DNA Puffs Research Articles

Chromatin structure and expression of transduced copies of [formula omitted][formula omitted] in drosophila melanogaster : [formula omitted] and [formula omitted]. Department of Biology, Washington University, St. Louis, MO, 63130, USA

Two differentiated sections (S1 and S2) of the salivary gland of Trichosia pubescens (Morgante) (Diptera : Sciaridae) have been examined by electron microscopy for fine structural alterations that occur in the cell cytoplasm during larval development. Such changes have been correlated with the puffing patterns of the polytene chromsomes. During stage 1 (end of the 3rd instar to mid 4th instar), the puffing pattern and the ultrastructure of S1 and S2 cells are rather constant. Nevertheless, marked differences are noted when the puffs and the fine structure of the 2 sections are compared. In S1, secretory material is concentrated and eliminated as membrane-bound granules, while in S2, secretory granules are not detected and the elimination of secretion seems to occur continuously. At stage 2 (end of the 4th instar), the puffing pattern undergoes considerable alterations simultaneously with the appearance of many ultrastructural modifications. In S1, the morphological aspect of the secretory granules is altered, while in S2 a decline in the secretory activity is detected. At stage 3 (older 4th-instar larvae), most of DNA puffs are active, there being no striking differences in the puffing pattern between S1 and S2. This stage is marked by the onset of gland histolysis, with the appearance of an intense autophagic activity of lysosomes in S1 and S2. As histolysis progresses during stage 4 (prepupae and early pupae) the activity of the polytene chromosomes decreases; most of the cells present a large number of autophagic vacuoles and an increasing disorganization of the cytoplasm, leading to the final lysis of the gland.

Segments of chromosomal DNA from Rhynchosciara americana that undergo additional rounds of DNA replication in the salivary gland DNA puffs have only weak ARS activity in yeast

We have constructed a library of recombinant phage containing DNA from salivary gland chromosomes of Rhynchosciara americana. We have isolated phage from this library that carry sequences homologous to cDNA clones that hybridise in situ to the DNA puffs at the polytene chromosome regions C3 and C8. This has enabled us to demonstrate a 16-fold amplification of the genomic DNA sequences at these regions during DNA-puffing. At the C8 site there is a sequence element that has characteristics of ‘scrambled’ moderately repetitive DNA. This is located within 3 kb from the gene encoding a 1.95-kb mRNA. We have assayed restriction fragments from the two DNA puffs for Ars activity in yeast. The only strong Ars activity is associated with a part of the moderately repetitive DNA element from the C8 puff which is not present at this site in all animals.

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and “DNA puffs,” which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.

Hormonal control of gene amplification and transcription in the salivary gland chromosomes of Trichosia pubescens

Trichosia pubescens larvae were ligated at different prepupal developmental stages in order to investigate whether ecdysterone acts just as a trigger or whether its presence in the hemolymph is constantly necessary for the induction of the late puffs, in particular the DNA puffs. The larvae were ligated in the middle of the body being sure that the salivary glands were divided in two halves. Some of the ligated larvae had the anterior portion of the body dissected immediately in order to determine the puff pattern at the time of ligature. These patterns were compared with those from the posterior half which were dissected at different times after ligature. The results obtained in these experiments can be summarized as follow: (1) the absence of ecdysterone source prevents the induction of late puffs even when ligature is carried out after the induction of the early ecdysterone-responsive puffs; (2) the lack of an ecdysterone source blocks the process of gene amplification when the ligature is carried out at the beginning of this process (L 3 stage); (3) the gene amplification process would not seem to be blocked but DNA puffs do not expand if the larvae are ligated some hours later (early L 4 stage); (4) both gene amplification and DNA puffs expansion occur normally when larvae are ligated at a more advanced stage (at late L 4 stage or thereafter). These results show that the presence of ecdysterone source is necessary for the induction of the late puffs up to a critical period at L 4 stage. They also show that gene amplification and transcription of amplified sequences at the DNA puffs are two independently controlled phenomena.

Effects of ecdysterone in polytene chromosomes of Trichosia pubescens

Injection of ecdysterone into mid-fourth instar larvae of Trichosia pubescens brought about drastic changes in the puffing pattern of the chromosomes of the salivary gland. The puff sequence induced by the hormone is quite similar to that which normally occurs at the end of larval life. Three classes of puffs were observed: the early induced, the late induced, and those repressed by the hormone. Among the late-induced puffs, several were DNA puffs. The protein synthesis inhibition prevents the induction of all late puffs and the process of gene amplification but does not affect the induction of the early puffs.

