Transfer RNA genes ofZea mays chloroplast DNA

A minimum of 37 genes corresponding to tRNAs for 17 different amino acids have been localized on the restriction endonuclease cleavage site map of theZea mays chloroplast DNA molecule. Of these, 14 genes corresponding to tRNAs for 11 amino acids are located in the larger of the two single-copy regions which separate the two inverted copies of the repeat region. One tRNA gene is in the smaller single-copy region. Each copy of the large repeated sequence contains, in addition to the ribosomal RNA genes, 11 tRNA genes corresponding to tRNAs for 8 amino acids. The genes for tRNA₂ ^Ile and tRNA^Ala map in the ribosomal spacer sequence separating the 16S and 23S ribosomal RNA genes. The three isoaccepting species for the tRNAs^Leu and the three for tRNAs^Ser, as well as the two isoaccepting species for tRNA^Asn, tRNA^Gly, tRNAs^Ile, tRNAs^Met, tRNAs^Thr, are shown to be encoded at different loci. Two independent methods have been used for the localization of tRNA genes on the physical map of the maize chloroplast DNA molecule: (a) cloned chloroplast DNA fragments were hybridized with radioactively-labelled total 4S RNAs, the hybridized RNAs were then eluted, and identified by two-dimensional polyacrylamide gel electrophoresis, and (b) individual tRNAs were³²P-labelledin vitro and hybridized to DNA fragments generated by digestion of maize chloroplast DNA with various restriction endonucleases.     

Associations between PRLR/AluI gene polymorphism with reproductive,growth, and meat traits in pigs

The aim of this study was to determine the associations between genotypes of the prolactin receptor gene (PRLR) and reproductive, growth, and meat traits in pigs. Genetic groups of sows were used: Large White (LW), Danish Landrace (LD), and Landrace × Yorkshire × Duroc hybrids (L × Y × D). Reproductive traits studied were the total number of born (TNB) and the number born alive (NBA). The growth traits were the number of days to 100 kg and the average daily gains (ADG). The meat traits were the average backfat thickness (BFT), half carcass weight (HCW), area of M. longissimus dorsi (MLT), and lean meat content (LM). The polymorphism was detected using the polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method. It was found that the genotype BB is associated with the best NBA. In sows LD, the AB genotype is associated with best number of days to 100 kg; in hybrids L × Y × D, the AA genotype is associated with better meat traits as compared to the genotype BB.     

The evolutionary pattern of aromatic amino acid biosynthesis and the emerging phylogeny of pseudomonad bacteria

Pseudomonad bacterial are a phylogenetically diverse assemblage of species named within contemporary genera that includePseudomonas, Xanthomonas andAlcaligenes. Thus far, five distinct rRNA homology groups (Groups I through V) have been established by oligonucleotide cataloging and by rRNA/DNA hybridization. A pattern of enzymic features of aromatic amino acid biosynthesis (enzymological patterning) is conserved at the level of rRNA homology, five distinct and unambiguous patterns therefore existing in correspondence with the rRNA homology groups. We sorted 87 pseudomonad strains into Groups (and Subgroups) by aromatic pathway patterning. The reliability of this methodology was tested in a blind study using coded cultures of diverse pseudomonad organisms provided by American Type Culture Collection. Fourteen of 14 correct assignments were made at the Group level (the level of rRNA homology), and 12 of 14 correct assignments were made at the finer-tuned Subgroup levels. Many strains of unknown rRNA-homology affiliation had been placed into tentative rRNA groupings based upon enzymological patterning. Positive confirmation of such strains as members of the predicted rRNA homology groups was demonstrated by DNA/rRNA hybridization in nearly every case. It seems clear that the combination of these molecular approaches will make it feasible to deduce the evolution of biochemical-pathway construction and regulation in parallel with the emerging phylogenies of microbes housing these pathways.     

Response of the population size and community structure of Paenibacillus spp. to different fertilization regimes in a long-term experiment of red soil

Background and aims

Paenibacillus spp. are widely considered to impact the fertility and health of soil. The aim of this study was to evaluate how different fertilization regimes affect the population size and community structure of Paenibacillus spp. over a long period of time in red soil.

