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Condensation of mouse C-heterochromatin is inhibited in cells of established mouse lines if treated with 33258 Hoechst. The fact that after such treatment these regions appear in mitotic chromosomes as longthreaded segments enabled us to localize, with the silver staining technique, the NORs outside the C-heterochromatin. The number of NORs per cell and their identification was determined.

We have examined the effect of 5-azacytidine (5-aza-C) induced hypomethylation of DNA on the time of replication and DNase I sensitivity of the X chromosomes of female Gerbillus gerbillus (rodent) lung fibroblast cells. Using in situ nick translation to visualise the potential state of activity of large regions of metaphase chromosomes we show that 5-aza-C causes a dramatic increase in the DNase-I sensitivity of the entire inactive X chromosome of female G. gerbillus cells and this increase in nuclease sensitivity correlates with a large shift in the time of replication of the inactive X chromosome from late S phase to early S phase. These effects of 5-aza-C on the inactive X chromosome are associated with a 15% decrease in DNA methylation. Our results indicate that DNA methylation concomitantly affects both the time of replication and the chromatin conformation of the inactive X chromosome.

Isolation of a wide variety of temperature-sensitive (ts) cell cycle mutants in mammalian cells has previously proved to be a very difficult task. The various procedures used for the isolation of such mutants included a mutant enrichment step based on exposure of the cells to the restrictive temperatures in order to kill the growing wild-type cells with agents that kill DNA-synthesizing cells. Hence, these methods favored the isolation of ts mutants that do not lose viability rapidly at the restrictive temperatures, We have treated cells of the Chinese hamster established cell line E36 with the mutagen ethyl-methane-sulfonate (EMS) and used a replicaplating technique that we developed to screen the ts mutants for growth. This technique enabled us to recover all ts mutants for growth including the ts cell cycle mutants. Screening of the ts cell cycle mutants among the ts mutants for growth was performed by the flow microfluorimetry technique and the premature chromosome condensation technique. Our results show that 1.3% of the survivors of the mutagenic treatment are ts mutants for growth. Six of 84 ts mutants analyzed were found to be ts cell cycle mutants. They include ts mutants arrested in phases G1, S, and G2. Many of the ts mutants for growth including the ts cell cycle mutants arrested in S and G2 lose viability very fast when incubated at the restrictive temperature. As a consequence they could not have been isolated by any method that includes a mutant enrichment step based on the exposure of the cells to the restrictive temperature.

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes in Microtus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. In Microtus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.

Condensation of human chromosomes in phase G2 and early mitosis is inhibited by the fluorochrome 33258-Hoechst. This inhibitory effect is most apparent in primary diploid fibroblasts and lymphoblasts and least pronounced in peripheral blood lymphocytes. Condensation of the human Y chromosome, which contains a large heterochromatic region rich in A-T base pairs, is drastically inhibited by 33258-Hoechst treatment of fibroblasts and lymphoblasts. The difference in sensitivity of human chromosomes in different cell types to 33258-Hoechst probably reflects differences in the cell-membrane permeabilities to 33258-Hoechst.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

The phenomenon of premature chromosome condensation has been applied to study the kinetics of condensation-inhibition exerted by the fluorochrome 33258-Hoechst (33258-H) on the centromeric heterochromatic regions of mouse chromosomes. Asynchronous mouse A-9 cells in culture were fused with mitotic HeLa cells in the presence of 33258-H. Pronounced condensation-inhibition of the c-heterochromatin was observed in prematurely condensed early G2, S and late G1 chromosomes in the 33258-H-treated cells. It is concluded that the c-heterochromatic regions begin to condense quite early in G2, decondense again late in G1 and remain decondensed in the S phase.

The virulent phage φ e of Bacillus subtilis which contains hydroxymethyluracil in its DNA requires host DNA polymerase III for its DNA replication. DNA polymerase III ts mutant cells infected with φ e at restrictive temperatures do not support phage DNA synthesis. However, φ e grows normally both at low and high temperatures in the mutant's parent strain and in spontaneous DNA polymerase III + revertants isolated from the mutant strain. Temperature-shift-down experiments with φ e -infected cells having thermosensitive DNA polymerase III ( pol III ts ) indicate that at 48 C the thermolabile DNA polymerase III is irreversibly inactivated and has to be synthesized de novo after the shift to 37 C, before phage DNA synthesis can begin. Temperature-shift-up experiments with φ e -infected mutant cells show that phage replication is arrested immediately after the temperature shift and indicate that φ e requires DNA polymerase III throughout its replication stage.

Mutants unable to grow on glutamate as the sole source of carbon and energy, isolated from glutamate-utilizing Escherichia coli K-12 strains, are described. One mutant had a very low aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1, formerly known as glutamate-oxaloacetate transaminase) activity (10% of wild-type activity). Another mutant lacked aspartate ammonialysae ( l -aspartate ammonia-lysae, EC 4.3.1.1, formerly known as aspartase) activity completely. These two mutants were unable to grow in a glutamate-minimal medium either at 30° or at 42°. A third mutant was selected for its inability to grow on glutamate at 42° but it could utilize glutamate for growth at 30°. This mutant was shown to have a thermolabile aspartate ammonia-lyase. All three mutants exhibited normal (wild-type) levels of glutamate dehydrogenase ( l -glutamate:NADP oxido-reductase (deaminating), EC 1.4.1.4) activity. On the basis of these data and earlier findings from this and other laboratories it is concluded that the major pathway of glutamate metabolism in E. coli is via trans-amination with oxaloacetate to give α-ketoglutarate and aspartate, and subsequent deamination of the aspartate to fumarate.

The glutamate permeation system in Escherichia coli K-12 consists of three genes: gltC, gltS , and gltR . The genes gltC and gltS are very closely linked, and are located between the pyrE and tna loci, in the following order: tna, gltC, gltS, pyrE; gltR is located near the metA gene. The three glt genes constitute a regulatory system in which gltR is the regulator gene responsible for the formation of repressor, gltS is the structural gene of the glutamate permease, and gltC is most probably the operator locus. The synthesis of glutamate permease is partially repressed in wild-type K-12 strains, resulting in the inability of these strains to utilize glutamate as the sole source of carbon. Derepression due to mutation at the gltC locus enables growth on glutamate as a carbon source both at 30 C and at 42 C. Temperature-sensitive gltR mutants capable of utilizing glutamate for growth at 42 C but not at 30 C were found to be derepressed for glutamate permease when grown at 42 C and partially repressed (wild-type phenotype) upon growth at 30 C. These mutants produce an altered thermolabile repressor which can be inactivated by mild heat treatment (10 min at 44 C) in the absence of growth.

Mycoplasmas are common contaminants of animal cells in cell cultures1–3. About 25 Mycoplasma and Acholeplasma species have been identified as cell culture contaminants but the most frequent are Mycoplasma hyorhinis, Mycoplasma orale, Mycoplasma arginini and Acholeplasma laidlawii3. These species are responsible for almost 85% of the contaminations in cell cultures3. We have devised a method for the selective killing of mycoplasmas in contaminated cell cultures, based on the differential nucleic acid metabolism of mycoplasma and mammalian cells. Mycoplasmas are unusual in that they have a nutritional requirement for nucleic acid precursors which can be met by purines and pyrimidine bases or by nucleosides4–6. Mammalian cells, on the other hand, incorporate very little free pyrimidines7,8 and for that reason incorporation of free bases such as uracil has been used to detect mycoplasmas in contaminated cell cultures9. One of the free base analogues which can be incorporated selectively into nucleic acids of mycoplasmas is 5-bromouracil (5-BrUra). Visible light induces breaks in 5-BrUra-containing DNA10 and this photosensitivity can be greatly increased by binding of the fluorochrome 33258-Hoechst to DNA11. Its unusually high content of A + T makes the mycoplasma DNA3 an excellent candidate for the induction of breakage by the combined action of 5-BrUra, 33258-H and light because 33258-H has a high affinity for A−T base pairs and an even higher affinity for A–BrUra base pairs12. We report here that treating them in this way provides a practical and simple method for the selective killing of contaminating mycoplasmas; they are probably killed by the breakage of their DNA.

Template-active regions of chromatin are structurally distinct from nontranscribing segments of the genome. Recently, it was suggested that the conformation of active genes which renders them sensitive to DNase I may be maintained even in fixed mitotic chromosomes. We have developed a technique of mitotic cell fixation and DNase I-directed nick-translation which distinguishes between active and inactive X chromosomes. We report here that Gerbillus gerbillus (rodent) female cells contain easily identified composite X chromosomes each of which includes the original X chromosome flanked by two characteristic autosomal segments. After nick-translation the active X chromosome in each cell is labelled specifically in both the autosomal and X-chromosomal regions. The inactive X chromosome is labelled only in the autosomal regions and in a small early replicating band within the late replicating 'original X' chromosome. Our technique opens the possibility of following the kinetics of X-chromosome inactivation and reactivation during embryogenesis, studying active genes in the inactive X chromosome and mapping tissue-specific gene clusters.

HUMAN–mouse cell hybrids usually exhibit preferential loss of human chromosomes1,2. After a time interval, which depends in part on the cell types used, most or sometimes all the human chromosomes segregate out from the hybrid cells3. In the course of investigation of the phenomenon of preferential loss of human chromosomes from hybrids between the mouse cell line RAG and human leukocytes, we isolated and analysed sixteen human–mouse (RH) hybrids. Seven of these hybrids had retained the human Y chromosome. Five of these had only one copy per cell of Y, one had two copies per cell and one, RH-28, had four copies of the Y in many cells. We decided to study more extensively this hybrid which seemed to have lost all the human chromosomes except number 17 and the Y.

We isolated Microtus agrestis-mouse somatic cell hybrid clones which had retained either the active or the inactive M. agrestis X chromosome. In both hybrid clones the X chromosomes retained their original chromatin conformation as studied by the in situ nick translation technique — the active X chromosome retained its high sensitivity to DNase I while the inactive one remained insensitive. A clone in which the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene had been spontaneously reactivated was isolated from the hybrid containing the inactive X chromosome. The in situ nick translation technique was used to study possible DNA conformation changes in the euchromatin of the inactive X chromosome with special reference to the reactivated HPRT locus. We found that the euchromatin in this X chromosome exhibited the same low sensitivity to DNase I as is characteristic of the inactive X chromosome.

The satellite DNA sequences located near the centromeric regions of mouse chromosomes replicate very late in S in both fibroblast and lymphocyte cells and are heavily methylated at CpG residues. F9 teratocarcinoma cells, on the other hand, contain satellite sequences which are undermethylated and replicate much earlier in S. DNA methylation probably plays some role in the control of satellite replication time since 5-azacytidine treatment of RAG fibroblasts causes a dramatic temporal shift of replication to mid S. In contrast to similar changes accompanying the inactivation of the X-chromosome, early replication of satellite DNA is not associated with an increase in local chromosomal DNase I sensitivity. Fusion of F9 with mouse lymphocytes caused a dramatic early shift in the timing of the normally late replicating lymphocyte satellite heterochromatin, suggesting that trans-activating factors may be responsible for the regulation of replication timing.

The fluorochrome 33258-Hoechst which binds to double-stranded DNA (dsDNA) has been previously shown to inhibit in several mammalian cell cultures the condensation of chromosomes in phase G2 and early mitosis. We have now found that this drug affects the cell cycle of Chinese hamster cells grown in vitro in several other ways. In cells treated with the drug, phase G2 is prolonged, the rate of DNA replication is drastically reduced and the cells are arrested most probably at very late S phase.

FUNCTIONAL AND CONCEPTUAL PHASES OF THE CELL CyCLE.... 390 Mitosis . . .. . . . . . . . . . . .. . . . . . . . . . . ... . . . . . . . . .. .. . . . . . . . . . . ... . . . . . . . . . . 391 GJ Phase . . . . . . .. . . .. . . . . . . . . 393 GO the Quiescent State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..... . . 393 S Phase . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . 394 G2 Phase . . . . . . . . . . . . . . . . . . . ... .. . . . . . . .... . . . . . . . .. .. . . . . . . . . . . . . . . . . . .. . . .. . 395 CELL CYCLE MUTANTS..... .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . ... . . .. .... . . . . . 396 The Concept of a Cell-Cycle Mutation . . . . . . . . . . . . . . . . . . . . ... . . . . . . . .. .. . . . . . . . . . . 396 Preliminary Characterization of a Cell-Cycle Mutant ..... . . . . ...... . . . . . . .... . . . . . . . . . . ... 396 Isolation of Temperature-Sensitive Cell-Cycle MlIIants . . . . . . . . . . . . . . . . . . . 398 Screening of Temperature-Sensitive Cell-Cycle Mutallls . . . . . ... . . . . . . . . .. . . . . . . . . . . . . . . . . . 399 Mitotic Mutallls. . . . . . . . . . . . . . . . . . . . . . . . . . 402 GO Mutants. . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . ..... . . . . . . . .. . . .. . . . . . . . . . 405 GI Phase Mutants . . . . . . . . . . ... . . . . . . . . . ..... 406 S Phase Mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . .. . . . . . . 409 G2 Phase Mutants . . . . . . . . . . ... . . . . . . . . . .. . . . . . . . . . ... . . . . . . . .. .. .. . 412 The GJ-less and G2-less Cell Cycle . .. . . . . . ... . . . . .. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . ... . . . . . . . . . 412 CONCLUSiONS . . . . . . . .. . . . . . . . . . . .. . . . . ..... 414

