Die intraspezifische Variabilität des heterochromatischen Armes eines Chromosoms bei der GattungCarabus L. (Coleoptera)

The chromosome complement ofC. auronitens Fabr. is 2n _♂=26+XY. One autosomal pair—called A-chromosomes—is relatively long.A-chromosomes consist of a euchromatic and a heterochromatic arm. Labelling of mitotic chromosomes with³H-thymidine shows that replication of the heterochromatic arm continues when it has ended in the euchromatic arm. In males and females the length of the heterochromatic arm varies intraindividually. In 47 of 99 males the heterochromatic arms were heteromorphic. Calculations of the quotient length of the euchromatic/length of the heterochromatic arm have shown that at least 6 different types of the A-chromosome exist. These types differ from each other in the number of heterochromatic sections separated by constrictions. The longest heterochromatic arm observed consisted of 8 such sections. The genetic significance of the heterochromatin in the genus ofCarabus is at present unknown (Zusammenfassung see p.305).      

Histone H4 Acetylation and Replication Timing in Chinese Hamster Chromosomes

The distribution of acetylated isoforms of histone H4 along Chinese hamster chromosomes has been studied by immunostaining with antibodies recognizing H4 acetylated at defined lysines in its N-terminal domain. The heterochromatic long arm of the X chromosome in both female (CHO) and male (DON) cell lines is underacetylated at three out of four lysines (5, 8, and 12). In contrast, the level of acetylation at lysine 16, which is the first to be acetylated in mammals, was similar in X chromosomes and autosomes. Labeling of the cells with bromodeoxyuridine (BrdU) to mark late-replicating chromosome domains, followed by double immunostaining with antibodies to BrdU and acetylated H4, showed a close, though not perfect, correlation between late replication and low levels of H4 acetylation. The results show that levels of histone acetylation are associated with the replication timing of defined domains on both the X chromosome and autosomes, but the exceptions we observe suggest that this link is not absolute or essential.     

C-banding and non-homologous associations

A chromosome complement formed by 16 autosomes and an Xy_p sex chromosome system was found in Epilachna paenulata Germar (Coleoptera: Coccinellidae). All autosomes were metacentric except pair 1 which was submetacentric. The X and the Y chromosomes were also submetacentric but the Y was minute. The whole chromosome set carried large paracentric heterochromatic C-segments representing about 15% of the haploid complement length. Heterochromatic segments associated progressively during early meiotic stages forming a large single chromocenter. After C-banding, chromocenters revealed an inner networklike filamentous structure. Starlike chromosome configurations resulted from the attachment of bivalents to the chromocenters. These associations were followed until early diakinesis. Thin remnant filaments were also observed connecting metaphase I chromosomes. Evidence is presented that, in this species, the Xy_p bivalent resulted from an end-to-end association of the long arms of the sex chromosomes. The parachute Xy_p bivalent appeared to be composed of three distinct segments: two intensely heterochromatic C-banded corpuscles formed the canopy and a V-shaped euchromatic filament connecting them represented the parachutist component. The triple constitution of the sex bivalent was interpreted as follows: each heterochromatic corpuscle corresponded to the paracentric C-segment of the X and Y chromosomes; the euchromatic filament represented mainly the long arm of the X chromosome terminally associated with the long arm of the Y chromosome. The complete sequence of the formation of the Xy_p bivalent starting from nonassociated sex chromosomes in early meiotic stages, and progressing through pairing of heterochromatic segments, coiling of the euchromatic filament, and movement of the heterochromatic corpuscles to opposite poles is described. These findings suggest that in E. paenulata the Xy_p sex bivalent formation is different than in other coleopteran species and that constitutive heterochromatic segments play an important role not only in chromosome associations but also in the Xy_p formation.     

Hoechst 33258 banding of Drosophila nasutoides metaphase chromosomes

Hoechst 33258 banding of D. nasutoides metaphase chromosomes is described and compared with Q and C bands. The C band positive regions of the euchromatic autosomes, the X and the Y fluoresce brightly, as is typical of Drosophila and other species. The fluorescence pattern of the large heterochromatic chromosome is atypical, however. Contrary to the observations on other species, the C negative bands of the large heterochromatic chromosome are brightly fluorescent with both Hoechst 33258 and quinacrine. Based on differences in the various banding patterns, four classes of heterochromatin are described in the large heterochromatic chromosome and it is suggested that each class may correspond to an AT-rich DNA satellite.     

