The Gordon Wilson Lecture: The new cell biology and its implications for medicine.
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Biomedical subjects
Publications and source records attributed to T T Puck.
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The application of the phenomenon of premature chromosome condensation for cell cycle analysis in HeLa and CHO cells has been examined. Random populations of HeLa and CHO cells pulse labelled with H3-TdR were separately fused with mitotic HeLa cells using U.V. inactivated Sendai virus. The resulting prematurely condensed chromosomes (PCC) were scored and classified into G1, S and G2-PCC on the basis of both morphological and autoradiographic data, The results of this study indicated that the G1, S and G2 phase cells are equally susceptible to virus-induced fusion with mitotic cells and subsequent induction into PCC. Hence the PCC method for cell cycle analysis is both practical and accurate. This study also revealed that the process of chromosome decondensation initiated during the telophase of mitosis continues throughout the G1 period reaching an ultimate state of decondensation by the end of G1, at which point the fusion of such cells with those in mitosis yield PCC with the most diffused morphology instead of the discrete single stranded structures characteristic of early G1-PCC. Thus, the decondensation of chromatin during G1 appears to be a prerequisite for the subsequent initiation of DNA synthesis.
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.
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 leads to 11pter; a2:11q13 leads to 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 leads to 11pter.
Additional evidence is presented for the previously proposed existence in normal fibroblasts of a cyclic AMP-dependent network of microtubules and microfilaments, which is connected with cell membrane elements on one end and with nuclear structures on the other and whose disorganization leads to malignant transformation. In the presence of cyclic AMP derivatives sufficient to promote integrity of this network, cell growth limitation in suspension, increased transport of alpha-[14C]aminobutyrate, and the relatively tranquilized membrane of the normal fibroblast are also achieved. A pattern of distribution of actin and tubulin has been demonstrated showing aggregated actin deposits which are presumably responsible for the oscillatory knob activity of cells with the transformed habitus. Specific orientations of microtubular and filamentous elements with respect to the nucleus can be demonstrated. The hypothesis that the microtubular-microfilamentous structure conveys growth-regulatory information from the cell membrane to the nucleus and that its disorganization can lead to malignancy has been extended to explain various cellular manifestations.
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Through hybridization of specific Chinese hamster cell auxotrophs with human cells and selection in media lacking the nutritional supplements required by the former cells, a series of stable hybrid clones can be prepared. These hybrids have genomes consisting of a common part--the complete or almost complete set of Chinese hamster chromosomes, plus a variable part--one or a few human chromosomes. The identity of the human chromosomes can be varied by utilizing different Chinese hamster auxotrophs and the appropriate selective media. The human chromosomes present can be determined by a combination of cytogenetic analysis with chromosome banding and testing for specific human marker genes. Hybrids containing single human chromosomes 11 and 12 and the combination of both 11 and 12 are described. The system appears to lend itself to various studies such as identification of human cell surface antigens, determination of their chromosomal loci, measurement of their distribution among cells of normal human tissues, study of interrelations among syntenic and asyntenic genes, and mutational analysis of the human genome.
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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 human cell-surface antigens whose expression is controlled by human chromosome 11, and are expressed by this hybrid, have been defined as the AL immunogenetic complex. Although one component of this immunogenetic complex (a1) is also expressed by human red blood cells, a second component (a2) is not. Killing of an a1+ hybrid by anti-a1 serum and complement can be completely inhibited by glycophorin, the major glycoprotein component of the human erythrocyte membrane. In the presence of complement, antiserum prepared against glycophorin will kill only those cells which express a1. The anti-a1 killing activity of the anti-glycophorin can be absorbed out only by those cells which express a1. Therefore, it is concluded that the a1 cell-surface antigen has at least one antigenic component in common with glycophorin.
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A population of 40,371 individuals consisting of every baby delivered at two Denver hospitals from 1964 to 1974 has been screened from aneuploidy of the sex chromosomes and chromosome 21. The pattern in time with which aneuploidy occurs suggests an epidemic component of the incidence superimposed on an approximately equal constant frequency. The epidemic incidence is most likely to be high for births from May to October, to persist for several consecutive years, and then to be absent for several consecutive years.
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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.
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