Targeted ablation of gonadotrophs in transgenic mice depresses prolactin but not growth hormone gene expression at birth as measured by quantitative mRNA detection

We previously reported that transgenic ablation of gonadotrophs results in impaired development of cells immunostainable for prolactin (PRL) but not of cells immunostainable for growth hormone (GH) or proopiomelanocortin (POMC) in pituitary of newborn mice. The question remained whether this reduction in PRL protein is a reflection of reduced PRL mRNA expression, or whether this regulation is only situated at the translational level. We therefore generated a new series of transgenic mice in which gonadotrophs were ablated by diphtheria toxin A targeting, and analyzed hormone mRNA levels instead of hormone protein around the day of birth. Pituitary mRNA expression levels of luteinizing hormone- (LH), PRL and GH were quantified using real-time TaqMan RT-PCR. Of the 13 transgenic mice obtained, 8 showed a clear-cut reduction (ranging from 62 to 98%) in LH mRNA levels. PRL mRNA values were significantly reduced in the transgenic mice (p=0.0034), while GH mRNA expression was unaffected (p=0.93). An additional observation was that female newborn mice produce 5 times more LH mRNA than male mice whereas no sex difference was observed for expression levels of PRL and GH mRNA. Moreover, in the wild-type mice, LH mRNA expression was 20-fold higher than GH mRNA expression which in turn was 500- to 1,000-fold higher than PRL mRNA expression, suggesting a low expression level of the PRL gene at birth. In conclusion, the present data support the hypothesis that embryonic development of PRL gene expression is stimulated by gonadotrophs.     

Damage formation and repair efficiency in the p53 gene of cell lines and blood lymphocytes assayed by multiplex long quantitative polymerase chain reaction

We examined ultraviolet (UV) irradiation and cisplatin treatment damage formation and repair efficiency in the p53 tumor suppressor gene of various cultured cell lines and lymphocytes using a nonradioactive multiplex long quantitative polymerase chain reaction (QPCR) assay, which amplified a 7-kb fragment of the target gene and a 500-bp fragment of the template control to successfully increase the sensitivity and reliability of the assay. The multiplex long QPCR detected a lesion frequency of 0.63 lesions/10kb/10J/m(2) in the p53 gene of fibroblast cells. In addition, the multiplex long QPCR assay detected pronounced differences in the repair of UV damage in the p53 gene among repair-proficient CRL-1475 cells and repair-deficient XP-A and XP-C cells. The multiplex long QPCR assay was also evaluated as a sensitive assay for the detection of DNA damage induced by cisplatin. The data indicated that the lesion frequency in the p53 gene was 1.27-1.75 times higher in the H23 cisplatin-sensitive cell than in the H1435 cisplatin-resistant cell at the IC(70) dose. After 8-h and 24-h repair periods, only 13 and 75% of cisplatin-induced damage had been removed in the H23 cells, whereas these values were 92 and 100% in the H1435 cells. In addition, our data indicate that multiplex long QPCR is a sensitive method for validly estimating repair in freshly isolated lymphocytes. The results suggest that the current protocol of the multiplex long QPCR method can be used to assess the damage formation and repair efficiency of various agents at biologically relevant doses and to allow a more precise determination of gene-specific repair in disease susceptibility and drug resistance in epidemiological studies.     

Quantitative microinjection of trehalose into mouse oocytes and zygotes,and its effect on development

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.     

Identification of quantitative trait loci and epistatic interactions for plant height and heading date in rice

Appropriate heading date and plant height are prerequisites for attaining the desired yield level in rice breeding programs. In this study, we analyzed the genetic bases of heading date and plant height at both single- locus and two-locus levels, using a population of 240 F_2:3 families derived from a cross between two elite rice lines. Measurements for the traits were obtained over 2 years in replicated field trials. A linkage map was constructed with 151 polymorphic marker loci, based on which interval mapping was performed using Mapmaker/QTL. The analyses detected six QTLs for plant height and six QTLs for heading date; collectively the QTLs for heading date accounted for a much greater amount of phenotypic variation than did the QTLs for plant height. Two-way analyses of variance, with all possible two-locus combinations, detected large numbers (from 101 to 257) of significant digenic interactions in the 2 years for both traits involving markers distributed in the entire genome; 22 and 39 were simultaneously detected in both years for plant height and heading date, respectively. Each of the interactions individually accounted for only a very small portion of the phenotypic variation. The majority of the significant interactions involved marker loci that did not detect significant effects by single-locus analyses, and many of the QTLs detected by single-locus analyses were involved in epistatic interactions. The results clearly demonstrated the importance of epistatic interactions in the genetic bases of heading date and plant height. Received: 5 May 2001 / Accepted: 3 August 2001     

Quantitative structure-retention relationships in affinity high-performance liquid chromatography

