JAK3 mediatesc-myc gene expression induced by interleukin-2

C myc gene expression can be rapidly induced by IL 2 through intracellular signal transduction which is triggered by the interaction between IL 2 and its receptor (IL 2R). JAK3 which associates to the intracellular domain of IL_2R γ may play a critical role in this process. To reveal the action of JAK3 in c myc induction, a chimeric receptor gene IL_2R α/γ/Δ NJAK3 is constructed which consists of extracellular domain derived from IL 2R α subunit (IL_2R α), the transmembrane sequence derived from IL_2R γ and the cytoplasmic domain derived from the catalytic domain of JAK3 (Δ NJAK3), and then transfected this chimeric gene into mouse fibroblast cell line L929 β which had been transfected with IL_2R β gene and stably expressed IL_2R β in high level. In the transfectants coexpressing IL_2R β and α/γ/Δ NJAK3, the stimulation of IL_2 could intensively induce c myc gene expression. Because the whole cytoplasmic domain of IL_2R γ which could recruit signaling molecules was replaced by JAK3 and the c myc could be still induced by IL_2 in this situation, the results here gave the direct evidence demonstrating that JAK3 plays an important role in c myc gene expression induced by IL_2.     

Follicles in pregnant rat ovary are incapable of steroidogenesis

Antral follicles are present in the ovaries throughout gestation. These follicles are “physically immature” and cannot ovulate under the induction of LH/hCG. The purpose of the present study is to examine whether follicles in pregnant rat ovaries are capable of steroidogenesis. StAR is believed to be the key regulator of steroid hormone biosynthesis. Antisense StAR probe and anti-StAR rabbit serum have been used to detect the StAR expression in ovarian follicles at various stages of pregnant rats. The results indicate that theca-interstitial cells and the membrane granulosa cells at the stages from the estrous or pre-estrous in the normal cycling rat ovary express StAR mRNA and its protein, whereas neither granulosa cells nor the theca cells in pregnant ovary throughout gestation express StAR. These results indicate that the pregnant follicles throughout gestation are incapable of steroidogenesis.     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

Cyclic AMP response element binding protein (CREB) participates in the heat-inducible expression of humanhsp90β gene

Human heat shock protein 90b gene ( hsp90b ) is a constitutively expressed heat shock gene existing in most of cell types tested that can be further induced by heat shock. Chloramphenical acetyl transferase (CAT) reporter plasmids driven by different regulatory fragments of hsp90b gene were constructed and transfected into Jurkat cells to explore the role of a cAMP response element (CRE) in the upstream of the gene. Results show that, in comparison with the wild type construct, a severe reduction (~2/3) in the increased folds of promoter activity induced by heat shock at 42℃ for 1 h was observed in a construct with CRE-containing fragment (-173/-91bp) deleted. Electrophoretic mobility shift assays (EMSA) showed that phosphorylated CRE-binding protein (CREB) in the nuclear extract of heat shocked Jurkat cells is specifically bound to the fragment. Additionally, both of the phosphorylation on CREB and the activity of protein kinase A (PKA) were found in Jurkat cells to be enhanced with extending time of heat shock treatment. Our results indicate that in addition to the intronic HSE/HSF pathway, phosphorylated CREB also participates in the heat shock induced expression of human hsp90b gene via its interaction with CRE which may be regulated by PKA-sig- naling pathway.     

Expression of human erythropoietin directed by mWAP promoter in mammary gland of transgenic mice

The present work has generated transgenic mice with a hybrid gene construct consisting of genomic sequences encodinghuman erythropoietin (hEPO) and governed by regulatory sequences of mousewhey acidic protein (mWAP). The construct proved effective by transient expression in lactating animal. After introducing hybrid gene construct into single-cell embryo via pronuclear microinjection, surviving embryo are reimplanted into pseudopregnant foster mother mouse. 58 mice of 86 generation zero mice obtained were identified to be positive by PCR-Southern blot and genomic DNA Southern blot methods. The integration rate is 67%.hEPO was expressed in the milk of 16 mice of 39 mice measured byhEPO ELISA kit The expression level gets over 15 μg/mL.     

