CHROMOSOMAL DNA SYNTHESIS IN DROSOPHILA MELANOGASTER

Analysis of labeling patterns in three chromosome segments of Drosophila melanogaster has shown that the replicative activity within chromosomes is temporally ordered. Moreover, specific labeling patterns on one chromosome occur with specific patterns on another chromosome with a very high degree of correlation. This circumstance leads to the conclusion that DNA synthesis among all the regions in the three chromosome segments studied is coordinated. The various labeling patterns observed in any one chromosome and the combinations of labeling patterns observed in all three chromosome segments can be arranged in ordered arrays, if one assumes that the DNA synthesis in each chromosome region will go to completion without stopping once it has started. Such arrays can serve as models for the temporal order of DNA synthesis among chromosome regions. They predict that in any one chromosome DNA replication begins and ends at very few loci and that synthesis at a larger number of points occurs at an intermediate time.     

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.     

Quinacrine fluorescence of specific chromosome regions

The pattern of intense fluorescence of interphase nuclei and metaphase chromosomes after staining with quinacrine is described in Samoaia leonensis. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated thymidine indicates that there is little or no overlap in the time of replication of the intensely fluorescing and weakly fluorescing regions. Autoradiographic analysis of metaphase figures after continuous labeling with tritiated thymidine shows that the intensely fluorescing regions are late replicating and establishes their order of replication. Autoradiographic analysis of interphase nuclei after pulse labeling with tritiated deoxycytidine and of metaphase figures after continuous labeling with this tracer show that there is little, if any, incorporation of deoxycytidine into those chromosome regions which fluoresce intensely after staining with quinacrine and quinacrine mustard. These results indicate that such chromosome regions are characterized chemically by an extremely high, if not exclusive, content of adenine and thymine.     

Replication pattern of a large homogenously staining chromosome region in antifolate-resistant Chinese hamster cell lines

We have investigated the replication pattern of a large, homogenously staining chromosome region (HSR) in two antifolate-resistant Chinese hamster cell lines. This region is believed to be the location of an amplified genetic sequence which includes at least the gene coding for dihydrofolate reductase and which may be present in as many as 200 copies. It is shown that the HSR in both cell lines is among the first chromosome regions to begin DNA synthesis after reversal of an early G1 block. In cells synchronized in the S period with hydroxyurea, it is also clear that the HSR in both cell lines begins replication at many sites within its length in early S. The replicons comprising the HSR therefore may respond to a common initiation signal in early S. In one cell line (A3), replication of the HSR requires, at most, 3 hours of a 7-hour S period; in a second line (MQ19), replication proceeds for approximately 5 hours. In neither line does replication of the HSR occur concomitantly with synthesis of characteristic late replicating regions. These results were confirmed in exponential cultures using a retroactive labeling technique. The significance of these findings is discussed with reference to the possible origin and arrangement of the amplified sequence in these two cell lines.     

Preferential derivation of abnormal human G-group-like chromosomes from chromosome 15

Summary The marked binding of antibodies specific for 5-methylcytidine to the short arm of chromosome 15 distinguishes this chromosome from the other human acrocentrics. This method has been used to study over 60 individuals including 12 who did not have Down's syndrome, but who did have an extra G-group sized acrocentric chromosome. In six cases the extra chromosome did not show intensive binding of anti-5-methylcytidine. In the other six cases, the extra chromosome contained a 5-methylcytidine rich band at each end indicating that both ends were derived from chromosome 15 and contained centromeric heterochromatin normally present on the short arm of chromosome 15. The duplication of short arm material in the abnormal chromosomes was confirmed in all cases by quinacrine staining, nucleolar organizer (Ag-AS) staining or C-banding. In three cases, the abnormal chromosome appeared to arise from two different chromosomes 15. Several possible mechanisms for the production of the abnormal chromosome are discussed. The individuals with this abnormal chromosome all showed some degree of mental retardation, but few common physical findings.     