Genetic control ofHylemya antiqua III. Differences in pupation ability between two strains

Some relevant traits of a wild (L) and a laboratory (C) strain of Hylemya antiqua (Meigen ), determining differences in their pupation ability under experimental conditions have been investigated in relation to genetic control. The wild strain showed an intrinsic higher pupation ability than the laboratory strain. The minimum feeding period was 0.6 days longer for the C strain. The minimum larval dry weight was different for the two strains. With a normal feeding period C larvae pupated on average 1.63 days later than L larvae. By the shortening of the larval feeding period an acceleration of the larval development of both strains was observed: the acceleration of development was more marked for the wild strain. This result has been contrasted with published works on Drosphila. The consequences of these differences as far as the competitive ability of each strain is concerned, have been discussed in relation to genetic control.     

Distribution of heterochromatin on the mitotic chromosomes of Musca domestica L. in relation to the activity of male-determining factors

In the housefly, male sex is determined by a dominant factor, M, located either on the Y, on the X, or on any of the five autosomes. M factors on autosome I and on fragments of the Y chromosome show incomplete expressivity, whereas M factors on the other autosomes are fully expressive. To test whether these differences might be caused by heterochromatin-dependent position effects, we studied the distribution of heterochromatin on the mitotic chromosomes by C-banding and by fluorescence in situ hybridization of DNA fragments amplified from microdissected mitotic chromosomes. Our results show a correlation between the chromosomal position of M and the strength of its male-determining activity: weakly masculinizing M factors are exclusively located on chromosomes with extensive heterochromatic regions, i.e., on autosome I and on the Y chromosome. The Y is known to contain at least two copies of the M factor, which ensures a strong masculinizing effect despite the heterochromatic environment. The heterochromatic regions of the sex chromosomes consist of repetitive sequences that are unique to the X and the Y, whereas their euchromatic parts contain sequences that are ubiquitously found in the euchromatin of all chromosomes of the complement. Received: 20 February 1998; in revised form: 11 May 1998 / Accepted: 23 May 1998     

Chromosome segregation and spindle structure in crane fly spermatocytes following Colcemid treatment

Chromosome segregation in primary spermatocytes of the crane fly Nephrotoma suturalis was studied after exposure to Colcemid at doses that did not completely inhibit spindle formation. Colcemid was added either to the medium in which larvae were cultured or to Tricine buffer in which isolated testes were incubated. Patterns of chromosome segregation were analyzed in fixed, Feulgen-stained smears of testes from Colcemid-treated larvae and in living cell preparations. Anomalies observed during the first meiotic division at higher than normal frequencies in Colcemid-treated spermatocytes included anaphase lagging of autosomes, chromosomal strands, tripolar and tetrapolar divisions, and unequal distribution of chromosomes to secondary cells. Following those doses of Colcemid that induced the above anomalies, the length of the birefringent spindle in primary spermatocytes was shorter than normal. This effect on spindle length also was apparent in Giemsastained preparations of fixed cells, in which the two centrosomes at the spindle poles were differentiated from the rest of the cytoplasm. The results indicate a correlation between the inhibition of spindle formation and the induction of anomalous patterns of chromosome segregation.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Meiotic differences between three triatomine species (Hemiptera,Reduviidae)

We have found the following differences in the male meiosis among three triatomine species: (1) The three largest autosomal bivalents ofTriatoma infestans are heterochromatic.Rhodnius prolixus has two autosomal bivalents with heterochromatic blocks.Triatoma rubrovaria does not show any heteropycnotic autosomes. (2) Sex chromosomes inT. infestans form a chromocenter. At early prophase terminal associations are seen between sex chromosomes inT. rubrovaria, and they maintain a close association until diakinesis. An intimate association between the X and Y chromosomes is observed during early prophase inR. prolixus, but a distant association is maintained by the sex chromosomes at diffuse and diplotene stages in this species. (3) Polyploid nuclei of the nutritive cells are quite distinct. Numerous chromocenters of different shapes and sized are seen in those ofT. infestans. InT. rubrovaria one chromocenter having two positively heteropycnotic elements is observed surrounded by homogeneous chromatin. Only one compact chromocenter is found amongst unevenly distributed chromatin, inR. prolixus.     

