Centromeric alpha satellite DNA amplification and translocation in an unusually large chromosome 14p+ variant

Summary We report cytogenetic and molecular studies on a family that carries, in the father, an unusually large chromosome 14p+ variant [WSi-var(14)(p+)] and, in one of his children, a translocation [DSi-der(14)] involving the variant chromosome. Increase in the size of WSi-var(14)(p+) was estimated to be approximately 35% that of a normal chromosome 14. Presence of extra chromosomal material in this variant chromosome was demonstrated by G-banding using trypsin and staining with Leishman, G-banding using bromodeoxyuridine (BrdU) and Giemsa, and R-banding using BrdU and Giemsa. This material was positive using C-banding with BaOH and staining with Giemsa and negative in DAPI/distamycin staining, suggesting that it contained repetitive DNA but probably not of the types found in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y. Staining of the nucleolus organiser region (NOR) with AgNO₃ indicated the retention of the NOR in WSi-var(14)(p+) but not in DSi-der(14). In situ hybridisation of metaphase cells with an alpha satellite DNA probe specific for human acrocentric chromosomes demonstrated a significantly increased amount of centromeric alpha sequences in WSi-var(14)(p+). Most or all of the extra alpha sequences were retained in DSi-der(14), indicating translocation near the very distal end of the enlarged region. The extra alpha satellite DNA material may have originated through amplification of some centromeric segments. The possible role of the amplified DNA in chromosomal translocations is discussed.     

A case of trisomy 22 in Pongo pygmaeus.

A behaviorally and clinically abnormal female orangutan was analyzed cytologically using general banding techniques and by an alkaline silver method for staining nucleolus organizer regions. The karyotype had 49 chromosomes, including an extra chromosome 22 (49,XX + 22). No variant chromosome types or heterozygous structural rearrangements were found. Nine of the 14 large acrocentric chromosomes, Nos. 11--17, and three of the five presumptive human G-group equivalents, i.e., two of three chromosomes 22, and one chromosome from pair 23, exhibited positive silver staining of the nucleolus organizer region (NOR).     

Supernumerary heterochromatic segments associated with the nucleolar chromosomes of Pyrgomorpha conica (Orthoptera) contain methylated rDNA sequences

The two nucleolus organizing chromosome pairs of the grasshopper Pyrgomorpha conica can carry a proximal supernumerary heterochromatic segment. We employed different cytological techniques to characterize and analyze the possible origin of this segment. The supernumerary segment and the nucleolus organizing regions (NORs) show similar responses after C-banding plus either Giemsa or acridine orange, and chromomycin A₃/distamycin A staining to detect GC-rich chromosome regions. Fluorescence in situ hybridization with a biotinylated rDNA probe demonstrated that the segment originated by amplification of the rDNA genes. However, as the silver staining indicates, the ribosomal genes present in the segment are not active since no nucleolus is formed. The use of in situ digestion with the isoschizomeric MspI and HpaII restriction endonucleases and subsequent Giemsa, ethidium bromide or chromomycin A₃/distamycin A staining, suggests that the segment has been inactivated by DNA methylation.     

Satellited Y chromosomes: Structure,origin, and clinical significance

Summary Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunction and/or to translocations of other chromosomes is discussed.     

Chromosome Rearrangements That Involve the Nucleolus Organizer Region in Neurospora

In ~3% of Neurospora crassa rearrangements, part of a chromosome arm becomes attached to the nucleolus organizer region (NOR) at one end of chromosome 2 (linkage group V). Investigations with one inversion and nine translocations of this type are reported here. They appear genetically to be nonreciprocal and terminal. When a rearrangement is heterozygous, about one-third of viable progeny are segmental aneuploids with the translocated segment present in two copies, one in normal position and one associated with the NOR. Duplications from many of the rearrangements are highly unstable, breaking down by loss of the NOR-attached segment to restore normal chromosome sequence. When most of the rearrangements are homozygous, attenuated strands can be seen extending through the unstained nucleolus at pachytene, joining the translocated distal segment to the remainder of chromosome 2. Although the rearrangements appear genetically to be nonreciprocal, molecular evidence shows that at least several of them are physically reciprocal, with a block of rDNA repeats translocated away from the NOR. Evidence that NOR-associated breakpoints are nonterminal is also provided by intercrosses between pairs of translocations that transfer different-length segments of the same donor-chromosome arm to the NOR.     

