Cloning,expression and characterization of a gene which encodes a topoisomerase I with positive supercoiling activity in pea

We have isolated and sequenced the full length cDNA for topoisomerase I. Using degenerate primers, based on the conserved amino acid sequences of five eukaryotic topoisomerase I, a 386 bp fragment was PCR amplified using pea cDNA as template. This fragment was used as a probe to screen a pea cDNA library. Two partial cDNA clones were isolated which were truncated at the 5 end. RACE-PCR was employed to isolate the remaining portion of the gene. The total size of the gene was 3055 bp with an open reading frame of 2676 bp. The deduced structure of pea topoisomerase I contain 892 amino acids with a calculated molecular weight of 100 kDa and an estimated pI of 9.3. A comparison of the deduced amino acid sequences of the pea topo I with the other eukaryotic topoisomerases clearly suggested that they are all related. Pea topoisomerase I has been overexpressed in E. coli system and the recombinant topoisomerase purified to homogeneity. The purified protein relaxes both positive and negative supercoiled DNA in the absence of divalent cation Mg²⁺. In the presence of Mg²⁺ ions the purified enzyme introduces positive supercoils a unique property not reported in any other organism except in archaebacterial topoisomerase I. Polyclonal antibodies were raised against recombinant topoisomerase I and western blotting with sub-cellular fractions indicated the localization of this topoisomerase in pea nuclei.     

Ascorbic acid effect on the onset of cell proliferation in pea root

The ability of ascorbic acid to induce cell proliferation of non-cycling cells was investigated in quiescent embryo root of Pisum sativum L. cv. Lincoln, as well as in the active plantlet root meristem, where a minor portion of the cells is non-proliferating. Quiescent embryo cells speeded up the G₀–G₁ transition during germination in the presence of ascorbic acid. In addition, proliferating cells present in the root tip of 3-day-old plantlets, arrested at the G₁/S boundary by hydroxyurea, resumed the cycle earlier than the control, when treated with ascorbic acid. In contrast, ascorbic acid was unable to induce the proliferation of non-cycling cells present in the active meristem. Therefore, these data suggest that the ability of ascorbic acid lo induce cell proliferation depends on the physiological status of the cell. In particular the data indicate that ascorbic acid is involved in cell proliferation as a factor necessary to enable already competent cells to progress through the cell cycle phases, but not as a factor able to induce non-competent cells to overcome proliferation arrest.     

Cloning and characterization of a cDNA encoding a cowpea seed defensin and analysis of its expression

We have previously isolated a cowpea seed defensin with potent antifungal activity. Here we report the cloning of its cDNA obtained from RT-PCR. The sequence of the deduced protein VUDEF exhibited homology to plant defensins. Northern analysis revealed that VUDEF mRNA is accumulated during cowpea seed development and its level increases in seedling tissues after exposure to fungal pathogen and cold.     

ABA content in shoots and roots of pea mutants af and tl as related to their growth and morphogenesis

The comparative study of shoot and root growth was carried out, and the level of ABA therein determined in the mutant af and tl and wild-type isogenic lines of pea. The recessive af mutation transformed the leaflets into tendrils, and the tl mutation transformed the tendrils into leaflets. These mutations did not affect the length and number of internodes. In all plants, the level of ABA in the leaves was 3–10 times greater than in the roots, and in the course of vegetative growth it rose in both organs. An increase in the shoot area of tl mutant did not change the dry weight of underground and above-ground parts; therefore, the ratio shoot/root in the mutant was identical to that in the wild-type plants. The maintenance of shoot dry weight in the tl mutant at the level of wild-type plant while its area considerably increased was accounted for by a decrease in the thickness of the leaflet and stipule blades. The level of ABA in the stipules of mutant plants was greater than in the wild-type plants. A decrease in the shoot area in the af mutant brought about a decline in its dry weight; however, the ratio root/shoot was maintained at the wild-type level due to a reduced accumulation of dry weight by the root. The level of ABA in the roots of the af mutant was twice greater than in the leafy forms. ABA was assumed to participate in the control over the root growth exerted by the shoot. The absence of leaflets in the af plants was partially compensated for by expanding stipules. The level of ABA therein was three times higher than in the plants of wild type and comparable with the level in the leaflets of the tl mutant and in the wild-type plants. The role of ABA in the growth and final size of leaf blades is discussed.     

