Structure of gut microbiota and characteristics of fecal metabolites in patients with lung cancer.

Objective: The gut micro-biome plays a pivotal role in the progression of lung cancer. However, the specific mechanisms by which the intestinal microbiota and its metabolites are involved in the lung cancer process remain unclear. Method: Stool samples from 52 patients with lung cancer and 29 healthy control individuals were collected and subjected to 16S rRNA gene amplification sequencing and non-targeted gas/liquid chromatography-mass spectrometry metabolomics analysis. Then microbiota, metabolites and potential signaling pathways that may play an important role in the disease were filtered. Results: Firmicutes, Clostridia, Bacteroidacea, Bacteroides, and Lachnospira showed a greater abundance in healthy controls. In contrast, the Ruminococcus gnavus(R.gnavus) was significantly upregulated in lung cancer patients. In this respect, the micro-biome of the squamous cell carcinoma(SCC)group demonstrated a relatively higher abundance of Proteobacteria, Gammaproteobacteria, Bacteroides,and Enterobacteriaceae, as well as higher abundances of Fusicatenibacter and Roseburia in adenocarcinoma(ADC) group. Metabolomic analysis showed significant alterations in fecal metabolites including including quinic acid, 3-hydroxybenzoic acid,1-methylhydantoin,3,4-dihydroxydrocinnamic acid and 3,4-dihydroxybenzeneacetic acid were significantly altered in lung cancer patients. Additionally, the R.gnavus and Fusicatenibacter of lung cancer were associated with multiple metabolite levels. Conclusion: Our study provides essential guidance for a fundamental systematic and multilevel assessment of the contribution of gut micro-biome and their metabolites in lung cancer,which has great potential for understanding the pathogenesis of lung cancer and for better early prevention and targeted interventions.

Comparative performance of microbiological methods for the detection of tuberculous meningitis pathogens in cerebrospinal fluid.

The aim of this study was to comprehensively evaluate metagenomic next-generation sequencing (mNGS), Acid-fast bacillus stain (AFB), MGIT960 culture, polymerase-chain-reaction (PCR), and Xpert MTB/RIF in the diagnosis of tuberculous meningitis (TBM). A cohort of 280 patients who presented with suspected TBM (ie, headache or altered mental status with clinical signs of meningism) were analyzed. The sensitivities of the 5 assays for the diagnosis of TBM ranged from 10.0% to 70.0%. The AFB had the lowest sensitivity of 10.0% (0.5-45.9), while mNGS and PCR had the highest sensitivity, both at 70.0% (35.4-91.9). mNGS demonstrated a distinct advantage in identifying a wider array of pathogens, including viruses, in CSF samples. PCR was a cost-effective option with excellent sensitivity and specificity. However, no single method was statistically significantly better than any other in the diagnosis of TBM. New diagnostic techniques are urgently needed for the independent, rapid and accurate detection of Mycobacterium tuberculosis to guide the diagnosis of TBM.

[Clinical Characteristics of Nontuberculous Mycobacterium Infection Cases in Sichuan, China in 2016-2021: A Retrospective Study].

Objective: To study the distribution of nontuberculous mycobacterium (NTM) strains, clinical characteristics and drug sensitivity data of NTM infections so as to provide support for the prevention and treatment of diseases caused by NTM infection in Sichuan. Methods: The clinical data of NTM infection cases treated at the Public Health Clinical Center of Chengdu between July 2016 and July 2021 were collected and the characteristics of the infections were retrospectively reviewed. Results: There were differences in sex, age and underlying diseases among the NTM infection cases in Sichuan. Specifically, young and middle-aged men aged between 20 and 40 were susceptible to AIDS, older men aged over 60 were susceptible to lung diseases, and middle-aged and older women over 40 were susceptible to bronchiectasis. Respiratory tract was the main route of NTM infection. The dominant strain in Sichuan was M. chelonae/ abscessus. The drug resistance rate of M. avium and M. chelonae/ abscessus were relatively higher. Conclusion: For NTM infection patients with different demographic characteristics and underlying diseases, the NTM infection sites, strains, and drug resistance are also different. Definite etiological diagnosis is essential to the treatment of NTM infection. We should highlight the importance of adopting individualized treatment for different NTM infections.