Cytological localization of ribosomal cistrons in polytene chromosomes of Phaseolus coccineus

Tritiated ribosomal RNA (rRNA) was prepared from hypocotyls of Phaseolus coccineus grown in liquid culture in the dark and in presence of 5-³H-uridine. A mixture of the 18S and 25S ³H-rRNA fractions was used for hybridization with DNA in the polytene chromosome cells of the embryo suspensor of P. coccineus. It was shown that the ribosomal cistrons (rDNA) are located in the nucleolus organizing system (satellite, nucleolar constriction and organizer) of the satellited chromosome pairs I (S₁) and V (S₂), in the proximal heterochromatic segment of the long arm of chromosomes S₁ and in the terminal heterochromatic segment of chromosome pair II. The micronucleoli which are produced by the satellite and nucleolus organizer of the chromosome pair S₁ contain rDNA; on the contrary, no rRNA-DNA hybridization is found in the DNA containing granules which are produced by the satellite and nucleolus organizer of chromosome pair S₂. The DNA which is amplified during production of DNA puffs at some chromosomal regions apparently does not code for ribosomal RNA (no detectable rRNA-DNA hybridization).Publication no. 62 from the Laboratorio di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche, Pisa. Part of the investigation was supported by Contract SC 001/076-69-1 BIAN between the European Atomic Energy Community and the University of Pisa, Institute of Genetics.     

The nucleolus organizer region of maize (Zea mays L.)

Maize plants were produced partially triploid for the heterochromatic segment of the nucleolus organizer region (NOR) or partially triploid or tetraploid for the site giving rise to the secondary constriction of the NOR. These partially hyperploid plants were characterized in terms of chromosome and/or nucleolar constitution by light microscopy at pachytene, diakinesis, and quartet stages of microsporogenesis. DNA's of the various partially hyperploid plants and appropriate controls were extracted and hybridized with ³H-rRNA. The heterochromatic segment of the NOR was found to contain most of the rRNA cistrons, but has little or no interaction with the nucleolus. In contrast with the heterochromatic segment, the site giving rise to the secondary constriction contains few rRNA cistrons but is active in nucleolar formation as viewed at pachytene, diakinesis and quartet stages.     

Position-dependent NOR activity in barley

Two types of intraspecific nucleolar dominance/suppression are described for barley,Hordeum vulgare L. When the nucleolus organizing regions (NORs) originally belonging to chromosomes 6 and 7 are combined by translocation in one chromosome, NOR 6 is dominant over NOR 7. Neither significant loss of rDNA nor its hypermethylation is the reason for the reduced nucleolus forming activity of NOR 7. Intrachromosomal NOR suppression probably does not occur in isochromosome 6s, which has two NORs 6 in one chromosome. Meiotic and somatic pairing of the homologous arms might be the reason for early fusion of their nucleoli and thus for the lower than expected maximum number of interphase nucleoli. Variable suppression of a partial NOR (6³) is described for descendants of crosses between translocation lines with split NORs 6 and 7. In these cases also, the reduced activity of the partial NOR 6³ is not due to deletion of rDNA as shown by in situ hybridization. Unstable methylation of NOR 6³ in heterozygous F₁ individuals is probably the cause of this phenomenon.     

Karyotype, C-banding, and Ag-NOR analysis in Diplodus bellottii (Sparidae, Perciforms). Intra-individual polymorphism involving heterochromatic regions.

