Heterochromatic Regions of Maize A- and B-Chromosomes

A collection of maize forms from the Vavilov All-Russia Institute of Plant Breeding (VIR) was studied. We compared variation in the following traits: the number and size of heterochromatic knob regions (HKs) of chromosomes of mother pollen cells at pachytene depending on the presence (B⁺) or absence (B⁰) of B-chromosomes; size of B-chromosomes (general, of heterochromatic part); and the frequency of B⁺ plants in groups of forms contrasting in the HK number (10–15, 4–10, 2–7, 1–4). It was shown that B chromosomes had statistically significantly different effects on HK polymorphism, relative heterochromatin content in the cell of multiknob and knobless forms, selection of plants for early ear flowering in these forms. The combination of maximum (and minimum) sizes of heterochromatic regions of A- and B-chromosomes was established; the genomic level of control of the trait is suggested. The role of the relationship of polymorphism at heterochromatic regions of A- and B-chromosomes between them and with the systems of maize reproduction is considered as a mechanism of maintenance of optimum plant heterozygosity via adaptive ontogenetic redistribution of heterochromatin among loci, chromosomes, and gametes.     

Differential staining of plant chromosomes with Giemsa

Simple Giemsa staining techniques for revealing banding patterns in somatic chromosomes of plants are described. The value of the methods in the recognition of heterochromatin was demonstrated using five monocotyledonous and two dicotyledonous species. In Trillium grandiflorum the stronger Giemsa stained chromosome segments were shown to be identical with the heterochromatic regions (H-segments) revealed by cold treatment. Preferential staining of H-segments was also observed in chromosomes from three species of Fritillaria and in Scilla sibirica. Under suitable conditions the chromosomes of Vicia faba displayed a characteristic banding pattern and the bands were identified as heterochromatin. The Giemsa techniques proved to be more sensitive than Quinacrine fluorescence in revealing a longitudinal differentiation of the chromosomes of Crepis capillaris, where plants with and without B-chromosomes were examined. Again all chromosome types had their characteristic bands but there was no difference in Giemsa staining properties between the B-chromosomes and those of the standard complement.     

玉米花粉单倍体植株染色体上异染色质的变异

我们用Giemsa BSG C-带技术检查了玉米花药培养获得的花粉单倍体植株根尖细胞染色体上异染色质的变异,观察结果表明,有的植株所显示的C-带数目是与供体植株的相一致,有的植株所显示的C-带数目则发生了显著变化,其中有的增加,有的减少。并讨论了异染色质发生变异的可能原因。还相应地观察到间期核中染色中心的变化是与中期染色体上C-带数目的变化相一致。     

Cytogenetic Analysis of A- and B-Chromosomes of Prochilodus Lineatus (Teleostei, Prochilodontidae) Using Different Restriction Enzyme Banding and Staining Methods

Different cytogenetic techniques were used to analyse the chromosomes of Prochilodus lineatus with the main objective of comparing the base composition of A- and B-chromosomes. The results of digestion of chromosomes with 10 different restriction endonucleases (REs), silver staining, CMA₃ staining and C-banding indicated the existence of different classes of highly repetitive DNA in the A-set and also suggested the existence of compositional differences between the chromatin of A- and B-chromosomes. The 5-BrdU incorporation technique showed a late replicating pattern in all B-chromosomes and in some heterochromatic pericentromeric regions of A-chromosomes. The cleavage with RE BamHI produced a band pattern in all chromosomes of P. lineatus which permitted the tentative pairing of homologues in the karyotype of this species. We concluded that the combined use of the above techniques can contribute to the correct identification of chromosomes and the karyotypic analysis in fishes. On the basis of the results, some aspects of chromosome structure and the origin of the B-chromosomes in P. lineatus are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Distribution and clustering of two highly repeated sequences in the A and B chromosomes of maize

Summary Clones from a family of highly repeated sequences present in a heterochromatin rich maize line have been characterized by sequencing and chromosome location. The repeats differ from each other in length and degree of sequence homology, and show areas rich in purine and pyrimidine. In situ hybridization experiments indicate that the repeats are mainly located in the knob heterochromatin of the A chromosomes and the centromeric heterochromatin of the B chromosome. However, in addition to previously published data, some copies are also distributed in euchromatic regions of the A chromosomes and in the distal heterochromatic block of the B chromosome. The results are discussed in relation to the centromeric activity of maize heterochromatin.Research work is sponsored by C.N.R. Italy, Special grant I.P.R.A., Subproject 1, Paper No. 300     

