Rate of replication of the murine immunoglobulin heavy-chain locus: evidence that the region is part of a single replicon.

We measured the temporal order of replication of EcoRI segments from the murine immunoglobulin heavy-chain constant region (IgCH) gene cluster, including the joining (J) and diversity (D) loci and encompassing approximately 300 kilobases. The relative concentrations of EcoRI segments in bromouracil-labeled DNA that replicated during selected intervals of the S phase in Friend virus-transformed murine erythroleukemia (MEL) cells were measured. From these results, we calculated the nuclear DNA content (C value; the haploid DNA content of a cell in the G1 phase of the cell cycle) at the time each segment replicated during the S phase. We observed that IgCH genes replicate in the following order: alpha, epsilon, gamma 2a, gamma 2b, gamma 1, gamma 3, delta, and mu, followed by the J and D segments. The C value at which each segment replicates increased as a linear function of its distance from C alpha. The average rate of DNA replication in the IgCH gene cluster was determined from these data to be 1.7 to 1.9 kilobases/min, similar to the rate measured for mammalian replicons by autoradiography and electron microscopy (for a review, see H. J. Edenberg and J. A. Huberman, Annu. Rev. Genet. 9:245-284, 1975, and R. G. Martin, Adv. Cancer Res. 34:1-55, 1981). Similar results were obtained with other murine non-B cell lines, including a fibroblast cell line (L60T) and a hepatoma cell line (Hepa 1.6). In contrast, we observed that IgCh segments in a B-cell plasmacytoma (MPC11) and two Abelson murine leukemia virus-transformed pre-B cell lines (22D6 and 300-19O) replicated as early as (300-19P) or earlier than (MPC11 and 22D6) C alpha in MEL cells. Unlike MEL cells, however, all of the IgCH segments in a given B cell line replicated at very similar times during the S phase, so that a temporal directionality in the replication of the IgCH gene cluster was not apparent from these data. These results provide evidence that in murine non-B cells the IgCH, J, and D loci are part of a single replicon.     

The beta-major and beta-minor globin genes in murine erythroleukemia cells replicate during the same early interval of the S phase

The time of replication in S phase was determined for the 7.3 kb EcoRI segment containing the beta-major globin gene and the 14 kb EcoRI segment containing the beta-minor globin gene in a murine Friend erythroleukemia virus transformed (MEL) cell line. Cells were obtained from different intervals of S phase by centrifugal elutriation to avoid artifacts of chemical synchronization. Newly synthesized DNA from different parts of S phase were obtained by isolation of 5-bromouracil (BU) labelled DNA from these cells. The BU-DNA synthesized during four different intervals of S was transferred to diazobenzyloxymethyl (DBM) paper and hybridized with a beta-globin cDNA probe. Quantitation showed that both beta-globin segments were replicated in the first quarter of the S phase with no significant difference in their time of replication in this MEL cell line.     

Nucleotide sequence and properties of the murine gamma 3 immunoglobulin heavy chain gene switch region: implications for successive C gamma gene switching

During B lymphocyte differentiation, immunoglobulin heavy chain constant region (CH) genes undergo a unique series of DNA recombination events culminating in the CH class switch. CH switch (S) regions are located 2 kb 5' of each CH gene except delta (i.e. mu, gamma 3, gamma 1, gamma 2b, gamma 2a, epsilon and alpha). We describe the structural features of the gamma 3 switch region. Hybridization experiments show that S gamma 3 has remarkable homology to both S mu and other S gamma regions while S mu possesses limited homology to the other S gamma sequences. However, S mu possesses extensive sequence homology with S epsilon and S alpha. The nucleotide sequence of S gamma 3 reveals higher densities of S mu repetitive sequences (GAGCT and GGGGT) and another S region common sequence (YAGGTTG) than observed for S gamma 1, S gamma 2b or S gamma 2a. In addition, the conservation of S mu like repetitive sequences in S gamma regions is correlated with the 5' leads to 3' gamma gene order (i.e. S gamma 3 greater than S gamma 1 greater than S gamma 2b greater than S gamma 2a). A model is presented which suggests that the unique features of S gamma 3 may allow for successive switches from C mu to any C gamma gene.     

Protein tyrosine kinase and protein phosphotyrosine phosphatase in normal and psoriatic skin

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the Eco RI 340 bp family (alpha RI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [3H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [3H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.     

Regulation of the replication of the murine immunoglobulin heavy chain gene locus: evaluation of the role of the 3' regulatory region.

