Translocations between Eu- and heterochromatic chromosomes,and spermatocytes lacking a heterochromatic set in male mealy bugs

In male mealy bugs the chromosomes of paternal origin become heterochromatic (H) early in embryogeny while those of maternal origin remain euchromatic (E). First instarPseudococcus obscurus Essig males which were irradiated with 3000 r of X-rays carried translocations between E and H chromosomes [T(E;H)'s] in their spermatocytes. In the T(E;H)'s the border between the E and H segments was usually quite sharp, but occasionally short E segments may have become either partially or completely heterochromatic. During the second meiotic division the E and H sets normally segregate to opposite poles. The T(E;H)'s often formed bridges in AII and TII, but in most of the cells they did succeed in reaching one of the poles. The segregation of the T(E;H)'s depended on the relative size of their E and H segments. When the E and H segments were of the same size, the T(E;H)'s segregated more often with the E chromosomes, even though the latter have been observed to be attached to their pole with fewer spindle fibers. Thirty-five of the 173 males analyzed had sectors in their testes which lacked an H set. The number of cysts per sector suggested that each sector was derived from a single irradiated cell. The karyotypes observed in some of the sectors indicated that the lack of an H set was the result of reversal of heterochromatization and not due to the loss of the H set and the endoduplication of the E set. Most of the cells lacking an H set divided normally during the first meiotic division. The second division, however, was abortive and resulted in the production of diploid sperm. Two possibilities for the origin of spermatocytes lacking an H set are considered: (i) that these spermatocytes resulted from X-ray induced reversal of heterochromatization in spermatogonia, and (ii) that these spermatocytes originated from presumptive cyst wall cells whose H set had undergone reversal prior to the irradiation.Supported by grant GB 6745 from the National Science Foundation, Washington, D. C.     

On the mechanism of chromatin loss induced by the B chromosome of maize

Knobbed regions of the regular maize complement frequently are eliminated at the second microspore division in spores which have two or more B chromosomes. Evidence is presented that no or little loss occurs in spores with one B and that the rate is not increased in spores with more than two B's.—The B chromosomes from an unrelated strain proved as effective in inducing loss as did the B's of the original high loss stock.—Chromatin loss induced by B's is restricted to knobbed A chromosomes and occurs only at the second microspore division. Knobbed chromosomes 3, 5, and 9 have been tested and all interact with B's to give loss. Chromosomes with large knobs are more frequently broken than are those with smaller knobs and knobless chromosomes show negligible loss.—Although knobs and B's are essential for chromatin elimination, modifying genes can markedly affect the rate of loss.——Two knobbed heterologous chromosomes undergo simultaneous loss more frequently than expected from independent events. The data indicate that joint loss occurs in competent cells and that preferential assortment of the two deficient chromosomes to specific poles is unlikely.—B chromosomes and deficient chromosomes assort independently at the second microspore anaphase.—Genetic data from crosses with marker genes in both arms of chromosome 3 show that breakage of the postulated dicentric bridge does not occur solely at the centric region since a variety of deficient chromosomes were recovered.—Nondisjunction of B chromosomes and elimination of knobbed chromatin take place during the second microspore mitosis. The argument is advanced that the two phenomena result from faulty replication of heterochromatic segments. The position of the nonreplicating segment in the two kinds of chromosomes determines whether nondisjunction or breakage takes place.—Finally, it is suggested that all of the reported effects of the B chromosome can be accounted for if the B is a parasitic entity having no genetic function other than controlling the replication of its proximal heterochromatic knob and increasing the ability of B-containing sperm cells to compete successfully for fertilization of the egg.     

