The JIL-1 kinase regulates the structure of Drosophila polytene chromosomes

The JIL-1 kinase localizes to interband regions of Drosophila polytene chromosomes and phosphorylates histone H3 Ser10. Analysis of JIL-1 hypomorphic alleles demonstrated that reduced levels of JIL-1 protein lead to global changes in polytene chromatin structure. Here we have performed a detailed ultrastructural and cytological analysis of the defects in JIL-1 mutant chromosomes. We show that all autosomes and the female X chromosome are similarly affected, whereas the defects in the male X chromosome are qualitatively different. In polytene autosomes, loss of JIL-1 leads to misalignment of interband chromatin fibrils and to increased ectopic contacts between nonhomologous regions. Furthermore, there is an abnormal coiling of the chromosomes with an intermixing of euchromatic regions and the compacted chromatin characteristic of banded regions. In contrast, coiling of the male X polytene chromosome was not observed. Instead, the shortening of the male X chromosome appeared to be caused by increased dispersal of the chromatin into a diffuse network without any discernable banded regions. To account for the observed phenotypes we propose a model in which JIL-1 functions to establish or maintain the parallel alignment of interband chromosome fibrils as well as to repress the formation of contacts and intermingling of nonhomologous chromatid regions. Electronic Supplementary Material Supplementary material is available for this article at and accessible for authorised users     

Late DNA replicaion of X chromosomes in female and pseudofemale cells of Microtus agrestis.

The late replication pattern of the short arms of the X chromosomes of Microtus agrestis was studied in female cells and in cells with 2 X chromosomes of male origin by means of the BUdR-Giemsa technique and of 3H-thymidine labelling. The light absorption of Giemsa stained chromosome sections which were unifilarly substituted with BUdR (labelled), was found to be 59.2% of that of unlabelled chromosomes. In female cells, asynchrony of DNA replication of both X chromosomes indicated the presence of facultative heterochromatin in the X2 and euchromatin in the X1. In the male cells only euchromatic X chromosomes were observed in diploid XX and XO cells as well as in triploid XXY, XX and XO cells. The results show that inactivation of an X chromosone in vitro, in cells with more than one originally active X chromosome does not occur even after a culture duration of several years.     

Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.     

Pattern of binding of tritiated actinomycin D to Phaseolus coccineus polytene chromosomes. II. The entire chromosome complement

Abstract

The pattern of labelling by tritiated actinomycin D (³H-AMD) on polytene chromosomes in the embryo suspensor cells of Phaseolus coccineus is described in preparations exposed for different times to photographic emulsion. It is observed that: a) each chromosome pair displays a typical labelling pattern; b) heterochromatin close to the centromere does not label in the same way in different chromosome pairs; c) in some chromosome pairs, different bundles of strands in one an the same chromosome segment display differences in ³H-AMD labelling. These results allow an improved characterization of the Phaseolus polytene chromosomes and are discussed in relation to the possible factors determining the differential binding of ³H-AMD to the chromatin.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

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.     

The COOH-terminal domain of the JIL-1 histone H3S10 kinase interacts with histone H3 and is required for correct targeting to chromatin

The JIL-1 histone H3S10 kinase in Drosophila localizes specifically to euchromatic interband regions of polytene chromosomes and is enriched 2-fold on the male X chromosome. JIL-1 can be divided into four main domains including an NH(2)-terminal domain, two separate kinase domains, and a COOH-terminal domain. Our results demonstrate that the COOH-terminal domain of JIL-1 is necessary and sufficient for correct chromosome targeting to autosomes but that both COOH- and NH(2)-terminal sequences are necessary for enrichment on the male X chromosome. We furthermore show that a small 53-amino acid region within the COOH-terminal domain can interact with the tail region of histone H3, suggesting that this interaction is necessary for the correct chromatin targeting of the JIL-1 kinase. Interestingly, our data indicate that the COOH-terminal domain alone is sufficient to rescue JIL-1 null mutant polytene chromosome defects including those of the male X chromosome. Nonetheless, we also found that a truncated JIL-1 protein which was without the COOH-terminal domain but retained histone H3S10 kinase activity was able to rescue autosome as well as partially rescue male X polytene chromosome morphology. Taken together these findings indicate that JIL-1 may participate in regulating chromatin structure by multiple and partially redundant mechanisms.     

