Chromosome-specific α-satellite DNA from the centromere of chimpanzee chromosome 4

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated α-satellite DNA. Comparison of the α-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human α-satellite DNA, we have amplified a chromosome-specific α-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones pαPTR4N and pαPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This α-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 α-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the α-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral α-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes. Received: 21 February 1997; in revised form: 26 February 1997 / Accepted: 27 February 1997     

Structure of the major block of alphoid satellite DNA on the human Y chromosome

Alphoid DNA is a family of tandemly repeated simple sequences found mainly at the centromeres of the chromosomes of many primates. This paper describes the structure of the alphoid DNA at the centromere of the human Y chromosome. We have used pulsedfield gradient gel electrophoresis, cosmid cloning and DNA sequencing to determine the organization of the alphoid DNA on each of the Y chromosomes present in two somatic cell hybrids. In each case there is a single major block of alphoid DNA. This is approximately 470,000 bases (475 kb) long on one chromosome and approximately 575 kb long on the other. Apart from the size difference, the structures of the two blocks and the surrounding sequences are very similar. However, one restriction enzyme, AvaII, detects two clusters of sites within one block but does not cleave the other. The alphoid DNA within each block is organized into tandemly repeating units, most of which are about 5.7 kb long. A few variant units present on one chromosome are about 6.0 kb long. These variants, like the AvaII site variants, are clustered. The 5.7 kb and 6.0 kb units themselves consist of tandemly repeating 170 base-pair subunits. The 6.0 kb unit has two more of these subunits than the 5.7 kb unit. Our results provide a basis for further structural analysis of the human Y chromosome centromeric region, and suggest that long-range structural polymorphisms of tandemly repeated sequence families may be frequent.     

Chromosome-specific subfamilies within human alphoid repetitive DNA

Nucleotide sequence data of about 20 X 10(3) base-pairs of the human tandemly repeated alphoid DNA are presented. The DNA sequences were determined from 45 clones containing EcoRI fragments of alphoid DNA isolated from total genomic DNA. Thirty of the clones contained a complete 340 base-pair dimer unit of the repeat. The remaining clones contained alphoid DNA with fragment lengths of 311, 296, 232, 170 and 108 base-pairs. The sequences obtained were compared with an average alphoid DNA sequence determined by Wu & Manuelidis (1980). The divergences ranged from 0.6 to 24.6% nucleotide changes for the first monomer and from 0 to 17.8% for the second monomer of the repeat. On the basis of identical nucleotide changes at corresponding positions, the individual repeat units could be shown to belong to one of several distinct subfamilies. The number of nucleotide changes defining a subfamily generally constitutes the majority of nucleotide changes found in a member of that subfamily. From an evaluation of the proportion of the total amount of alphoid DNA, which is represented by the clones studied, it is estimated that the number of subfamilies of this repeat may be equal to or exceed the number of chromosomes. The expected presence of only one or a few distinct subfamilies on individual chromosomes is supported by the study, also presented, of the nucleotide sequence of 17 cloned fragments of alphoid repetitive DNA from chromosome 7. These chromosome-specific repeats all contain the characteristic pattern of 36 common nucleotide changes that defines one of the subfamilies described. A unique restriction endonuclease (NlaIII) cleavage site present in this subfamily may be useful as a genetic marker of this chromosome. A family member of the interspersed Alu repetitive DNA was also isolated and sequenced. This Alu repeat has been inserted into the human alphoid repetitive DNA, in the same way as the insertion of an Alu repeat into the African green monkey alphoid DNA.     

