Klaus Hägele

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.

Satellite DNA of Drosophila nasuta nasuta and D. n. albomicana: localization in polytene and metaphase chromosomes.

The DNA from the two Drosophila nasuta races, D. n. nasuta and D. n. albomicana was investigated by CsCl density gradient centrifugation. D. n. nasuta has one major AT-rich satellite DNA sequence with a density of 1.664g/cm3, while D. n. albomicana has at least three satellites with densities of 1.674g/cm3, 1.665g/cm3 and 1.661 g/cm3. The isolated satellite sequences hybridize in situ to all heterochromatic regions of all metaphase chromosomes of both races. In polytene chromosomes the satellite sequences hybridize exclusively to the chromocenter. All chromosomal regions hybridizing with the satellites show also bright quinacrine fluorescence.

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.

Different hybrid effects in reciprocal crosses betweenChironomus thummi thummi andCh. th. piger including spontaneous chromsome aberrations and sterility

Hybrids from reciprocal erosses between twoChironomus thummi thummi andCh. th. piger laboratory stocks show four abnormalities in comparison to the parental stocks. One cross direction (Ch. th. thummi ♂ x Ch. th. piger ♀) is characterized by chromosome aberrations, reduced hatchability and malformations, whereas in reciprocal hybrids both sexes are sterile. Sterility is the consequence of rudimentary or non developed gonads.InCh. th. thummi ♂ x Ch. th. piger ♀ crosses chromosome aberrations were analysed in salivary gland nuclei. These aberrations are all somatic in origin, and they are induced during the first 40 h of embryonic development, prior to the onset of polytenization. The chromosomes of both subspecies are equally affected. In all four chromosomes breaks occur preferentially at specific regions. Reduced hatchability and malformations are presumably caused by chromosome mutations because within egg-masses a correlation exists between the rate of salivary gland chromosome aberrations and the rates of hatchability and malformations.

Komplementäre DNA-Replikationsmuster bei Drosophila melanogaster

In D. melanogaster salivary gland chromosomes continuous and discontinuous labeling patterns have been described. Arcos-Teran (1972) has devided those of the X-chromosome in ♂♂ into a 6 class sequence beginning with continuous labeling and ending with labeling restricted to a few heavy bands. Labeling patterns reverse to the described ones have not been reported. — In the present communication new types of discontinuous labeling patterns are described for the X of melanogaster females. One type shows unlabeling of the majority of heavy bands and of the chromocenter whereas interbands and faint bands are labeled. In other types the number of unlabeled heavy bands is decreased and the chromosome is nearly totally labeled. These new labeling patterns are the reverse of Arcos-Terans patterns II, IV, and V. It must therefore be assumed that the continuous pattern of labeling corresponds to the (early) middle of the replication cycle and that replication in melanogaster females starts with the labeling patterns described here.In D. melanogaster salivary gland chromosomes continuous and discontinuous labeling patterns have been described. Arcos-Terán (1972) has devided those of the X-chromosome in ♂♂ into a 6 class sequence beginning with continuous labeling and ending with labeling restricted to a few heavy bands. Labeling patterns reverse to the described ones have not been reported. — In the present communication new types of discontinuous labeling patterns are described for the X of melanogaster females. One type shows unlabeling of the majority of heavy bands and of the chromocenter whereas interbands and faint bands are labeled. In other types the number of unlabeled heavy bands is decreased and the chromosome is nearly totally labeled. These new labeling patterns are the reverse of Arcos-Teráns patterns II, IV, and V. It must therefore be assumed that the continuous pattern of labeling corresponds to the (early) middle of the replication cycle and that replication in melanogaster females starts with the labeling patterns described here.