CHROMOSOMAL BASIS OF DOSAGE COMPENSATION IN DROSOPHILA : III. Early Completion of Replication by the Polytene X-Chromosome in Male: Further Evidence and Its Implications

Thymidine-³H labeling patterns on the X (section 1 A to 12 E of Bridges' map) and 2 R (section 56 F to 60 F of Bridges' map) segments in the salivary gland chromosomes of Drosophila melanogaster have been analyzed in male and female separately. The observed patterns fit, with a few exceptions, in a continuous to discontinuous labeling sequence. In nuclei with similar labeling patterns on the 2R segment in both sexes, the number of labeled sites on the X in male is always less than in female X's. The labeling frequency of the different sites on the male X is considerably lower than those on the female X's, while the sites on the 2R segment have very similar frequency in the two sexes. The rate of thymidine-³H incorporation (as judged by visual grain counting) is relatively higher in male X than in female X's. It is concluded that the model sequence of replication in polytene chromosomes follows a continuous to discontinuous labeling sequence, and that the single X in male completes its replication earlier than either the autosomes in male or the X's in female. This asynchronous and faster rate of replication by the polytene X-chromosome in male substantiates the hypothesis of hyperactivity of the single X in male as the chromosomal basis of dosage compensation in Drosophila.     

Differential sex chromosome replication and dosage compensation in polytene trichogen cells of Lucilia cuprina (Diptera: Calliphoridae)

Non banded sex chromosome elements have been identified in polytene trichogen cells of Lucilia cuprina using Y-autosome translocations, C-banding and Quinacrine fluorescence. The X chromosome is an irregular granular structure while the much smaller Y chromosome has both a dense darkly stained and a loosely organised segment. The X and Y chromosomes are underreplicated in polytene cells but comparison of C- and Q-banding characteristics of sex chromosomes in diploid and polytene tissues indicates that selective replication of non C-banding material occurs in both the sex chromosomes. Brightly fluorescing material in the Y chromosome is replicated to such an extent that it consists of half the polytene element, while the C-banding material, which makes up most of the diploid X chromosome, is virtually unreplicated. Differential replication also occurs in autosomes. In XXY males, and in males carrying a duplication of the X euchromatic region, a short uniquely banded polytene chromosome is formed. It is suggested that in males carrying two doses of X euchromatin a dosage compensation mechanism operates in which genes in one copy are silenced by forming a banded polytene chromosome.     

Meiosis in the male mouse. An autoradiographic investigation

Meiosis in the male mouse has been studied autoradiographically in air-dried preparations. Information has been obtained on the relative rates of DNA synthesis and the lengths of the S-periods in spermatogonia and spermatocytes. The average rate of synthesis in the spermatocyte is lower, and the S-period is of longer duration than the preceding spermatogonial generations. The labelling pattern of the sex-chromosomes and autosomes observed at diakinesis and metaphase II in cells labelled at the spermatocyte S-period appears to be similar to that found in cells labelled during the spermatogonial S-periods. Replication in the autosomes commences before the sex-chromosomes. Late replicating autosomal centric regions show a marked degree of asynchrony in labelling both between and within bivalents. The Y chromosome starts and finishes replication later than the X. There is a short, late-replicating, segment of the X in the vicinity of the centromere. There is a short, early-replicating segment of the Y in the vicinity of the centromere which may represent the euchromatic short arm. The X and Y appear to associate at diakinesis by the distal ends of their long arms.     

Studies of X inactivation and isodisomy in twins provide further evidence that the X chromosome is not involved in Rett syndrome.