Histone H3 lysine 9 acetylation pattern suggests that X and B chromosomes are silenced during entire male meiosis in a grasshopper

The facultative heterochromatic X chromosome in leptotene spermatocytes of the grasshopper Eyprepocnemis plorans showed marked hypoacetylation for lysine 9 in the H3 histone (H3-K9) with no sign of histone H2AX phosphorylation. Since H3-K9 hypoacetylation precedes the meiotic appearance of phosphorylated H2AX (gamma-H2AX), which marks the beginning of recombinational DNA double-strand breaks (DSBs), it seems that meiotic sex-chromosome inactivation (MSCI) in this grasshopper occurs prior to the beginning of recombination and hence synapsis (which in this species begins later than recombination). In addition, all constitutively heterochromatic chromosome regions harbouring a 180-bp tandem-repeat DNA and rDNA (B chromosomes and pericentromeric regions of A chromosomes) were H3-K9 hypoacetylated at early leptotene even though they will synapse at subsequent stages. This also suggests that meiotic silencing in this grasshopper might be independent of synapsis. The H3-K9 hypoacetylated state of facultative and constitutive heterochromatin persisted during subsequent meiotic stages and was even apparent in round spermatids. Finally, the fact that B chromosomes are differentially hypoacetylated in testis and embryo interphase cells suggests that they might be silenced early in development and remain this way for most (or all) life-cycle stages.     

Differential DNA synthesis in heterochromatic and euchromatic chromosome sets of Planococcus citri

In males of the mealy bug Planococcus citri, Nur (1966) counted five heterochromatic (H) and about 5, 10, 20, 40, or 80 euchromatic (E) chromosomes in testis sheath nuclei which were undergoing endomitosis. He suggested that the H chromosomes were not replicating and that the nuclei were becoming polyploid as a result of successive cycles of replication of only the E chromosomes. This hypothesis was tested using autoradiography with H³-thymidine to detect DNA synthesis and microspectrophotometric measurements of the Feulgen reaction in nuclei to detect quantitative changes in DNA. — The integrated absorbance of the whole nucleus and of the isolated clump of heterochromatic chromosomes (H body) in polyploid testis sheath nuclei were measured using the mechanical scanner of the CYDAC system. The absorbance of the H body was similar in all testis sheath nuclei examined and was not significantly different from the absorbance of a haploid set of H chromosomes measured after meiosis. The absorbance of the euchromatic component varied in different sheath nuclei, the values closely corresponding to the terms of the series 2c, 4c, 8c. This series is expected if the DNA in the E chromosomes is exactly doubled at each cycle of replication. — Autoradiographs showed that most labeled sheath nuclei had silver grains localized exclusively over euchromatin. With one exception, the remainder of the labeled nuclei had silver grains over both euchromatin and the H body. The observation that euchromatin was much more heavily labeled than the H body and that labeled H bodies occurred at a low frequency and only in the presence of labeled euchromatin suggests that the H body did not incorporate the label and that the silver grains over the H body were the result of -particles which originated in proximal euchromatin.     

Asynchrony of DNA replication and mitotic spiralization along heterochromatic portions of Chinese hamster chromosomes

Sequence of DNA synthesis and mitotic chromosome spiralization along heterochromatic portions of the sex (X₁X₂) and of some marker chromosomes in cultured Chinese hamster cells were studied, employing two methods: study of segmentation pattern caused in chromosomes with colcemid, and autoradiography with tritiated thymidine. The heterochromatic portions of all chromosomes studied were characterized by striking internal asynchrony of DNA replication. In particular, they had segments that replicated relatively early. The short arm of the X₂ chromosome, heterochromatic in female somatic cells, had at least three such segments. Replication patterns of the long arms of the X₁ and X₂ chromosomes were different. In X₁ this arm contains several segments showing relatively early replication. The long arm of X₂ had no similar segments. The possible significance of the data obtained is discussed with regard to the problem of genetic inertness of heterochromatin. At the terminal stage of the S period, H³-thymidine seems to be incorporated into condensed chromatin of interphase nuclei. On the basis of the data obtained, it is proposed that during replication of heterochromatin consecutive despiralization of parts of it takes place.     

