Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution

The characteristic patterns of dynamic banding (replication banding) were analysed. Extremely high resolution (850 to 1,250 bands per genome) G- and R-band patterns were obtained after 5-bromo-2-deoxyuridine (BrdUrd) incorporation either during the early or the late S-phase. We synchronized human lymphocytes with high concentrations of thymidine or BrdUrd as blocking agents, followed by low concentrations of BrdUrd or thymidine respectively as releasing agents, and obtained R- or G-band patterns respectively. The dynamic R-and G-band patterns were complementary for all chromosomes, even for the late-replicating X chromosome. There was no overlapping and every part of each chromosome was positively stained by one of the two banding procedures. The complementarity of the two patterns shows that both high thymidine and high BrdUrd concentrations blocked S-phase progression near the R-band to G-band replication transition in the middle of S-phase. Some bands of the inactive X chromosome replicate before this transition concurrently with R-band replication. The 48 different telomeric regions could be classified into 5 distinct morphotypes based upon the distribution of early and late-replicating DNA in each telomeric region. The dynamic band patterns are particularly useful for the study of the structural and physiological organization of chromosomes at high resolution and should prove invaluable for assessing the replication behavior of rearranged chromosomes.     

The discriminating fluorescence patterns of the chromosomes of Drosophila melanogaster

Mitotic and salivary gland chromosomes of D. melanogaster show striking fluorescent patterns when stained with Quinacrine. In the salivary gland chromosomes there are up to five strongly fluorescing bands located on the fourth chromosome and at the proximal end of the X chromosome.—In mitotic cells the Y chromosome shows four fluorescent segments and other fluorescent regions are found proximally on the third pair and on the X chromosome. It is, therefore, possible to distinguish male and female interphase cells by their patterns of fluorescence.—A comparison between the position of heterochromatic, late replicating and fluorescing segments in the mitotic chromosomes, shows differences which demonstrate, for the first time, the chemical, morphological and genetical diversity of these three types of segments.     

Studies on early replication patterns in the marsupial Sminthopsis crassicaudata: no evidence for XY replication homologies

We present here the first detailed replication banding study of a marsupial species using the BrdU-replication technique. A comparison of the structural and replication bands of the chromosomes of Sminthopsis crassicaudata clearly demonstrates that the replication behavior is the same as the described for the chromosomes of eutherians. The early replicating segments correspond to R-bands, whereas the late-replicating regions tend to be situated within Q- and C-bands. Use of this technique clearly reveals an early and late replicating X chromosome. The very small Y chromosome can be subdivided into two replication segments, but no replication homologies can be demonstrated between the X and Y chromosomes of S. crassicaudata.     

Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy.

The genetically inactive, late-replicating human female X chromosome can be effectively distinguished from its more active, earlier-replicating homologue, when cells are grown according to the appropriate BrdU-33258 Hoechst protocol. Results obtained from a fluorescence analysis of DNA replication in X chromosomes are consistent with those from previous autoradiographic studies, but reflect additional sensitivity and resolution offered by the BrdU-Hoechst methodology. Both qualitative and quantitative differences in 33258 Hoechst fluorescence intensity, reflecting alterations in replication kinetics, can be detected between the two X chromosomes in female cells. The pattern of replication in the single X chromosome in male cells is indistinguishable from that of the early female X. Intercellular fluctuations in the distribution of regions replicating early or late in S phase, particularly with reference to the late female X, can be localized to structural bands, suggesting multifocal control of DNA synthesis in X chromosomes.     

Late replicating bands of human chromosomes demonstrated by fluorochrome and Giemsa staining.

The addition of thymidine (TdR) to cells growing in a medium containing 5-bromodeoxyuridine (BUdR) at the end of the first replication cycle results in the incorporation of TdR into the late replicating DNA regions. These sites can be visualized by staining the metaphase chromosomes with the fluorescent dye "33258 Hoechst" or a "33258 Hoechst" Giemsa procedure. A sequence of late replication patterns has been established in metaphase chromosomes of cultured human peripheral lymphocytes. The patterns are in agreement with those obtained by the standard autoradiographic procedures, but are more accurate. As is known from autoradiography, late replicating bands are in the position of G or Q bands. The "33258 Hoechst" Giemsa staining procedure of chromosomes which have replicated in the presence of BUdR first and in TdR for the last 2 hrs of the S phase is preferable to the currently used Giemsa banding techniques: the method yields very well banded metaphases in all preparations examined, as the chromosome structure is not disrupted by the pretreatment. The bands are very distinct, even in the "difficult" chromosomes (e.g. No. 4, 5, 8 and X). In female cells the late replicating X chromosome can be identified by its size and staining pattern. In addition to the replication asynchrony, the sequence of replication within both X chromosomes in female cells is not absolutely identical. The phenomenon of a phase difference in replication between the homologues is not a peculiarity of the X chromosome, but can be found in all autosomes as well as in homologous positions on the chromatids of individual chromosomes.     