On the mechanism of differential Giemsa staining of bromodeoxyuridine-substituted chromosomes

Summary Experiments were performed to find out whether different mechanisms are involved in FPG-(fluorescent plus Giemsa) staining for the demonstration of replication patterns and sister chromatid differentiation (SCD) after bromodeoxyuridine (BrdU)-substitution of V79 Chinese hamster chromosomes. The influence of variations of the staining procedure on the quality of both SCD and replication patterns was comparatively investigated and differences in the demonstration of these two phenomena within the same chromosome were studied using various BrdU-labeling protocols. The results show that at least graduated differences exist. For a good differentiation of replication patterns a stronger FPG-treatment is necessary than it is for SCD. Partial BrdU substitution only leads to replication patterns in the next mitosis. A further round of replication either in the presence or absence of BrdU causes a reduced staining of the complete chromatid and three-way differentiation is seen in third generation mitoses. These results support the view that alterations of chromosomal proteins during BrdU-incorporation and replication of BrdU-substituted DNA are decisive for differential staining.     

Isolabeling of the long arm of the human Y chromosome demonstrated by the FPG technique

Isolabeling segments were found in the distal region of the long arm of Y chromosomes derived from human leukocytes grown through two replication cycles in medium containing BrdU and stained by the FPG technique. Three main types of Y chromosome staining patterns were demonstrated: I-Y chromosome with typical SCD, II-Y chromosome with weakly stained distal regions of long arms (isolabeling segments), III-Y chromosome with both terminal regions displaying SCD interrupted by one isolabeled segment. The existence of different types of Y chromosome staining patterns was explained on the basis of the previously described hypothesis of unequal distribution of thymine residues between two DNA polynucleotide chains in the distal part of the long arms of human Y chromosomes.     

Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina

The recent discovery and characterization of several proteins that purify with endogenous, bound retinoid have given rise to the suggestion that these proteins, which are abundant in retina, perform a role in transport and function of vitamin A. Immunocytochemical techniques were used to localize two retinoid-binding proteins in the retina of four species. Antisera to cellular retinal-binding protein (CRALBP) and an interphotoreceptor retinoid-binding protein (IRBP) were obtained from rabbits immunized with antigens purified from bovine retina. Antibodies from each antiserum reacted with a single component in retinal homogenates and supernatants which corresponded to the molecular weight and charge of the respective antigen (non-SDS and SDS PAGE, electrophoretic transfer to nitrocellulose, immunochemical staining). Immunocytochemistry controls were antibodies from nonimmune serum and antibodies absorbed with purified antigen. Antigens were localized on frozen-sectioned bovine, rat, monkey, and human retina using immunofluorescence and the peroxidase-antiperoxidase technique. Specific staining with anti-IRBP was found in the space that surrounds photoreceptor outer segments, with heaviest labeling in a line corresponding to the retinal pigment epithelium (RPE) apical surface. Cone outer segments were positive. Staining with anti-CRALBP was found in two cell types in all species: the RPE and the Muller glial cell. Within the RPE, labeling filled the cytoplasm and was heaviest apically, with negative nuclei. Labeling of Muller cells produced Golgi- like silhouettes with intense staining of all cytoplasmic compartments. Staining of the external limiting membrane was heavy, with labeled microvilli projecting into the interphotoreceptor space. Localization of IRBP to this space bordered by three cell types (RPE, photoreceptor, and Muller) is consistent with its proposed role in transport of retinoids among cells. Localization of CRALBP in RPE corroborates previous biochemical studies; its presence in the Muller cell suggests that this glial cell may play a hitherto unsuspected role in vitamin A metabolism in retina.     

A rare variant of chromosome 1 in the house mouse: the presence of 2 supernumerary heterochromatin segments

An abnormal chromosome 1 with two extra interstitial heterochromatin segments was found in the karyotype of a house mouse from the Maritime Territory. Until recently, variants of the abnormal chromosome 1 with the only extra C-block were known in house mouse of some European populations. Sizes of the abnormal chromosome 1 in a house mouse of the Maritime Territory are increased almost by 50%, in comparison with the normal homologue. C-banding showed that extra segments were localized in the area of D and F segments of the standard karyotype in house mouse, and stained homogeneously.     