Apoptosis-like programmed cell death occurs in procambium and ground meristem of pea (Pisum sativum) root tips exposed to sudden flooding

BACKGROUND AND AIMS: Pea (Pisum sativum) primary roots form long vascular cavities when grown under wet or flooded conditions at 25 degrees C. It is thought that the cavities are a form of aerenchyma. At 25 degrees C short roots continue to grow after flooding. After roots reach 10 cm long flooding causes rapid cessation of growth, and root tips often become curled. In longer roots the cavities do not extend into the base of the roots, perhaps rendering them ineffective as aerenchyma. It was hypothesized that the resulting growth arrest was due to programmed cell death (PCD) rather than necrosis. METHODS AND KEY RESULTS: Histological examination by light microscope showed that some cells in the primary meristem (elongation) zone of the primary root tips had morphological abnormalities, including misshapen and fragmented nuclei, and cytoplasmic shrinking and fragmentation. Transmission electron microscopy revealed lobing, invagination and chromatin aggregation in nuclei. The affected cells were positive for terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling. Extracted DNA formed a "DNA ladder" during electrophoresis. Cell death usually began in procambium at one or two protoxylem poles and seemed to spread out to nearby tissues, which asymmetrically inhibited growth and resulted in tip curling. CONCLUSIONS: The above are symptoms of apoptosis-like PCD. Programmed root tip death may rapidly reduce oxygen demand and sink strength, allowing more rapid diversion of resources to lateral roots growing in more permissive conditions.     

Cloning,characterization and expression of a cDNA encoding a granulin-like polypeptide in Ciona savignyi

Previous study in our laboratory confirmed that a novel polypeptide, CS5931 derived from Ciona savignyi possesses potent antitumor activity. In the present study, the full length cDNA of CS5931 precursor, termed Cs-pgrn-1 was cloned. The complete cDNA sequence of this gene consists of 685 bp containing an open reading frame (ORF) of 522 bp (173 amino acid residues). In silico analysis revealed that the polypeptide consists of two identical domains, similar with granulin (GRN) found in other species, and each of the domain encodes a polypeptide identical with CS5931. Phylogenetic analysis confirmed that CS5931 shares high homology with Ciona intestinalis GRN and is conserved during evolution. The polypeptide also shows high similarity with human GRN A, B, and C. Prediction of 3D protein structure revealed the 3D structure of CS5931 is very similar with human GRN A. The CS5931 was expressed using a prokaryotic expression system and the purified polypeptide inhibited the growth of several tumor cell lines in vitro via apoptotic pathway. Our study revealed that CS5931 has the potential to be developed as a novel antitumor agent.     

Cloning and expression of cDNA encoding a neural calcium-dependent cell adhesion molecule: its identity in the cadherin gene family

The neural cadherin (N-cadherin) is a Ca2+-dependent cell-cell adhesion molecule detected in neural tissues as well as in non-neural tissues. We report here the nucleotide sequence of the chicken N-cadherin cDNA and the deduced amino acid sequence. The sequence data suggest that N-cadherin has one transmembrane domain which divides the molecule into an extracellular and a cytoplasmic domain; the extracellular domain contains internal repeats of characteristic sequences. When the N-cadherin cDNA connected with virus promoters was transfected into L cells which have no endogenous N-cadherin, the transformants acquired the N-cadherin-mediated aggregating property, indicating that the cloned cDNA contained all information necessary for the cell-cell binding action of this molecule. We then compared the primary structure of N-cadherin with that of other molecules defined as cadherin subclasses. The results showed that these molecules contain common amino acid sequences throughout their entire length, which confirms our hypothesis that cadherins make a gene family.     

