Sequence heterogeneity within the human alphoid repetitive DNA family.

We have cloned and determined the base-sequence and genome organization of two human chromosome-specific alphoid DNA fragments, designated L1.26, mapping principally to chromosomes 13 and 21, and L1.84, mapping to chromosome 18. Their copy number is estimated to be approximately 2,000 per haploid genome. L1.84 has a double-dimer organization, whereas L1.26 has a much less defined higher order tandem organization. Further, we present evidence that the restriction-site spacing within the alphoid DNA family is chromosome specific. From sequence analysis, clones L1.26 and L1.84 are found to consist of 5 and 4 tandemly duplicated 170 bp monomers. Cross-homology between the various monomers is 65-85%. The analysis suggests that the evolution of tandem-arrays does not take place via a defined 340 bp unit, as was inferred by others, but via circularly permutated monomers or multimers of the 170 bp unit.     

Structural analysis of alphoid DNA of primates. I. Heterogeneity of nucleotide sequence of alphoid repeats in human DNA

The nucleotide sequence of two cloned fragments of human alphoid DNA was established. These fragments were earlier characterized in our laboratory as molecular markers of the 3rd (pHS05) and 11th (pHS53) chromosomes. Fragment pHS53 (2546 bp) contains alphoid repeats tandemly arranged and organized into three highly homologous pentamers. The heterogeneity of monomeric sequences within individual pentamers reaches 24-33%. Structural analysis of EcoRI subfragment pHS05 showed that this alphoid tetramer consists of two dimers 340 bp long. These dimers differ up to 16% from each other and from the so-called consensus sequence of the EcoRI-340 bp-restriction fragments family reported earlier by Wu and Manuelidis. The primary structure of four cloned fragments of EcoRI-340 bp-family was established. The data show that human alphoid DNA is highly heterogeneous. This conclusion is opposite to the view suggesting that alphoid DNA is a highly homogeneous class of reiterated sequences of the human genome.     

Study of alpha-satellite DNA in cosmid libraries, specific for chromosomes 13, 21, and 22, using fluorescence in situ hybridization

Fluorescent in situ hybridization (FISH) was employed in mapping the alpha-satellite DNA that was revealed in the cosmid libraries specific for human chromosomes 13, 21, and 22. In total, 131 clones were revealed. They contained various elements of centromeric alphoid DNA sequences of acrocentric chromosomes, including those located close to SINEs, LINEs, and classical satellite sequences. The heterochromatin of acrocentric chromosomes was shown to contain two different groups of alphoid sequences: (1) those immediately adjacent to the centromeric regions (alpha 13-1, alpha 21-1, and alpha 22-1 loci) and (2) those located in the short arm of acrocentric chromosomes (alpha 13-2, alpha 21-2, and alpha 22-2 loci). Alphoid DNA sequences from the alpha 13-2, alpha 21-2, and alpha 22-2 loci are apparently not involved in the formation of centromeres and are absent from mitotically stable marker chromosomes with a deleted short arm. Robertsonian translocations t(13q; 21q) and t(14q; 22q), and chromosome 21p-. The heterochromatic regions of chromosomes 13, 21, and 22 were also shown to contain relatively chromosome-specific repetitive sequences of various alphoid DNA families, whose numerous copies occur in other chromosomes. Pools of centromeric alphoid cosmids can be of use in further studies of the structural and functional properties of heterochromatic DNA and the identification of centromeric sequences. Moreover, these clones can be employed in high-resolution mapping and in sequencing the heterochromatic regions of the human genome. The detailed FISH analysis of numerous alphoid cosmid clones allowed the identification of several new, highly specific DNA probes of molecular cytogenetic studies--in particular, the interphase and metaphase analyses of chromosomes 2, 9, 11, 14, 15, 16, 18, 20, 21-13, 22-14, and X.     

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome ΔYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from ΔYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.     

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.     

Isolation and comparative mapping of a human chromosome 20-specific alpha-satellite DNA clone.

We have isolated and characterized a human genomic DNA clone (PZ20, locus D20Z2) that identifies, under high-stringency hybridization conditions, an alphoid DNA subset specific for chromosome 20. The specificity was determined using fluorescence in situ hybridization. Sequence analysis confirmed our previously reported data on the great similarity between the chromosome 20 and chromosome 2 alphoid subsets. Comparative mapping of pZ20 on chimpanzee and gorilla chromosomes, also performed under high-stringency conditions, indicates that the alphoid subset has ancestral sequences on chimpanzee chromosome 11 and gorilla chromosome 19. However, no hybridization was observed to chromosomes 21 in the great apes, the homolog of human chromosome 20.     