Transcriptomic analysis reveals the regulatory role of quorum sensing in the Acinetobacter baumannii ATCC 19606 via RNA-seq.

BACKGROUND: Acinetobacter baumannii has emerged as the major opportunistic pathogen in healthcare-associated infections with high-level antibiotic resistance and high mortality. Quorum sensing (QS) system is a cell-to-cell bacterial communication mediated by the synthesis, secretion, and binding of auto-inducer signals. It is a global regulatory system to coordinate the behavior of individual bacteria in a population. The present study focused on the QS system, aiming to investigate the regulatory role of QS in bacterial virulence and antibiotic resistance. METHOD: The auto-inducer synthase gene abaI was deleted using the A. baumannii ATCC 19606 strain to interrupt the QS process. The RNA-seq was performed to identify the differentially expressed genes (DEGs) and pathways in the mutant (△abaI) strain compared with the wild-type (WT) strain. RESULTS: A total of 380 DEGs [the adjusted P value < 0.05 and the absolute value of log2(fold change) > log21.5] were identified, including 256 upregulated genes and 124 downregulated genes in the △abaI strain. The enrichment analysis indicated that the DEGs involved in arginine biosynthesis, purine metabolism, biofilm formation, and type VI secretion system (T6SS) were downregulated, while the DEGs involved in pathways related to fatty acid metabolism and amino acid metabolism were upregulated. Consistent with the expression change of the DEGs, a decrease in biofilm formation was observed in the △abaI strain compared with the WT strain. On the contrary, no obvious changes were found in antimicrobial resistance following the deletion of abaI. CONCLUSIONS: The present study demonstrated the transcriptomic profile of A. baumannii after the deletion of abaI, revealing an important regulatory role of the QS system in bacterial virulence. The deletion of abaI suppressed the biofilm formation in A. baumannii ATCC 19606, leading to decreased pathogenicity. Further studies on the role of abaR, encoding the receptor of auto-inducer in the QS circuit, are required for a better understanding of the regulation of bacterial virulence and pathogenicity in the QS network.

Determination of protoplast growth properties using quantitative single-cell tracking analysis.

BACKGROUND: Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. RESULTS: High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). CONCLUSIONS: AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

Analysis of the spatial characteristics and influencing factors of agricultural eco-efficiency: evidence from Anhui Province, China, during the period 2011-2018.

Sustainable agriculture is important for preserving environmental health and simultaneously gaining economic profits while maintaining social and economic equity. One way to evaluate sustainable agriculture is by studying agricultural eco-efficiency (AEE). Hence, this study constructed a data-driven method to evaluate and optimize AEE with the aim of providing a basis for improving the sustainable development of regional agriculture. Sixteen cities in Anhui Province, China, were considered in the study, and the variables used were agricultural resource inputs, environmental pollution, and agricultural economic development. Agricultural non-point source pollution (NPSP) emissions were considered the undesired output to build an AEE evaluation index system. Furthermore, a data envelopment analysis (DEA) model was established to analyse AEE from the static and dynamic perspectives. The spatial development and the temporal and spatial characteristics of AEE were also analysed. In addition, we applied a random effect (RE) panel Tobit model to quantitatively analyse the influencing factors of AEE from the input perspective and then proposed reasonable suggestions for improving the sustainable development of regional agriculture. Our findings show that the overall agricultural development in the 16 cities in Anhui Province has been continuously improving, even though there is an agglomeration of spatial development in some regions. In conclusion, this study provides suggestions and references for policy makers and agricultural practitioners regarding how to improve regional AEE and promote the sustainable development of the regional agricultural economy.

Transcriptional Profiling of Human Peripheral Blood Mononuclear Cells Stimulated by Mycobacterium tuberculosis PPE57 Identifies Characteristic Genes Associated With Type I Interferon Signaling.

