Molecular cloning and transgenic expression of a synthetic human erythropoietin gene in tobacco

Erythropoietin (EPO) is a hormone belonging to a group of hematopoietic growth factors that control the proliferation and differentiation of bone marrow cells. It induces the production of erythrocytes, thereby increasing the amount of circulating hemoglobin and oxygen. Previous attempts to transgenically express human EPO in plants failed to succeed because the plants exhibited abnormal morphology and infertility. In the present work, we describe the generation of fertile transgenic tobacco plants able to express a synthetic version of human EPO. A 582-bp fragment of the human EPO gene was synthesized using a PCR-based method and ligated into pCR-Blunt. After sequencing, the human EPO fragment was transferred to pWUbi.tm1 and the expression cassette was then transferred to the binary vector pWBVec4a. After Agrobacterium-mediated transformation of Nicotiana tabacum SR1 plants, integration of the transgene into T₀ and T₁ plant genomes was confirmed by PCR. The human EPO gene was found to be expressed in tobacco leaves at the mRNA and protein levels. Self-crossing allowed us to obtain T₁ plants exhibiting Mendelian segregation of the transgene. None of the plants presented any kind of malformation or deformity.     

Molecular cloning and expression of Bacillus thuringiensis subsp. galleriae insecticidal crystal protein genes in Escherichia coli

The location of the toxin gene of B. thuringiensis subsp. galleriae (H5ab) on the Mr-130Md plasmid is determined by molecular cloning. Double digestion fragments (BamHI and SalI) and PstI restriction fragments as well, from the 130 Md plasmid, of B. thuringiensis subsp. galleriae, are ligated with the cloning vector pAT 153 respectively and transformed into E. coli strain HB 101. Out of 208 transformants, three colonies (FG2, FG9, FG19) give positive hybridization reaction using the HD-1 delta-endotoxin gene as a probe. They are presumed to contain the delta-endotoxin gene of B. thuringiensis subsp. galleriae. Western blot assays indicate that Mr-130 kDal and 68 kDal, crystal proteins produced by clone FG2 react with anticrystal protein antibody. The protein extracts of clone FG2 are lethal to Ostrinia furnacalis (Guenee). This is the first report with regard to the cloning and expression of the B. thuringiensis subsp. galleriae (H5ab) delta-endotoxin gene.     

cDNA cloning,expression and bioinformatical analysis of Tssk genes in tree shrews

Tree shrews are more closely related to primate animals than rodents in many aspects.In addition, they also possess several advantageous characteristics including small body size, high brain-to-body mass ratio, low cost of feeding and maintenance, short reproductive cycle and life span, which make them promising novel laboratory animals to replace more precious larger primate animals. Testis-specific serine/threonine kinase (Tssk) plays important roles in spermatogenesis and/or the regulation of sperm function. However, studies on Tssk in tree shrews have not been reported yet. In the present study, the full-length sequences of five members of the Tssk family in tree shrews were cloned and their CDS region sequences were analyzed by basic bioinformatics. The phylogenetic tree and prokaryotic protein expression system of Tssk gene of tree shrews were constructed. The mRNA expressions of Tssk genes in 11 tissues/organs from tree shrews were studied. The results showed that: 1. the length of the CDS region of tree shrew Tssk gene for Tssk1B, Tssk2, Tssk3 (variant X1 / X2), Tssk4 (variant X1 / X2) and Tssk6 is 1080bp, 1077bp, 867 / 807bp, 1014 / 984bp, 822bp, respectively, encoding 359, 358, 288/268, 337/327 and 273 amino acids, respectively; the cloned sequences of Tssk genes have been submitted to GenBank with the following accession numbers: KX091161(Tssk1B), KX091162(Tssk2), KX091163(Tssk3 variant X1)/KX091164(Tssk3 variant X2), KX091165(Tssk4variant X1)/KX091166(Tssk4variant X2), KX091160(Tssk6). 2. All tree shrew Tssk proteins distribute in cytoplasm, indicating that they are hydrophilic and non-secretory proteins, with multiple phosphorylation sites of serine and/or threonine. In addition, they are all mixed proteins with similar tertiary structures sharing a highly conserved functional domain of S_TKc (Serine/Threonine protein kinases, catalytic domain). 3.The molecular phylogenetic tree of five Tssk genes in tree shrews indicates that they are neither rodent nor primate animal, but are closely related to primate animals. 4. Five members of the Tssk recombinant proteins in tree shrews were successfully obtained using the constructed prokaryotic protein expression system. 5. Five Tssk genes are specifically expressed in the testis and/or sperm of tree shrews. Additionally, small amount of Tssk1B was expressed in several tissues other than testis and sperm. Limited mRNA levels of Tssk2 and Tssk4 were expressed in the brain, while mRNA of Tssk3 or Tssk6 could only be detected in the testis and sperm.This study will provide fundamental data on reproductive biology of tree shrews, which paves a way for further studying Tssk’s biological function in this novel model animal.     

