Methylation status of ribosomal RNA gene clusters in the flow-sorted human acrocentric chromosomes

Southern blot analysis of the human acrocentric chromosomes that were flow-sorted from B-lymphoblastoid cell line GM130B revealed that the sensitivity of the ribosomal RNA (rDNA) gene clusters to the restriction enzyme NotI differs among these rDNA-containing chromosomes: the rDNA clusters of Chromosomes (Chr) 13, 14, and 15 are much more sensitive to NotI digestion than those of Chrs 21 and 22 in this particular cell line. Detailed analysis by use of methylation-sensitive enzymes HpaII and HhaI and methylation-insensitive enzyme MspI confirmed the significant variation in the methylation status of rDNA clusters among these chromosomes. Quantitative analysis by fluorescent in situ hybridization (FISH) indicated that copy number of rDNA varies among individual chromosomes, but the average copy number in the acrocentric Chrs 21 and 22 is significantly greater than that of the Chrs 13, 14, and 15 in GM130B cells. Similar analysis reveals that the methylation status of rDNA clusters in another B-lymphoblastoid cell line GM131 was different from that of GM130B. These data together indicate that the copy number and methylation patterns of rDNA clusters differ among individual acrocentric chromosomes in a given cell line, and they are different among cell lines.     

Further studies on polyploid amphibians (Ceratophrydidawe)

Odontophrynus cultripes Reinhardt and Lutken, 1862 has 22 chromosomes in its diploid complement. Spermatocyte I contained 11 ring bivalents and metaphase II exhibited 11 chromosomes. Odontophrynus americanus (Duméril and Bibron) 1882 has 44 chromosomes in somatic as well as germ cells, these can be sorted into 11 groups of homologues. Metaphase I showed varying numbers of quadrivalents and metaphase II exhibited 22 dyads. Ceratophrys dorsata Wied., 1824 has 104 chromosomes in somatic and germ cells; these 104 chromosomes comprise 8 each of 13 kinds of homologues. The spermatocyte I contained ring octovalents and other multivalents, and metaphase II 52 chromosomes. The above findings indicate that evolution by polyploidization occurred in South American frogs belonging to the family Ceratophrydidae.This work was supported by a grant (GM-14577-01) from the National Institute of General Medical Sciences U. S. Public Health Service.     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Karyomorphological studies of theIxeris dentata complex on Mount Ishizuchi

The karyotypes of four taxa in theIxeris dentata complex collected from Mt. Ishizuchi were investigated.I. dentata subsp.dentata var.dentata f.dentata had 2n=21 in all the examined individuals and its karyotypes were classified into four different types. Var.dentata f.amplifolia showed 2n=21 in all individuals and had three different karyotypes.I. dentata subsp.dentata var.albiflora showed 2n=21 and two different karyotypes.I. dentata subsp.alpicola showed three different karyotypes with 2n=21 and four different ones with 2n=28. Judging from the results of the karyotype analyses, the morphological variation of chromosomes in theI. dentata complex is assumed to be in the process of increasing asymmetry in the shape of chromosomes. Following Levitzky's principle, it can be said that the a-, b-, and c-chromosomes exist in different advanced stages in the process of chromosome variation. The c-group chromosomes are at the most advanced stage in morphological variation, the b-group in the second and the a-group in the least advanced stage. The above-mentioned structural diversity of chromosomes may be related to apomictic reproduction of theI. dentata complex.     

Non-random association of trypsin-banded human acrocentric chromosomes

Summary This paper deals with a computer-aided study of the associations between acrocentric chromosomes as well as between those other chromosomes which in our investigations were revealed to be significantly closer to each other than random. The chromosome pairs were identified by a trypsinbanding technique. The method used has been elaborated previously with the specific aim of determining associations in a manner that avoids all subjective criteria.The tendency for association between homologous chromosomes is in decreasing order: 21 and 13>1>14, 18 and 19>17. Among the nonhomologous acrocentric chromosomes the significant tendencies for associations are between D-D: 13–14>13–15>14–15; between D-G: 13–21>14–21>13–22>15–22.The implication of the different tendencies to associate are dicussed in terms of aetiology of numerical and structural chromosome abnormalities.     