Cloning of cDNA encoding the sweet-tasting plant protein thaumatin and its expression in Escherichia coli

The structural gene of the sweet-tasting plant protein (prepro)thaumatin was cloned and expressed in Escherichia coli. Expression was effected under control of lac and trp promoter/operator systems and through the use of bacterial ribosome-binding sites. The naturally occurring thaumatin II represents a processed form. The primary translation product, preprothaumatin, of the cloned mRNA-derived cDNA contains extensions at both the amino terminus and the carboxy terminus. The amino terminal extension of 22 amino acids is hydrophobic and very much resembles an excretion-related signal sequence. The six amino acids-long carboxy terminal extension is very acidic in character, in contrast to the overall highly basic thaumatin molecule. The possible role of such an acidic tail with respect to compartmentalization is discussed.     

Cloning of rat olfactory bulb tubulin tyrosine ligase cDNA: a dominant negative mutant and an antisense cDNA increase the proliferation rate of cells in culture

In this paper we describe the cloning of rat olfactory bulb tubulin tyrosine ligase (TTL) cDNA, and investigate the physiological role of TTL in cultured CHO-K1 cells. Comparison of the deduced amino acid sequence of rat TTL cDNA with those of bovine and pig showed approximately 90% of identity. Transient transfection of CHO-K1 cells with a dominant negative mutant of TTL that contains the binding site to the substrate (tubulin) but not the catalytic domain, significantly decreased the endogenous TTL activity as determined in vitro. Similar results were obtained using a construction encoding for the antisense sequence of TTL. The reduction in TTL activity is not accompanied by a decrease in the tyrosination levels of microtubules, as judged by immunofluorescence analysis. Strikingly, the number of cells in the plates transfected with the mutant TTL or the antisense TTL cDNA was, after 72 h of culture, two and three times higher, respectively, than the number of cells in the control plates. These results support the hypothesis that TTL may play a role in the regulation of the cell cycle in living cells.     

人干细胞生长因子-α基因的克隆、表达及其协同rhGM-CSF对人脐带间充质干细胞的增殖作用

为了研究hSCGF-α对人脐带间充质干细胞 (Human umbilical cord mesenchymal stem cells,hUCMSCs) 的作用,采用基因工程技术获得重组人干细胞生长因子-α (Recombinant human Stem Cell Growth Factor-α,rhSCGF-α)。针对SCGF基因的高GC含量,采用PCR两步法获得hSCGF-α基因,插入pET-28a(+) 载体质粒,构建重组质粒pET-28a-SCGF-α,转化大肠杆菌BL21(DE3) 获得表达菌株。低温20 ℃诱导24 h,目标重组蛋白人干细胞生长因子-α以可溶性表达为主。通过Ni2+-NTA柱纯化,获得目标重组蛋白,电泳谱带扫描分析蛋白纯度可达90％以上。以巨噬细胞/粒细胞(Granulocyte/macrophage,GM)集落形成实验鉴定重组蛋白的生物学活性,并协同重组人巨噬细胞/粒细胞集落刺激因子(Recombinant human GM-colony stimulating factor,rhGM-CSF) 研究其对人脐带间充质干细胞的影响。结果显示,纯化的重组蛋白rhSCGF-α具有生物学活性;hSCGF-α及rhGM-CSF对hUCMSCs均有刺激增殖活性,但协同作用效果最强。     

Cloning,sequence analysis and expression of a cDNA encoding active phosphoenolpyruvate carboxylase of the C3 plant Solanum tuberosum

A cDNA coding for phosphoenolpyruvate carboxylase (PEPC) was isolated from a cDNA library from Solanum tuberosum and the sequence of the cDNA was determined. It was inserted into a bacterial expression vector and a PEPC^- Escherichia coli mutant could be complemented by the cDNA construct. A functional fusion protein could be synthesized in E. coli. The properties of this PEPC protein clearly resembled those of typical C3 plant enzymes.