Replication of the origin region of simian virus 40 DNA in permeabilized monkey cells

Simian virus 40 (SV40) DNA replication was studied in monolayers of infected monkey CV-1 cells, permeabilized with lysolecithin, by incubation with [alpha-32P]dTTP, the other dNTPs and rNTPs and an ATP-regenerating system. Analysis of the labeled SV40 DNA by sedimentation in alkaline sucrose gradients showed that about 30% of the material synthesized by the permeable cells in the course of 60 min consisted of covalently closed circular SV40 DNA (form I), with the remainder sedimenting as relaxed circles (form II) and replicative intermediates between 18 S and 4 S. The synthesis of SV40 DNA in the permeabilized cell system required the presence of all four dNTPs and was completely inhibited by aphidicolin, consistent with the involvement of DNA polymerase alpha. A detailed analysis of the distribution of radioactivity in the DNA synthesized involved cleavage with BstNI restriction endonuclease, followed by polyacrylamide gel electrophoresis and radioautography. The extent of labeling of all restriction fragments was nearly proportional to their length, suggesting that the entire SV40 chromosome was being replicated. This was confirmed by the careful comparison of the rate of labeling of a DNA fragment which includes the replication origin, and a fragment which includes the replication terminus. Their labeling was proportional to their size, regardless of the time for which the labeling was carried out. This demonstrated that the replication of the entire SV40 chromosome occurred in a steady state and that the start and termination of replication continuously occurred throughout the labeling period. The availability of an in vitro system in which replication of SV40 DNA undergoes multiple replication cycles should be of considerable value in the analysis of the mechanism of replication of this viral genome.     

Timing of DNA replication of translocated Y chromosome sections in somatic cells of Drosophila melanogaster

The chronology of DNA replication was studied in cultured somatic cells of three stocks of Drosophila melanogaster marked by the presence of translocations between the Y chromosome and the X, 2nd and 3rd autosome, respectively. In all translocations the Y chromosome is split into two portions differently located. The different Y chromosome segments are always replicating later than euchromatin, but their timing of replication varies independently of the eu- or heterochromatic nature of the adjoining chromosome sections. This variation could be formally described as a position effect without spreading effect. It is concluded that there is evidence for the existence of factors controlling the timing of replication of the Y which are located on the chromosome itself.This is contribution No. 497 of the Euratom Biology Division. In Milano and Pavia this work was supported in part by grants of the Consiglio Nazionale delle Ricerche, Roma.     

Differential replication of male and female X-chromosomes in Drosophila

The replication patterns of larval salivary gland chromosomes of D. hydei and D. melanogaster were studied by autoradiography with tritiated thymidine injected in mid third instar larvae. The male X chromosome showed a different replication behavior in comparison to that of the female X chromosome and autosomes. It is concluded that the male X chromosome finishes its replication earlier than the female X chromosome. Moreover, the time needed for a complete replication cycle of individual identical replication units was found to be shorter in the male than in the female X chromosome. Although the whole X chromosomes behave different there were no differences observed in the sequence of the discontinuous labeling patterns of the two types of X chromosome. One autosomal replication unit was observed which showed a different replication behavior in males and females. The possible origin of the differential behavior of the two X chromosomes is discussed in terms of their difference in degree of polyteny.     

Analysis of DNA replication patterns of human fibroblast chromosomes the replication map

Summary A replication map of human fibroblast chromosomes from two diploid human female fibroblast lines, 46,XX and 46,X, del (X)(q13), was determined using the fluorescent plus Giemsa (FPG) technique. Each chromosome was found to stain homogeneously dark when thymidine was incorporated for the entire S phase of that particular cell. As the duration of exposure to thymidine progressively decreased by increasing the incubation time in bromodeoxyuridine, the staining intensity of chromosomes decreased and, concurrently, gaps in the staining began to appear. These gaps coincide with R bands and represent the earliest areas to complete DNA synthesis. As these areas widen and increase in frequency, first Q and G bands appear, and finally C bands.Homologous X chromosomes were easily differentiated by either a comparison of the bands present or their staining intensity. The replication kinetics of the structurally abnormal heterocyclic X chromosome were very similar to those of the normal heterocyclic X chromosome. The X chromosome with deletion of a portion of the long arm was consistently late in replication.     

