demonstrate that HJURP exhibits a centromere expansion activity that is separable from its CENP-A–binding activity. We also observed centromere expansion surrounding natural centromeres after HJURP overexpression. We propose that this centromere expansion activity reflects the functional properties of HJURP, which uses this activity to contribute to the plastic establishment of a centromeric chromatin structure.