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Hybrids of CHO cells were constructed consisting of either a 1:1 or 1:2 ratio of alpha-amanitin-resistant and sensitive cells, respectively. The resistance of such hybrids to killing by the drug was similar but slightly less than that of the resistant parent. The hybrids contained both resistant and wild-type RNA polymerase II, in amounts related to the expected gene dosage. The alpha-amanitin marker therefore is expressed codominantly.

Single-step mutants were isolated from the murine metastatic MDAY-D2 cell line after selection in toxic concentrations of wheat-germ agglutinin. They were partially characterized by measuring their relative level of resistance to WGA, PHA, Con A, RIC, and LCA (Lec phenotype), and by comparing their karyotype and their ability to produce metastases upon transplantation into syngeneic DBA/2 mice. Based on their Lec phenotype, a total of 19 independent isolates were ranked into 10 distinct classes. Among them, two EMS-induced mutants were nontumorigenic (Lec II, Lec III), one nonmetastatic (Lec IV), and one spontaneous mutant (Lec I) failed to produce blood-borne metastases. Other spontaneous mutants belonging to Lec I, Lec II, and other classes were as metastatic as their parents. The Lec IV phenotype was found to segregate independently from metastatic potential in somatic hybrids. Metastatic ability was recovered in mutants expressing the Lec IV phenotype, after further selection for resistance to RIC. Our results strongly suggest that the loss or reduction of the invasive property of tumor cells is associated with only few Lecr1 phenotypes and, therefore, that a restricted number of cell surface glyconjugates are essential for this particular function.

The hypothesis of functional hemizygostiy at the emetine-resistant (Emtr, a non-X-linked recessive marker) locus in Chinese hamster ovary (CHO) cells has been examined by segregation analysis. The frequencies and the rates of segregation of the Emtr and Thgr (thioguanine-resistant, an X-linked recessive mutation) markers were determined from hybrids constructed between an Emtr-Thgr CHO cell line and various other Chinese hamster lines (V79, M3-1, CHO, GM7S, CHW and CHL). Thgr segregants were obtained at similar frequencies (10(-2)-10(-3)) from all the hybrids. The frequency of segregation of the Emtr marker, however, was similar to that of Thgr only in the CHO x CHO hybrids and was much lower (10(-4)-10(-6)) than the CHO x other Chinese hamster hybrids. Similar results were obtained when the segregation rates for the two markers from various hybrids were determined. These results are consistent with the hypothesis that in CHO cells, the gene responsible for Emtr is present in a single (functional) copy, whereas two copies of this gene are present in other Chinese hamster lines examined.

Stable mutants resistant to the protein synthesis inhibitors cryptopleurine and tylocrebine can be isolated in Chinese hamster ovary (CHO) cells, in a single step. The frequency of occurrence of cryptopleurine (CryR) and tylocrebrine (TylR) resistant mutants in normal and mutagenized cell populations is similar to that observed for emetine resistant (EmtR) mutants. The CryR, TylR, and EmtR mutants exhibit strikingly similar cross-resistance to the three drugs used for selection, to tubulosine and also to two emetine derivatives cephaeline and dehydroemetine, based on assays of in vivo cytotoxicity and on assays of protein synthesis in cell-free extracts. The identity of cross-resistance patterns of the CryR, TylR, and EmtR mutants indicates that the resistance to all these compounds results from the same primary lesion, which in the case of EmtR cells has been shown to affect the 40S ribosomal subunit. This conclusion is strongly supported by the failure of EmtR, TylR, and CryR mutants to complement each other in somatic cell hybrids. Based on these results it is suggested that the above group of compounds possesses common structural determinants which are responsible for their activity. The above mutants, however, do not show any cross-resistance to other inhibitors of protein synthesis such as cycloheximide, trichodermin, anisomycin, pactamycin, and sparsomycin, either in vivo or in vitro, indicating that the site of action of these inhibitors is different from that of the emetine-like compounds.

Chinese hamster ovary cell lines which are resistant to an amino acid analog, beta-aspartyl hydroxamate, have been isolated and characterized. Mutants resistant to 100-150 microM beta-aspartyl hydroxamate arose from ethyl methane sulfonate-treated parental lines at frequencies of 3.4 x 10(-6) to 1.3 x 10(-7). The mutants fell into at least two genetic classes: 18% of the mutants behaved codominantly in hybrids, the others recessively. Complementation studies indicated that all the recessive mutants belonged to the same class. Mutants selected after one step of mutagenesis overproduce the enzyme asparagine synthetase constitutively with four- to sixfold increases in specific activities over the basal levels of the parental lines. beta-Aspartyl hydroxamate-resistant cell lines with up to 20-fold elevations in asparagine synthetase activity have been isolated after two steps of mutageneis. In addition, highly resistant lines have been selected by long-term growth of a dominant mutant in increasing concentrations of the drug. Resistance in the latter appears to be due not only to overproduction of asparagine synthetase but also to an alteration in the affinity of the enzyme for beta-aspartyl hydroxamate.

The simian virus 40 (SV40)-pBR322 recombinant, pSV2, carrying the origin of SV40 replication and the gpt gene of Escherichia coli, has been stably introduced into Chinese hamster ovary hprt- cells. All gpt-transformed cell lines were found to contain one or more insertions of pSV2 sequences exclusively associated with high-molecular-weight DNA. Additional analyses showed that at least one integrated copy in each cell line retained an intact gpt gene and flanking SV40 sequences required for expression of xanthine-guanine phosphoribosyltransferase. Most cell lines contained pSV2 sequences which had integrated with partial sequence duplication. Upon fusion with COS-1 cells, a simian cell line permissive for autonomous pSV2 replication, most gpt-transformed cell lines produced low-molecular-weight DNA molecules related to pSV2. The majority of these replicating DNAs were indistinguishable from the original transfecting plasmid in both size and restriction enzyme cleavage pattern. In addition, the recovered DNA molecules were able to confer ampicillin resistance to E. coli and to transform mouse L cells and Gpt- E. coli to a Gpt+ phenotype. These studies indicate that all of the genetic information carried by this SV40-plasmid recombinant can be introduced into and retrieved from the genome of mammalian cells., link_to_OA_fulltext

Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPURA exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.

The cellular property of being able to grow on agar (aga + ) or to show anchorage independence has been transferred by means of metaphase chromosomes from CHO cells to BHK and other permanent transformed hamster lines unable to grow on agar. As with other genetic markers, the transferents are unstable when grown under non-selective conditions. The aga + transferents are tumorigenic, providing further evidence for the association between the ability to grow in agar or anchorage independence and tumorigenicity. Evidence has been obtained in these experiments for the existence of at least two discrete events in the transformation of normal into tumorigenic cells. The ability to transfer and select for the aga + marker in recipient cells indicates that tumorigenicity behaves dominantly phenotypically.

The conditions necessary to achieve high frequency transfer of the thymidine kinase and dihydrofolate reductase genes from hamster cells into mouse cells were investigated. Of the parameters examined, the length of adsorption time, input gene dosage, and treatment with dimethylsulfoxide (DMSO) were found to significantly alter the transfer frequency using either metaphase chromosomes or purified DNA as the transfer vehicle. With the mouse cell line as a recipient, the optimal adsorption period for DNA or chromosomes from MtxRIII cells was found to vary from 8 to 16 h in those experiments where the recipient cells were subsequently treated with DMSO. Without DMSO, similar frequencies could be obtained by extending the period of adsorption. Increasing the dosage of DNA or chromosomes resulted in an almost linear increase in the number of transformants. The optimal conditions for transfer did not significantly differ for the two genes studied. On the average, the optimal conditions yielded 1.5 x 10(3) transformants per 10(7) recipient cells with chromosomes; with DNA an average of only 60 transformants were observed. In general, DNA transformants grown in the absence of methotrexate were unstable; whereas, under the same conditions about 20% of the transformants from the chromosome experiments were stable.

Second-step mutants highly resistant to the protein synthesis inhibitor emetine (Emt(RII) have been selected from emetine resistant (Emt(RI)) Chinese hamster ovary cells described earlier. The frequency of the Emt(RII) mutants was increased 50- to 75-fold after mutagenesis, and none of these highly resistant mutants could be selected in one step using wild-type cells. Like the Emt(RI) mutants, the increased resistance of Emt(RII) mutants results from another lesion in the polyribosomal fraction, as measured by the effects of emetine in fractionated extracts. As with the first-step mutants, the Emt(RII) isolates behave recessively in somatic cell hybrids. Segregation studies have shown that the Emt(R) lesions are not on the X chromosome, and in at least one isolate there is evidence that the Emt(RI) and Emt(RII) mutations may occur at different sites.

Stable mutants highly resistant to the protein synthesis inhibitor diphtheria toxin (dipr) have been selected in human diploid fibroblast cells at a frequency of 1-8 X 10(-6). Treatment of cells with mutagens, (e.g., ethylmethanesulfonate, nitrosoguanidine, and ICR-170), increased the frequencies of dipr mutants by 50- to 500-fold in different experiments, and the optimal expression time for dipr mutation was about 5 days. All mutants examined thus far have bred true, and no effects of cell density or cross feeding have been observed on the selection. Fluctuation analysis showed that the dipr mutation occurs in these fibroblasts at the rate of 5-6 X 10(-7) mutations per cell per generation. Protein synthesis in mutant extracts was resistant to diphtheria toxin, indicating that the dipr lesion in such mutants lies in the protein synthesis machinery. The characteristics of the dipr marker should make this system particularly useful for studies of quantitative mutagenesis in human diploid cells.

The molecular basis of resistance to the protein synthesis inhibitor emetine has been examined in cell-free, protein-synthesizing extracts derived from normal and emetine-resistant (Emt R ) mutants. We had earlier shown that protein synthesis in extracts of the mutant cells was resistant to the inhibitory action of the emetin. When extracts from a wild-type and mutant cell line were fractionated into supernatant (S-100) and polyribosome fractions and mixed in different combinations, resistance to emetine was found to be associated with the mutant polyribosome fraction. Further fractionation of wild-type and mutant polyribosomes into 40S and 60S ribosomal subunits and mixing them in various combinations with an S-100 fraction from the wild-type cell indicates that resistance of mutant cells to emetine involves an alteration in the 40S ribosomal subunit. The behavior of Emt R has also been examined in somatic cell hybrids. Studies of Emt R × Emt S hybrid cell lines in vivo and in vitro show that Emt R is phenotypically recessive to Emt S , which is consistent with the ribosomal location of the genetic change.

Cell lines that have high levels of resistance to beta-aspartylhydroxamate and elevated levels of asparagine synthetase activity were selected in two steps from Chinese hamster ovary cells. Resistance to beta-aspartylhydroxmate was transferred into sensitive cells by using total genomic DNA derived from the dominant two-step mutants. The surviving colonies were characterized as transferants on the basis of transfer frequency, degree of resistance to beta-aspartylhydroxamate, increased level of asparagine synthetase activity, expression of the donor form of asparagine synthetase, codominance in hybrids, and instability of the phenotype in the absence of selection.

The amino acid analog, albizziin, which acts as a competitive inhibitor of asparagine synthetase with respect to glutamine was used to isolate mutants of Chinese hamster ovary cells with alterations in levels of the target enzyme. These mutant lines have been characterized biochemically and genetically. Mutants selected in a single step are up to 40-fold more resistant to the drug than the parental line, express levels of asparagine synthetase activity 6-17-fold greater than that of wild type cells, and act co-dominantly in hybrids. Several classes of mutations can be distinguished on the basis of cross-resistance to beta-aspartyl hydroxamate, another amino acid analog. Studies on asparagine synthetase indicate that resistance to albizziin may be due to altered regulation of asparagine synthetase, structural mutations of the enzyme, and gene amplification.

Thymidine auxotrophs of Chinese hamster ovary cells have been isolated through selection for resistance to the drug 1-β-D-arabinofuranosyl cytosine (ara C). Deoxycytidine and deoxyuridine, but not the other deoxyribonucleosides or ribonucleosides, are also able to supply the nutritional requirement of these mutants. The frequency of isolation of the auxotrophs in mutagenized cultures is about 10−5, and the spontaneous frequency is 5–10 times lower. However, the reversion frequency to thymidine prototrophy is high. Analysis of the revertants suggests that the ara C resistance and the thymidine auxotrophy are the consequence of a single mutation. In hybrid cells, the thymidine auxotrophy can be complemented by wild-type cells or another thymidine-requiring cell line deficient in folate metabolism, while the ara C resistance behaves in a codominant manner. The pool of dCTP in the ara C-resistant thymidine auxotrophs is elevated, accounting for the ara C resistance. Upon thymidine starvation, the pools of dTTP and dGTP decline and the rate of DNA synthesis decreases immediately. We suggest that the complex phenotype and the genetic and biochemical properties of these mutants can be best explained by an inability to reduce UDP to dUDP.

Chinese hamster ovary (CHO) cells resistant to the cytolytic effects of Sindbis virus infection have been selected in one step from mutagenized wild-type cells. The mutants were present at frequencies as high as 8.8 × 10 −4 , and were obtained virus free following the incubation of the isolates in the presence of anti-Sindbis virus antibody. The mutant phenotypes behaved recessively in somatic cell hybrids, and there were at least two complementation groups among the isolates tested. In addition to the virus-resistant phenotype, the mutants clearly have surface changes as demonstrated by their altered morphology, increased sensitivity to phytohemagglutinin, and increased resistance to diphtheria toxin. Analyses of revertants from one mutant indicated that all of the phenotypes of the mutant resulted from a single genetic alteration. All isolates were partially defective in viral adsorption. Comparison of viral replication in WT and one mutant cell strain suggested that the major effect on viral replication in the mutant studied is at the level of viral mRNA translation.

Mutants resistant to the protein synthesis inhibitor trichodermin have been selected in Chinese hamster ovary (CHO) cells. The mutants vary in their stability from those which rapidly lose their resistance to others which are relatively stable after prolonged growth in nonselective medium. Protein synthesis in extracts from the latter class of mutants (Trir) is resistant to the inhibitory action of trichodermin as compared to similar extracts from wild-type cells. After dissociation into subunits, the ability of the 60S ribosomal subunits from Trir cells to function in a protein-synthesizing system is greatly diminished. This subunit also shows reduced binding of [acetyl-14C]TRICHODERMIN. The lesion in Trir mutants therefore seems to have affected this ribosomal subunit. Trir X Tris hybrids are sensitive to trichodermin indicating that the Trir mutation behaves recessively to Tris in hybrids. The Emtr and Trir markers segregate independently from hybrid cells showing that the Trir mutation is probably not linked to the Emtr locus, which as we have shown earlier affects the 40S ribosomal subunit.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levels of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W. F., Davidson, S. V., and Siminovitch, L. (1976) Somatic Cell Genet. 2, 245–261). By selecting for increased resistance from these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In this report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and a class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

DNA synthesis in two mutants of Chinese hamster overy cells, ts 13A and ts 15C, which were temperature sensitive for growth, was found to be shut off rapidly at the nonpermissive temperature. The mutants did not complement each other and the ts lesion was not located on the X chromosome. Both isolates were found to be considerably more sensitive to the alkylating agents, ethylmethanesulfonate (EMS) and methylmethanesulfonate (MMS), as compared to the parental cells, but showed normal sensitivity to UV irradiation. The mutants also showed interesting differences in their response to EMS-induced mutation frequencies at the ouabain-resistant and thioguanine-resistant loci. At high survival (50%) the frequencies of mutations at these genetic loci were markedly low in the ts mutants as compared to the parental cells. In ts+ revertants isolated from the mutants, the ts phenotype and the increased sensitivity to EMS and MMS were affected simultaneously, indicating that both these characteristics resulted from a single genetic lesion.

Chinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8--10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (RicRII) behaves dominantly. Tests for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complimentation groups can be delineated among the phenotypically recessive mutants.

Stable mutants resistant to the nucleoside analog 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), which interferes with RNA synthesis, have been selected in Chinese hamster ovary (CHO) and human diploid fibroblasts. In CHO cells, upon treatment with the mutagen ethyl-methane sulfonate (EMS), a linear dose--response between the concentration of mutagen and the frequency of DrbR mutants was observed in the range of 20--300 micrograms/ml. The selection system did not show cell density or cross-feeding effects, and the optimal expression time following mutagenesis was found to be 2--3 days for CHO cells and 5--6 days for human fibroblasts. The DrbR mutation behaved codominantly in DrbR x DrbS hybrids. Addition of DRB affected nucleoside uptake to a similar extent in both wild-type and mutant cells, indicating that the drug was able to enter the mutant cells. The failure of DrbR mutants to show any cross-resistance to other toxic nucleoside analogs examined suggests that the action of DRB does not involve the initial phosphorylation step. DRB addition did not cause any marked inhibition of either RNA polymerase I or RNA polymerase II activity from both wild-type and mutant cells in vitro, indicating that its effect on RNA synthesis may be indirect.

Histidinol-resistant (HisOHR) mutants with up to a 30-fold increase in histidyl-tRNA synthetase activity have been isolated by stepwise adaptation of wild-type Chinese hamster ovary (CHO) cells to increasing amounts of histidinol in the medium. Immunoprecipitation of [35S]methionine-labeled cell lysates with antibodies to histidyl-tRNA synthetase showed increased synthesis of the enzyme in histidinol-resistant cells. The histidinol-resistant cell lines had an increase in translatable polyadenylated mRNA for histidyl-tRNA synthetase. A cDNA for CHO histidyl-tRNA synthetase has been cloned, using these histidyl-tRNA synthetase-overproducing mutants as the source of mRNA. Southern blot analysis of wild-type and histidinol-resistant cells with this cDNA showed that the histidyl-tRNA synthetase DNA bands were amplified in the resistant cells. These HisOHR cells owed their resistance to histidinol to amplification of the gene for histidyl-tRNA synthetase.

We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells. In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.

A MoMLV-based retroviral vector capable of transmitting and expressing both the human hypoxanthine phosphoribosyltransferase (hprt) coding sequence and the Herpes simplex type 1 thymidine kinase (tk) gene has been constructed. After infection of a rat cell line, cell clones were selected on the basis of expressing both markers. They were subsequently found to contain a single provirus of the expected topology. The ease with which loss of expression of the markers can be monitored has allowed us to make observations on the stability of proviral genes. In particular, we have found indirect evidence of strong position effects on proviral gene expression by comparing the characteristic frequency of marker loss in different clonal proviral lines. Effects of the selection protocol on the apparent frequency of variants have also been noted. Finally, a combination of molecular and genetic observations lead us to invoke chromosome loss as the major factor influencing marker stability in this system.

Forty-seven independent clonal isolates of cells obtained from polyoma virus infected hamster embryo cultures 10 weeks or more after infection were found to possess a broad spectrum of heritable, stable colony morphologies. All produced rapidly growing tumours in 20-day-old hamsters. The cellular properties of nine of the transformed clonal lines, believed to be representative of the entire morphological spectrum, were studied in considerable detail and were compared with the properties of freshly explanted hamster embryo cells. Many differences in properties were found both between different lines of transformed cells and between these and hamster embryo cells. The significance of these findings with respect to the mechanism of viral carcinogenesis is discussed. Emphasis was placed in this study on the discovery of differences in properties between transformed and normal cells which might allow the selective growth of a few transformed cells when present in a multitude of normal hamster embryo cells. Properties common to all the transformed lines suggested techniques that would select for all types of transformed cells, whereas other properties peculiar to just a few of the lines similarly suggested techniques that would be selective for transformed cells with these properties only.

Suppression of erythropoiesis by transfusion of animals of genotype W/Wv was found to prevent the development of macroscopic spleen colonies following injection of normal coisogenic marrow cells. This inhibition of colony-formation was not due to a failure of colony-forming cells to proliferate in the absence of erythropoietic stimulation, since the growth rate of normal colony-forming cells in plethoric animals did not differ significantly from that seen in anemic hosts. It is likely that, in plethoric hosts, insufficient differentiated erythroblasts were produced to permit the development of macroscopically visible spleen colonies. Evidence was obtained that granulocytic differentiation proceeded during the growth of the transplanted colony-forming cells, and that this mode of differentiation was not affected by the suppression of erythropoiesis. These results indicate that both granulocytic differentiation and the process of selfrenewal by which colony-forming cells increase in numbers are controlled independently of the control of erythropoiesis. These experiments provide additional support for the view that colony-forming cells differ from erythropoietin-sensitive cells.

The phenotypic characteristics of a series of temperature-sensitive, suppressor-sensitive, and defective mutants of λ bacteriophage located in the left-hand arm of the conventional chromosome map were examined by electron microscopy and the concentrations of viral-specific products were determined. Two morphologically distinct types of tubular heads as well as a variety of other aberrant head forms were observed in lysates of mutants mapping in cistrons B and C.

A technic capable of estimating the fraction of hemopoietic colony-forming progenitor cells in DNA synthesis in vivo has been described. The technic is based on the ability of tritiated thymidine to inhibit the growth of those colony-forming cells which, by virtue of their presence in the S-phase during a 20-minute incubation in vitro, in the presence of 500 µc./ml. of H3TdR, have incorporated large amounts of the nucleoside. The method has been applied to transplanted colony-forming cells proliferating in spleens of heavily irradiated recipients as well as to cells from normal fetal liver, normal marrow, and normal spleen. In situations where the hemopoietic system is expanding (fetal liver and regenerating transplants), a large fraction, 40-65 per cent, of the stem cells are in DNA synthesis. In the steady-state situations (adult marrow and spleen), the fraction of cells in DNA synthesis is almost imperceptible using this technic. It is concluded that control mechanisms which govern the rate of hemopoiesis operate, at least in part, by altering the generative cycle of blood-forming progenitor cells.

Analytical separation of the proteins of λ bacteriophage has been made possible by treatment with sodium dodecyl sulfate (SDS) at neutral pH and high temperature followed by electrophoresis in acrylamide gels containing SDS. The λ virion consists of three major proteins accounting for 57, 19, and 19% of the total protein. The largest of these proteins (estimated M.W. 37,500) is the major constituent of heads, accounting for approximately 95% of the head protein. The second largest protein (estimated M.W. 32,500) is the principal constituent of λ tails, and accounts for 90% of the tail protein. What is most probably an “internal protein” of λ is found in neither heads nor tails and has an estimated M.W. of 11,000. There are, in addition, 3–6 minor components accounting for about 5% of the phage protein. The principal head protein is coded for by gene E while gene V controls the synthesis of the principal tail protein.

Progenitor cells that are recognized by their ability to form colonies of descendants in the spleens of irradiated mice have the capacity for self-renewal. The distribution of new colony-forming cells per colony is extremely heterogeneous, indicating lax control of self-renewal. The capacities of colony-forming cells for self-renewal, for extensive proliferation, and for giving rise to differentiated descendants, fulfill three requirements for studies of stem cells. Thus, colony-forming cells may be considered to be class (though not necessarily the only class) of such progenitor cells, and the spleen colony method is a quantitative method for their detection. Journal of Cellular and Comparative Physiology copyright 1963 Wiley-Liss, Inc., A Wiley Company (www.interscience.Wiley.com).

The spleen colony technique' has proved to be a useful tool for the enumeration of hemopoietic stem cells in the mouse and for the study of their properties. However, since the method depends on colony formation in the spleens of heavily irradiated' or genetically anemic2 mice, it has certain inherent limitations. For example, events occurring early in the growth of colonies cannot be observed, since they are obscured by the splenic environment; further, the spleencolony method has not been applied successfully to the study of human cells3, 4 and, therefore, the information obtained by use of this method cannot be applied directly to clinical problems. These limitations might be avoided by the use of cell-culture methods. Recently, Pluznik and Sachs' and Bradley and Metcalf6 have reported that cells derived from mouse hemopoietic tissue will form colonies in culture if they are suspended in dilute agar over a firm agar base containing a source of suitable "conditioning factor."7-9 If a relationship were established between the cells that give rise to these colonies in culture and the hemopoietic stem cells responsible for colony formation in the spleen, the two techniques might complement each other. Results obtained by the use of the culture technique might be related through the spleen-colony method to normal and abnormal hemopoiesis invivo, and the information gained in the mouse by using the spleen-colony technique might be extended to other species, including man, by using the culture method. The existence of a relationship between hemopoietic stem cells and the cells that give rise to colonies in culture is indicated by the properties of the latter: they are capable of extensive proliferation,1? 11 self-renewal,5 and granulopoietic differentiation.6 10 Further, they are found in the same organs as hemopoietic colony-forming cells and in approximately the same relative numbers.5, 6 In order to establish a relationship more directly, we have employed two approaches. First, we have used a chromosome marker techniquel2 to determine whether or not the cells that give rise to colonies in culture can belong to the same clone as cells that form colonies in vivo. Second, we have used both techniques in assaying cell suspensions derived from individual spleen colonies for their colonyforming activity. Previous work had shown that the distribution of spleen colony-forming cells among spleen colonies is very heterogeneous, with a 1000fold difference between colonies containing little and those containing much colony-forming potential.", 14 If individual colonies yielded similar results in both assay systems, a close relationship between the cells responsible for each activity might be assumed. The results of these experiments are presented in this report. They provide 1209

1. 1. The time elapsed between induction of B. megatherium 899 (1) by irradiation with ultraviolet light, and formation of the first intra- and extra-cellular bacteriophages has been measured at 27 and 37°C. The latent period is prolonged by an increase in the inducing dose of ultraviolet light. 2. 2. A study of the biochemical modifications occuring after induction of lysogenic B. megatherium has shown that, concomitant with the residual growth, respiration increases, and ribonucleic acid is synthesized. The synthesis of desoxyribonucleic acid is blocked during approximately the first third of the latent period. Rapid synthesis then commences and continues until the moment of lysis. 3. 3. The biochemical modifications during development of temperate phages in induced lysogenic bacteria have been compared with those found in bacteria infected with virulent phages, which block growth completely. Whereas growth, ribonucleic acid and enzyme synthesis does, or does not, occur, depending on the phage-host system studied, the pattern of desoxyribonucleic synthesis seems to be a common property of all phage-host systems examined heretofore.

