Relationship of developmental stage to initiation of replication in polytene nuclei

The various types of autoradiographic patterns occurring in salivary gland nuclei ofD. melanogaster following a short pulse of tritiated thymidine have been described and the probable order of their sequence within a polytenic replication cycle assigned. On the basis of that assignment, the distribution of those patterns in the nuclei of salivary glands of 160 staged larvae and prepupae has been assessed and the following interpretations made: Throughout the third larval instar and prepupal period there is a continuing decrease in frequency of labeled nuclei. Within the larval period that decrease is reflected in a proportional decrease in label patterns characteristic of both initiation and propagation phases of the replication cycle. At the beginning of the prepupal period there is a rise in the proportion of labeled cells displaying initiation type patterns followed by an abrupt cessation of such patterns. It is proposed that termination of initiation of polytenic replication is amongst the physiologic manifestations of the induction of pupation.     

Control of polytenic replication in dipteran larvae. II. Effect of growth temperature

A comparison has been made, by Feulgen photometry, of the polytene nuclei of the salivary glands of a wild-type strain of Drosophila melanogaster grown at 17°C and 25°C, respectively. Despite the fact that the time period of the larval stage was more than doubled at the lower temperature, the DNA values were the same in magnitude and similar in distribution of replication classes at each of the stages studied. The data have been interpreted as indicating that, so long as the larval state prevails, initiation of polytenic replication occurs upon completion of the previous cycle.     

DNA synthesis in polytenic chromosomes of Anopheles atroparvus

DNA synthesis in polytenic chromosomes of Anopheles atroparvus has been studied during the third and fourth larval stages. The beginning of the replication cycle coincides with the moult. With increasing larval age the frequency of nuclei in synthesis decreases continuously to reach 1 per cent in the fourth instar at 48 hours of age. The beginning of synthesis is characterized by asynchrony both among larvae and among nuclei within the same larva. At the beginning of replication the polytenic chromosomes have a discontinuous labelling pattern. The same pattern was observed at the end of reduplication. Whole nuclei and single chromosomal regions seem to reach their maximum degree of polyteny during the third larval stage.     

Cytophotometric DNA determinations and autoradiographic studies in salivary gland nuclei from larvae with different karyotypes in Drosophila melanogaster

Cytophotometric DNA determinations in Feulgen stained mitotic diploid chromosome sets of neuroblasts from larvae of Drosophila melanogaster stocks, which possess different karyotypes, show significant differences between the 4C values, caused by an additional or deficient X- and Y-chromosome depending on the karyotype. The ranges of polytenic DNA size classes are theoretically expected to be doublings of the corresponding 4C mean value of each karyotype. The extinction integral data of nuclei with completely duplicated 4C quantities exclusively fall into the range of the expected size classes. Not all data falling into the range of a size class necessarily originate from duplicated nuclei, because the limits of the DNA size classes cannot be determined by measurements, but must be estimated from the confidence limits of the corresponding 4C mean value. The validity of the mitotic 4C values of the karyotypes X/X and X/Y is tested using data from non-labeled interphase nuclei, where extinction integral data accumulate in two groups. The larger values (= G₂-nuclei) confirm the 4C values of mitotic chromosome sets, and the lower values (= G₁-nuclei) are just half of these. Extinction integrals from individual, ³H-thymidine non-incorporating polytene salivary gland nuclei accumulate in distinct, non-overlapping groups which are always complete doublings of the preceding smaller group. In each karyotype, the most frequent data of each group are in accord with the 4C doublings. The data from labeled nuclei alternate with those from unlabeled nuclei. The measured DNA values of individual polytene nuclei that did not incorporate any ³H-thymidine, demonstrate that all chromosomal DNA replicates completely during polytenization of the chromosomes in the larval salivary gland nuclei of Drosophila melanogaster. Specifically, this would mean that the heterochromatic Y-chromosome replicates as well as the partially heterochromatic X-chromosome along with the autosomes. There is no indication of underreplicating heterochromatin.     

