Biosurfer for systematic tracking of regulatory mechanisms leading to protein isoform diversity.

Long-read RNA sequencing has shed light on transcriptomic complexity, but questions remain about the functionality of downstream protein products. We introduce Biosurfer, a computational approach for comparing protein isoforms, while systematically tracking the transcriptional, splicing, and translational variations that underlie differences in the sequences of the protein products. Using Biosurfer, we analyzed the differences in 32,799 pairs of GENCODE annotated protein isoforms, finding a majority (70%) of variable N-termini are due to the alternative transcription start sites, while only 9% arise from 5' UTR alternative splicing. Biosurfer's detailed tracking of nucleotide-to-residue relationships helped reveal an uncommonly tracked source of single amino acid residue changes arising from the codon splits at junctions. For 17% of internal sequence changes, such split codon patterns lead to single residue differences, termed "ragged codons". Of variable C-termini, 72% involve splice- or intron retention-induced reading frameshifts. We found an unusual pattern of reading frame changes, in which the first frameshift is closely followed by a distinct second frameshift that restores the original frame, which we term a "snapback" frameshift. We analyzed long read RNA-seq-predicted proteome of a human cell line and found similar trends as compared to our GENCODE analysis, with the exception of a higher proportion of isoforms predicted to undergo nonsense-mediated decay. Biosurfer's comprehensive characterization of long-read RNA-seq datasets should accelerate insights of the functional role of protein isoforms, providing mechanistic explanation of the origins of the proteomic diversity driven by the alternative splicing. Biosurfer is available as a Python package at https://github.com/sheynkman-lab/biosurfer.

Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes.

Protein-coding genes in trypanosomes occur in polycistronic transcription units (PTUs). How RNA polymerase II (Pol II) initiates transcription of PTUs has not been resolved; the current model favors chromatin modifications inducing transcription rather than sequence-specific promoters. Here, we uncover core promoters by functional characterization of Pol II peaks identified by chromatin immunoprecipitation sequencing (ChIP-seq). Two distinct promoters are located between divergent PTUs, each driving unidirectional transcription. Detailed analysis identifies a 75-bp promoter that is necessary and sufficient to drive full reporter expression and contains functional motifs. Analysis of further promoters suggests transcription initiation is regulated and promoters are either focused or dispersed. In contrast to the previous model of unregulated and promoter-independent transcription initiation, we find that sequence-specific promoters determine the initiation of Pol II transcription of protein-coding genes PTUs. These findings in Trypanosoma brucei suggest that in addition of chromatin modifications, promoter motifs-based regulation of gene expression is deeply conserved among eukaryotes.

Clinical significance of telomerase reverse transcriptase promoter mutation in radioactive iodine refractory papillary thyroid cancer / 中华核医学与分子影像杂志

Objective:To evaluate the influence of telomerase reverse transcriptase (TERT) promoter mutation on radioiodine uptake status of radioactive iodine refractory papillary thyroid cancer (RAIR-PTC) and radioiodine therapy response by analyzing the mutation frequency of TERT promoter in RAIR-PTC.Methods:A total of 37 patients with RAIR-PTC (15 males, 22 females, age (49.8±16.1) years) and 40 PTC patients with effective radioiodine therapy (13 males, 27 females, age (39.8±10.9) years) between January 2005 and June 2020 in JiangYuan Hospital Affiliated to Jiangsu Institute of Nuclear Medicine were retrospectively analyzed. TERT promoter mutation and B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E mutation of patients were observed. The differences across genotype patterns on radioiodine uptake status and therapy response were compared. The Fisher′s exact test and independent-sample t test were used for data analysis. Results:The incidence rate of TERT promoter mutation in the RAIR-PTC group was 40.54% (15/37, all C228T), which was significantly higher than that in the effective radioiodine therapy group (0, 0/40; P<0.001). No statistically significant difference was found for the mutation rate of BRAF V600E between the RAIR group (64.86%, 24/37) and the effective radioiodine therapy group (72.50%, 29/40; P=0.858). Patients with TERT promoter mutation were older ( t=3.76, P=0.001) and the non-intake rate of radioiodine in distant metastases of those patients was higher ( P=0.037). Furthermore, 2/3 of patients who received targeted therapies and 3/4 deaths had TERT promoter mutation. Among 35 patients with negative thyroglobulin antibody (TgAb), 11/14 of patients with TERT mutation had a rising stimulated thyroglobulin (sTg), while the percentage of the non-TERT mutation group was 57.1% (12/21; P=0.357). Conclusion:The TERT promoter mutation rate is significantly increased in RAIR-PTC patients and can serve as a prognostic predictor in RAIR.