Distribution of gamma satellite DNA on the human X and Y chromosomes suggests that it is not required for mitotic centromere function

The bulk of the DNA found at human centromeres is composed of tandemly arranged repeats, the most abundant of which is alpha satellite. Other human centromeric repetitive families have been identified, one of the more recent being gamma satellite. To date, gamma satellite DNAs have been reported at the centromeres of human chromosomes 8 and X. Here, we show that gamma-X satellite DNA is not interspersed with the major DZX1 alpha-X block, but rather is organised as a single array of approximately 40-50 kb on the short-arm side of the alpha satellite domain. This repeat array is absent on two mitotically stable Xq isochromosomes. Furthermore, a related repeat DNA has been identified on the human Y chromosome. Fluorescence in situ hybridisation has localised this satellite DNA to the long arm side of the major DYZ3 alpha-Y domain, outside the region previously defined as that required for mitotic centromere function. Together, these data suggest that while blocks of highly related gamma satellite DNAs are present in the pericentromeric regions of both human sex chromosomes, this repeated DNA is not required for mitotic centromere function.     

Recent evolution of DNA sequence homology in the pericentromeric regions of human acrocentric chromosomes

A search for genes located on human chromosome 21 resulted in the isolation of a HeLa cDNA clone, pUNC724, which hybridized to 3.7 and 2.5 kilobase (kb) EcoRI fragments on each of the human acrocentric chromosomes. In situ hybridization further localized pUNC724 to the pericentromeric region of the human acrocentrics. Two other EcoRI fragments that hybridized to pUNC724 were assigned to the long arms of chromosomes 1 and 18. The pUNC724 sequence does not appear to be related to ribosomal or satellite DNA sequences. The juxtaposition of DNA sequences homologous to pUNC724 and ribosomal DNA sequences presumably occurred within the past thirty-five million years, following the divergence of the lines leading to man and the New World owl monkey, Aotus trivirgatus--pUNC724 is not syntenic with the single chromosome containing ribosomal DNA sequences in the owl monkey.     

Isolation and characterization of a human telomere.

A method is described that allows cloning of human telomeres in S. cerevisiae by joining human telomeric restriction fragments to yeast artificial chromosome halves. The resulting chimeric yeast-human chromosomes propagate as true linear chromosomes, demonstrating that the human telomere structure is capable of functioning in yeast and suggesting that telomere functions are evolutionarily conserved between yeast and human. One cloned human telomere, yHT1, contains 4 kb of human genomic DNA sequence next to the tandemly repeating TTAGGG hexanucleotide. Genomic hybridizations using both cloned DNA and TTAGGG repeats have revealed a common structural organization of human telomeres. This 4 kb of genomic DNA sequence is present in most, but not all, human telomeres, suggesting that the region is not involved in crucial chromosome-specific functions. However, the extent of common features among the human telomeres and possible similarities in organization with yeast telomeres suggest that this region may play a role in general chromosome behavior such as telomere-telomere interactions. Unlike the simple telomeric TTAGGG repeats, our cloned human genomic DNA sequence does not cross-hybridize with rodent DNA. Thus, this clone allows the identifications of the terminal restriction fragments of specific human chromosomes in human-rodent hybrid cells.     

The Bam repeats of the mouse genome belong in several superfamilies the longest of which is over 9 kb in size.

Mouse DNA contains two equally abundant, homologous subfamilies of MspI 3.6 and 5 kb repeated fragments. The first subfamily corresponds to the previously described (1) Bam 4 kb repeats, the second one to Bam repeated fragments of higher molecular weight. These subfamilies account for the vast majority of long Bam repeats and are linked with contiguous short Bam 0.5 kb repeats. A minority of these composite Bam repeats extend, on the 0.5 kb side, into R repeats. In turn, a fraction of the composite Bam/R repeats extend further, for at least 3 kb, into other repeated sequences contiguous to the R repeats. The long Bam repeats belong, therefore, in at least three superfamilies of repeats, the longest one being over 9 kb in size. Some general properties of these superfamilies are discussed.     

Structure and variability of human chromosome ends.