DNA replication in polytene chromosomes: Similarity of termination patterns in somatic and germ-line derived polytene chromosomes of Anopheles stephensi liston (Diptera: Culicidae)

DNA replication is initiated in the ovarian follicles of the anautogenous Culicid Anopheles stephensi in response to a blood meal. The chronology of nurse cell polytene chromosome³H-thymidine labelling patterns has been defined and the terminal labelling patterns of the nurse cell and larval salivary gland polytene X-chromosomes have been compared and found to be essentially similar.     

localisation of non-replicating heterochromatin in polytene cells of Drosophila nasuta by fluorescence microscopy

A simple fluorescence technique is decribed to localise in situ the non-replicating alpha heterochromatin in the chromocentre region of Drosophila nasuta polytene nuclei. After incorporating 5-bromodeoxyuridine in larval salivary gland cells for one or two cycles of replication, the polytene nuclei are examined for Hoechst 33258 flourescence at pH 7.O. The nonreplicating alpha heterochromatin remains brightly fluorescing as it does not incorporate any 5-bromodeoxyuridine while the rest of the replicating chromatin shows dull fluorescence due to the quenching of Hoechst 33258 fluorescence by the bromodeoxyuridine substituted DNA.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

The properties of hybrids formed between the P-group plasmid RP1 and various plasmids from Pseudomonas aeruginosa

Summary Ribosomal DNA content has been determined in several adult and larval tissues of Drosophila melanogaster. Underreplication of rRNA genes was observed in polytenic salivary glands of larvae. On the contrary, polytenic/polyploid ovaries showed no decrease in rDNA. It is concluded that polyteny is not necessarily associated with underreplication of rDNA. No other tissue examined displayed any change in rDNa redundancy. Third-instar-larvae showed a decrease in rDNA amount which might be partly accounted for by underreplication of rDNA in salivary glands. No such decrease was seen in pupae. Bobbed genotypes were essentially similar to wild type in all tissues except salivary glands. In this case, it was found that the extent of underreplication is less in bobbed as compared to wild genotypes.Ribosomal DNA activity was examined in various tissues of Drosophila melanogaster. The rates of rRNA synthesis vary greatly between various tissues. It is concluded that a control at the level of gene activity operates as differences in the amount of precursor rRNA synthesized can be observed both in flies of varying rDNA contents as well as in various tissues of the same genotype.     

The effect of chromosomal polytenization on the rates of RNA synthesis and decay in the salivary glands of the blowfly, Calliphora erythrocephala

The rates of total RNA synthesis and accumulation have been measured in the polytenic salivary gland cells of the blowfly, Calliphora erythrocephala, by three methods: (1) injecting larvae with [2-³H]adenosine and determining its flow into the cellular ATP pool and RNA, (2) measuring the increase in glandular RNA optically, and (3) measuring the rate of flow of ATP out of the cellular pool. The size of the steady-state pool of rapidly turning over RNA and its half-life, were calculated from these kinetic data and, also, by an independent measurement of the steady-state content of nuclear RNA. These parameters were compared at a number of developmental stages which differed in degree of chromosomal polytenization. The results indicate that these polytenic cells synthesize RNA at a rate approximately 10³ times those of other diploid eukaryotic cells. This rate is independent of the increase in chromosomal polyteny that accompanies larval development. Approximately 67% of the newly synthesized salivary gland RNA is an unstable component with an average first-order half-life of 20–25 min. The remainder is a long-lived species with an estimated average first-order half-life of about 30 hr.     