Construction and transcriptional activity of human CREG promoter reporter gene plasmid / 解放军医学杂志

Objective To construct the promoter enhanced green fluorescent protein (pEGFP1) reporter gene vector of different truncated fragments of human cellular repressor of E1A-stimulated genes (hCREG), and compare the transcriptional activity of each promoter to determine the hCREG core promoter region. Methods The promoter fragment with length of 2003 (–1925/+78) bp was obtained by querying the hCREG sequence from US National Center of Biotechnology Information (NCBI) database and combining with the characteristics of the promoter. Five promoter fragments were truncated by PCR and double enzyme digestion and cloned into pEGFP1 to construct pEGFP1_hCREG_2003, pEGFP1_hCREG_945, pEGF P1_hCREG_586, pEGFP1_ hCREG_478 and pEG FP1_hCREG_358 reporter gene vector plasmid. The 293T cells were transiently co-transfected with the internal reference plasmid pGL4.73 [hRluc/SV40] for 48 hours. The green fluorescence expression of pEGFP1_hCREG promoter reporter gene was observed under fluorescence microscope, and the mRNA expression of each promoter was detected by real-time quantitative PCR, and the core promoter region was determined. Bioinformatics was used to predict the transcription factors that might bind to the core promoter region. Results Five hCREG promoter reporter gene vectors were successfully constructed by double enzyme digestion and gene sequencing. The results showed that the transcription activity of pEGFP1_ hCREG_586 was the highest (P0.05), implying that –867/ –509 bp is a negative regulatory region, and there existed enhancer sequences in –400/–281 bp and –508/–401 bp, so the core promoter region of hCREG gene is located in the upstream sequence of –508/–281 bp. Bioinformatics predicted that the possibly bound transcription factors in key promoter region –508/–281 bp were Pax5/P53, C/EBPβ, GR-β, GATA-1, GR-α, c-Jun, PRB/ PRA, YY1, RXR-α, AP-2, FOXP3, GR, TFIID, STAT4 and c-Ets-1. Conclusion The recombinant plasmid of hCREG gene promoter has been successfully constructed, the core promoter of which is located in –508/–281 bp, where several transcription factors might be bound.

Nucleosomes positioning around transcriptional start site of tumor suppressor (Rbl2/p130) gene in breast cancer.

Dynamic positioning of nucleosomes is pivotal in determining level of genes expression especially on or around transcription start site (TSS) of a gene. Purpose of the current study was to determine nucleosome position around TSS of Rbl2/p130. We investigated Rbl2/p130 expression in connection to nucleosome positions around its TSS among breast tumors and their adjacent normal control tissues (ANCT) using micrococcal nuclease (MNAse) digestion assay and ChIP-PCR analysis. Three fold reduced Rbl2/p130 expression in these tumor tissues were noticed compared to their control tissues. DNA obtained from MNAse digested chromatin was used as PCR template. Region between - 137 to + 140 around TSS was scanned using 3 primer pairs (P1 = - 137 to + 69; P2 = - 90 to + 69; P3 = - 33 to + 140). ~ 66% breast tumors and ~ 26% ANCT samples were positive for P1. The difference was found statistically significant (p = 0.000) with an odd ratio (OD) of 9.143, suggesting that nucleosome formation in this region is ~ 9 times more probable in tumor samples. ~ 73% of the tumor and 60% ANCT were positive for P2, which although is significant (p = 0.035) with OD = 3.250, but less preferable than P1. However, P3 was not found to be a preferred area for nucleosome occupancy (p = 0.670; OD = 1.2). Negative correlations for nucleosome positions were observed especially for P1. Our results indicate that nucleosome are present slightly downstream of TSS in routine, while in case of breast carcinogenesis nucleosomes slides 55 bases upstream of the TSS, aligning + 1 position at the center of nucleosome, hence hindering access to the transcriptional machinery.