Mammalian telomeres are thought to be composed of a tandem array of TTAGGG repeats. To further define the type and arrangement of sequences at the ends of human chromosomes, we developed a direct cloning strategy for telomere-associated DNA. The method involves a telomere enrichment procedure based on the relative lack of restriction endonuclease cutting sites near the ends of human chromosomes. Nineteen (TTAGGG)n-bearing plasmids were isolated, two of which contain additional human sequences proximal to the telomeric repeats. These telomere-flanking sequences detect BAL 31-sensitive loci and thus are located close to chromosome ends. One of the flanking regions is part of a subtelomeric repeat that is present at 10 to 25% of the chromosome ends in the human genome. This sequence is not conserved in rodent DNA and therefore should be a helpful tool for physical characterization of human chromosomes in human-rodent hybrid cell lines; some of the chromosomes that may be analyzed in this manner have been identified, i.e., 7, 16, 17, and 21. The minimal size of the subtelomeric repeat is 4 kilobases (kb); it shows a high frequency of restriction fragment length polymorphisms and undergoes extensive de novo methylation in somatic cells. Distal to the subtelomeric repeat, the chromosomes terminate in a long region (up to 14 kb) that may be entirely composed of TTAGGG repeats. This terminal segment is unusually variable. Although sperm telomeres are 10 to 14 kb long, telomeres in somatic cells are several kilobase pairs shorter and very heterogeneous in length. Additional telomere reduction occurs in primary tumors, indicating that somatic telomeres are unstable and may continuously lose sequences from their termini.     

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.     

Functional genomic mapping of an early-activated centromeric mammalian origin of DNA replication.

Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV-1 cells) nascent DNA from active replication bubbles. It contains a region of alpha-satellite extending 168-bp from the 5'-end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle-dependent manner, and hybridizes at the centromeric region of six CV-1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growing CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion.     

A chromosome 14-specific human satellite III DNA subfamily that shows variable presence on different chromosomes 14.

We describe a new subfamily of satellite III DNA (pTRS-63), which, by a combination of in situ hybridization to human metaphase chromosomes and analysis of a panel of somatic cell hybrids, is shown to be specific for human chromosome 14. This DNA has a basic 5-bp repeating unit of diverged GGAAT which is tandemly repeated and organized into either one of two distinct higher-order structures of 5 kb (designated the "L" form) or 4.8 kb (designated the "S" form). In addition, a third (Z) form, representing no detectable levels of this satellite III subfamily, is found. Results from five somatic cell hybrid lines and from a number of informative human individuals suggest that, on any one chromosome 14, only one of the three forms may exist. Subchromosomally, this sequence has been mapped to the p11 region and is distal to the domain occupied by another previously described satellite III subfamily (pTRS-47) found on chromosome 14. The pTRS-63 sequence described adds to the understanding of the structural organization of the short arm of human chromosome 14 and should be useful for the investigation of the molecular etiology of the frequently occurring t(13q14q) and t(14q21q) Robertsonian translocations.     

Structures of two HaeIII-type genes in the human salivary proline-rich protein multigene family

Two members of the human salivary proline-rich protein (PRP) multigene family have been isolated and completely sequenced. These PRP genes, PRH1 and PRH2, are of the HaeIII-type subfamily and code for acidic PRP proteins. Both genes are approximately 3.5 kilobase pairs (kb) in length and contain four exons. Exon 3 encodes the proline-rich part of the protein and includes five 63-base pair (bp) repeats. CAT and ATA boxes and several possible enhancer sequences occur in a 1-kb region 5' to exon 1. Two sets of repeats occur in the sequenced region in addition to the 63-bp repeats: one pair of about 140 bp flanks 500 bp of DNA in the first intervening sequence, and the other pair of 72 bp is tandemly repeated 1.4 kb 5' to the PRH1 gene. The 4-kb region of sequenced DNA from PRH1 differs by an average of 8.7% from the same region in PRH2, but the nucleotide sequences of the exon 3 of the two genes differ by only 0.2%. This result suggests the occurrence of a recent gene conversion event. The regions containing the 5-fold repeated sequences of 63 bp are identical in the two genes, PRH1 and PRH2. A comparison of the human HaeIII and BstNI subfamily repeats and a comparison of the human, mouse, and rat repeats suggest that the individual repeats have evolved in a concerted fashion within each gene and within the PRP gene family as a whole.     