A karyotype analysis was carried out in nine specimens of the Sparid species Diplodus bellottii using conventional staining, as well as C-banding and Ag-NOR banding techniques, showing, respectively, 2n = 46 and fundamental number (FN) = 54, and scarce heterochromatic areas irregularly distributed and up to four NOR active regions that were C positive. When compared with the karyotypes of other related species, one centric fusion giving rise to a large metacentric pair and several pericentric inversions seem to have been involved in the karyotype evolution. An intra-individual polymorphism was detected in one specimen, resulting in two karyotypic forms in roughly identical proportion, owing to a larger C-band by the NOR regions, appearing either in a terminal position of the short arms of pair 2 or in telomeric position of pair 3. These findings suggest that the extra heterochromatic segment responsible for the heteromorphism apparently only involves associated heterochromatin and not the NORs themselves. This C-positive block seems to have eventually been transferred between heterologous NOR chromosomes by a somatic event, facilitated by the physical proximity of NOR pairs in the nucleolus.     

Chromosomal constitution of nucleolus-associated chromatin in man

Summary Use of specific stains permits analysis of the frequency of nucleolus-associated heterochromatin in chromosomes 1 and 9 from human fibroblasts. In 81% of interphase nuclei the heterochromatic segment of both No. 1 chromosomes is associated with the nucleolus, while in 19% only one heterochromatic segment shows such an association with the other occupying a random position in the nucleoplasm. The nucleolar association of chromosome 9 heterochromatin is less constant: in 42.3% of the nuclei both segments are associated with the nucleolus, in 39% of the nuclei only one heterochromatic segment presents such an association, and in 18.7% neither of the two heterochromatic segments is in nucleolar association. In 6% of the cells, one or two chromosome 9 heterochromatic segments are in contact with the nuclear membrane.In situ hybridization using tritium-labeled 28S and 18S RNA shows that in the interphase nucleus the acrocentric short arms, carriers of ribosomal cistrons, are associated with the nucleolus.These observations demonstrate the complexity of the nucleolus-associated chromatin which, in addition to segments of chromosomes 1, 9, 13, 14, 15, 21 and 22, may include the Y chromosome. They also confirm that the nucleolus constitutes one of the orientation points determining the relative localization of chromosomes in the interphase nucleus.     

Heterochromatin accumulation,disposition and diversity in Gibasis karwinskyana (Commelinaceae)

C-banding differences within Gibasis karwinskyana (Roem & Schult.) Rohw. were reassessed using dual fluorochrome staining. Pronounced differences in C-band pattern between two subspecies with identical basic karyotypes were due to different chromosomal locations of AT-rich and GC-rich heterochromatin. The AT-rich component had an equilocal distribution in the karyotype and has evidently been accumulated at telomeres, as shown by its prevalence in supernumerary segments and B chromosomes. The GC-rich component also varied in amount, but was limited to nucleolus organizing regions (NORs) and centromeres. Centromeres and telomeres are suggested to constitute separate, although perhaps interdependent, centres of heterochromatin amplification. The possible role of nuclear architecture in determining the accumulation, distribution and spread of these sequences is discussed.Abbreviations H Hoechst 33258 - CMA chromomycin A3 - NOR nucleolus organizing region - SS supernumerary segment - Q quinacrine dihydrochloride - H⁺ H etc. indicate enhanced (+) and quenched (-) fluorescence with the stated fluorochrome by H.C. Macgregor     

A new approach to the auchenorrhyncha (Hemiptera, Insecta) cytogenetics: chromosomes of the meadow spittlebug Philaenus spumarius (L.) examined using various chromosome banding techniques

