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Cotton fiber is an economically important seed trichome and the world's leading natural fiber used in the manufacture of textiles. As a step toward elucidating the genomic organization and distribution of gene networks responsible for cotton fiber development, we investigated the distribution of fiber genes in the cotton genome. Results revealed the presence of gene-rich islands for fiber genes with a biased distribution in the tetraploid cotton (Gossypium hirsutum L.) genome that was also linked to discrete fiber developmental stages based on expression profiles. There were 3 fiber gene-rich islands associated with fiber initiation on chromosome 5, 3 islands for the early to middle elongation stage on chromosome 10, 3 islands for the middle to late elongation stage on chromosome 14, and 1 island on chromosome 15 for secondary cell wall deposition, for a total of 10 fiber gene-rich islands. Clustering of functionally related gene clusters in the cotton genome displaying similar transcriptional regulation indicates an organizational hierarchy with significant implications for the genetic enhancement of particular fiber quality traits. The relationship between gene-island distribution and functional expression profiling suggests for the first time the existence of functional coupling gene clusters in the cotton genome.

Seed storage proteins (SSPs) are important for seed germination and early seedling growth. We studied the accumulation and degradation profiles of four major Arabidopsis 12S SSPs using a 2-DE scheme combined with mass spectrometric methods. On the 2-DE map of 23 dpa (days post anthesis) siliques, 48 protein spots were identified as putative full-length or partial alpha, beta subunits. Only 9 of them were found in 12 dpa siliques with none in younger than 8 dpa siliques, indicating that the accumulation of 12S SSPs started after the completion of cell elongation processes both in siliques and in developing seeds. The length and strength of transcription activity for each gene determined the final contents of respective SSP. At the beginning of imbibition, 68 SSP spots were identified while only 2 spots were found at the end of the 4 d germination period, with alpha subunits degraded more rapidly than the beta subunits. The CRC alpha subunit was found to degrade from its C-terminus with conserved sequence motifs. Our data provide an important basis for understanding the nutritional value of developing plant seeds and may serve as a useful platform for other species.

Proteins accumulated in dry, stratified Arabidopsis seeds or young seedlings, totaled 1100 to 1300 depending on the time of sampling, were analyzed by using immobilized pH gradient 2-DE gel electrophoresis. The molecular identities of 437 polypeptides, encoded by 355 independent genes, were determined by MALDI-TOF or TOF-TOF mass spectrometry. In the sum, 293 were present at all stages and 95 were accumulated during the time of radicle protrusion while another 18 appeared in later stages. Further analysis showed that 226 of the identified polypeptides could be located in different metabolic pathways. Proteins involved in carbohydrate, energy and amino acid metabolism constituted to about 1/4, and those involved in metabolism of vitamins and cofactors constituted for about 3 % of the total signal intensity in gels prepared from 72 h seedlings. Enzymes related to genetic information processing increased very quickly during early imbibition and reached highest level around 30 h of germination.

The origin of non-coding sequences, especially introns,is an outstanding issue that has been receiving continuous debate for the last two decades. In the current work we use a mathematical model to characterize DNA sequences and find that the 3-tuple distributions in different reading frames of a given coding sequence differ sharply from each other, while they are almost identical to each other in introns or other non-coding sequences. SREs (Symmetric relative entropies) decrease progressively from coding sequences of primitive prokaryotes to those of advanced eukaryotes and from non-coding sequences of low eukaryotes to those of high eukaryotes with a correlation coefficient of 0.86. In silico evolution experiments show that SREs typical of higher eukaryotic introns can be achieved from prokaryotic coding sequences as the mutation ratio reaches 2/100. The fact that (a total of 25 introns) from all three different genomes S. pombe, C. elegans and H. sapiens searched are found to share high sequence identity with coding regions indicates that at least some introns may have come directly from CDS (coding sequences). We suggest that SREs may be a useful feature for evolutionary study.

