Invasion of the human albumin-alpha-fetoprotein gene family by Alu, Kpn, and two novel repetitive DNA elements

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.     

Characterisation of the telomeres at opposite ends of a 3 Mb Theileria parva chromosome.

Bacteriophage lambda clones containing Theileria parva genomic DNA derived from two different telomeres were isolated and the nucleotide sequences of the telomeric repeats and adjacent telomere-associated (TAS) DNA were determined. The T.parva telomeric repeat sequences, a tandem array of TTTTAGGG or TTTAGGG interspersed with a few variant copies, showed a high degree of sequence identity to those of the photosynthetic algae Chlamydomonas reinhardtii (97% identity) and Chlorella vulgaris (87.7% identity) and the angiosperm Arabidopsis thaliana (84.4% identity). Unlike most organisms which have been studied, no significant repetitive sequences were found in the nucleotide sequences of TAS DNA located centromere-proximal to the telomeric repeats. Restriction mapping and hybridisation analysis of lambda EMBL3 clones containing 16 kilobases of TAS DNA derived from one telomere suggested that they did not contain long regions of repetitive DNA. The cloned TAS DNAs were mapped to T.parva Muguga genomic SfiI fragments 8 and 20, which are located at opposite ends of the largest T.parva chromosome. A 126 bp sequence located directly centromere-proximal to the telomeric repeats was 94% identical between the two cloned telomeres. The conserved 126 bp sequence was present on all T.parva Muguga telomeric SfiI fragments.     

Initial analysis of tandemly repetitive sequences in the genome of Zhikong scallop (Chlamys farreri Jones et Preston).

Tandemly repetitive sequences are widespread in all eukaryotic genomes, but data on tandem repeats are limited in Zhikong scallop (Chlamys farreri). In the present study, paired-end sequencing of 2016 individual fosmid clones resulted in 3646 sequences. A total of 2,286,986 bp of genomic sequences were generated, representing approximately 1.84 per thousand of the Zhikong scallop genome. Using tandem repeats finder (TRF) software, a total of 2500 tandem repeats were found, including 313 satellites, 1816 minisatellites and 371 microsatellites. The cumulative length of tandem repeats was 552,558 bp, accounting for 24.16% of total length. Specifically, the length of microsatellites, minisatellites and satellites was 9425, 336,001 and 207,132 bp, accounting for 1.71, 60.81 and 37.49% of the length of tandem repeats, and 0.41, 14.69 and 9.06% of total length, respectively. The detailed information on the characteristic of all repeat units was also represented, which will provide a useful resource for physical mapping and better utilization of the existing genomic information in Zhikong scallop.     

DNA profiling by detection of repetitive nucleotide sequences on human chromosome 6

Genetic/genomic polymorphism, i.e. variations in DNA sequences are ideally assayed by direct nucleotide sequencing of a gene region or other homologous segment of the genome. An easier and cheaper approach, however, if the variants are analyzed by hybridization technology using restriction fragment length polymorphisms (RFLPs) or by detection of the number of tandem repeats (VNTR) of small DNA segments, the "minisatellites". In this study we describe results of the DNA analysis of repetitive sequences of human 6th chromosome by the application of a chemiluminescent labeled probes. The allele frequency distribution of polymorphic DNA sequences has been determined in unrelated individuals. The isolated genomic DNA was cut with Pst I restriction enzyme, size fractionated on agarose gel and hybridized with a chemiluminescent labeled D6 S132 probe. At this locus the Pst I cleaved DNA fragments are ranging from 1841 to 6098 base pairs (bp). Specific genetic pattern was characterized by more frequent fragments (3313 and 3884 bp), and the rarely occurring ones (clustered between 1841-2595 and 5227-6098 bp). Our study provides a further possibility for characterization of individual genomic patterns.     

Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11.

We have shown that many of the Alu repeats found in the GenBank database are polymorphic and that this polymorphism can be detected by a simple technique, single-strand conformation polymorphism (SSCP) analysis, after polymerase chain reaction (PCR) amplification of each repeat from DNA of individuals. Here, we describe a method for collecting many anonymous Alu repeats and their flanks in a chromosome-specific phage library and cloning them into plasmids. The flanking single-copy sequences of each repeat in the plasmid were then determined, and 20mer to 30mer segments of these sequences were used as primers for the PCR-SSCP analysis. Many new polymorphic DNA markers on chromosome 11 were obtained with this method. These markers can also serve as sequence-tagged sites for physical mapping of the genome.     