Analysis of Y chromosome heterochromatin in Rumex thyrsiflorus

The Y chromosome heterochromatin in Rumex thyrsiflorus has been analyzed. In natural populations the Y chromosome shows a higher morphological variability than the X chromosome. The total duration of replication of Y chromosomes is about 2 hrs longer than that of euchromatin. Autoradiography with tritiated thymidine showed that chromocentres formed by Y chromosomes in interphase nuclei retain their heterochromatic form during DNA replication. — Y chromosome heterochromatin in interphase nuclei is stained pink, while the rest of the nucleus stains green after fast green-eosin staining for histones. — During the premeiotic stage of PMC development Y chromosomes are no longer visible as compact bodies and become more fuzzy in appearance. A diffuse state of Y coincides with intense RNA synthesis. Therefore genetic activity of Y chromosomes or their parts during premeiotic stage of microsporogenesis is postulated.     

Nonreplication of heterochromatic chromosomes in a mealy bug,Planococcus citri (Coccoidea: Homoptera)

In males of mealy bugs with the lecanoid chromosome system, the paternal set of chromosomes becomes heterochromatic in early embryogeny. In males of the mealy bug, Planococcus citri, the heterochromatic (H) set in testis sheath cells and in most of the oenocytes apparently did not replicate while the euchromatic (E) set was undergoing several cycles of endoreplication. In third instar males, testis sheath cells in endoanaphase and endotelophase exhibited 5H and either 40 or 80E chromosomes. The increase in the number of E chromosomes was attributed to the replication of only the E chromosomes. Oenocytes of third instar males had 0, 5, or 10H chromosomes and from 10 to 240E chromosomes. The oenocytes with 5H chromosomes had a mean of 50.8E chromosomes, and those with 10H chromosomes had a mean of 155.6E chromosomes. Nuclear and cell fusion was considered as a means of producing the various numbers of H and E chromosomes in oenocytes, and it was concluded that although nuclear fusion probably took place, the differences between the number of H and E chromosomes was at least in part due to replication of only the E chromosomes. The size of the H chromosomes was about the same in all the testis sheath cells and the oenocytes irrespective of the level of endopolyploidy for the E set. These H chromosomes apparently did not increase in polyteny, because they were only about half the size of the H chromosomes in prophase I of spermatogenesis. The significance of the nonreplication of the H set and the control of nonreplication are briefly discussed.This study was aided by a grant (GB-1585) from the National Science Foundation, Washington, D.C.     

Kinetics of DNA replication in the Indian muntjac chromosomes as studied by quantitative autoradiography

DNA replication patterns of individual chromosomes and their various euchromatic and heterochromatic regions were analyzed by means of quantitative autoradiography. The cultured cells of the skin fibroblast of a male Indian muntjac were pulse labeled with ³H-thymidine and chromosome samples were prepared for the next 32 h at 1–2 h intervals. A typical late replication pattern widely observed in heterochromatin was not found in the muntjac chromosomes. The following points make the DNA replication of the muntjac chromosomes characteristics: (1) Heterochromatin replicated its DNA in a shorter period with a higher rate than euchromatin. (2) Two small euchromatic regions adjacent to centromeric heterochromatin behaved differently from other portions of euchromatin, possessing shorter Ts, higher DNA synthetic rates and starting much later and ending earlier their DNA replication. (3) Segmental replication patterns were observed in the chromosomes 2 and 3 during the entire S phase. (4) Both homologues of the chromosome 3 showed a synchronous DNA replication pattern throughout the S phase except in the distal portion of the long arms during the mid-S phase.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

Replication variants of the human inactive X chromosome

Replication variants of the inactive X chromosome were investigated in lymphocytes from six donors by means of terminal BrdU or thymidine incorporation. There were interindividual differences in the incidence of particular variants. In endoreduplicated and tetraploid cells both allocyclic X chromosomes showed the same replication sequence. The Xp22 band of the allocyclic X chromosome seemed to replicate later than the homologous material in some cells. Initiation time of DNA synthesis within the inactive X chromosome was found to be stable; termination time, however, varied greatly relative to the other chromosomes. Early completion of replication within the heterochromatic X chromosome could be demonstrated preferentially for the Xq25–27 terminal sequence, but other variants expressed the phenomenon also. A variable replication rate of the inactive X chromosome is believed to be responsible for its asynchronous, independent replication. The biological significance of the phenomenon is discussed with respect to cell differentiation.     

