T. Timothy Crocker

Nuclear-cytoplasmic relationships in human cells in tissue culture. IV. A study of some aspects of nucleic acid and protein metabolism in enucleate cells.

Enucleate pieces of cytoplasm were prepared from human amnion cells in tissue culture by a method of microsurgery. Vital activities of the fragments were retained for 10–30 h. One of several amino acids or RNA precursors labeled with 14C was added to the culture medium just before or just after preparation of a group of enucleate cytoplasmic fragments. When about half the fragments had “died”, the surviving enucleates were fixed and examined by autoradiography for evidence of incorporation of the labeled compound. Amino acids were incorporated as actively by enucleate cytoplasm as by cytoplasm of complete cells. This incorporation could be blocked by excess label-free L-isomers of the corresponding labeled amino acid but not by the label-free D-isomer. RNA precursors were not incorporated by enucleate cytoplasmic fragments in detectable amount, although neighboring intact cells incorporated the precursors actively into cytoplasm. n nThe conclusions reached are (a) that protein synthesis continues in enucleate cytoplasm of mammalian cells, but (b) that RNA synthesis stops or is so slight as to escape detection by these methods. These data add to the evidence that cytoplasmic RNA is derived chiefly from the nucleus. n nThe labeled precursors used were: [14C]adenine, [14C]uridine, L-[14C]glutamic acid, L-[14C]leucine, L-[14C]phenylalenine and DL-[14C]tryptophan; [8-14C] adenylic acid was also used.

Nuclear-cytoplasmic relationships in human cells in tissue culture: II. The microscopic behavior of enucleate human cell fragments☆

Abstract Enucleation of mammalian cells in tissue culture has been performed by cutting a narrow cytoplasmic strand connecting the nucleated portion of a cell from a non-nucleate cytoplasmic mass. Conditions influencing “survival” are discussed and data for duration of survival of 301 enucleate cytoplasmic fragments are compiled. A structural organization within enucleated cytoplasmic pieces has been described as a “granular ring”, possessing remarkable constancy of size and rigidity within otherwise mobile cytoplasm. Preservation of motility, motion of cell surfaces, and pinocytosis in enucleate mammalian cells is contrasted with loss of these normal functions in enucleate amoebae.

DNA-associated acidic proteins

Abstract After previous removal of acid-soluble nuclear proteins and of RNA, 0.5 M hot perchloric acid extracts DNA together with associated acidic proteins from Ehrlich ascites cell nuclei. The specific activity of these DNA-associated proteins, after pulse-labeling of cells kept in minimal medium, is higher than the specific activity of other nuclear proteins. Their specific activity is proportional to the synthesis of RNA with DNA-like base composition and is not correlated with synthesis of ribosomal precursor RNA and DNA. Centrifugation in CsCl showed that labeled amino acids are associated with DNA in the form of protein molecules and not in the form of amino acids. Labeling experiments indicated that DNA-associated proteins are a mixture of proteins which are labeled independently of each other. Despite their close association with DNA, the synthesis of these proteins is inhibited to the same extent as synthesis of all other proteins by actinomycin D, puromycin and ribonuclease. This indicates that DNA-associated proteins are synthesized in the usual way, by translation of RNA on the ribosomal system. The presence of these proteins is not limited to Ehrlich ascites cells; DNA-associated proteins were also found in various organs of rats. Their specific activity is low in liver in vivo during synthesis of ribosomal precursor RNA. In the liver of starved animals where RNA synthesis is switched to synthesis of RNA with a DNA-like base composition the specific activity of DNA-associated proteins is substantially increased. All of these findings support the proposition that DNA-associated proteins are involved in the control of DNA-like RNA synthesis on DNA template.

Poliovirus : Growth in Non-Nucleate Cytoplasm.

Cytoplasmic fragments were produced by micromanipulation of cells from a human amnion cell line cultured on coverslips. The cultures were infected with type 1 (Mahoney) poliovirus, and incubated for 7 hours with tritiated uridine (H3U). Fluorescent antibody to the poliovirus indicated antigenic sites in a number of non-nucleate fragments. By autoradiography the incorporation of H3U was demonstrated at some of the same sites. The occurrence of poliovirus antigen at the same site as induced synthesis of RNA in non-nucleate cytoplasm of mammalian cells indicates that poliovirus infection and growth occurred independently of immediate contribution from the nucleus.

The inhibition of nuclear RNA synthesis by added RNA.

Abstract The addition of RNA to Ehrlich ascites cells resulted in rapid suppression of labeling of nuclear RNA with 3 H-uridine. This suppression started shortly after the addition of RNA and persisted for about 6 hr. After 8 hr the normal rate of RNA labeling was re-established. Cellular respiration as well as the citric acid cycle were not inhibited by addition of RNA. Labeled RNA penetrated into cell nuclei. Approximately 3 per cent of the added RNA could be recovered immediately as RNA from the nuclei but only about 0.3 per cent from the cytoplasm. Deoxyribonucleoprotein isolated from Ehrlich ascites cells contains 4 per cent of RNA. Added labeled RNA is recovered with the deoxyribonucleoprotein. After separation into DNA and protein the major portion of the RNA is found in the protein fraction. Based on present data and on results of other authors it is proposed that added RNA interferes with RNA synthesis on DNA template.

Subcellular Cultivation of a Virus: Growth of Ornithosis Virus in Nonnucleate Cytoplasm.

Abstract Cytoplasmic fragments, prepared by microdissection of cells from a human amnion cell line, are motile and undergo pinocytosis. They incorporate amino acids but do not incorporate those ribonucleic acid precursors so far tested. Cultures were infected with ornithosis virus before or after preparation of nonucleate cytoplasmic pieces and (1) stained (Giemsa or acridine orange), or (2) labeled with tritiated nucleosides, digested with ribonuclease, stained by the Feulgen method and covered with autoradiographic stripping film. Attachment, penetration, initiation of virus growth and moderately advanced maturation of virus inclusions occurred whether the prospective cytoplasmic fragment was part of an intact cell at early points in this sequence or was already amputated before infection. Synthesis of viral deoxyribonucleic acid was demonstrated by autoradiography. Nuclear participation in the infectious process was not required at any time during the events studied here, but may be necessary for renewal of cytoplasmic materials which are equally essential to functional survival of cytoplasm alone.