Study on the Chromosome N-Banding in Plants

The present paper is dealing with the investigation of the chromosome N-banding technique and the N-banding patterns in Hordeum vulgare, Triticum aestivum, Secale cereale, Vicia faba and Allium eepa. Two N-banding techniques were applied. First, the chromosome slides were stained with Giemsa solution. Second, the slides were treated in 1 M NaH2PO4 solution at 92—94 ℃ for 3.5—8.5 min. After rinsing in tap water they were stained with Giemsa solution. The experiments have demonstrated that the N-banding technique is simple and rapid and the banding patterns are distinctive. The data of N-banding patterns indicated that the N bands did not display the nucleolus organisers exclusively. The comparison of the N-banding patterns of these plants with their C-banding patterns shows that in some of these plants although some regions of N-bands and C-bands correspond, there are a number of instances where regions show N-bands but no C-bands and vice-versa. Therefore, a combination of the N-banding and C-banding techniques should be valuable in the cytological identification of plant chromosome. Like the C-bands, the N-bands are also useful markers in cytogenetics.     

Characterization of heterochromatin in Schistocerca gregaria by C- and N-banding methods

Mitotic and meiotic chromosomes of Schistocerca gregaria were C-, mild N- and strong N-banded. After C-banding, three out of eleven autosomes show, in addition to the centromeric C-bands, a second C-band. — The mild N-banding method produces a single N-band in each of only four chromosomes. With the exception of one N-band these mild N-bands correspond to the non-centromeric, second C-bands, indicating the heterochromatic character of at least three mild N-band regions. — The strong N-banding technique produces bands both at the C- and mild N-band positions and additionally a third band in one chromosome (M₈), not present after C- or mild N-banding. — The N-bands do not correspond to the nucleolus organizer regions. Because of the mechanisms of the N-banding methods, it is concluded that the centromeric heterochromatin, as well as the non-centromeric N-band regions, contain high quantities of non-histone proteins. Presumably a specific difference exists between the non-histone proteins in the centromeric and non-centromeric N-band regions because the centromeres are banded by the strong N-banding technique, but not after mild N-banding. It is concluded that the N-band regions (two exceptions) contain a heterochromatin type which has the following features in common with the -heterochromatin of Drosophila: C- as well as N-banding positive, high nonhistone protein content, repetitive and late replicating DNA. It is discussed whether the N-banded heterochromatin regions of Schistocerca contain that DNA fraction which is, like the Drosophila -heterochromatin, underreplicated in polyploid nuclei.     

Selective staining of the same set of nucleolar phosphoproteins by silver and Giemsa

Gel electrophoresis of nucleolar isolates from Zajdela ascites hepatoma cells followed by various staining procedures revealed a common set of bands that stained selectively with silver and Giemsa. The gel bands, corresponding to molecular weights of 104, 78, 37, and 29 kilodaltons (kd), appeared to contain phosphoproteins that were at least partly associated with oligo-deoxyribonucleotides. Enzyme digestion studies showed that the Giemsastainability was due to the phosphorylated state of the proteins. The positive selective silver-staining reaction in gels could be most likely attributed to the high content of carboxyl groups present in these phosphoproteins. The significance of these findings in relation to cytological results produced by selective silver staining of nucleolus organizing regions (NORs) and by Giemsa N-banding is discussed.     

Recognition of Two Types of Positive Staining Chromosomal Material by Manipulation of Critical Steps in the N-Banding Technique

The nucleolar regions on chromosomes 1B and 6B of Triticum aestivum L. cv Chinese Spring wheat can reliably be observed after careful control of the Giemsa N-banding technique. Identification of rye (Secale cereaie) chromosomes using N-banding is demonstrated and compared to a simple C-banding method. The N-banding in rye chromosomes and the nucleolar sites on IB and 6B of wheat differ from the normal N-banding sites of wheat chromosomes. Further, the banding of these nucleolar regions and of the rye chromosomes does not reappear in preparations that have been retreated with hot acid buffer. These differences provide evidence for at least two types of chromatin that stain darkly (positively) using N-banding. The critical procedures in the N-banding technique and the use of alternatives to 1 M NaH₂PO₄ buffer are discussed along with the possible basis of N-band formation.     

