Mapping of the pattern of DNA replication in polytene chromosomes from Chironomus thummi using monoclonal anti‐bromodeoxyuridine antibodies

Abstract

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae.This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands. later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.Key terms: DNA replication, 5-bromodeoxyuridine, monoclonal anti-BrdUrd antibodies, polytene chromosomes Successive endoreduplication cycles during the larval growth of dipteran insects give rise to the formation in certain tissues of giant polytenic chromosomes (3,23). In the salivary gland cells of Chironomus species, 12 to 13 rounds of DNA replication during embryonic and larval development (15) result in the formation of polytene chromosomes with up to Z13 chromatids. Due to their size, the constancy of their band-interband pattern, the equation of bands with genes, and the structural changes that accompany gene activation (puffing), these chromosomes present a unique opportunity for studying in situ the structural organization of active and inactive chromatin, the distribution of chromosomal proteins and of particular types of DNA sequences, the mechanisms of gene activation, and the dynamics of DNA replication (3,251.DNA replication in polytene chromosomes of dipteran larvae has been the subject of numerous studies (1,2,4,5,8,17,20,21), which have made use of autoradiographic techniques to detect the incorporation of 3H-thymidine. Various patterns of DNA synthesis have been described and tentatively ordered on a time scale. Patterns characteristic of early S-phase display more or less continuous labeling, while late S-phase patterns are of a discontinuous type. In Drosophila, a discontinuous pattern with label confined to puffs and interbands was observed by Hagele and Kalisch (10,19) and assigned to the very beginning of the replication phase. It was concluded from these experiments that the various ...

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

“…The concept that the S-phase in eukaryotic cells seems to consist of a number of successive synchronous initiations of replication clusters is concordant with various studies on chromosomal banding-patterns in synchronized cell populations (1,2,6,13,31). Allison et al (l), using a BrdUrd labeling technique, recently described six successively appearing banding patterns in insect polytene chromosomes: E l , confined to puffs and interbands (Sl?…”

“…[1][2][3]. Figure 1 shows sections of rat colon tissues and transplants of the MCR83 rat mammary carcinoma (33) labeled in vivo.…”

“…This is particularly clear from studies on polytene chromosomes of dipteran larvae, describing various patterns of DNA synthesis, tentatively assigned to specific periods of the S-phase (1,2,28). In general, GC-rich euchromatin replicates early, whereas AT-rich heterochromatic DNA replicates late (13); these early and late DNA synthesizing sites roughly correspond to R-and G-bands on trypsinGiemsa-stained chromosomes, respectively (6).…”

DNA Replication and differentiation in rat myoblasts studied with monoclonal antibodies against 5‐bromodeoxyuridine, actin, and α2‐macroglobulin

“…these cells were exposed to BrdUrd for 21'5 h 7 it was not possible to whether the B'/d?M-cells ceased DNA synthesis prior to turning off uptake of a2M. However, 60% of those cells FIG 1. Analysis of in vitro myogenesis.…”

“…Inhibition of DNA synthesis for 20 h did not affect uptake of a2M in the final 2 h of incubation. and map patterns of DNA replication in salivary gland chromosomes (1). We have standardized the use of BrdUrd labeling and compared the sensitivity and time course of incorporation of BrdUrd into DNA with 3Hthymidine incorporation using clone 76.7 anti-BrdUrd antibody conjugated with fluorescein (17).…”