Synaptonemal complex analysis in spermatocytes and oocytes of tertiary trisomic Ts(512)31H mice with male sterility

Whole-mount preparations of silver-stained spermatocytes and oocytes from Ts(512)31H mice were examined in the electron microscope. The 5(12) chromosome was associated with the XY bivalent in the large majority of spermatocytes, whereas in about one-half of the oocytes, the 5(12) was associated with either unpaired chromosomes or heterochromatic parts of chromosomes or showed self-synapsis. There was a tendency for 5(12) chromosomes to be more fully heterochromatic in oocytes than in spermatocytes. A large proportion of oocytes (50%) and a much smaller proportion of spermatocytes exhibited various errors of chromosome pairing, but these proportions were only marginally greater than in control gametocytes from mice with normal karyotypes. It is concluded that the observed errors of pairing bear no simple relation to the almost complete breakdown of spermatogenesis and the marked impairment of oogenesis that occur in tertiary trisomic Ts(5(12))31H mice.     

DNA REPLICATION PATTERNS AND CHROMOSOMAL PROTEIN SYNTHESIS IN OPOSSUM LYMPHOCYTES IN VITRO

DNA replication patterns were determined in the autosomes and sex chromosomes of phytohemagglutinin-stimulated lymphocytes from the opossum (Didelphis virginiana) by employing thymidine-³H labeling and high-resolution radioautography. Opossum chromosomes are desirable experimental material due to their large size, low number (2n = 22), and morphologically distinct sex chromosomes. The autosomes in both sexes began DNA synthesis synchronously and terminated replication asynchronously. One female X chromosome synthesized DNA throughout most of the S phase. Its homologue, however, began replication approximately 3.5 hr later. The two X's terminated DNA synthesis synchronously, slightly later than the autosomes. This form of late replication, in which one X chromosome begins DNA synthesis later than its homologue but completes replication at the same time as its homologue, is apparently unique in the opossum. The male X synthesized DNA throughout S while the Y chromosome exhibited late-replicating characteristics. The two sex chromosomes completed synthesis synchronously, slightly later than the autosomes. Grain counts were performed on all chromosomes to analyze trends in labeling intensity at hourly intervals of S. By analyzing the percent of labeled mitotic figures on radioautographs at various intervals after introduction of arginine-³H, chromosomal protein synthesis was found not to be restricted to any portion of interphase but to increase throughout S and into G₂.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Two new chromodomain-containing proteins that associate with heterochromatin in <Emphasis Type="Italic">Sciara coprophila</Emphasis> chromosomes

We report here the molecular and cytological characterization of two proteins, ScoHET1 and ScoHET2 (for Sciara coprophila heterochromatin), which associate to constitutive heterochromatin in the dipteran S. coprophila. Both proteins, ScoHET1 of 37 kDa and ScoHET2 of 44 kDa, display two chromodomain motifs that contain the conserved residues essential for the recognition of methylated histone H3 at lysine 9. We raised antibodies to analyze the chromosomal location of ScoHET1 and ScoHET2 in somatic and germline cells. In S. coprophila polytene chromosomes, both proteins associate to the pericentromeric regions and to the heterochromatic subterminal bands of the chromosomes. In germinal nuclei, ScoHET1 and ScoHET2 proteins distribute to the heterochromatic regions of the regular chromosome complement and are abundantly present along the heterochromatic germline-limited “L” chromosomes. We investigated histone methylation modifications and found that all heterochromatic regions enriched in ScoHET1/ScoHET2 proteins exhibit high levels of di- and tri-methylated histone H3 at lysine 9. Taken together, our results support that the association of ScoHET1/ScoHET2 to heterochromatin is mediated by histone H3K9 methylation. Using 5-methylcytosine antibodies, we proved the cytological detection of DNA methylation in S. coprophila. From our observations in L germline chromosomes, heterochromatin in S. coprophila is highly enriched in DNA 5-methylcytosine residues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

