Michael G. Murray

Phaseolin gene from bean is expressed after transfer to sunflower via tumor-inducing plasmid vectors.

Sequences coding for the bean seed protein phaseolin were inserted into transferred DNA regions of tumor-inducing plasmids. Constructions were devised in which the coding region of phaseolin was fused in the correct reading frame with the coding region of octopine synthase and placed under the transcriptional control of the octopine synthase promoter. Other plasmids were prepared to permit expression of the phaseolin-encoding sequences from the flanking phaseolin promoter region. The RNA transcribed in sunflower cells transformed with these constructions was characterized by hybridization procedures, SI nuclease mapping, and by translation in vitro of extracted RNA. These tests showed that the genomic intervening sequences were correctly excised. Immunoreactive phaseolin polypeptides were detected by enzyme-linked immunosorbent assay and by antibody hybridization to electrophoretically separated protein extracts of sunflower tissues isolated from crown gall tumors and of transformed sunflower cells grown in tissue culture. These results demonstrate the expression of a plant gene after transfer to a taxonomically distinct botanical family.

Poliovirus antigenic hybrids simultaneously expressing antigenic determinants from all three serotypes

We have constructed six hybrid polioviruses (PVs) modified to express PV type 2 and type 3 antigenic determinants on a PV type 1 (Mahoney) capsid. The hybrids were modified in neutralizing antigenic site (NAg) I and/or NAgII. They were viable, but impaired for growth in comparison to PV1 (Mahoney). Some hybrids modified to express type 2 and type 3 NAgI determinants simultaneously displayed some type 2 but no type 3 antigenicity (in addition to type 1 antigenicity associated with other antigenic sites). Hybrids modified to express a type 2 NAgI determinant and a type 3 NAgII determinant, or vice versa, displayed antigenic characteristics of all three serotypes, although expression of the modified NAgII determinant was weak. We conclude that it is possible to construct a viable hybrid PV simultaneously modified in NAgI and NAgII which expresses antigenic determinants of all three serotypes.

Use of a recombination-deficient phage lambda system to construct wheat genomic libraries

The poor cloning efficiency of wheat (Triticum aestivum cv. Yamhill) DNA in conventional cloning vectors has previously prevented the preparation of complete genomic libraries. We show here that while wheat DNA does not clone efficiently using the vector Ch4A, it can be cloned efficiently using Ch32. Ch32 clones are red- gam+ and therefore can be propagated on recombination-deficient hosts. These results suggest that instability of wheat sequences in conventional lambda vector systems has frustrated previous attempts to prepare libraries.

Phenotypic characterization of antigenic hybrids of poliovirus

Three poliovirus hybrids, modified in neutralization antigenic sites (NAgs) I or II, were characterized for several phenotypic traits. The modifications to the capsid interfered with some stage of the life-cycle of the virus, since all three hybrids were growth-impaired in comparison to poliovirus type 1 (Mahoney) [PV1 (M)], the wild-type parent virus. All hybrids exhibited a reduced growth rate and a small-plaque phenotype, but they were not temperature sensitive. Furthermore, only one hybrid was slightly less stable to heating than the parent virus; the other two were as stable as the parent. Therefore, decreased thermal stability of the capsid is not an important cause of the poor growth characteristics of these hybrids.

Chemical Synthesis of Poliovirus Peptides and Neutralizing Antibody Responses

Poliomyelitis, a disease caused by polio virus, remains a serious health problem throughout the world. Only in developed countries where the existing vaccines are regularly administered is the incidence of the disease low. However, occasional outbreaks of poliomyelitis in developed countries, albeit rare, are evidence of the present danger of infection with neurovirulent strains that may be imported from other places in the world. Moreover, the existing live vaccines produce a very low level of disease in vaccine recipients and/or persons coming in contact with vaccine recipients. During 1975–1984, the incidence of vaccine related poliomyelitis in the United States of America was one case per 3.22 × 106 doses of live vaccine administered to immunologically normal recipients (CDC., 1986). Even such low incidence has aroused much public discussion recently (Chasan, 1986). Organizations concerned with public health, such as the World Health Organization (WHO), are interested therefore in developing programs that may eventually lead to the worldwide eradication of poliomyelitis. It should be stressed that the existing live (OPV) and inactivated (IPV) vaccines have proven to be excellent vaccines whenever applied properly. However, investigation to identify and eliminate those properties of the OPV strains that are responsible for reversion to the neurovirulent phenotype or research to explore the possibility of augmenting a protective immune response by the development of synthetic antigens has become an important subject for the WHO: Although much effort may have to be expended, a synthetic vaccine may be developed that possesses the required potency and that can be produced at such low cost, that it can be considered as an alternative to those vaccines now in existence. This in turn may serve as a model to combat other enteroviral disease such as hepatitis caused by HAV.