Studies on polytene chromosomes of Smittia parthenogenetica (Chironomidae,Diptera)

In polytene chromosome II of Smittia parthenogenetica a heterochromatin insertion has been studied which is derived from a germ-line limited chromosome section (Bauer, 1970). This insertion is C-banding positive, late replicating, inactive in RNA synthesis, fluoresces brightly with quinacrine and is polytenized. After N-banding a major part of the heterochromatin insertion is N-banding negative, whereas in the centre of the insertion a N-banding positive body is present. The properties of the N-positive and N-negative parts of the inserted heterochromatin section are compared with the properties of the heterochromatin of Chironomus melanotus and Drosophila melanogaster. It is concluded that the heterochromatin insertion consists of two different heterochromatin types and it is discussed whether the N-banding positive part within the insertion represents a heterochromatin type which is underreplicated during polytenization.Dedicated to Professor Dr. Hans Bauer in honour of his 75th birthday on September 27, 1979     

Somatic karyotype,heterochromatin distribution,and nature of chromosome differentiation in common wheat,Triticum aestivum L. em Thell

Heterochromatin distribution and structural differentiation of somatic chromosomes of five common wheat cultivars — Chinese Spring, Wichita, Cheyenne, Timstein, and Hope — were studied by an acetocarmine/N-banding technique. Detailed morphological observations on acetocarmine stained somatic chromosomes of Chinese Spring were made on all A genome chromosomes (except 1A), all B genome chromosomes, and chromosomes 1D, 2D, and 7D. N-banding patterns of chromosomes 2A, 3A, 5A, 6A, 1D, 2D, and 7D were described for the first time. Substitution lines of 21 individual chromosomes each of Cheyenne, Timstein, and Hope in Chinese Spring were analyzed by N-banding. A high frequency of N-band polymorphism was observed, especially for most of the B genome chromosomes. Chromosomes 3A, 5A, 2D, and 7D showed a constant banding pattern. Three cases of doubtful substitutions, Hope 2A, 2B, and Timstein 7A, and several cases of incomplete and chromosomally modified substitutions were observed. The reduced level of chromosome pairing that is often observed in intercultivar hybrids of wheat may be due to heterochromatic differentiation, genic and structural heterozygosity, or hybrid dysgenesis.     

Identification of wheat-barley addition lines with N-banding of chromosomes

The seven chromosomes of barley (Hordeum vulgare) have been identified individually by their distinctive N-banding pattern. Furthermore all of the barley chromosome N-banding patterns were found to be recognizably different from those exhibited by wheat chromosomes, making it possible to identify individual barley chromosomes when present in a wheat background. N-banding has therefore been used to identify the individual barley chromosomes present in (a) reciprocal wheat-barley F₁ hybrids, including some with abnormal chromosome constitution, and (b) a set of wheat-barley addition lines produced in this laboratory. The value of N-banding for detecting translocations between wheat and barley chromosomes and for isolating lines possessing a pair of barley chromosomes substituted for a particular pair of wheat chromosomes is also demonstrated.     

Comparison of the karyotypes ofPsathyrostachys juncea andP. huashanica (Poaceae) studied by banding techniques

The karyotypes ofP. juncea (Elymus junceus) andP. huashanica (both outbreeders) were investigated by Feulgen-staining and by C-, N-, and Agbanding, based on a single plant in cach case. Both species have 2n=2x=14 and large chromosomes, possibly a generic character. The karyotype ofP. juncea has 8 metacentrics and 6 SAT-chromosomes with minute, heterochromatic satellites while that ofP. huashanica has 9 metacentrics and 5 SAT-chromosomes only, 2 of which with small, heterochromatic satellites. The C-banding patterns ofP. juncea chromosomes comprise from one to five, mostly small, bands at distal, and terminal positions, while those ofP. huashanica chromosomes are characterized by large telomeric bands in most arms. Banding patterns and chromosome morphology allow identification of the homologues of the seven chromosome pairs inP. juncea, but of two pairs inP. huashanica only. The patterns of both taxa are polymorphic, supporting that both taxa are outbreeders. The karyotypic characters suggest thatP. juncea is more closely related toP. fragilis than either is toP. huashanica. N-banding stains weakly. Silver nitrate staining demonstrates that nucleolus organizers of both species have different nucleolus forming capacities. The presence of micronucleoli suggests that both species have an extra unidentified chromosome with nucleolus forming capacity.     

Chromosomal repatterning in crocodiles: C,G and N-banding and the in situ hybridization of 18S and 26S rRNA cistrons

The chromosomes of Crocodylus porosus, C. johnstoni and Caiman crocodilus have been analysed using C, G and N-banding techniques and the in situ hybridization of 18S+26S rRNA cistrons derived from Xenopus. It is clear that in addition to the gross structural changes (robertsonian rearrangements and pericentric inversions) which are known to distinguish these crocodiles, numerous other modes of repatterning have occurred. These involved both the heterochromatic and euchromatic portions of the genome. They appear to be associated with the gross structural changes which have been established, and involve two distinct forms of chromatin transformation.In addition, the in situ hybridization of 18S+26S rRNA cistrons onto these crocodilian chromosomes has localized the site of nucleolus organizer activity to the C-band positive G-band negative secondary constrictions present in all three species. The significance of these results is discussed.     

