Late DNA replication pattern of human chromosomes determined by means of 3H-Deoxycytidine

Summary In the earlier investigations of the late labeling patterns of human chromosomes, ³H-thymidine was used for labeling. Since the quinacrine-positive areas of the chromosomes which were generally shown to correspond to the late replicating segments are composed of A+T-rich DNA, preferential incorporation of ³H-thymidine alone might result in the formation of this labeling pattern. When ³H-deoxycytidine was used, however, the same, but less distinct late labeling patterns were obtained as after ³H-thymidine incorporation. Obviously, the late labeling pattern is enhanced by ³H-thymidine incorporation in the quinacrine-positive areas. The dull fluorescent secondary constrictions of the chromosomes Nos. 1 and 16 were more frequently labeled when ³H-deoxycytidine was used.

---

Zusammenfassung Bei den früheren Untersuchungen über das Spät-Replikationsmuster der menschlichen Chromsomen wurde ³H-Thymidin zur Markierung verwendet. Da die quinacrinpositiven Areale der Chromosomen, die im allgemeinen mit den spätreplizierenden Segmenten zusammenfallen, durch einen erhöhten A+T-Gehalt gekennzeichnet sind, könnten diese Muster allein durch bevorzugten Einbau von ³H-Thymidin entstehen.Bei Verwendung von ³H-deoxy-Cytidin werden jedoch identische, allerdings weniger deutliche Replikationsmuster erhalten. Offenbar wird das Spätreplikationsmuster durch verstärkten Einbau von ³H-Thymidin in die quinacrinpositiven Areale verstärkt. Die nur schwach fluorescierenden sekundären Constrictionen der Chromosomen Nr. 1 und 16 waren nach Verwendung von ³H-deoxy-Cytidin häufiger markiert als nach Markierung mit ³H-Thymidin.

     

Insect sex chromosomes. IV DNA replication in the chromosomes of Gryllotalpa fossor

In Gryllotalpa the cell cycle duration in the hepatic caecae in vivo is about 12.5 h and of various phases are, G2 + P about 10 h, S about 2.5--3.5 h, and G1 appears negligible or absent. These estimates of the cell cycle are the only ones available in Gryllotalpidae. In the female Gryllotalpa, as in mammals, there is asynchronous DNA replication between the two euchromatic arms of the two X chromosomes. The other arm is constitutively heterochromatized and as expected is late replicating. Thus, a regulatory mechanism for dosage compensation by X chromosome inactivation appears to be operating in Gryllotalpa. This we believe, is the first cytogenetic demonstration of such a mechanism outside mammals.     

DNA replication and inactivation patterns in structural abnormality of sex chromosomes

Summary High resolution chromosome analysis and bromodeoxyuridine (BrdUrd) incorporation have been applied to study patterns of chromosomal replication (inactivation) in two cases of unbalanced X-autosome translocations, seven cases of X and Y chromosome rings or fragments, and five cases of dicentric isochromosomes (Xq). Our results indicate the following: (1) In (X-A) translocations, detailed replicational analysis of the translocated autosomal segment is informative. Absence of spreading effect and partial-incomplete spreading effect are the most common observations. (2) Sex chromosome derived fragments and rings can be differentiated based on their replicational features. (3) Dicentric isochromosomes (Xq) can be classified based on intercentromeric distances, replicational asynchrony, and centromere inactivation. (4) A correlation between intercentromeric distance and degree of 45,X mosaicism was observed in dicentric i(Xq) chromosomes.Evidence for spreading effect based on our results and on the review of the literature has been critically analyzed and general rules in evaluating spreading effects (SE) proposed. The cytologic detection of active regions on the late replicating X chromosome and the inactivation capacity of the juxtacentromeric region of Xp is evaluated. It is proposed that centromere suppression and underreplication are related phenomena. Finally, the analysis of informative replicational stages is emphasized and the application of their analysis in basic and clinical cytogenetics demonstrated.     

The pattern of DNA replication in the chromosomes of Puschkinia libanotica

The uptake of H³-thymidine into the chromosomes of Puschkinia libanotica has been studied in plants possessing or lacking a heterochromatic B chromosome. The pattern of H³-thymidine uptake by the A chromosomes at the end of the S phase is similar in plants of both genotypes. Regions around the centromere take up more H³-thymidine at the end of S than do more distal regions. The rate of uptake into the heterochromatin of the B chromosome increases towards the end of S, but there is no evidence that synthesis in the B chromosome carries on after the completion of DNA synthesis in the euchromatic A complement. It is proposed that at the end of the S phase more replicons in the heterochromatin of the B chromosome are engaged in DNA synthesis than in euchromatin.     

