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Biomedical subjects

T T Puck

Publications and source records attributed to T T Puck.

At least 55 records · Page 3Linked to original sources
PubMed1982

Phosphorylation changes induced by cAMP derivatives in the CHO cell and selected mutants.

Phosphorylation analysis of CHO mutants selected for unresponsiveness to the Reverse Transformation reaction of cAMP derivatives has been carried out by 2D gel electrophoresis in hopes of finding mutants differing from the wild-type cell in a minimal number of such phosphorylations. Seven differences in protein phosphorylations in the parental CHO cell have been identified as a result of treatment with db-cAMP. db-cAMP-unresponsive mutants of two kinds have been found: One type has lost all seven of the phosphorylation changes induced by db-cAMP in the wild-type cell. The other type which is equally resistant to the Reverse Transformation response differs from the parental cell in only one (or possibly two) phosphorylation(s) involving a 55 000 D protein. This protein may, therefore, be directly related to the events of Reverse Transformation.

Animals↗
PubMed1982

Genetic fine-structure mapping in human chromosome 11 by use of repetitive DNA sequences.

A method is described for mapping of the DNA fragments of a human chromosome produced by restriction enzyme treatment of the total DNA from a hybrid cell containing a single human chromosome. The method involves production and selection of somatic cell mutants containing deletions of the human chromosome and application of a hybridization probe consisting of an individual member copy of a repetitive human DNA family. A linear map has been constructed of 19 marker DNA fragments and 5 immunological and biochemical markers on human chromosome 11, selected as a model chromosome for these studies. This approach appears to be widely applicable, is independent of cytogenetic analysis, promises to be capable of revealing the existence of rearrangements as well as deletions, appears to be amenable to further increase in resolving power, and offers potential application in various human genetic problems.

Cell Line↗
PubMed1981

Reverse transformation of vole cells transformed by avian sarcoma virus containing the src gene.

Vole cells transformed by avian sarcoma virus carrying the src gene lose their fibroblastic morphology, the organized cytoskeletal system of the normal fibroblastic cell, the typical fibronectin deposit around the cell membrane, and the ability to shut off multiplication when suspended in liquid medium. All of these transformation characteristics are reversed by treatment with cAMP derivatives. Moreover, the cAMP treatment does not cause loss of activity of the src gene product. These data imply that cAMP exerts its effect at or after the point in the metabolic pathway affected by the src gene product, pp60src. Presumably, the decision to adopt the transformed or the normal state is determined by the degree to which the src gene or cAMP-mediated kinase activities respectively predominante in the cell. The development of all four transformation characteristics as a result of introduction of the src gene, and their coordinate reversal by cAMP derivatives, supports the previous thesis that in the normal vole or CHO fibroblast all four properties are part of a common regulatory system.

Animals↗
PubMed1981

Two-dimensional electrophoresis of human-CHO cell hybrids containing human chromosome 11.

Two-dimensional electrophoresis-electrofocusing with polyacrylamide gels is described in which the pattern of peptide spots obtained from hybrid cells of CHO containing the single human chromosome 11 are compared with those from the hybrid which has undergone reversion so as to lose this chromosome. At least eight distinct spots unique to the cell containing chromosome 11 have been located on the electrophoretogram. Experiments are described demonstrating applicability of the method to hybrids containing specific deletion mutants of chromosome 11, an approach which makes possible regional mapping of the loci responsible for specific peptides. The methodology appears applicable to study of gene expression under the influence of hormones and other agents and to the comparison of normal and disease situations.

Animals↗
PubMed1981

Some new developments in genetic analysis of somatic mammalian cells.

The in vitro study of somatic cell genetics originally made possible a new approach to human genetics, which by passed the need for mating and thus permitted enormous expansion of the genetics of man and other mammals. More recently, by increasing the variety of cell mutants obtainable, by the production of hybrids containing single or small numbers of human chromosomes, by development of the immunogenetics of macromolecules in differentiated cell membranes, and by combining the techniques of somatic cell genetics with those of recombinant DNA, new approaches are available to the problem of regulation of mammalian gene expression. Experiments are presented offering the possibility of application of these techniques to human gene mapping, detection of mutagenic actions, identification of genes involved in differentiation, and a beginning of molecular understanding of the pathology of the aneuploid diseases.

Animals↗
PubMed1980

Deposition of fibronectin in the course of reverse transformation of Chinese hamster ovary cells by cyclic AMP.

The Chinese hamster ovary (CHO) cell, like other transformed cells, has lost the fibronectin deposit around its membrane. Treatment with cyclic AMP derivatives restores the typical fibroblastic deposit of fibronectin. Thus, the reverse transformation process induced by cyclic AMP (cAMP) in the CHO cell restores this important property as well as other morphological, biochemical, and growth behavioral characteristics of the normal fibroblastic state. The fibronectin deposit occurs significantly later in time than do other characteristics of the reverse transformation reaction and may therefore reflect a metabolic action that requires other cAMP effect to precede it. The restoration of fibronectin deposition in response to cAMP derivatives is also exhibited by vole cells transformed by avian sarcoma virus, but it is not by HeLa cell. Addition of Colcemid, which disrupts microtubules, to CHO cells containing a fibronectin deposit induced by cAMP derivatives causes little or no erosion of the deposit, but cytochalasin B, which disrupts 5-nm microfilaments, eliminates it completely. Thus, various features of the action of cAMP derivatives on CHO and related cells require integrity of the cellular microfibrils--in some cases microtubules only, in some cases 5-nm microfilaments only, and in some cases both classes of fibrils.