Familial transmission of a translocation Y/14

Summary A translocation of heterochromatic material, brightly fluorescent after actinomycin D-DAPI staining, to the short arm of chromosome 14 was prenatally detected during cytogenetic examination of cells obtained by amniocentesis on the indication of advanced maternal age. Besides this abnormal chromosome, 43 autosomes and two X chromosomes were present. Silver staining made clear that an active nucleolus-organizing region was included in the translocation product. Both the intense fluorescence and the size of the translocated extra heterochromatic block were indicative of a Yq origin. Upon cytogenetic investigation of the parents, the mother appeared to carry the same t(Y;14) chromosome. Therefore, we expected a normal girl to be born. This was confirmed after birth.     

DNase I sensitivity in facultative and constitutive heterochromatin

In situ nick translation allows the detection of DNase I sensitive and insensitive regions in fixed mammalian mitotic chromosomes. We have determined the difference in DNase I sensitivity between the active and inactive X chromosomes inMicrotus agrestis (rodent) cells, along both their euchromatic and constitutive heterochromatic regions. In addition, we analysed the DNase I sensitivity of the constitutive heterochromatic regions in mouse chromosomes. InMicrotus agrestis female cells the active X chromosome is sensitive to DNase I along its euchromatic region while the inactive X chromosome is insensitive except for an early replicating region at its distal end. The late replicating constitutive heterochromatic regions, however, in both the active and inactive X chromosome are sensitive to DNase I. In mouse cells on the other hand, the constitutive heterochromatin is insensitive to DNase I both in mitotic chromosomes and interphase nuclei.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Differential response of constitutive and facultative heterochromatin in the manifestation of mitomycin induced chromosome aberrations in Chinese hamster cells in vitro

The distribution of chromatid aberrations induced by mitomycin C among the individual chromosomes of female and male Chinese hamster cells in vitro was studied. The aberrations were found to be non-randomly distributed. Among the autosomes, the chromosomes possessing constitutive heterochromatin were more often involved in aberrations as well as in homologous exchanges. The inactivated X chromosomes in the female cells offer a situation where the short arm is facultatively heterochromatic and the long arm constitutively heterochromatic, thus enabling an analysis of their response for aberration formation. The short arm was seldom found to be involved in the aberration. The long arm of the inactivated X was more often affected (5 to 10 times) than the long arm of the functional X though both are constitutively heterochromatic. The possible role of (a) structure of heterochromatin, (b) the chromocenter formation and their association, (c) allocycly, and (d) the qualitative differences in the DNA of different types of heterochromatin are discussed in relation to the formation of chromatid aberrations.     

Differential chromosome contraction at the pachytene stage of meiosis in alfalfa (Medicago sativa L.)

Using the length of the total chromosome complement as a measure of the pachytene stage of a cell, most of the variation from cell to cell in chromosome lengths can be accounted for. Significant regression equations were obtained for chromosome and arm lengths upon the cell stage and these provide estimates of the relative contraction rates of the chromosomes. The regression lines for chromosomes 1 and 2 were quadratic whereas they were linear for the remaining six chromosomes. The contraction rates were different for each chromosome as well as for the short and long arms of chromosomes 1 to 5. The relative contraction rates for the heterochromatic short arms of chromosomes 3, 4 and 5 were very low and therefore arm ratios as well as relative lengths of chromosomes could vary with the cell stage examined. The differences in chromosome numbering systems reported by alfalfa researchers are mainly attributed to the pachytene stages observed and the small numbers of observations per study.     

The behavior and morphology of the X and Y chromosomes during prophase I in the Sitka deer mouse (Peromyscus sitkensis)

Surface-spread, silver-stained primary spermatocytes from individuals of the Sitka deer mouse (Peromyscus sitkensis) were analyzed by electron microscopy. Pairing of the X and Y chromosomes is initiated at early pachynema and is complete by mid pachynema. The pattern of sex chromosome pairing is unique in that it is initiated at an interstitial position, with subsequent synapsis proceeding in a unidirectional fashion towards the telomeres of the homologous segments. One-third the length of the X and two-thirds the length of the Y are involved in the synaptonemal complex of the sex bivalent. Various morphological complexities develop in the heteropycnotic (unpaired) segments as pachynema progresses, but desynapsis is not initiated until diplonema. Analysis of C-banded diakinetic nuclei indicated that sex chromosome pairing involves the heterochromatic short arm of the X and the long arm of the heterochromatic Y. An interstitial chiasma between the X and Y was observed in the majority of the diakinetic nuclei. The observation of a substantial pairing region and chiasma formation between the sex chromosomes of these deer mice is interpreted as indicating homology between the short arm of the X and the long arm of the Y.     