The karyotype of the meadow spittlebug Philaenus spumarius (L.) was studied using conventional chromosome staining, C- and AgNOR- banding, and fluorescent CMA3- and DAPI- techniques. This is the first report on differential staining of the holocentric chromosomes of Auchenorrhyncha. The karyotype of Ph. spumarius includes 2n = 22 + XX/X0. The autosomal pair 1 is large and carries a gap in every homologue. After silver staining, NORs were revealed in both this chromosome pair and a middle-sized pair, most likely 6 or 7. In spermatocyte meiosis, the majority of bivalents formed one chiasma each. The bivalent 1 showed from 1 to 4 chiasmata, the value of 1 or 2 being prevalent. Two further bivalents also showed two chiasmata in some cells. After C-banding, terminal and interstitial dot-type C-heterochromatic blocks were revealed in the chromosomes. In 4 of 11 studied males, the autosomal pair 1 was polymorphic for an extra segment attached to one of the homologues. The segment consisted of both heterochromatic and euchromatic portions. No defined signals were observed in any chromosome treated with DAPI. After CMA3- staining, bright fluorescent signals were obtained in the NOR-bearing chromosomes, suggesting GC-rich DNA bound to the NORs.     

Cytological localization of the genes for the four classes of ribosomal RNA (25S, 18S, 5.8S and 5S) in polytene chromosomes of Phaseolus coccineus

Homologous tritiated 25S, 18S and 5.8S rRNAs were used separately for in situ hybridization to the polytene chromosomes of the embryo suspensor cells of Phaseolus coccineus. Hybridization occurred at the same chromosomal sites which were labeled in previous in situ hybridization experiments with 25+18S rRNAs in the same material (Avanzi et al., 1972), namely: nucleolus organizing system (satellite, nucleolar constriction and organizer) of chromosome pairs I (S₁) and V (S₂), proximal heterochromatic segment of the long arm of chromosome pair I, and terminal heterochromatic segment of chromosome pair II. Competition hybridization experiments confirmed for P. coccineus the high sequence homology between 25S and 18S rRNA already known for other plants.Homologous ¹²⁵I-5S rRNA was found to hybridize to three sites in the polytene chromosomes of P. cocdneus: the proximal heterochromatic segment in the long arm of chromosome pair I (which also bears the sequences complementary to 25S, 18S and 5.8S RNAs), most of the proximal heterochromatic segment plus a small portion of adjoining euchromatin in the long arm of chromosome pair VI and the large intercalary heterochromatic segment in the same chromosome pair. Simultaneous labeling of the two 5S RNA sites in chromosome VI was quite rare (3%), the rule being labelling of one site to the exclusion of the other, with a labeling frequency of 43.7% and 53.3% for sites no. 1 and no. 2 respectively. These results are interpreted as being due to differential hybridizability of chromosomal sites such as described in other materials.     

Chromosome banding in Amphibia

The mitotic and meiotic chromosomes of the marsupial frog Gastrotheca riobambae were analysed with various banding techniques. The karyotype of this species is distinguished by considerable amounts of constitutive heterochromatin and unusual, heteromorphic XY sex chromosomes. The Y chromosome is considerably larger than the X chromosome and almost completely heterochromatic. The analysis of the banding patterns obtained with GC- and AT-base-pair-specific fluorochromes shows that the constitutive heterochromatin in the Y chromosome consists of at least three different structural categories. The only nucleolus organizer region (NOR) of the karyotype is localized in the short arm of the X chromosome. This causes a sex-specific difference in the number of NOR: female animals have two NORs in diploid cells, male animals one. No cytological indications were found for the inactivation of one of the two X chromosomes in the female cells. In male meiosis, the heteromorphic sex chromosomes form a characteristic sex-bivalent by pairing their telomeres in an end-to-end arrangement. The significance of the XY/XX sex chromosomes of G. riobambae for the study of X-linked genes in Amphibia, the evolution of sex chromosomes and their specific DNA sequences, and the significance of the meiotic process of sex chromosomes are discussed.     

Cytological and molecular evidence of deletion of ribosomal RNA genes in chromosome 6 of barley (Hordeum vulgare).