Initial phases of DNA synthesis in Drosophila melanogaster

Replication patterns of the X chromosomes and autosomes in D. melanogaster male and female larvae during the discontinuously labeled initial and end phases of DNA synthesis were compared. In female larvae X and autosomes behaved correspondingly during all the replication stages. In males, however, the X chromosome shows a differential replication behavior from that of the autosomes already during the discontinuously labeled initial stage.—In those nuclei of both sexes, in which the autosomes correspond in their initial replication patterns, significantly more labeled regions are to be found over the male X than over the female X. The complementary behavior during the end phases (Berendes, 1966), i.e. the reverse of that above, leads to an earlier completion of the replication cycle in most of the labeled regions of the male X chromosome. The differential replication revealed in the autoradiograms is interpreted as a consequence of the polytene structure in giant chromosomes.

Identification of a polytene chromosome band containing a male sex determiner of Chironomus thummi thummi

Hybrid males of Chironomus thummi piger ♀ x Ch. th. thummi ♂ crosses were backcrossed with females of both parental stocks. Fourth-instar larvae of these backcrosses showed sex specific differences in the pairing behavior of region D3d-g in chromosome arm F of salivary gland chromosome III. — Analysis of the banding pattern of region D3d-g after RB and quinacrine staining demonstrated that in piger ♀ x thummi ♂ hybrid males a single selectively stained band occurs within this region in the heterozygous condition at map position D3e1. This band could only be found in the thummi chromosome partner, it is heterochromatic and contains AT-rich DNA. In female hybrid larvae, however, such a selectively stained band is present in neither the thummi nor the piger chromosome region D3d-g. From these results it is concluded that the selectively stained band D3e1 represents the male sex determiner of our Ch. th. thummi stock and that the male is the heterogametic sex.

N-banding in polytene chromosomes of Chironomus and Drosophila

The N-banding patterns of the polytene chromosomes of Drosophila melanogaster, Chironomus melanotus, Ch. th. thummi and Ch. th. thummi x Ch. th. piger were studied. In Chironomus the polytene N-banding patterns correspond to the polytene puffing patterns. This is revealed by comparison of the puffing and N-banding patterns of identical chromosomes. Size and staining intensity of the N-bands reflect the size of the puffs as shown by puff induction. There is no evidence that the N-bands are also located in Chironomus heterochromatin or are restricted to the nucleolar organizer regions. In Drosophila the α-heterochromatin is strongly N-positive, whereas the β-heterochromatin, as well as the Chironomus heterochromatin is not N-banded. Contrary to Chironomus, the puffs in Drosophila polytene chromosomes do not give rise selectively to well stained N-bands. — The N-banding method is interpreted to stain specifically non-histone protein which is (1) accumulated in genetically active chromosome regions and (2) present in a specific type of heterochromatin (α-heterochromatin of Drosophila).

Prolongation of replication time after doublings of the DNA content of polytene chromosome bands of Chironomus

Using 3H-thymidine autoradiography, labeling frequency of homologous asynapsed chromosome bands of the hybrid of Chironomus th. thummi and Chironomua th. piger has been studied. In a number of these bands the DNA content of the thummi bands is 2, 4, 8 or 16 times as large as that of the homologous piger bands (Keyl, 1965). Those bands of Ch. th. thummi which show one doubling of their DNA content in comparison with the homologous piger bands are also labeled two times more frequently than piger. In contrast to this such a correlation between increase of labeling frequency (i.e. prolongation of replication time) and doubling of the DNA content is not observed, when thummi bands have 4, 8 or 16 times more DNA than their homologues in piger. In these cases replication time is also prolonged after each doubling. Duration of DNA synthesis increases linearly but always by a smaller factor as the corresponding DNA content is increased.

Correspondence of banding patterns to 3h-thymidine labeling patterns in polytene chromosomes.

Abstract3H-thymidine labeling frequencies over X chromosomal region 1A-4E of Drosophila melanogaster, were analysed with reference to chromosome sections with and without prominent bands. A correspondence was found between band sections and late start of silver grain labeling at the initial stage in combination with late labeling at the end stage of replication. A complementary situation is always to be found over puff/interband sections, where an early start of labeling at the initial stage is generally combined with early labeling completion at the end stage of replication.