A cDNA that was rapidly induced upon abscisic acid, cold, drought, mechanical wounding and to a lesser extent, by high salinity treatment, was isolated from Arabidopsis seedlings. It was classified as DREB subfamily member based on multiple sequence alignment and phylogenetic characterization. Since it encoded a protein with a typical ERF/AP2 DNA-binding domain and was closely related to the TINY gene, we named it TINY2. Gel retardation assay revealed that TINY2 was able to form a specific complex with the previously characterized DRE element while showed only residual affinity to the GCC box. When fused to the GAL4 DNA-binding domain, either full-length or its C-terminus functioned effectively as a trans-activator in the yeast one-hybrid assay while its N-terminus was completely inactive. Our data indicate that TINY2 could be a new member of the AP2/EREBP transcription factor family involved in activation of down-stream genes in response to environmental stress.

To investigate the imunogenicity and protective efficacy of the divalent DNA vaccine encoding the antigens MPT83 and MPT64 of Mycobacterium tuberculosis.Two genes of the M. tuberculosis, MPT83 and MPT64, were amplified and cloned into the vector pJW4303. The vector containing the fusion gene DNA-MM and pJW4303 blank vector were transfected into CO57 cells. The expression of DNA-MM in the supernatant was detected by Western blotting. Thirty-six C57BL/6 mice were divided into 3 equal groups to be injected subcutaneously with the vector containing the fusion gene DNA-MM, pJW4303 blank vector, or bacillus of Calmette-Guerin vaccine (BCG) once a week for 3 weeks. Twelve mice were used as non-immunized controls. Blood samples were collected from the mice before and after every immunization to detect the titer of antibody by ELISA. Three weeks after the 3rd immunization 3 mice from each were killed and their spleens were taken out. The splenocytes were cultured and stimulated by the recombinant antigen to detect the concentrations of interferon (IFN)-gamma and interleukin (IL)-4. Six weeks after the 3rd immunization M. tuberculosis of the line H37Rv was injected intravenously into the mice. Eight weeks later, the mice were killed and their lungs and spleens were taken out. The tissues were cultured to calculate the numbers of M. tuberculosis.Western blotting showed that the CO57 cells transfected with the vector containing the fusion gene expressed DNA-MM. ELISA showed that no antibody titer was detected before immunization in every group. The titer of MPT83 and MPT64 were increased to 1:200, 1:800, and 1:6400 3 weeks after the 1st, 2nd, and 3rd immunization respectively in the divalent group, increased to 1:800 3 weeks after the 3rd immunization in the BCG group, and remained 0 at any time points in the blank vector group. The IFN-gamma titer of the divalent group was 7520 pg/ml, significantly higher than that of the BCG group (6675.6 pg/ml, P0.05). The IL-4 concentration was basically the same and at the ng level in all groups. After challenge of H37Rv the bacterial loading in the lung was (2.21 +/- 0.032) x 10(5) in the divalent group, one thousandth of that in the blank vector group, and one tenth of that in the BCG group.The divalent vaccine containing the antigens MPT83 and MPT64 effectively heightens the protective efficiency of vaccine against tuberculosis.

Genes encoding enzymes involved in biosynthesis of very long chain fatty acids were significantly up-regulated during early cotton fiber development. Two cDNAs, GhKCR1 and GhKCR2 encoding putative cotton 3-ketoacyl-CoA reductases that catalyze the second step in fatty acid elongation, were isolated from developing cotton fibers. GhKCR1 and 2 contain open reading frames of 963 bp and 924 bp encoding proteins of 320 and 307 amino acid residues, respectively. Quantatitive RT-PCR analysis showed that both these genes were highly preferentially expressed during the cotton fiber elongation period with much lower levels recovered from roots, stems and leaves. GhKCR1 and 2 showed 30%-32% identity to Saccharomyces cerevisiae Ybr159p at the deduced amino acid level. These cotton cDNAs were cloned and expressed in yeast haploid ybr159wΔ mutant that was deficient in 3-ketoacyl-CoA reductase activity. Wild-type growth rate was restored in ybr159wΔ cells that expressed either GhKCR1 or 2. Further analysis showed that GhKCR1 and 2 were co-sedimented within the membranous pellet fraction after high-speed centrifugation, similar to the yeast endoplasmic reticulum marker ScKar2p. Both GhKCR(s) showed NADPH-dependent 3-ketoacyl-CoA reductase activity in an in vitro assay system using palmitoyl-CoA and malonyl-CoA as substrates. Our results suggest that GhKCR1 and 2 are functional orthologues of ScYbr159p.