DNA replication in mammalian cells is a precisely controlled physical and temporal process, likely involving cis-acting elements that control the region(s) from which replication initiates. In B cells, previous studies showed replication timing to be early throughout the immunoglobulin heavy chain (Igh) locus. The implication from replication timing studies in the B-cell line MPC11 was that early replication of the Igh locus was regulated by sequences downstream of the C alpha gene. A potential candidate for these replication control sequences was the 3' regulatory region of the Igh locus. Our results demonstrate, however, that the Igh locus maintains early replication in a B-cell line in which the 3' regulatory region has been deleted from one allele, thus indicating that replication timing of the locus is independent of this region. In non-B cells (murine erythroleukemia cells [MEL]), previous studies of segments within the mouse Igh locus demonstrated that DNA replication likely initiated downstream of the Igh gene cluster. Here we use recently cloned DNA to demonstrate that segments located sequentially downstream of the Igh 3' regulatory region continue to replicate progressively earlier in S phase in MEL. Furthermore, analysis by two-dimensional gel electrophoresis indicates that replication forks proceed exclusively in the 3'-to-5' direction through the region 3' of the Igh locus. Extrapolation from these data predicts that initiation of DNA replication occurs in MEL at one or more sites within a 90-kb interval located between 40 and 130 kb downstream of the 3' regulatory region.     

Replication and subnuclear location dynamics of the immunoglobulin heavy-chain locus in B-lineage cells

The murine immunoglobulin heavy-chain (Igh) locus provides an important model for understanding the replication of tissue-specific gene loci in mammalian cells. We have observed two DNA replication programs with dramatically different temporal replication patterns for the Igh locus in B-lineage cells. In pro- and pre-B-cell lines and in ex vivo-expanded pro-B cells, the entire locus is replicated early in S phase. In three cell lines that exhibit the early-replication pattern, we found that replication forks progress in both directions through the constant-region genes, which is consistent with the activation of multiple initiation sites. In contrast, in plasma cell lines, replication of the Igh locus occurs through a triphasic pattern similar to that previously detected in MEL cells. Sequences downstream of the Igh-C alpha gene replicate early in S, while heavy-chain variable (Vh) gene sequences replicate late in S. An approximately 500-kb transition region connecting sequences that replicate early and late is replicated progressively later in S. The formation of the transition region in different cell lines is independent of the sequences encompassed. In B-cell lines that exhibit a triphasic-replication pattern, replication forks progress in one direction through the examined constant-region genes. Timing data and the direction of replication fork movement indicate that replication of the transition region occurs by a single replication fork, as previously described for MEL cells. Associated with the contrasting replication programs are differences in the subnuclear locations of Igh loci. When the entire locus is replicated early in S, the Igh locus is located away from the nuclear periphery, but when Vh gene sequences replicate late and there is a temporal-transition region, the entire Igh locus is located near the nuclear periphery.     

Studies of mammalian chromosome replication

Sister chromatids of metaphase chromosomes can be differentially stained if the cells have replicated their DNA semiconservatively for two cell cycles in a medium containing 5-bromodeoxyuridine (BrdU). When prematurely condensed chromosomes (PCC) are induced in cells during the second S phase after BrdU is added to the medium, the replicated chromosome segments show sister chromatid differential (SCD) staining. Employing this PCC-SCD system on synchronous and asynchronous Chinese hamster ovary (CHO) cells, we have demonstrated that the replication patterns of the CHO cells can be categorized into G₁/S, early, early-mid, mid-late, and late S phase patterns according to the amount of replicated chromosomes. During the first 4 h of the S phase, the replication patterns show SCD staining in chains of small chromosome segments. The amount of replicated chromosomes increase during the mid-late and late S categories (last 4 h). Significantly, small SCD segments are also present during these late intervals of the S phase. Measurements of these replicated segments indicate the presence of characteristic chromosome fragment sizes between 0.2 to 1.2 m in all S phase cells except those at G₁/S which contain no SCD fragments. These small segments are operationally defined as chromosome replicating units or chromosomal replicons. They are interpreted to be composed of clusters of molecular DNA replicons. The larger SCD segments in the late S cells may arise by the joining of adjacent chromosomal replicons. Further application of this PCC-SCD method to study the chromosome replication process of two other rodents, Peromyscus eremicus and Microtus agrestis, with peculiar chromosomal locations of heterochromatin has demonstrated an ordered sequence of chromosome replication. The euchromatin and heterochromatin of the two species undergo two separate sequences of decondensation, replication, and condensation during the early-mid and mid-late intervals respectively of the S phase. Similar-sized chromosomal replicons are present in both types of chromatin. These data suggest that mammalian chromosomes are replicated in groups of replicating units, or chromosomal replicons, along their lengths. The organization and structure of these chromosomal replicons with respect to those of the interphase nucleus and metaphase chromosomes are discussed.     

