Safe biotechnology 9: values in risk assessment for the environmental application of microorganisms. The Safety in Biotechnology Working Party of the European Federation of Biotechnology.

Risk assessment for the deliberate release of microorganisms into the environment is traditionally carried out on a case-by-case basis. In a similar approach to that used when assessing human pathogenicity, we propose an alternative approach by introducing risk classes to facilitate or complement this type of risk assessment. These consider several sets of scenarios that address the different values that need to be protected. Examples of this approach include risk-class definitions for soil fertility and biodiversity.

Safe biotechnology. 8. Transport of infectious and biological materials. Working Party "Safety in Biotechnology" of the European Federation of Biotechnology.

The transport of infectious and biological material is regulated by a number of international organizations. This mini-review has been compiled to increase awareness within the scientific community of problems caused by differences in terminology (such as infectious materials/substances, biological products, diagnostic specimens, genetically modified microorganisms) and certain technical aspects of the main international guidelines, and to assist policy makers in the creation of harmonized guidelines. A list of relevant Internet resources has been compiled.

Safe biotechnology. 7. Classification of microorganisms on the basis of hazard. Working Party "Safety in Biotechnology" of the European Federation Biotechnology.

The current systems for classifying human pathogens on the basis of hazard are well developed and their basic criteria are in general agreement one with another. Of more importance, the safety practices based on these classifications have generally been successful. They have enabled extensive research activities, medical practice and industrial production to be conducted on an ever-increasing scale, involving dangerous microorganisms (e.g. in vaccine production and treatment of infected patients) with a very low incidence of adverse effects on the workers involved and the general public. Although the EU has adopted a harmonised list of agents in groups 1-4 there is as yet no complete agreement among member states and individual microbiologists. The purpose of this paper is to present a historical survey and to discuss the current processes for identifying and classifying the hazards posed by the use of microorganisms in research and technology. This is essential in the design of appropriate methods of counteracting potential risks.

Constructing expression cDNA libraries using unphosphorylated adaptors.

Making libraries of DNA fragments in various cloning vehicles is a basic experimental procedure in molecular biology, yet the methods involved often yield disappointing results, especially for the beginner. When screening for rare DNA molecules, large libraries must be constructed, and the efficiency of each reaction becomes critical. For cDNA libraries, three steps are necessary: cDNA must first be synthesized from poly(A)(+) RNA, the double-stranded cDNA must then be inserted into a suitable cloning vehicle, and, finally, cells must be transformed with the chimeric vector/cDNA molecules. Recent improvement in cDNA synthesis protocols (1) and in the quality of commercially available reverse transcriptase enables double-stranded cDNA to be made with an overall yield of about 50% from poly(A)(+) RNA. At the other end of the procedure, Escherichia coli strains can be made competent for transformation with plasmid DNA with efficiencies of more than 10(8) transformants/µg of supercoiled DNA (2). Frequently, however, the process of cloning DNA fragments into a plasmid vector reduces this potential to disappointing levels. If fragments are cloned directly into the vector by blunt-end ligation or by sticky-end ligation after attaching linkers, the efficiency is reduced by the need to treat the vector with alkaline phosphatase to prevent recircularization without insert.

Efficient construction of cDNA libraries in plasmid expression vectors using an adaptor strategy.

We describe a method for the construction of large DNA fragment libraries in plasmid vectors, in which complementary, single-stranded extensions are ligated onto both vector and insert DNA using un-phosphorylated adaptor oligonucleotides. Special consideration has been taken of the requirements of expression screening as follows: cDNA synthesis using random oligonucleotide primers is described which maximises the probability of obtaining open reading frame fragments from large mRNA molecules, the adaptors use codons found in high abundance E. coli proteins to minimise problems of premature termination when using strong promoters, and the sequence encoded by the adaptors, when cloned into the bacterial expression vector pEX1, promotes a surface location for the foreign antigenic determinant where it is accessible to antibodies used for screening.

The messenger RNA for alcohol dehydrogenase in Drosophila melanogaster differs in its 5' end in different developmental stages.

Alcohol dehydrogenase (EC 1.1.1.1) of Drosophila melanogaster is coded by a single structural gene, active in both larvae and adults. The major larval and adult transcripts of Adh differ in their 5'-untranslated regions. The major larval mRNA is about 1100 bases long, some 50 bases shorter than the major adult transcript. The 5' end of the larval mRNA is colinear with the genomic sequence immediately adjacent to the coding region, starting 70 base pairs (bp) upstream of the initiation codon. By contrast, the adult mRNA shares only 36 of its 123 5'-untranslated bases with the larval mRNA; the remaining 87 are encoded by a sequence 654 bp upstream. Both initiation sites are preceded by a TATA box some 24 bp upstream. The developmental specificity of Adh expression is seen, therefore, to have a counterpart in the specificity of transcription initiation at the two separate promoter regions.

The structural and infected cell polypeptides of influenza B virus.

Structural and virus-induced infected cell polypeptides of several strains of influenza B virus were examined by high resolution polyacrylamide gel electrophoresis and shown to be directly analogous to those of the influenza A viruses. Eight structural polypeptides, P1, P2, P3, HA1, HA2, NA, NP and M were observed in purified virus and at least two additional polypeptides, HA and NS could be detected in infected MDCK cells. The three P proteins plus NP were shown to be associated with RNA-dependent RNA polymerase activity and HA, HA1, HA2 and NA were shown to be glycosylated. Like the influenza A viruses, migrational differences of some of the infected cell polypeptides could be observed between different B strains. Investigation of a time course of virus replication failed to show any temporal control of protein synthesis in the infected cell.