In this report the affinity high-performance liquid chromatography data, which were determined on silica-based human serum albumin, alpha1-acid glycoprotein, keratin, collagen, melanin, amylose tris(3,5-dimethylphenylcarbamate), and basic fatty acid binding protein columns, are discussed. Using a quantitative structure-retention relationship (QSRR) approach the affinity data were interpreted in terms of structural requirements of specific binding sites on biomacromolecules. The unique chromatographic properties of immobilized artificial membrane and cholesterol stationary phases were also analyzed from the point of view of mimicking biological processes. It has been demonstrated that chemometric processing of appropriately designed sets of chromatographic data derived in systems comprising biomolecules provides information of relevance for molecular pharmacology and rational drug design.     

Generalized character process models: estimating the genetic basis of traits that cannot be observed and that change with age or environmental conditions

The genetic analysis of characters that change as a function of some independent and continuous variable has received increasing attention in the biological and statistical literature. Previous work in this area has focused on the analysis of normally distributed characters that are directly observed. We propose a framework for the development and specification of models for a quantitative genetic analysis of function-valued characters that are not directly observed, such as genetic variation in age-specific mortality rates or complex threshold characters. We employ a hybrid Markov chain Monte Carlo algorithm involving a Monte Carlo EM algorithm coupled with a Markov chain approximation to the likelihood, which is quite robust and provides accurate estimates of the parameters in our models. The methods are investigated using simulated data and are applied to a large data set measuring mortality rates in the fruit fly, Drosophila melanogaster.     

Biosynthesis of the sesquiterpene hodgsonox from the New Zealand liverwort Lepidolaena hodgsoniae

The incorporation of [1-13C] labelled glucose into hodgsonox, a sesquiterpene epoxide with a unique, doubly allylic ether functionality has been studied in axenic cultures of the liverwort Lepidolaena hodgsoniae. Quantitative 13C NMR spectroscopic analysis showed that the isoprene units are derived exclusively from the methylerythritol phosphate pathway.     

Molecular marker dissection of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plant architecture under temperate and tropical climates

Rice (Oryza sativa L.) plants develop vertically with shoot elongation and horizontally with tillering. The purpose of this study was to identify and characterize genomic regions influencing the rice plant architecture by quantitative trait locus (QTL) analysis for the component traits: culm length (CL), panicle length (PnL), panicle number (PnN) and tiller number (TN). For this QTL analysis, 191 recombinant inbred lines (F₇) derived from a cross of Milyang 23 (M23) and Akihikari (AK) were grown in 1995, 1996 and 1997 (May–Oct) in Joetsu, Japan (temperate climate), and in the 2000 dry season (Jan–Apr), the 2000 wet season (Jun–Oct) and the 2001 dry season in Los Baños, The Philippines (tropical climate). Results showed that rice plant architecture was influenced by 19 genomic regions categorized into five groups. In Group I, two regions (on chrs. 6 and 11) affected shoot elongation (CL and PnL) and tillering (PnN and TN) in opposite directions more significantly in Los Baños than in Joetsu. In Group II, two regions (chrs. 3 and 12) affected shoot elongation, whereas in Group III, five regions [chrs. 1 (two), 2, 3 and 9] affected only culm length (CL). Expressions of four regions of Group III were influenced by either tropical or temperate environments. In Group IV, seven regions (chrs. 1, 2, 4, 5, 6, 8 and 9) controlled panicle development (PnN or PnL), and in Group V, three regions (chrs. 1, 2 and 3) regulated tillering (PnN or TN). Characterizing these 19 genomic regions provided a detailed analysis of rice plant architecture with emphasis on the multiple effect and environmental responsive regions.Communicated by D. Mackill     

Quantification of total mitochondrial DNA and the 4977-bp common deletion in Pearson's syndrome lymphoblasts using a fluorogenic 5'-nuclease (TaqMan) real-time polymerase chain reaction assay and plasmid external calibration standards

This study describes a multiplex real-time polymerase chain reaction (PCR) assay that quantifies total mitochondrial DNA (mtDNA(total)) and mtDNA bearing the 4977-base pair 'common deletion' (deltamtDNA4977) in lymphoblasts derived from an individual diagnosed with Pearson's syndrome. The method is unique in its use of plasmids as external quantification standards and its use of multiplex conditions. Standards are validated by comparison with purified mtDNA amplification curves and by the fact that curves are largely unaffected by nuclear DNA (nucDNA). Finally, slopes of standard curves and unknowns are shown to be similar to each other and to theoretical predictions. From these data, mtDNA(total) in these cells is calculated to be 3258 (+723/-592) copies per cell while deltamtDNA4977 averages 232 (+136/-86) copies per cell or 7% (+4.65/-2.81).