Rett syndrome (RS), a progressive encephalopathy with onset in infancy, has been attributed to an X-linked mutation, mainly on the basis of its occurrence almost exclusively in females and its concordance in female MZ twins. The underlying mechanisms proposed are an X-linked dominant mutation with male lethality, uniparental disomy of the X chromosome, and/or some disturbance in the process of X inactivation leading to unequal distributions of cells expressing maternal or paternal alleles (referred to as a "nonrandom" or "skewed" pattern of X inactivation). To determine if the X chromosome is in fact involved in RS, we studied a group of affected females including three pairs of MZ twins, two concordant for RS and one uniquely discordant for RS. Analysis of X-inactivation patterns confirms the frequent nonrandom X inactivation previously observed in MZ twins but indicates that this is independent of RS. Analysis of 29 RS females reveals not one instance of uniparental X disomy, extending the observations previously reported. Therefore, our findings contribute no support for the hypothesis that RS is an X-linked disorder. Furthermore, the concordant phenotype in most MZ female twins with RS, which has not been observed in female twins with known X-linked mutations, argues against an X mutation.     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Genetic and developmental analysis of X-inactivation in interspecific hybrid mice suggests a role for the Y chromosome in placental dysplasia

It has been shown previously that abnormal placental growth, i.e., hyper- and hypoplasia, occurs in crosses and backcrosses between different mouse (Mus) species. A locus that contributes to this abnormal development has been mapped to the X chromosome. Unexpectedly, an influence of fetal sex on placental development has been observed, in that placentas attached to male fetuses tended to exhibit a more pronounced phenotype than placentas attached to females. Here, we have analyzed this sex dependence in more detail. Our results show that differences between male and female placental weights are characteristic of interspecific matings and are not observed in intraspecific Mus musculus matings. The effect is retained in congenic lines that contain differing lengths of M. spretus-derived X chromosome. Expression of the X-linked gene Pgk1 from the maternal allele only and lack of overall activity of two paternally inherited X-linked transgenes indicate that reactivation or lack of inactivation of the paternal X chromosome in trophoblasts of interspecific hybrids is not a frequent occurrence. Thus, the difference between male and female placentas seems not to be caused by faulty preferential X-inactivation. Therefore, these data suggest that the sex difference of placental weights in interspecific hybrids is caused by interactions with the Y chromosome.     

Dosage Compensation Regulatory Proteins and the Evolution of Sex Chromosomes in Drosophila

In the fruitfly Drosophila melanogaster, the four male-specific lethal (msl) genes are required to achieve dosage compensation of the male X chromosome. The MSL proteins are thought to interact with cis-acting sites that confer dosage compensation to nearby genes, as they are detected at hundreds of discrete sites along the length of the polytene X chromosome in males but not in females. The histone H4 acetylated isoform, H4Ac16, colocalizes with the MSL proteins at a majority of sites on the D. melanogaster X chromosome. Using polytene chromosome immunostaining of other species from the genus Drosophila, we found that X chromosome association of MSL proteins and H4Ac16 is conserved despite differences in the sex chromosome karyotype between species. Our results support a model in which cis-acting regulatory sites for dosage compensation evolve on a neo-X chromosome arm in response to the degeneration of its former homologue.     

Interline differences in morphology of the precentromeric region of polytene X-chromosome in Drosophila melanogaster salivary glands]

Morphology of the Drosophila melanogaster polytene X chromosome section 20 in normal flies, in strains carrying inversions that break pericentric heterochromatin at different points, and at the background of the Su(UR)ES mutation has been examined. In all of the strains carrying the Su(UR)ES mutation section 20 displayed a distinct banding pattern till to the section 20F, while in the wild-type strains this region was represented by beta-heterochromatin. The strains carrying different inversions substantially differed in the number and morphology of bands forming section 20. In the Su(UR)ES mutants the most proximal X chromosome euchromatin gene, su(f), is mapped to the boundary between sections 20E and F, while rDNA forming the middle part of the X chromosome mitotic heterochromatin is located in the proximal part of section 20F. All large bands observed in section 20 of the w; Su(UR)ES strain were also present in In(1)sc4; Su(UR)ES, which breaks heterochromatin in the distal part. Hence, the bands of polytene chromosome section 20 are virtually devoid of mitotic heterochromatin.