Structural Organization of Multiple Alphoid Subsets Coexisting on Human Chromosomes 1, 4, 5, 7, 9, 15, 18, and 19

FISH experiments on metaphase chromosomes, interphase nuclei, and extended chromatin were performed to investigate the structural organization of alphoid subsets coexisting on human chromosomes 1, 4, 5, 7, 9, 15, 18, and 19. Results indicate that multiple subsets present on chromosomes 5, 7, 15, 18, and 19 are organized in structurally distinct and contiguous domains, while those on chromosomes 4 and 9 give perfectly overlapping signals. Chromosome 1 shows a peculiar organization: probe pAL1, specific for this chromosome, detects two distinct domains separated by the subset identified by probe pZ5.1. The order along the chromosome of alphoid subsets lying on chromosomes 5, 7, 15, 18, and 19, organized in distinct blocks, has also been established. The relationship between the structural organization of these alphoid sequences and their evolutionary history in great apes is discussed.     

Correlation of polymorphism of heterochromatin segments with hybridization on chromosome preparations of various cloned repeated human DNA sequences

Comparison between results of measurements of heterochromatic regions detected by differential C and DA/DAP1 staining and the hybridization data of two cloned repeated human DNA sequences one alphoid (pH S05) and the other the satellite DNA III (pPD18) on chromosome preparations was made. A positive correlation of heterochromatic region sizes on several chromosomes and the amount of label over them detected after hybridization with both alphoid and satellite sequences was shown, the correlation with the latter being more pronounced.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Evolutionary implications of the human aldolase-A, -B, -C, and -pseudogene chromosome locations.

The aldolase genes represent an ancient gene family with tissue-specific isozymic forms expressed only in vertebrates. The chromosomal locations of the aldolase genes provide insight into their tissue-specific and developmentally regulated expression and evolution. DNA probes for the human aldolase-A and -C genes and for an aldolase pseudogene were used to quantify and map the aldolase loci in the haploid human genome. Genomic hybridization of restriction fragments determined that all the aldolase genes exist in single copy in the haploid human genome. Spot-blot analysis of sorted chromosomes mapped human aldolase A to chromosome 16, aldolase C to chromosome 17, the pseudogene to chromosome 10; it previously had mapped the aldolase-B gene to chromosome 9. All loci are unlinked and located on to two pairs of morphologically similar chromosomes, a situation consistent with tetraploidization during isozymic and vertebrate evolution. Sequence comparisons of expressed and flanking regions support this conclusion. These locations on similar chromosome pairs correctly predicted that the aldolase pseudogene arose when sequences from the aldolase-A gene were inserted into the homologous aldolase location on chromosome 10.     

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.     

Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Different subfamilies of alphoid repetitive DNA are present on the human and chimpanzee homologous chromosomes 21 and 22.

The alphoid repeat DNA on chimpanzee chromosome 22 was compared with alphoid repeat DNA on its human homologue, chromosome 21. Hybridization of different alphoid probes under various conditions of stringency show that the alphoid repeats of chimpanzee chromosome 22 are not closely related to those of human chromosome 21. Sequence analysis of cloned dimer and tetramer EcoRI fragments from chimpanzee chromosome 22 confirm the low overall level of homology, but reveal the presence of several nucleotide changes which are exclusive to the chromosome 21 subfamily of human alphoid DNA. Southern blot analysis of alphoid repeat DNA on the chimpanzee X chromosome suggests this subfamily has been strongly conserved during and since the separation of chimpanzee and man although the two subfamilies can be distinguished on the basis of Taq I restriction fragments.     

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.     