INHIBITION OF MYOBLAST FUSION AFTER ONE ROUND OF DNA SYNTHESIS IN 5-BROMODEOXYURIDINE

The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-³H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-³H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-³H for one S period do not fuse with normal myotubes.     

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.     

Differential sensitivity to 5-bromodeoxyuridine during the S phase of synchronized myogenic cells

Synchronized myogenic cell cultures have been used to demonstrate differential sensitivity to BUdR during segments of the S period. Synchronization of the cells was achieved by two methods. First, cells were initiated in medium containing FUdR, an inhibitor of DNA synthesis. Following FUdR blockade reversal with TdR after 19 hr in vitro, the synchronized cells were allowed to replicate their DNA with BUdR for periods corresponding to early and late S. Determinations of percentage labeled cells during synchronization with FUdR indicate that about 90% of the cycling population of cells accumulates at the G1/S interface of the cell-cycle and that the duration of the S period following blockade reversal with TdR is not altered. Since BUdR is pulsed to these cultures immediately after the point of synchronization, a high degree of synchrony is obtained. In the second method of synchrony, cohorts of cells which had been in G2, late S, or early S during a BUdR pulse were collected in metaphase arrest with Colcemid and selectively removed from the cultures. With the mitotic selection method the point of synchronization occurred several hours after the BUdR pulse. In both methods the cells were allowed to resume myogenesis and scored for percentage fused nuclei after approx 50 hr in vitro. With both methods of synchrony, BUdR incorporation into early replicating DNA results in a striking decline in myoblast fusion, whereas incorporation into late replicating DNA is without effect. The results cannot be attributed to a disproportionate uptake of nucleotide during early S. Further fractionation of the 4-hr S phase into 1-hr periods indicates that the BUdR sensitive target is replicated during the second hr of DNA synthesis.     

Replication timing of DNA sequences associated with human centromeres and telomeres.

The timing of replication of centromere-associated human alpha satellite DNA from chromosomes X, 17, and 7 as well as of human telomeric sequences was determined by using density-labeling methods and fluorescence-activated cell sorting. Alpha satellite sequences replicated late in S phase; however, the alpha satellite sequences of the three chromosomes studied replicated at slightly different times. Human telomeres were found to replicate throughout most of S phase. These results are consistent with a model in which multiple initiations of replication occur at a characteristic time within the alpha satellite repeats of a particular chromosome, while the replication timing of telomeric sequences is determined by either telomeric origins that can initiate at different times during S phase or by replication origins within the flanking chromosomal DNA sequences.     

A nuclear DNA-binding protein expressed during early stages of B cell differentiation interacts with diverse segments within and 3' of the Ig H chain gene cluster.

We have used electrophoretic mobility shift assays (EMSA) to detect B cell lineage-specific nuclear proteins that bind to diverse segments within and 3' of the Ig H chain gene cluster. DNA binding sites include sequences 5' of each of the following C region genes: mu, gamma 1, gamma 2a, epsilon, and alpha. For the most part, these binding sites lie 5' of CH-associated tandem repeats. Binding sites for the same B cell lineage-specific proteins have also been defined in the region 3' of C alpha, close to a recently described B cell-specific enhancer element. Cross-competition of EMSA indicates that the B cell lineage-specific nucleoprotein is indistinguishable from those described previously by others: S alpha-BP and BSAP. Because of the diverse sequences recognized by this protein, we term it NF-HB, B-lineage-specific nuclear factor that binds to Ig H gene segments. EMSA using segments 5' of S gamma 2a (5'S gamma 2a-176) and 3' of C alpha (3' alpha-88) shows multiple binding complexes, two of which are B cell lineage specific. The B cell-specific complex with fastest mobility contains only NF-HB, and the one with slowest mobility contains NF-HB together with a ubiquitous DNA-binding protein(s). The ubiquitous binding protein is different for 5' S gamma 2a-176 and for 3' alpha-88, representing the formation of protein-NF-HB complexes specific for these particular Ig DNA regions. Spleen cells show a single band upon EMSA with either 5'S gamma 2a-176 or 3' alpha-88. Upon LPS stimulation, additional binding complexes of slower mobility were formed resulting in a pattern comparable to those detected in pro-B, pre-B, and B cell lines. We hypothesize that NF-HB may promote physical interactions between the 3' alpha-enhancer and segments of the Ig H gene cluster.     