Understanding of the cancer process in chemical terms has been seriously hampered by the difficulty of interpreting results of biochemical comparisons between masses of tumor and of normal tissue. Normal tissue consists of a variety of cell types and tumors may originate from one or more of these. As whole masses, therefore, normal tissues cannot serve as adequate controls for experiments on any single tumor. Tumor cell populations, even those arising from a single cell type, are themselves cytogenetically and continually undergoing changes during growth (progression). It is thus difficult, if not impossible, to separate the relevant from the irrelevant biochemical features of malignancy.Progress in this field requires means of dealing with the problem of biological heterogeneity. Several biochemical approaches that are free from the hazards of heterogeneity and which have already yielded valuable results, or appear promising, are indicated. These include: (1) The use of ascites tumors for studying the biochemical machinery of cells. No normal tissue exists, however, that could serve as satisfactory control. (2) Biochemical comparisons between pairs of tumor lines which differ by only one inherited characteristic of malignancy. These might reveal a biochemical basis for the biological properties of tumor cells with different degrees of malignancy. (3) Elucidation of normal growth-controlling mechanisms between cells, e.g. action of hormones at the cellular level, and within cells, e.g. mechanism of feed-back control of enzymes and metabolic pathways. (4) Further research into the biochemistry of plant tumor induction in vitro. Here biochemical changes associated with inherited changes leading to nutritional autonomy and uncontrolled growth have already been demonstrated. (5) Studies on the biochemical events during induction of malignancy by viruses in clonal cultures of animal cells in vitro. These could serve as useful models of the whole process of carcinogenesis.

Mutants of mouse L-cells which are temperature-sensitive for growth have been obtained by using both selective and nonselective isolation procedures on populations treated with the mutagen nitrosoguanidine. Selective isolation was carried out by utilizing a five-day treatment with 3H-TdR and ara-C as selective agents at the nonpermissive temperature. Nonselective isolation was performed by isolating 1400 clones in the absence of selective agents and then testing them for temperature-sensitivity. From this experiment we obtained a minimum estimate of 6 × 10−3 for the frequency of mutants in the mutagentreated population. The mutants were characterized by their plating efficiencies, growth in suspension culture, and uptake of isotopic precursors of DNA, RNA, and protein. A range in phenotypes was observed, and there appeared to be some differences between the mutants obtained by the two types of isolation procedures. In uptake experiments the most marked reductions in the rates of precursor incorporation were seen with 3H-TdR, rather than 3H-UR or 3H-Leu. Different mutant lines showed considerable variation in the rate of cessation of DNA synthesis as well as the time required for termination of cell division. These experiments suggest that both types of isolation procedures are feasible for obtaining temperature-sensitive mutants having a range of phenotypes.

A two-stage procedure has been used to obtain hemopoietic spleen colonies derived from single precursor cells containing radiation-induced chromosomal markers. Of a total of 46 colonies examined, 17 were found to contain cells with abnormal karyotypes. In each of the 17 marked colonies, 90% or more of the dividing cells in the colony carried the same marker. Cell suspensions prepared from each of the individual colonies were tested for their content of dividing cells possessing recognizable differentiated functions. Metaphase cells with peroxidase-positive granules in their cytoplasm were considered to be members of the granulopoietic series, while metaphase cells which contained Fe55 were considered to be members of the erythropoietic series. Results were obtained for 12 of the marked colonies, and in nine of these, the percentage of metaphases lacking the marker was less than the percentage of metaphases which were scored as erythropoietic, and also was less than the percentage of metaphases scored as granulopoietic. This is the result which would be expected if the marker were present in both erythropoietic and granulopoietic cells. These results provide support for the view that colony forming hemopoietic stem cells are multipotent, and that differentiation along more than one pathway can occur during the formation of macroscopic splenic colonies.

The relationship between hematopoietic colony-forming stem cells and cells in the thymus and lymph nodes of unirradiated mice has been investigated using a chromosome-marker technique. It was found that a high proportion of cells in the thymus may belong to the same clone as normal hematopoietic colony-forming cells. It was also found that cells belonging to the same clone as colony-forming cells may reach the lymph nodes, and that nodes containing such cells can participate in an immunological response against sheep red cells. Either the precursors of cells in thymus and lymph node are identical with hematopoietic colony-forming cells, or they are both descendants of a common precursor which has not yet been identified. The results are compatible with the view that cells of the hematopoietic system and the immune system may be derived from the same stem cell.