Sequence organization of repetitive sequences enriched in small polydisperse circular DNAs from HeLa cells

A total of 36 clones were randomly selected from a recombinant DNA library of small polydisperse circular DNA (spcDNA) molecules from HeLa cells and were shown to contain repetitive sequences of different reiteration frequencies that ranged from several hundred to several hundred thousand per genome. Sequencing of representative clones revealed tandem repeats of alphoid (alpha) satellite DNA, clustered repeats of the Alu family, KpnI family sequences, tandem repeats of an alpha satellite DNA specific to the X chromosome (alpha X), and A + T-rich segments carrying short stretches of poly(A) or poly(T). DNA rearrangement was frequently found in the repetitive sequences enriched in these spcDNA clones. Short regions of homology that were patchy and inverted were often found, especially at the novel joint where spcDNA sequences are circularized. The presence of these inverted repeats suggests that HeLa spcDNAs are formed by a mechanism that involves looping out of the spcDNA region and joining of the flanking DNA by illegitimate recombination.     

Occurrence of reiterated sequences in an untranslated region of Simian virus 40 DNA determined by nucleotide sequence analysis.

An earlier report (Subramanian, Dhar, and Weissman, 1977c) presented the nucleotide sequence of Eco RII-G fragment of SV40 DNA, which contains the origin of DNA replication. The nucleotide sequence of Eco RII-N fragment located next to Eco RII-G on the physical map of SV40 DNA is presented in this report. Eco RII-N is found to be a tandem duplication of the last 55 nucleotides of Eco RII-G. This tandem repeat is immediately preceded by two other reiterated sequences occurring within Eco RII-G, one of them being a tandem repeat of 21 nucleotides and the other a nontandem repeat of 10 nucleotides. These repetitive sequences occur in close proximity to the origin of DNA replication which is known to contain other specialized sequences such as a few palindromes (one of which is 27 long and possesses a perfect 2-fold axis of symmetry), one "true" palindrome, and a long A/T-rich cluster. The repeats (and the replication origin) occur within an untranslated region of SV40 DNA flanked by (the few) structural genes coding for the "late" proteins on the one side and that (those) coding for the "early" protein(s) on the other side. The reiterated sequences are comparable in some respects to repetitive sequences occurring in eucaryotic DNAs. Possible biological functions of the repeats are discussed.     

Localization and characterization of members of a family of repetitive sequences in the goat beta globin locus.

We have characterized a family of repetitive DNA elements in the beta-globin locus of the goat. These sequences are structurally analogous to the Alu families of repeats of other mammals. Repetitive elements are located both in the intervening sequences and in the intergenic regions of the goat beta-globin locus. Nucleotide sequence analysis of five repetitive elements located within the large intervening sequence of the beta-like globin genes, and four repeats located 5' to the major developmentally regulated beta-globin genes has resulted in the definition of a consensus sequence for this family of repeats.     

Survey of human and rat microsatellites

Length variations in simple sequence tandem repeats (microsatellite DNA polymorphisms) are finding increasing usage in mammalian genetics. Although every variety of short tandem repeat that has been tested has been shown to exhibit length polymorphisms, little information on the relative abundance of the different repeat motifs has been collected. In this report, summaries of GenBank searches for all possible human and rat microsatellites ranging from mononucleotide to tetranucleotide repeats are presented. In humans, the five most abundant microsatellites with total lengths for the runs of repeats of greater than or equal to 20 nucleotides contained repeat sequences of A, AC, AAAN, AAN, and AG, in order of decreasing abundance, where N is C, G, or T. These five groups comprised about 76% of all microsatellites. Many other human simple sequence repeats were found at low frequency. In the 745 kb of human genomic DNA surveyed, one microsatellite of greater than or equal to 20 nucleotides in length was found, on average, every 6 kb. Only 12% of the human microsatellites had total lengths greater than or equal to 40 nucleotides. Roughly 80% of the A, AAN, and AAAN microsatellites and 50% of the AT microsatellites, but few of the other human microsatellites, were found to be associated with interspersed, repetitive Alu elements. In rats, the five most abundant microsatellites contained AC, AG, A, AAAN, and AAGG sequences, respectively. Rat microsatellites were generally longer than human microsatellites, with 43% of the rat sequences greater than or equal to 40 nucleotides.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.     

Integration site preferences of the Alu family and similar repetitive DNA sequences.

Numerous flanking nucleotide sequences from two primate interspersed repetitive DNA families have been aligned to determine the integration site preferences of each repetitive family. This analysis indicates that both the human Alu and galago Monomer families were preferentially inserted into short d(A+T)-rich regions. Moreover, both primate repeat families demonstrated an orientation specific integration with respect to dA-rich sequences within the flanking direct repeats. These observations suggest that a common mechanism exists for the insertion of many repetitive DNA families into new genomic sites. A modified mechanism for site-specific integration of primate repetitive DNA sequences is provided which requires insertion into dA-rich sequences in the genome. This model is consistent with the observed relationship between galago Type II subfamilies suggesting that they have arisen not by mere mutation but by independent integration events.     

Repetitive DNA sequences in the human corticotropin-beta-lipotrophin precursor gene region: Alu family members.

Repetitive DNA sequences in the human corticotropin-beta-lipotropin precursor gene region have been studied by blot hybridization analysis and DNA sequencing. Six repetitive sequences are present in this gene region; five of them are Alu family members with an approximate length of 300 base pairs, and the other consists of a portion of an Alu family sequence. Two of these Alu family members are located in the 5'-flanking region of the gene, and the remaining four within the intervening sequences. These Alu family sequences constitute inverted repeats in the intervening sequences as well as in the 5'-flanking region of the gene.     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

Extensive Direct-Tandem Organization of a Long Repeat DNA Sequence on the Y Chromosome of Chinook Salmon (Oncorhynchus tshawytscha)

A long repetitive DNA sequence (OtY8) has been cloned from male chinook salmon and its genomic organization has been characterized. The repeat has a unit length of 8 kb and is present approximately 300 times per diploid male nucleus. All internal fragments within the 8-kb repeat segregate from father to son, suggesting that the entire repeat unit is located on the Y chromosome. The organization of this sequence into an 8-kb repeat unit is restricted to the Y chromosome, as are several male-specific repeat subtypes identified on the basis of restriction-site variation. The repeat possesses only weak internal sequence similarities, suggesting that OtY8 has not arisen by duplication of a smaller repeat unit, as is the case for other long tandem arrays found in eukaryotes. Based on a laddered pattern arising from partial digestion of genomic DNA with a restriction enzyme which cuts only once per repeat unit, this sequence is not dispersed on the Y chromosome but is organized as a head-to-tail tandem array. Pulse-gel electrophoresis reveals that the direct-tandem repeats are organized into at least six separate clusters containing approximately 12 to 250 copies, comprising some 2.4 Mb of Y-chromosomal DNA in total. Related sequences with nucleotide substitutions and DNA insertions relative to the Y-chromosomal fragment are found elsewhere in the genome but at much lower copy number and, although similar sequences are also found in other salmonid species, the amplification of the repeat into a Y-chromosome-linked tandem array is only observed in chinook salmon. The OtY8 repetitive sequence is genetically tightly associated with the sex-determination locus and provides an opportunity to examine the evolution of the Y chromosome and sex determination process in a lower vertebrate. Received: 4 April 1997 / Accepted: 22 July 1997     

Repetitive sequences and their organization on genomic clones of Zea mays

Fourteen recombinant clones from Zea mays were studied with regard to their composition of unique and repetitive sequences. Southern hybridization experiments were used to classify restriction fragments of the clones into a unique, middle or highly repetitive class of reiteration frequency. All three classes were often found on the same genomic clone. Crosshybridization studies between clones showed that a given repeat might be present on several clones, and thus four families of highly repetitive elements were established. Heteroduplex analysis was used to show the arrangement and size of repeats common between several clones. A short interspersion pattern of unique, middle and highly repetitive DNA was found. The dispersed repetitive elements were 300-1300 bp in length. Analysis of the pattern produced by a given repeat in genomic Southern experiments suggests that some small dispersed repeats may also exist as part of a larger repeating unit elsewhere in the genome.     

Repetitive DNA sequences near three human beta-type globin genes.

Five repetitive DNA sequences, of average length 259 bp, have been identified in the intergenic regions which flank three human beta-tupe globin genes. A pair of inverted repeat sequences, separated by 919 bp, was found 1.0 kb to the 5' side of the epsiln-globin gene. Each contains a homologous Alu I site. Another repetitive sequence, with the same orientation as the inverted repeat sequence closest to the epsilon-globin gene, lies about 2.2 kb to the 5' side of the delta-globin gene. A pair of inverted repeat sequences, with the same relative orientations as the other pair and separated by about 800 bp, was found about 1.5 kb to the 3' side of the beta-globin gene.     

