Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction

Gene Splicing by Overlap Extension or "gene SOEing" is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. By modifying the sequences incorporated into the 5'-ends of the primers, any pair of polymerase chain reaction products can be made to share a common sequence at one end. Under polymerase chain reaction conditions, the common sequence allows strands from two different fragments to hybridize to one another, forming an overlap. Extension of this overlap by DNA polymerase yields a recombinant molecule. This powerful and technically simple approach offers many advantages over conventional approaches for manipulating gene sequences.     

The carboxy-terminal 41 amino acids of herpes simplex virus type 1 glycoprotein B are not essential for production of infectious virus particles.

Glycoprotein B (gB) is a virally encoded protein that is found in the envelope of herpes simplex virus type 1 and membranes of cells infected with herpes simplex virus type 1. It is essential for the production of infectious virus particles. An amber mutation was introduced into the gB gene by oligonucleotide-directed mutagenesis at the codon for amino acid 863 of the protein. Virus carrying this mutation should synthesize gB molecules lacking the last 41 amino acids of the cytoplasmic domain. Immunoprecipitation of infected cell extracts demonstrated the synthesis of appropriately truncated gB molecules. Characterization of the mutant virus indicated that the loss of the carboxy-terminal 41 amino acids has little effect on gB function.     

从“湖北光敏感核不育水稻”的未受精子房和花药培养出单倍体植株

采用10种诱导培养基,培养湖北光敏感核不育水稻农垦58品种的未受精子房和花药。共培养未受精子房2790个,获得胚囊愈伤组织17块,最高诱导频率达3.33%,其中2块分化出绿苗。培养花药16740个,获得花药愈伤组织15块,最高诱导频率为0.92%,其中3块分化出苗,2丛白苗,1株绿苗。胚囊植株和花粉植株经根尖染色体检查为单倍体,2n=x=12。实验证明,液体培养、2,4-D0.2-0.5 mg/1、低温预处理对诱导胚囊愈伤组织及花粉愈伤组织的形成具良好效果。     

灭幼脲引起两种幼虫表皮组织病变的显微观察

本文研究了灭幼脲引起黄粉(虫甲)(Tenebrio mclitor)和粘虫(Mythimna separata)幼虫的中毒征象和组织学病变.低剂量能引起幼虫蜕皮障碍,但看不到明显的组织学病变.高剂量处理,不仅引起了严重的中毒征象,而且伴有明显的组织学病变:内表皮生长停滞,真皮细胞排列异常,在内表皮和真皮细胞之间出现附加层和球状颗粒.对这些现象进行了较细致的讨论.     

蚤数量与宿主数量关系

无论在自然条件下或在人为条件下,蚤指数和染蚤率的高低与宿主密度的高低是一致的.宿主密度的升降,会导致其寄生蚤指数和染蚤率的升降. 本文讨论了宿主数量下降导致其寄生蚤数量下降的原因,仅是分析推测,提出几方面的看法.     

Hydrolysis of phosphatidylcholine is stimulated by Ras proteins during mitogenic signal transduction.

We have used a dominant inhibitory ras mutant (Ha-ras Asn-17) to investigate the relationship of Ras proteins to hydrolysis of phosphatidylcholine (PC) in the transduction of mitogenic signals. Expression of Ha-Ras Asn-17 inhibited NIH 3T3 cell proliferation induced by polypeptide growth factors or phorbol esters. In contrast, the mitogenic activity of PC-specific phospholipase C (PC-PLC) was not inhibited by Ha-Ras Asn-17 expression. Similarly, cotransfection with a cloned PC-PLC gene bypassed the block to NIH 3T3 cell proliferation resulting from expression of the inhibitory ras mutant. Hydrolysis of PC can therefore induce cell proliferation in the absence of normal Ras activity, suggesting that PC-derived second messengers may act downstream of Ras in mitogenic signal transduction. This was substantiated by the finding that Ha-Ras Asn-17 expression inhibited growth factor-stimulated hydrolysis of PC. Taken together, these results indicate that PC hydrolysis is a target of Ras during the transduction of growth factor-initiated mitogenic signals.     

Calcium-dependent protease of the cyanobacterium Anabaena: molecular cloning and expression of the gene in Escherichia coli, sequencing and site-directed mutagenesis

Summary It has been suggested that a calcium-dependent intracellular protease of the cyanobacterium, Anabaena sp., participates in the differentiation of heterocysts, cells that are specialized for fixation of N₂. Clones of the structural gene (designated prcA) for this protease from Anabaena variabilis strain ATCC 29413 and Anabaena sp. strain PCC 7120 were identified via their expression in Escherichia coli. The prcA gene from A. variabilis was sequenced. The genes of both strains, mutated by insertion of a drug resistance cassette, were returned to these same strains of Anabaena on suicide plasmids. The method of sacB-mediated positive selection for double recombinants was used to achieve replacement of the wild-type prcA genes by the mutated forms. The resulting mutants, which lacked Ca²⁺-dependent protease activity, were not impaired in heterocyst formation and grew on N₂ as sole nitrogen source.     

Immunocytochemical Localization of Glutamine Synthetase in Organs of Phaseolus vulgaris L

Glutamine synthetase was localized in nodules, roots, stems, and leaves of red kidney bean (Phaseolus vulgaris L.) by immunocytochemistry. Affinity purified antibodies reactive with glutamine synthetase were prepared using purified nodule-enhanced glutamine synthetase. Immunogold labeling was observed in the cell cytoplasm in each plant organ. In nodules, the labeling was more intense in the infected cells than in the uninfected cells. No labeling was observed in nodule bacteroids, peribacteroid spaces, or in peribacteroid membranes, while previous reports of glutamine synthetase immunolabeling of legume nodules showed labeling in the bacteroid fraction. Significant labeling was observed in nodule proplastids which contained starch granules. Substantial labeling was also observed in leaf chloroplasts. No labeling was observed in other organelles including mitochondria, peroxisomes, and endoplasmic reticulum. Preimmune IgGs did not bind to any structure in the tissues examined.     

A mechanistic perspective on bacterial metabolism of chlorinated methanes

Chlorinated methanes are important environmental pollutants, which can be metabolized by bacteria. The biotransformation of chlorinated methanes by bacteria has been shown to be due either to gratuitous metabolism (cometabolism) or their use as a source of carbon and energy. The reactions which result in carbon-halogen bond cleavage include substitutive, reductive, oxygenative, and gem-elimination mechanisms. Certain methylotrophic bacteria can use dichloromethane as a source of carbon and energy. Dichloromethane dehalogenase catalyzes the first substitutive reaction in this metabolism. The enzyme shows a 10¹⁰-fold rate enhancement over the reaction of the bisulfide anion with dichloromethane in water. Pseudomonas putida G786 synthesizes cytochrome P-450_CAM which catalyzes the gratuitous reduction of chlorinated methanes. These studies with purified enzymes are beginning to reveal more detailed mechanistic features of bacterial chlorinated methane metabolism.Abbreviations DNA deoxyribonucleic acid - k_cat catalytic first order rate constant for an enzyme catalyzed reaction - K_M Michaelis constant for an enzyme catalyzed reaction - MNDO modified neglect of diatomic overlap - PIMA pattern induced multialignment - DCMD dichloromethane dehalogenase