The synthesis of λ-coded proteins has been examined in infected cells in which host protein synthesis was depressed by previous irradiation with uv light. Labeled proteins were analyzed by electrophoresis in polyacrylamide gels containing sodium dodecylsulfate, and the pattern of radioactive bands was visualized by autoradiography of dried longitudinal gel slices. More than 30 viral proteins were identified; 15 of these were assigned to cistrons in the morphogenetic region, three to cistrons in the b region (or silent region) and three were under control of the early genetic region between the att site and cistron N. Among the 15 late proteins assigned to cistrons, nine were structural components of the phage particle. A kinetic analysis of the amounts of gene products, as measured by microdensitometry, indicated that the synthesis of individual late proteins in phage λ seems to be initiated in an orderly fashion, related to the distance of the genes from the late main promoter between genes Q and S. Absence of gene Q product resulted in a sevenfold depression in the synthesis of late proteins, including the late proteins of the b region. Only three polar effects were demonstrated in the left arm. These included the group of cistrons EF, VGTH and LK. The number of copies of gene products synthesized by the different genes in the morphogenetic region was not related in a simple way to the distance of the genes from the main late promoter and may vary by up to a factor of 870. This indicates the presence of a remarkable control mechanism for gene expression of the left arm. Evidence has also been obtained for the conversion of one late protein during λ development, probably by proteolytic cleavage. Measurement of the molecular weights of the assigned late proteins has allowed the construction of a composite genetic, physical and molecular map of the left arm of phage λ. On this basis it is predicted that the gene products of the genes for which no corresponding electrophoretic band has yet been found will be of small molecular weight.

SummaryWhen cell suspensions containing normal mouse spleen or lymph node cells mixed with sheep erythrocytes are injected into heavily-irradiated mice, foci of hemolysin-producing cells are formed in the spleens of the recipients. A method is described for demonstration of these foci, and evidence is presented that they result from the proliferation of antigen-sensitive precursors of hemolysin-producing cells. The demonstration of the existence of “antigen-sensitive” cells provides strong support for the view that cell proliferation is involved in the generation of hemolysin-producing cells.

Buoyant density distributions of hemopoietic colony-forming units (CFU) from normal mouse marrow were determined by equilibrium density gradient centrifugation in bovine serum albumin (BSA) gradients. The distributions were compared with those obtained for the total population of nucleated cells from normal mouse marrow. The buoyant density distribution for CFU was found to differ from the density distribution for the total nucleated cell population, and the portion of the total cell population with densities much less than the mean value was found to contain up to a 30-fold greater proportion of CFU than an uncentrifuged control. These results provide a preliminary approach to the purification and characterization of normal hemopoietic colony-forming stem cells.

Somatic cell mutants resistant to ouabain, which inhibits the plasma membrane Na/K ATPase, have been isolated from mouse L and Chinese hamster ovary (CHO) cells. Ouabain at concentrations ≥ 1 mM with 5–6 mM K + , or ≥ 0.1 mM with 0.5 mM K + , inhibits the growth of the wild-type cells and is ultimately cytotoxic. Clones 2- to 100-fold more resistant than wild type in terms of dose can be obtained by single-step selection from a wild-type population in the presence of ouabain. The phenotypes of ouabain-resistant (OUA R ) clones are reproducible with high fidelity and stable over long intervals of growth in the absence of the selecting drug. Wild-type and OUA R L cell clones were compared with respect to their susceptibility to ouabain inhibition of 42 K uptake by whole cells and of Na/K ATPase activity in isolated plasma membranes. In both respects the OUA R cells are less sensitive to the drug than are wild-type cells. Conditions for optimal ATPase activity in the absence of ouabain were indistinguishable for the wild-type and one OUA R clone examined in detail and were comparable to requirements reported for ATPase preparations from other source materials. The frequency of OUA R cells in a wild-type population can be substantially increased, to approximately 10 −4 per viable cell, by exposure to the chemical mutagen EMS. The spontaneous mutation rate to 10-fold increase in ouabain-resistance is estimated by Luria-Delbruck fluctuation analyses to be 5–6 × 10 −8 per cell per generation for both L and CHO cells. Cell-cell hybridization experiments utilizing OUA R and wild-type CHO cells indicate that resistance to ouabain behaves as a codominant trait, and that this marker can be useful for selection of somatic cell hybrids.