B-Chromosomen beiChironomus

In the population “Herrenmühle” ofChironomus plumosus 11% of the individuals contain one supernumerary chromosome. This B-chromosome is present both in germ-line and somatic cells. — InChironomus melanotus 6% of the larvae of the population “Falkau” carry supernumerary chromosomes. These B-chromosomes cannot be found in all nuclei of testis and soma, their number varies between cells within the individual. In both species the B-chromosomes represent centromeric fragments of chromosome IV as can be shown by their structure and pairing behaviour. — The polytene B-chromosome ofCh. plumosus exhibits a banding pattern in the salivary gland nuclei. Furthermore it is able to form an additional nucleolus in the nuclei of the malpighian tubules. InCh. melanotus band structures can be seen only in the B-chromosome of malpighian tubules. The larvae ofCh. melanotus, carrying B-chromosomes, show heterochromatic bodies in the salivary gland nuclei, varying in number and size in the nuclei of the same gland. These bodies are interpreted to be polytenic B-chromosomes divided into subunits.

---

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Anomalous development and differential DNA replication in the X-Chromosome of a Drosophila hybrid

Male hybrids of the cross D. azteca x D. athabasca are larger (hybrid giant males) than their parents, whereas hybrid females are of the same size as the parental species. Microspectrophotometric measurements have shown that the larval polytene salivary gland chromosomes of hybrid giant males undergo one more endoreplication than those of their sisters or parents. Replication patterns of the larval salivary gland chromosomes were compared after pulse labeling with ³H-thymidine and autoradiography. In females of either species as well as of hybrids X-chromosomes and autosomes are equally labeled, i.e. all chromosome arms replicate synchronously. In males, however, often fewer sites are labeled on the X-chromosome than on the autosomes. In addition, in a significant number of nuclei from D. athabasca males and also from hybrid giant males the converse can also be observed: i.e. more sites are labeled on the X-chromosome than on the autosomes. The modified labeling patterns are interpreted as an indication of a time-shift in the replication of hemizygous X-chromosomes in males, in relation to the autosomes.     

Characteristics of the endocrine system in Drosophila melanogaster 1 (2)gl mutants, differing in time of death]

A comparative study of the state of the endocrine system and one of the larval organs, the salivary gland, has been carried out in larvae homozygous for the 1(2)gl gene. They differ in time of death (death at the third larval instar--larval allele, and at the prepupal state--prepupal allele). It is shown that homozygotes for the larval and prepupal allele have underdeveloped prothoracal glands. Corpora allata in homozygotes for the larval allele does not differ from the norm. Corpora allata in homozygotes for the prepupal allele is decreased proportionally to the decrease of prothoracal glands. A decrease of gland size is due to a decrease of the volume of cell but not to their number; this decrease is accompanied by the decrease of their relative DNA content. Salivary glands in homozygotes are reduced and comprise 80% of the normal size in homozygotes for the prepupal allele and 50% for homozygotes for the larval allele. Polyteny level in the salivary gland nuclei is much decreased as compared with the normal level. DNA level is more reduced in larvae homozygous for the larval allele.     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

Polysaccharides associated with chromosomes and their behavior in the cell cycle

Summary The interphase nuclei, especially of the latest stages (G₂ or early prophase), in the mouse and rat livers were stained blue in the histochemical demonstration of acidic polysaccharide according to the method of Mowry, while the mitotic chromosomes (meta-, ana- and telophase) in the livers, sea urchin embryos as well as root tips of broad beans were stained red, suggesting the presence of neutral polysaccharide. The giant polytenic interphase chromosome of the salivary gland of Chironomus larvae was stained blue in the puffing and nucleolar regions while stained red in the condensed part of the chromosome. ³H-Glucosamine as well as ³H-glucose incorporations into the regenerating rat liver nuclei reached a peak at 30 h after partial hepatectomy when the highest mitosis is seen. These results suggest that the nuclear acid mucopolysaccharide present in the swollen chromosomes may be converted to or replaced with the neutral polysaccharide in the condensed chromosomes such as mitotic chromosomes or polytenic giant interphase chromosomes.     

