Co-amplification of rRNA genes with CAD genes in N-(phosphonacetyl)-L-aspartate-resistant Syrian hamster cells.

The amplified CAD genes in N-(phosphonacetyl)-L-aspartate (PALA)-resistant Syrian hamster cells are located in an expanded chromosomal region emanating from the site of the wild-type gene at the tip of the short arm of chromosome B-9. The terminus of B-9 in PALA-sensitive cells contains a cluster of rRNA genes (i.e., a nucleolus organizer, rDNA). We have used a molecular clone containing sequences complementary to Syrian hamster 28S rRNA to investigate whether rDNA is coamplified with CAD genes in the PALA-resistant mutants. In situ hybridization of this probe to metaphase chromosomes demonstrates that rDNA and CAD genes do coamplify in two independently isolated PALA-resistant mutants. The tight linkage of CAD and rDNA genes was demonstrated by their coordinate translocation from B-9 to the end of the long arm of chromosome C-11 in one mutant. Blot hybridization studies substantiate the in situ hybridization results. Both types of analysis indicate that only one or two rDNA genes, on the average, are coamplified per CAD gene. The data are consistent with the model that unequal exchanges between rDNA genes mediate the amplification of CAD genes in the Syrian hamster mutants that were analyzed.     

Physical mapping of the 5S ribosomal RNA genes in Arabidopsis thaliana by multi-color fluorescence in situ hybridization with cosmid clones

The 5S ribosomal RNA genes were mapped to mitotic chromosomes of Arabidopsis thaliana by fluorescence in situ hybridization (FISH). In the ecotype Landsberg erecta, hybridization signals appeared on three pairs of chromosomes, two of which were metacentric and the other acrocentric. Hybridization signals on one pair of metacentric chromosomes were much stronger than those on the acrocentric and the other pair of metacentric chromosomes, probably reflecting the number of copies of the genes on the chromosomes. Other ecotypes, Columbia and Wassilewskija, had similar chromosomal distribution of the genes, but the hybridization signals on one pair of metacentric chromosomes were very weak, and detectable only in chromosomes prepared from young flower buds. The chromosomes and arms carrying the 5S rDNA were identified by multi-color FISH with cosmid clones and a centromeric 180 bp repeat as co-probes. The metacentric chromosome 5 and its L arm carries the largest cluster of the genes, and the short arm of acrocentric chromosome 4 carries a small cluster in all three ecotypes. Chromosome 3 had another small cluster of 5S rRNA genes on its L arm. Chromosomes 1 and 2 had no 5S rDNA cluster, but they are morphologically distinguishable; chromosome 1 is metacentric and 2 acrocentric. Using the 5S rDNA as a probe, therefore, all chromosomes of A. thaliana could be identified by FISH. Chromosome 1 is large and metacentric; chromosome 2 is acrocentric carrying 18S-5.8S-25S rDNA clusters on its short arm; chromosome 3 is metacentric carrying a small cluster of 5S rDNA genes on its L arm; chromosome 4 is acrocentric carrying both 18S-5.8S-25S and 5S rDNAs on its short (L) arm; and chromosome 5 is metacentric carrying a large cluster of 5S rDNA on its L arm.     

Dual-color FISH karyotype analyses using rDNAs in three Cucurbitaceae species

Dual-color fluorescence in situ hybridization (FISH) analysis of three Cucurbitaceae species from different genera was conducted using 5S and 45S rDNA probes. In Benincasa hispida (Thunb.) Cogn. (2n=24), the 45S rDNA probe hybridized on two chromosomes, one in the short arm of a medium-sized metacentric chromosome and another at the satellite of a chromosome. The 5S rDNA hybridized at a site proximal to the centromere of the same short arm of the 45S rRNA gene locus that occupied almost the entire short arm. For Citrullus lanatus (Thunb.) Matsum & Nakai (2n=22), the 45S rDNA probe hybridized at sites in the short arms of two chromosomes and the 5S rDNA probe was co-localized with the 45S rRNA locus at the region proximal to the centromere in one chromosome. The 45S rRNA loci occupied almost all of the short arms in both chromosomes. In Cucurbita moschata Duch. (2n=40), the 45S rDNA probe hybridized in five chromosomes in which the 45S rRNA genes occupied almost two-thirds of the chromosomes in two large chromosomes and the entire short arm of a medium-sized chromosome. Two other loci were present in two medium-sized chromosomes, one in the proximal region in the short arm of a chromosome and another at the tip of the long arm of a chromosome. Chromosomes of B. hispida were relatively larger than those of the other two species. The karyotype of B. hispida is composed of two metacentrics and 10 submetacentrics, while that of C. lanatus is composed of seven metacentrics and four submetacentrics and that of C. moschata is composed of 18 metacentrics and two submetacentrics. Comparative chromosome evolution among the three Cucurbitaceae species was attempted using the karyotypes and the chromosomal distribution patterns of the 5S and 45S rDNAs. The results presented herein will be useful in elucidating the phylogenetic relationships among Cucurbitaceae species, and will provide basic data for their breeding programs.     