Expression,purification and spectra characterization of neuroglobin

The expression, purification and spectra characterization of recombinant human neuroglobin （NGB） are reported. The pET3a plasmid with the gene of NGB was transformed to E. coli BL21 （DE3） plys cells and expressed in TB culture medium. The results indicated that the expression amount of NGB is about 10 percent of the total protein in cells. The NGB protein was purified by ammonium sulfate precipitation, DEAE-Sepharose anion exchange column, Hiload 16/60 superdex 75 size exclusion chromatography and a Hiprep 16/10 Q FF anion exchange column, and a red soluble protein was obtained which showed a single band in electrophoresis. Electrospray ionization mass spectrometry （ESI-MS） showed that its molecular weight is 16930.0 Da. UV-spectra indicated that the reduced NGB has a strong absorption peak at 425 nm, and two weak peaks at 531 and 559 nm, which can be assigned to γ, β and α bands of porphyrin, respectively, and the oxidized NGB has a strong absorption peak at 413 nm which corresponds to the transition of π electrons in the porphyrin ring. The fluorescence maximal excitation wavelength is at 281 nm and its maximal emission wavelength is at 338 nm. CD spectra indicated that its secondary structure is a typical α helix, and has a positive peak at 410 nm induced by heme, The NGB protein is stable when the pH is higher than 4.     

Evaluation and characterization of an endosperm-specific sbeⅡa promoter in wheat

Starch,the main component of the wheat grain,is the product of a complex biochemical pathway. The sbeⅡα gene plays a key role in controlling the synthesis of starch, in particular, the biosynthesis of amylopectin,in maturing wheat grain.To investigate its regulatory mechanisms and endosperm-specific expression pattern, the sbeⅡα promoter (3094 bp in length) was cloned using APCR and sequenced.The effect of a series of deletions was studied using a GUS transient assay system. Results showed that the 3094 bp sequence (sbe.g construct) exhibited full stable promoting activity and that the activities of 5′ or 3′ deletions reduced levels of GUS expression. Some constructs with internal deletions showed only weak activity, however,sbe.e, with a deletion from -1579—--1210 bp resulted in higher levels of expression than the full-length promoter sequence, sbe.g. This indicates that motifs such as the -300 bp element, G-box and/or P-box act as positive elements and are necessary in determining the promoter‘s endosperm-specific pattern and that negative repressor elements or motifs may also be present within the -1579—-1210 bp sequence. The age of wheate ndosperm tissue used in the GUS-transient assay system is shown to be of significant importance.     

The foxtail millet Si69 gene is a Wali7 (wheat aluminum-induced protein 7) homologue and may function in aluminum tolerance

We isolated a clone, named Si69, from a foxtail millet immature seed cDNA library. The protein encoded by Si69 contains a conserved Wali7 (wheat aluminum induced protein 7) domain and shares high-level homology with aluminum-induced proteins from other species including rice and Arabidopsis. The Si69 gene presents as a single locus in foxtail millet genome and is globally expressed in all tissues examined. Its expression is up-regulated by aluminum. The sequence feature and expression pattern suggest that the Si69 gene is involved in aluminum tolerance or detoxification. To confirm its biological functions, Si69 controlled by the CaMV35S promoter was introduced into Arabidopsis. Transgenic plants did not show any visible morphological changes compared to wild-type plants under normal growth conditions. However, when treated with 20 or 50 μmol/L Aluminum (Al), the root apices of wild-type plants were heavily stained by hematoxylin, whereas those of Si69 transgenic plants were not stained when treated with 20 μmol/L Al and slightly stained when treated with 50 μmol/L Al. Scanning electron microscopy (SEM) results further demonstrated that the damage of the root apices was severer in wild-type plants than in transgenic plants. Inhibition of root growth and accumulation of malondialdehyde (MDA), an indicator of lipid peroxidation, were lower in transgenic plants than in wild-type plants. The results show that overexpression of Si69 may increase Al tolerance in transgenic plants, indicating that a series of Wali7-containing genes may play similar roles in Al tolerance/detoxification.     