Methods

Soil samples were collected from a long-term experiment and were then analyzed using real-time PCR and PCR-DGGE. The correlation analysis, PCA and RDA were used to explore the relationships among Paenibacillus spp. population, community structure and soil properties in different treatments.

Results

The pH was seriously decreased only by the application of chemical fertilizer. The largest population of Paenibacillus spp. was found in the soil treated with organic fertilizer application, while the richest diversity was observed in the soil treated only with the chemical fertilizer. The Paenibacillus spp., Paenibacillus alkaliterrae, Paenibacillus campinasensis, and Paenibacillus xylanilyticus were found in all treatments. Paenibacillus castaneae was found in the soil treated with NPK, and Paenibacillus pabuli was specifically observed in the lime-amended treatment. Paenibacillus taichungensis and Paenibacillus prosopidis were detected in the soil treated with only chemical fertilizer. Except for the ammonium and pH, all the tested soil fertility parameters (total C, total N, nitrate, available K and available P) could significantly affect both the Paenibacillus spp. population number and diversity. The soil pH was significantly correlated with Paenibacillus spp. diversity only.

Conclusions

Our results indicate that the different long-term fertilization regimes have varied impact on both the Paenibacillus spp. population size and the diversity of the community associated with the soil properties tested. These results can help to enrich the information on the response of beneficial soil microbes to different long-term fertilization regimes.     

Location of the genes coding for 18S and 28S ribosomal RNA in the human genome

³H-rRNA obtained from Xenopus laevis tissue cultured cells, or a ³H-cRNA made from Xenopus ribosomal DNA, was used for heterologous in situ hybridisation with human lymphocyte metaphase chromosomes. Prior to hybridisation, chromosome spreads were stained with Quinacrine and selected cells showing good Q-banding photographed; the same cells were then rephotographed after autoradiography and pairs of photographs for each cell were used to make dual karyotypes. The chromosomes within each karyotype were divided into equal sized segments (approx. 0.7 μ), with a fixed number of segments for each chromosome type. The distribution of silver grains between segments showed that the ³H-RNAs hybridised specifically to the nucleolar organising regions of the D and G group chromosomes with no other sites of localised labelling in the complement. Control experiments showed no localisation, with insignificant labelling, when metaphase spreads were incubated in a mixture containing Xenopus ³H-rRNA and competing cold human (HeLa) rRNA. Filter hybridisation experiments on isolated human DNA showed that the Xenopus derived ³H-RNAs hybridised to a fraction of human DNA which was on the heavy side of the main DNA peak and that these RNAs were competed out in the presence of excess cold human rRNA, confirming the specificity of the heterologous hybridisation. In situ hybridisation experiments were also carried out on cells from individuals with one chromosome pair showing heteromorphism for either a very long stalk (nucleolar constriction) subtending a satellite, or a large satellite. It was shown that the chromosome with the large stalk hybridised four times as much ³H-rRNA as its homologue, whereas differences in the sizes of the subtended satellites did not materially affect hybridisation levels indicating that rDNA is located in the stalks and not the satellites. The amount of ³H-rRNA hybridised differs between chromosomes and individuals; these differences are heritable and rDNA can be detected by in situ hybridisation in all three chromosomes number 21 in cells from Down's patients and in translocated chromosomes conta.ining a nucleolar constriction. Different D and G group chromosomes which hybridised equal amounts of ³H-rRNA participated in rosette associations at metaphase in a random fashion in some individuals and in a non-random fashion in others. In all individuals studied chromosomes with large amounts of rDNA were not found to be preferentially involved in association. It was therefore concluded that the probability of a chromosome being involved in the formation of a common nucleolus is not a simple function of its rDNA content and other possible factors are considered.     