Biological molecules found to serve as prostate cancer specific promoters during construction of oncolytic adenovirus / 肿瘤

Prostate cancer is a common malignant disease in elder men, and it is the second leading death cause in male cancer patients. The incidence rate of prostate cancer has been increasing year by year, and it is ranked the third in the incidence rate of urologic cancers in China. The oncolytic adenovirus carrying specific promoter has been used as a novel target of antitumor therapy. The adenovirus has specific effects on tumor cells and can express downstream therapeutic genes to inhibit the growth of tumor cells, but it can't influence the normal cells or only has weak influence. At present, oncolytic adenovirus carrying specific promoter which has been used widely in basic and clinical studies has showed unique advantages in great antitumor effect and medication safety. This paper reviews the several biological molecules, which have been found during the construction of oncolytic adenovirus, may potentially become to be prostate cancer-specific promoters.

Association of tumor necrosis factor-α gene promoter polymorphisms with generalized pustular psoriasis / 中华皮肤科杂志

Objective To investigate the association of tumor necrosis factor-ot (TNF-α) gene promoter polymorphisms with generalized pustular psoriasis.Methods Totally,91 patients of Han nationality with generalized pustular psoriasis (generalized pustular psoriasis group) and 102 health checkup examinees (healthy control group) were enrolled into this study.PCR and direct sequencing were performed to analyze the-238,-308 and-857 polymorphic sites of the TNF-α promoter.Results The frequency of the A allele at TNF-α-238 site was significantly higher in the generalized pustular psoriasis group than in the healthy control group (P =0.003,OR =4.819,95％ CI:1.581-14.694),so was the frequency of GA/AA genotype (P =0.006,OR =4.455,95％ CI:1.410-14.077).However,no significant differences were observed in the frequencies of G/A alleles (P =0.794) and GG/GA/AA genotypes (P =0.786) at TNF-o-308 site,or in the frequencies of C/T alleles (P =0.474) and CC/CT/TT genotypes (P =0.453) at TNF-α-857 site,between the generalized pustular psoriasis group and healthy control group.Conclusion TNF-α-238G ＞ A polymorphisms may be associated with the occur-rence of generalized pustular psoriasis.

Constructing the dual luciferase reporter vector containing human DRD1 promoter region / 中国组织工程研究

BACKGROUND:The polymorphisms of dopamine receptor in promoter region wil affect the expression of the receptor, thereby affecting the dopaminergic neurotransmitter, final y lead to related diseases. OBJECTIVE:To construct the dual luciferase reporter vector containing human DRD1 promoter region and determine its activity, which could provide the basic tool for studying the transcriptional regulation of DRD1 gene. METHODS:DRD1 promoter sequence was amplified by PCR using the human blood genomic DNA and cloned into pGM-T vector. After sequencing, the correctly constructed vectors were ligated to the firefly luciferase reporter plasmid pGL3-Basic. The cloned pGL3-Basic vectors were transfected into HEK293 using cationic liposome method. In the meanwhile, PGL3-Basic vector with no promoter was co-transfected with pGL3-TK plasmid as negative control group. The relative fluorescence intensity was measured by chemiluminescence. RESULTS AND CONCLUSION:(1) Recombinant luciferase reporter gene vectors were confirmed by restriction analysis and sequencing. (2) Compared with the negative control group, the HEK293 cel s transfected by recombinant vectors presented transcriptional activity. (3) In conclusion, luciferase reporter gene vectors containing DRD1 promoter region are successful y constructed and can provide the basic tool for further study on the transcriptional regulation of DRD1.

