Chromosome-specific alpha satellite DNA: nucleotide sequence analysis of the 2.0 kilobasepair repeat from the human X chromosome.

The pericentromeric region of the human X chromosome is characterized by a tandemly repeated family of 2.0 kilobasepair (kb) DNA fragments, initially revealed by cleavage of human DNA with the restriction enzyme BamHI. We report here the complete nucleotide sequence of a cloned member of the repeat family and establish that this X-linked DNA family consists entirely of alpha satellite DNA. Our data indicate that the 2.0 kb repeat consists of twelve alpha satellite monomers arranged in imperfect, direct repeats. Each of the alpha X monomers is approximately 171 basepairs (bp) in length and is 60-75% identical in sequence to previously described primate alpha satellite DNAs. The twelve alpha X monomers are 65-85% identical in sequence to each other and are organized as two adjacent, related blocks of five monomers, plus an additional two monomers also related to monomers within the pentamer blocks. Partial nucleotide sequence of a second, independent copy of the 2.0 kb BamHI fragment established that the 2.0 kb repeat is, in fact, the unit of amplification on the X. Comparison of the sequences of the twelve alpha X monomers allowed derivation of a 171 bp consensus sequence for alpha satellite DNA on the human X chromosome. These sequence data, combined with the results of filter hybridization experiments of total human DNA and X chromosome DNA, using subregions within the 2.0 kb repeat as probes, provide strong support for the hypothesis that individual human chromosomes are characterized by different alpha satellite families, defined both by restriction enzyme periodicity and by chromosome-specific primary sequence.     

Concerted evolution of alpha satellite DNA: Evidence for species specificity and a general lack of sequence conservation among alphoid sequences of higher primates

We investigated relationships among alpha satellite DNA families in the human, gorilla, chimpanzee, and orangutan genomes by filter hybridization with cloned probes which correspond to chromosome-specific alpha satellite DNAs from at least 12 different human chromosomes. These include representatives of both the dimer-based and pentamer-based subfamilies, the two major subfamilies of human alpha satellite. In addition, we evaluated several high-copy dimer-based probes isolated from gorilla genomic DNA. Under low stringency conditions, all human probes tested hybridized extensively with gorilla and chimpanzee alpha satellite sequences. However, only pentameric and other non-dimeric human alphoid probes hybridized with orangutan alpha satellite sequences; probes belonging to the dimer subfamily did not cross-hybridize detectably with orangutan DNA. Moreover, under high stringency conditions, each of the human probes hybridized extensively only with human genomic DNA; none of the probes cross-hybridized effectively with other primate DNAs. Dimer-based gorilla alpha satellite probes hybridized with human and chimpanzee, but not orangutan, sequences under low stringency hybridization conditions, yet were specific for gorilla DNA under high stringency conditions. These results indicate that the alpha satellite DNA family has evolved in a concerted manner, such that considerable sequence divergence is now evident among the alphoid sequences of closely related primate species.     

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

Summary Non-isotopic in situ hybridization of chromosome-specific alphoid DNA probes has become a potent tool in the study of numerical aberrations of specific human chromosomes at all stages of the cell cycle. In this paper, we describe approaches for the rapid generation of such probes using the polymerase chain reaction (PCR), and demonstrate their chromosome specificity by fluorescence in situ hybridization to normal human metaphase spreads and interphase nuclei. Oligonucleotide primers for conserved regions of the alpha satellite monomer were used to generate chromosome-specific DNA probes from somatic hybrid cells containing various human chromosomes, and from DNA libraries from sorted human chromosomes. Oligonucleotide primers for chromosome-specific regions of the alpha satellite monomer were used to generate specific DNA probes for the pericentromeric heterochromatin of human chromosomes 1, 6, 7, 17 and X directly from human genomic DNA.     

A human alphoid DNA clone from the EcoRI dimeric family: Genomic and internal organization and chromosomal assignment

We isolated an alpha satellite DNA clone (pC1.8), 17 kb long, which is composed exclusively of tandemly repeated 340-bp EcoRI fragments. Hybridization studies using 37 random EcoRI dimers subcloned from pC1.8 showed that they are heterogeneous. The sequence of 5 dimers, 3 of them adjacent, confirmed this observation and showed that the heterogeneity is more accentuated among the second monomers. The chromosomal assignment under high stringency conditions showed that this alphoid subset is located on chromosomes 1, 5, and 19. No conditions that eliminate the hybridization on any one of those chromosomes were found. This suggests that, in contrast to many other chromosome-specific alpha satellite subsets, the single chromosome subsets of this family are virtually indistinguishable by hybridization techniques.     