Different structure of polytene chromosome ofPhaseolus coccineus suspensors during early embryogenesis 3. Chromosome pair VI

Different regions of polytene chromosomes pair VI have been characterized by: 1. morphological observations, 2. incorporation of3H-thymidine and3H-uridine, 3. cytophotometry of DNA and associated proteins, 4. hybridization with satellite DNA and highly repeated DNA sequences. The collected data indicate that DNA and RNA puffs are organized by heterochromatic segments. DNA puffs show often a clustered pattern of labeling by3H-thymidine and RNA puffs are always labeled by3H-uridine. Each heterochromatic segment is characterized by a definite ratio between DNA and associated fast-green stainable proteins. Satellite DNA binds mostly to heterochromatic blocks at centromere, highly repeated DNA sequences bind, with approximately the same frequency, to centromeric heterochromatin and to the main intercalary heterochromatic band. The telomeric portions of euchromatin seem to be also enriched in highly repeated DNA sequences. The results indicate that heterochromatic chromosome segments might be sites of intense localized DNA replication. The same chromosome regions are also engaged in an active transcription process. The response to hybridization suggests that heterochromatic blocks of chromosome pair VI are heterogeneous in nucleotide sequences. The present studies also indicate that DNA and RNA puffs organized by different chromosome sites are specific of particular steps of embryo differentiation. The observed metabolic aspects of the suspensors's polytene chromosomes are discussed in relation to the synthesis of growth regulators which is known to occur in the suspensor.

The allometric relationship between nucleus and cytoplasm in the salivary glands of Bradysia hygida

The volumetric relationship between nucleus and cytoplasm of the Bradysia hygida salivary gland cells was studied during development using an allometric relation. This relationship during the fourth larval stage is defined by a simple allometric relation. Cell growth, differentiation, and maturation during the fourth larval stage takes place in two distinct steps separated by an interphase occurring between the 6th and 8th day after the third moult. This interphase marks a critical time of development and coincides with the occurrence of DNA puffs on the polytene chromosomes of the glandular cells. Although the allometric coefficient does not change over two distinct developmental steps (k = 1), our observations support the hypothesis that the interphase observed in the allometric relations is subject to the appearance of DNA puffs and is related to functional changes in the gland.

Gene amplification in Rhynchosciara salivary gland chromosomes.

Late in the fourth larval instar, several regions of the Rhynchosciara americana salivary gland chromosomes undergo "DNA puffing." We have constructed a library of cloned cDNAs synthesized from poly(A)+RNA isolated from salivary glands during the period of development when the DNA puffs are active. From this library we have studied clones representative of three genes active during this period but not active at earlier developmental periods of the gland. One of these genes is not amplified during the developmental process and encodes a 0.6-kilobase RNA molecule. The other two genes are located within the DNA-puff sites C3 and C8 and encode 1.25-kilobase and 1.95-kilobase RNA molecules, respectively. We estimate from the quantitation of transfer hybridization experiments that each of these genes undergoes 16-fold amplification during DNA puffing.

Inverse relationship between transcriptional activity and 5-methylcytosine content of DNA in polytene chromosomes of Sciara coprophila

There is an inverse relationship between RNA transcriptional activity and DNA methylation in DNA puffs, RNA puffs, bands and interbands of polytene salivary gland chromosomes of Sciara corprophila.

Alcune caratteristiche strutturali dei cromosomi politenici del sospensore embrionale di Phaseolus coccineus in due momenti dell'embriogenesi

Abstract Some features of polytene chromosomes of Phaseolus coccineus suspensor during two stages of early embryogenesis. – The distribution of DNA and RNA puffs in the whole genome of the giant cells of the Phaseolus coccineus embryo suspensor has been detected in two stages of embryo development. The collected data show that the chromosome regions showing the highest frequency of DNA puffs in both analysed stages are the following: i) band B (the fraction proximal to secondary constriction) of chromosome pair I and band E of chromosome pair V. When the two stages of development are however compared, it is seen that the % of DNA puffs in chromosome pair is at least double in suspensors dissected from the first stage of embryo development (86% in the first stage; 41% in the second stage). As to chromosome pair V band E organizes DNA puffs in 36% and in 50% of observed chromosomes in the first and second stage respectively; ii) band A of chromosome pair II, with a frequency of 52% (first stage) and 27% (se...

Amplification of genes for chorion proteins during oogenesis in Drosophila melanogaster.

The endochorion and exochorion of Drosophila eggs are synthesized by the ovarian follicle cells during a brief period of about 5 hr. In this terminal phase of egg chamber development, the structural genes for several abundant chorion proteins are expressed at high levels according to a temporally regulated program. The female-sterile mutation ocelliless maps at the site of the genes for two of these proteins, the 36,000- and 38,000-dalton chorion proteins (c36 and c38), which are closely linked. The mutation results in a cis-acting reduction in the amounts of c36 and c38 that accumulate in late-stage egg chambers. We have investigated the mechanism that underlies this decreased production by using cDNA clones complementary to these gene sequences. Unexpectedly, it was found that, in normal females, the genes for c36, c38, and at least one other chorion protein are specifically amplified more than 10-fold in the DNA of late-stage egg chambers. The extra replication involves at least some adjacent chromosomal sequences and begins prior to the onset of mRNA and protein synthesis. The additional DNA remains stable after gene expression has ceased. The behavior of these genes is thus reminiscent of the properties of the DNA puffs that have been described in several groups of Diptera. The extent of amplification of c36 and c38, but not of the 18,000-dalton chorion protein c18 (which is unlinked), was decreased in the egg chambers of flies homozygous for ocelliless, suggesting that altered gene dosage may be responsible for the decreased synthesis of chorion proteins in the mutant.