DNA replication patterns in somatic chromosomes of Leptodactylus ocellatus (Amphibia,Anura)

The morphology and pattern of replication in the somatic chromosomes of Leptodactylus ocellatus (Amphibia, Anura) was studied by means of H³-thymidine autoradiography. A total of 300 metaphases from leukocyte cultures and 200 metaphases from spleen cell cultures were analysed.The diploid chromosome number in Leptodactylus ocellatus is 22. The pairs 1, 2, 3, 4, 7 and 8 could be easily identified on the basis of their size, centromere position, and location of secondary constrictions. In 30% of metaphases the pair 10 could be recognized on account of an end-to-end homologous association, which originated from a satellite fusion.The continuous H³-thymidine labelings carried out in the last 10, 5 and 3 hours of a culture indicated that the G₂ period was 3.5 hours. The labeled metaphases were divided in two groups. In the first one all those cells showing radioactivity along the entire length of every chromosome were included. The second group was formed by metaphases with extensive unlabeled chromosome regions. The former and the latter group were identified as representatives of the intermediate and final stages of the S period, respectively.The pattern of chromosome labeling indicates that secondary constrictions are associated with late replicating regions. However, the presence of chromosome areas, which in spite of being late in finishing duplication did not bear any kind of constriction, suggests that regions other than those associated with constrictions also may replicate late. No interchromosomal asynchrony of replication at the end of the S period was noticed. However, very often in pair 10 one chromosome had about two times as much labeling as its homologue. No sex-linked differences in chromosome morphology or in patterns of chromosome replication could be noticed.     

Symmetrical replication patterns and sex chromatin bodies formation of an idic(X)(p22.3::p22.3) chromosome

Summary The morphologic and staining characteristics of the sex chromatin bodies and the DNA replication patterns were studied in a patient with a 45,X/46,X,idic(X)(p22.3::p22.3) karyotype and in a normal woman. The analysis showed a relatively high frequency of bipartite Barr bodies as well as some variation of the distance, staining intensity, and size relationship between their halves. Regarding the DNA replication studies, in 71% of the cells the abnormal X chromosome showed a synchronous pattern, and in the remaining 29%, in which a slight asynchrony was present, an almost equal proportion of early and late functional and nonfunctional centric halves was observed. Furthermore, the atypical chromosome had a quite similar replication pattern to the late replicating X chromosome of the normal woman, suggesting that its sequence of DNA synthesis was not altered.Supported in part by grant No. 1479 from the Programa Nacional de la Salud, Conacyt (México)     

MAMMALIAN CHROMOSOMES IN VITRO : XVIII. DNA Replication Sequence in the Chinese Hamster

The complete DNA replication sequence of the entire complement of chromosomes in the Chinese hamster may be studied by using the method of continuous H³-thymidine labeling and the method of 5-fluorodeoxyuridine block with H³-thymidine pulse labeling as relief. Many chromosomes start DNA synthesis simultaneously at multiple sites, but the sex chromosomes (the Y and the long arm of the X) begin DNA replication approximately 4.5 hours later and are the last members of the complement to finish replication. Generally, chromosomes or segments of chromosomes that begin replication early complete it early, and those which begin late, complete it late. Many chromosomes bear characteristically late replicating regions. During the last hour of the S phase, the entire Y, the long arm of the X, and chromosomes 10 and 11 are heavily labeled. The short arm of chromosome 1, long arm of chromosome 2, distal portion of chromosome 6, and short arms of chromosomes 7, 8, and 9 are moderately labeled. The long arm of chromosome 1 and the short arm of chromosome 2 also have late replicating zones or bands. The centromeres of chromosomes 4 and 5, and occasionally a band on the short arm of the X are lightly labeled.     

