Polytene Chromosome Puffs Research Articles

Gene amplification in dipteran chromosomes.

The giant polytene chromosomes characteristic of many Dipteran cells allow fundamental nuclear processes occurring during development to be directly visualized. Polytene chromosomes afford a detailed picture of “large-scale” structures within developmentally regulated interphase cells — features extending over more than about 5–10 kb of chromosomal DNA. Structures, such as chromomeres, constrictions, and puffs, must be disrupted for biochemical studies and cannot currently be reconstituted. However, following Wolfgang Beermann’s recognition that polytene chromosome puffs were manifestations of developmentally regulated gene expression, much progress has been made in understanding the molecular basis of gene transcription. Genetic studies have identified small regulatory regions located near tissue-specific genes that mediate developmental control (see Rubin 1985). However, the functional significance of associated large-scale structures, such as puffs, remains poorly understood.KeywordsSalivary GlandPolytene ChromosomeFollicle CellSalivary Gland ChromosomeDifferential ReplicationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Localization of nuclear proteins related to high mobility group protein 14 (HMG 14) in polytene chromosomes.

An antibody was raised against "high mobility group" nuclear protein 14 (HMG 14) from calf thymus, known to be associated with actively transcribed chromatin. By means of indirect immunofluorescence, it was shown to react with the nuclei of mouse fibroblasts and of brain cells from Xenopus and Drosophila, but not of Xenopus erythrocytes. The antibody was used to detect immunologically related proteins in giant chromosomes of the midge, Chironomus pallidivittatus. Indirect immunofluorescence with anti-HMG 14 antibody in polytene nuclei was restricted to the active puffs. Giant puffs (Balbiani rings) exhibited especially intense fluorescence in their peripheral regions. An inducible puff site, the Balbiani ring 6 locus, showed no reaction with the antibody prior to induction. When puff formation began, the chromosome site assumed a very intense fluorescence, which disappeared again when the Balbiani ring was recondensed. - Protein extracts of salivary gland nuclei were found on immunoblots to contain one major protein fraction that reacted with the anti-HMG 14 antibody. The electrophoretic mobility of this fraction was similar to that of calf thymus HMG 17. - It is concluded that actively transcribed puffs in polytene chromosomes contain HMG 14-related protein(s) that are not present in potentially active gene loci prior to induction.

Genetic interpretation of polytene chromosomes banding pattern.

This mini-review covers new data regarding the problem of the functional organization of polytene chromosomes: The localization of RNA synthesis in the polytene chromosome puffs, diffuse bands and interbands; The relative stability of banding pattern and its functional value; The informational content of bands.

Molecular analysis of a gene in a developmentally regulated puff of Drosophila melanogaster.

An increase in the concentration of the steroid hormone ecdysone in late larval life triggers a profound change in the pattern of polytene chromosome puffs in the Drosophila melanogaster salivary gland. One of the preexisting puffs that regress as the ecdysone concentration increases is located at the 3C11-12 bands, the site of the Sgs-4 gene, which codes for the sgs-4 protein, one of the proteins in the salivary glue secretion. We have isolated cloned segments of chromosomal DNA that define a 60-kilobase region containing the 0.9-kilobase Sgs-4 gene, and we have determined its position and orientation within this region. Fine structure restriction endonuclease mapping shows that approximately 45% of this gene consists of tandemly repeated sequences of 21 base pairs that occupy most of its 5' half, indicating that most of the amino-terminal half of the sgs-4 protein consists of tandemly repeated amino acid sequences of seven residues. We also report on the amount of the Sgs-4 mRNA as a function of developmental stage and in nine different strains, four of which produce little or no sgs-4 protein. Three of the null strains produce minute amounts of the mRNA and one yields none, whereas the five sgs-4 producing strains yield abundant amounts. The mRNAs frm these strains exhibit different lengths, which correlate with different gene lengths that appear to result from different numbers of the repeated sequences in their tandem arrays.

Concanavalin A binds to puffs in polytene chromosomes

CHANGES in transcriptional activity at defined loci are often correlated with significant local structural changes in the genome(1), and in polytene chromosomes, such changes are thought to be associated with compositional or conformational changes in the protein complement at these particular bands(2,3). Thus, various studies on Balfoiani rings and specific 'puffs' in such chromosomes are useful for elucidating the role of defined chromosomal components in both chromosome structure and gene activity. Such studies require specific probes which will allow in situ localisation of a chromosomal component during the various stages of puffing. Antibodies specific to purified histone fractions(4-7), HMG proteins(8), RNA polymerase(9) and non-histone protein subfractions(10) have been used in studies on chromatin and chromosome structure. We reported previously that concanavalin A (Con A) specifically binds to three types of non-histone proteins present in chromatin purified from rat liver nuclei and suggested that derivatives of Con A might serve as specific probes to study the in situ organisation of these non-histone proteins(11). We have now reacted fluorescein-labelled Con A with polytene chromosomes isolated from different developmental stages of Chironomus thummi and visualised the location of the bound Con A by fluorescence microscopy. We observed that the fluorescent lectin, which has an affinity for glucose- and mannose-containing molecules, specifically bound to the transcriptionally active regions of chromosome IV. The extent of binding of Con A to the Balbiani rings present in regions b and c of chromosome IV is proportional to the size of the respective ring. Our results indicate that glucose- or mannose-containing molecules are present in these Balbiani rings and that the availability of these sugars to interact with Con A can be correlated with the developmental stage of a puff. We suggest that lectins can be useful cytological tools with which to study the in situ organisation of defined chromosomal components during various functional states of the genome.

