Nuclear protein matrix in giant nuclei from salivary glands of <Emphasis Type="Italic">Chironomus plumosus</Emphasis>

Polythene chromosomes from salivary glands of Chironomus plumosus were treated in situ in order to reveal residual nuclear protein matrix (NPM). It was shown that after the removal of H1-histones by 0.6 M NaCl the general morphology of chromosomes is preserved, revealing distinct banding pattern. Further treatment of chromosomes with 2 M NaCl and DNase completely disorganized the structure of chromosome bodies and patterns of banding. Preliminary treatment of salivary glands with 2 mM CuCl₂ resulted in stabilization of the structure of polythene chromosome in every stage of histone and DNA extractions. Stabilized chromosomes maintained their morphology and banding patterns observed by phase contrast or after the staining with Brilliant blue. Thus, after the removal of histones and DNA, stabilized chromosomes retain their morphological features, which depend on the presence of NMP. In stabilized polythene chromosomes, in spite of the absence of histones and DNA, topoisomerase IIα retains its localization, typical for untreated chromosomes.     

DNS-Replikationsmuster der Speicheldrüsen-Chromosomen von Chironomiden

The pattern of DNA-synthesis of the salivary gland chromosomes of Chironomus thummi thummi, Ch. th. piger, Ch. annularius, Ch. plumosus and Ch. melanotus was studied using H³-thymidine-autoradiography. Contrary to the previous conception the bands of the salivary gland chromosomes of Chironomus do not begin replication simultaneously. H³-thymidine incorporation in bands of high DNA content begins later than in bands with a lesser amount of DNA. This difference in time is very small in bands outside the kinetochore regions and not comparable to the asynchrony in replication of typical heterochromatin in the salivary gland chromosomes of Chironomus melanotus. Differences in the amount of DNA in homologous bands do not affect the onset of replication. — Bands of high DNA content are replicating during a longer time than those having less DNA. However, certain chromosome regions behave differently. In these regions bands of very low DNA content are synthesizing DNA during the whole replication cycle. Since no excessive increase of DNA could be observed in these regions it is supposed that in addition to the duplication of structural DNA an extra DNA is synthesized which disappears immediately from the chromosome. — At the end of the replication cycle in the salivary gland nuclei of the hybrid Chironomus th. thummi X Ch. th. piger a labeling pattern is found in the chromosomes of Ch. th. thummi which differs from that in the parental subspecies Ch. th. thummi.     

Satellite DNA associated with heterochromatin in Rhynchosciara

The DNA of Rhynchosciara hollaenderi was examined using isopycnic centrifugation in neutral CsCl. Two low density minor bands (collectively termed satellite DNA) were detected in addition to the main band DNA. Main band DNA has a buoyant density of 1.695 g/cm³. The larger of the two minor bands has a buoyant density of 1.680 g/cm³ while the smaller of the two minor bands has a buoyant density of about 1.675 g/cm³. Thermal denaturation studies have confirmed the presence of the two minor classes of DNA.—The satellite and main band DNAs were isolated in relatively pure form and were transcribed in vitro using DNA-dependent RNA polymerase from Escherichia coli. Annealing of the two complementary RNAs (cRNAs) with main band and satellite DNA was examined using filter hybridization techniques.—The chromosomal distribution of the satellite DNA was determined by in situ molecular hybridization of satellite-cRNA with Rhynchosciara salivary gland chromosomes. Satellite-cRNA hybridized with the centromeric heterochromatin of each of the four chromosomes (A, B, C, and X) and with certain densely staining bands in the telomere regions of the A and C chromosomes. Main band-cRNA annealed with many loci scattered throughout the chromosomes including areas containing satellite DNA.     

Measurements of the total solid material in the bands and interbands of the polytene chromosomes in living cells of chironomus

Summary The refractive indices of a number of bands and interbands of the polytene chromosomes in the living salivary gland cells of two species of Chironomus were measured with an interference microscope, and values were obtained for the total solid material present in each region. To make these measurements it was necessary to compress the cells until virtually no cytoplasm lay above or below the nucleus, but this produced no deleterious effects on the cells as a whole.The results indicated that there is not less than 25% of solid material in the band regions of these chromosomes, and not more than 15% of solids in the interbands. The nuclear sap of these cells was found to contain about 12% of solid material.Similar refractive index measurements were made on the bands and interbands of isolated chromosomes mounted in saline and in a 12% isotonic saline/protein solution of the same refractive index as the nuclear sap. In both these media the bands were again found to contain at least 10% more material than the interbands, but their refractive indices were lower in the saline than they were in the intact cells and markedly higher in the saline/protein. It is therefore concluded that the isolated chromosomes are, to some extent, permeable, and can permit the entry of protein molecules from the mounting medium and the egress of some or their own material.     