A cloned fragment of HeLa DNA containing consensus sequences of satellite II and III DNA hybridizes with the Drosophila P-element and with the 1.8 kb family of human KpnI fragments

We have cloned a repetitive EcoRI fragment from the human genome which displays weak homologies with the Drosophila melanogaster transposable P-element. This cloned DNA appeared not to be a mobile element but, instead, a divergent member of human satellite II or III DNAs. We present here the first complete nucleotide sequence of a 1.797 kilobase pair (kb) satellite-like DNA. Moreover, this EcoRI satellite monomer contains a unique sequence of 49 basepairs (bp) that is devoid of the satellite consensus repeat 5'TTCCA3'. Southern hybridization analysis revealed that the cloned insert is closely related to a highly repetitive 1.8 kb KpnI family of tandemly organized satellite DNAs. Thus, the relationships among these satellite DNA families appear to be complex and may be a factor in their copy number, position and spatial organization.     

Sequence, higher order repeat structure, and long-range organization of alpha satellite DNA specific to human chromosome 8.

We have characterized alphoid repeat clones derived from a chromosome 8 library. These clones are specific for human chromosome 8, as demonstrated by use of a somatic cell hybrid mapping panel and by in situ hybridization. Hybridization of the clones to HindIII digests of human genomic DNA reveals a complex pattern of fragments ranging in size from 1.3 to greater than 20 kb. One clone, which corresponds in size to the most prevalent genomic HindIII fragment, appears to represent a major higher order repeat in the chromosome 8 centromere. The DNA sequence of this clone reveals a dimeric organization of alphoid monomers. Restriction analysis of two other clones indicates that they are derivatives of this same repeat unit. The chromosome 8 alphoid clones hybridize to EcoRI fragments of genomic DNA ranging up to 1000 kb in length and reveal a high degree of polymorphism between chromosomes. Distribution of higher order repeat units across the centromere was examined by two-dimensional gel electrophoresis. Repeat units of the same size class tended to cluster together in restricted regions of centromeric DNA.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Localization of the human gene for the type I cyclic GMP-dependent protein kinase to chromosome 10.

We have recently characterized cDNAs and genomic DNA fragments for human type I cGMP-dependent protein kinase (cGK). By probing human x hamster hybrid cell lines with a 1.2-kb intron fragment from the human type I cGK gene, we identified a 5.9-kb BglII restriction fragment and localized it to human chromosome 10. In situ hybridization analyses using 3H-labeled cDNA and genomic DNA probes for the human type I cGK to human metaphase chromosomes supported the somatic cell hybrid data and indicated that the gene (PRKG1B; protein kinase, cGMP-dependent) maps to 10p11.2----q11.2.     

Isolation and characterization of two families of satellite DNA with repetitive units of 135 bp and 2.5 kb in the ant Monomorium subopacum (Hymenoptera, Formicidae)

Analyzing the satellite DNA in the ant species Monomorium subopacum we found two unrelated families of satellite DNA. Because these satellite DNA families were isolated using the two enzymes HaeIII and EcoRI we called the two families HaeIII and EcoRI family, respectively. The HaeIII family proved to be organized in a 135-bp basic unit repeat, the EcoRI family in a 2.5-kb basic unit repeat. The latter represents perhaps the longest satellite DNA isolated up to now in insects. The HaeIII family apparently comprises about 10% of the total genomic DNA whereas the EcoRI family represents only about 1-2%. A comparative analysis of the two satellite DNA sequences showed no homology between the two families although both sequences possessed long A and T stretches. Eight of the 34 chromosomes showed hybridization with the HaeIII family and hybridization signals are visible in six chromosomes with the EcoRI family. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that the HaeIII family is only slightly curved. However, the unit of the EcoRI satellite DNA family has curvature, especially the first 1000 bp of the monomeric repeat, in which this DNA is AT rich and has numerous A and T stretches. There are also internal inverted subrepeats in each family. The sequences of satellite DNA families found in Monomorium subopacum are different from the sequences of other satellite DNAs cloned in insects, including other species of ants.     

Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.     

Saturation of human chromosome 3 with unique sequence hybridization probes

We have generated chromosome 3-specific recombinant libraries in both lambda and cosmid cloning vectors starting with somatic cell hybrids (hamster/human) containing either an intact chromosome 3 or a chromosome 3 with an interstitial deletion removing 75% of long-arm sequences. The libraries contained between 2 X 10(5) and 5 X 10(6) independent recombinants. Approximately 2% of the recombinants in these libraries contain inserts of human DNA. These were identified by hybridizing the recombinants to radioactively labeled total human DNA. Over 2500 recombinants containing human DNA were isolated from these various libraries and DNA was prepared from each of them. This represents 80,000 kb of cloned chromosome 3 sequences. One-third of the DNAs were digested with EcoRI or HindIII, and fragments free of repetitive sequences were radioactively labeled using random hexanucleotide primers and tested as unique sequence hybridization probes. Over 6500 of the fragments were tested and of these 758 were unique sequence probes with minimal or no background hybridization. Their hybridization only to chromosome 3 was verified. These probes, which were derived from 452 independent recombinants, should provide an effective saturation of human chromosome 3.     

Genomic content and new insights on the origin of the B chromosome of the cichlid fish <Emphasis Type="Italic">Astatotilapia latifasciata</Emphasis>

B chromosomes are additional chromosomes widely studied in a diversity of eukaryotic groups, including fungi, plants and animals, but their origin, evolution and possible functions are not clearly understood. To further understand the genomic content and the evolutionary history of B chromosomes, classical and molecular cytogenetic analyses were conducted in the cichlid fish Astatotilapia latifasciata, which harbor 1–2 B chromosomes. Through cytogenetic mapping of several probes, including transposable elements, rRNA genes, a repeated DNA genomic fraction (C ₀ t − 1 DNA), whole genome probes (comparative genomic hybridization), and BAC clones from Oreochromis niloticus, we found similarities between the B chromosome and the 1st chromosome pair and chromosomes harboring rRNA genes. Based on the cytogenetic mapping data, we suggest the B chromosome may have evolved from a small chromosomal fragment followed by the invasion of the proto-B chromosome by several repeated DNA families.     

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.     

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.     

Cytological and biochemical characterization of the in situ endonuclease digestion of fixed Tenebrio molitor chromosomes

Cytological and biochemical experiments were undertaken in order to characterize the action of several restriction enzymes on fixed chromosomes of Tenebrio molitor (Coleoptera). EcoRI cuts the satellite DNA of this organism into suunit monomers of 142 bp in naked DNA and acts on fixed chromosomes cleaving and extracting these tandemly repeated sequences present in median centromeric heterochromatin. AluI, in contrast, is unable to attack the satellite sequences but does cut the main band DNA both in naked DNA and in fixed chromosomes. These enzymes therefore permit the in situ localization of satellite DNA or main band DNA in T. molitor. Other enzymes such HinfI or Sau3A do not produce longitudinal differentiation in chromosomes because of the extraction of DNA from satellite and main band DNA regions. In situ hybridization with a satellite DNA probe from T. molitor confirms that the DNA extracted from the chromosomes is the abundant and homogenous highly repeated DNA present in pericentromeric regions. These results plus the analysis of the DNA fractions retained on the slide and solubilized by the action of the restriction enzymes in situ provide evidence that: (a) as an exception to the rule EcoRI (6 bp cutter) is able to produce chromosome banding; (b) the size of the fragments produced by in situ digestion of satellite DNA with EcoRI is not a limiting factor in the extraction; (c) there is a remarkable accord between the action of EcoRI and AluI on naked DNA and on DNA in fixed chromosomes, and (d) the organization of specific chromosome regions seems to be very important in producing longitudinal differentiation on chromosomes.by E.R. Schmidt     

The organization of the evolutionarily conserved GATA/GACA repeats in the mouse genome

