Abstract:Eukaryotic initiation factor 5 (eIF-5) is an important factor in translation initiation. It acts as a GTPase activating factor to mediate the hydrolysis of GTP binding to eIF-2 which is essential for the composition of functional 80S complex. Besides,eIF-5 binds eIF-2 and eIF-3 at the same time, so induces the construction of translation initiation factors complex.

Abstract:Ankyrin repeat (ANK) is a widely used sequence motif in organisms. An individual ankyrin repeat has an L-shaped structure consisting of a β hairpin followed by two α helices, namely β₂α₂ pattern. These ANK repeats form a compact domain stabilized through hydrogen-bonding interaction and hydrophobic stacking. Massive of structurally similar but functionally diverse proteins containing ANK repeats can be formed by virtue of assembly of the tandem array motifs. The central role of ANK repeat is to mediate protein-protein interaction, so that proteins can make sophisticated functions by interacting with a variety of ligands. This review focuses on structure, function and related diseases of the ANK repeats and their complexes from several members of the ANK family.

Abstract:The exosome is a conserved complex which identified in Saccharomyces cerevisiae and has 3′ exoribonuclease activity in vitro. The exosome contains ten core subunits, and all except Csl4p/Ski4p are 3′ exoribonucleases in vitro. The exosome acts important role in several pathways of RNA metabolism. The exosome is a universal complex in eukaryote and is related to expression of eukaryotic genes.

Abstract:Calcium/calmodulin-dependent serine protein kinase（CASK）is a member of membrane associated guanylate kinase (MAGUK) family. CASK has multiple protein-binding domains which can interact with other kinds of proteins. CASK acts as a molecular scaffold organizing multiprotein complexes at special areas of the plasma membrane. Mediating a direct link between extracellular matrix and actin cytoskeleton of plasma, CASK might help some proteins to localize to the functional places and be involved in multiple functions of signal transduction, neurotransmitter release in the synapses, nuclear translocation, and transcriptional control.

Abstract:Yeast surface display system had been developed greatly since the phage display was invented. It is well suitable for displaying mammalian cell-surface and secreted proteins that require endoplasmic reticulum-specific post translational processing for efficient folding and activity. Yeast cells are large enough particles, unlike phage, that can be screened and separated using flow cytometer. Yeast adhesion receptor, a agglutinin or α agglutinin, have been used as a surface display scaffold. Yeast surface display has been successfully applied in protein directed evolution and alive oral vaccines.

Abstract:RNA interference is something of posttranscriptional gene silencing, specifically mediated by double-stranded RNA. With the development of this technology and wider applications in the study of Drosophila, dsRNA has come to be a potent and efficacious inhibitor of gene activity in Drosophila. RNAi now promises to be a powerful reverse-genetic method to determine gene function in Drosophila.

Abstract:O⁶-methylguanine-DNA methyltransferase (MGMT) is a specific DNA repair protein that exist in cells and tissues from bacteria to mammalias. The function of the repair protein is to catalyzing and transfering the alkyl group from the O⁶ position of guanine of DNA to an internal cysteine residue of MGMT protein which repairs the lesion by reversion the guanine of DNA. Therefore the proper expression of MGMT is useful to repair O⁶-methylguanine-DNA adducts formed by the induction of alkyl agents. The amount and activity of MGMT are not only regulated by various factors at genetic level, but also associated with the directly effects of some drugs. It has important significance to regulate the activity of MGMT in the cells during the prevention of tumor-geneses and overcoming the drug resistance or marrow toxicity.

Abstract:The potential importance of ubiquitin-proteasome pathway (UPP) as a mechanism for a series of processes including cell cycle progression, apoptosis, immune and inflammatory responses, tumorigenesis and metastasis, as well as some genetic and neurodegenerative diseases has long been recognized. Nevertheless, roles of UPP in different reproductive events are only beginning to be clarified. UPP has recently been shown to be related to a variety of male reproductive events, such as spermatogenesis, spermiogenesis, as well as many female reproductive events, including tissue remodeling of the endometrium during normal menstrual cycle and early pregnancy and the degradation of the steroid receptors. Combined with domestic situation, the emerging roles of UPP in reproductive tissues are also presented.