Mitochondrial divergence between slow- and fast-aging garter snakes.

Mitochondrial function has long been hypothesized to be intimately involved in aging processes--either directly through declining efficiency of mitochondrial respiration and ATP production with advancing age, or indirectly, e.g., through increased mitochondrial production of damaging free radicals with age. Yet we lack a comprehensive understanding of the evolution of mitochondrial genotypes and phenotypes across diverse animal models, particularly in species that have extremely labile physiology. Here, we measure mitochondrial genome-types and transcription in ecotypes of garter snakes (Thamnophis elegans) that are adapted to disparate habitats and have diverged in aging rates and lifespans despite residing in close proximity. Using two RNA-seq datasets, we (1) reconstruct the garter snake mitochondrial genome sequence and bioinformatically identify regulatory elements, (2) test for divergence of mitochondrial gene expression between the ecotypes and in response to heat stress, and (3) test for sequence divergence in mitochondrial protein-coding regions in these slow-aging (SA) and fast-aging (FA) naturally occurring ecotypes. At the nucleotide sequence level, we confirmed two (duplicated) mitochondrial control regions one of which contains a glucocorticoid response element (GRE). Gene expression of protein-coding genes was higher in FA snakes relative to SA snakes for most genes, but was neither affected by heat stress nor an interaction between heat stress and ecotype. SA and FA ecotypes had unique mitochondrial haplotypes with amino acid substitutions in both CYTB and ND5. The CYTB amino acid change (Isoleucine → Threonine) was highly segregated between ecotypes. This divergence of mitochondrial haplotypes between SA and FA snakes contrasts with nuclear gene-flow estimates, but correlates with previously reported divergence in mitochondrial function (mitochondrial oxygen consumption, ATP production, and reactive oxygen species consequences).

Location of DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 in colonel cancer cell line SW480 cells by inverse-PCR / 肿瘤研究与临床

Objective To precious localize DNase Ⅰ hypersensive sites exactly in the promoter region of CD133 of cell line SW480 by inverse-PCR.Methods The colonel cancer cell SW480 nuclei were suspended in digested buffer,treated with DNase Ⅰ at the concentration of 10 U/ml for 10 min.The inversePCR was performed as follows.DNA treated by DNase Ⅰ was purified,fragmented with restricted enzyme EcoRI and Xmal Ⅰ.Then the ends were blunted,ligated by T4 ligase.PCR was performed,and production was sequenced.The restricted enzymes cut sites were near DNase Ⅰ cleavage sites.Results 9 DNase Ⅰ cut sites were identified in CD133 promoter region.The DNaseI hypersensitive sites all distributed in a region -300 bp--700 bp up to transcription start site.Conclusion The DNase Ⅰ cleavage sites could identified preciously by application of inverse-PCR.These sites locate in a region of-300 bp--700 bp up to transcription start site.

Methylation status of IGF -Ⅱgene P4 promoter in hepatocellular carcinoma / 中国基层医药

Objective To investigate the alternation of insulin -like growth factor -Ⅱ( IGF-Ⅱ) gene promoter P4 methylation status in hepatocellular carcinoma ( HCC) and explore its relationship with expression of P4 mRNA levels.Methods Liver specimens of 43 patients with HCC and normal liver specimens of 9 control patients were collected in operation .Tissue DNA and total RNA were extracted from these specimens .IGF-Ⅱ P4 methylation status and P4 mRNA expression levels were detected .Results (1)The incidence of IGF -ⅡP4 methyl-ation in HCC group was significantly lower than that in normal liver specimens (16.28%vs 88.89%,χ2 =19.12,P<0.01).(2)The expression level of IGF -ⅡP4 mRNA in HCC group was significantly higher than that that in normal liver specimens[(0.96 ±0.74) vs (0.25 ±0.19),t=5.48,P<0.01].(3)In HCC group,the IGF-Ⅱ P4 mRNA expression level with hypomethylation gene was significantly higher than that without hypomethylation gene [(1.18 ± 0.76) vs (0.32 ±0.27),t=5.28,P<0.01].Conclusion The hypomethylation alternation of IGF -Ⅱ P4 gene promoter which is accomplished by up -regulate P4 mRNA expression has a close relationship with HCC .