Animals↗
PubMed1980

Isolation and localization of DNA segments from specific human chromosomes.

Recombinant DNA techniques have been combined with somatic cell genetic methods to identify, isolate, and amplify fragments of human DNA localized at specific regions of human chromosome 11 selected as a model system. A library of genomic DNA segments has been constructed, in lambda Charon 4A bacteriophage, from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background. Using a nucleic acid hybridization technique that distinguishes human and Chinese hamster interspersed, repetitive DNA, we have been able to distinguish recombinant phages carrying DNA segments of human origin from recombinant phages carrying DNA segments of Chinese hamster origin. We have isolated 50 human DNA segments thus far and have characterized 5 in detail. For each DNA segment characterized, a subsegment that carries no repetitive human DNA sequences has been identified. These segments have been used as hybridization probes in experiments that localize the DNA fragment on the chromosome. In each case an unequivocal chromosomal localization has been obtained with reference to a panel of hybrid cell clones each of which carries a deletion of a portion of the short arm of chromosome 11. At least one DNA segment has been identified which maps to each of the four regions on the short arm defined by the panel of hybrid cell clones used. The approaches described here appear to be general. They can be extended to produce a fine structure map of human chromosome 11 and other human chromosomes. This approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.

Animals↗
PubMed1979

Role of the microfibrillar system in knob action of transformed cells.

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5-4 micron in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15-60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: 1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; 2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; 3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reverse-transformed cells or normal fibroblasts; 4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; 5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.

Animals↗
PubMed1979

Studies on cell transformation.

Seven different transformation stigmata of the transformed CHO cell line, including morphological characteristics, growth behavior, cell membrane biochemical properties, and failure of fibronectin deposition, are reversed by addition of cAMP derivatives to the medium. Simultaneously the microtubular pattern changes from a sparse, relatively random set to an orderly arrangement of tubules largely parallel to each other and to the long axis of the resulting fibroblastic cell. Agents like colcemid and cytochalasin B, respectively disorganizing microtubular and particular microfilamentous structures, prevent at least certain aspects of the reverse transformation reaction induced by cAMP in interphase cells. It is proposed that malignant transformation can be effected by damage to the microtubular and microfilamentous structures which changes cell constitution and behavior in two ways: (1) chromosomal instability is introduced which promotes continuous selection for variants better able to resist environmental signals to limit reproduction and (2) a variety of metabolic defects in biochemical processes such as specific membrane functions are introduced which may alter the growth responses of the cell. This picture offers a reasonable explanation for a number of aspects of normal and malignant cell behavior.

Animals↗
PubMed1979

Precise localization of human beta-globin gene complex on chromosome 11.

Cloned DNA probes were used in combination with a panel of five hybrid cell clones containing a series of different terminal deletions in human chromosome 11 to map precisely the human hemoglobin beta and delta chain structural genes contained on this chromosome. The region of deletion in each clone of the panel has been defined by biochemical, immunologic, and cytogenetic markers. DNA from clones containing successively larger terminal deletions was tested with appropriate DNA probes to determine the point on the chromosome at which DNA for these two closely linked hemoglobin genes is deleted. These genes, and by inference the closely linked G gamma and A gamma globin genes as well, have been assigned to the intraband region 11p1205 leads to 11p1208 on the short arm of chromosome 11, an interval containing approximately 4500 kilobases of DNA. The approach appears to have potential for even greater resolution and reasonably wide applicability for gene mapping.

Animals↗
PubMed1979

Measurement of mutagenesis in mammalian cells.

A method using mammalian cells in vitro for detection and quantitation of mutagenic actions that appears to be useful for screening for carcinogenesis and genetic damage by environmental agents is presented. The method involves use of stable human--Chinese hamster ovary hybrid cells that have retained a single human chromosome not necessary for cell reproduction. Forward mutations are detected in genes necessary for production of specific human cell surface antigens. Such mutants form colonies in the presence of specific antisera and complement that destroy the unmutagenized cells. Use of the method is illustrated for the action of x-irradiation, N-methyl-N'-nitro-N-nitrosoguanidine, and caffeine. The method appears to be unique in that it permits assessment of lesions that cause loss of all or most of the chromosome as well as various localized gene mutations. The former action is particularly important because of the major involvement of chromosomal lesions in an extremely important class of human genetic disease.

Amniotic Fluid↗
PubMed1978

The role of butyrate in the reverse transformation reaction in mammalian cells.

The reverse transformation reaction of Chinese hamster ovary cells from compact, epithelial-like, randomly growing, heavily knobbed, lectin reactive cells into stretched, tighly adherent, smooth-surfaced, lectin resistant, fibroblast-like cells normally elicited by dibutyryl cAMP can be produced to its complete extent by N6-monobutyryl cAMP or 8-bromo-cAMP, O2'-monobutyryl cAMP is ineffective as is cAMP itself in the absence of an inhibitor of phosphodiesterase activity. In the presence of a phosphodiesterase inhibitor, cAMP is fully effective. These results indicate that the role of the butyryl groups of dibutyryl cAMP and, especially, the N6-butyryl, in the reverse transformation reaction is protection of the cAMP analogue from degradation. Butyrate at concentrations of about 1 mM does produce a response which to some extent mimics that of cAMP analogues. The cells, however, fail to assume a fibroblastic-like shape, but rather become flattened. The butyrate effect is much slower and less readily reversible than that evoked by cAMP analogues. Butyrate produces an approximately 2-fold increase in intracellular cAMP levels. These results are consistent with the hypothesis that butyrate effects, in part, are mediated by AMP.

Butyrates↗