AP2/EREBP transcription factors (TFs) play functionally important roles in plant growth and development, especially in hormonal regulation and in response to environmental stress. Here we reported verification and correction of annotation through an exhaustive cDNA cloning and sequence analysis performed on 145 of 147 gene family members. A RACE analysis performed on genes with potential in-frame up-stream ATG codon resulted in identification of At2g28520 as an authentic AP2/EREBP member and corrected ORF annotations for three other members. A further phylogenetic analysis of this updated and likely complete family divided it into three major subfamilies. The expression patterns of the AP2/EREBP family members among the 11 organ or tissue types were examined using an oligo microarray and their hormonal and environmental responsiveness were further characterized using cDNA custom macroarrays. These detailed expression profile results provide strong support for a role for AP2/EREBP family members in development and in response to environmental stimuli, and a foundation for future functional analysis of this gene family.

We tested the effectiveness of combined DNA vaccines encoding antigens Ag85B, MPT64 and MPT83 from Mycobacterium tuberculosis on cattle. Our results showed that calves treated with combined DNA vaccines in the presence of dimethyldioctyldecyl ammonium bromide (DDA) or saline elicited a strong gamma interferon (IFN-gamma) response 1 or 2 months after the third vaccination. All three antigens induced substantial levels of IFN-gamma production 1 month after the bacterial challenge, when the BCG-driven IFN-gamma levels dropped to less than one third of their peak values. Animals receiving combined DNA vaccines produced highest amounts of IgG antibody titer 2 months after the third vaccination. Steady state low IgG levels were found 2 months after bacterial challenge. A few small lung and lymph node lesions were detected in 1/5 animals treated with combined DNA vaccines, whereas 3/5 of BCG-treated and 5/5 of vector-control calves showed larger and significantly more lesions. About 70- to 100-fold fewer bacteria were found in the lungs and lymph nodes of combined DNA vaccine-treated animals compared to those of the control group. Histopathological analyses showed that vaccinated calves possessed substantially improved post-infection lung and lymph node pathology relative to the controls. Our data indicate that combined DNA vaccines may be used in cattle to combat bovine tuberculosis.

This study evaluated the adjuvant Dimethyldioctyldecyl Ammonium Bromide (DDA) effect on the protective immunity induced by a combination of plasmids containing genes encoding antigens Ag85B, MPT-83, and ESAT-6 from Mycobacterium tuberculosis. The combined DNA vaccines in DDA resulted in significant increases in both specific IgG and splenic T-cell-derived Th1-type cytokine gamma interferon (IFN-gamma) production in response to the three purified antigens when compared to that of combined DNA vaccines in saline. Vaccines in DDA increased the protective efficacy of mice challenged with M. tuberculosis H37Rv as measured by reduced relative CFU counts in their lungs. Mice immunized with the combined DNA vaccines were shown to limit the growth of tubercle bacilli both in lungs and in spleens. Histopathological analyses showed that vaccinated mice had substantially improved postinfection lung pathology relative to the controls. We suggest that our combination of antigens together with DDA formulation may provide a new insight into tuberculosis prevention.