Interleukin-2 mediates the peripheral analgesia of interferon alpha

Interferon-α (IFN-α) and interleukin-2 (IL-2) are crucial cytokines in immune system. They also play an important role in nerve system. It has been reported that IL-2 can induce the central analgesia. It is demonstrated that IFN-α also can induce the peripheral analgesia, which can be blocked by naloxone as IL-2. Furthermore, the analgesic effect of IFN-α is reversible by monoclonal anti-IL-2 antibody. In vitro experiments show that IFN-α significantly increases the production of IL-2 in a dose dependent manner. These data suggest that IL-2 mediates the peripheral analgesia of IFN-α.     

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. TheGFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of theGFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.     

A tumor suppressor genefhit prokaryotic expression and identification

The recombinant expression vector pGEMD-fhit which contains full encoding region offhit gene was constructed. The recombinant was introduced into the BL21 (DE3) strain ofE. coli and induced by 1 mmol/L IPTG to express a 29×10³ polypeptide offhit fusion protein. And the 29×10³ protein was sensitive and specific in reaction with anti-fhit antibody in Western blot. Foundation item: Supported by the National Natural Science Foundation of China (39770373) Biography: SUN Yan (1975−), female, Master of science, Research direction: gene engineering     

An exact expression for fractional absorption in Mossbauer spectroscopy

The fractional resonance absorption ε(0) in transmission Mossbauer spectroscopy is defined as a relative number of the absorbed γ-ray, and regarded as a measure of Mossbauer effect. The absorption linewidth Λ _a , as it is usually suggested, is nearly equal to the emission linewidth, Λ _s , and such an approximation leads to a extremely simplified expression ε(0), depending on neither Λ _s nor Λ _a . We consider the general case Λ _s ≠Λ _a , and obtain an exact expression for ε(0) which is given in the present paper. This expression ε(0), as a function oft _a, Λ _s , Λ _a , is figured and discussed. Biography: Chen Yi-long (1934-), male, Professor, research interest: Mossbauer effect and its applications.     

Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

An interspecies conserved antigen ofPlasmodium falciparum containing clusters of asparagine residues

An interspecies conservedPlasmodium asparagine rich antigen, designated as ARK26, was isolated by immunoscreeningP. falciparum genomic DNA expression library with mouse convalescent anti-P. yeolii serum. Partial DNA sequence analysis reveals that ARK26 contains clusters of asparagines and no randomly repeated amino acid sequence motifs are observed. A 65×10³ GST fusion protein is expressed by recombinant plasmid PGEX-5X-1 (ARK26) inE. coli C strain ABLE-K. Computer programs predict that two asparagine rich regions are among the possible antigenic epitopes of p37 encoded by ARK26. Interestingly, the sequence of ARK26 displays significant similarity to yeast and several other species’ mitochondrial genes, and its possible function is discussed. Supported by a fellowship offered by International Center for Genetic Engineering & Biotechnology(ICGEB) Ma Donghui: born in 1969, Graduate student     

Synthesis and properties of novel gemini surfactant with short spacer

Cationic gemini surfactant dimethylene-1,2-bis(dodecyldiethylammonium bromide), referred to as C12C2C12(Et) was synthesized, and its surface property and aggregation behavior in aqueous solution were studied. The value of γ at the critical micelle concentration (γcmc) is much smaller than that of the surfactant homologues with longer spacer. Spherical and elongated micelles were formed in the aqueous solution of this gemini surfactant,and the spherical micelles were absolutely dominant compared to the elongated micelles at our studied concentration quantitatively.     

Evolution of the serum resistance-associated SRA gene in African trypanosomes

Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and sequenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the ‘donor VSG’, which after duplication became inserted into the expression site by recombination. Under natural selection, SRAbc could reform into SRA following mosaic formation. Supported by National Natural Science Foundation of China (Grant Nos. 30570245, 30670275), Changjiang Scholars and Innovative Research Team in University (Grant No. DPCKSCU/IRT0447), International Foundation for Science of Sweden (Grant No. B/4318-1), Grant Agency of the Czech Republic (Grant No. Z60220518) and Education Foundation of the Czech Republic (Grant No. 2B06129)