Abstract:Synaptic vesicle docking and fusion at release sites require the association of proteins on both vesicle and plasma membranes. Syntaxin interacts with synaptobrevin/VAMP and SNAP-25, constituting the SNARE core complex. This complex's formation comprises the minimal molecular requirement for membrane fusion in vitro. However, the Ca²⁺-triggered synaptic vesicle fusion with presynaptic plasma membrane can be regulated rapidly and tightly when the main SNARE intermediate is so stable. Some synaptic proteins, which regulate the accessibility of SNARE components by interacting with the individual SNARE proteins, might tightly control assembly of functional fusion machinery. Thus, the identification of regulators or molecular switches involved in the assembly of the fusion core complexes is critical for elucidating the molecular mechanisms underlying neurotransmitter release and synaptic plasticity.

Abstract:Helicobacter pylori(Hp) causes gastritis and peptic ulcer disease of a human being, and is associated with certain types of gastric cancer. In recent years, much attention has been focuced on its moclecular mechanisms of H.pylori infection. As an important human pathogen, Hp genome has been sequenced. A detail summary of the literature, significant genome structure feature, gene expression regulation, and indentificaton of virulence factors is provided.

Abstract:DNAs with catalytic potential isolated from a ssDNA pool with random-sequence in vitro selection, were called deoxyribozymes or DNAzyme.Deoxyribozymes that exhibit RNA transesterification ,DNA transesterification,porphyrin metalation and peroxidase activities,DNA kinase activity and DNA ligase activity have recently been reported. Deoxyribozyme 10～23 might act as a sequence-specific RNA endoribonuclease in vitro,and could be used to inactivate target cellular RNA in vivo.

Abstract:Gut-enriched Kruppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract, the expression of which is associated with growth arrest. GKLF expression in the tumor was compared to normal mucosa in esophageal squamous cancer patients, using semi-quantitative RT-PCR. Expression of GKLF mRNA was detected all of 17 cases of esophageal squamous cancer. Decreased expression of GKLF mRNA was observed in 14 of esophageal squamous cancer patients compared to normal esophageal tissues adjacent to tumor from the same patient. Serum deprivation induced overexpression of GKLF was observed in the primary culture of human fibroblast and this induction was decreased in an esophageal squamous cancer cell line EC9706. Sequencing of GKLF cDNA from EC9706 showed no mutations in the coding region. The results demonstrate that GKLF expression in esophageal squamous cancer is down-regulated.

Abstract:A nasopharyngeal carcinoma(NPC) related gene, named NAG73 which mapped to 3p25～26 was identified by positional candidate cloning. Structural analysis reveals that NAG73 has an intronless genomic sequence, which is homologous with the fourth exon and 3′UTR of Homo sapiens Growth Hormone Secretagogue Precursor Gene (GHRELIN). NAG73 has no 5′ promoter sequence, and has putative polyA. The results suggest that NAG73 might be a processed pseudogene of GHRELIN. It has been showed that NAG73 is actively transcribed in some cells and tissues examined. There is expression difference between normal nasopharynx epithelia and nasopharyngeal carcinoma epithelia. And there is potential for a NAG73 encoded translation product. So, it is possible that NAG73 is translated and the product might act on the tumorigenesis of nasopharyngeal carcinoma.

Abstract:After more and more genome sequencing projects, like the “Human Genome Project”, the prediction of genes, including their coding region and their regulatory region, has received a lot of attention. Softwares such as GENSCAN and GeneMark are powerful, but still do not meet the requirement of the practical application. The GENSCAN predicts exons accurately, if the sequences predicted does not have insertions and deletions in their coding regions. But if it does have, even only one, the prediction could be disturbed seriously and satisfactory results can not be obtained. A hidden Markov model with states of deletions, insertions and main state is introduced to find the error of deletions and insertions. The result shows that sensitivity and specificity in exon level are both higher than 84% on the Burset/Guigò test data set.

Abstract:Biotin-linked bradykinin was synthesized by solid phase peptide synthesis for development of a functionalized peptide and study on structure-relevant bioactivity in biological system. In PC12 cell system, bioactivity of the synthetic peptide was evaluated and found to be controllable in the presence of neutravidin and free biotin. The controlling mechanism had been discussed and could be ascribed to steric hindrance induced by binding of neutravidin to the linked biotin. Moreover, influence of competitive binding between the free biotin and the linked biotin to the neutravidin had also investigated into and could be employed for switching the bioactivity on and off.