Heterochromatic differentiation in barley chromosomes revealed by C- and N-banding techniques

Summary Heterochromatin distribution in barley chromosomes was investigated by analyzing the C- and N-banding patterns of four cultivars. Enzymatic maceration and air drying were employed for the preparation of the chromosome slides. Although the two banding patterns were generally similar to each other, a clear difference was observed between them at the centromeric sites on all chromosomes. Every centromeric site consisted of N-banding positive and C-banding negative (N⁺ C^–) heterochromatin in every cultivar examined. An intervarietal polymorphism of heterochromatin distribution was confirmed in each of the banding techniques. The appearance frequencies of some bands were different between the two banding techniques and among the cultivars. The heterochromatic differentiation observed is discussed with respect to cause.     

Chromosomal Banding Analysis of Monosomics in Wheat

N-banding analysis has been used to identify the univalents of all 21 monosomics at diakinesis or metaphase Ⅰ. The univalents of nine wheat monosomics which are monosomic lB to 7B, 4A and 7A have shown distinctive N-banding patterns. These banding patterns appear to be identical in meiotic and mitotic chromosomes. The method is simple and speedy. The research probably provides a new way for cytological identification of monosomics in wheat and offers a technique for genome analysis of hybrids in wheat.     

The karyotype ofFestucopsis serpentini (Poaceae Triticeae) from Albania studied by banding techniques and in situ hybridization

The karyotypes of two populations ofFestucopsis serpentini (2n = 2x = 14) endemic to Albania were investigated in detail by Giemsa C- and N-banding, AgNO₃ staining, and in situ hybridization with an rDNA probe. The complements consisted of 14 large chromosomes, 10 metacentric and 4 SAT-chromosomes, a metacentric and a submetacentric pair. SAT-chromosomes from one population carried exclusively minute satellites, whereas SAT-chromosomes from another population also carried larger polymorphic satellites, suggesting a geographical differentiation. The existence of four chromosomes with nucleolus forming activity was established through AgNO₃ staining; however, the rDNA probe additionally hybridized to intercalary positions in the short arms of two metacentric chromosomes revealing two inactive rDNA sites. C-banding patterns comprised from zero and up to four very small to larger, generally telomeric bands per chromosome giving low levels of constitutive heterochromatin. Similarities in chromosome morphology and C-banding patterns identified the homologous relationships of all chromosomes in one population, but of three pairs only in the other. Reliable identification of homologous chromosomes between plants was only possible for the SAT-chromosomes. A comparison between the C-banded karyotypes ofF. serpentini andPeridictyon sanctum supports their position in two genera.     

Karyotypes of Elymus scabrifolius (Poaceae: Triticeae) from South America studied by banding techniques and in situ hybridization

Karyotypes of 4 accessions of Elymus scabrifolius (2n = 4x = 28) were investigated by Giemsa C- and N-banding, GAA-banding (one accession), AgNO3-staining and in situ hybridization with the rDNA probe pTa71. Two additional accessions were studied in less detail. The chromosomes were large (9-14 microns). The complements included 11 pairs of metacentrics, one with conspicuous satellites on the short arms, and 3 pairs of submetacentrics. Two of 4 accessions from Eastern Argentina and Uruguay had minute or small satellites on a submetacentric pair. No such satellites were observed in the other two accessions. In two accessions from the Cordoba province, a non-homologous submetacentric pair had very long satellites. AgNO3-staining established the presence of 4 nucleoli, two larger and two small ones, in 5 accessions. The C-banding patterns comprised from one to 12 conspicuous bands per chromosome at no preferential positions. The amount of constitutive heterochromatin (19-21%) was the highest hitherto established in the Triticeae. Similarities in banding patterns and chromosome morphology identified homologous and discriminated between non-homologous chromosomes within and, except for two chromosomes, between plants. Heteromorphic chromosome pairs were identified in satellite-carrying chromosomes only. N-banding produced conspicuous bands overall at the same positions as C-banding. GAA-banding patterns were similar to N-banding patterns. The rDNA probe hybridized to chromosome segments at nucleolar constrictions only. The production of C- and N-banding patterns in both genomes of E. scabrifolius suggests the presence of two H genomes and the absence of the pivotal St genome of Elymus. On account of the uncertain identity of one genome, and the overall similar gross morphology of E. scabrifolius and other tetraploid South American species referred to Elymus, E. scabrifolius is retained in Elymus.     

