A hierarchic arrangement of the repetitive sequences in the Balbiani ring 2 gene of Chironomus tentans

One cloned cDNA sequence, pCt63, was used to characterize the repeated structure of the Balbiani ring 2 gene in Chironomus tentans. Although small in size (0.63 kb), the cDNA insert corresponds to a large portion (25 kb) of the BR2 gene (37 kb). Southern blotting experiments suggested that a large part of the BR2 gene consists of tandemly repeated units, each about 215 bp. Sequence analysis of the cDNA confirmed the repeated nature of the BR2 gene and revealed the internal structure of the repeat unit. Each such unit is composed of two regions of approximately equal length; one is highly ordered and built from about six 18 bp repeats, each consisting of a slightly diverged 9 bp duplication. The recorded hierarchic arrangement of the repetitive sequences in the BR2 gene and a specific pattern of base substitutions along the gene have enabled us to propose how a major part of the giant BR2 gene has evolved from a short primordial sequence, 110-120 bp in length.     

Sequence finishing and gene mapping for Candida albicans chromosome 7 and syntenic analysis against the Saccharomyces cerevisiae genome

The size of the genome in the opportunistic fungus Candida albicans is 15.6 Mb. Whole-genome shotgun sequencing was carried out at Stanford University where the sequences were assembled into 412 contigs. C. albicans is a diploid basically, and analysis of the sequence is complicated due to repeated sequences and to sequence polymorphism between homologous chromosomes. Chromosome 7 is 1 Mb in size and the best characterized of the 8 chromosomes in C. albicans. We assigned 16 of the contigs, ranging in length from 7309 to 267,590 bp, to chromosome 7 and determined sequences of 16 regions. These regions included four gaps, a misassembled sequence, and two major repeat sequences (MRS) of >16 kb. The length of the continuous sequence attained was 949,626 bp and provided complete coverage of chromosome 7 except for telomeric regions. Sequence analysis was carried out and predicted 404 genes, 11 of which included at least one intron. A 7-kb indel, which might be caused by a retrotransposon, was identified as the largest difference between the homologous chromosomes. Synteny analysis revealed that the degree of synteny between C. albicans and Saccharomyces cerevisiae is too weak to use for completion of the genomic sequence in C. albicans.     

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

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

A minisatellite with fold-back structure is included in the 5'-flanking region of the Adh gene of Scaptodrosophila lebanonensis

A tandem repetitive sequence with a repeat unit of 12 bp has been found 1.3 kb upstream of the Adh gene of Scaptodrosophila lebanonensis. This repetitive sequence extends over 4.3 kb and consists of two inverted arrays (a fold-back segment). The repeated unit with a consensus sequence GAATACAGAATA is highly conserved and the nucleotide substitutions are not distributed randomly among the 12 bp. In situ hybridization in S. lebanonensis polytene chromosomes revealed two signals, one at the 60A section, the Adh locus, and a second site in the same chromosome at the 60C section close to the telomere. This same pattern of hybridization is obtained in all the analyzed strains including the subspecies S. lebanonensis casteeli. The minisatellite sequence accounts for about 0.03-0.04% of the S. lebanonensis genome and showed intraspecific variability in tandem repeat numbers. Possible functions of this sequence are discussed.     

Repetitive DNA sequences in Mycoplasma pneumoniae.

Two types of different repetitive DNA sequences called RepMP1 and RepMP2 were identified in the genome of Mycoplasma pneumoniae. The number of these repeated elements, their nucleotide sequence and their localization on a physical map of the M. pneumoniae genome were determined. The results show that RepMP1 appears at least 10 times and RepMP2 at least 8 times in the genome. The repeated elements are dispersed on the chromosome and, in three cases, linked to each other by a homologous DNA sequence of 400 bp. The elements themselves are 300 bp (for RepMP1) and 150 bp (for RepMP2) long showing a high degree of homology. One copy of RepMP2 is a translated part of the gene for the major cytadhesin protein P1 which is responsible for the adsorption of M. pneumoniae to its host cell.     

