Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Localization of α-amylase isozymes within the endomembrane system of barley aleurone

Summary The localization of -amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts was studied using electron microscope immunocytochemistry. Antibodies were raised against total barley -amylase, i.e., -amylase containing both highisoelectric point (high-pI) and low-pI isoforms, as well as against purified high- and low-pI isoforms. All antibodies localized -amylase to the endoplasmic reticulum (ER) and Golgi apparatus (GApp) of the aleurone cell, and various controls showed that the labeling was specific for -amylase. Labeling of protein bodies and spherosomes, which are the most abundant organelles in this cell, was very low. There was no evidence that -amylase isoforms were differentially distributed within different compartments of the endomembrane system. Rather, both high- and low-pI isoforms showed the same pattern of distribution in ER and in the cis, medial, and transregions of the GApp. We conclude that in the Himalaya cultivar of barley, all isoforms of -amylase are transported to the plasma membrane via the GApp.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - GApp Golgi apparatus - PBS phosphate buffered saline - PCR partially coated reticulum - PM plasma membrane - TBS Tris buffered saline - TGN trans-Golgi network     

The inheritance of β-amylase null in storage roots of sweet potato,Ipomoea batatas (L.) Lam.

Summary Several sweet potato genotypes were found to lack completely or to have only traces of-amylase in their storage roots. Such genotypes do not increase in sweetness during cooking because, without a sufficient amount of-amylase, the normal hydrolysis of starch to maltose does not occur in the cooking process. In order to study the inheritance of this biochemical variant in the genotype, 41 families were generated. The following conclusions were drawn from analyzing these families. (1) This trait is controlled by one recessive allele (designated-amy) (2) It is inherited in a hexasomic or tetradisomic manner, but not disomically or tetrasomically. This conclusion supports previous cytological data that sweet potato is an autohexaploid or has two identical genomes plus one genome which is somewhat different. (3) The-amy allele appears to exist at a high frequency in cultivated germplasm. (4) Breeding sweet potato for low-amylase activity is relatively easy. New types of sweet potato without normal-amylase activity have great potential for processing and as a staple food.     

Optimization of delivery of foreign DNA into higher-plant chloroplasts

We report here an efficient and highly reproducible delivery system, using an improved biolistic transformation device, that facilitates transient expression of -glucuronidase (GUS) in chloroplasts of cultured tobacco suspension cells. Cultured tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 or pBI101.3 (negative controls), pBI505 (positive nuclear control) or a chloroplast expression vector (pHD203-GUS), and were assayed for GUS activity. No GUS activity was detected in cells bombarded with pUC118 or pBI101.3. Cells bombarded with pBI505 showed high levels of expression with blue color being distributed evenly throughout the whole cytosol of the transformants. pHD203-GUS was expressed exclusively in chloroplasts. We base this conclusion on: i) the procaryotic nature of the promoter used in the chloroplast expression vector; ii) delayed GUS staining; iii) localization of blue color within subcellular compartments corresponding to plastids in both shape and size; and iv) confirmation of organelle-specific expression of pHD203-GUS using PEG-mediated protoplast transformation. Chloroplast transformation efficiencies increased dramatically (about 200-fold) using an improved helium-driven biolistic device, as compared to the more commonly used gun powder charge-driven device. Using GUS as a reporter gene and the improved biolistic device, optimal bombardment conditions were established, consistently producing several hundred transient chloroplast transformants per Petri plate. Chloroplast transformation efficiency was found to be increased further (20-fold) with supplemental osmoticum (0.55 M sorbitol and 0.55 M mannitol) in the bombardment and incubation medium. This system provides a highly effective mechanism for introducing and expressing plasmid DNA within higher-plant chloroplasts, and the fact that GUS functions as an effective marker gene now makes many genetic studies possible which were not possible before.     

Diversity,metabolic types and δ13C carbon isotope ratios in the grass flora of Namibia in relation to growth form,precipitation and habitat conditions

The grass flora of Namibia (374 species in 110 genera) shows surprisingly little variation in ¹³C values along a rainfall gradient (50–600 mm) and in different habitat conditions. However, there are significant differences in the ¹³C values between the metabolic types of the C4 photosynthetic pathway. NADP-ME-type C4 species exhibit the highest ¹³C values (–11.7 ) and occur mainly in regions with high rainfall. NAD-ME-type C4 species have significantly lower ¹³C values (–13.4 ) and dominate in the most arid part of the precipitation regime. PCK-type C4 species play an intermediate role (–12.5 ) and reach a maximum abundance in areas of intermediate precipitation. This pattern is also evident in genera containing species of different metabolic types. Within the same genus NAD species reach more negative ¹³C values than PCK species and ¹³C values decreased with rainfall. Also in Aristida, with NADP-ME-type photosynthesis, ¹³C values decreased from –11 in the inland region (600 mm precipitation) to –15 near the coast (150 mm precipitation), which is a change in discrimination which is otherwise associated by a change in metabolism. The exceptional C3 species Eragrostis walteri and Panicum heterostachyum are coastal species experiencing 50 mm precipitation only. Many of the rare species and monotypic genera grow in moist habitats rather than in the desert, and they are not different in their carbon isotope ratios from the more common flora. The role of species diversity with respect to habitat occupation and carbon metabolism is discussed.     

大肠癌中p53基因突变的研究

应用聚合酶链反应(PCR)──单链构型多态性(SSCP)结合银染法对14例大肠癌p53基因的第4、第5─6和第7外显子进行了点突变的研究,结果共检测出6例点突变,而且发现各外显子的突变频率存在差异。另外,利用购自ATCC的两个探针 (p53cDNA探针和pYNZ22探针)对大肠癌中p53基因的杂合性失去进行了研究,在14例大肠癌中共检出6例杂合性丢失。将点突变检测结果同杂合性丢失结果进行比较分析, 并着重探讨了大肠癌中p53基因失活导致肿瘤的作用方式。 Abstract:The exons 4-7 of p53 gene were examined in 14 colorectal Cancer patients by using PCR-SSCP-silver staining method.The results showed 6 cases of point mutation and the mutation frequencies of exons were different from each other.p53 cDNA and pYNZ22 VNTR were used as probes to examine LOH(Loss of heterozygosity)of 14 colorectal cancers.6 cases with LOH were found.The results of present research suggest that mutation and LOH of p53 gene are critical events in the progress and development of Cancer.There were different kinds of inactivation model of p53 gene in the process of development of cancer and transformation of cells.     

Thermal response in murine L929 cells lacking αB-crystallin expression and αB-crystallin expressing L929 transfectants

We investigated the role of B-crystallin expression in the development of thermotolerance in murine L929 cells. An initial heat-shock of 10 min at 45°C induced thermotolerance in these cells to a heat challenge at 45°C administered 24 h later. The thermotolerance ratio at 10^–1 isosurvival was 1.7. Expression of B-crystallin gene was not detected during the 24 h incubation at 37°C following heat shock by either northern or western blots. In contrast, inducible HSP70 synthesis was observed during this time period. Thus, this cell line provided an unique system in which to examine the effects of transfected B-crystallin on thermoresistance and thermotolerance. Cells stably transfected with B-crystallin under the control of an inducible promoter did not show a significant increase in the ability to develop thermotolerance. However, a stably transfected L929 clone expressing high levels of constitutive B-crystallin exhibited an approximately 50% increase in thermal resistance over parental and control cells. Though expression of B-crystallin is not requisite for the development of thermotolerance in L929 cells, overexpression of transfected B-crystallin can contribute to increased thermoresistance.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline