A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Identification of Porto-1, a new repeated sequence that localises close to the centromere of chromosome 2 of Drosophila melanogaster

We have used the polymerase chain reaction (PCR) technique to search the Drosophila melanogaster genome for the presence of sequences with homology to mammalian and yeast centromeric DNA. Using primers based on the human CENP-B box present in α-satellite DNA and part of the Saccharomyces cerevisiae CDEIII centromeric sequence, a number of specific DNA fragments were amplified from total genomic DNA. In situ hybridization to polytene and mitotic chromosomes showed these fragments to localise to centromeric and pericentromeric regions. Direct cloning of the amplified fragments into conventional plasmids proved unsuccessful. However, a recombinant P1 clone containing D. melanogaster genomic DNA that supports PCR amplification by the primers was identified. Molecular characterisation of this clone revealed a DNA fragment that localises primarily to the centromere of chromosome 2. Sequence analysis indicated that this fragment contains at least four different repeats, including Rsp, transposable elements, Bari-1 and a new AT-rich repeated sequence that we have designated Porto-1. Detailed fluorescence in situ hybridization analysis shows that Porto-1 is localised very close to the primary constriction of chromosome 2. Sequence analysis suggests that this repeat was specifically amplified by our primers, although limited homology to the CENP-B box or CDEIII elements was found. In situ hybridization to a number of Drosophila species shows Porto-1 to be present only in D. melanogaster. Received: 13 April 1996; in revised form: 25 June 1996 / Accepted: 6 July 1996     

DNA repeat arrays in chicken and human genomes and the adaptive evolution of avian genome size

Background

Birds have smaller average genome sizes than other tetrapod classes, and it has been proposed that a relatively low frequency of repeating DNA is one factor in reduction of avian genome sizes.

Results

DNA repeat arrays in the sequenced portion of the chicken (Gallus gallus) autosomes were quantified and compared with those in human autosomes. In the chicken 10.3% of the genome was occupied by DNA repeats, in contrast to 44.9% in human. In the chicken, the percentage of a chromosome occupied by repeats was positively correlated with chromosome length, but even the largest chicken chromosomes had repeat densities much lower than those in human, indicating that avoidance of repeats in the chicken is not confined to minichromosomes. When 294 simple sequence repeat types shared between chicken and human genomes were compared, mean repeat array length and maximum repeat array length were significantly lower in the chicken than in human.

Conclusions

The fact that the chicken simple sequence repeat arrays were consistently smaller than arrays of the same type in human is evidence that the reduction in repeat array length in the chicken has involved numerous independent evolutionary events. This implies that reduction of DNA repeats in birds is the result of adaptive evolution. Reduction of DNA repeats on minichromosomes may be an adaptation to permit chiasma formation and alignment of small chromosomes. However, the fact that repeat array lengths are consistently reduced on the largest chicken chromosomes supports the hypothesis that other selective factors are at work, presumably related to the reduction of cell size and consequent advantages for the energetic demands of flight.     

Origin of human chromosome 2 revisited

Similarities in chromosome banding patterns and hornologies in DNA sequence between chromosomes of the great apes and humans have suggested that human chromosome 2 originated through the fusion of two ancestral ape chromosomes. A lot of work has been directed at understanding the nature and mechanism of this fusion. The recent availability of the human chrornosome-2-specific alpha satellite DNA probe D2Z and the human chromosome-2p-specific subtelomeric DNA probe D2S445 prompted us to attempt cross-hybridization with chromosomes of the chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orangutan (Pongo pygmaeus) to search for equivalent locations in the great apes and to comment on the origin of human chromosome 2. The probes gave different results. No hybridization to the chromosome-2-specific alpha satellite DNA probe was observed on the presumed homologous great ape chromosomes using both high-stringency and low-stringency post-hybridization washes, whereas the subtelomeric-DNA probe specific for chromosome 2p hybridized to telomeric sites of the short arm of chromosome 12 of all three great apes. These observations suggest an evolutionary difference in the number of alpha satellite DNA repeat units in the equivalent ape chromosomes presumably involved in the chromosome fusion. Nevertheless, complete conservation of DNA sequence of the subtelomeric repeat sequence D2S445 in the ape chromosomes is demonstrated.     

An improved method for detecting Y chromosomal DNA

Summary The DNA probe Y97 was derived from a repeat sequence in the human Y centromere, a region which must be present in a mitotically functional Y chromosome. We have demonstrated that Y97, which detects a Y-specific 5.5-kb Eco RI fragment by Southern analysis, is very useful for the molecular detection of small amounts of Y-derived material and represents a significant improvement over previous tests for molecular diagnosis of sex. The male-female difference in hybridization was unequivocal even when only 25 ng of total DNA was used per lane. Furthermore, in mixing experiments the 5.5-kb Eco RI fragment was detectable even when only 5% of the total DNA was male. By increasing hybridization stringency, we have developed a rapid, sensitive, and accurate method to detect Y chromosomal DNA in unrestricted samples.     

Physical mapping of sequences homologous to an endogenous retrovirus LTR on human chromosome 19

The human genome contains multiple copies of sequences related to the HERV-K family of endogenous retroviruses, homologous to the B-type mouse mammary tumour virus. A DNA fragment closely resembling an HERV-K long tandem repeat (LTR) was detected in a library of hncDNA clones enriched for sequences from human chromosome 19. Sites showing homology to the sequence of this fragment have been identified on human chromosome 19 by hybridization to previously mapped chromosome 19 cosmids. Thus the distribution of LTR sequences on a specific human chromosome has been mapped for the first time. We estimate the total number of such sites on human chromosome 19 to be at least 110. Many of these sites are located in the vicinity of known genes. The precise localizations (to specific cosmids) of LTR-homologous sequences on chromosome 19 can serve as a reference source and will automatically provide further insight into LTR-gene relationships as new genes are mapped onto the chromosome.     

Single base discrimination of CENP-B repeats on mouse and human Chromosomes with PNA-FISH

The satellite repeat structure of the mammalian centromere contains the CENP-B protein binding site. Using the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH), we show by direct PNA-DNA binding that all detectable CENP-B sites in a mammalian genome might have the same sequence. Two species-specific PNA 17-mers, pMm and pMc, were identified from CENP-B binding sites of Mus musculus and M. caroli, respectively. Fluorescence in situ hybridization confirmed that pMc hybridized to M. caroli centromeres only; however, pMm cross-hybridized to M. musculus and human centromeres. By using a series of CENP-B PNA 17-mers containing 1, 2, 3, 5, and 7 base-pair mismatches to their DNA counterparts, we further demonstrate that PNA-FISH can discriminate between two CENP-B DNA sequences that differ by a single base-pair in mouse and human centromeres, suggesting the degree of conservation of CENP-B sequences throughout the genome. In comparison with DNA oligonucleotides, PNA oligomers demonstrate the higher sequence specificity, improved stability, reproducibility, and lower background. Therefore, PNA oligomers have significant advantages over DNA oligonucleotide probes in analyzing microsatellites in a genome. Received: 16 June 1998 / Accepted: 3 September 1998     

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.     

A novel avian W chromosome DNA repeat sequence in the lesser black-backed gull (Larus fuscus)

The phenol emulsion reassociation technique was used to isolate and clone a female specific, repetitive DNA sequence fromLarus fuscus. The repeat, designated P2000-17, is restricted to the W chromosome, although related sequences occur elsewhere in the genome ofL. fuscus. Similar sequences were detected in the genome of six other bird species from outside the genus Laridae, but the sequence occurs less frequently and to a similar extent in both sexes. The 298 bp DNA sequence of P2000-17 was determined and found to have extensive sequence identity to the rabbit dihydropyridine (DHP) receptor calcium channel. P2000-17 is represented once within a larger 8.6 kb tandem repeat (LfW-1), which has a complex internal DNA sequence. LfW-1 is highly conserved between repeat motifs and may comprise 3% of the female genome. The possible evolutionary origin of LfW-1 is discussed in relation to the repeat types found on the W and Y chromosomes of other species.     

Non xenopus-like DNA sequence organization in the Chironomus tentans genome

Summary We have examined the sequence organization of Chironomus tentans DNA by means of optical and hydroxyapatite renaturation kinetics of total DNA fragment sizes of 0.36, 2.6 and 13.5 kilobases (kb) as well as isolated middle repeat DNA at sizes of 0.36 and 13.5 kb. 90% of the DNA renatured as unique sequences of a genome of 0.20 pg with the balance of DNA renaturing as middle repetitive sequences present on average 90 times per haploid genome. At a DNA fragment length of 13.5 kb, 35% of the DNA was trapped on the hydroxyapatite as middle repetitive fraction. We concluded C. tentans DNA to have a mean repeat length of about 4.3 kb distributed through out at least 35% of the genome with an inter repeat spacing of at least 13.5 kb but possibly being distributed throughout the whole genome with an inter repeat spacing of 36 kb. This shows C. tentans DNA organization not to follow the almost ubiquitous Xenopus model but to be similar to the organization of Drosophila melanogaster DNA.     

Alu-splice PCR: a simple method to isolate exon-containing fragments from cloned human genomic DNA

We have developed a simple, straightforward procedure to isolate exons from cloned human genomic DNA. The method is PCR based and relies upon the conservation of splice-site sequences and the frequency of Alu repeat elements in the genome to capture coding sequences. We designed two different sets of primers: a primer from each end of the Alu element and primers with the 5′ or 3′ splice-site consensus sequences. Putative exons were amplified by PCR using YAC DNA as starting material. We applied Alu-splice PCR to two overlapping YACs, 72H9 and 860G11, from human chromosome 21. Sequence and northern analysis of 37 initial clones resulted in the identification of five novel exons. Received: 17 July 1997 / Accepted: 28 August 1997     

Identification of paralogous HERV-K LTRs on human chromosomes 3, 4, 7 and 11 in regions containing clusters of olfactory receptor genes

A locus harboring a human endogenous retroviral LTR (long terminal repeat) was mapped on the short arm of human chromosome 7 (7p22), and its evolutionary history was investigated. Sequences of two human genome fragments that were homologous to the LTR-flanking sequences were found in human genome databases: (1) an LTR-containing DNA fragment from region 3p13 of the human genome, which includes clusters of olfactory receptor genes and pseudogenes; and (2) a fragment of region 21q22.1 lacking LTR sequences. PCR analysis demonstrated that LTRs with highly homologous flanking sequences could be found in the genomes of human, chimp, gorilla, and orangutan, but were absent from the genomes of gibbon and New World monkeys. A PCR assay with a primer set corresponding to the sequence from human Chr 3 allowed us to detect LTR-containing paralogous sequences on human chromosomes 3, 4, 7, and 11. The divergence times for the LTR-flanking sequences on chromosomes 3 and 7, and the paralogous sequence on chromosome 21, were evaluated and used to reconstruct the order of duplication events and retroviral insertions. (1) An initial duplication event that occurred 14-17 Mya and before LTR insertion - produced two loci, one corresponding to that located on Chr 21, while the second was the ancestor of the loci on chromosomes 3 and 7. (2) Insertion of the LTR (most probably as a provirus) into this ancestral locus took place 13 Mya. (3) Duplication of the LTR-containing ancestral locus occurred 11 Mya, forming the paralogous modern loci on Chr 3 and 7.     

A new human alphoid-like repetitive DNA sequence

A new tandemly repetitive sequence family, having the 170 bp basic repeat characteristic of alphoid sequences, has been identified in the human genome. Its organization in the whole genome and on chromosome 21 is different from that of any of the previously described alphoid families. Members of this new family are unusually heterogeneous in sequence, and there are a number of variant sequence classes. Some of the variant classes exist in separate genomic domains, and even on a single chromosome the members of such a class are not significantly intermixed with members of another class.     

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.     

Phylogenetic relationships among transposon-like elements in human and primate DNA

A THE-1 sequence in intron 7 of the human dystrophin gene has been found to represent a new subfamily of THE-1 elements. The sequence is closely related to the MstII family of repetitive sequences and is more like single-copy sequences found in the galago genome than any other THE-1 sequence previously reported. This new THE-1 sequence has been compared with two other complete THE-1 sequences and three related long-terminal repeat elements that we have previously found in intron 7 of the dystrophin gene, and with members of the same family from elsewhere in the primate genome. Parsimony and deletion analysis show that the cluster of THE-1 sequences in intron 7 of the dystrophin gene has arisen from at least three individual insertion events, rather than from the insertion and duplication of a single progenitor sequence. Correspondence to: G.B. Petersen     

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.     

Interhomologue sequence variation of alpha satellite DNA from human chromosome 17: Evidence for concerted evolution along haplotypic lineages

Alpha satellite DNA is a family of tandemly repeated DNA found at the centromeres of all primate chromosomes. Different human chromosomes 17 in the population are characterized by distinct alpha satellite haplotypes, distinguished by the presence of variant repeat forms that have precise monomeric deletions. Pairwise comparisons of sequence diversity between variant repeat units from each haplotype show that they are closely related in sequence. Direct sequencing of PCR-amplified alpha satellite reveals heterogeneous positions between the repeat units on a chromosome as two bands at the same position on a sequencing ladder. No variation was detected in the sequence and location of these heterogeneous positions between chromosomes 17 from the same haplotype, but distinct patterns of variation were detected between chromosomes from different haplotypes. Subsequent sequence analysis of individual repeats from each haplotype confirmed the presence of extensive haplotype-specific sequence variation. Phylogenetic inference yielded a tree that suggests these chromosome 17 repeat units evolve principally along haplotypic lineages. These studies allow insight into the relative rates and/or timing of genetic turnover processes that lead to the homogenization of tandem DNA families. Correspondence to: H.F. Willard     

Construction of a YAC library from a Beta vulgaris fragment addition and isolation of a major satellite DNA cluster linked to the beet cyst nematode resistance locus Hs1 pat−1

A YAC library was constructed from the Beta vulgaris fragment addition AN5-203b. This monosomic fragment addition harbors an approximate 12-Mbp fragment of B.patellaris chromosome 1 accomodating the Hs1 ^pat-1 conferring resistance to the beet cyst nematode (Heterodera schachtii). The YAC library consists of 20,000 YAC clones having an average size of 140 kb. Screening with organelle-specific probes showed that 12% of the clones contain chloroplast DNA while only 0.2% of the clones hybridizes with a mitochondrial specific probe. On the basis of a sugar beet haploid genome size of 750 Mbp this library represents 3.3 haploid genome equivalents. The addition fragment present in AN5-203b harbors a major satellite DNA cluster that is tightly linked to the Hs1 ^pat-1 locus. The cluster is located on a single 250-kb EcoRI restriction fragment and consists of an estimated 700–800 copies of a 159-bp core sequence, most of which are arranged in tandem. Using this core sequence as a probe, we were able to isolate 1 YAC clone from the library that contains the entire 250-kb satellite DNA cluster.Abbreviations YAC Yeast artificial chromosome - BCN beet cyst nematode - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism     

Cloning and sequencing of an Escherichia coli K12 gene which encodes a polypeptide having similarity to the human ferritin H subunit

Summary Using lambda phage clones containing segments of the Escherichia coli K12 chromosome as hybridization probes, we found one gene at 42 min on the E. coli chromosome map, the expression of which was affected by RNase III. The sequence of the DNA fragment containing this gene (gen-165) revealed the presence of an open reading frame encoding a polypeptide of 165 amino acid residues. The amino acid sequence deduced from the nucleotide sequence exhibited a remarkable similarity to that of the human ferritin H chain.     

Finishing the finished human chromosome 22 sequence

Background

Although the human genome sequence was declared complete in 2004, the sequence was interrupted by 341 gaps of which 308 lay in an estimated approximately 28 Mb of euchromatin. While these gaps constitute only approximately 1% of the sequence, knowledge of the full complement of human genes and regulatory elements is incomplete without their sequences.

Results

We have used a combination of conventional chromosome walking (aided by the availability of end sequences) in fosmid and bacterial artificial chromosome (BAC) libraries, whole chromosome shotgun sequencing, comparative genome analysis and long PCR to finish 8 of the 11 gaps in the initial chromosome 22 sequence. In addition, we have patched four regions of the initial sequence where the original clones were found to be deleted, or contained a deletion allele of a known gene, with a further 126 kb of new sequence. Over 1.018 Mb of new sequence has been generated to extend into and close the gaps, and we have annotated 16 new or extended gene structures and one pseudogene.

Conclusion

Thus, we have made significant progress to completing the sequence of the euchromatic regions of human chromosome 22 using a combination of detailed approaches. Our experience suggests that substantial work remains to close the outstanding gaps in the human genome sequence.