Simple repeated GATA and GACA sequences which were originally isolated from sex-specific snake satellite DNA have been found subsequently in all eukaryotes studied. The organization of these sequences within the mouse genome was investigated here by using synthetic oligonucleotide probes as a novel tool in comparison with conventional hybridization probes. Southern blot hybridization showed sex-specific patterns with both the (GATA)₄ and (GACA)₄ oligonucleotide probes, as previously described with conventional probes. The quantitative analysis of two mouse DNA phage libraries and of 25 isolated GATA-positive phage clones revealed intensive interspersion of GATA sequences with GACA, and with other repetitive and single-copy sequences. Ubiquitous interspersion and homogeneous genomic distribution of GATA and GACA sequences were confirmed by hybridization in situ of the oligonucleotide probes to metaphase chromosomes. The lengths of the GATA and GACA stretches were found to vary considerably in the individual phage clones. DNA inserts from 20 phages were assigned to autosomes and sex chromosomes and three genomic fragments were found to be confined to the Y chromosome. The organization of GATA and GACA sequences is discussed in the context of their evolutionary potential and possible conservation mechanisms.     

Assignment of the alpha 1-antitrypsin gene and a sequence-related gene to human chromosome 14 by molecular hybridization

alpha 1-Antitrypsin is a major plasma protease inhibitor synthesized in the liver. Genetic deficiency of this protein predisposes the affected individuals to development of infantile liver cirrhosis or chronic obstructive pulmonary emphysema. The human chromosomal alpha 1-antitrypsin gene has been cloned and shown to contain three introns in the peptide-coding region. When the cloned alpha 1-antitrypsin gene was used as a hybridization probe to analyze Eco RI-digested genomic DNA from different individuals, two distinct bands of 9.6 kilobases (kb) and 8.5 kb in length were observed in every case. Further analysis using only labeled intronic DNA as the hybridization probe has indicated that the authentic alpha 1-antitrypsin gene resides within the 9.6-kb fragment. Thus the 8.5-kb fragment must contain another gene that is closely related in sequence to the alpha 1-antitrypsin gene. Using a series of human-Chinese hamster somatic cell hybrids containing unique combinations of human chromosomes, the alpha 1-antitrypsin gene as well as the sequence-related gene have been assigned to human chromosome 14 by Southern hybridization and synteny analysis.     

Organization of delta-crystallin genes in the chicken.

Double-stranded DNA was synthesized from delta-crystallin mRNA prepared from lens fibers of 15-day-old chick embryos and cloned at the Pst I site of the plasmid pBR322. Using the cloned cDNA and single-stranded cDNA as hybridization probes, a number of genomic DNA fragments containing delta-crystallin gene sequences have been cloned from the partial and complete EcoRI digests of chick brain DNA. One of the clones from the partial digests contains a DNA fragment that consists of four EcoRI fragments of 7.6 kb, 4.0 kb, 2.6 kb, and 0.8 kb. The gene sequences reside in the (5')7.6 kb - 0.8 kb - 4.0 kb (3') fragments. Electron microscopy has provided evidence that the cloned DNA fragment includes the entire gene sequences complementary to delta-crystallin mRNA except for the 3' terminal poly(A) tail, and that the delta-crystallin gene is interrupted by at least 13 intervening sequences. Another clone contains a genomic fragment that consists of two EcoRI fragments of 3.0 kb and 11 kb. The DNA fragment in the latter clone represents a different delta-crystallin gene, as judged by restriction endonuclease mapping and by electron microscopy.     

Long tandem arrays of complex repeat units in Chironomus telomeres.

A cloned 340-bp DNA fragment excised by EcoRI from the Chironomus pallividittatus genome has been localized to the telomeres by in situ hybridization as well as to connectives between telomeres. No hybridization was observed in other regions of the chromosomes. Another cloned EcoRI fragment, 525 bp long has also been studied. This represents a partial duplication of the 340-bp sequence. Genomic blot hybridization experiments show that the 340-bp sequence is a representative monomeric unit of tandemly repeated arrays which account for 1.2% of the Chironomus genome, on average 300 kb per telomere. The repeat unit contains two types of subrepeats each present twice per repeat unit. Northern blot hybridization experiments show that the telomere-associated sequences are transcribed into a discrete RNA species approximately 20 kb in size. The evolution of this telomere-associated DNA is discussed.