Cotton fibers are differentiated epidermal cells originating from the outer integuments of the ovule. To identify genes involved in cotton fiber elongation, we performed subtractive PCR using cDNA prepared from 10 days post anthesis (d.p.a.) wild-type cotton fiber as tester and cDNA from a fuzzless-lintless (fl) mutant as driver. We recovered 280 independent cDNA fragments including most of the previously published cotton fiber-related genes. cDNA macroarrays showed that 172 genes were significantly up-regulated in elongating cotton fibers as confirmed by in situ hybridization in representative cases. Twenty-nine cDNAs, including a putative vacuolar (H+)-ATPase catalytic subunit, a kinesin-like calmodulin binding protein, several arabinogalactan proteins and key enzymes involved in long chain fatty acid biosynthesis, accumulated to greater than 50-fold in 10 d.p.a. fiber cells when compared to that in 0 d.p.a. ovules. Various upstream pathways, such as auxin signal transduction, the MAPK pathway and profilin- and expansin-induced cell wall loosening, were also activated during the fast fiber elongation period. This report constitutes the first systematic analysis of genes involved in cotton fiber development. Our results suggest that a concerted mechanism involving multiple cellular pathways is responsible for cotton fiber elongation.

Three proteins secreted from Mycobacterium tuberculosis, Ag85B, ESAT-6 and MPT63 were selected as antigens for making combined DNA vaccine by immunizing mice. The immune response induced by the vaccine and its protective efficacy were studied. It was demonstrated that when mice were immunized with the combined DNA vaccine, the titer of antibody for Ag85B in serum increased to more than 10(5), but the titers of ESAT-6 and MPT63 specific antibodies were undetectable. After the final immunization, the level of gamma specific for Ag85B, ESAT-6 and MPT63 reached (17.0+/-7.0) u/ml, (6.0+/-0.8) u/ml and (11.9+/-8.0) u/ml, respectively. Mice, that were inoculated with the empty eukaryotic expression vector pJW4303 DNA, produced negligible amounts of antigen-specific INF-gamma. The combined DNA vaccine resulted also in significantly reduced amount of bacteria in the lungs of experimental mice. Microphotographs showed clearly that these lungs were better protected against Mycobacterium tuberculosis challenge than control mice. The combined DNA vaccine reported in this work shed new lights on the prophylactic protection against tuberculosis.

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

Using cDNA representational difference analysis (cDNA RDA), we isolated a cDNA named GDA-1 from a cDNA library constructed with mRNA from short-day (SD) grown G2 pea apical tissue. The amino acid sequence deduced from GDA-1 shares partial identity with the B2 protein which is expressed during embryogenesis of carrot cells. Northern analysis showed that GDA-1 mRNA is abundant in SD-grown G2 pea apical buds. In long-day (LD) conditions, there was almost no detectable GDA-1 mRNA. When LD-grown G2 peas were kept in continuous darkness for 24 h, the GDA-1 mRNA content reached a level equivalent to about 50% of that in the SD samples. On the other hand, when SD-grown peas were transferred into the light for 24 h, the amount of hybridizable GDA-1 mRNA dropped to the same as that of LD-grown plants. GDA-1 expression was found to be independent of flower initiation time. GA3 application in vitro resulted in rapid accumulation of GDA-1 mRNA in LD-grown G2 pea apical buds, which is compatible with its delaying effect on apical senescence. Time-course experiments revealed that GDA-1 is induced within 15 min of GA3 application. Exogenous GA3 did not influence the expression of GDA-1 in SD-grown G2 peas. Since both photoperiod and GA induce the expression of GDA-1, we speculate that they may activate similar signal transduction pathways in G2 peas. Our work also shows that photoperiod may change the efficiency of gibberellin perception by plants.

DNA methylation controls many aspects of plant growth and development. Here, we report a novel annual growth potential change that may correlate with changes in levels of the major DNA demethylases and methyltransferases in cotton ovules harvested at different times of the year. The abundances of DNA demethylases, at both the mRNA and protein levels, increased significantly from February to August and decreased during the remainder of the 12-month period, with the opposite pattern observed for DNA methyltransferases. Over the course of one year, substantial changes in methylcytosine content was observed at certain CHH sites (H = A, C, or T) in the promoter regions of the ETHYLENE RESPONSIVE FACTOR 6 (ERF6), SUPPRESSION OF RVS 161 DELTA 4 (SUR4) and 3-KETOACYL-COA SYNTHASE 13 (KCS13), which regulate cotton fiber growth. Three independent techniques were used to confirm the annual fluctuations in DNA methylation. Furthermore, in homozygous RNAi lines specifically targeting REPRESSOR OF SILENCING 1 (ROS1, a conserved DNA demethylase domain), promotion of DNA methylation significantly reduced fiber growth during August.

The cucumber mosaic virus coat protein (CMV-CP) gene and a modified Bacillus thuringiensis δ-endotoxin (Bt toxin) gene were cloned into plant expression vector pE3. Tobacco (Nicotiana tabacum cv. G28) leaf discs were transformed with Agrobacterium tumefaciens A12 carrying recombinant pE14. Transgenic r0 and R1 tobacco plants expressing CMV-CP and Bt toxin genes were protected from CMV infection as well as feeding damage of Manduca Sexta (tobacco hornworm) larvae. These results demonstrate that it is feasible to breed new cultivars with multiple resistances via genetic engineering.

Metabolism of [(14)C]gibberellin (GA) A(12) (GA(12)) and [(14)C]gibberellin A(12)-aldehyde (GA(12)-aldehyde) was examined in cotyledons and seed coats from developing seeds of pea (Pisum sativum L.). Both were metabolized to only 13-hydroxylated GAs in cotyledons but to 13-hydroxylated and non-13-hydroxylated GAs in seed coats. The metabolism of [(14)C]GA(12) was slower in seed coats than in cotyledons. [(14)C]GA(12)-aldehyde was also metabolized to conjugates in seed coats. Seed coat [(14)C]-metabolites produced from [(14)C]GA(12)-aldehyde were isolated by high-performance liquid chromatography (HPLC). Conjugates were base hydrolyzed and the free GAs reisolated by HPLC and identified by gas chromatography-mass spectrometry. [(14)C]GA(53)-aldehyde, [(14)C]GA(12)-aldehyde conjugate, and [(14)C]GA(53)-aldehyde conjugate were major metabolites produced from [(14)C]GA(12)-aldehyde by seed coats aged 20-22 days or older. The dilution of (14)C in these compounds by (12)C, as compared to the supplied [(14)C]GA(12)-aldehyde, indicated that they are endogenous. Feeding [(14)C]GA(53)-aldehyde led to the production of [(14)C]GA(53)-aldehyde conjugate in seed coats and shoots and also to 13-hydroxylated GAs in shoots. Labeled GAs, recovered from plant tissue incubated with either [(14)C]GA(12), [(14)C]GA(12)-aldehyde, or [(3)H]GA(9), were used as appropriate markers for the recovery of endogenous GAs from seed coats or cotyledons. These GAs were purified by HPLC and identified and quantified by gas chromatography-mass spectrometry. GA(15), GA(24), GA(9), GA(51), GA(51)-catabolite, GA(20), GA(29), and GA(29)-catabolite were detected in seed coats, whereas GA(9), GA(53), GA(44), GA(19), GA(20), and GA(29) were found in cotyledons. The highest GA levels were for GA(20) and GA(29) in cotyledons (783 and 912 nanograms per gram fresh weight, respectively) and for GA(29) and GA(29)-catabolite in seed coats (1940 and > 1940 nanograms per gram fresh weight, respectively).