Chromosome studies of Saguinus midas niger (Callithricidae,Primates) from Tucurui,Para, Brazil: Comparison with the karyotype of Callithrix jacchus

The karotype of Saguinus midas niger was studied by G-, C-, and nuclear organizer region (NOR)-banding techniques. Variations in C-banding patterns were observed in some chromosomes. The banding patterns obtained were compared with those previously described for Callithrix jacchus. The two species differ by a reciprocal translocation involving pairs 9 and 16; by a paracentric inversion in chromosomes 1, 13, 14, 18, and 22; and by a pericentric inversion in at least four subtelocentric pairs (chromosomes 19, 20, 21, and 22), dislocating the nucleolar organizer region from the small short arm in C. jacchus to the proximal segment of the long arm in S. m. niger (or vice versa). The amount of constitutive heterochromatin is greater in S. m. niger than in C. jacchus, especially in chromosomes 4, 7, and 14. The Y chromosome is smaller in C. jacchus than in S. m. niger.     

Cytogenetic,FISH and DNA studies in 11 individuals from a family with two siblings with dup(21q) Down syndrome

A Spanish family has previously been described with two siblings with dup(21q) Down syndrome. The father has a normal karyotype. The mother has a microchromosome. Cytogenetic, fluorescence in situ hybridization and DNA studies have now been carried out on the family. Findings include that the mother has three different chromosome anomalies, viz. (1) a chromosome 22 with an unusual pericentromeric region that contains alphoid DNA from chromosomes 21/13 and chromosome 22, (2) an isochromosome 21p in the frequent cell line and (3) an isochromosome 21q in a rare second cell line. A possible explanation is that the mother developed from a zygote with trisomy 21 and that mitotic error in early development resulted in the formation of two cell lines with karyotypes of 47,XX,+i(21p) and 47,XX,+i(21q), respectively. The unusual chromosome 22 represents a hitherto undescribed chromosome anomaly and one possible explanation is a translocation of the short arms between chromosomes 21/13 and 22 in the ancestry of the family. The relationship between the unusual chromosome 22 and the isochromosome formation in the mother is not known. However, all three chromosome anomalies involve the alphoid DNA of chromosome 21/13, indicating that this is not a chance finding.     

Chromosome painting of Y chromosomes and isolation of a Y chromosome-specific repetitive sequence in the dioecious plant Rumex acetosa

The dioecious plant Rumex acetosa has a multiple sex chromosome system: XX in female and XY₁Y₂ in male. Both types of Y chromosome were isolated from chromosome spreads of males by manual microdissection, and their chromosomal DNA was amplified using degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). When the biotin-labeled DOP-PCR product was hybridized with competitor DNA in situ, the fluorescent signal painted the Y chromosomes. A library of Y chromosome DNA was constructed from the DOP-PCR product and screened for DNA sequences specific to the Y chromosome. One Y chromosome-specific DNA sequence was identified and designated RAYSI (R. acetosa Y chromosome-specific sequence I). RAYSI is a tandemly arranged repetitive DNA sequence that maps to the 4’,6-diamidino-2-phenylindole bands of both Y chromosomes. Received: 22 December 1998; in revised form: 22 March 1999 / Accepted: 23 March 1999     

Mosaic structure of the DNA molecules of the human chromosomes 21 and 22

We calculated nucleotide distribution curves along the DNA molecules of the human chromosomes 21 and 22, their correlations in more than 10000 equidistant positions, and subjected the correlations to cluster analysis. The cluster analysis demonstrated that both DNA molecules were composed of two types of segments exhibiting qualitatively different correlations. The segments differed most in the correlation of the distribution curves of cytosine and guanine, which was very high in type I segments but weak in type II segments. The type I and II segments also significantly differed in the correlations of the distribution curves of adenine with thymine. In addition, adenine strongly anticorrelated with cytosine but this anticorrelation was uniform along both chromosomes and, therefore, it did not contribute to the distinction of the two types of segments. The segments were up to 100 kbp long but they had nothing in common with isochores. Building blocks of the mosaic structure of the DNA molecules of the human chromosomes 21 and 22 are very similar but different in several interesting aspects from those of E. coli.     