Incomplete sequence homogenization in 45S rDNA multigene families: intermixed IGS heterogeneity within the single NOR locus of the polyploid species Medicago arborea (Fabaceae).

BACKGROUND AND AIMS: Ribosomal sequences have become the classical example of the genomic homogenization of nuclear multigene families. Despite theoretical advantages and modelling predictions that support concerted evolution of the 45S rDNA, several reports have found intragenomic polymorphisms. However, the origins and causes of these rDNA polymorphisms are difficult to assess because seed plants show a wide range of 45S rDNA loci number variation, especially in polyploids. Medicago arborea is a tetraploid species that has a single 45S rDNA locus. This feature makes this species a suitable case study to assess the fate of ribosomal IGS homogenization in polyploid species showing nucleolus organizer region (NOR) reduction. METHODS: The intergenic spacer (IGS) region was amplified by long PCR and the fragments were cloned and sequenced by a primer-walking strategy. The physical mapping of the whole and partial IGS variants was assessed by fluorescent in situ hybridization (FISH) and fibre-FISH methods on mitotic chromosomes and extended DNA fibres, respectively. KEY RESULTS: Two IGS fragments of 4·8 and 3·5 kb were obtained showing structural features of functional sequences. The shorter variant appears to be a truncated copy of the 4·8 kb fragment that lacks the duplication of the transcription initiation site region and the entire D region. The physical localization of the two IGS variants on metaphase chromosomes and extended DNA fibres using FISH corroborated their joint presence within the same locus. In addition, no spatial structure of the two variants was detected within the NOR. CONCLUSIONS: The results suggest that full sequence homogenization is not operating within the NOR locus of M. arborea. The structure of the NOR locus reported here departs from the models of IGS heterogeneity present in plants and caution against assuming the widespread belief that intragenomic ribosomal heterogeneity is mainly due to sequence variation between paralogous loci.

Dynamic CCAAT/enhancer binding protein-associated changes of DNA methylation in the angiotensinogen gene.

DNA methylation patterns are maintained in adult somatic cells. Recent findings, however, suggest that all methylation patterns are not preserved. We demonstrate that stimulatory signals can change the DNA methylation status at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site and activate expression of the angiotensinogen gene (AGT). A CEBP binding site in the human AGT promoter was hypomethylated in tissues with high expression of AGT, but not in those with low expression. The transcriptional activity of AGT promoter sequences cloned into a reporter plasmid depended on DNA methylation. In cultured human cells, interleukin 6 stimulation caused DNA demethylation around a CEBP binding site and a transcription start site; demethylation was accompanied by increased CEBP-ß recruitment and chromatin accessibility of the AGT promoter. DNA methylation activity decreased in the nucleus. Excess circulating aldosterone upregulated AGT expression and was accompanied by DNA hypomethylation around a CEBP binding site and a transcription start site in human visceral adipose tissue. High salt intake led to upregulation of Agt expression, DNA hypomethylation around 2 CEBP binding sites and a transcription start site, and decreased DNA methylation activity in rat visceral adipose tissue. Taken together, CEBP binding initiates chromatin relaxation and transcription, which are followed by DNA demethylation around a CEBP binding site and a transcription start site in the AGT promoter. Decreased DNA methylation activity in the nucleus may play a role in DNA demethylation. DNA demethylation switches the phenotype of AGT expression from an inactive to an active state.