Initial phases of DNA synthesis in Drosophila melanogaster

Replication patterns of the X chromosomes and autosomes in D. melanogaster male and female larvae during the discontinuously labeled initial and end phases of DNA synthesis were compared. In female larvae X and autosomes behaved correspondingly during all the replication stages. In males, however, the X chromosome shows a differential replication behavior from that of the autosomes already during the discontinuously labeled initial stage.—In those nuclei of both sexes, in which the autosomes correspond in their initial replication patterns, significantly more labeled regions are to be found over the male X than over the female X. The complementary behavior during the end phases (Berendes, 1966), i.e. the reverse of that above, leads to an earlier completion of the replication cycle in most of the labeled regions of the male X chromosome. The differential replication revealed in the autoradiograms is interpreted as a consequence of the polytene structure in giant chromosomes.     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

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.     

Prometaphase banding of human chromosomes with basic fuchsin

Summary A technique is described for the production of detailed and richly contrasting G-band patterns in human prometaphase chromosomes with the aid of the triphenylmethane dye basic fuchsin. The usefulness of this method is illustrated by its application for the precise analysis of two chromosome 11 rearrangements. It is also demonstrated that high-resolution banding with basic fuchsin can reveal bands not present in the international standard idiogram of human prophase chromosomes (ISCN 1981). The technique described can also be used for easy recognition of the late replicating X chromosome, which stains darker than its early replicating homologue. A preliminary analysis of the late replicating X chromosomes in a 49,XXXXY individual suggests that the three supernumerary X chromosomes do not necessarily replicate synchronously.     

Chromosome banding in Amphibia

High-resolution replication banding patterns were induced in prometaphase and prophase chromosomes of Xenopus laevis by treating kidney cell lines with 5-bromodeoxyuridine (BrdU) and deoxythymidine (dT) in succession. Up to 650 early and late replicating bands per haploid karyotype were demonstrated in the very long prophase chromosomes. This permits an exact identification of all chromosome pairs of X. laevis. Late replicating heterochromatin was located by analysing the time sequence of replication throughout the second half of S-phase. Neither heteromorphic sex chromosomes nor sex chromosome-specific replication bands were demonstrated in the heterogametic ZW females of X. laevis. A detailed examination of the BrdU/dT-labelled prometaphases and prophases revealed that the X. laevis chromosomes can be arranged in groups of four (quartets), most of which show conspicuous similarities in length, centromere position, and replication pattern. This is interpreted as further evidence for an ancient allotetraploid origin of X. laevis.by H.C. MacgregorThis paper is dedicated to Prof. Wolfgang Engel on the occasion of his 50th birthday     

Strukturverändernde Wirkung von FUdR auf polytäne Chromosomen und Beziehungen zwischen Replikationsdauer und Bruchhäufigkeit von Querscheiben

Larvae of Chironomus th. thummi at the age of 10 hours after hatching were treated for 20 hours with 10^–4 M FUdR. The salivary gland chromosomes were studied at fourth instar. FUdR induces chromosomal constrictions and partial breakage of various diameters ranging from 1/2 to less than 1/16 of the total cross section of the polytene chromosomes. Breaks were predominantly found in chromosome regions containing bands of high DNA content. By H³-thymidine-autoradiography it is demonstrated that bands which are frequently broken are late replicating. This is shown by histograms correlating the distribution of breaks over the chromosomes with labeling patterns obtained in late S.—As bands with a great amount of DNA do not only replicate late but also spend the longest time in DNA synthesis, it is assumed that they also represent the largest replicons. It is discussed if this is the reason why FUdR induces breaks preferentialy in bands of high DNA content.     

Karyotype,DNA replication and origin of sex chromosomes in Anopheles atroparvus

Anopheles atroparvus has two pairs of autosomes similar in length and morphology and two sex chromosomes with equal, heterochromatic, late replicating long arms with homologous C-, G-, and Q-bands. The short arm of the Y is shorter than that of the X and both are euchromatic. The mean number of chiasmata per cell in the male is 3.2. During mitosis there is a high grade of somatic pairing but X and Y, which form a heteropycnotic mass in the interphase nucleus, have a differential behaviour. The chronology of DNA replication was studied in spermatogonia and brain cells by autoradiography. It is hypothesized that the present sex chromosomes of A. atroparvus evolved by accumulation of sex determining factors and gene deterioration resulting in heterochromatinization of the long arms, followed by structural rearrangements.—The homology of the two sex chromosomes requires limited dosage compensation which is achieved either as in Drosophila by modifier genes or by accumulation on the short arm of the X, only of female determining factors which do not require dosage compensation.     