On immunoglobulin heavy chain gene switching: two gamma 2b genes are rearranged via switch sequences in MPC-11 cells but only one is expressed

During B lymphocytes differentiation, switches in the expression of heavy chain immunoglobulin constant region (CH) genes occur by a novel DNA recombination mechanism. We have investigated the requirements of the CH gene switch by characterizing two rearranged gamma 2b genes from a gamma 2b producing mouse myeloma (MPC-11). One of the two gamma 2b genes is present in 2-3 copies per cell (gamma 2b strong hybridizer) while the other is present in approximately 1 copy per cell (gamma 2b weak hybridizer). Genomic clones of the gamma 2b strongly hybridizing gene indicate that this is an abortive switch event between the S gamma 3 and S gamma 2b regions. However, clones of the gamma 2b weakly hybridizing gene suggest a functional rearrangement due to the presence of VH, JH and S mu sequences. The switch-recombination sites of these rearranged gamma 2b genes and those of other CH genes show a high degree of preference for the sequence AGGTTG 5' of either the S mu donor site or the appropriate CH S acceptor site. AGGTTG and its analogs are rare in the S mu region, are somewhat prevalent in s alpha and in the case of S mu are found 5' of a tandemly repeated DNA sequence (GAGCT, GGGGT) comprising most of S mu.     

Cell cycle-dependence of HL-60 cell deformability.

In this study, the role of cytoskeleton in HL-60 deformability during the cell cycle was investigated. G1, S, and G2/M cell fractions were separated by centrifugal elutriation. Cell deformability was evaluated by pipette aspiration. Tested at the same aspiration pressures, S cells were found to be less deformable than G1 cells. Moreover, HL-60 cells exhibited power-law fluid behavior: mu = mu c(gamma m/ gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material constant. At a given shear rate, S cells (mu c = 276 +/- 14 Pa.s, b = 0.51 +/- 0.03) were more viscous than G1 cells (mu c = 197 +/- 25, b = 0.53 +/- 0.02). To evaluate the relative importance of different cytoskeletal components in these cell cycle-dependent properties, HL-60 cells were treated with 30 microM dihydrocytochalasin B (DHB) to disrupt F-actin or 100 microM colchicine to collapse microtubules. DHB dramatically softened both G1 and S cells, which reduced the material constants mu c by approximately 65% and b by 20-30%. Colchicine had a limited effect on G1 cells but significantly reduced mu c of S cells (approximately 25%). Thus, F-actin plays the predominate role in determining cell mechanical properties, but disruption of microtubules may also influence the behavior of proliferating cells in a cell cycle-dependent fashion.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

Immunoglobulin heavy chain switch region recombination within a retroviral vector in murine pre-B cells.

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a substrate for detecting the presence of immunoglobulin heavy chain constant region (CH) gene switch (S) recombination activity in murine pre-B cells. ZN(Smu/S gamma 2b)tk1 contains a neomycin (neo) resistance gene in addition to the herpes simplex virus thymidine kinase (Htk) gene which is positioned between murine Smu and S gamma 2b sequences. Stable acquisition of the ZN(Smu/S gamma 2b)tk1 vector was selected in G-418 and switch region recombination within these proviruses was selected by resistance to the drug bromodeoxyuridine (BUdR). Fluctuation analyses of ZN(Smu/S gamma 2b)tk1 infected 18-8tk- and 38B9tk- pre-B lines revealed Htk gene inactivations with apparent frequencies of 5 X 10(-5) and 1 X 10(-5) events/cell/generation, respectively, while G-418 resistant Ltk- fibroblasts lost the HTK phenotype at an apparent rate of 4 X 10(-8). Southern blot analysis demonstrated that switch recombination caused the deletion of the Htk gene in all pre-B clones examined while the loss of Htk in Ltk- clones was not mediated by S region recombination. In 21 out of 24 pre-B clones, the recombinations involved the tandemly repetitive portions of the Smu and S gamma 2b sequences. These results demonstrate that the CH gene S region segments inserted into ZN(Smu/S gamma 2b)tk1 are sufficient for B-cell-specific recombination/deletion within the S region tandem repeats.     