Construction and ldentification of Cell Division Cycle 2 Promoter Reporter Gene Vector / 天津医药

Objective To construct the luciferase reporter gene vector of cell division cycle 2 (Cdc2) gene promoter and determine its transcriptional activity. Methods Primers were designed based on human Cdc2 promoter sequence from UCSC software. Then Cdc2 promoter from human genome DNA was replicated. After pGL3-Basic vector and Cdc2 promoter were digested with restriction enzymes SacⅠand XhoⅠseparately, Cdc2 promoter was inserted into pGL3-Basic vector. The recombinant plasmid named pGL3-Cdc2-promoter was transiently co-transfected into U2OS cells with control vector pRL-SV40, and then the activity of dual luciferase was detected. Results pGL3-Cdc2-promoter was constructed successfully. The restriction analysis and sequencing proved the entirely correct sequencing results. The luciferase activity was higher in pGL3-Cdc2-promoter/pRL-SV40 group than that of pGL3-Basic/pRL-SV40 group (1.591 5±0.199 8 vs 0.049 9±0.010 4). Conclusion pGL3-Cdc2-promoter can be transcribed and activated in U2OS cells. This study provided an important basis for screening and evaluation of anticancer drugs.

Identification and characterization of a nanog homolog in Japanese flounder (Paralichthys olivaceus).

The homeodomain-containing transcription factor nanog plays a key role in maintaining the pluripotency and self-renewal of embryonic stem cells in mammals. Stem cells offered as a significant and effective tool for generation of transgenic animals and preservation of genetic resources. The molecular genetic organization and expression of nanog gene in marine fish have not been reported yet. In this study, we isolated and characterized the flounder nanog gene as a first step towards understanding the mechanism of the plurpotency of fish stem cells and develop a potential molecular marker to identify the stem cells in vivo and in vitro. Phylogenetic, gene structure and chromosome synteny analysis provided the evidence that Po-nanog is homologous to the mammalian nanog gene. Protein sequence comparison showed that flounder Nanog shared low similarity with other vertebrate orthologs except for a conserved homeodomain. Quantitative RT-PCR analysis showed that flounder nanog was maternally expressed, and the transcripts were present from the one-cell stage to the neurula stage with the peaking at blastula stage. Whole mount in situ hybridization analyses demonstrated that the transcripts were present in all blastomeres of the early embryo. Tissue distribution analysis indicated that nanog was detectable only in gonads. Further, the expression was significantly high in ovary than in testis. In situ hybridization revealed that the transcripts were located in the cytoplasm of the oogonia and oocytes in ovary, only in the spermatogonia but no spermatocytes or spermatids in testis. The promoter region was also analyzed to have several basal core promoter elements and transcription factor binding sites. All these results suggest that Po-Nanog may have a conservative function between teleosts and mammals.

Current challenges in bacterial transcriptomics.

Over the past decade or so, dramatic developments in our ability to experimentally determine the content and function of genomes have taken place. In particular, next-generation sequencing technologies are now inspiring a new understanding of bacterial transcriptomes on a global scale. In bacterial cells, whole-transcriptome studies have not received attention, owing to the general view that bacterial genomes are simple. However, several recent RNA sequencing results are revealing unexpected levels of complexity in bacterial transcriptomes, indicating that the transcribed regions of genomes are much larger and complex than previously anticipated. In particular, these data show a wide array of small RNAs, antisense RNAs, and alternative transcripts. Here, we review how current transcriptomics are now revolutionizing our understanding of the complexity and regulation of bacterial transcriptomes.

Current Challenges in Bacterial Transcriptomics

Over the past decade or so, dramatic developments in our ability to experimentally determine the content and function of genomes have taken place. In particular, next-generation sequencing technologies are now inspiring a new understanding of bacterial transcriptomes on a global scale. In bacterial cells, whole-transcriptome studies have not received attention, owing to the general view that bacterial genomes are simple. However, several recent RNA sequencing results are revealing unexpected levels of complexity in bacterial transcriptomes, indicating that the transcribed regions of genomes are much larger and complex than previously anticipated. In particular, these data show a wide array of small RNAs, antisense RNAs, and alternative transcripts. Here, we review how current transcriptomics are now revolutionizing our understanding of the complexity and regulation of bacterial transcriptomes.

