Carol Jones

Cell surface antigens

A stable human-Chinese hamster ovary cell hybrid has been produced which, in addition to the complement of Chinese hamster ovary (CHO-K1) chromosomes, contains only one human chromosome, No. 11. The h

Genetics of cell-surface antigens: regional mapping of three components of the human cell-surface antigen complex, AL, on chromosome 11.

Cytogenetic analysis has been performed on a series of deletion mutations on human chromosome 11 of AL hybrid clones in which specific markers have been lost as a result of treatment with mutagenic agents. Such analysis has localized the three previously identified components of the AL cell-surface antigen complex to the indicated regions of chromosome 11: a1 and a3:11p13 → 11pter; a2: 11q13 → 11qter. Using these methodologies human lactic dehydrogenase A localization on the short arm as reported by others has been confirmed. Evidence is presented provisionally assigning this gene to 11p13 → 11pter.

Genetics of somatic cell surface antigens. III. Further analysis of the AL marker

The AL antigen present on the surface of various human somatic cells, and on those hybrids of human and Chinese hamster ovary cells which have retained human chromosome number 11, has been resolved into at least two separate antigenic activities, a1 and a2. Specific antisera active against each antigen separately have been prepared. By treatment of the original AL+ hybrid with mutagenic agents and selection in particular antisera, stable clones are preparable whose phenotypic behavior corresponds to the antigenic compositions a1+ a2−, a1− a2+, and a1− a2−. The adsorption behavior of these variants for specific antisera is consistent with their phenotypic assignments.

Further studies on hybrid cell-surface antigens associated with human chromosome 11.

A new human immunogenetic cell-surface activity associated with human chromosome 11 in the AL human-Chinese hamster ovary cell hybrid is described. Like a1 but not a2, it is present on the human erythrocyte. By mutagenesis and selection, specific, stable, variants of the AL hybrid have been prepared exhibiting various combinations of a1 a2, a3, and lactic dehydrogenase A activities. The antigens of the AL system can be demonstrated by the horseradish peroxidase system which offers a promising approach to scanning of tissue cells.

Analysis of human chromosome 11 by somatic cell genetics: Reexamination of derivatives of human-hamster cell line J1

Through the fusion of a CHO cell population to a human cell population, a hybrid cell line which has lost all human chromosomes except chromosome 11 was derived. This cell line, J1, does not appear to segregate human chromosome 11 during growth. A series of deletion segregants were isolated from J1 which had lost a portion of either the long, short, or both arms of chromosome 11. This panel of deletion segregants was used for mapping a number of genetic markers on the short arm of chromosome 11. Karyotypic analysis led to the interpretation that derivatives of J1 selected for the loss of cell surface antigens encoded by genes on the short arm of the chromosome had simple terminal deletions of this chromosome arm. More recently, we have applied recombinant DNA and in situ hybridization techniques to the analysis of the structure of chromosome 11. In the course of this analysis, we have obtained data that indicate that all J1 deletion segregants retain a small chromosomal segment containing the structural genes for insulin and HRAS1. Analysis of in situ hybridization data indicates that in cell lines in which a chromosome 11 fragment cannot be identified by karyotype analysis, human DNA has been translocated to a Chinese Hamster chromosome. These results suggest that the original interpretation of the karyotypes of deletion segregants derived from J1 as simple terminal deletions is not correct. A reanalysis of gene localization studies based on these deletion segregants suggests that some assignments of genes to specific bands on chromosome 11 should be reconsidered. In particular, data on additional deletion segregants are consistent with localization of the Β-globin gene complex to band 11p15. The data presented here suggest that in several hybrid derivatives of J1, a continuous DNA segment of approximately 107base pairs in length which includes the insulin and HRAS1 (cellular homolog of retroviral oncogene Harvey ras) genes has been isolated from the remainder of the human genome. We propose that the stability of chromosome 11 in the original hybrid was due to complementation of a genetic defect in the original CHO cell parent by a gene located in close physical proximity to the insulin and HRAS1 genes on chromosome 11. Data are presented which test and support this hypothesis.

Isolation of mutants lacking branched-chain amino acid transaminase

Variants of the Chinese hamster ovary cell have been isolated which can no longer grow when valine, leucine, or isoleucine is replaced in the culture medium by its respective α-keto acid: α-ketoisovaleric acid, α-ketoisocaproic acid, or α-keto-β-methylvaleric acid. These variants lack branched-chain amino acid transaminase activity. Evidence is presented indicating these variants to be single gene mutants. Genetic evidence is also presented confirming previous biochemical evidence that a single enzyme carries out transaminase functions on valine, leucine, and isoleucine. The branched-chain transaminase-deficient (trans−)mutants can be reverted to wild-type behavior by treatment with mutagenic agents. These mutants promise to be useful in exploring regulatory mechanisms in biochemical, genetic, and cancer research.

Isolation of a lactic dehydrogenase-A-deficient CHO-K1 mutant by nylon cloth replica plating

A mutant Chinese hamster ovary cell deficient in lactate dehydrogenase A activity has been isolated using a nonselective technique. The method uses histochemical staining to examine colonies directly for enzyme activity and nylon cloth replica plating to recover particular clones. The mutant cell has an apparent Km (pyruvate to lactate) that is nearly tenfold higher than the parental cell, while itsVmax has been reduced more than 80-fold. In mutant cell extracts with added porcine LDH-B enzyme, molecular dissociation and recombination of subunits produces two new active LDH tetramers (A1B3, A2B2). The electrophoretic mobility of at least one of the tetramers (A1B3) was different from those formed in the parental extracts. The evidence suggests the variant cell contains a mutation in the structural gene for LDH-A.

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation, selection, and characterization of a mutant lacking hypoxanthine-guanine phosphoribosyltransferase activity by nutritional means.

Mutants of the Chinese hamster ovary cell derived from CHO-KI have been selected for lack of hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) (HGPRT) without the use of a drug-resistance protocol. The procedure depends on the use of a parental strain carrying a mutation making it unable to synthesize purines and thus dependent upon exogenously added purines for growth. The standard “BUdR-visible-light” procedure is then used to select those cells which can use adenine but cannot use hypoxanthine as a purine source. These cells are shown to be thioguanine resistant, to be unable to incorporate exogenously added hypoxanthine into purine nucleotides, to complement our other adenine-specific purine auxotrophs, Ade−H and Ade−I but not to complement a cell isolated by virtue of thioguanine resistance, and to lack the activity of HGPRT. The use of such multiply marked mutants and cells related to them for further analysis of purine nucleotide biosynthesis and interconversion is discussed.

Chapter 5 Immunogenetics of Mammalian Cell Surface Macromolecules

Publisher Summary This chapter describes a method for the analysis of human cell surface antigens, which makes possible the identification of specific cell membrane macromolecules and simultaneous identification of the human genes controlling their biosynthesis. Studies of many different kinds indicate that cell surface molecules play a crucial role in the control of growth, differentiation, development, and communication in mammalian cells. Methods for the elucidation of the genetic and genetic biochemical aspects of cell membrane macromolecules are needed to illuminate regulatory control mechanisms. The study of the genetics of the mammalian cell surface is complex in part because of the large number of genes that can participate in the production of surface structures. In addition, classical genetic operations with their attendant mating procedures are particularly difficult for human situations. For these reasons, somatic cell methodologies in vitro are used for the genetic analysis of these structures. The chapter discusses the use of cultured human and Chinese hamster ovary cells and their somatic cell hybrids. This allows simplification of the study of human cell surface antigens by means of procedures that permit study of membrane macromolecules due to individual or small groups of chromosomes.