Replication of the rRNA and legumin genes in synchronized root cells of pea (Pisum sativum): evidence for transient EcoR I sites in replicating rRNA genes

Summary The temporal pattern of replication of the rRNA and legumin genes differs in synchronized pea root cells. The relative number of rRNA genes replicated hourly during the first five hours of S phase ranges between 5 and 10 percent. In late S phase, during hours six through nine, the number of rRNA genes replicated increases reaching a maximum of about 25 percent at the ninth hour. Unlike the rRNA genes, the legumin genes have a wave-like pattern of replication peaking in early S phase at the third hour and again in late S phase at the eighth hour.Replicating rDNA, isolated by benzoylated naphthoylated DEAE-column chromatography, has EcoR I restriction sites that are absent in non-replicating rDNA sequences. The cleavage of these sites is independent of the time of rDNA replication. The transient nature of the EcoR I sites suggests that they exist in a hemimethylated state in parental DNA.The two Hind III repeat-size classes of rDNA of var. Alaska peas are replicated simultaneously as cells progress through S phase. Thus, even if the 9.0 kb and 8.6 kb repeat classes are located on different chromosomes, their temporal order of replication is the same.     

Two new isotype-specific switching activities detected for Ig class switching

Ig class switch recombination (CSR) occurs by an intrachromosomal deletional process between switch (S) regions in B cells. To facilitate the study of CSR, we derived a new B cell line, 1.B4.B6, which is uniquely capable of mu --> gamma3, mu --> epsilon, and mu --> alpha, but not mu --> gamma1 CSR at its endogenous loci. The 1.B4.B6 cell line was used in combination with plasmid-based isotype-specific S substrates in transient transfection assays to test for the presence of trans-acting switching activities. The 1.B4.B6 cell line supports mu --> gamma3, but not mu --> gamma1 recombination, on S substrates. In contrast, normal splenic B cells activated with LPS and IL-4 are capable of plasmid-based mu --> gamma1 CSR and demonstrate that this S plasmid is active. Activation-induced deaminase (AID) was used as a marker to identify existing B cell lines as possible candidates for supporting CSR. The M12 and A20 cell lines were identified as AID positive and, following activation with CD40L and other activators, were found to differentially support mu --> epsilon and mu --> alpha plasmid-based CSR. These studies provide evidence for two new switching activities for mu --> gamma1 and mu --> epsilon CSR, which are distinct from mu --> gamma3 and mu --> alpha switching activities previously described. AID is expressed in all the B cell lines capable of CSR, but cannot account for the isotype specificity defined by the S plasmid assay. These results are consistent with a model in which isotype-specific switching factors are either isotype-specific recombinases or DNA binding proteins with sequence specificity for S DNA.     

Effects of controlled exposure of L cells to bromodeoxyuridine: II. Turnover rates and activity profiles during cell cycle of bromodeoxyuridine-sensitive and -resistant enzymes

Fluorodeoxyuridine (FUdR)-synchronized mouse L cells were allowed to incorporate 5-bromodeoxyuridine (BUdR) at restricted intervals in the S phase and the effects of the selective incorporation of BUdR in DNA on the activities of seven randomly chosen enzymes (five dehydrogenases and two phosphatases) were analysed. Reductions to 56.9 and 83.3 % of the control levels were noted for glucose-6-phosphate dehydrogenase (G6PD) and alcohol dehydrogenase (ADH) activities respectively, when cells were exposed to BUdR during the 1st h of S. Acid phosphatase (AcP) activity was reduced to 81.9% of the control level following exposure to the analogue during the 3rd h of S. Exposure of cells to BUdR for the entire S period failed to increase the magnitude of the reductions in activity for any of these three enzymes. Alternately, when cells were allowed to synthesize DNA in the presence of thymidine for the 1st h of S and the remainder in the presence of BUdR, the activities of G6PD and ADH were comparable to those found in untreated cells. Exposure of cells to thymidine for the 3rd h of S, combined with exposure to BUdR for the preceding and subsequent hours of S, provided complete protection against the BUdR-mediated reduction in AcP activity. The activities of lactate dehydrogenase (LDH), 6-phosphogluconate dehydrogenase (6pGD), isocitrate dehydrogenase (IDH) and alkaline phosphatase (A1P) were found to be insensitive to treatment with BUdR, even when the period of analogue exposure encompassed the entire S period.Additional investigations carried out with G6PD for characterization of the nature of the BUdR effects suggest that the BUdR-mediated reductions in enzyme activities are not caused by the increased rates of degradation of the enzymes, formation of enzyme inhibitors or by the disproportionate replication of A-T base pairs during BUdR treatment. The alterations of enzyme activities appear to result from decreased rates of synthesis of enzymes in BUdR-treated cells. The results of the present study clearly suggest that pulse labelling of cells with BUdR at various intervals of the S phase may provide a useful approach for determining temporal localization of replication time of DNA segments that are critical for the synthesis or regulation of specific gene products.