Heterochromatin composition and nucleolus organizer activity in four canid species

Sequential staining with a counterstain-contrasted fluorescent banding technique (chromomycin A3-distamycin A-DAPI) revealed the occurrence of distamycin A-4,6-diamidino-2-phenylindole (DA-DAPI) staining heterochromatin in the centromeric regions of chromosomes 33, 36, 37, and 38 in the wolf (Canis lupus pallipes) and of chromosomes 13, 16, and 23 in the blue fox (Alopex lagopus). The red fox (Vulpes vulpes) lacked such regions. Staining with DAPI--actinomycin D produced a QFH-type banding pattern with clearcut differences in the staining behaviour of DA-DAPI positive regions between these three canid species. Staining with the fluorochrome D 287/170 did not preferentially highlight any of the DA-DAPI positive regions in any of them. Counterstain-enhanced chromomycin A3 R-banding and studies of nucleolus organizer region location and activity confirmed a close relationship between the karyotype of the wolf and the domestic dog. Few heterochromatic marker bands were encountered in these two species, but heterochromatin polymorphism was evident in the blue fox.     

The heterochromatic B chromosome of maize: the segments affecting recombination

The B chromosome of maize is known to increase recombination in specific regions of the genome. In an attempt to determine the portion of the B responsible for crossover enhancement, translocations between the B and A chromosomes were used to dissect the B into four distinct segments. The effects of the segments on crossing over were studied in a sensitive region composed of chromatin from chromosomes 3 and 9. The relatively euchromatic chromomeres terminating the B lacked enhancement activity, but the remaining segments, all of which possess large amounts of heterochromatin, were capable of elevating recombination. There was no localization of activity to a specific heterochromatic region, however.     

Obervations on the Induction of Position Effect Variegation of Euchromatic Genes in Drosophila Melanogaster

In the T(1;2)dor(var7) translocation, the 1A-2B7-8 segment of the X chromosome is brought to the vicinity of 2R-chromosome heterochromatin resulting in position effect variegation of dor, BR-C and more distal genes, as well as compaction of chromatin in this segment. By irradiation of T(1;2)dor(var7), nine reversions (rev) to a normal phenotype were recovered. In two cases (rev27, rev226), the 1A-2B7-8 section is relocated to the 19A region of the X chromosome, forming free duplications (1A-2B7-8/19A-20F-X-het). Modifiers of position effect do not change the normal expression of the BR-C and dor genes in these duplications. In five reversions (rev3, rev40, rev60, rev167, rev175), free duplications have formed from the 1A-2B7-8 fragment and X chromosome heterochromatin. In these rearrangements, modifiers of position effect (low temperature, removal of Y and 2R-chromosome heterochromatin and a genetic enhancer (E-var(3)201) induce position-effect again. Two reversions (rev45 and rev110) are associated with additional inversions in the original dor(var7) chromosomes. The inversions relocate part of the heterochromatin adjacent to the 1A-2B7-8 section into new positions. In T(1;2)dor(rev45), position-effect is seen in the 2B7-8-7A element as compaction spreading from 2B7-8 proximally in some cases as far as the 5D region. Thus, in rev45 the pattern of euchromatin compaction is reciprocal to that of the initial dor(var7) strain. Apparently, it is due to the same variegation-evoking center near the 2R centromere in both cases. In all nine revertants, weakening or complete disappearance of the position-effect is observed despite retention of the 20- kb heterochromatic segment adjacent to the 1A-2B7-8 region. Thus, a 20-kb heterochromatic sequence does not inactivate euchromatin joined to it.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

Unusual occurrence of a ZZ/ZW sex-chromosome system and supernumerary chromosomes in Characidium cf. fasciatum (Pisces, Characiformes, Characidiinae)

Individuals of two populations of the fish Characidium cf. fasciatum were cytogenetically studied and showed a basic diploid number of 50 chromosomes. Some fishes were found to have 51 to 54 chromosomes due to the presence of one to four small subtelocentric/acrocentric supernumerary chromosomes. When analyzed by conventional Giemsa staining, male and female specimens of C. cf. fasciatum from the Quinta stream and Pardo River presented the same basic karyotypic macro- and microstructure, consisting of 32 metacentric and 18 submetacentric chromosomes. Ag-NORs were terminally located on the long arms of two submetacentric chromosome pairs. Constitutive heterochromatin was identified by C-banding as small pericentromeric blocks in the majority of the chromosomes, and B-chromosomes were found to be heterochromatic. The occurrence of one totally heterochromatic submetacentric chromosome restricted to females and considered as an unusual feature in fish karyotypes led to the identification of a ZZ/ZW sex-chromosome system. The implications of chromosomic differentiation observed in the genus Characidium are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

NOR Sites Detected by Ag-DAPI Staining of an Unusual Autosome Chromosome of Bradysia Hygida (Diptera:Sciaridae) Colocalize with C-banded Heterochromatic Region

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.     