Late DNA replication in rhesus monkey (Macaca mulatta) chromosomes

A study of the pattern of late DNA replication in rhesus monkey chromosomes showed evident similarities with man. This must be a consequence of the evolutionary conservation of replication patterns in primate chromosomes, as it has been demonstrated in the great apes, in Cebus, and man. However, the pattern of late replication of the allocyclic X chromosome in lymphocytes of female rhesus monkey was identical with the fibroblast pattern in man, and with the pattern found in only 5 to 20% of human lymphocytes.     

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.     

Initiation of DNA replication in human chromosomes

DNA replication within the first 10 min of the S phase was studied using synchronized human diploid cells. It appeared that every chromosome in the human genome, including late-replicating X, had segment(s) which initiated DNA replication within the first 10 min of the S phase. The position, the shape and the size of these segments corresponded to those of Q(G)-negative bands suggesting that each of them constitutes a basic unit of initiation of DNA replication.     

Initiation of DNA replication in yeast chromosomes

A family of DNA fragments from the yeast genome has properties that suggest that chromosome replication starts at specific DNA sequences. These elements (autonomously replicating sequences: ARS) have a bipartite structure: a small (less than 20 base pairs) AT-rich region essential for function, flanked by larger regions important for maximal activity of the replicator. In an attempt to identify proteins involved in initiation of replication, yeast mutants that show an enhanced ability to replicate minichromosomes with defective ARSS have been isolated.     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Asynchrony of DNA replication in the chromosomes of Luzula

Seedlings of Luzula purpurea (2n=6) were placed in contact with H³-thymidine for 30 minutes. After removal of the isotope the roots and leaf primordia were fixed at intervals between 0 and 14 hours. The percentage of labelled mitoses follows a very close curve in roots and leaf primordia. In both tissues the value of G₂ is approximately 3 to 4 hours and of S circa 8 hours. DNA replication in the chromosomes of L. purpurea is asynchronous. The discontinuous DNA synthesis discloses that Luzula chromosomes are composed of many segments replicating independently of each other. The results support a polycentric rather than a completely diffuse kinetochore system in this species.     

DNA replication sequence of human chromosomes in blood cultures

Summary The pattern of labelling over the chromosomes, the chronology of chromosome duplication and the duration of the S and G ₂ periods in the leukocytes from 6 normal females and 5 normal males, have been studied by using a combination of pulse and continuous tregtments with thymidine-H³. According to the criteria used to analyse the results it is suggested that the S period begins 15 to 20 hours and finishes 5 to 3 hours before the cells reach the metaphase stage. The S period could be subdivided into the four phases S₁ to S₄.The first chromosomes to replicate were Nos. 1, 3, 5 and X followed by the Nos. 2, 4 and several chromosomes of groups 6–12, 13–15 and 19–20. Later the pairs 16, 17, 18 and the chromosomes of group 21–22 replicated. Chromosome Y in the male was the last to replicate, beginning its duplication when all the other chromosomes had reached the intermediate S stage.The earliest chromosomes to finish the duplication were Nos. 19, 20 and 21 followed by Nos. 16, 17, 18, 22 and the chromosomes of group 13–15. Afterward and at about the same time the replication of pairs 2, 4, 6, 8, the X and Y chromosomes in the male and one X chromosome in the female concluded. The other X chromosome in the female was the last to end its duplication appearing totally labelled until the final stage of the S period.Replication of the long and medium size chromosomes begins at localised regions, then extends over the total length of the chromosome and at the end of the S stage takes place only in small zones different from those replicating early.Asynchrony between homologous chromosomes was observed at the beginning and at the end of the S period.     

Microfluorometric analysis of DNA replication in human X chromosomes

BUdR-sensitive fluorescence of the dye 33258 Hoechst allows microfluorometric analysis of replication in human chromosomes. Comparison of the fluorescence patterns of male and female X chromosomes obtained with this technique reveals differences that may reflect regional alterations in DNA synthesis kinetics.