Treatment of human pancreatic cancer with adenovirus-mediated fusion gene system driven by KDR promoter in nude mice / 中华肝胆外科杂志

Objective To investigate the curative effect of the adenovirus-mediated fusion gene system driven by KDR promoter (AdKDR-CDglyTK) on a model of pancreatic cancer. Methods By using transplantation of the cultivated cells, human pancreatic cell line Capan-2 was injected subcutaneously on the back of nude mice to establish the animal model of the pancreatic cancer. Twenty nude mice were divided randomly and equally into four groups. The mice in group Ⅰ were injected with AdKDR-CDglyTK and 5-FC/GCV, those in group Ⅱ were injected with 5-FC/GCV, those in group Ⅲwere injected with AdKDR-CDglyTK and those in group Ⅳ received no any injection. AdKDR-CDglyTK was injected directly into the tumor and 5-FC/GCV was given by intraperitoneal injection. The observing parameters included common status, tumor bulk, tumor weight, inhibition rate of tumor growth, pathology, immunohistochemistry and treatment effect in each group. Electron microscopy was performed to observe the pathological changes of cells. The apoptotic cells in tumor were detected using the TUNEL assay. The expression of CDglyTK in tumors from each group was examined by RT-PCR. Results Tumor growth was dramatically inhibited in group Ⅰ. Tumor growth has no significant difference among groupⅡ , group Ⅲ and group Ⅳ. The apoptotic rate (34.20±4.60)% was significantly increased in group Ⅰ (F= 243. 22, P= 0. 00) and it had no significant difference among groupⅡ , group Ⅲ and group Ⅳ (P＞0.05). Conclusion AdKDR-CDglyTK with 5-FC/GCV can obviously inhibit the growth of human KDR-expressing pancreatic cell line Capan-2 and induce the cell apoptosis in vivo. The probable molecular mechanism lies in the facts that the system can cause a decline in the level of Bcl-2.

Tumor-specific promoters and their application in cancer gene targeted therapy / 国际肿瘤学杂志

Tumor-specific promoters can induce high-efficiency and specific expression of exogenous genes in tumor cells. At present, commonly used promoters include alpha-fetoprotein promoter, carcinoembryonic antigen promoter,prostate specific antigen promoter,human telomerase reverse transcriptase promoter and multidrug resistance gene promoter and etc. Dual promoters, enhancer, regulatory element and other physical or chem-ical factors could be used to reconstruct or modify promoters to increase the expression and location of exogenous genes in tumor cells. Tumor-specific promoters play an important role in cancer gene targeted therapy by cou-pling with suicide gene, anti- oncogene, antiseuse nucleic acid, apoptosis gene and RNA interference.

Suillus bovinus glutamine synthetase gene organization, transcription and enzyme activities in the Scots pine mycorrhizosphere developed on forest humus.

• Glutamine synthetase (GS) expression and activity is of central importance for cellular ammonium assimilation and recycling. Thus, a full characterization of this enzyme at the molecular level is of critical importance for a better understanding of nitrogen (N) assimilation in the mycorrhizal symbiosis. • Genomic and cDNA libraries of Suillus bovinus were constructed to isolate the GS gene, glnA, and corresponding cDNAs. The transcription initiation site was identified and transcription and enzyme activities were characterized in pure culture mycelium and mycorrhiza, and extramatrical mycelium samples harvested from Scots pine-Suillus bovinus microcosms grown on forest humus. • Pure culture mycelium, mycorrhiza and extramatrical mycelium all exhibited equivalent levels of GS transcription, translation and enzyme activities. However, levels of transcription and enzyme activity did not correlate as a large majority of detectable transcripts showed specific 5'-end truncation. • Our data suggest that GS is constitutively expressed and not directly affected by environmental conditions of the symbiotic N uptake. Any changes in the intracellular ammonium level are most likely handled by regulatory flexibility of GS at enzyme level.