Use of polymerase chain reaction (PCR) in the development of a recombinant organism.

Tryptic mapping of a purified recombinant antibody expressed in a mammalian cell line demonstrated low level heterogeneity in the heavy chain. Amino acid analysis and N-terminal sequencing of these fragments revealed that a conversion of tyrosine (Y) to glutamine (Q) had occurred at residue 376. DNA sequencing of 30 clones of the original expression construct indicated that the chance of the Y376Q variant being present in the original plasmid was low, < 5%. Once the gene had been cloned into a mammalian cell system, sequencing of 30 clones is considered ineffective for screening large numbers of subclones. Consequently, an alternative method was developed to directly probe total mammalian DNA for the presence of cloned variant. The method described here uses a polymerase chain reaction (PCR) based assay optimizing the ability of Taq polymerase to distinguish whether the 3' end of primers have completely annealed with template DNA during PCR cycling. Ten percent of the subclones producing high levels of the variant were identified and experiments indicated that the Y to Q conversion had developed during transfection of the antibody gene into the mammalian cell system. PCR was shown to be useful as a quick screen for verification of a cloned variant. Tryptic mapping is still considered the most sensitive and appropriate analytical tool for assessing genetic heterogeneity of recombinant cell lines.

Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

Structure and developmental expression of the nerve growth factor receptor in the chicken central nervous system.

Chicken nerve growth factor (NGF) receptor cDNAs have been isolated and sequenced in an effort to identify functionally important receptor domains and as an initial step in determining the functions of the NGF receptor in early embryogenesis. Comparisons of the primary amino acid sequences of the avian and mammalian NGF receptors have identified several discrete domains that differ in their degree of conservation. The highly conserved regions include an extracellular domain, likely to be involved in ligand binding, in which the positions of 24 cysteine residues and virtually all negatively charged residues are conserved; a transmembrane region, including flanking stretches of extracellular and cytoplasmic amino acids, which has properties suggesting it interacts with other proteins; and a cytoplasmic PEST sequence, which may regulate receptor turnover. Transient expression of NGF receptor mRNA has been seen in many regions of the developing CNS. Experiments suggest that both NGF and its receptor help regulate development of the retina.

Amino acid sequence and distribution of mRNA encoding a major skeletal muscle laminin binding protein: an extracellular matrix-associated protein with an unusual COOH-terminal polyaspartate domain.

Two cDNAs encoding an abundant chicken muscle extracellular matrix (ECM)-associated laminin-binding protein (LBP) have been isolated and sequenced. The predicted primary amino acid sequence includes a probable signal peptide and a site for N-linked glycosylation, but lacks a hydrophobic segment long enough to span the membrane. The COOH terminus consists of an unusual repeat of 33 consecutive aspartate residues. Comparison with other sequences indicates that this protein is different from previously described LBPs and ECM receptors. RNA blot analysis of LBP gene expression showed that LBP mRNA was abundant in skeletal and heart muscle, but barely detectable in other tissues. Blots of chicken genomic DNA suggest that a single gene encodes this LBP. The amino acid sequence and mRNA distribution are consistent with the biochemical characterization described by Hall and co-workers (Hall, D. E., K. A. Frazer, B. C. Hahn, and L. F. Reichardt. 1988. J. Cell Biol. 107:687-697). These analyses indicate that LBP is an abundant ECM-associated muscle protein with an unusually high negative charge that interacts with both membranes and laminin, and has properties of a peripheral, not integral membrane protein. Taken together, our studies show that muscle LBP is a secreted, peripheral membrane protein with an unusual polyaspartate domain. Its laminin and membrane binding properties suggest that it may help mediate muscle cell interactions with the extracellular matrix. We propose the name "aspartactin" for this LBP.

Molecular cloning and characterization of ribosomal RNA genes from the brine shrimp.

A library of genomic DNA from the brine shrimp, Artemia, has been constructed with the Charon 4A phage vector, utilizing EcoRI passenger fragments. Screening this library with purified Xenopus laevis cloned rDNA genes has resulted in the identification and plaque purification of a recombinant containing a complete Artemia (18 S + 26 S) rDNA repeat unit. A physical map derived from the analysis of restriction endonuclease digests of the repeat unit, which measures 13.9 kilobase pairs, is similar to the map derived from genomic DNA. In common with several other species, the 26 S rRNA gene terminates with a HindIII recognition site.

Future of computerised electrocardiography.

The advent of computerised electrocardiography has been of prime importance for the storage and retrieval of data, but none of the available systems is of universal application for analysis of patterns. Future needs require hierarchical systems of increasing degrees of complexity, depending on the source of requests, and there should be appropriate provision for review by cardiologists before the final report is issued.

An automated ECG-system in a large hospital: coding, storage and retrieval of tracings.

This paper describes an automated ECG-system as it is used in the 1000-bed University Hospital Utrecht, The Netherlands. The system involves a "hybride" approach, combining computer analysis of the ECG by means of the Pipberger program with the reading by a cardiologist via a specially developed coding system. Up until now (since January 1, 1972) 35,000 ECGs have been handled systematically at a rate of approximately 100 ECGs per working day. All the ECGs, together with the ECG-diagnoses and other relevant data of the patient are stored and can be retrieved whenever wanted. The system enables comparison of computer analysis and cardiologist's reading of the ECG. The boundary between reliable computer analysis and the necessity of human reading and verification lies with the normal ECGs. This apparently meagre result of the computer ECG-analysis for hospital use is, however, a great achievement for its use in epidemiological studies.