Evolution of 5S rDNA units and their chromosomal localization in Allium cepa and Allium schoenoprasum revealed by microdissection and FISH

Allium cepa and Allium schoenoprasum each possess 5S rDNA units of two different sizes. The evolution of the two repeat units and their chromosomal localization were investigated. A. cepa has 5S rDNA loci in the proximal and distal regions of the short arm of chromosome 7. When the proximal and distal segments of the short arm of chromosome 7 were microdissected separately, and used as templates for PCR, the short and long 5S rDNA fragments were amplified predominantly from the proximal and distal segments, respectively. The nucleotide sequence of the long 5S rDNA unit resulted from partial duplication of a non-transcribed spacer (NTS) and the insertion of a unique sequence. FISH using a probe consisting of the unique sequence demonstrated that the long unit was distally localized. In A. cepa, the long 5S rDNA unit is only present distally and the short unit is predominantly located proximally on the short arm of chromosome 7. In A. schoenoprasum, the NTSs of the two different-sized 5S rDNAs had quite different sequences. The two 5S rDNA loci were localized very close together in the interstitial region of chromosome 6. FISH, using long and short 5S rDNA unit probes with a competitor of a 120-bp sequence of the 5S rRNA gene, indicated that the long 5S rDNA unit was localized proximally and the short unit distally. Although the NTSs of the 5S rDNA of A. cepa and A. schoenoprasum had quite different nucleotide sequences, the long 5S rDNA units of A. cepa and A. schoenoprasum share a common 75-bp sequence. This sequence might act in the formation of the long 5S rDNA unit in Allium species.     

18-26S rDNA在4种重楼属植物中的定位

为探讨rDNA在重楼属Paris L.中的分布规律,利用荧光原位杂交（FISH）对4种重楼属植物 的18-26S rDNA进行了定位。所有植物均为二倍体,基因组由A、B、C、D和 E5条染色体构成。（1）滇重楼P.polyphylla var.yunnanensis:2n=10=6m+4t,C和D染色体的 短臂上各有1个18-26S rDNA位点;（2）长柱重楼P.forrestii:2n=10=6m+4t,B染色体的长臂 、C和D染色体的短臂上各有1个位点;（3）五指莲P.axialis:2n=10=6m（2sat）+4t（2sat） +1-2B,C和D染色体的短臂上各有1个位点;在有1个B染色体的细胞中,B染色体没有信号点, 而有2个B染色体的细胞中,只有1个B染色体上有信号点,表明B染色体上有基因存在且其分裂 不均等;（4）大理重楼P.daliensis:2n=10=4m+2sm+2st+2t,C染色体的短臂上有1个位点。1 8-26S rDNA位点不仅出现在染色体的次缢痕上,也出现在非次缢痕位点。另外,4个种中C染 色体短臂末端均有18-26S rDNA。     

Regulation of Ribosomal RNA Gene Multiplicity in DROSOPHILA MELANOGASTER

The ribosomal RNA (rRNA) genes of Drosophila melanogaster can undergo a disproportionate replication of their number. This occurs when the cluster of rRNA genes (rDNA) of one chromosome is maintained with a homologous chromosome that is completely or partially deficient in its rDNA. Under appropriate genetic conditions, it appears that disproportionate rDNA replication can be generated at the level of both somatic and germ line cells. In the latter case, mutants partially deficient for rDNA can increase their rRNA gene number to the wild type level and transmit this new genotype to successive generations.     