Analysis of the expression of two phosphoglycerate kinase genes in a mouse cultured cell line during activation and inactivation of the c-myc gene

The effect of a change in the concentration of c-myc protein on the expression of genes for two phosphoglycerate kinase isozymes was investigated. The steady state levels of messenger ribonucleic acids (mRNAs) for sperm-type and non-sperm-type proteins were determined by blot hybridization using the RNA of the mouse cell line 38-2 containing the inducible rat c-myc gene cultured under various conditions. Without induction the c-myc gene. mRNA for non-sperm-type protein was detected at a level that remained essentially constant during both activation and inactivation of the c-myc gene. mRNA for sperm-type protein was not detected in 38-2 cells cultured under any conditions used. Change in the amount of c-myc protein alone does not appear to bring about a switch of the expression of the two phosphoglycerate kinase genes during spermatogenesis in mouse testis.     

Molecular cloning of clathrin assembly protein gene (rCALM) and its differential expression to AP180 in rat brain

Binding of clathrin assembly protein to clathrin triskelia induces their assembly into clathrin-coated vesicle (CCV) in neurons. The clathrin assembly protein gene (rCALM) was cloned from rat brain cDNA library. rCALM deduced 69 kD molecule has overall 73% amino acid homology compared with that of AP180 protein. The N-terminal domain, where amino acid sequences are very similar with AP180, harbours binding sites for clathrin and inositides, as well as possible phosphorylation sites, but the proline rich C-terminal domain is different from that of AP180. The mRNA expression of rCALM and AP180 by in situ hybridization histochemistry revealed that the rCALM mRNA was more intensely expressed than that of AP180, and the distribution patterns were different from each other. These results suggest that the rCALM mediates the assembly of clathrin in neural and supporting cells of brain, and regulates the clathrin coated-vesicle formation through phosphorylation and inositide metabolism.     

Molecular structure of σ3λ5

Institute for Organic Chemistry, Academy of Sciences of the USSR. Translated from Zhurnal Strukturnoi Khimii, Vol. 32, No. 5, pp. 117–128, September–October, 1991.     

LEA proteins in higher plants: structure, function, gene expression and regulation

Late embryogenesis abundant (LEA) proteins are mainly low molecular weight (10–30 kDa) proteins, which are involved in protecting higher plants from damage caused by environmental stresses, especially drought (dehydration). These findings and the fact that the breeding of drought tolerant varieties would be of great value in agriculture, form the basis of search for anti-drought inducible genes and their characterization. LEA proteins are generally classified into six groups (families) according to their amino acid sequence and corresponding mRNA homology, which are basically localized in cytoplasm and nuclear region. LEA protein synthesis, expression and biological activities are regulated by many factors (e.g. developmental stages, hormones, ion change and dehydration), signal transduction pathways and lea genes. No tissue-specific lea gene expression has been considered as one main regulatory mechanism on the basis of extensive studies with the model plant, Arabidopsis thaliana. The study of the regulatory mechanism of lea gene expression is an important feature of modern plant molecular biology.     

Molecular structure of 3-trifluoroacetamidobenzoyltrifluoroacetone enol form

3-Trifluoroacetamidobenzoyltrifluoroacetone formed in the reaction of 3-aminoacetophenone with an excess of methyl trifluoroacetate crystallizes in the cis-enol form with a hydroxy group located at the carbon atom bound with the trifluoromethyl substituent. Analysis of geometric characteristics indicate the presence of both intramolecular hydrogen bond in the enol fragment and intermolecular H...O contacts in the crystal of the compound.     

Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes

Ergot alkaloids are toxins and important pharmaceuticals which are produced biotechnologically on an industrial scale. They have been identi?ed in two orders of fungi and three families of higher plants. The most important producers are fungi of the genera Claviceps, Penicillium and Aspergillus (all belonging to the Ascomycota). Chemically, ergot alkaloids are characterised by the presence of a tetracyclic ergoline ring, and can be divided into three classes according to their structural features, i.e. amide- or peptide-like amide derivatives of D-lysergic acid and the clavine alkaloids. Signi?cant progress has been achieved on the molecular biological and biochemical investigations of ergot alkaloid biosynthesis in the last decade. By gene cloning and genome mining, gene clusters for ergot alkaloid biosynthesis have been identi?ed in at least 8 different ascomycete species. Functions of most structure genes have been assigned to reaction steps in the biosynthesis of ergot alkaloids by gene inactivation experiments or biochemical characterisation of the overproduced proteins.     

Molecular structure of eight-membered cyclic sulfites with two planar fragments

Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 9, pp. 2033–2036, September, 1988.     

Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis

PCR primers designed to selectively amplify the unique C-methyltransferase domain of fungal polyketide synthases were used to selectively clone a polyketide synthase gene involved in the biosynthesis of the squalene synthase inhibitor squalestatin S1 , heterologous expression of which led to the biosynthesis of the squalestatin side-chain.     

Molecular,crystal, and electronic structure of 3-aminocarbonyl-1-phenyl-5-chloro-2-iminopyridone

Riga Polytechnical Institute. Institute of Organic Synthesis, Academy of Sciences of the Latvian SSR. Translated from Zhurnal Strukturnoi Khimii, Vol. 29, No. 5, pp. 169–172, September–October, 1988.