Contribution to the study on the reparative effect of exogenous DNA in the irradiated meristem ofVicia faba

The present report is focused on the study of participation of exogenous DNA in the process of postirradiation reparation of meristematic cells ofVicia faba primary roots. It is aimed at comparison of the positive reparative effect of isologous DNA with postirradiation action of heterologous DNA in its native, thermally denatured and DNAase-degraded forms, or DNA degraded by ultrasound, and with the effect of other biologically important macromolecules (RNA, histone, heparin, and dextran sulphate). For this purpose, the roots ofVicia faba irradiated by 150 r exposure were cultivated in solutions containing the above substances for an appropriate time interval. In squash slides both mitotic activity of the investigated cell population and frequency of postmetaphase chromosomal aberrations induced by radiation were evaluated. It was shown that a stimulation of cell division and reparation of chromosome damages were supported exclusively by isologous DNA. On the contrary, exogenously applied heterologous DNA increased postirradiation frequency of aberrations; maintenance of native structure of applied DNA was an essential condition for the above effect. Other macromolecules investigated on the course of postirradiation reparation ofVicia faba meristematic cells were without effect.     

Occurrence of Phytophthora plurivora and other Phytophthora species in oak forests of southern Poland and their association with site conditions and the health status of trees

Phytophthora plurivora and other Phytophthora species are known to be serious pathogens of forest trees. Little is known, however, about the presence of P. plurivora in Polish oak forests and their role in oak decline. The aims of this study were to identify P. plurivora in healthy and declining Quercus robur stands in southern Poland and to demonstrate the relationship between different site factors and the occurrence of P. plurivora. In addition, the virulence of P. plurivora and other Phytophthora species was evaluated through inoculations using 2-year-old oak seedlings. Rhizosphere soil was investigated from 39 oak stands representing different healthy tree statuses. The morphology and DNA sequences of the internal transcribed spacer regions (ITS) of the ribosomal DNA and the mitochondrial cox1 gene were used for identifications. P. plurivora, an oak fine root pathogen, was isolated from rhizosphere soil samples in 6 out of 39 stands. Additionally, Phytophthora cambivora, Phytophthora polonica and Phytophthora rosacearum-like were also obtained from several stands. The results showed a significant association between the presence of P. plurivora and the health status of oak trees. Similar relationships were also observed for all identified Phytophthora species. In addition, there was evidence for a connection between the presence of all identified Phytophthora species and some site conditions. Phytophthora spp. occurred more frequently in declining stands and in silt loam and sandy loam soils with pH?≥?3.66. P. plurivora and P. cambivora were the only species capable of killing whole plants, producing extensive necrosis on seedling stems.     

Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.     

Association between Fok I vitamin D receptor gene (VDR) polymorphism and impulsivity in alcohol-dependent patients

Vitamin D appears to have an important role in the modulation of the central nervous system. Vitamin D exerts its biological effects through its interaction with the vitamin D receptor (VDR). Located on chromosome 12 (12q13.1), the VDR gene has many different polymorphisms. Some of them are known to affect the VDR function, such as FokI (rs2228570, T/C) single nucleotide polymorphism. We aimed to explore a potential relationship between FokI VDR polymorphism and impulsiveness in alcohol-dependent (AD) patients. The study population consisted of 148 patients diagnosed with alcohol dependence (DSM-IV criteria) and 212 healthy controls. DNA was extracted from whole blood samples using the standard procedure. Genotypes were analyzed using a real-time PCR method. We found that FokI VDR gene polymorphism was associated with impulsivity [Barratt Impulsiveness Scale (BIS)-11 total score; P = 0.014], and with attentional impulsivity (BIS-11 subscale; P = 0.002) in the male AD patients. Our results suggest that CC FokI genotype of the VDR gene is associated with a higher level of impulsivity in these patients. This finding supports the hypothesis that impulsiveness, which significantly contributes to development of alcohol dependence, has a genetic background.     

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.     

The level of genetic variability of cells in prolonged suspension culture of Arabidopsis thaliana

The level of heterogeneity and genetic variability of cells in a suspension of Arabidopsis thaliana cultured in vitro for more than seven years was studied. The considerable heterogeneity of the suspension in cell size was shown. As revealed by nuclear DNA cytophotometry, the suspension culture was mixoploid and the amount of DNA in the cells varied from 4 to 16 C. However, PCR with 6 RAPD- and 4 ISSR-primers and their intragroup combinations showed the lowest degree of variability of DNA markers. The genetic distances of clones obtained from a suspension culture of the parent plant were only 1.5%. Differences between the clones were identified with only one pair of 31 primer combinations tested, indicating low level of genetic heterogeneity of the suspension. The results showed that variations in the amount of DNA in the suspension culture cells are not accompanied by significant changes in the DNA sequence.     