High polymorphism level of genomic sequences flanking insertion sites of human endogenous retroviral long terminal repeats

The polymorphism at the multitude of loci adjacent to human endogenous retrovirus long terminal repeats (LTRs) was analyzed by a technique for whole genome differential display based on the PCR suppression effect that provides selective amplification and display of genomic sequences flanking interspersed repeated elements. This strategy is simple, target-specific, requires a small amount of DNA and provides reproducible and highly informative data. The average frequency of polymorphism observed in the vicinity of the LTR insertion sites was found to be about 12%. The high incidence of polymorphism within the LTR flanks together with the frequent location of LTRs near genes makes the LTR loci a useful source of polymorphic markers for gene mapping.     

Novel DNA polymorphism in the mouse tumor necrosis factor receptors type 1 and type 2

The introduction of the polymerase chain reaction (PCR) provides an entirely new means of analyzing DNA polymorphism and makes practical the analysis of length variation in simple-sequence tandem repeats of dinucleotides. In the process of cloning and sequencing the mouse genomic DNA for tumor necrosis factor (TNF) receptors type 1 and type 2, we identified two simple dinucleotide repeats within the noncoding regions of TNF receptor type 1 and three such sequences within TNF receptor type 2. PCR analysis of these sequences, using genomic DNA from 21 different inbred and wild mouse strains, as demonstrated by running the amplified products on sequencing gels, showed that the repeats are highly polymorphic. We identified seven alleles of TNF receptor type 2 and five alleles of TNF receptor type 1. Using these polymorphic markers in two sets of recombinant inbred strains of mice, the chromosomal localization of Tnfr-1 was mapped to mouse chromosome 6 and Tnfr-2 was located to the distal portion of mouse chromosome 4.     

Serial Alu sequence transposition interrupting a human B creatine kinase pseudogene.

We have isolated, sequenced, and characterized a single-copy B creatine kinase pseudogene. The chromosomal assignment of this gene is 16p13 and a unique sequence probe from this locus detects EcoRI restriction fragment length polymorphisms of 7.8 and 5.4 kb. In 26 unrelated individuals, the frequencies for the 7.8- and 5.4-kb B creatine kinase pseudogene alleles were calculated to be 17.3 and 82.7%, respectively. The B creatine kinase pseudogene is interrupted by a 904-bp DNA insertion composed of three Alu repeat sequences in tandem flanked by an 18-bp direct repeat, derived from the pseudogene sequence. Nucleotide sequence analysis of the Alu elements suggests that the Alu sequences were incorporated into this locus in three separate integration events. Several complex clustered Alu repeat sequences without defined integration borders have been previously identified at different genomic loci. This is the first evidence that complex tandem Alu elements can integrate in an apparently serial manner in the human genome and supports the contention that Alu repeats integrate nonrandomly into the human genome.     

Variable numbers of tandem repeat loci in genetically homogeneous Haemophilus influenzae strains alter during persistent colonisation of cystic fibrosis patients

Serial sputum isolates of Haemophilus influenzae (n = 69) were obtained from eight patients suffering from cystic fibrosis. For two of these patients all strains were analysed for polymorphism in the major outer membrane protein profile. For all patients the strains were genetically characterised by random amplification of polymorphic DNA analysis. All strains were included in a survey for polymorphism in regions containing moieties of repetitive DNA as well. A single locus containing trinucleotide repeat units, three loci harbouring tetranucleotides, one region comprising pentanucleotide units and two hexanucleotide repeat unit-containing loci were analysed for repeat number variability. Most of the regions were previously shown to be directly adjacent to or even within virulence genes. All regions behaved as genuine variable number of tandem repeat loci in the sense that genetic polymorphism based on the presence of varying numbers of repeat units could be demonstrated among different strains. Interestingly, several of the repeats showed variation in the absence of the variability as assessed by major outer membrane protein or random amplification of polymorphic DNA analysis. These observations indicate that the repeat loci may vary independently from major chromosomal polymorphism. Consequently, H. influenzae appears to modify its virulence gene regions of the chromosome during persistent colonisation of the lung in cystic fibrosis patients.