Different structure of polytene chromosomes ofPhaseolus coccineus suspensors during early embryogenesis

The pattern of DNA puffs in two pairs of polytene chromosomes (S1 and S2, both involved in nucleolar organization) is described in the suspensor ofPhaseolus coccineus during the heart-like and early cotyledon stages of embryo development. Collected data show that: 1. a structural variability indicating differential DNA replication of bands bearing ribosomal cistrons occurs in the two chromosome pairs and within the same chromosome; 2. within chromosome S1, a positive correlation is found between extra replication (amplification) of band p 13 and under-replication of band p 12; 3. within chromosome S2, bands q 21 and q 22 often evidence a simultaneous DNA amplification; 4. a negative correlation is observed between amplification of band p 13 of chromosome S1 and amplification of bands q 21 and q 22 of chromosome S2; 5. within chromosome S2, no correlation is detectable between amplification of bands p 11 and q 11 and amplification of bands q 21 and q 22.

The transcript from a DNA puff of Rhynchosciara and its migration to the cytoplasm

Electrophoretic analysis of 3H-RNA obtained from the proximal sections of Rhynchosciara salivary glands at two distinct developmental periods, one characterized by the presence and the other by the absence of the giant B-2 DNA puff, revealed that the appearance of a 14S poly(A) + RNA is correlated with the opening of this puff. That this RNA species is transcribed from this puff is indicated by the fact that it is found in RNA extracted from B-2 puffs obtained by microdissection. This is confirmed by the specific hybridization of the 14S poly(A) + RNA to the B-2 locus. Our data indicate that the polyadenylation process takes place at the chromosome level, and that the nuclear sap is not an important compartment in the transport of polyadenylated RNA from the chromosome to the cytoplasm. The kinetics of migration of the 14S species to the cytoplasm were studied; the data indicate that this process is very rapid and, in addition, that the 14S RNA is unstable.

Nucleic acid synthesis in Rhynchosciara hollaenderi polytene chromosomes: I. Dose response and temporal sequence after injection of 20-hydroxy-ecdysone

Injection of various doses of 20-hydroxyecdysone into 30-day old Rhynchosciara hollaenderi female larvae resulted in enhancement of DNA synthesis in salivary gland chromosomes with doses 0.002 μg/mg or greater and in the induction of DNA puffs in the same tissue with doses of 0.01 μg/mg or greater. Higher incorporation of label at the DNA puff sites was observed only with doses 0.02 μg/mg or greater. In all cases, a lag period of 6–10 hr was observed before the response to the injected 20-hydroxyecdysone was detected. The number of cells responding and the length of time spent in synthesis were dose dependent. Malpighian tubules were refractory to 20-hydroxyecdysone.

Translation of messages transcribed from the “DNA puffs” of Rhynchosciara

Summary “In vitro” translation of RNA extracted from the different segments of the salivary gland of R. americana indicates that the 21,000 M.W. and 29,000 M.W. peptides produced by the gland and which were previously assigned to the B-2 and C-3 “DNA puffs”, are coded by [poly(A) + ]RNA. Sucrose gradient fractionation of the [poly(A) + ]RNA extracted from the proximal segment of the gland, where the B-2 puff is most active, indicates that the 21,000 M.W. peptide is translated from the fractions which contain the RNA transcribed from this puff. The evidence obtained also indicates that the high M.W. peptides synthesized by the gland might be translated from [poly(A) − ]RNA.

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with 3H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.

Relationships between newly synthesized proteins and DNA puff patterns in salivary glands of Rhynchosciara americana

An analysis of the polypeptides synthesized in the salivary glands of late fourth instar R. americana larvae was accomplished by the use of electrophoresis followed by fluorography of in vitro labelled proteins. It was possible to detect five polypeptides which are synthesized only when the giant DNA puffs occur. One of these polypeptides was tentatively assigned to DNA puff B2 and another to the DNA puff C3. This assignment was based on correlations of polypeptide labelling, puffing patterns and RNA synthesizing capacity of the puffs in different sections of the gland during development. The possible meanings and implications of these DNA puffs are discussed.

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi.

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both trna and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. - Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puggs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the tdrosophila-based model may not be completely applicable to Rhynchosciara.

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.

Variation in Dry Mass Concentrations and Protein Contents in DNA Puffs

SUMMARYVariations on total dry mass concentrations and relative amounts of histones and non-histone proteins were estimated in the chromosome C of Bradysia hygida salivary glands by means of interferometric, cytophotometric and cytofluorometric procedures.Unpuffed region, C3, used as a control, showed a dry mass value equal to 29.3 × 10−12 g in the various larval phases studied. The regions C4, C5, and C7 displayed dry mass values increased during formation of the DNA puffs and subsequently decreased during puff regression. The region C4 exhibited the largest dry mass values, namely 41.73 times; 10−12 g (m5 phase), 184,50 × 10−12 g (m7+ phase), and 86,53 × 10−12 g (m7+ phase). In this case there was a 53.1% dry mass loss during puff regression, but proteins are not involved in such phenomenon. The data are discussed in terms of the function ascribed for the non-histone proteins concerned with gene activity and chromosome structure.