The influence of the genes An1, An2, and An4 on the activity of the enzyme UDP-glucose:Flavonoid 3-O-glucosyltransferase in flowers of Petunia hybrida

A relation between gene dosage and UDP-glucose:flavonoid 3-O-glucosyl-transferase (UFGT) activity was found in homozygous dominant and recessive parental lines and their F₁ progeny for both of the genes An1 and An2. In both F₂ crosses, progeny plants could be classified as belonging to groups showing either a low or a medium to high UFGT activity. Test crosses showed that heterozygous and homozygous dominant plants were present throughout the medium- to high-active group. The dosage relation in F₂ plants is most probably confounded by the segregation of modifiers. Thermal inactivation experiments indicated that structurally different UFGT enzymes are formed in homozygous dominant lines as well as in lines homozygous recessive for either An1 or An2. Lines homozygous recessive for the gene An4 contain a UFGT with a half-life time at 55° C of less than 8 min, whereas UFGTs from lines homozygous dominant for An4 show a half-life time of 25 min or above, with one exception. This relation was confirmed in the F₂ progeny; heterozygotes for An4 showed an intermediate half-life time. It is concluded that An4 might be the structural gene for the enzyme; An1 and An2 are both regulatory genes. UFGT activity in flowerbuds of An4/An4 plants seems to be lower than in an4/an4 plants. Anthers of flowers of an4/an4 lines, however, are virtually devoid of UFGT activity.     

Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences

Flower pigmentation patterns were scored in 185 senseChalcone synthase (Chs) transgenotes and 85 antisenseChs transgenotes; upon first flowering, 139 (75%) of sense transgenotes were found to be phenotypically altered, as were 70 (82%) of the antisense transgenotes. The observed patterns document the range of phenotypic variations that occur, as well as confirm and extend the finding that senseChs constructs produce several types of morphologybased based flower pigmentation patterns that antisenseChs constructs do not. Long-term monitoring for epigenetic variations in one population of 44 senseChs transgenotes showed that 43 (98%) were capable of producing a cosuppression phenotype. The primary determinant of sense-specific patterns of cosuppression ofChs was found to be the repetitiveness and organization pattern of the transgene, not position effects by, or readthrough from, flanking plant DNA sequences. The degree of cosuppression observed in progeny of transgenotes carrying multiple, dispersed copies as compared to that observed with a single copy of the transgene suggests that sense cosuppression ofChs is subject to a transgene dosage effect.     

The dosage effect of the wildtype GBSS allele is linear for GBSS activity but not for amylose content: absence of amylose has a distinct influence on the physico-chemical properties of starch

A gene-dosage population was obtained by crossing two genotypes that were duplex for the GBSS allele. Nulliplex, simplex, duplex or triplex/quadruplex plants could be identified by monitoring the segregation of red and blue microspores after staining with iodine. GBSS activity was significantly different for all groups and showed an almost linear dosage effect for the wildtype GBSS gene. A dosage effect was found for amylose content that was not linear. The amylose content was similar for both the duplex and triplex/quadruplex group. Within the simplex group, differences in amylose content were found, which might be due to a different genetic background. There was no linear correlation between GBSS activity and amylose content. A certain level of GBSS activity led to a maximum amount of amylose, and further increase in GBSS activity did not result in a further increase in amylose content. The presence of one or more wildtype GBSS allele(s), and therefore the presence of amylose in the starch granules, had a great influence on the physico-chemical properties of the starch suspensions.     

An SAR sequence containing 395 bp DNA fragment mediates enhanced,gene-dosage-correlated expression of a chimaeric heat shock gene in transgenic tobacco plants

A 395 bp fragment located downstream from the soybean heat shock geneGmhsp 17.6-L exhibits several characteristics of scaffold attachment region (SAR) sequences. It contains matrix consensus elements, a topoisomerase II binding sequence and it associates with the isolated nuclear scaffold of soybeanin vitro. Chimaeric genes containing the SAR_L fragment either at one side (5 or 3) or at both sides of a heat shock promoter-regulated -glucuronidase reporter gene were constructed. A five-to nine-fold increase of heat-inducible -glucuronidase activity was observed in transgenic tobacco plants containing constructs with SAR_L fragments either at both sides or with at least one SAR_L copy located upstream from the reporter gene. The gene copy number is positively correlated with the level of heat-inducible reporter gene activity in these. plants but positional effects are not entirely eliminated. Thus, SAR sequences may potentially be used to increase gene expression, via as yet unknown mechanisms, and to reduce adverse effects on the expression of multiple gene copies in transgenic plants.     

Control of tryptophan synthetase amplified by varying the numbers of composite plasmids in Escherichia coli cells.

Using pSC101, RSF1010, RSF2124 and RP4 plasmids as vectors and bacteriophage lambdatrpD-A60-3 DNA as a source of the Escherichia coli whole tryptophan operon, composite plasmids of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were constructed in vitro with EcoRI restriction endonuclease and DNA ligase. Each composite plasmid could be maintained stably in E. coli cells. The copy number of pSC101-trp, RSF1010-trp, RSF2124-trp and RP4-trp were 4.2, 11.2, 11.9 and 1.6 per chromosome respectively. The tryptophan synthetase activities in cells containing pSC101-trp, RSF1010-trp, RSF2124-trp aand RP4-trp plasmid were found to be 2.1, 6.0, 5.0 and 2.5 times compared with the level in chromosomal trp+ cells when they were grown in a minimal medium. By partial derepression with indolylacrylic acid, the enzyme levels were elevated to 10.1, 16.3, 15.3, 12.3 times, respectively, that of the control cells. The tryptophan synthetase activities did not increase in proportion to the copy number of the plasmids, but were strongly affected by the repression system of host cells.     

Trisomic analysis of isozymic loci in tomato species: Segregation and dosage effects

Five isozymic loci were localized in the tomato (Lycopersicon esculentum) genome by trisomic analysis. Results revealed the following locations: Aps-1 on chromosome 6, Est-1 and Prx-2 on chromosome 2, Prx-4 on chromosome 10, and Prx-7 on chromosome 3. Three genes—Aps-1, Prx-2, and Prx-4—showed an arithmetic increase in allozyme concentration in direct proportion to the increase of gene dosage in respective primary trisomics. In contrast, no increase in relative Est-1 isozyme concentration was observed for any primary trisomic type. The phenotypes of the Aps-1, Prx-2, and Est-1 genes showed a pattern of banding intensity proportional to the allelic ratio (+/+/a vs. + /a/a) in primary trisomics; zymotypes of these differential trisomic heterozygotes appeared as converse images of each other.This research was performed under the auspices of NSF Grants BMS75-03024 and DEB77-02248 to C. M. Rick.     

基因拷贝数对重组毕赤酵母产谷氨酰胺转胺酶的影响

基于毕赤酵母核糖体DNA序列 (rDNA),构建多拷贝谷氨酰胺转胺酶基因表达载体pPICZα-rDNA- mtg,并转化到表达前导肽 (Pro peptide或pro) 的宿主菌pGAP9-pro/GS115,得到共表达菌株pro/rDNA-mtg (GS115)。实时荧光定量PCR (qPCR) 分析了4株阳性表达菌株中mtg基因拷贝数,进一步研究了不同基因拷贝数对重组毕赤酵母产酶的影响及高产菌株在3 L发酵罐高密度发酵。结果表明,被检测的4株阳性表达菌株中mtg拷贝数分别为2.21、3.36、5.72和7.62 (mtg-2c、mtg-3c、mtg-6c和mtg-8c),其发酵产酶能力和蛋白质表达水平为mtg-3c>mtg-2c>mtg-6c>mtg-8c;高密度发酵较低和较高拷贝数的两株菌mtg-3c和mtg-6c,发酵上清的最高酶活和单位菌体酶活分别为3.12 U/mL、52.1 U/g湿重和2.07 U/mL、36.5 U/g湿重,其中单位菌体酶活mtg-3c是mtg-6c的1.4倍;mtg-3c纯化酶的最高酶活达到7.21 U/mL,蛋白浓度为437.2 μg /mL。通过分析拷贝数对重组毕赤酵母产酶的影响,发现mtg-3c适合pro/rDNA-mtg中pro和mtg共表达,MTG高酶活与菌株较高分泌蛋白有关。     

Bayesian modeling of skewed X inactivation in genetically diverse mice identifies a novel Xce allele associated with copy number changes

Female mammals are functional mosaics of their parental X-linked gene expression due to X chromosome inactivation (XCI). This process inactivates one copy of the X chromosome in each cell during embryogenesis and that state is maintained clonally through mitosis. In mice, the choice of which parental X chromosome remains active is determined by the X chromosome controlling element (Xce), which has been mapped to a 176-kb candidate interval. A series of functional Xce alleles has been characterized or inferred for classical inbred strains based on biased, or skewed, inactivation of the parental X chromosomes in crosses between strains. To further explore the function structure basis and location of the Xce, we measured allele-specific expression of X-linked genes in a large population of F1 females generated from Collaborative Cross (CC) strains. Using published sequence data and applying a Bayesian “Pólya urn” model of XCI skew, we report two major findings. First, inter-individual variability in XCI suggests mouse epiblasts contain on average 20–30 cells contributing to brain. Second, CC founder strain NOD/ShiLtJ has a novel and unique functional allele, Xce^g, that is the weakest in the Xce allelic series. Despite phylogenetic analysis confirming that NOD/ShiLtJ carries a haplotype almost identical to the well-characterized C57BL/6J (Xce^b), we observed unexpected patterns of XCI skewing in females carrying the NOD/ShiLtJ haplotype within the Xce. Copy number variation is common at the Xce locus and we conclude that the observed allelic series is a product of independent and recurring duplications shared between weak Xce alleles.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

FOLFOX方案联合西妥昔单抗治疗转移性结直肠癌的近期疗效及安全性分析

目的：探讨FOLFOX方案联合西妥昔单抗治疗转移性结直肠癌的近期临床疗效及安全性。方法：选择2009年2月～2011年2月本院诊治的42例转移性结直肠癌患者为研究对象,采用随机数字表法将其随机分入对照组与观察组,其中对照组20例,观察组22例。对照组患者接受FOLFOX方案治疗,每2周重复1次,治疗3周期;观察组患者给予FOLFOX方案联合西妥昔单抗治疗。比较两组的近期疗效及毒副反应。结果：观察组的客观缓解率和疾病控制率均显著高于对照组,差别具有统计学意义（P〈0．05）;骨髓抑制、消化道反应、神经毒性是两组常见的毒副反应,两组患者骨髓抑制、消化道反应、神经毒性、脱发及肝功能损害发生率无显著差别（P〉0．05）,观察组痤疮样皮疹的发生率显著高于对照组（36．4％VS0,P〈0．05）。结论：西妥昔单抗联合FOLFOX方案可提高转移性结直肠癌患者的近期疗效,毒副反应可耐受。