Replication pattern of the X and Y chromosomes in partially synchronized human lymphocyte cultures

The replication pattern of the X and Y chromosomes at the beginning of the synthetic phase was studied in human lymphocyte cultures partially synchronized by the addition of 5-fluoro-2-deoxyuridine (FUdR). The data were evaluated statistically by an analysis of the distribution of silver grain counts over the X and Y chromosomes. —In cells from normal females, one of the X chromosomes began replication later than any other chromosomes of the complement. The short arm of the late replicating X chromosome started replication earlier than the long arm. The telomeric region of the short arm was a preferential site of DNA synthesis at the beginning of replication. —In partially synchronized lymphocyte cultures from a patient with the XXY syndrome, the Y chromosome started replication together with the late replicating X chromosome. The Y chromosome most frequently replicated synchronously with the short arm of the X. The centromeric region of the Y chromosome initiated synthesis before the telomeric region and appeared to replicate synchronously with the telomeric region of the short arm of the X. These findings are discussed with reference to the pairing of the X and Y chromosomes at meiosis.Supported in part by the National Institute of Health Research Grant HD-01979 and National Foundation Birth Defects Research Grant CRCS-40. Dr. Knight was a predoctoral fellow under National Institute of Health Training Program HD-00049-09.     

Electron microscopical analysis of Drosophila polytene chromosomes

Replication studies on prophasic human Y chromosomes reveal 4 early replicating segments in the euchromatic portion. The distal segment of Yp replicates first. After replication of the euchromatic part is almost finished 3 to 5 segments start replication in the heterochromatic portion of Yq. These segments exhibit considerable intraindividual variation with respect to the origin of onset of replication. While the location of these bands — once they are differentiated — is fixed within one individual, the number of these bands varies interindividually.Dedicated to Professor Dr. Ulrich Wolf on the occasion of his 50the birthday     

Relation between the SCE points and the DNA replication bands

A method for obtaining a combination of differential sister chromatid staining and DNA replication banding is described. Using this method the SCE points can be precisely localized to particular bands of individual chromosomes. It was shown, that SCEs occur not only in the regions of early DNA replication bands (=euchromatic segments=negative G-bands), but also in the regions of late DNA replication bands (=heterochromatic segments=positive G-bands). SCEs occurred about three times more frequently in the euchromatic segments than in the heterochromatic segments. Furthermore, more SCEs were observed in the early replicating X-chromosome than in the late replicating X-chromosome.     

Replication kinetics of X chromosomes in fibroblasts and lymphocytes

Summary The kinetics of replication for early and late replicating X chromosomes in karyotypically normal fibroblasts and lymphocytes was studied using terminal bromodeoxyuridine (BrdU) treatment followed by Hoechst/light/Giemsa staining. Although the order of band appearance differs between the two tissues, the programme (order and interval between band appearances) for early replicating bands (dark R-bands) is identical in the two homologues. This is probably also the case for later replicating bands (dark G-bands) though the criteria for derermining mean band appearance times are less reliable for these bands when terminal BrdU treatment is used. This means that the late X has a delayed start but thereafter proceeds at the same pace as its early counterpart.     

DNase I sensitivity in facultative and constitutive heterochromatin

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 inMicrotus 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. InMicrotus 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.     

Distribution of heterochromatin in a reconstructed karyotype of Vicia faba as identified by banding- and DNA-late replication patterns

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 marker and 18 additional bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.     

BrdU-33258 Hoechst analysis of DNA replication in human lymphocytes with supernumerary or structurally abnormal X chromosomes

BrdU-33258 Hoechst techniques have been used to characterize DNA replication patterns in lymphocytes from human females with supernumerary or structurally abnormal X chromosomes. Fluorescence analysis permits identification of late replicating X chromosomes in a very high proportion of cells and affords a high resolution method for determining the interchange points of X-X and X-autosome translocations. Asynchrony among terminal replication patterns of multiple late replicating X chromosomes within an individual cell can occasionally be demonstrated. The arms of isochromosomes usually exhibit symmetrical fluorescence patterns, with replication terminating in bands Xq21 and Xq23 (predominant pattern) or in bands Xq25 and Xq27 (alternative pattern) in both arms. In the vast majority of lymphocytes containing a balanced X-13 or X-19 translocation, the normal X is late replicating. However, DNA synthesis in the translocation products occasionally appears somewhat delayed relative to that expected for an early replicating X, consistent with possible position effects on replication kinetics.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.