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.

     

Microdissection and sequence analysis of pericentric heterochromatin from the Drosophila melanogastermutant Suppressor of Underreplication

In the Suppressor of Underreplication( SuUR) mutant strain of Drosophila melanogaster, the heterochromatin of polytene chromosomes is not underreplicated and, as a consequence, a number of beta-heterochromatic regions acquire a banded structure. The chromocenter does not form in these polytene chromosomes, and heterochromatic regions, normally part of the chromocenter, become accessible to cytological analysis. We generated four genomic DNA libraries from specific heterochromatic regions by microdissection of polytene chromosomes. In situ hybridization of individual libraries onto SuUR polytene chromosomes shows that repetitive DNA sequences spread into the neighboring euchromatic regions. This observation allows the localization of eu-heterochromatin transition zones on polytene chromosomes. We find that genomic scaffolds from the eu-heterochromatin transition zones are enriched in repetitive DNA sequences homologous to those flanking the suppressor of forked gene [ su(f) repeat]. We isolated and sequenced about 300 clones from the heterochromatic DNA libraries obtained. Most of the clones contain repetitive DNA sequences; however, some of the clones have unique DNA sequences shared with parts of unmapped genomic scaffolds. Hybridization of these clones onto SuUR polytene chromosomes allowed us to assign the cytological localizations of the corresponding genomic scaffolds within heterochromatin. Our results demonstrate that the SuUR mutant renders possible the mapping of heterochromatic scaffolds on polytene chromosomes.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

The quinacrine-fluorescence patterns of the chromosomes of Allium carinatum

Quinacrine staining of somatic chromosomes in Allium carinatum shows intense fluorescence patterns which allow their recognition and the study of their degree of heterozygosity. This makes possible the study of chromosome polymorphism at a level until now impossible to achieve. The intense fluorescence patterns correspond to heterochromatic segments visible as darkly stained regions in prophase chromosomes. Interphase nuclei show fluorescent chromocentres of the same size and distribution as in conventionally stained preparations, and there is a good correlation between intense fluorescence patterns, late replicating DNA and heterochromatin.     

Chromosome banding in Amphibia

The distribution and quantity of constitutive heterochromatin and of the nucleolus organizer regions (NORs) on the chromosomes of 22 species of bufonids and hylids (Amphibia, Anura) was investigated. Three different kinds of constitutive heterochromatin were found and the frequency of brightly fluorescing heterochromatic regions was remarkably high. On almost all chromosomes there is centric and telomeric heterochromatin. Quantitative estimates of heterochromatin demonstrate that large DNA differences among closely related species can not be attributed to differing quantities of constitutive heterochromatin. In all species investigated, only one homologous pair of NORs was found, which lies preferentially in the proximal and interstitial segments of the long chromosome arms. The NORs are always associated with constitutive heterochromatin on both sides. The size variability between homologous NORs is very high. In the euchromatic regions of the metaphase chromosomes, neither Q- nor G-bands can be demonstrated; this can be attributed to an extremely strong contraction of the anuran chromosomes. On the basis of these results various mechanism of the chromosomal evolution in Anura are discussed.     

Cytogenetic studies of the Australian rodent,Uromys caudimaculatus,a species showing extensive heterochromatin variation

The Australian rodent, Uromys caudimaculatus, consists of two chromosome races, a) The Southern Race is characterized by the possession of two to twelve B-chromosomes. These vary considerably in size, morphology, and C- and G-banding characteristics, they behave as univalent at meiosis and are inherited in a random manner, b) The Northern Race lacks Bchromosomes, but possesses large blocks of distal C-positive heterochromatin on between 18 and 28 of the 46 chromosomes. The C-blocks may be entirely G-positive, entirely G-negative, or G-banded, suggesting heterogeneity within the C-blocks. There is extensive variation both between and within populations of the northern race in the number and size of the distal heterochromatic blocks. There is no apparent difference between the races in chiasma frequency. The northern race does have a much higher proportion of interstitial versus distal chiasmata, although it is probable that this is merely a reflection of lack of crossing over in the heterochromatic blocks rather than an actual shift of chiasma localisation within the euchromatin. Despite the extensive differences between the races in the amount and organization of constitutive heterochromatin, hybrids show no abnormalities at meiosis and they are fully fertile.     