Strukturverändernde Wirkung von FUdR auf polytäne Chromosomen und Beziehungen zwischen Replikationsdauer und Bruchhäufigkeit von Querscheiben

Larvae of Chironomus th. thummi at the age of 10 hours after hatching were treated for 20 hours with 10^–4 M FUdR. The salivary gland chromosomes were studied at fourth instar. FUdR induces chromosomal constrictions and partial breakage of various diameters ranging from 1/2 to less than 1/16 of the total cross section of the polytene chromosomes. Breaks were predominantly found in chromosome regions containing bands of high DNA content. By H³-thymidine-autoradiography it is demonstrated that bands which are frequently broken are late replicating. This is shown by histograms correlating the distribution of breaks over the chromosomes with labeling patterns obtained in late S.—As bands with a great amount of DNA do not only replicate late but also spend the longest time in DNA synthesis, it is assumed that they also represent the largest replicons. It is discussed if this is the reason why FUdR induces breaks preferentialy in bands of high DNA content.     

Nuclear protein matrix of giant nuclei of polythene chromosome organization of midge <Emphasis Type="Italic">Chironomus plumosus</Emphasis> determinates

Giant nuclei from salivary glands of the midge Chironomus plumosus were treated in situ with 2M NaCl detergent and nucleases to reveal residual nuclear matrix proteins (NMP). It was shown that, after the prestabilization of nonhistone proteins with 2 mM CuCl₂, the polythene chromosome body preserved its morphologic integrity and banding pattern, even after the extraction of all histones and DNA. The stabilized NPM of polythene chromosomes can be observed in both light and electron microscopy; no interchromatin fibrillary-granular structures are revealed in the nucleus except for peripheral lamina. Using the immunocytochemical method, in polythene chromosomes, we managed to detect major nonhistone proteins (topoisomerase IIα and SMC 1) and some RNA-components. Besides, in giant nuclei of larvae of early stages there is observed BrDU incorporation visualizing sites of DNA synthesis, which also are connected with NPM of polythene chromosomes. Thus, it can be concluded that structure of NPM of giant nuclei of Chironomus plumosus has all properties of NPMs of usual interphase nuclei; furthermore, this NPM determines specific structure of the polythene chromosome.     

Polytene chromosomes of Oxytricha: Biochemical and morphological changes during macronuclear development in a ciliated protozoan

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120±2 polytene chromosomes and each chromosome has an average of 81 bands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus. — During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 g/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50×10⁶ daltons to about 2×10⁶ daltons.This paper is No. VI in the series, DNA of Ciliated Protozoa.     

Two puff-specific proteins bind within the 2.5 kb upstream region of theDrosophila melanogaster Sgs-4 gene

TheDrosophila nuclear proteins Bj6 and Bx42 characterized previously are detected in a series of developmentally active puffs on salivary gland chromosomes. Here the binding of both proteins at puff 3C11-12 containing the glue protein geneSgs-4 is described in more detail. By deletion analysis we show that both proteins bind within a chromosomal segment containing 17–19 kb of DNA surrounding theSgs-4 gene. They are detectable at this site during the intermoult stages, before the puff regresses in response to the moulting hormone ecdysone. If theSgs-4 gene together with flanking DNA sequences is brought into a different chromosomal position by P element transfer, both proteins are detected at this new location. Both proteins are bound to the chromosome within the range of 2.5 kb DNA upstream of theSgs-4 gene. A strain containing a 52 bp deletion within this region fails to bind Bx42 protein suggesting that the missing DNA, which overlaps a hypersensitive region, may be required for the binding of the Bx42 protein.     