Chromosomes and DNA of Mus: Terminal DNA synthetic sequences in three species

The DNA replication patterns of the terminal S phase of three species of Mus were analyzed by tritiated thymidine autoradiography. The centromeric heterochromatin of M. fulvidiventris is the latest component to finish DNA synthesis. The Y chromosome finishes replication earlier than the centromeric heterochromatin. The centromeric heterochromatin of M. musculus, on the other hand, is not the latest component to finish DNA synthesis. At the very late S phase, grains are found in the euchromatic arms instead of the heterochromatic areas. The hot X and the hot Y can be identified in the majority of, but not all, cases. The heterochromatic short arms of the autosomes in M. dunni finish DNA replication earlier than many areas in the euchromatic long arms and the heterochromatin of the sex chromosomes. This indicates that in M. dunni there are at least two types of heterochromatin. The late-replicating zones in the euchromatic long arms are distinctly banded. This banded grain pattern can be seen in all Mus species observed, but in M. dunni it is most exaggerated. Late-replicating chromosome segments can be demonstrated also by 2+ cycles of BUdR incorporation and Giemsa staining.     

Chromosome nondisjunction and instabilities in tapetal cells are affected by B chromosomes in maize

Abnormal mitosis occurs in maize tapetum, producing binucleate cells that later disintegrate, following a pattern of programmed cell death. FISH allowed us to observe chromosome nondisjunction and micronucleus formation in binucleate cells, using DNA probes specific to B chromosomes (B's), knobbed chromosomes, and the chromosome 6 (NOR) of maize. All chromosome types seem to be involved in micronucleus formation, but the B's form more micronuclei than do knobbed chromosomes and knobbed chromosomes form more than do chromosomes without knobs. Micronuclei were more frequent in 1B plants and in a genotype selected for low B transmission rate. Nondisjunction was observed in all types of FISH-labeled chromosomes. In addition, unlabeled bridges and delayed chromatids were observed in the last telophase before binucleate cell formation, suggesting that nondisjunction might occur in all chromosomes of the maize complement. B nondisjunction is known to occur in the second pollen mitosis and in the endosperm, but it was not previously reported in other tissues. This is also a new report of nondisjunction of chromosomes of the normal set (A's) in tapetal cells. Our results support the conclusion that nondisjunction and micronucleus formation are regular events in the process of the tapetal cell death program, but B's strongly increase A chromosome instability.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

Early replication banding in <Emphasis Type="Italic">Leporinus</Emphasis> species (Osteichthyes,Characiformes) bearing differentiated sex chromosomes (ZW)

There are few examples of differentiated sex chromosomes in fishes. In the genus Leporinus, seven species present a highly differentiated ZW system, derived from heterochromatinization process. Cytogenetic analyses carried out in three of these fish species, Leporinus obtusidens, L. elongatus and L. reinhardti, through RBG-banding, showed late replication bands, coincident with heterochromatic regions in both Z and W chromosomes. A similar interstitial early replication segment was observed in the complex heterochromatic region along the Wq arms in the three species, which might correspond to a pseudoautosomal segment (SD, sex determining locus). Asynchrony related to the replication pattern among different Z chromosomes was not observed. When the identification of nuclear organizer regions by silver nitrate was performed over chromosomal preparations previously exposed to 5-bromo-2′-deoxyuridine (BrdU), remarkable positive signals at interstitial and telomeric position were observed on the q arms of W chromosomes in the species L. elongatus and L. reinhardti. The absence of 18S ribosomal RNA gene loci in this region, formerly demonstrated by FISH, indicates that this argentophilic behavior is putatively due to heterochromatin decondensation caused by BrdU incorporation, favoring such Ag+ reaction. Early and late replication bands were also observed in the heterochromatic portions of Z and W chromosomes, indicating that euchromatic and heterochromatic regions are interspersed. The present data suggest a significant level of heterochromatic complexity in the sex chromosomes of each species. On the other hand, the replication pattern shared by them supports a monophyletic origin.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.