CYTOLOGICAL ANALYSIS OF ANTHURIUM ANDRAEANUM (ARACEAE), ITS RELATED TAXA AND THEIR HYBRIDS

Karyotypes and meiotic configurations of Anthurium andraeanum and closely related taxa were analyzed. The karyotypes of A. andraeanum, A. caperatum, A. formosum, A. kamemotoanum, A. lindenianum, A. roseospadix, A. cf. sanctifidense, A. subsignatum, A. garagaranum, and an unidentified Anthurium sp. commonly consisted of four large metacentric or submetacentric chromosomes, two fairly large acrocentric chromosomes, two satellite chromosomes, and 22 smaller chromosomes. Variation in the karyotypes of A. nymphaeifolium and A. ochranthum suggested chromosomal rearrangement in the genus. All taxa showed 15 pairs of chromosomes at prometaphase I of meiosis in pollen mother cells. Four large chromosomes appeared as ring bivalents, and the rest of the chromosomes appeared as either ring or rod bivalents. Regular bivalent formation at prometaphase I of meiosis in pollen mother cells of species hybrids suggested close genomic relationships among parental taxa. On the other hand, reduction of pollen fertility estimated by pollen stainability in those hybrids suggested genetic divergence of species.     

Structural Organization of the Human Genome: Distribution of Nucleotides,AluRepeats, and Exons in Chromosomes 21 and 22

Analysis of DNA sequences of the human chromosomes 21 and 22 performed using a specially designed MegaGene software allowed us to obtain the following results. Purine and pyrimidine nucleotide residues are unevenly distributed along both chromosomes, displaying maxima and minima (waves) with a period of about 3 Mbp. Distribution of G+C along both chromosomes has no distinct maxima and minima, however, chromosome 21 contains considerably less G+C than chromosome 22. Both exons and Alurepeats are unevenly distributed along chromosome 21: they are scarce in its left part and abundant in the right part, while MIR elements are quite monotonously spread along this chromosome. The Alurepeats show a wave-like distribution pattern similar for both repeat orientations. The number of the Alurepeats of opposite orientations was equal for both studied chromosomes, and this may be considered a new property of the human genome. The positive correlation between the exon and Aludistribution patterns along the chromosome, the concurrent distribution of Alurepeats in both orientations along the chromosome, and the equal copy numbers for Aluin direct and inverted orientations within an individual chromosome point to their important role in the human genome, and do not fit the notion that Alurepeats belong to parasitic (junk) DNA.     

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.     

Rejoining between 9q+ and Philadelphia chromosomes results in normal-looking chromosomes 9 and 22 in Ph1-negative chronic myelocytic leukemia

Summary Rearrangement of the breakpoint cluster region (bcr) and the chromosomal location of c-abl and 3-bcr were studied in two patients with Philadelphia chromosome (Ph¹)-negative chronic myelocytic leukemia (CML). One patient (patient 1) had a normal karyotype and the other (patient 2), 46,XY,inv(3)(q21q26). Both patients showed the bcr rearrangement by Southern blot analysis with a 1.2 kb 3-bcr probe. In situ hybridization studies demonstrated the location of the homologous sequences of bcr on chromosome 22 in patient 1, and on chromosomes 9 and 22 in patient 2. These findings indicate that the morphologically normal-looking chromosomes 9 and 22 in patient 2 are the result of a retranslocation between chromosomes 9q+ and 22q-, abnormalities which were first formed by a standard Ph¹ translocation.     

AFLP markers in a molecular linkage map of maize: codominant scoring and linkage group ditsribution

We exploited the AFLP technique to saturate a RFLP linkage map derived from a maize mapping population. By using two restriction enzyme, EcoRI and PstI, differing in methylation sensitivity, both in combination with MseI, we detected 1568 bands of which 340 where polymorphic. These were added to the exitsing RFLP marker data to study the effects of incorporation of AFLPs produced by different restriction-enzyme combinations upon genetic maps. Addition of the AFLP data resulted in greater genome coverage, both through linking previously separate groups and the extension of other groups. The increase of the total map length was mainly caused by the addition of markers to telomeric regions, where RFLP markers were poorly represented. The percentage of informative loci was significantly different between the EcoRI and PstI assays. There was also evidence that PstI AFLP markers were more randomly distributed across chromosomes and chromosome regions, while EcoRI AFLP markers clustered mainly at centomeric regions. The more-random ditsribution of PstI AFLP markers on the genetic map reported here may reflect a preferential localisation of the markers in the hypomethylated telomeric regions of the chromosomes. Received: 22 December 1998 / Accepted: 25 March 1999     

A molecular genetic approach to the identification of isochromosomes of chromosome 21