Expression of GroES TB antigen in tobacco and potato

As the world races towards a plant-based bioeconomy, plants known to be ideal and economical bioreactors are being harnessed for the production of recombinant proteins. The major immunodominant 10 kDa GroES TB antigen (Chaperonin 10) gene from Mycobacterium tuberculosis was selected for expression in plants as a putative tuberculosis (TB) subunit vaccine candidate. Two crops, tobacco and potato, were engineered by stable plant transformation for expression of the 10 kDa GroES TB antigen using non-viral binary vectors. The integration of the GroES TB gene into the genomes of tobacco and potato was confirmed by PCR and Southern blotting. The expression of the GroES TB antigen in tobacco was 0.04–1.2 % of the total soluble protein (TSP). However, the expression of the same TB antigen in the Indian potato cv. Kufri bahar was comparatively low (0.033 % of TSP). The recombinant GroES plant derived protein was characterised and confirmed by MALDI-TOF–TOF and ELISA. This is the first report of the expression of the 10 kDa chaperonin in tobacco and potato.     

Recombinant DNA Technology for Melanoma Immunotherapy: Anti-Id DNA Vaccines Targeting High Molecular Weight Melanoma-Associated Antigen

Anti-idiotypic MK2-23 monoclonal antibody (anti-Id MK2-23 mAb), which mimics the high molecular weight melanoma-associated antigen (HMW-MAA), has been used to implement active immunotherapy against melanoma. However, due to safety and standardization issues, this approach never entered extensive clinical trials. In the present study, we investigated the usage of DNA vaccines as an alternative to MK2-23 mAb immunization. MK2-23 DNA plasmids coding for single chain (scFv) MK2-23 antibody were constructed via the insertion of variable heavy (V _H) and light (V _L) chains of MK2-23 into the pVAC-1mcs plasmids. Two alternative MK2-23 plasmids format V _H/V _L, and V _L/V _H were assembled. We demonstrate that both polypeptides expressed by scFv plasmids in vitro retained the ability to mimic HMW-MAA antigen, and to elicit specific anti-HMW-MAA humoral and cellular immunoresponses in immunized mice. Notably, MK2-23 scFv DNA vaccines impaired the onset and growth of transplantable B16 melanoma cells not engineered to express HMW-MAA. This pilot study suggests that optimized MK2-23 scFv DNA vaccines could potentially provide a safer and cost-effective alternative to anti-Id antibody immunization, for melanoma immunotherapy.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

NDRG1 expression is related to the progression and prognosis of gastric cancer patients through modulating proliferation,invasion and cell cycle of gastric cancer cells

N-myc downstream-regulated gene 1 (NDRG1) has been proposed as a tumor suppressor gene in many different types of tumors, but its potential function and corresponding mechanism are not yet fully elucidated. This study aims to detect the possible function of NDRG1 in gastric cancer progression. In this study, 112 paired gastric cancer tissues and corresponding nonmalignant gastric tissues were utilized to identify the differential protein expression of NDRG1 by immunohistochemistry and its clinical significance was analyzed. Furthermore, 49 of 112 paired gastric specimens were used to detect the differential mRNA expression by real-time PCR. The over expression of NDRG1 in human gastric cancer cell line AGS by PcDNA3.1–NDRG1 transfection was utilized to detect the role of NDRG1 in regulating the biological behavior of gastric cancer. NDRG1 expression was significantly decreased in primary gastric cancer tissues, compared with its corresponding nonmalignant gastric tissues (p < 0.05), and its decreased expression was significantly associated with lymph node metastasis (p < 0.01), invasion depth (p < 0.01) and differentiation (p < 0.05). Additionally, the overall survival rate of gastric cancer patients with high expression of NDRG1 was higher than those with low expression during the follow-up period. NDRG1 overexpression suppressed cells proliferation, invasion and induced a G1 cell cycle arrest in gastric cancer. Furthermore, the down-regulation of NDRG1 in gastric cancer metastatic progression was correlated to E-cadherin and MMP-9. Our results verify that NDRG1 acts as a tumor suppressor gene and may play an important role in the metastasis progression and prognosis of gastric cancer.