Chromosome specificity of satellite DNAs: short- and long-range organization of a diverged dimeric subset of human alpha satellite from chromosome 3

The human alpha satellite DNA family, like many highly repeated satellite DNAs in eukaryotic genomes, is organized in distinct chromosome-specific subsets. As part of investigations into the molecular and evolutionary basis for the chromosome-specific nature of such subsets, we report the isolation and characterization of alpha satellite sequences specific for human chromosome 3. This subset is characterized by a predominant tandemly arranged 2.9 kb higher-order repeat unit which, in turn, consists of 17 tandem diverged monomer repeat units of 171 bp. Nucleotide sequence analysis reveals that the chromosome 3 higher-order repeat units are comprised, at least in part, of diverged dimeric ( 340 bp) sub-repeats and that this divergence accounts for the chromosome-specific behavior of this subset. Pulsed-field gel electrophoresis demonstrates that the chromosome 3 higher-order repeat units are localized in large domains, at least 1000 kb in length. Familial restriction fragment length polymorphisms associated with the satellite subset can be detected by pulsed field gel electrophoresis and may facilitate molecular analysis of interchromosomal variation.     

Species-specific repetitive extragenic palindromic (REP) sequences in Pseudomonas putida

Pseudomonas putida KT2440 is a soil bacterium that effectively colonises the roots of many plants and degrades a variety of toxic aromatic compounds. Its genome has recently been sequenced. We describe that a 35 bp sequence with the structure of an imperfect palindrome, originally found repeated three times downstream of the rpoH gene terminator, is detected more than 800 times in the chromosome of this strain. The structure of this DNA segment is analogous to that of the so-called enterobacteriaceae repetitive extragenic palindromic (REP) sequences, although its sequence is different. Computer-assisted analysis of the presence and distribution of this repeated sequence in the P.putida chromosome revealed that in at least 80% of the cases the sequence is extragenic, and in 82% of the cases the distance of this extragenic element to the end of one of the neighbouring genes was <100 bp. This 35 bp element can be found either as a single element, as pairs of elements, or sometimes forming clusters of up to five elements in which they alternate orientation. PCR scanning of chromosomes from different isolates of Pseudomonas sp. strains using oligonucleotides complementary to the most conserved region of this sequence shows that it is only present in isolates of the species P.putida. For this reason we suggest that the P.putida 35 bp element is a distinctive REP sequence in P.putida. This is the first time that REP sequences have been described and characterised in a group of non-enterobacteriaceae.     

A novel centromeric repetitive DNA from human chromosome 22

A recombinant DNA clone localized in the centromeric region of chromosome 22 was isolated from a flow-sorted human chromosome 22 DNA library. When the original insert of about 1.9 kb was used to probe Southern blots of EcoRI-digested genomic DNA it revealed at least 40 fragments. A comparable pattern was obtained with each of the three subclones (800, 700, and 380 bp). In situ hybridization showed signals clustered in the region 22cen. DNA sequence analysis using the 380 bp fragment subcloned in pTZ18/19 (p22hom48.4) revealed eight copies of a 48 bp repeat and the size of hybridizing restriction fragments indicated that this tandemly repeated sequence is spread over a region of a few hundred kilobases. Whereas this novel DNA, termed D22Z3, displayed no sequence homology to rodent and monkey genomes cross-homology was discernible for DNA from two great ape species.     

Modular structure in developmentally eliminated DNA in Tetrahymena may be a consequence of frequent insertions and deletions

The work reported here describes insertion-deletion (Indel) polymorphisms in two internally eliminated sequences (IESs, that are deleted during development in Tetrahymena): a 1.8 kb Indel at one end of the 1.1 kb H1 IES and a 0.5 kb Indel inside the 1.4 kb calmodulin (C) IES. These two IESs are located in the proximity of the H1 histone and calmodulin genes, respectively, and are among the ten IESs that have been fully sequenced out of an estimated total of 6000. Three hundred base-pairs of the 1.8 kb H1 Indel are retained in the macronucleus. Both the +Indel and the -Indel variants of the H1 and C IESs that occur in different strains are eliminated during development. Thus, a drastic change involving over half of the deleted sequence and 300 bp of flanking sequence does not disable developmental elimination of the H1 IES, which may indicate a lack of requirement for specific sequences on the Indel side of the IES. The H1 Indel is a composite of three sequence elements: a unique segment and two other sections containing members of different repeat families. One of these, a 0.5 kb repetitive component, is 75% similar to another 0.5 kb sequence that constitutes the C Indel, a sequence present in the middle of the calmodulin IES in some strains, but not in others. Therefore, the C Indel sequence is likely to have been part of a mobile unit, even though it has no obvious features of a transposon. However, sequences similar to the C Indel are present in about 100 copies in the genome. The results suggest that IESs may consist, at least in part, of relatively short modules of repeated sequences that are the source of insertion-deletion polymorphisms among strains of Tetrahymena thermophila.     