Summary The largest class of de novo chromosomal rearrangements in Down syndrome are rea(21q21q). Classically, these rearrangements have been termed Robertsonian translocations, implying an attachment of two different chromosome 21 homologues. Additionally, a Robertsonian translocation between two chromosomes 21 cannot be distinguished from an isochromosome composed of genetically identical arms by cytogenetic analyses. Therefore, we have used molecular techniques to differentiate between true Robertsonian translocations and isochromosomes. Samples were obtained from 12 probands, ascertained for de novo rearrangements between homologous chromosomes 21 [11 rea(21q21q) and 1 rea (21;21)(q22;q22)], their parents (n = 24) and available siblings (n = 7). The parental origins of the de novo rearrangements were assigned using molecular and cytogenetic analyses. Although not statistically significant, there was a two-fold increase in the number of paternally derived de novo rearrangements (n = 8) as compared with maternally derived rearrangements (n = 4). To distinguish between rob(21q21q) and i(21q), we used restriction fragment length polymorphisms (RFLPs) spanning the length of chromosome 21. Using all informative and partially informative RFLPs, we used the method of maximum likelihood to assign the most likely rearrangement definition (i or rob) and parental origin in each family. The maximum likelihood estimates indicated that all rearrangements tested (n = 8) were isochromosomes. C-banding revealed two centromeres in three cases indicating that a U-type exchange occurred between sister chromatids in these rearrangements. Our results suggest that the majority of de novo rea(21q21q) are isochromosomes derived from a single parental chromosome 21.     

Cytogenetic studies on <Emphasis Type="Italic">Metasequoia glyptostroboides</Emphasis>, a living fossil species

The chromosome morphology and meiotic pairing behavior in the pollen mother cells (PMCs) of Metasequoia glyptostroboides were investigated. The results showed that: (1) The chromosome number of the PMCs was 2n=22. (2) The PMCs developed in the successive manner, and the nucleoids in the dynamic development were similar to those of the other gymnosperms. (3) At prophase, most of the chromosomes were unable to be identified distinctively because the chromosomes were long and tangled together. The chromosome segments were paired non-synchronously. At pachytene, the interstitial or terminal regions of some bivalents did not form synapsis and the paired chromosomes showed difference in sizes, indicating that there were structure differences between the homologous chromosomes. (4) At diakinesis, the ring bivalents showed complicated configurations due to the differences in location and number of chiasmata. In addition, there were cross-linked bivalents. (5) At metaphase I, the chromosome configuration of each cell was 8.2II ⁰ + 1.1II + 1.3II ⁺ + 0.8I. Most of the chromosomes were ring bivalents, but some were cross-linked bivalents, rod bivalents, or univalents. (6) 15\% PMCs at anaphase I and 22\% PMCs at anaphase II presented chromosome bridges, chromosome fragments, micronuclei, and lagging chromosomes. Twenty seven percent microspores finally moved into one to three micronuclei. Twenty five percent pollens were abortive. The results indicated that the observed individual of M. glyptostroboideswas probably a parpcentric inversion heterozygote, and there were structural and behavioral differences between the homologous chromosomes. The chromosomal aberration of M. glyptostroboidesmay play an important role in the evolution of this relict species, which is known as a living fossil. Further evidence is needed to test whether the differences between homologous chromosomes were due to hybridization.     

Genome relationships between Hordeum procerum (6x) and H. lechleri (6x)

Investigations on the meiotic behaviour of chromosomes in interspecific hybrids (2n=6x=42) between Hordeum lechleri (6x) and H. procerum (6x) and in their component haploids have been utilized to assess the nature of pairing and the extent of genome homology between the two species. In the F₁ hybrids an average of 25 (60%) chromosomes associated at metaphase I, mostly as bivalents. A majority (60%) of the pollen mother cells (PMCs) in H. procerum haploids (2n=3x=21) displayed 21 univalents and even in the remainder, a maximum of two rod bivalents were formed resulting in an average of 0.52 bivalents per cell. In haploids of H. lechleri (2n=3x=21) however, 30% of chromosomes pair. The sum of the chromosomal associations in the component haploids represents only 17% of the complement, far below the observed frequency (60%) in the hybrids. Thus, the pairing displayed in hybrids between H. lechleri and H. procerum was mostly allosyndetic and suggestive of two genomes being common in these species.In haploid H. procerum 1/3 of the PMCs displayed a tripolar organisation of chromosomes leading to triad and hexad formation after divisions I and II respectively. The significance of hexad formation in the trihaploid H. procerum and a possible suppression of homoeologous pairing in H. procerum haploids are discussed.     

Meiosis in polyhaploid Hordeum: hemizygous ineffective control of diploid-like behaviour in a hexaploid?