N-banding in polytene chromosomes of Chironomus and Drosophila

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

Evolution of the rat oocyte nucleolus during follicular growth

The ultrastructural evolution of the nucleolus was followed during follicular growth by means of a silver staining procedure. The oocyte nucleolus in the primordial and primary follicles consists of strands of dense fibrillar silver-stained component and aggregates of granules which are devoid of silver grains. Small fibrillar centres are also recognized and appear to have less silver stainability. At the secondary follicle stage, a new nucleolar component appears in the reticulated oocyte nucleolus. This component is devoid of silver grains. During follicle growth, at the antral follicle stage, this new component seems to fuse and the nucleolus becomes constituted of a compact homogeneous mass which exhibits a vacuole at the end of the oocyte maturation. The results obtained suggest that this nucleolar mass is essentially made of proteins and particularly of acidic proteins.     

Nucleolar 4s ribonucleic acid in dipteran salivary glands in the presence of inhibitor

1. Salivary glands of insect larvae accumulate newly made transfer RNA in the nucleolus when maintained in the presence of nucleoside antagonists that inhibit RNA synthesis preferentially at the chromosome. 2. The nucleus contains precursor transfer RNA, which, on the basis of the general evidence, may originate in the chromosome and then be methylated in the nucleolus. 3. The maturation of precursor ribosomal RNA is blocked in the nucleolus during inhibition. 4. The transport of nuclear RNA to cytoplasm is also blocked. 5. It is suggested that, if the transfer RNA accumulated in the nucleolus does indeed originate in the chromosome, the accumulation may result from the blockage of an obligatory transient association of the RNA with the nucleolus.     

The chromosomes of two Drosophila races: D. nasuta nasuta and D. n. albomicana

Heterochromatin distribution and differentiation in metaphase chromosomes of two morphologically identical Drosophila races, D. nasuta nasuta and D. n. albomicana, have been studied by C- and N-banding methods. — The total heterochromatin values differ only slightly between these races. However, homologous chromosomes of the two Drosophila forms show striking differences in the size of heterochromatin regions and there is an alternating pattern in D. n. nasuta and D. n. albomicana of chromosomes which contain more, or respectively less heterochromatin than their counterparts in the other race. — Three different N-banding patterns could be obtained depending on the conditions of the method employed: One banding pattern occurs which corresponds to the C-banding pattern. Another pattern is the reverse of the C-band pattern; the euchromatic chromosome regions and the centromeres are stained whereas the pericentric heterochromatin regions remain unstained. In the Y chromosomes of both races and in chromosome 4 of D. n. albomicana, however, the heterochromatin is further differentiated. In the third N-banding pattern only the centromeres are deeply stained. Furthermore, between the races, subtle staining differences in the pericentric heterochromatin regions can be observed as verified in F₁ hybrids. On the basis of C- and N-banding results specific aspects of chromosomal differences between D. n. nasuta and D. n. albomicana are discussed.Dedicated to Prof. W. Beermann on the occasion of his 60th birthday     

Structural organization of ribosomal cistrons in human nucleolar organizing chromosomes

The variable morphology of nucleolar organizer regions (NORs) of human acrocentric chromosome 13 was studied in detail by the N-banding technique. It was demonstrated that NOR may become further segmented and several hypotheses have been proposed for structural variability. The polymorphic nature of NORs may have an important significance in human evolution.     

An N-band marker for gene Lr18 for resistance to leaf rust in wheat

The leaf rust resistance gene, Lr18, of common wheat cultivars has been derived from Triticum timopheevi and is located on chromosome arm 5BL. Chromosome banding (N-banding) analyses revealed that in the wheat cultivars carrying Lr18 that were examined, which had been bred in 6 different countries, chromosome arm 5BL possessed a specific terminal band not carried by their susceptible parental cultivars. It was suggested that this terminal N-band was introduced from T. timopheevi together with Lr18. N-banding analysis of a T. timopheevi strain showed that one of two timopheevi chromosomes had provided Japanese wheat lines containing Lr18 with the terminal band.     

提莫菲维小麦C—带和N—带异染色质的比较研究

利用C-带和N-带分别及连续处理技术对提莫菲维小麦(Triticum timopheevi Zhuk.)的染色体带型及异染色质的类型与分布进行比较分析。结果表明,提莫菲维小麦的4A染色体以及G-染色体组带型丰富,并具有明显的端带,其异染色质是多样化的。4G和6G为随体染色体,随体显带明显。在提莫菲维小麦的染色体中异染色质类型有:(1)只有C~ N~ 型(4A、4G、6G和7G染色体);(2)只有C~ N~-型(1A、5A、6A和7A染色体);(3)C~ N~ 和C~ N~-型(2A、3A、1G、2G、3G和5G染色体)。在C-带和N-带连续处理中,N-带异染色质的消失部位在1G、2G、3G和5G染色体的端部,3A染色体的着丝点附近以及染色体1A、2A、5A、6A和7A的着丝点附近及端部。本文还讨论了C-带和N-带异染色质的异同以及端部异染色质在G染色体组进化中的可能作用。