The barley chromosomal mutant T-35, in which only one pair of satellite chromosomes is apparent, was analyzed using a range of cytological and molecular techniques. Using conventional Feulgen staining, Giemsa and silver banding, in situ hybridization, and Southern blot analysis, unequivocal cytological and molecular evidence was obtained that T-35 is a homozygous deletion of rRNA genes residing in the nucleolus organizer region (NOR) of chromosome 6. According to the criteria of arm ratio and Giemsa-banding pattern of this chromosome, the deletion involved the whole NOR, one of the breakpoints being localized in the short arm proximally to the NOR-associated heterochromatic band, the other probably in the satellite of the chromosome. As a result of this deletion, an increased activity of the rRNA genes (as indicated by the size of the silver bands) on the other NOR-bearing chromosome (chromosome 7) was observed. The possible reasons for this phenomenon are discussed.     

Nucleolar organizing chromosomes ofRicinus

Summary Pachytene chromosome morphology was compared in nine races ofRicinus communis L. (2n = 20), using pollen mother cells (PMCs) and light microscopy. Of the ten bivalents, only the two possessing nucleolar organizing regions (NORs), chromosomes 2 and 7, exhibit structural variations among the races. The NORs are located in the short arms of these two chromosomes. Most of the observed structural variations affect these short arms, which are similar morphologically and consist largely of heterochromatic segments. The PMCs contain a single nucleolus and this is associated with the NOR of each of the two chromosomes at a particular frequency in each race. In eight races, a nucleolar constriction (NC) is present in either chromosome 2 or chromosome 7. In these races, the nucleolus is associated with the chromosome possessing an NC at a frequency of 100% and with the chromosome lacking an NC at a frequency ranging between 5.6 and 100%, depending upon the race. No microscopically visible NC is present in the ninth race. In this race, the nucleolus is associated with both chromosomes 2 and 7 at a frequency of 100%. The association of the nucleolus with a chromosome possessing an NC is at the NC and with a chromosome lacking an NC is at the terminal heterochromatic segment of the short arm. Several interpretations are offered to account for the variations in frequency of association between the nucleolus and each of the nucleolar organizing chromosomes. It is suggested that the two non-linked NORs have evolved through some intragenomic changes rather than polyploidy, that this species is highly intolerant to structural variations other than those occurring in or near the NORs, and that structural variations in the nucleolar organizing chromosomes are not associated with racial variations in plant phenotype.Paper of the Journal Series, New Jersey Agricultural Experiment Station     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes.

We performed a detailed karyotype analysis in the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), the key pest of pome fruit in the temperate regions of the world. The codling moth karyotype consisted of 2n = 56 chromosomes of a holokinetic type. The chromosomes were classified into 5 groups according to their sizes: extra large (3 pairs), large (3 pairs), medium (15 pairs), small (5 pairs), and dot-like (2 pairs). In pachytene nuclei of both sexes, a curious NOR (nucleolar organizer region) bivalent was observed. It carried 2 nucleoli, each associated with one end of the bivalent. FISH with an 18S ribosomal DNA probe confirmed the presence of 2 clusters of rRNA genes at the opposite ends of the bivalent. In accordance with this finding, 2 homologous NOR chromosomes were identified in mitotic metaphase, each showing hybridization signals at both ends. In highly polyploid somatic nuclei, females showed a large heterochromatin body, the so-called sex chromatin or W chromatin. The heterochromatin body was absent in male nuclei, indicating a WZ/ZZ (female/male) sex chromosome system. In keeping with the sex chromatin status, pachytene oocytes showed a sex chromosome bivalent (WZ) that was easily discernible by its heterochromatic W thread. To study molecular differentiation of the sex chromosomes, we employed genomic in situ hybridization (GISH) and comparative genomic hybridization (CGH). GISH detected the W chromosome by strong binding of the Cy3-labelled, female-derived DNA probe. With CGH, both the Cy3-labelled female-derived probe and Fluor-X labelled male-derived probe evenly bound to the W chromosome. This suggested that the W chromosome is predominantly composed of repetitive DNA sequences occurring scattered in other chromosomes but accumulated in the W chromosome. The demonstrated ways of W chromosome identification will facilitate the development of genetic sexing strains desirable for pest control using the sterile insect technique.