Direct association of Balbiani ring 75S RNA with membranes of the endoplasmic reticulum.

PUFFS in polytene chromosomes probably represent active genes (see, for example, ref. 1). Cytogenetic investigations with large tissue-specific puffs, the Balbiani rings (BR) in Chironomus salivary gland cells, revealed a correlation between the BR and salivary export proteins2,3. Biochemical analysis of the BR showed that in Chironomus tentans RNA of a defined size is synthesised and transported to the cytoplasm4. This RNA, which sediments as 75S RNA, has properties of a messenger RNA, that it, it contains poly A5 and is located in polysomes6. If the BR-RNA directs synthesis of export proteins it would be expected to associate with the membranes of the endoplasmic reticulum (ER). Here we show that such an association exists and that it is independent of ribosomes and polysomal structures.

Transcription and chromatin structure.

Control of gene expression in eukaryotes appears to be exerted, at least in part, through selective transcription. Cytological and biochemical evidence indicates that the synthesis of RNA at selected loci is associated with chemical and structural features of the interphase chromosomes. Thus RNA synthesis occurs in extended euchromatic fibrils and not in condensed, heterochromatic masses. Similarly, cytological observations tend to correlate RNA synthesis with loops of amphibian lampbrush chromosomes and with puffs in insect polytene chromosomes. The concept that active and inactive portions of the genome may differ in chemical and physical terms has provided an impetus for the development of biochemical procedures for fractionating interphase chromosomes. The desirability of accomplishing such a goal rests upon two overriding considerations. It is probable that in many eukaryotic cells, the majority of the genome is repressed at any one time. Therefore, biochemical or physical studies of total chromatin refer primarily to the...

Puffing patterns in Drosophila melanogaster and related species.

The major objective of this review is to summarize our knowledge of the behaviour of polytene chromosome puffs in Drosophila melanogaster and some of its more closely related species. It is hoped that this review will provide a sufficient background of information for further analysis of puffing in D. melanogaster for, as will become apparent later in this review, the wealth of our knowledge of the genetics of this species, and the relative ease with which it can be subjected to “genetic engineering”, far outweigh its disadvantages as an object of cytological examination.

Induction of puffs in polytene chromosomes of in vitro cultured salivary glands of Drosophila melanogaster by ecdysone and echysone analogues.

THE hormone ecdysone1 induces specific sequences of genes in the polytene chromosomes of Dipteran larvae2,3. Gene activation is visualized by the appearance of puffs at certain sites in these polytene chromosomes4. We now describe an experimental system for the analysis of the response to ecdysone at the level of gene transcription which affords an opportunity (1) to determine the molecular specificity required for the hormone's action; (2) to analyse the cellular interactions with ecdysone leading to the genetic response; and (3) to determine the basis of sequential induction, that is, the nature of the regulatory circuits between the genes induced.

RNA and DNA puffs in polytene chromosomes of rhynchosciara: inhibition by extirpation of prothorax.

In the giant polytene chromosomes, gene amplification is made visible by formation of DNA puffs, and gene transcription is made visible by formation of RNA puffs. Ligation of the anterior portion of the larva at the end of the fourth larval instar inhibited the formation of the DNA puffs that normally develop at this stage and caused regression of RNA puffs.

Aufnahme von 22Na in die Zellkerne der Speicheldrüsen von Chironomus thummi

The in vivo and in vitro uptake of 22Na from the hemolymph into cell nuclei of larval salivary glands was measured and compared. The uptake of 22Na in vivo follows approximately a saturation curve. The respective in vitro curve has a much steeper slope during the first 8 min and this phase is followed by an interval during which the 22Na content of the nuclei decreases (8 to 16 min). After the 16th min it increases again to reach a nuclear 22Na content approximately twice as high as that of nuclei in vivo at 60 minutes. The uptake curves are discussed in relation to recent findings on the induction of puffs in polytene chromosomes by inorganic ions.

The fine structure of the kinetochore of a mammalian cell in vitro.

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 A in diameter surrounded hy a less dense zone 200–600 A wide. The dense core consists of a pair of axial fibrils 50–80 A in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oocytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.