A variant tandemly repeated nucleotide sequence in Balbiani ring 2 of Chironomus tentans

pCtBR2-2 is a genomic clone from Chironomus tentans that hybridized in situ to Balbiani ring 2 (BR2) on salivary gland polytene chromosome IV. DNA sequencing indicated that the insert contained nearly four copies of a 180 bp tandemly repeated nucleotide sequence that was distinctly different from a previously reported BR2 repeat. Sequence titration experiments detected about 70 copies of the 180 bp repeat per haploid genome, which would correspond to approximately 34% of a 37 kb BR2 gene. Each 180 bp repeat included a conserved 90 bp segment whose sequence was internally nonrepeating (INR), and a variable 90 bp repeated (R) segment comprised of three 30 bp repeats that may have evolved from a 9 bp consensus sequence. Results presented here raise the distinct possibility that other BR genes may contain significantly different repeated sequences that have not been identified.     

Organization and evolutionary progress of a dispersed repetitive family of sequences in widely separated rodent genomes

The genomes of Mus musculus and other rodent species share a long conserved family of sequences that are dispersed and abundant (approx. 20,000 copies), and that have several novel features of organization and evolution. EcoR1 restriction of M. musculus DNA reveals a prominent 1350 bp² set of sequences. Two nonhomologous sequences of 850 and 500 bp, representing almost the total population of the 1350 bp repeats, were used to examine the detailed organization of the dispersed family and its surrounding sequences using a combination of restriction analysis and “Southern” hybridization. The 1350 bp sequence is contained within a longer repeating unit of approximately 3 kb that is dispersed amongst a wide variety of non-homologous and seemingly non-repetitive sequences. At some sites within the 3 kb repeat, considerable sequence heterogeneity has been found between members of the family, such that the family can be divided into largely non-overlapping subsets (or “segments”) according to the positioning of HinIII sites. Underlying the segmental organization there is a low background overlap of each segment with every other. Some but not all members of the family and its variants have been located on the X-chromosome in a Chinese hamster, M. musculus, X chromosome cell line: suggesting a wide genomic dispersion of the family. Homologous repeated sequences to the M. musculus 1350 bp repeat have been identified in species of Mus and Apodemus, with strikingly similar features of organization and dispersion. In M. spretus a 1350 bp sequence is contained within a dispersed repeat of at least 2·9 kb. However, the majority of M. spretus repeats contain an additional restriction site not present in the equivalent M. musculus array, suggesting a mechanism of widespread substitution or “conversion” of one variant by another in each genome. Apodemus sylvaticus possesses two dispersed and homologous families of 1350 bp and 1850 bp repetition, respectively, which contain sequences that have diverged from M. musculus to differing extents. A. mystacinus possesses only one family of dispersed and homologous repeats of 1850 bp. The majority of members within each Apodemus homologous family also contain characteristic variant restriction-site arrangements. The mechanisms underlying the spread of such variants within each array; the generation of segmental patterns; and the evolutionary conservation of this mouse interspersed family (MIF-1) are discussed in relation to the present knowledge of the organization and activity of other dispersed sequence families.     

Cloning and characterization of an amplified DNA sequence in chromosomal DNA of Streptomyces aureofaciens 2201

In strain 2201 of Streptomyces aureofaciens, a high copy number amplified DNA sequence (ADS-Sa2201) was found and characterized. The amplified sequence in the chromosomal DNA of this strain forms a stretch of about 10 kb tandemly repeated 100-500 times. In this strain also, extrachromosomal copies of the repeated unit of the ADS-Sa2201 were found. In cloning experiments any autonomous replicon was found on ADS-Sa2201 and it thus can be presumed that the presence of the extrachromosomal copies of the repeated unit of ADS-Sa2201 is only a result of its excision from the chromosome. A part of the repeated unit of ADS-Sa2201 was also found in chromosomal DNA of strain 13 of S. aureofaciens. In this strain no part of ADS-Sa2201 is present extrachromosomally.     

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.     

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.     

In vivo cleavage of Escherichia coli BIME-2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site

The Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3' end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3' exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.     

Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli

Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes. REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria. These REP sequences may participate in the folding of the bacterial chromosome. In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence. We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically. It is estimated that there are about 70 RIP elements in E. coli. Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome. The possible function of the RIP element is discussed.