C-banding patterns in chromosomes and sperm of Strophosoma capitatum (De Geer, 1775) (Coleoptera: Curculionidae, Brachyderinae)

An analysis was made of the C-banded karyotype of Strophosoma capitatum (Deg.). The results indicate that the chromosome number is 2n = 22 and n Male = 10 + Xyp. The examined karyotype shows a pericentromeric position of constitutive heterochromatin in all autosomes. The shorter arm of the X chromosome is heterochromatic while the y chromosome is wholly euchromatic. Successive stages of spermatogenesis were analysed.     

Meiotic disjunction and embryonic lethality in sex-linked double-translocation heterozygous males of the onion fly,Hylemya antiqua (Meigen)

Summary The frequencies of disjunction types in double-translocation heterozygous males (2⁶2^Y6²6XY²) in Hylemya antiqua have been established in MII cells and eggs of testcrosses.Several disjunction types occurred but four predominated. A correlation was found between the frequencies of the disjunction types and the relative position of the centromeres. The frequency of numerical non-disjunction (NND) was 4%. Differences in frequency of NND between sex-linked and autosomal translocations of H. antiqua are discussed. A good correspondence between the frequencies of unbalanced karyotypes, and embryonic and larval mortality was found. The total genetic load which can be induced by the T14/T61 males is estimated to be 60–65%. Some duplication/deficiency karyotypes appeared to be viable in pupal and even adult stages. In 2⁶2⁶2^Y6²6²X males a regular coorientation between 2^Y and X was observed, in spite of non-homologous centromeres and a complicated synapsis of 2^Y. Application possibilities of the present material for genetic control of H. antiqua are discussed.     

Molecular Cytogenetic Characterization of the Dioecious Cannabis sativa with an XY Chromosome Sex Determination System

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution.     

Cytomolecular analysis of the male sex chromosome of Tropidacris cristata grandis (Thunberg, 1824) (Orthoptera: Romaleidae)

Many species of grasshopper have an XX/XO sex chromosome system, including Tropidacris cristata grandis (23, XX/XO). The X chromosome behaves differently from the autosomes, but little is known about its origin and molecular composition. To better understand the genomic composition and evolutionary processes involved in the origin of the sex chromosomes, we undertook an analysis of its meiotic behavior, heterochromatin distribution and microdissection in T. c. grandis. Analysis of meiotic cells revealed a difference in the behavior of the X chromosome compared to the autosomes, with different patterns of condensation and cellular arrangement. Heterochromatic terminal blocks were predominant. The chromosome painting revealed a bright block in the centromeric/pericentromeric region of the X chromosome and slight markings in the other regions. In the autosomes, the X chromosome probe hybridized in the centromeric/pericentromeric region, and hybridization signals on terminal regions corresponding to the heterochromatic regions were also observed. The results showed that the X chromosome contains a significant amount of repetitive DNA. Based on the hybridization pattern, it is possible that the autosomes and sex chromosomes of T. c. grandis have a similar composition of repetitive DNAs, which could mean that the X chromosome has an autosomal origin.     

Banding pattern analysis of initial structural chromosome alterations induced by n-methyl-n'-nitro-n-nitrosoguanidine in Syrian hamster cells

Cultured secondary Syrian hamster embryo cells exposed to 0.5 N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) microgram/ml medium exhibited chromatid type of aberrations consisting of gaps, breaks and exchanges. Although no specific chromosome or chromosome segment was preferentially affected, chromosomes belonging to the larger groups tended to more often involved. G-band analysis demonstrated that 80% of the lesions occurred in negative bands, 9% involved the centromere, 3% were on non-banded heterochromatin, and approximately 8% of the lesions could not be definitely categorized by G-band analysis. Whether the lesions occur at positive bands or at the interface between negative and positive bands is difficult to discern by the G-band resolution. The Y chromosome compared to autosomes of similar size rarely had lesions. X chromosome damage was found in both the euchromatic and heterochromatic arms. However, both sex chromosomes, as well as an autosome (E20) which is heterochromatic on its long arm, were not found joined to the chromatids of other chromosomes, further emphasizing that chromosomes with large heterochromatic areas are isolated in terms of chromatid exchange events. The analysis of MNNG induced chromosome damage indicates that the negative bands are the primary site of damage and points of exchange.