Immune response and protective efficacy for the combination of four tuberculosis DNA vaccines were evaluated in this study. We obtained 1: 200 antibody titers against Ag85B 21d after mice were vaccinated for the first time by four recombinant eukaryotic expression vectors containing coding sequences for Ag85B, MPT-64, MPT-63 and ESAT-6. The titers of Ag85B were elevated to 1: 102400 after the second injection and decreased to 1: 12800 after the third injection. Antibody titers for MPT-64 and MPT-63 reached 1: 25600 21 d after the first vaccination, and were then decreased following the second and third injections. No antigen-specific antibody titer against ESAT-6 was detected in sera harvested from immunized mice at any time. These DNA vaccines evoked specific IFN-gamma responses in the spleens of vaccinated mice as well. When challenged with M. tuberculosis H37Rv, we found that the lungs of the vaccinated mice produced 99.8% less bacterial counts than that of the empty-vector control group and the bacterial counts were also significantly less than that of the BCG group. Histopathological analyses showed that the lungs of vaccinated mice produced no obvious caseation while over 50%-70% of the pulmonary parenchyma tissue produced central caseation in the vector control group. Our results indicated that the combination of four tuberculosis DNA vaccines may generate high levels of immune responses and result in better animal protection.

To interpret the metabolism of radiolabeled gibberellins A(12)-aldehyde and A(12) in shoots of pea (Pisum sativum L.), the identity of the radiolabeled peaks has to be determined and the endogenous presence of the gibberellins demonstrated. High specific activity [(14)C]GA(12) and [(14)C]GA(12)-aldehyde were synthesized using a pumpkin endosperm enzyme preparation, and purified by high performance liquid chromatography (HPLC). [(14)C]GA(12) was supplied to upper shoots of pea, line G2, to produce radiolabeled metabolites on the 13-OH pathway. Endogenous compounds copurifying with the [(14)C]GAs on HPLC were analyzed by gas chromatography-mass spectrometry. The endogenous presence of GA(53), GA(44), GA(19) and GA(20) was demonstrated and their HPLC peak identity ascertained. The (14)C was progressively diluted in GAs further down the pathway, proportional to the levels found in the tissue and inversely proportional to the speed of metabolism, ranging from 63% in GA(53) to 4% in GA(20). Calculated levels of GA(20), GA(19), GA(44), and GA(53) were 42, 8, 10, and 0.5 nanograms/gram, respectively.

While gibberellin (GA) A12 and GA12-7-aldehyde have been well established as intermediates in GA biosynthesis, their presence in higher plants has not been conclusively demonstrated, with the exception of GA12-aldehyde in mature pea seeds. High specific activity [14C] GA12 and [14C]GA12-aldehyde were synthesized enzymatically from [4,5-14C]mevalonic acid, using a pumpkin endosperm preparation, and purified by HPLC. These were added to developing pea seeds during homogenization and the radioactive material purified by HPLC. Mass spectra of GA12 and GA12-aldehyde were obtained following gas chromatography. The ratio of the 12C to 14C mass ions for the radio-labelled GAs, and the seed extract with added radio-labelled GAs as internal standards, was examined. There was a 123 and a 579% enrichment of the 12C present in the GA12 and GA12-aldehyde spectra, respectively, relative to 14C, proving the presence of endogenous GA12 and GA12-aldehyde in the developing pea seeds. The amounts present were very low, namely 1.2 ng/g fresh weight for GA12 and 8.4 ng/g fresh weight for GA12-aldehyde.

BARD1 (BRCA1 associated RING domain protein 1), as an important animal tumor suppressor gene associated with many kinds of cancers, has been intensively studied for decades. Surprisingly, homolog of BARD1 was found in plants and it was renamed AtROW1 (repressor of Wuschel-1) according to its extremely important function with regard to plant stem cell homeostasis. Although great advances have been made in human BARD1, the function of this animal tumor-suppressor like gene in plant is not well studied and need to be further elucidated. Here, we review and summarize past and present work regarding this protein. Apart from its previously proposed role in DNA repair, recently it is found essential for shoot and root stem cell development and differentiation in plants. The study of AtROW1 in plant may provide an ideal model for further elucidating the functional mechanism of BARD1 in mammals.