Satellite DNA of Anopheles stephensi liston (Diptera: Culicidae)

Four satellite DNAs in the Anopheles stephensi genome have been defined on the basis of their banding properties in Hoechst 33258-CsCl density gradients. Two of these satellites, satellites I and II, are visible on neutral CsCl density gradients as a light density peak forming approximately 15% of total cellular DNA. Hoechst-CsCl density gradient profiles of DNA extracted from polytene tissues indicates that these satellites are underreplicated in larval salivary gland cells and adult female Malpighian tubules and possibly also in ovarian nurse cells. The chromosomal location of satellite I on mitotic and polytene chromosomes has been determined by in situ hybridisation. Sequences complementary to satellite I are present in approximately equal amounts on a heterochromatic arm of the X and Y chromosomes and are also present, in smaller amounts, at the centromere of chromosome 3. A quantitative analysis of the in situ hybridisation experiments indicates that sequences complementary to satellite I at these two sites differ in their replicative behaviour during polytenisation: heterosomal satellite I sequences are under-replicated relative to chromosome 3 sequences in polytene larval salivary gland and ovarian nurse cell nuclei.     

Comparative study of the function of polytene chromosomes in laboratory stocks of Drosophila melanogaster and the l(3)tl mutant (lethal tumorous larvae)

A study of the puffing pattern of the salivary gland autosomes of D. melanogaster was performed through the last 24 hours of larval development and 0-hour prepupae. Since both prominent and small puffs were taken into account, the total puff number amounted to 275. Of these, 116 are almost constant in size during the 24 hours observation period, 106 increase in size or appear before pupation. 37 puffs are active in 96 hour larvae and disappear or decrease sharply in size by 115–118 hours. 12 biphasic puffs have been found with higher activity in 96 hour larvae and 0-hour prepupae and lower activity by 115–118 hours. Three extremely irregular puffs have been detected in chromosome 4. The data obtained evidence that a larger number of D. melanogaster polytene chromosome loci are active during larval development than it has been thought earlier. It has also been shown that only 38% of autosomal puffs change before the beginning of metamorphosis. The functional significance of small puffs and strain specificity of puffs are discussed.     

Veränderungen im Puffmuster und das Wachstum der Riesenchromosomen in Speicheldrüsen von Drosophila melanogaster aus spätlarvalen und embryonalen Spendern nach Kultur in vivo

Salivary glands from late third instar larvae of Drosophila melanogaster were transplanted into the abdomens of adult female and male flies and were kept in this medium from 6 to 120 h. Changes in the puffing pattern of chromosome arm III L were studied after the culture in vivo. Two noticeable puffs are induced. They are located in 68 B and 78 E. Neither of these loci show activity during normal development. — Front halves of embryos (6 to 9 h of age) were also transferred into adults. After 5 to 13 days in vivo they are able to develop and differentiate larval structures. Salivary glands, imaginal discs, fat body, Malpighian tubules and muscle fibers could be identified. Even 4 h old embryos can form polytene salivary gland chromosomes after a 13 day culture. These chromosomes can reach sizes comparable with the maximal size in normal development. In some nuclei an extensive growth leads to “supergiant” chromosomes. The puffs in 68B and 78E are formed in the polytenic chromosomes from embryonic implants as in cultured larval salivary gland chromosomes.     

Quantitative analysis of the DNA content in salivary gland chromosomes of Chrironomus thummi in the larval and prepupal stages]

Relative DNA content during the polytenization of the salivary gland nuclei of Chironomus thummi was measured by cytophotometric and cytofluorometric methods. To estimate polyteny degree, DNA content was calculated in hemocyte and spermatocyte nuclei. Chromosome polytenization is associated with the 10th-12th replication rounds. There are 4-5 replication rounds in the 1st instar, 2-3 rounds in the 2nd instar, the 3rd and the 4th instars have 1-2 rounds each. From early postembryonic development, larvae already have salivary gland nuclei representing two polyteny classes (2-2-2-4c), this heterogeneity being retained in all instars. Approximate DNA content is 0.51-0.58 picogram per a diploid set.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor ^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann