Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors.

The suitability of yeast artificial chromosome vectors (YACs) for cloning human Y chromosome tandemly repeated DNA sequences has been investigated. Clones containing DYZ3 or DYZ5 sequences were found in libraries at about the frequency anticipated on the basis of their abundance in the genome, but clones containing DYZ1 sequences were under-represented and the three clones examined contained junctions between DYZ1 and DYZ2. One DYZ3 clone was quite stable and had a long-range structure corresponding to genomic DNA. All other clones had long-range structures which either did not correspond to genomic DNA, or were too unstable to allow a simple comparison. The effects of the transformation process and host genotype on YAC structural stability were investigated. Gross structural rearrangements were often associated with re-transformation of yeast by a YAC. rad1-deficient yeast strains showed levels of instability similar to wild-type for all YAC clones tested. In rad52-deficient strains, DYZ5 containing YACs were as unstable as in the wild-type host, but DYZ1/DYZ2 or DYZ3 containing YACs were more stable. Thus the use of rad52 hosts for future library construction is recommended, but some sequences will still be unstable.     

A cloned repeated DNA sequence in human chromosome heteromorphisms

A sequence derived by ECoRI restriction of human satellite DNA III has been cloned in lambda gt WES. The cloned DNA was used as a template for in vitro synthesis of cRNA, which was hybridized in situ to preparations of human metaphase chromosomes with a range of heterochromatic polymorphisms. Most of the hybridization was found on chromosome 1, and the amount of hybridization was related to the size of the C-band on this chromosome. Hybridization to other chromosomes was not related to the C-band size, although hybridization of total satellite DNA is proportional to C-band size. Total satellite DNAs contain a mixture of sequences, some of which are predominantly located on only one pair of chromosomes. Hybridization in situ is able to discriminate between such chromosome-specific sequences and the bulk of satellite DNA. Further analysis of satellite DNAs may identify sequences specific for every chromosome pair.     

Regional mapping of six cloned DNA sequences on human chromosome 7.

The regional localization of six cloned DNA sequences on human chromosome 7 was assessed by molecular hybridization to human/rodent cell hybrid DNAs. The allelic distribution and familial segregation of two frequent polymorphisms revealed by two probes are presented.     

Presence of various rye-specific repeated DNA sequences on the midget chromosome of rye.

The chromosome 1R of rye, or the midget chromosome, is necessary for plump, viable seed development and fertility restoration in the alloplasmic line with rye cytoplasm and a hexaploid wheat nucleus. The midget chromosome of rye represents 1/15th of the physical length of the chromosome 1R of rye. C-banding analysis indicated that the centromeric and pericentric region (approximately 30% physical length) of the midget chromosome is heterochromatic and the distant 70% physical length is euchromatic. These data suggest that the midget chromosome may represent the pericentric region of the long arm of chromosome 1R. In contrast with earlier reports, our results indicate that an array of rye-specific repeated sequences (both dispersed and tandem) are present on the midget chromosome. Various rye-specific repeated DNA sequences that are present on the midget chromosome will be useful in constructing a long-range map and studying the genomic organization of the midget chromosome. It is unclear if any of these repeated DNA sequences are involved in the origin of the midget chromosome.     

高等植物DNA重复序列的主要类型和特点

高等植物核基因组的一个显著特征是其内含有大量的ＤＮＡ重复序列,因此它们在核基因组结构和功能研究中居于举足轻重的地位。一些ＤＮＡ重复序列已日趋广泛地作为分子民用于构建遗传图谱、鉴别品种、研究进化和分离目标基因等。主要介绍高等植物几类重要ＤＮＡ重复序列,如卫星ＤＮＡ、微卫星ＤＮＡ、核糖体ＲＮＡ基因、端粒重复序列和转座子等的若干特点和用途。     

Characterization of human heterochromatin by in situ hybridization with satellite DNA clones

Biotinylated DNA from two satellite-related, repetitive DNA clones, pHuR 98 and pHuR 195 (specific for chromosomes 9 and 16, respectively), and from a Y-specific clone, pY-3.4A, were hybridized to human metaphase chromosomes using fluoresceinated avidin to detect binding. The chromosomes were simultaneously counterstained with distamycin-DAPI to identify the AT-rich heterochromatin of chromosomes 1, 9, 15, 16, and the Y chromosome. With this method, clear results were obtained under both normal and low stringency conditions, allowing hybridization between molecules sharing 80-85% and 60-65% identity, respectively. Thus, additional sites related to the probes could be identified. A close relationship was shown between the heterochromatin of chromosomes 1 and 16, both hybridizing with clone pHuR 195 under low stringency. Hybridization with clone pHuR 98 was highly specific for chromosome 9, even under low stringency. A relationship between chromosomes 9, 15, and the Y chromosome, however, was shown by hybridization with clone pY-3.4A. The chromosomal distribution of the three repetitive DNA clones used in this study, and data from the literature, are in accordance with the distribution of the heterochromatin types characterized by staining with different fluorescent dyes and dye combinations. Furthermore, our sequence data for clones pHuR 98 and pHuR 195 may explain the fluorescent properties on which the cytogenetic classification of the heterochromatin is based.     

Replication pattern of human repeated DNA sequences

Either aphidicolin- or thymidine-synchronized human HL-60 cells were used to study the replication pattern of a family of human repetitive DNA sequences, the EcoRI 340 bp family (αRI-DNA), and of the ladders of fragments generated in total human DNA after digestion with XbaI and HaeIII (alpha satellite sequences). DNAs replicated in early, middle-early, middle-late and late S periods were labelled with BUdR or with [³H]thymidine. The efficiency of the cell synchronization procedure was confirmed by the transition from a high-GC to a high-AT average base composition of the DNA synthesized going from early to late S periods. By hybridizing EcoRI 340 bp repetitive fragments to BUdR-DNAs it was found that this family of sequences is replicated throughout the entire S period. Comparing fluorograph densitometric scans of [³H]DNAs to the scans of ethidium bromide patterns of total HL-60 DNA digested with XbaI and HaeIII, it was observed that DNA synthesized in different S periods is characterized by approximately the same ladder of fragments, while the intensity of each band may vary through the S phase; in particular, the XbaI 2.4 kb fragment becomes undetectable in late S.     

Localization of repeated DNA sequences in CsCl gradients by hybridization with complementary RNA

Single-stranded ends of mouse satellite DNA sequences exposed during the extraction of nuclear DNA, or by mechanical shear, can hybridize with highly labelled RNA, complementary to satellite DNA. In neutral CsCl gradients, the hybrid sediments as a sharp peak at the density of mouse satellite DNA (1.691 g/cm³). The possibility of exploiting this observation in locating and isolating cryptic repetitive DNAs from nuclear DNA or the DNA of heterochromatin is discussed.     

Use of a cloned alphoid repetitive sequence of human DNA in studying the polymorphism of heterochromatin regions of chromosomes

Chromosomal location of the cloned fragment pHS05 of alphoid DNA from the collection of human PstI restricts has been studied in 38 individuals by in situ hybridization. Pericentromeric localization of the DNA fraction studied was found in practically all chromosomes of the set. Significant interchromosomal and poorly expressed interindividual differences were detected in a number of the copies of the sequence class investigated. The majority of the label (approx. 27%) was observed over the pericentromeric region of chromosome 3. No relationship was discovered between hybridization results and the pattern of Q-polymorphism.     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.     

Molecular hybridization analysis of cloned Vicia faba minicircular DNA sequences

Summary Three types of minicircular DNA isolated from Vicia faba mitochondria were cloned in pBR322 plasmids. The correspondence of cloned sequences to the original molecules was verified by coelectrophoresis and by DNA-DNA hybridization with total mitochondrial DNA preparations. It was found that the cloned sequences hybridized not only with the minicirclar DNAs and their derivates, but also with some discrete classes of higher molecular weight DNA and with the major DNA. The data obtained from restriction enzyme analysis of complementary sequences suggested that the majority of them were represented by the minicircular DNAs and by the oligomers. The physical maps of cloned sequences were also prepared. These maps differed significantly. However, reciprocal DNA-DNA hybridization showed the existence of sequence homology between minicircular DNAs.     

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

Summary The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.     

Quantitative analysis of chromosomal localization of two human cloned satellite DNA III sequences by in situ hybridization

Chromosomal location of two cloned human satellite DNA III sequences pPD9 and pPD18 has been studied in 30 individuals by in situ hybridization. Pericentromeric localization of the DNA subsets studied was found in practically all chromosomes of the set. The majority of label was observed over the pericentromeric region of chromosome 9 (38.3% for pPD18 clone and 26.2% for pPD9), the short arm of chromosome 15 (17.2% - the pPD9 clone and 10.6% - the pPD18 clone) and the distal part of the long arm of Y chromosome (19.6% - the pPD9 clone and 15.4% - the pPD18 clone). Besides significant interchromosomal differences, moderately pronounced interindividual differences were also detected in the number of grains over the regular sites of the chromosomal location. Pretreatment of slides with DA/DAPI induced differences in the results of quantitative analysis is described.     

Rapid methods to visualize Y-specific repeated DNA sequences in cytological preparations

We described rapid methods to detect Y-specific repeated DNA sequences in cytological preparations using in situ hybridization. A human Y chromosome specific DNA probe with an insert equivalent to that in pHY2.1 was labelled with [alpha-32P]dCTP or photobiotin, and hybridized to chromosome preparations. Signals were visualized specifically on Y chromosomes after 1 day's autoradiography or a couple of hours treatment with streptavidin alkaline phosphatase/BCIP/NBT. These methods are useful for molecular confirmation of Y-autosomal translocations.     

A male-specific DNA probe detects heterochromatin sequences in a familial Yq- chromosome.

Using a recombinant DNA probe, we have demonstrated the presence of residual 3.4-kilobase (kb) repeat sequences in a family with a Yq- chromosome. The heterochromatin of this Y variant was not readily detectable with conventional chromosome-banding techniques. These data suggest that the breakpoint of the deletion occurs at the heterochromatin region proximal to the euchromatin/heterochromatin junction.     

Simple repeated sequences in human satellite DNA.

In an extensive analysis, using a range of restriction endonucleases, HinfI and TaqI were found to differentiate satellites I, II and III & IV. Satellite I is resistant to digestion by TaqI, but is cleaved by HinfI to yield three major fragments of approximate size 770, 850 and 950bp, associated in a single length of DNA. The 770bp fragment contains recognition sites for a number of other enzymes, whereas the 850 and 950bp fragments are "silent" by restriction enzyme analysis. Satellite II is digested by HinfI into a large number of very small (10-80bp) fragments, many of which also contain TaqI sites. A proportion of the HinfI sites in satellite II have the sequence 5'GA(GC)TC. The HinfI digestion products of satellites III and IV form a complete ladder, stretching from 15bp or less to more than 250bp, with adjacent multimers separated by an increment of 5bp. The ladder fragments do not contain TaqI sites and all HinfI sites have the sequence 5'GA(AT)TC. Three fragments from the HinfI ladder of satellite III have been sequenced, and all consist of a tandemly repeated 5bp sequence, 5'TTCCA, with a non-repeated, G+C rich sequence, 9bp in length, at the 3' end.