E36ts24 is a temperature-sensitive (ts) cell cycle mutant derived from the Chinese hamster lung cell line E36. At the restrictive temperature for growth (40.3 degrees C), the mutant cells are arrested at early S phase. We have microinjected poly(A)+ RNA isolated from the wild-type E36 cells into the cytoplasm of E36ts24 cells arrested at early S phase after 24 hr of incubation at 40.3 degrees C. The ts mutation was transiently complemented in a significant fraction of the microinjected cells as evidenced by the incorporation of [3H]thymidine assayed by autoradiography. Microinjection of mRNA fractionated by methylmercuric hydroxide/agarose gel showed that the mRNA capable of transiently complementing the mutation in E36ts24 contains about 940 nucleotides. Hence, it can code at most for a protein containing about 230 amino acids. We estimate that the partial purification by fractionation of the mRNA active in the transient complementation is on the order of 100- to 200-fold.

We have shown that in fixed mitotic chromosomes from female G. gerbillus cells the inactive X chromosome is distinctly less sensitive to DNAase I than the active X chromosome, as demonstrated by in situ nick translation. These results indicated that the specific chromatin conformation that renders potentially active genes sensitive to DNAase I is maintained in fixed mitotic chromosomes. We increased the sensitivity and accuracy of in situ nick translation using biotinylated dUTP and a specific detection and staining procedure instead of radioactive label and autoradiography and now show that in both human and CHO chromosomes, the DNAase I sensitive and insensitive chromosomal regions form a specific dark and light banding pattern. The DNAase I sensitive dark D-bands usually correspond to the light G-bands, but not all light G-bands are DNAase I sensitive. Identifiable regions of inactive constitutive heterochromatin are in a DNAase I insensitive conformation. Our methodology provides a new and important tool for studying the structural and functional organization of chromosomes.

The fluorochrome Hoechst 33258 which binds preferentially to A-T base pairs, drastically inhibits the condensation of A-T-rich centromeric heterochromatin regions in mouse cell lines. The condensation of all other regions of these chromosomes is also inhibited to some extent. The human Y chromosome contains a large heterochromatic region, which is also rich in A-T base pairs. This chromosome is not affected by Hoechst 33258 in human leukocyte cell cultures. On the other hand, condensation of the multiple copies of human Y chromosome in the mouse-human cell hybrid RH-28Y-23 is inhibited and the chromosomes appear distorted in Hoechst 33258-treated cells.

E36 ts24 is a temperature-sensitive cell cycle mutant which has been derived from the Chinese hamster lung cell line E36. This mutant is arrested in phase S when incubated at the restrictive temperature (40.3 degrees C) for growth. At this temperature, proliferation of the mutant cells ceases after 10 h. About 2 h earlier, DNA synthesis is arrested. These kinetic studies indicate that the execution point of the mutant cells is in early S phase well beyond the G1/S boundary. The pattern of replication bands in E36 ts24 cell grown for 9 h at 40.3 degrees C strengthen the kinetic studies and map the execution point to early S phase. The exact point of arrest of the mutant cells in phase S was mapped in early S phase near the execution point. At the point of arrest the cells continue to synthesize DNA at at a high rate but practically all of the newly synthesized DNA is degraded. This high rate of DNA degradation is limited to nascent DNA at the point of arrest. In the presence of 5-bromodeoxyuridine (5-BudR), the last E36 ts24 cells which reach mitosis at the restrictive temperature for growth show asymmetric replication bands which illustrate DNA degradation and resynthesis occurring in these cells at 40.3 degrees C.