Differential staining of polytene chromosome bands in Chironomus by Giemsa banding methods

Two Giemsa banding methods (C banding and RB banding) are described which selectively stain the centromere bands of polytene salivary gland chromosomes in a number of Chironomus species. — By the C banding method the polytene chromosome appearance is changed grossly. Chromosome bands, as far as they are identifiable, are stained pale with the exception of the centromere bands and in some cases telomeres, which then are intensely stained reddish blue. — By the RB method the centromere bands are stained bright blue, whereas the remainder of the polytene bands stain red to red-violet. — Contrary to all other species examined, in Chironomus th. thummi numerous interstitial polytene chromosome bands, in addition to the centromere regions, are positively C banded and blue stained by RB banding. In the hybrid of Ch. th. thummi x Ch. th. piger only those interstitial thummi bands which are known to have a greater DNA content than their homologous piger bands are C banding positive and blue stained by the RB method whereas the homologous piger bands are C banding negative and red stained by RB banding. Ch. thummi and piger bands with an equal amount of DNA both show no C banding and stain red by RB banding. — It seems that the Giemsa banding methods used are capable of demonstrating, in addition to centromeric heterochromatin, heterochromatin in those interstitial polytene chromosome bands whose DNA content has been increased during chromosome evolution.     

The amounts of nuclear DNA and the duration of DNA synthetic period (S) in related diploid and autotetraploid species of oats

The relative amounts of nuclear DNA of root meristematic cells of two related diploid Avena species, A. strigosa 2x and A. pilosa, which have different karyotypes, and an autotetraploid of one, A. strigosa 4x, were measured by Feulgen microspectrophotometry. The durations of various periods of their mitotic cycles were studied by autoradiography of cells pulse-labeled with tritiated thymidine. The results show that the autotetraploid, with twice the amount of nuclear DNA of its diploid, has the same duration of S period as the diploid, while A. pilosa, with intermediate nuclear DNA content, has a longer S period. These results support the hypothesis that homologous chromosomes or genomes require similar duration for their DNA synthesis and suggest that the structures of chromosomes are involved in temporal control of the DNA synthesis in cells.     

Behavior of polytene chromosomes of rhynchosciara angelae at different stages of larval development

Summary Polytene chromosomes in cells of salivary gland, Malpighian tubules and intestine of Rhynchosciara angelae are very favorable for study. The polytene chromosomes of the salivary gland are among the largest available for cytogenetics work. The ones in Malpighian tubules and in some parts of the intestine are as large and as favorable for cytological studies as the salivary chromosomes of many species of Drosophila.Two additional characteristics of Rhynchosciara make these flies excellent material for studies on the development of polytene chromosomes. 1.It is possible to observe the banding pattern of the polytene chromosomes at many stages of the larval life for at least 30 days before pupation, and 2. since the gregarious larvae develop simultaneously, one can sample the group at any stage desired. Sampling the group every day, it is possible to follow the development of the chromosomes as though one studied a single individual by observing it every day.We have followed in detail the behavior of the bands in two sections of chromosome B and in one section of chromosome C, at different stages of larval development. Some regions of the chromosomes which are represented by typical euchromatic bands at one stage of the larval development may develop in enormous bulbs, and later on may return to the banded stage again.The formation of the bulbs is not uniform in different sections of the same or of different chromosomes. In section 2 of chromosome B a certain locus swells enormously and then develops an enormous bulb, and later returns to the banded stage. At the point where the bulb was formed there is an accumulation of DNA, in amounts probably several times greater than before the bulb formation. In section 3 of chromosome B and section 3 of chromosome C the extra accumulation of DNA preceeds the formation of the bulb and is maintained during and after it. In the bulb formed in section 3 of chromosome C a single band seems to be responsible for the process.As shown by several authors, experimental evidence suggests that a gene is located within a band. The bulb formation in polytene chromosomes may then be morphological evidence of gene activities. This type of bulb formations and of return to the banded stage is a property of many chromosomes bands, during larval development. This type of behavior of many bands in polytene chromosomes is related to the process of nucleolus formation. However, this behavior may be found in almost all (if not in all) bands of the polytene chromosomes. If so, the behavior of the nucleolus organizer region is only a special case of this general process.The accumulation of DNA in different parts of the chromosome in cells of the same or of different tissues may be an argument against the theory of the constancy of the amount of DNA in all cells of a species. The bulb formations is not peculiar to R. angelae but occurs in several other Diptera.     

IN SITU DEMONSTRATION OF DNA HYBRIDIZING WITH CHROMOSOMAL AND NUCLEAR SAP RNA IN CHIRONOMUS TENTANS

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA.     