Evolution of alpha-satellite DNA on human acrocentric chromosomes

In situ hybridization of five new and one previously described alpha-satellite sequences isolated from chromosome 21 libraries gave the following chromosomal distribution patterns: (a) two sequences (pTRA-1 and -4) hybridizing to chromosomes 13, 14, 15, 21, and 22 (also 19 and 20); (b) one sequence (pTRA-7) hybridizing to chromosome 14; and (c) three sequences (pTRA-2, -11 and -15) hybridizing to chromosomes 13, 14, and 21, with significant but weaker signals on 15 and 22. These results suggested the sharing of alphoid domains between different acrocentric chromosomes and the coexistence of multiple domains on each chromosome. Analysis of somatic cell hybrids carrying a single human acrocentric chromosome using pTRA-2 demonstrated a higher-order repeating structure common to chromosomes 13, 14, and 21, but not to 15 and 22, providing direct evidence for sequence homogenization in this domain among the former three chromosomes. We present a model of evolution and genetic exchange of alpha sequences on the acrocentric chromosomes which can satisfactorily explain these and previous observations of (a) two different alphoid subfamilies, one common to chromosomes 13 and 21 and the other common to chromosomes 14 and 22, (b) a different alphoid subfamily on chromosome 22, and (c) nonrandom participation of chromosomes 13 and 14, and 14 and 21 in Robertsonian translocations.     

Alpha satellite DNAs on chromosomes 10 and 12 are both members of the dimeric suprachromosomal subfamily, but display little identity at the nucleotide sequence level

We have investigated the organization and complexity of alpha satellite DNA on chromosomes 10 and 12 by restriction endonuclease mapping, in situ hybridization (ISH), and DNA-sequencing methods. Alpha satellite DNA on both chromosomes displays a basic dimeric organization, revealed as a 6- and an 8-mer higher-order repeat (HOR) unit on chromosome 10 and as an 8-mer HOR on chromosome 12. While these HORs show complete chromosome specificity under high-stringency ISH conditions, they recognize an identical set of chromosomes under lower stringencies. At the nucleotide sequence level, both chromosome 10 HORs are 50% identical to the HOR on chromosome 12 and to all other alpha satellite DNA sequences from the in situ cross-hybridizing chromosomes, with the exception of chromosome 6. An 80% identity between chromosome 6- and chromosome 10-derived alphoid sequences was observed. These data suggest that the alphoid DNA on chromosomes 6 and 10 may represent a distinct subclass of the dimeric subfamily. These sequences are proposed to be present, along with the more typical dimeric alpha satellite sequences, on a number of different human chromosomes.     

Cloned fragment of human alphoid DNA--a molecular marker of the pericentromeric region of chromosome 18

From the library of cloned fragments of human DNA we have isolated two recombinant plasmids containing alphoid DNA sequences pBRHS13, pBRHS65. Both cloned sequences hybridized in situ predominantly to pericentromeric regions of chromosome 18 and with less intensity to pericentromeric regions of chromosomes 2, 9, 20, and were characterized by populational copy number polymorphism in homologous chromosomes. These sequences may appear very useful in the diagnostics and cytogenetic analysis of chromosomal aberrations and in studies of polymorphisms of heterochromatic regions of human chromosomes.     

p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

The African trypanosome genome

The haploid nuclear genome of the African trypanosome, Trypanosoma brucei, is about 35 Mb and varies in size among different trypanosome isolates by as much as 25%. The nuclear DNA of this diploid organism is distributed among three size classes of chromosomes: the megabase chromosomes of which there are at least 11 pairs ranging from 1 Mb to more than 6 Mb (numbered I-XI from smallest to largest); several intermediate chromosomes of 200-900 kb and uncertain ploidy; and about 100 linear minichromosomes of 50-150 kb. Size differences of as much as four-fold can occur, both between the two homologues of a megabase chromosome pair in a specific trypanosome isolate and among chromosome pairs in different isolates. The genomic DNA sequences determined to date indicated that about 50% of the genome is coding sequence. The chromosomal telomeres possess TTAGGG repeats and many, if not all, of the telomeres of the megabase and intermediate chromosomes are linked to expression sites for genes encoding variant surface glycoproteins (VSGs). The minichromosomes serve as repositories for VSG genes since some but not all of their telomeres are linked to unexpressed VSG genes. A gene discovery program, based on sequencing the ends of cloned genomic DNA fragments, has generated more than 20 Mb of discontinuous single-pass genomic sequence data during the past year, and the complete sequences of chromosomes I and II (about 1 Mb each) in T. brucei GUTat 10.1 are currently being determined. It is anticipated that the entire genomic sequence of this organism will be known in a few years. Analysis of a test microarray of 400 cDNAs and small random genomic DNA fragments probed with RNAs from two developmental stages of T. brucei demonstrates that the microarray technology can be used to identify batteries of genes differentially expressed during the various life cycle stages of this parasite.