Comparative cytogenetics of human chromosome 3q21.3 reveals a hot spot for ectopic recombination in hominoid evolution

Fluorescence in situ hybridization mapping of fully integrated human BAC clones to primate chromosomes, combined with precise breakpoint localization by PCR analysis of flow-sorted chromosomes, was used to analyze the evolutionary rearrangements of the human 3q21.3-syntenic region in orangutan, siamang gibbon, and silvered-leaf monkey. Three independent evolutionary breakpoints were localized within a 230-kb segment contained in BACs RP11-93K22 and RP11-77P16. Approximately 200 kb of the human 3q21.3 sequence was not present on the homologous orangutan, siamang, and Old World monkey chromosomes, suggesting a genomic DNA insertion into the breakpoint region in the lineage leading to humans and African great apes. The breakpoints in the orangutan and siamang genomes were narrowed down to 12- and 20-kb DNA segments, respectively, which are enriched with endogenous retrovirus long terminal repeats and other repetitive elements. The inserted DNA segment represents part of an ancestral duplication. Paralogous sequence blocks were found at human 3q21, approximately 4 Mb proximal to the evolutionary breakpoint cluster region; at human 3p12.3, which contains an independent orangutan-specific breakpoint; and at the subtelomeric and pericentromeric regions of multiple human and orangutan chromosomes. The evolutionary breakpoint regions between human chromosome 3 and orangutan 2 as well their paralogous segments in the human genome coincide with breaks of chromosomal synteny in the mouse, rat, and/or chicken genomes. Collectively our data reveal reuse of the same short recombinogenic DNA segments in primate and vertebrate evolution, supporting a nonrandom breakage model of genome evolution.     

Structural and transcriptional analysis of a human subtelomeric repeat

A human subtelomeric repeat (designated as the HST repeat) has been isolated and characterized from a yeast artificial chromosome containing one human telomere. This repeat is located immediately adjacent to the telomeric T2AG3 repeats at the extreme termini of the human chromosomes. The DNA sequence of 3.6 kb of the HST repeat has been determined. The HST repeat spans over 3.6 kb in length, and contains one evolutionarily conserved CpG-rich region. The copy number of the HST repeat varies among telomeres. Genomic hybridization experiments suggest that the HST repeat consists of two distinct segments, and the distal portions of the HST repeat are also distributed elsewhere in the genome. In HeLa cells, the HST repeat sequence appears to be transcribed into a 6 kb polyadenylated RNA and a variety of non-polyadenylated RNA species.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

利用粗线期染色体和DNA纤维的FISH分析水稻端粒序列

利用粗线期染色体和DNA纤维的荧光原位杂交(FISH)技术分析了水稻广陆矮四号(Oryzasativassp.indicacv.GuangluaiNo.4)的端粒序列。粗线期染色体荧光原位杂交结果表明,大多数染色体的末端都有端粒串联重复,但信号的强度在不同染色体上是不同的。伸展DNA纤维荧光原位杂交结果显示,端粒最长的线状信号长度为6.55μm,最短的为1.82μm,依据2.51kb/μm的标准,它们分别相当于16.44kb和4.56kb。端粒的平均信号长度为3.62±1.32μm,相当于9.09±3.31kb。由此可以估计,最长的、最短的和平均长度的端粒拷贝数约为2349、651和1298±473。     

Chromosome-size variation in Giardia lamblia: the role of rDNA repeats.

Giardia lamblia trophozoites contain at least five sets of chromosomes that have been categorized by chromosome-specific probes. Pulsed field separations of G. lamblia chromosomes also demonstrated minor bands in some isolates which stained less intensely with ethidium than the major chromosomal bands. Two of the minor bands of the E11 clone of the ISR isolate, MBa and MBb, were similar to each other and to chromosomal band I by hybridization to total chromosomal DNA and by hybridization of specific probes. In order to determine the extent of this similarity, I have developed a panel of probes for many of the Pacl restriction fragments and have shown that most of the Pacl and Notl fragments found in MBa are also present in MBb. The differences are found in both telomeric regions. At one end, MBb contains a 300 kb region not found in MBa. At the other end of MBb is a 160 kb region containing the rDNA repeats which is bounded on one end by the telomeric repeat and on the other by sites for multiple enzymes that do not digest the rDNA repeats. The corresponding region of MBa is 23 kb in size. The size difference is consistent with the eightfold greater number of rDNA repeats in MBb than MBa and suggests that 30% of the size difference is accounted for by different numbers of copies of the rDNA repeat. MBa of another ISR clone (ISR G5) is 150 kb larger in size than MBa of ISR E11. The data suggest that MBa and MBb are homologous chromosomes of different sizes and that a portion of the size difference is accounted for by different copy numbers of the rDNA repeat.     

Nature and distribution of chromosomal intertwinings in Saccharomyces cerevisiae.

To elucidate yeast chromosome structure and behavior, we examined the breakage of entangled chromosomes in DNA topoisomerase II mutants by hybridization to chromosomal DNA resolved by pulsed-field gel electrophoresis. Our study reveals that large and small chromosomes differ in the nature and distribution of their intertwinings. Probes to large chromosomes (450 kb or larger) detect chromosome breakage, but probes to small chromosomes (380 kb or smaller) reveal no breakage products. Examination of chromosomes with one small arm and one large arm suggests that the two arms behave independently. The acrocentric chromosome XIV breaks only on the long arm, and its preferred region of breakage is approximately 200 kb from the centromere. When the centromere of chromosome XIV is relocated, the preferred region of breakage shifts accordingly. These results suggest that large chromosomes break because they have long arms and small chromosomes do not break because they have small arms. Indeed, a small metacentric chromosome can be made to break if it is rearranged to form a telocentric chromosome with one long arm or a ring with an "infinitely" long arm. These results suggest a model of chromosomal intertwining in which the length of the chromosome arm prevents intertwinings from passively resolving off the end of the arm during chromosome segregation.     

Molecular evidence of Y-autosomal translocations in owl monkeys

Probe pDP1007, which contains highly conserved DNA sequences from the sex-determining region of the human Y chromosome, cross-hybridized with owl monkey EcoRI restriction fragments of 1.8 kb and 6.6 kb. Southern transfer analysis of owl monkey (karyotype VI)--rodent somatic cell hybrids localized the 1.8-kb fragment on the owl monkey X chromosome and the 6.6-kb fragment, which is male specific, on chromosome 14/Y. Regional in situ chromosome mapping of pDP1007 revealed specific sites of hybridization: the distal short arm of the X chromosome of karyotypes IV, VI, and VII; the small metacentric Y of karyotype IV; the C-band positive region on the short arm of chromosome 17/Y (karyotype VII); and the C-band positive region on the long arm of chromosome 14/Y (karyotype VI). These molecular findings reinforce cytological evidence that Y-chromosomal material has been transferred to autosomes 14 and 17 in owl monkeys of karyotypes VI and VII, respectively, in which there are no independently segregating Y chromosomes.     

Interruption of a human nuclear sequence homologous to mitochondrial DNA by a member of the KpnI 1.8 kb family.

We reported that several DNA sequences homologous to mitochondrial DNA (mtDNA) are present in the human nuclear genome (Tsuzuki et al. (1983) Gene 25, 223-229). Detailed Southern blot analyses revealed that one of such sequences is interrupted by a repetitive sequence about 1.8 kb long, and that the insert is one member of the dispersed repeated DNA sequences of the KpnI 1.8 kb family. Nucleotide sequence analysis showed that the KpnI 1.8 kb DNA is flanked with imperfect 15-base pair (bp) direct repeats of mtDNA. This KpnI 1.8 kb DNA has an A-rich sequence at its 3'-end, and has a considerable homology with one of the published cDNA sequences homologous to one of the human KpnI families and also to one of the African green monkey KpnI families, KpnI-LS1. These structural features suggest that the KpnI 1.8 kb DNA is a movable element and is inserted within the mtDNA-like sequence by an RNA-mediated process.