Physical mapping of rRNA genes by fluorescent in-situ hybridization and structural analysis of 5S rRNA genes and intergenic spacer sequences in sugar beet (Beta vulgaris)

A digoxigenin-labelled 5S rDNA probe (pTa-794) and a rhodamine-labelled 18S-5.8S-25S rDNA probe (pTa71) were used for double-target in-situ hybridization to root-tip metaphase, prophase and interphase chromosomes of cultivated beet,Beta vulgaris L. After in-situ hybridization with the 18S-5.8S-25S rDNA probe, one major pair of sites was detected which corresponded to the secondary constriction at the end of the short arm of chromosome 1. The two rDNA chromosomes were often associated and the loci only contracted in late metaphase. In the majority of the metaphase plates analyzed, we found a single additional minor hybridization site with pTa71. One pair of 5S rRNA gene clusters was localized near the centromere on the short arm of one of the three largest chromosomes which does not carry the 18S-5.8S-25S genes. Because of the difficulties in distinguishing the very similarly-sizedB. vulgaris chromosomes in metaphase preparations, the 5S and the 18S-5.8S-25S rRNA genes can be used as markers for chromosome identification. TwoXbaI fragments (pXV1 and pXV2), comprising the 5S ribosomal RNA gene and the adjacent intergenic spacer, were isolated. The two 5S rDNA repeats were 349 bp and 351 bp long, showing considerable sequence variation in the intergenic spacer. The use of fluorescent in-situ hybridization, complemented by molecular data, for gene mapping and for integrating genetic and physical maps of beet species is discussed.     

Molecular Cytogenetic Analysis of the European Hake Merluccius merluccius (Merlucciidae,Gadiformes): U1 and U2 snRNA Gene Clusters Map to the Same Location

The European hake (Merluccius merluccius) is a highly valuable and intensely fished species in which a long-term alive stock has been established in captivity for aquaculture purposes. Due to their huge economic importance, genetic studies on hakes were mostly focused on phylogenetic and phylogeographic aspects; however chromosome numbers are still not described for any of the fifteen species in the genus Merluccius. In this work we report a chromosome number of 2n = 42 and a karyotype composed of three meta/submetacentric and 18 subtelo/telocentric chromosome pairs. Telomeric sequences appear exclusively at both ends of every single chromosome. Concerning rRNA genes, this species show a single 45S rDNA cluster at an intercalary location on the long arm of subtelocentric chromosome pair 12; the single 5S rDNA cluster is also intercalary to the long arm of chromosome pair 4. While U2 snRNA gene clusters map to a single subcentromeric position on chromosome pair 13, U1 snRNA gene clusters seem to appear on almost all chromosome pairs, but showing bigger clusters on pairs 5, 13, 16, 17 and 19. The brightest signals on pair 13 are coincident with the single U2 snRNA gene cluster signals. Therefore, the use of these probes allows the unequivocal identification of at least 7 of the chromosome pairs that compose the karyotype of Merluccius merluccius thus opening the way to integrate molecular genetics and cytological data on the study of the genome of this important species.     

Does differential selection on the 5S rDNA explain why the rainbow trout sex chromosome heteromorphism is not linked to the SEX locus?

Many but not all rainbow trout strains have morphologically distinguishable sex chromosomes. In these strains, the short arm of the X has multiple copies of 5S rDNA and a bright DAPI band near the centromere, both of which are missing from the Y chromosome, which has a very small short arm. We examined the presence of these markers using fluorescence in situ hybridization (FISH) in four different YY clonal lines derived from different strains and compared the results with sexed fish of the Donaldson strain with the normal X/Y heteromorphism. The Y chromosome in two of the YY clonal lines (Arlee and Swanson) is indistinguishable from the X chromosome and it is positive for 5S rDNA and the DAPI bright band. On the other hand, both 5S rDNA sequences and the DAPI band were not found on the Y chromosome in Hot Creek and Clearwater which have the normal Y. Thus the presence of these two cytogenetic markers may account for the size difference between the short arm of the X and Y chromosome found in most rainbow trout strains. In fishes the expression of one type of 5S rRNA is restricted to oocytes and previous work suggests that although XX males are fairly common, XY females are rare, implying a selective disadvantage for XY females. A hypothesis is presented to explain why this sex chromosome heteromorphism is not closely linked to the SEX locus, which is found on the long arm of the Y chromosome in rainbow trout.     

Sequence comparison of distal and proximal ribosomal DNA arrays in rice (Oryza sativa L.) chromosome 9S and analysis of their flanking regions

Rice (Oryza sativa ssp. japonica cv. Nipponbare) harbors a ribosomal RNA gene (rDNA) cluster in the nucleolar-organizing region at the telomeric end of the short arm of chromosome 9. We isolated and sequenced two genomic clones carrying rice rDNA fragments from this region. The rice rDNA repeat units could be classified into three types based on length, which ranged from 7,928 to 8,934 bp. This variation was due to polymorphism in the number of 254-bp subrepeats in the intergenic spacer (IGS). Polymerase chain reaction (PCR) analysis suggested that the rDNA units in rice vary widely in length and that the copy number of the subrepeats in the IGS ranges from 1 to 12 in the rice genome. PCR and Southern blot analyses showed that most rDNA units have three intact and one truncated copies of the subrepeats in the IGS, and distal (telomere-side) rDNA units have more subrepeats than do proximal (centromere-side) ones. Both genomic clones we studied contained rDNA-flanking DNA sequences of either telomeric repeats (5′-TTTAGGG-3′) or a chromosome-specific region, suggesting that they were derived from the distal or proximal end, respectively, of the rDNA cluster. A similarity search indicated that retrotransposons appeared more frequently in a 500-kb portion of the proximal rDNA-flanking region than in other subtelomeric regions or sequenced regions of the genome. This study reveals the repetitive nature of the telomeric end of the short arm of chromosome 9, which consists of telomeric repeats, an rDNA array, and a retrotransposon-rich chromosomal region.Sequence accession numbers in DDBJ assigned for OSJNOa063K24 and OSJNBb0013K10 are AP009051 and AP008245, respectively.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

Physical mapping of 5S and 18S-26S ribosomal RNA gene families inAllium victorialis var.platyphyllum

A physical map of the 5S and 18S–26S rRNA genes was determined using bi-color fluorescencein situ hybridization technique inA. victorialis var.platyphyllum. 5S rRNA genes were positioned in the intercalary regions of the short arms in homologous chromosomes 6. Two major loci of the 18S-26S rRNA genes were detected in the secondary constrictions flanking with a pair of satellite and terminal region of short arm in chromosome 4. And two additional minor loci were heterotype, representing one signal on the terminal region of the short arm in one homolog of chromsome 2, and other on one homolog of chromosome 6 with linked 5S rRNA loci. In addition chromomycin A₃ (CMA,) fluorescent banding method was used to identify the relation between Nucleolus Organizer Region (NOR) sites and CMA, positive heterochromatin sites. In homologous chromosome 4 showing 18S–26S rDNA hybridization signals revealed also distinct CMA, positive band.     

Physical mapping of the 5S rDNA gene complex in rice (Oryza sativa).

This study was designed to use biotin labelling in situ hybridization to physically map the 5S rDNA genes to a chromosome arm location in rice. Chromosome preparations were made using an improved protoplast technique, which resulted in more mitotic cells with less overlying cytoplasmic and cellular debris. Cells in which both chromatids were labelled were observed. The hybridization detection level for the 5S rDNA gene complex was 17.22%. The results established that the 5S rDNA gene complex of rice is located at the end of the short arm of chromosome 9 in rice cultivar IR36. The similarities and differences of the 5S rDNA gene complex location between rice and other cereals and advantages of in situ hybridization for physical mapping are discussed.     

Assessment of genome organization among diploid species (2n = 2x = 14) belonging to primary and tertiary gene pools of pearl millet using fluorescent in situ hybridization with rDNA probes.

Two contrasting forms of Pennisetum belonging to the primary and tertiary gene pools were assessed for genomic organization using in situ hybridization with rDNA probes (18S-5.8S-25S and 5S) on metaphase and interphase cell nuclei. The primary gene pool is represented by diploid (2n = 2x = 14) cultivated pearl millet (Pennisetum glaucum) and its close wild relatives (Pennisetum violaceum and Pennisetum mollissimum). Pennisetum schweinfurthii (2n = 2x = 14) was taken as representative of the tertiary gene pool, owing to its diploid status and its similarity to the accessions of the primary gene pool in chromosome number. Using the 18S-5.8S-25S probe, we observed two sites of distribution in the four species but at different locations. Within the primary gene pool, signals were detected on the telomeric part of the short arm of chromosome pair VI and at the nucleolar organizing region (NOR) of the satellited chromosome pair (VII). Signals were observed at the NOR of the two satellited chromosome pairs (I and IV) of P. schweinfurthii. The 5S probe was detected at the telomeric part of the short arm of metacentric chromosome pair IV of the three species of the primary gene pool, while it occured in an intercalary position on the short arm of chromosome pair II of P. schweinfurthii. These results showed a chromosomal similarity of rDNA sequence locations within the primary gene pool and are in agreement with the high genetic identity between wild and cultivated forms of pearl millet previously revealed by allozyme studies. Implications of genomic organization for genetic resource enhancement are discussed. Key words : Pennisetum, in situ hybridization, rDNA probes, genomic organization.     

Physical mapping of rDNA sequences in four karyotypes of Ranunculus silerifolius (Ranunculaceae)

The chromosomal locations of the 18S-5.8S-26S rDNA and 5S rDNA sequences were examined in four cytotypes of Ranunculus silerifolius (the Matsuyama, Mugi, Otaru, and Karatsu types) using fluorescence in situ hybridization (FISH). Using the 18S-5.8S-26S rDNA probe, one pair of probe hybridization sites was detected by FISH in the interstitial region corresponding to the secondary constriction on the short arm of a satellite chromosome (chromosome pair 6) in all four karyotypes. FISH using 5S rDNA identified one pair of sites. The 5S rDNA locus was on different chromosomes in the four karyotypes: in the interstitial region of the short arm of the largest metacentric chromosome (chromosome pair 1) in the Matsuyama type, in the interstitial region of the short arm of the subtelocentric chromosome (pair 2) in the Mugi and Otaru types, and in the interstitial region of the short arm of the metacentric chromosome (pair 2) in the Karatsu type. This physical mapping of the 5S rDNA provides valuable information about karyotype evolution in R. silerifolius. Possible mechanisms of chromosome evolution are discussed.     

Chromosomal location of rRNA genes in Diprion pini detected by in situ hybridization

Diprion pini belongs to a small family of sawflies with n = 7 as the modal chromosome number. This species has 14 acrocentric chromosomes and one carries a satellite. In situ hybridization to mitotic chromosomes of a Drosophila rDNA probe was carried out according to two protocols. The biotinylated probe was detected with peroxidase-conjugated extravidin and diaminobenzidine or with fluorescein-conjugated extravidin (FISH). These two techniques showed that only one chromosome per haploid complement responds to the probe used. The centric fission hypothesis is consequently more likely than polyploidization in order to explain the chromosome number doubling in D. pini. The sites of probe hybridization are located on the satellite and its carrier short arm, which are heterochromatin-rich. The propensity of rDNA and heterochromatin for self duplication and accretion most likely created the satellite and the short arms. This process is also known to be one of the mechanisms by which extra segments may arise.     

A primate transfer RNA gene cluster and the evolution of human chromosome 1.

The localisation of tRNA(Asn) gene clusters in the karyotypes of primates has been studied by means of in situ hybridisation. In the human and orangutan (Pongo pygmaeus) karyotypes there are two such gene clusters, one each on the long and short arms of chromosome 1. Old World monkeys, however, contain both gene clusters on their equivalent of the human chromosome 1 short arm, which can be explained by a pericentric inversion which (amongst other chromosome changes) distinguishes the human and Old World monkey chromosomes 1. The capuchin (Cebus appella), however, a New World monkey, has only one tRNA(Asn) gene cluster, at least on the elements equivalent to human chromosome 1. This cluster is located proximal to the centromere on a chromosome that has been tentatively identified (by others) as the equivalent of the long arm of human chromosome 1. Should this prove to be correct, it would indicate that the large primate metacentric came into being in the form found today in the great apes, rather than in the form currently found in Old World monkeys. These data further show that the tRNA(Asn) gene cluster has been split in two since before the Old World monkeys and hominids diverged, i.e., over 30 million years ago, and also that the original transfer of these genes from one arm of chromosome 1 to the other was unlikely to have involved a pericentric inversion but, rather, some form of replicative transposition.     

Mapping of the nucleolus organizer region on chromosome 4 inArabidopsis thaliana

InArabidopsis thaliana the ribosomal RNA genes (rRNA genes or rDNA) are clustered in tandemly repeated blocks in two nucleolus organizer regions (NORs). Cytogenetic analysis has shown that the NORs are localized on chromosome 2 (NOR 2) and 4 (NOR 4). Recently the map position of NOR 2 was determined using a RFLP which was larger than 100 kb. In the course of a fingerprint analysis of differentArabidopsis ecotypes we have detected four rDNA polymorphisms between the ecotypes Landsberg (La) and Niederzenz (Nd). Mapping of these polymorphisms using established segregating F₂ populations reveals that all polymorphisms detected are dominant. Three of them map to the locus on the second chromosome that has been shown to harbour the NOR 2. The fourth polymorphism can be unambigously assigned to the upper arm of the fourth chromosome. This is the first polymorphism found which originates in the second rDNA cluster ofArabidopsis thaliana. It enables localization of NOR 4 and thus completes the mapping of rDNA genes in the NORs ofArabidopsis.