Abstract:The change of apoptosis and apoptotic regulatory gene Bcl-2 and Bax expression in pregnant rabbit placenta were investigated. Nuclear DNA fragmentation analysis indicated that DNA ladder, which is characteristic of apoptosis was detected in D18 and D26 pregnant rabbit placenta and the scan results showed that DNA fragmentation increased significantly in D18 and D26 pregnant rabbit placenta compared with that in D12 pregnant rabbit placenta. TUNEL test and immunolocalization of active caspase-3 demonstrated that apoptosis occurred in D12 pregnant rabbit placenta, and apoptotic cells localized predominantly in syncytiotrophoblast throughout pregnancy. Immunoblot analysis showed an increase of Bcl-2 and Bax expression throughout pregnancy, and the change of expression of these two proteins resulted in an increase in Bax to Bcl-2 ratio. The results demonstrated that apoptosis occurred in syncytiotrophoblast in rabbit pregnancy and apoptotic cell death increased from early to the end of gestation period, moreover Bax to Bcl-2 ratio may be related to apoptosis in placenta.

Abstract:A mathematical description of the differentiation and development from DP thymocytes to SP thymocytes in the thymus is explored. According to the theory of a combination of instructive and stochastic model, a mathematical model is established. The results demonstrate that as the sites (expressing MHCⅠ/peptide) of thymic stromal cells(TSCs) increase, the CD4⁺8⁺ DP thymocytes lessen but CD4^-8⁺ thymocytes increase. And as the avidity of DP thymocytes with sites of TSCs increases, more DP thymocytes differentiate into SP thymocytes. How the thymic stromal cells mediate the differentiation from DP thymocytes to SP thymocytes is described.

Abstract:Ce³⁺，Cd²⁺ and Pb²⁺ could influence the activity of amylase from porcine pancreas and competitively replace Ca²⁺ from amylase. The results showed that the activity of amylase was enhanced under the treatment of Ce³⁺，Cd²⁺ and Pb²⁺ respectively at low concerntration, but inhibited at the high concerntration. Both replacement and inhibition showed that the action was in the order: Pb²⁺＞Cd²⁺＞Ce³⁺.

Abstract:ATP-sensitive K⁺ channels play an important role in coupling membrane excitability with intracellular metabolic stress. To characterize such K_ATP channels from rat brain, the inside-out mode of patch-clamp technique was applied to freshly dissociated hippocampal CA1 pyramidal neurons of adult rat. One type of K⁺ permeable channel was recorded only at the presence of Ca²⁺ in the internal solution and it could be inhibited by application of 1～3 mmol/L ATP and 1 mmol/L tolbutimade, a K_ATP channel blocker. Both of channel open probability and the ATP induced-inhibition displayed a voltage-dependent fashion. When both sides of the excised membrane were in symmetrical 140 mmol/L K⁺, the I-V relation was linear with a conductance of 204 pS and reversal potential was 3.57 mV. Unlike the previously reported “classical” K_ATP channel, this large-conductance K_ATP channel (L-K_ATP) was regulated by membrane potential, intracellular Ca²⁺ and ATP, indicating a new subtype of K_ATP channel presents in hippocampus neurons. These results demonstrate that at least two distinct K_ATP channels exist in rat hippocampal neurons and suggest that metabolic state may be continuously sensed in neurons via different K_ATP channels with resulting alterations in neuronal membrane excitability.

Abstract:Two retrovirus vectors: pSIR-EGFP containing enhance green fluorescence protein (EGFP) reporter gene and pSIR-EGFP-870 with 870 bp p16 promoter were constructed on the base of self-inactivating retrovirus vector pSIR. Then retrovirus vectors were transducted into package cells for virus production and 2BS cells were infected with virus. During the course of reverse transcription, the 5′LTR lost its promoter activity.The transcriptional activity of p16 promoter in 2BS cells obviously increased during the aging process. The results demonstrate that self-inactivating retrovirus vector can be used for gene transcription regulation research during the aging process.

Abstract:The small heat shock protein Hsp16.3 of Mycobacterium tuberculosis was shown to be a trimer-of-trimers. Hsp16.3 proteins were denatured under three kinds of strong denaturing conditions by heat treatment (at 100℃, 15 min) or chemical reagents (12 mol/L urea or 8 mol/L guaridine, 4 hours) and then were renatured by cooling or dialysis. The secondary, tertiary and quaternary structures of Hsp16.3 were investigated by using far- and near- UV circular dichroism as well as pore-gradient polyacrylamide gel electrophoresis, respectively. The data clearly showed that the renatured Hsp16.3 proteins almost completely regained its native conformation, thus suggesting the strong ability of Hsp16.3 to refold and reassembly to its native conformation.

