Peter G. Sanders

Genetic engineering of hybridoma glutamine metabolism

The murine hybridoma PQXB1/2 cannot be adapted to grow in culture media containing < 0.5 mM glutamine. Transformants selected following electroporation of PQXB1/2 cells with vectors containing a Chinese hamster glutamine synthetase (GS) cDNA under the control of the SV40 early promoter also failed to grow in the absence of glutamine in the culture medium. PQXB1/2 cells have, however, been transformed to glutamine independence following electroporation with a vector containing this glutamine synthetase cDNA under the control of the human cytomegalovirus immediate early promoter. In these cells, sufficient active glutamine synthetase was expressed from one vector per cell to enable growth in glutamine-free media. The specific activity of glutamine synthetase in two transformed cell lines producing parental levels of antibody was increased by 128 and 152%, respectively (0.57 and 0.63 mumol min-1 per 10(6) cells in transformants compared with parental levels of 0.25 mumol min-1 per 10(6) cells). This reprogramming of glutamine synthetase expression and glutamine metabolism is important for developing strategies to deal with ammonia toxicity and the production of cell lines with improved metabolic processes.

Nucleotide sequences and expression of cDNAs for a bovine anti-testosterone monoclonal IgG1 antibody

cDNAs coding for the heavy and light chains of a bovine anti-testosterone IgG1 monoclonal antibody have been cloned and sequenced. These cDNAs are the first to be reported for functionally rearranged bovine immunoglobulin genes. Testosterone binding by the antibody encoded by the cDNAs has been verified by expression of the cDNAs in COS-1 cells and detection of anti-testosterone antibodies in transfected cell media using an ELISA specific for bovine anti-testosterone IgG. The derived protein sequence of the variable domains have suggested a possible binding model for the interaction between the antibody and testosterone. The derived protein sequence of the constant domains has been used to identify residues which could be involved in the selective transport of bovine IgG1 from blood plasma into colostrum at the time of parturition.

Purification, crystallization and preliminary X-ray analysis of human recombinant cytosolic serine hydroxymethyltransferase

As an enzyme of the thymidylate synthase cycle, serine hydroxymethyltransferase (SHMT) has a key role in nucleotide biosynthesis. Elevated activities of SHMT have been correlated with the increased demand for nucleotide biosynthesis in tumors of human and rodent origin, making this enzyme a novel target for cancer chemotherapy. Here the purification and crystallization of recombinant human cytosolic SHMT are reported. Crystals belong to space group P6222 or P6422 with cell parameters a = b = 155.0, c = 235.5 A and diffract to at least 3.0 A resolution.

Characterisation of a human serine hydroxymethyltransferase pseudogene and its localisation to 1p32.3-33

The conversion of serine and tetrahydrofolate to glycine and 5,10 methylene tetrahydrofolate by serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is the major route for the provision of one-carbon units for biosynthetic reactions. SHMT cDNAs have been cloned from both rabbit and man, and a human mitochondrial SHMT gene sequence has recently been reported. We have isolated phage clones containing human genomic sequences homologous to cytosolic SHMT and have found these to contain a processed pseudogene (SHMT-psl) with a 90% identity to cloned SHMT cDNAs. SHMT ps1 contains 2335 nt that are homologous to SHMT but it has an additional leader sequence of 203 nt of unknown origin. The complete SHMT-ps.1 sequence of 2538 nt is bounded by two 16 nt direct repeats that are characteristic of retroelement insertion sites. Two phage clones containing SHMT-ps1 have been mapped by fluorescence in situ hybridisation to 1p32.3–33. ln addition, an SHMT cDNA clone hybridized to the same region and to 17p11.2–12. The latter is consistent with a previous localisation of the gene for cytosolic SHMT.

GENETIC MODIFICATION OF HYBRIDOMA GLUTAMINE METABOLISM: PHYSIOLOGICAL CONSEQUENCES

Transformation of the hybridoma cell line PQXB1/2 with a cloned glutamine synthetase gene has resulted in the ability to grow in the absence of glutamine. Other phenotypic effects resulting from this genetic manipulation have been investigated particularly with regard to culture duration, maximum cell density, antibody production, glucose utilisation, and the production of ammonia and lactate.

Sequence variation in 5' termini of rubella virus genomes : changes affecting structure of the 5' proximal stem-loop

SummaryVariation within a 523 nucleotide region proximal to the 5′ terminus of seven rubella virus strains has been anlysed. Compared to the Therien strain twenty sites of nucleotide variation have been identified, three of which are in the 5′ untranslated region. Individual strains have between three and nine nucleotide differences, only three of which result in amino acid substitutions. TO-336 has a serine for threonine at amino acid (aa) 42 and CM arginine for histidine at aa 159. RA27/3 has arginine for lysine at aa 3 and serine for threonine at aa 42. Nucleotide differences which affect a stem-loop structure reported to be important for binding of host cell proteins have been identified.

PRODUCTION OF RECOMBINANT RUBELLA ANTIGENS

The E1 and E2 glycoprotein coding regions of the rubella genome have been cloned, sequenced and modified for expression in mammalian cells as both membrane bound and secreted proteins.

Regulation of Mammalian Serine Hydroxymethyltransferase

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5’ phosphate dependent enzyme that directs serine towards nucleotide synthesis by catalysing the interconversion of serine and glycine. The products of this reaction, glycine and 5, 10-methylene tetrahydrofolate, are involved directly and indirectly in pyrimidine and purine biosynthesis. SHMT is therefore an important target for enzyme directed anticancer agents, as inhibition of the enzyme would block DNA synthesis by action on the two parallel biosynthetic pathways resulting in a combination chemotherapeutic effect from a single inhibitor. Both cytosolic and mitochondrial isozymes of SHMT exist in the cell. However the individual contributions of each isozyme to nucleotide biosynthesis remains to be elucidated.