Autonomous replication of human chromosomal DNA fragments in human cells.

We have examined whether a human chromosome has distinct segments that can replicate autonomously as extrachromosomal elements. Human 293S cells were transfected with a set of human chromosomal DNA fragments of 8-15 kilobase pairs that were cloned on an Escherichia coli plasmid vector. The transfected cells were subsequently cultured in the presence of 5-bromodeoxyuridine during two cell generations, and several plasmid clones labeled in both of the daughter DNA strands were isolated. Efficiency of replication of these clones, as determined from the ratios of heavy-heavy and one-half of heavy-light molecules to total molecules recovered from density-labeled cells, was 9.4% per cell generation on the average. Replication efficiency of control clones excluded during the selection was about 2.2% and that of the vector plasmid alone was 0.3%. A representative clone p1W1 replicated in a semiconservative manner only one round during the S phase of the cell cycle. It replicated extrachromosomally without integration into chromosome. The human segment of the clone was composed of several subsegments that promoted autonomous replication at different efficiencies. Our results suggest that certain specific nucleotide sequences are involved in autonomous replication of human segments.     

DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI

It is generally believed that DNA replication in most eukaryotes proceeds according to a precise program in which there is a defined temporal order by which each chromosomal region is duplicated. However, the regularity of this program at the level of individual chromosomes, in terms of both the relative timing and the size of the DNA domain, has not been addressed. Here, the replication of chromosome VI from synchronized budding yeast was studied at a resolution of ∼ 1 kb with DNA combing and fluorescence microscopy. Contrary to what would be expected from cells following a rigorous temporal program, no two molecules exhibited the same replication pattern. Moreover, a direct evaluation of the extent to which the replication of distant chromosomal segments was coordinated indicates that the overwhelming majority of these segments were replicated independently. Importantly, averaging the patterns of all the fibers examined recapitulates the ensemble-averaged patterns obtained from population studies of the replication of chromosome VI. Thus, rather than an absolutely defined temporal order of replication, replication timing appears to be essentially probabilistic within individual cells, exhibiting only temporal tendencies within extended domains.     

Staining of Ultraviolet Irradiated Chromosomes by the Gomori Alkaline Phosphatase Technique

When a chromosome segment is selectively irradiated with an ultraviolet micro-beam, the chromosome(s), which normally appear black by medium-dark phase-contrast microscopy, become “pale” in the irradiated region (decrease in refractive index). Previous ultraviolet absorption and Feulgen staining studies indicated that all or most of the deoxyribonucleic acid is lost in this region. After fixation, the irradiated area appears pale with most of the usual staining methods. The residual material in the paled spot, however, can be stained with the Comori alkaline phosphatase technique and is seen to be directly continuous with the nonirradiated segments. With the bright field microscope, there appears to be no decrease or increase in chromosome width. It is concluded that staining by the Gomori technique is independent of the presence or absence of deoxyribonucleic acid. Positive staining of chromosomes by the nonenzymatic peroxide method of Danielli indicated that staining was due to nonspecific precipitation of calcium phosphate rather than to enzymatic activity.     

Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures

Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are not formed. These changes in replication staining, which reflect changes in timing of replication, are different between these two tissues. However, in both tissues, the delayed onset of replication in the heterocyclic, inactive X is shortened by 5- azaC . A correlation is thus suggested between the induced temporal change to earlier DNA replication, and induced hypomethylation and gene activation. The temporal effect on chromosome replication in 5- azaC -treated cells depends on the portion of the S-period studied. Toward the beginning of S, early-replication patterns are increased in both lymphocytes and fibroblasts. Toward the end of S, late-replication patterns are increased only in lymphocytes, suggesting a differential effect of 5- azaC in: (1) early-vs. late-S, and (2) lymphocytes vs. fibroblasts. Generally, 5- azaC has its greatest effect on the inactive chromosome regions that are typically late-replicating prior to 5- azaC treatment. These observed changes in replication band staining suggest that DNA methylation may modify regional groups of genes in concert.