Proline-glutamic acid (PE)- and proline-proline-glutamic acid (PPE)-containing proteins are exclusive to Mycobacterium tuberculosis (MTB), the leading cause of tuberculosis (TB). In this study, we performed global transcriptome sequencing (RNA-Seq) on PPE57-stimulated peripheral blood mononuclear cells (PBMCs) and control samples to quantitatively measure the expression level of key transcripts of interest. A total of 1367 differentially expressed genes (DEGs) were observed in response to a 6 h exposure to PPE57, with 685 being up-regulated and 682 down-regulated. Immune-related gene functions and pathways associated with these genes were evaluated, revealing that the type I IFN signaling pathway was the most significantly enriched pathway in our RNA-seq dataset, with 14 DEGs identified therein including ISG15, MX2, IRF9, IFIT3, IFIT2, OAS3, IFIT1, IFI6, OAS2, OASL, RSAD2, OAS1, IRF7, and MX1. These PPE57-related transcriptomic profiles have implications for a better understanding of host global immune mechanisms underlying MTB infection outcomes. However, more studies regarding these DEGs and type I IFN signaling in this infectious context are necessary to more fully clarify the underlying mechanisms that arise in response to PPE57 during MTB infection.

Nonenzymatic ß-Carotene Degradation in Provitamin A-Biofortified Crop Plants.

Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency, representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of ß-carotene were supplemented. The current notion that ß-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We quantitatively analyzed numerous plant food items and concluded that neither the amounts of apocarotenoids nor ß-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We also investigated ß-carotene degradation pathways over time. This reveals a substantial nonenzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized ß-carotene polymers that form in all plant tissues investigated.

In vitro analysis of the plastid terminal oxidase in photosynthetic electron transport.

The plastid terminal oxidase PTOX catalyzes the oxidation of plastoquinol (PQH2) coupled with the reduction of oxygen to water. In vivo PTOX is attached to the thylakoid membrane. PTOX is important for plastid development and carotenoid biosynthesis, and its role in photosynthesis is controversially discussed. To analyze PTOX activity in photosynthetic electron transport recombinant purified PTOX fused to the maltose-binding protein was added to photosystem II-enriched membrane fragments. These membrane fragments contain the plastoquinone (PQ) pool as verified by thermoluminescence. Experimental evidence for PTOX oxidizing PQH2 is demonstrated by following chlorophyll fluorescence induction. Addition of PTOX to photosystem II-enriched membrane fragments led to a slower rise, a lower level of the maximal fluorescence and an acceleration of the fluorescence decay. This effect was only observed at low light intensities indicating that PTOX cannot compete efficiently with the reduction of the PQ pool by photosystem II at higher light intensities. PTOX attached tightly to the membranes since it was only partly removable by membrane washings. Divalent cations enhanced the effect of PTOX on chlorophyll fluorescence compared to NaCl most likely because they increase connectivity between photosystem II centers and the size of the PQ pool. Using single turnover flashes, it was shown that the level of reactive oxygen species, generated by PTOX in a side reaction, increased when the spacing between subsequent double flashes was enlarged. This shows that PTOX generates reactive oxygen species under limited substrate availability.

Functional and molecular characterization of plastid terminal oxidase from rice (Oryza sativa).

The plastid terminal oxidase (PTOX) is a plastohydroquinone:oxygen oxidoreductase that shares structural similarities with alternative oxidases (AOX). Multiple roles have been attributed to PTOX, such as involvement in carotene desaturation, a safety valve function, participation in the processes of chlororespiration and setting the redox poise for cyclic electron transport. We have investigated a homogenously pure MBP fusion of PTOX. The protein forms a homo-tetrameric complex containing 2 Fe per monomer and is very specific for the plastoquinone head-group. The reaction kinetics were investigated in a soluble monophasic system using chemically reduced decyl-plastoquinone (DPQ) as the model substrate and, in addition, in a biphasic (liposomal) system in which DPQ was reduced with DT-diaphorase. While PTOX did not detectably produce reactive oxygen species in the monophasic system, their formation was observed by room temperature EPR in the biphasic system in a [DPQH2] and pH-dependent manner. This is probably the result of the higher concentration of DPQ achieved within the partial volume of the lipid bilayer and a higher Km observed with PTOX-membrane associates which is ≈47mM compared to the monophasic system where a Km of ≈74µM was determined. With liposomes and at the basic stromal pH of photosynthetically active chloroplasts, PTOX was antioxidant at low [DPQH2] gaining prooxidant properties with increasing quinol concentrations. It is concluded that in vivo, PTOX can act as a safety valve when the steady state [PQH2] is low while a certain amount of ROS is formed at high light intensities.