The fluorochrome 33258-Hoechst which binds to DNA and preferentially to A-T-rich regions, inhibits drastically the condensation of the centromeric heterochromatic regions in mouse cell lines. Condensation of all other regions of the chromosomes is also inhibited to some extent. The kinetics of condensation-inhibition of the C-heterochromatin indicates that these regions are being condensed at specific time intervals in the G2 period with a specific order of condensation. The C-heterochromatic regions of mouse chromosomes nos. 9, 12, 14, 15 and 16 condense late in G2 and complete their condensation about 30 min before metaphase. Condensation in G2 of Chinese hamster chromosomes is also inhibited by 33258-H treatment.

The Chinese hamster temperature-sensitive cell-cycle mutant ts24 was analyzed biochemically in order to determine the nature of this lesion. The inability of these cells to proceed through S phase at the restrictive temperature could be complemented by the addition of asparagine to the growth medium, and enzymological analysis showed that this line contains a temperature-sensitive asparaginyl-tRNA synthetase. Normal asparaginyl-tRNA synthetase activity was restored in cells transfected with cloned genomic DNA that overcomes the mutational defect. In corroboration with these results it was shown that a different temperature-sensitive asparaginyl-tRNA synthetase mutant isolated in another laboratory was blocked in S phase in a manner similar to that of ts24. While the mechanism by which asparaginyl-tRNA synthetase affects cell-cycle progression has not been elucidated, it can be shown that it is not mediated through alteration in overall levels of protein synthesis.

Hypoplasia and retardation of the fetus are prominent developmental features in trisomies of the mouse. They are caused by disturbances and depression of growth in vivo. In a study of growth parameters of two trisomic mice. Ts12 and Ts19, cell cultures were initiated from Ts12, Ts19 and normal embryos at late developmental stages. Kinetics of growth of these cells under different conditions show that growth is not significantly affected in either of the trisomies. These results indicate that the trisomy-dependent growth impairment in vivo is not expressed under the experimental growth conditions used in vitro. It remains possible, though, that other autosomal trisomies of the mouse may show a different behaviour in this respect. If so, this may be considered specific to the tested trisomic chromosome and not as a general phenomenon of any trisomy.

Publisher Summary This chapter describes isolation and characterization of mutants that are defective in biochemical, cellular, or developmental processes provide a powerful tool in the analysis of the regulation of these processes at the molecular and cellular levels. A special group is the conditional mutants—usually, temperature sensitive. Temperature-sensitive ( ts ), mainly heat-sensitive, mutants have been isolated in various organisms and also in cultured cell lines. Such mutants have been proven very useful in physiological studies of mammalian cells grown in culture. As a result, all those ts mutants that lose viability rapidly when they become arrested at the high temperature are lost. To recover all potential ts mutants, any selection step that involves exposure of cells to the restrictive temperature is avoided. Screening of conditional mutants by replica plating is a well-known procedure in microorganisms. However, applying similar techniques for mammalian cells is much harder because of technical problems.

Arrest of host DNA synthesis in Bacillus subtilis infected with the virulent phage ∅e is dependent on the synthesis of a phage-coded protein ( Marcus and Newlon, 1971 ). The mechanism of host DNA arrest was studied in toluene-treated B. subtilis cells. The results showed that preparations of toluenized phage infected cells manifest faithfully the kinetics and specificity of host DNA arrest and the appearance of phage DNA synthesis. The arrest of host DNA synthesis is not caused by the production in the host DNA of single strand breaks that can be repaired by the DNA polymerase repair enzyme (Pol I). Neither hydroxymethyl deoxyuridylate (HMdUMP) nor probably any other small molecule is responsible for host DNA arrest.

After infection of Bacillus subtilis 3610 with bacteriophage ∅e, which contains hydroxymethyluracil in place of thymine in its DNA, bacterial DNA synthesis is arrested. Our results show that the phage-coded deoxythymidine triphosphate hydrolase (TTPase) is not essential for the arrest. A phage-coded protein made in the first 13 min after infection appears to be involved in arresting host DNA synthesis. Phage mutants defective in this function have been isolated. The inhibition of host DNA synthesis does not appear to take place by detachment of bacterial DNA from the cell membrane or by inhibition of DNA polymerase. A phage фe mutant defective in TTPase, with thymine replacing up to 20% of the HMU in its DNA, can grow and transfer the thymine-containing DNA to progeny normally. The role of HMU in this phage remains unexplained.

No correlation was found between glutamate decarboxylase (GAD) activity and the ability of Escherichia coli K-12 strains to grow on glutamate. A gene, gad , determining GAD activity maps near gltC , which controls glutamate permease.