Study of the organization of polytene chromosomes in the nuclei of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> pseudonurse cells with the use of the DNA library of the <Emphasis Type="Italic">Drosophila orena</Emphasis> chromocenter

The location of the Drosophila orena chromocenter in polytene chromosomes of pseudonurse cells of the D. melanogaster ovaries (the otu11 mutation) and salivary glands has been studied. Numerous sites of location of the D. orena chromocenter DNA have been found throughout the length of D. melanogaster chromosomes. The specific distribution of the binding sites for the DNA probe has made it possible to identify chromosomes and analyze their mutual positions in the three-dimensional space of the nuclei of pseudonurse cells. The mutual positions of chromosomes have been found to vary, the pericentromeric regions of different chromosomes differing from one another in associative ratios.     

The discriminating fluorescence patterns of the chromosomes of Drosophila melanogaster

Mitotic and salivary gland chromosomes of D. melanogaster show striking fluorescent patterns when stained with Quinacrine. In the salivary gland chromosomes there are up to five strongly fluorescing bands located on the fourth chromosome and at the proximal end of the X chromosome.—In mitotic cells the Y chromosome shows four fluorescent segments and other fluorescent regions are found proximally on the third pair and on the X chromosome. It is, therefore, possible to distinguish male and female interphase cells by their patterns of fluorescence.—A comparison between the position of heterochromatic, late replicating and fluorescing segments in the mitotic chromosomes, shows differences which demonstrate, for the first time, the chemical, morphological and genetical diversity of these three types of segments.     

Two AT-rich satellite DNAs in the chironomid Glyptotendipes barbipes (Staeger)

Two AT-rich satellite DNAs are present in the genome of Glyptotendipes barbipes. The two satellites have densities of 1.680 g/cm³ (=21% GC) and of 1.673 g/cm³ (=13% GC) in neutral CsCl-density gradients. The main band DNA has a density of 1.691 g/cm³ (=32% GC). This value is in agreement with the 33% GC-content of G. barbipes DNA calculated from thermal denaturation (T_M=83° C). — In brain DNA as well as in salivary gland DNA the two satellite sequences together comprise 12–15% of the total G. barbipes DNA. Comparisons of the density profiles of DNA extracted from polytene and non-polytene larval tissue gave no hints for underreplication of the satellite DNAs during polytenization. — The two satellite DNAs have been isolated from total DNA by Hoechst 33258-CsCl density centrifugation and then localized in the polytene salivary gland chromosomes by in situ hybridization. Both satellite sequences hybridize to all heterochromatic centromere bands of all four chromosomes of G. barbipes. Satellite I (1.673 g/cm³) hybridizes mainly with the middle of the heterochromatin, satellite II (1.680 g/cm³) hybridizes with two bands at the margin of the heterochromatin. In situ hybridization with polytene chromosomes of Chironomus thummi revealed the presence of G. barbipes satellite sequences also in the Ch. thummi genome at various locations, mainly the centromere regions.     

An AT-rich DNA component in the genomes of Chironomus thummi thummi and Chironomus thummi piger

A DNA fraction has been isolated from total Chironomus thummi thummi DNA which is discernible from the bulk Ch. th. thummi DNA by a lower thermal stability. In situ hybridizations with polytene salivary gland chromosomes of Ch. th. thummi and Ch. th. piger made localization of this DNA fraction possible. Hybridizations with bands which contain different amounts of DNA in the two subspecies indicate that the isolated DNA fraction mostly consists of those sequences which represent the genetical difference between thummi and piger.This paper is dedicated to Professor Dr. H. Bauer on the occasion of his 75th birthday     

Isolation and distribution of a Drosophila protein preferentially associated with inactive regions of the genome

The distribution patterns of chromosomal proteins from Drosophila can be observed by immunofluorescent staining of the polytene chromosomes from larval salivary glands. We have purified a non-histone chromosomal protein of M_r=69 000 molecular weight which has a high affinity for DNA with little sequence specificity. Immunofluorescent staining indicates that this protein is preferentially associated with the inactive portions of the genome, including the centric heterochromatin and the condensed bands within the euchromatic arms of the chromosomes. Observation of both the heat shock loci 87A and 87C and the developmentally regulated loci 74EF and 75B shows an inverse correlation between immunofluorescent staining for the M_r=69 000 protein and for RNA polymerase. The presence of this protein appears to be correlated with the packaging of the chromatin in an inactive form.