Reaction specificities of the ε-ionone-forming lycopene cyclase from rice (Oryza sativa) elucidated in vitro.

Lycopene cyclases responsible for the formation of ε-ionone rings (LCYe) mark a plant-specific bifurcation of carotenogenesis. We investigated purified rice LCYe (OsLCYe) in a liposome-based biphasic assay system. OsLCYe depends on reduced flavin cofactors stabilizing a transient state formed during the non-redox cyclization reaction. In contrast to OsLCYb, OsLCYe produces predominantly monocyclic products and monocyclic carotene intermediates are not suitable substrates. Determination of the OsLCYe reaction specificities and the combined use of OsLCYb allow the characterization of the reaction sequence leading to heterocyclic carotenoids. It was also found that 5-cis-lycopene, which was thought to be decisive for ε-cyclization, was not involved in the reaction, with OsLCYe acting as an exclusion filter for this naturally occurring isomer.

On the structure and function of the phytoene desaturase CRTI from Pantoea ananatis, a membrane-peripheral and FAD-dependent oxidase/isomerase.

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C(40) hydrocarbon substrate.

Plant carotene cis-trans isomerase CRTISO: a new member of the FAD(RED)-dependent flavoproteins catalyzing non-redox reactions.

The carotene cis-trans isomerase CRTISO is a constituent of the carotene desaturation pathway as evolved in cyanobacteria and prevailing in plants, in which a tetra-cis-lycopene species, termed prolycopene, is formed. CRTISO, an evolutionary descendant of the bacterial carotene desaturase CRTI, catalyzes the cis-to-trans isomerization reactions leading to all-trans-lycopene, the substrate for the subsequent lycopene cyclization to form all-trans-α/ß-carotene. CRTISO and CRTI share a dinucleotide binding motif at the N terminus. Here we report that this site is occupied by FAD in CRTISO. The reduced form of this cofactor catalyzes a reaction not involving net redox changes. Results obtained with C(1)- and C(5)-deaza-FAD suggest mechanistic similarities with type II isopentenyl diphosphate: dimethylallyl diphosphate isomerase (IDI-2). CRTISO, together with lycopene cyclase CRTY and IDI-2, thus represents the third enzyme in isoprenoid metabolism belonging to the class of non-redox enzymes depending on reduced flavin for activity. The regional specificity and the kinetics of the isomerization reaction were investigated in vitro using purified enzyme and biphasic liposome-based systems carrying specific cis-configured lycopene species as substrates. The reaction proceeded from cis to trans, recognizing half-sides of the symmetrical prolycopene and was accompanied by one trans-to-cis isomerization step specific for the C(5)-C(6) double bond. Rice lycopene ß-cyclase (OsLCY-b), when additionally introduced into the biphasic in vitro system used, was found to be stereospecific for all-trans-lycopene and allowed the CRTISO reaction to proceed toward completion by modifying the thermodynamics of the overall reaction.

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

The lycopene cyclase CrtY from Pantoea ananatis (formerly Erwinia uredovora) catalyzes an FADred-dependent non-redox reaction.

The cyclization of lycopene generates provitamin A carotenoids such as beta-carotene and paves the way toward the formation of cyclic xanthophylls playing distinct roles in photosynthesis and as precursors for regulatory molecules in plants and animals. The biochemistry of lycopene cyclization has been enigmatic, as the previously proposed acid-base catalysis conflicted with the possibility of redox catalysis as predicted by the presence of a dinucleotide binding site. We show that reduced FAD is the essential lycopene cyclase (CrtY) cofactor. Using flavin analogs, mass spectrometry, and mutagenesis, evidence was obtained based on which a catalytic mechanism relying on cryptic (net) electron transfer can be refuted. The role of reduced FAD is proposed to reside in the stabilization of a transition state carrying a (partial) positive charge or of a positively charged intermediate via a charge transfer interaction, acid-base catalysis serving as the underlying catalytic principle. Lycopene cyclase, thus, ranks among the novel class of non-redox flavoproteins, such as isopentenyl diphosphate:dimethylallyl diphosphate isomerase type 2 (IDI-2) that requires the reduced form of the cofactor.