Meiotic chromosome behaviour was studied in the hexaploid Hordeum parodii (2n=6x=42) and in six haploids (2n=3x=21) obtained from a cross between H. parodii and H. bulbosum (2n=2x=14) whereby all bulbosum chromosomes were selectively eliminated. The alloploid nature of H. parodii was evident from the exclusive bivalent formation at the hexaploid level and the low and variable number of bivalents in its haploid derivatives. In haploids, both nonhomologous (intragenomic) and homoeologous (intergenomic) chromosomes paired at prophase. Foldbacks in single chromosomes, bivalents and trivalents were observed at prophase and metaphase I. At diakinesis, the associations involved a maximum of 20 chromosomes which decreased to 12 by metaphase I. This decrease was attributed to the failure of the non-homologous associations to persist until metaphase I. A hemizygous-ineffective control for the diploid-like behaviour of the hexaploid parodii is proposed to explain the homeologous chromosome pairing in its haploid derivatives.     

Genomic in situ hybridization (GISH) analyses of Thinopyrum intermedium, its partial amphiploid Zhong 5, and disease-resistant derivatives in wheat

Genomic in situhybridization (GISH) to root-tip cells at mitotic metaphase, using genomic DNA probes from Thinopyrum intermedium and Pseudoroegneria strigosa, was used to examine the genomic constitution of Th. intermedium, the 56-chromosome partial amphiploid to wheat called Zhong 5 and disease-resistant derivatives of Zhong 5, in a wheat background. Evidence from GISH indicated that Th. intermedium contained seven pairs of St, seven J^S and 21 J chromosomes; three pairs of Th. intermedium chromosomes with satellites in their short arms belonging to the St, J, J genomes and homoeologous groups 1, 1, and 5 respectively. GISH results using different materials and different probes showed that seven pairs of added Th. intermedium chromosomes in Zhong 5 included three pairs of St chromosomes, two pairs of J^S chromosomes and two pairs of St-J^S reciprocal tanslocation chromosomes. A pair of chromosomes, which substituted a pair of wheat chromosomes in Yi 4212 and in HG 295 and was added to 21 pairs of wheat chromosomes in the disomic additions Z1, Z2 and Z6, conferred BYDV-resistance and was identical to a pair of St-J^S tanslocation chromosomes (StJ^S) in Zhong 5. The StJ^S chromosome had a special GISH signal pattern and could be easily distinguished from other added chromosomes in Zhong 5; it has not yet been possible to locate the BYDV-resistant gene(s) of this translocated chromosome either in the St chromosome portion belonging to homoeologous group 2 or in the J^S chromosome portion whose homoeologous group relationship is still uncertain. Among 22 chromosome pairs in disomic addition line Z3, the added chromosome pair had satellites and belonged to the St genome and homoeologous group 1. Disomic addition line Z4 carried a pair of added chromosomes which was composed of a group-7 J^S chromosome translocated with a wheat chromosome; this chromosome was different to 7 Ai-1, but was identical to 7 Ai-2. The leaf rust and stem rust resistance genes were located in the distal region of the long arm, whereas the stripe rust resistance gene(s) was located in the short arm or in the proximal region of the long arm of 7 Ai-2. A pair of J^S-wheat translocation chromosomes, which originated from the WJ^S chromosomes in Z4, was added to the disomic addition line Z5; the added chromosomes of Z5 carried leaf and stem rust resistance but not stripe rust resistance; Z5 is a potentially useful source for rust resistance genes in wheat breeding and for cloning these novel rust-resistant genes. GISH analysis using the St genome as a probe has proved advantageous in identifying alien Th. intermedium in wheat. Received: 17 May 1999 / Accepted: 22 June 1999     

Prometaphase chromosomes of the howler monkey (Aloutta caraya): G,C, NOR,and restriction enzyme (RES) banding

Prometaphase lymphocyte chromosomes from eight adult argentinian Alouatta caraya females were characterized using sequential G-C banding techniques, Ag-NOR bands and bands obtained with the restriction enzymes Hae III, Eco RI, Alu I and Sau 3A. The cytogenetic analysis showed 2n = 52, with four, five, or six NOR chromosomes. Digestion with Hae III and Eco RI produced G-like-bands. Centromere regions and two interstitial C-bands (in chromosomes number 16 and 21) showed intraindividual or interindividual heterochromatic polymorphisms. Alu I digestion produced C-like bands with gaps in the centromere regions, and Sau 3A produced C-like bands. The karyotypes and banding patterns of A. caraya, A. palliata, A. belzebul, and A. seniculus are compared, based on whole chromosome and whole arm homeologies. © 1994 Wiley-Liss, Inc.