The biotechnology and applications of antibody engineering

The exquisite specificity of monoclonal antibodies (MAb) has long provided the potential for creating new reagents for the in vivo delivery of therapeutic drugs or toxins to defined cellular target sites or improved methods of diagnosis. However, many difficulties associated with their production, affinity, specificity, and use in vivo have largely confined their application to research or in vitro diagnostics. This situation is beginning to change with the recent developments in the applied molecular techniques that allow the engineering of the genes that encode antibodies rather than the manipulation of the intact antibodies themselves. Techniques, such as the polymerase chain reaction, have provided essential methods with which to generate and modify the genetic constituents of antibodies, allow their conjugation to toxins or drugs, provide ways of humanizing murine antibodies, and allow discrete modular antigen binding components to be produced. More recent developments of in vitro expression systems and powerful phage surface display technologies will without doubt play a major role in future antibody engineering and in the successful development of new diagnostic and therapeutic antibody-based reagents.     

Specific monitoring by PCR amplification and bioluminescence of firefly luciferase gene-tagged bacteria added to environmental samples

Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 10², by PCR amplification; 10³, by whole cell luminescence; and 10³−10⁴, by measurement of luminescence in cell extracts.     

Chlorsulfuron resistance in Daucus carota cell lines and plants:Involvement of gene amplification

Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level.     

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5 end of the cDNA was amplified by a 5-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.     

Microbes in food processing technology

Abstract: There is an increasing understanding that the microbial quality of a certain food is the result of a chain of events. It is clear that the microbial safety of food can only be guaranteed when the overall processing, including the production of raw materials, distribution and handling by the consumer are taken into consideration. Therefore, the microbiological quality assurance of foods is not only a matter of control, but also of a careful design of the total process chain. Food industry has now generally adapted quality assurance systems and is implementing the Hazard Analysis Critical Control Point (HACCP) concept. Rapid microbiological monitoring systems should be used in these cases. There is a need for rapid and simple microbiological tests which can be adapted to the technology and logistics of specific production processes. Traditional microbiological methods generally do not meet these high requirements. This paper discusses the tests, based on molecular biological principles, to detect and identify microbes in food-processing chains. Tests based on DNA technology are discussed, including in vitro DNA amplification like the polymerase chain reaction (PCR) method and identifications based on RFLP, RAPD and DNA fingerprinting analysis. PCR-haled methodology can be used for the rapid detection of microbes in food manufacturing environments. In addition, DNA fingerprinting methods are suitable for investigating sources and routes of microbial contamination in the food cycle.     

Magnification in Drosophila: evidence for an inducible rDNA-specific recombination system

An experiment is described that provides evidence for an exchange mechanism to explain the increase in ribosomal gene number that occurs during bobbed magnification. We show that bobbed and bobbed-lethal alleles do not magnify in closed X chromosomes, but that a spontaneous ring opening restores normal magnification. The results provide strong evidence that the elementary magnifying event is unequal sister chromatid exchange, and can be interpreted in the framework of an inducible rDNA-specific recombination system as the basis of ribosomal gene magnification. Correspondence to: S.A. Endow at the above address     

旋毛虫肌幼虫ES抗原的基因克隆及高效表达

作者对编码旋毛虫肌幼虫ES抗原的部分结构基因进行了克隆、鉴定和表达。用RNA PCR技术直接从旋毛虫肌幼虫总RNA中反转录并扩增出0．7kh的靶DNA,酶切分析后将其克隆到融合表达载体pEx3lC中。SDS—PAGE电泳表明,含重组子的大肠杆菌能够表达出一分子量为37kDa的融合蛋白(P37),后者占菌体总蛋白的22%以上,并以包含体形式存在于菌体中。经对纯化后表达蛋白的ELlSA检测,证明它能被猪旋毛虫病阳性血清和抗旋毛虫单克隆抗体识别。研究结果揭示,重组蛋白P37对于研制旋毛虫病诊断抗原和免疫抗原具有潜在的应用价值。