Survey of differentially methylated promoters in prostate cancer cell lines.

DNA methylation and copy number in the genomes of three immortalized prostate epithelial and five cancer cell lines (LNCaP, PC3, PC3M, PC3M-Pro4, and PC3M-LN4) were compared using a microarray-based technique. Genomic DNA is cut with a methylation-sensitive enzyme HpaII, followed by linker ligation, polymerase chain reaction (PCR) amplification, labeling, and hybridization to an array of promoter sequences. Only those parts of the genomic DNA that have unmethylated restriction sites within a few hundred base pairs generate PCR products detectable on an array. Of 2732 promoter sequences on a test array, 504 (18.5%) showed differential hybridization between immortalized prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, there were eight (CD44, CDKN1A, ESR1, PLAU, RARB, SFN, TNFRSF6, and TSPY) previously observed in prostate cancer and 13 previously known methylation targets in other cancers (ARHI, bcl-2, BRCA1, CDKN2C, GADD45A, MTAP, PGR, SLC26A4, SPARC, SYK, TJP2, UCHL1, and WIT-1). The majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, and GG2-1, representing a rich new source of candidate genes used to study the role of DNA methylation in prostate tumors.

"Promoter array" studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells.

DNA microarrays of promoter sequences have been developed in order to identify the profile of genes bound and activated by DNA regulatory proteins such as the transcription factors c-Jun and ATF2 as well as DNA-modifying methylases. The arrays contain 3083 unique human promoter sequences from +500 to -1000 nts from the transcription start site. Cisplatin-induced DNA damage rapidly leads to specific activation of the Jun kinase pathway leading to increased phosphorylation of c-Jun and ATF2-DNA complexes at hundreds of sites within 3 hours. Using three statistical criteria, approximately 269 most commonly phosphorylated c-Jun/ATF2-DNA complexes were identified and representative cases were verified by qPCR measurement of ChIP-captured DNA. Expression was correlated at the mRNA and protein levels. The largest functional cohort was 24 genes of known DNA repair function, most of which exhibited increased protein expression indicated coordinate gene regulation. In addition, cell lines of prostate cancer exhibit stable methylation or copy number changes that reflect the alterations of the corresponding primary tumors. 504 (18.5%) promoters showed differential hybridization between immortalized control prostate epithelial and cancer cell lines. Among candidate hypermethylated genes in cancer-derived lines, eight had previously been observed in prostate cancer, and 13 were previously determined methylation targets in other cancers. The vast majority of genes that appear to be both differentially methylated and differentially regulated between prostate epithelial and cancer cell lines are novel methylation targets, including PAK6, RAD50, TLX3, PIR51, MAP2K5, INSR, FBN1, GG2-1, representing a rich new source of candidate genes to study the role of DNA methylation in prostate tumors. Earlier studies using prototype promoter arrays examine approximately 7% of the proximal regulatory sequences while the current gene regulatory events surveyed here occur on a large scale and may rapidly effect the coordinated expression of a large number of genes.

Cloning of the promoter region of plasma membrane aquaporinBnPIP1 fromBrassica napus and its functional analysis.

A 1.6 kb upstream regulatory sequence (GenBank accession no. AF472487) of plasma membrane aquaporinBnPIP1 gene fromBrassica napus was obtained by genomic walking based on ligation-mediated PCR method. Sequence analysis indicated that this fragment contained seed germination specific and vascular specific sequences. The 1.6 kb upstream sequence and various 5' end deleted sequences were fused withuidA gene and constructed into plant expression vectors which were used for tobacco transformation. GUS histochemical assay showed that the 1.6 kb fragment had high levels of promoter activity and the GUS staining was mainly distributed in vascular systems and tissues with rapid expanding and proliferating cells. Promoter deletion analysis showed that the deletion of -1610 - -1030 bp resulted in a dramatic reduction in GUS activity. It was assumed that there might be cis-acting element(s) existing in this region. Whereas, the region located at -1030 - -902 bp strongly inhibited the expression ofgus and probably contained negative regulatory element(s). The fragment of -902 - -19 bp could also directgus expression at high level.