The modification of crossing over in maize by extraneous chromosomal elements

Summary Five regions of the maize genome were tested for their response to endogenous factors influencing recombination. These included heterochromatic B chromosomes and abnormal chromosome 10 as well as the sex in which recombination occurred.The frequency of recombination in the proximal A ₂-Bt and Bt-Pr segments of chromosome 5 was increased in the presence of B chromosomes, with the male meiocytes showing a greater response than the female meiocytes. In addition, experiments involving 0, 1, 2 and 4 B's revealed a dosage effect of B chromosomes on crossing over in chromosome 5. Recombination in the proximal Wx-Gl ₁₅ interval of chromosome 9 was found to be slightly higher than normal in male flowers when two B chromosomes were present. This increase was accompanied by a decrease in the adjacent Sh-Wx segment. Crossing over in the distal C-Sh segment and in the C-Sh-Wx-Gl ₁₅ regions of female flowers was unaffected by B's.Comparisons of plants heterozygous for abnormal chromosome 10 (K10 k10) and homozygous for the standard chromosome 10 (k10 k10) showed that abnormal 10 greatly enhances crossing over in the A ₂-Bt and Bt-Pr segments of chromosome 5. In contrast to the finding with B's, the effect is greater in female than in male sporocytes. K10 showed no significant effect on recombination in the C-Sh-Wx-Gl ₁₅ region of chromosome 9 except in male sporocytes, where there was a slight increase in the Sh-Wx region of 0 B K10 k10 plants and a possible interaction with B chromosomes to raise the level of recombination between Wx and Gl ₁₅. The fact that the regions adjacent to the centromere of chromosome 9 show little or no response to the presence of K10 indicates that the proximal heterochromatin of this chromosome differs qualitatively from that of other maize chromosomes. This conclusion is supported by a comparison of the effects of B chromosomes, K10 and sex on crossing over in chromosomes 5 and 9.Dedicated to Dr. M. M. Rhoades on the occasion of his seventieth birthday.     

Interaction between rye B-chromosomes and wheat genetic systems controlling homoeologous pairing

The interactive effect on homoeologous pairing of rye B-chromosomes with the absence of both pairing suppressor (3A, 3D, 5B) and promotor (3B, 5A, 5D) chromosomes of common wheat (Triticum aestivum L.) is analyzed by comparison of pairing at Metaphase I of 27-, 27+2B, 28- and 28+2B-chromosome plants. These plants were obtained from crosses between the respective wheat monosomics (2n=41) and rye plants (Secale cereale L.) carrying or not carrying two B-chromosomes (2n=14 or 14+2Bs). —The effect of rye B-chromosomes on pairing depends on the function of the wheat chromosome which is absent in the appropriate hybrids, i.e., rye B-chromosomes have a suppressor effect on pairing when the pairing suppressing wheat chromosomes 3A, 3D or 5B are absent, while they behave as promotors when the pairing promoting chromosomes 3B, 5A or 5D are absent.     

Accentuated polymorphism of heterochromatin and nucleolar organizer regions in Astyanax scabripinnis (Pisces, Characidae): tools for understanding karyotypic evolution

Astyanax scabripinnis has been considered a species complex because it presents high karyotypic and morphological variability among its populations. In this work, individuals of two A. scabripinnis populations from different streams in the same hydrographic basin were analyzed through C‐banding and AgNOR. Although they present distinct diploid numbers, they show meta and submetacentric chromosome groups highly conserved (numerically and morphologically). Other chromosomal characteristics are also shared by both populations, as the pattern of constitutive heterochromatin distribution (large blocks in the telomeric regions of subtelocentric and acrocentric chromosomes) and some nucleolar chromosomes. Inter‐individual variations both in the number and size of heterochromatic blocks, and in the number and localization of NORs were verified in the studied populations, characterizing them as polymorphics for these regions. The mechanisms involved in the dispersion of heterochromatin and NORs through the karyotypes, as well as the possible events related to the generation of polymorphism of those regions are discussed. Furthermore, relationships between these populations and within the context of the scabripinnis complex are also approached. This revised version was published online in July 2006 with corrections to the Cover Date.