Abstract:The xanthophyll cycle and non-radiative energy dissipation in detached maize (Zea mays L.) leaves produced significant changes under illumination. With the raising of light intensity, zeaxanthin(Z) content evidently rose, violaxanthin(V) content significantly decreased, antheraxanthin(A) content significantly increased in low and middle level lights, and then decreased slightly in strong lights. The xanthophyll cycle components pool size V+A+Z only enlarged a little. Under the same conditions，the non-radiative energy dissipation of leaves apparently enhanced，which was shown that non-photochemical quenching (NPQ) rapidly rose，while F_v/F_m apparently reduced. It was analysed that (Z+0.5A)/(V+A+Z) had exhibited apparently positive linear correlation with NPQ，and showed a negative correlation with F_v/F_m. Compared to (Z+0.5A)/(V+A+Z), (V+0.5A)/(V+A+Z) had showed contrary results. Based on the results, it was deduced that Z epoxidation and V deepoxidation had been significantly related to non-radiative energy dissipation and PSⅡ light energy conversion in detached maize leaves.

Abstract:To investigate new genes related to renal damage of diabetic hypertension rats for exploring its mechanism,the model of diabetic hypertension rats was established by streptozotocin(STZ) injection. Urine protein of this model rats continued positive and ultrastructure of their renal cortex changed. Fluorecence-labelled differential display reverse transcription polymerase chain reaction and Northern blot were used to isolate and identify the genes that showed transcription changes in the renal cortex between the unitary hypertension and diabetic hypertension rats.Four differential fragments were obtained, two of which were novel whereas the other two showed significant similarity with rat amyloidogenic glycoprotein and CDK109 respectively according to the public database of Genbank. Via the application of bioinformatics the altered renal mRNA expression of these genes associated with diabetic hypertension suggested that they were the candidates for a role in the development of the nephropathy.

Abstract:Chicken anemia virus (CAV) encodes a small protein, VP3. A fusion gene construct which can express the fusion protein of enhanced green fluorescence protein (EGFP) and VP3 in eukaryotic cells, was transfected into five cell lines, human hepatoma cell strain Smmu7721, HepG2, rat hepatoma cell strain HTC, human embryo kidney cell stain 293 and mouse fibroblast cell stainΨ2.The green fluoresence was observed within the nuclei of the strains. While the control plasmid, which can express only enhanced green fluorescence protein in eukaryotic cells, was transfected into these cell strains，the green fluorescence was observed around the whole cytosol. This means that VP3 has the function of nuclear localization in these cell strains. After the analysis of the amino acid sequence of VP3, a domain of the amino acid sequence of the protein that has the character of nuclear localization sequences (NLS) was found. Deletion of this domain from VP3 led to the deprivation of nuclear location in human hepatoma cell strain Smmu7721. This domain was subcloned and fused to EGFP. The fusion gene construct was transfected to human hepatoma cell strains. The green fluorescence could be seen again to locate mainly in the nuclei. Deletion of the basic amino acids region near the C end of VP3 also led to the deprivation of nuclear location. The prediction of the secondary structure of VP3 shows that the basic amino acid region near the C end can form a β-sheet. This means that the basic amino acids region near the C end is very important for VP3 nuclear localization. The domain maybe the NLS of VP3. After a vector that could only express VP3 was transfected into Smmu7721 and stained the cells with propidium iodide (PI), the primary evidence of VP3 inducing apoptosis was obtained.

Abstract:After two-step chromatography of Sepharose Q Fast Flow and Superdex 30, a small protein with molecular size of 7.2 ku was purified from Escherichia coli. The purity examined by SDS-PAGE showed as a single band. Its molecular mass measured by mass spectrum and the amino acid sequence of N terminal were in consistence with CspC, one of the cold shock proteins, in E.coli. Subsequently, the content of its secondary structure was estimated from the circular dichroism spectra, and moreover, its stability in high temperature and the conformational change after binding with single strand DNA were monitored with a CD spectrophometer.

Abstract:Poly(3-hydroxybutyrate) (PHB) films were biodegraded by DS9701. The degradation process was monitored by using SEM. It was shown that the PHB degradation occurred firstly in the amorphous part of PHB and then in the crystalline part, especially from the center of PHB spherulites. PHB deplymerase produced by DS9701 mainly attacked the second ester bond of PHB and the degraded product was dimmer, determined by using mass spectrometer.

Abstract:The cis-acting elements were scanned within 11.5 kb of the 3′juxtaposed region of the Yunnanese (Aγδβ)⁰-thalassemia deletion using the luciferase report gene system. A 1.7 kb fragment immediately downstream of the 3′ breakpoint of the deletion was found to increase expression of the luciferase gene driven by Gγ-globin gene promoter by 3.8 to 4.0 fold in K562 cells and MELGM979 cells and 1.5 fold in HeLa cells. A 1.4 kb fragment that is located 10 kb downstream of the 3′ breakpoint was found to increase the luciferase expression by 2.4 to 2.9 fold in K562 cells and MELGM979 cells but no enhancement in HeLa cells. The results suggested that the two fragments contain enhancer-like elements and they function in a certain erythroid-specific manner. Furthermore, a 430 bp region that contains several putative motifs for known transacting factors binding within the 1.7 kb fragment was showed to include the most of the enhancer activity of the fragment. These results provided experimental proof for the hypothesis that the importation of enhancer-like sequences into the vicinity of Gγ globin gene may be responsible for the reactivation of the Gγ-globin gene in the Yunnanese (Aγδβ)⁰-thalassemia mutant.

Abstract:To express chick MMP-2 C-terminal hemopexin-like fragment in E.coli and to explore whether it has the biological effect on the inhibition of angiogenesis, a 630 bp of MMP-2 C-terminal coding fragment (PEX) was cloned into prokaryotic expression vector pCal-n from primary cultured day 10 chicken embryo fibroblasts using RT-PCR. The relatively pure inclusion bodies of CBP/PEX fusion protein were denatured and renatured by guandine·HCl method from PEX/pCal-n plasmid transformed log phase E.coli BL21 (Des) pLys induced with IPTG. Growth curve of human umbilical vein endothelial cells (HUVEC) and chick embryo chorioallantoic membrane (CAM) assay were performed to examine the effects on the inhibition of angiogenesis. The prokaryotic expressed CBP/PEX fusion protein has the biological activity and can inhibit the growth of HUVEC in vitro and the angiogenesis on chick CAM in vivo. These results suggest that the expressed PEX is to be a novel potential therapeutic inhibitor of diseases associated with angiogenesis.

Abstract:The concept of compact structural domain (CSD) is proposed which means a maximal compactly - packing - fold composed of α helices and β sheets in secondary structure sequence. There are three kinds of CSDs, namely α domain, β domain and α/β domain. Then five classes of protein are defined. The mainly- α protein is constructed from one or several α domains, the mainly-β protein is constructed from one or several β domains, the α/β protein is constructed from one or several α/β domains, the multi-domain protein is constructed from two or more kinds of CSDs, and the ζ-class protein does not contain any CSD. A database of 1 261 globular proteins is classified into five classes. The classification is compared with SCOP's. The analysis of redundancy-deletion has been done. A prediction rule on structural class is given which is the generalization of previous work. The successful rate of the prediction is higher than 82%.

Abstract:Biological effects of microgravity on brain cells were simulated by rotating chicken embryos in a clinostat during their hatch. Continuous fluorometry was used to study dynamic release of glutamate from brain cells of the embryonic chicks. The initial release rate, the induced release rate, the release content by KCl or by a single electric pulse and the total intracellular glutamate concentration have been compared between rotated and control groups. The results showed that there was no obvious difference in initial release rate between both rotated and controls. The total intracellular glutamate concentration increased significantly (P＜0.01) when E10 embryos were rotated for 4 h, and KCl- induced release rate and release content from brain cells of E10 embryos rotated for 24 h were higher than those from controls. However, there was no significant difference in the total intracellular glutamate concentration and the KCl- induced release rate, release content from brain cells of E13 embryos between rotated and their control groups. These results suggest that the releasing behaviors of neuro-transmitters from brain cells can be affected by microgravity and the influence is related to the age of the embryos. The dynamic process of glutamate release from brain cells induced by a singlee electric pulse shows that the release is related with a rapid increase of the intracellular ［Ca²⁺］_{i following the pulse.}

Abstract:In order to study the possible mechanism of the cellular proteins involved in the process of replication of HCV RNA. UV cross-linking experiment was used to identify the cellular proteins which bind to the 3′-end of HCV intermediate negative-strand RNA. Results showed that a protein with approximate molecular mass of 45 ku(p45) cross-linked with the 3′-end 131～278 nt of HCV intermediate negative-strand RNA. The p45 presented in various cell lines of different origins. The amounts of RNA-protein complexes increased with increasing amounts of cellular extracts. Non-homologous proteins and RNA transcripts could not compete for the binding between p45 and the 3′-end of HCV intermediate negative-strand RNA. These results suggested that the cellular protein p45 can specifically bind to the secondary structure of the 3′-end of HCV intermediate negative-strand RNA, thus p45 may play an important role in HCV RNA replication.

Abstract:By the action of xanthine - xanthine oxidase reaction (X+XO) as well as methyl viologen (MV) respectively, it was studied that chlorophyll fluorescence quenching induced by superoxide anion in lettuce (Lactuca sativa L.) chloroplasts. The results showed that the production of superoxide anion increased photochemical quenching(qP) and non-photochemical quenching(qN) evidently. As superoxide dismutase (SOD) was inhibited by sodium diethyldithiocarbamate (DDC) in chloroplasts, qP decreased and qN increased in the quenching of chlorophyll fluorescence induced by X+XO, while the increasing extent of qP was not great and qN rose insignificantly in the quenching of chlorophyll fluorescence induced by MV. When iodoacetamide (JAA) inhibited carbon assimilation, qP decreased and qN increased. Uncoupler NH₄Cl promoted the permeability of across thylakoid membrane proton, which made qP rising and qN decreasing. Under uncoupling conditions, MV raised qP and qN insignificantly. It was analyzed that the producing and scavenging of superoxide anion in time was important to maintain photosynthetic electron transport and to improve transmembrane ΔpH. It contributed to the conversion and dissipation of photon energy absorbed in chloroplasts, and reduced the harm of photoinhibition to a certain extent led by excess photon energy.

Abstract:Ribosomal protein L6 (RpL6, also called Taxreb107) possesses at least three nuclear localization signal (NLS)-like motifs. The activity of these motifs for their ability to mediate protein nuclear translocation was analyzed by using a NLS trapping system established. The full length or different fragments of RpL6/Taxreb107 cDNA was inserted to the cloning site of NLS trapping vector and the resulting constructs were used for transformation of host yeast. The result showed that the first two NLS-like motifs of RpL6/Taxreb107 induced the fusion protein to be transfected into the nucleus while the third one did not. This conclusion was confirmed by transfection of cultured cells with (EGFP) fused with different RpL6/Taxreb107 fragments. The results also showed that the first two NLS-like motifs of RpL6/Taxreb107 have nucleolus localization activity. When expressed in cultured cells, the RpL6/Taxreb107 fragments containing the first two NLS preferentially induced the fusion protein to be transfected into the nucleoli. These results are helpful for understanding of the nuclear translocation of RpL6/Taxreb107, and also confirmed that the NLS-trapping system is useful for searching NLS in proteins.

Abstract:The 300 bp upstream fragment of cpcβ gene which encodes the β-subunit of phycocyanin and the 1.4 kb fragment of glnA gene encoding glutamine synthetase were obtained by polymerase chain reaction (PCR) from genomic DNA of marine cyanobacterium Synechococcus sp. PCC 7002. Then, integrative expression vector pKGC-MT, which contained promoter Pcpcβ, mMT-Ⅰ gene and integrative platform glnA, was constructed and introduced into Synechococcus sp. PCC 7002 via natural transformation. Selected by ampicillin, the stable transgenic cyanobacterium was obtained. PCR analysis indicated the integration of mMT-Ⅰ gene in genomic DNA of Synechoccus sp. PCC 7002 and Western blotting demonstrated the expression of mMT-Ⅰ in the cyanobacterium. According to the result of ELISA, the amount of the expressed mMT-Ⅰ was about 800 μg/g fresh cells.

Abstract:Microarray hybridization is a powerful technique in life science since it allows a relatively straightforward determination of gene expression, in respect to distribution as well as quantification. Large-scale hybridization-based mRNA expression mapping projects call for robust and high efficient microarray hybridization techniques that are amenable to automation. However, a major drawback of microarray is the fact that all standard protocols available include time consuming optimization steps of several critical parameters such as DNA fixation to surface of the slides, hybridization conditions and washing procedures. The aim of this investigation was a rational design of DNA probes which were adapted to the standard protocol and could therefore be used without changing any parameter. A rapid microarray technique for quantitative analysis has been recently introduced. For highly repetitive DNA probes the hybridization time and the number of washing steps were reduced considerably by formamide or equivalent denaturing chemical agents. Due to low stringency conditions major and minor binding sites of the probes showed visible hybridization signals well suited for quantitative image-screening. The discrimination of minor and major binding sites to surface of glass slide was possible by automated image-processing. With respect to the optimization it was necessary to verify two sensitive parameters (hybridization time and temperature, spotting buffer) of the given microarray protocol. By comparation of several candidate conditions, the optimized standard protocol was constructed.