Linkage of two distinct AT-rich minisatellites at multiple loci in the genome of Theileria parva

Minisatellite tandem repeat elements are well known components of vertebrate genomes, but have not yet been extensively characterized in lower eukaryotes. We describe two unusual, AT-rich minisatellites of the protozoan parasite Theileria parva whose sequences are unrelated to the G/C-rich `chi minisatellite superfamily' of vertebrate and plant genomes. The T. parva tandem repeats, one with a conserved sequence T_2-5ACACA (6–17 copies), and the other with a 6-bp core sequence of either ACTATA or TATACT associated with additional variable sequences in repeats of 10–17 bp (3–7 copies), were closely linked at more than 20 sites in the T. parva genome, separated by 390, 510 and 660 bp at three loci analysed in detail. Such linkage is without precedent in minisatellites so far analysed in other organisms. The minisatellite loci were widely dispersed on 13 out of 33 genomic SfiI fragments, on all four T. parva chromosomes and did not exhibit a telomeric bias in their distribution. Analysis of flanking sequences revealed no obvious conserved sequences between the five loci, or other multicopy repeat sequences outside the minisatellite regions. The T_2-5 ACACA minisatellite was highly effective as a multilocus fingerprinting probe for discrimination of T. parva isolates. Analysis of two individual minisatellite loci revealed variation between the genomic DNAs of two T. parva isolates in the copy number of the constituent repeats within the array, similar to that typical of vertebrate minisatellites.     

Identification of a variable region of the African swine fever virus genome that has undergone separate DNA rearrangements leading to expansion of minisatellite-like sequences

Nucleotide sequencing identified a tandemly repeated sequence array 22 x 10(3) base-pairs from the right-hand DNA terminus of the African swine fever virus (ASFV) genome. The sequence of the repeat array and sequences closely flanking it were compared in the genomes of four groups of ASFV isolates that had very different restriction enzyme site maps. Arrays present in one group of ASFV isolates from East Zambia/Malawi varied in length and contained between 8 and 38 copies of a 17-nucleotide repeat unit. Repeat arrays in a second group of ASFV isolates from Europe were less variable in length but consisted of different types of repeat unit that were divergent in sequence. A third genetically diverse ASFV isolate. LIV 13 from a South Zambia Game Park, contained repeat unit types that were similar to those of European viruses. MFUE6 isolate from an East Zambia Game Park contained a shorter version of the European repeat unit. An eight-base-pair core sequence was conserved between the East Zambia/Malawi and European and LIV 13 repeat units. These tandemly repeated sequence arrays share a number of properties with chromosomal minisatellite DNA. Similar tandem repeat arrays have not been described in poxviruses.     

A unique AT-rich hypervariable minisatellite 3' to the ApoB gene defines a high information restriction fragment length polymorphism

A DNA restriction fragment length polymorphism has been found immediately 3' to the human apoB gene. Digestion of many different human DNAs at sites flanking the region and Southern blotting analysis reveal that this region can vary in length by approximately 300 base pairs with five alleles readily distinguishable. The length polymorphism is due to a unique AT-rich minisatellite that consists primarily of a 30-base pair tandem repeat with two structurally related subunit sequences, x (ATAATTAAATATTTT) and y (ATAATTAAAATATTT). In general, the sequences repeat in an x-y order. The AT-rich region also contains variant x and y sequences that result from C or G for A substitution. Sequence analysis of one large allele revealed the expected increased number of xy repeats. In addition, similar analysis of three different smaller alleles with the same apparent size on Southern blotting analysis showed that all were of slightly different size due to minor differences in the number of xy repeats. The heterogeneity of this AT-rich minisatellite provides the basis for a highly informative restriction fragment length polymorphism of the apoB gene and should be very useful in association and linkage analysis studies of the contribution of this locus to atherosclerosis susceptibility.     

中国明对虾基因组小卫星重复序列分析

通过对中国明对虾基因组随机DNA片断的测序 ,我们获得了总长度约 6 4 10 0 0个碱基的基因组DNA序列 ,从中共找到 172 0个重复序列。其中 ,小卫星序列的数目为 398个 ,占重复序列总数目的 2 3 14 %。这些小卫星序列的重复单位长度为 7- 16 5个碱基 ,集中分布于 7- 2 1个碱基范围内 ,其中以重复单位长度为 12个碱基的重复序列数目最多 ,为 5 8个 ,占小卫星重复序列总数目的 14 5 7%。不同拷贝数目所对应的重复序列的数目情况为 :拷贝数目为 2的重复单位所组成的重复序列数目最多 ,为 137个 ;其次是拷贝数目为 3的重复序列 ,为12 2个 ,且随着拷贝数目的增加 ,由其所组成的重复序列的数目呈递减的趋势。其中一部分序列见GeneBank数据库 ,登录号为AY6 990 72 -AY6 990 76。 398个重复序列分别由 398种重复单位所组成 ,因而小卫星重复序列的类型很多 ,我们初步分成三类 :两种碱基组成类别、三种碱基组成类别和四种碱基组成类别 ,并进一步根据各个重复序列中所含有的碱基种类的数量从大到小排列这些碱基而分成若干小类。从这些分类中可以看出 ,中国明对虾基因组中的小卫星整体上是富含A T的重复序列 ,并具有一定的“等级制度” ,揭示了其与微卫星重复序列之间的关系 ,即一部分小卫星重复序列可能起源于微卫星     

A novel minisatellite repeat expansion identified at FRA16B in a Japanese carrier

Previously, the allelic expansion of a 33-bp AT-rich minisatellite repeat has been reported to cause FRA16B, a distamycin A-inducible fragile site. Here, we identified a novel 35-bp minisatellite repeat at FRA16B in a Japanese carrier. The nucleotide sequence of the 35-bp minisatellite was highly AT-rich and nearly identical to the 33-bp one but with insertion of two nucleotides, thymine and adenine. The copy number of the AT-rich minisatellite was 21 in total in the carrier, while only a few copies of the 33-bp minisatellite were present in a non-carrier Japanese subject. These results suggest that the molecular mechanism involved in the allelic expansion of the minisatellite repeat in FRA16B recognizes both minisatellites, the 33-bp one and the 35-bp one, as an amplicon. These observations were different from the ones at folate-sensitive fragile sites, where the CCG triplet repeat was commonly involved in the allelic expansion. Although a slight reduction in AT content (95% > 90%) in the region of minisatellite expansion in the carrier subject was observed, both AT-content and length of the highly AT-rich region seem to play important roles in the cytogenetic expression of the distamycin A-inducible fragile site. In another normal subject, without fragile site expression, allelic expansion involving the 33-bp minisatellite was observed, and the length of the AT-rich DNA region was increased up to approximately 1000 bp. Since the length of the AT-rich minisatellite region was increased up to approximately 1,100-bp in the carrier subject, the threshold length for the cytogenetic expression of the AT-rich DNA region may be between about 1,000-bp and 1,100-bp.     

Genetic mapping of hypervariable minisatellite sequences in rice (Oryza sativa L.)

Minisatellites, or DNA fingerprinting sequences, have been utilized in animal linkage studies for several years but have not been used as markers for plant genome mapping. In animal genome mapping they have resulted in limited success because they are evenly dispersed in some species but are often clustered near telomeric regions, as observed on human chromosomes. The purpose of the present study was to generate DNA fingerprints utilizing several rice-derived minisatellites containing different core sequences and numbers of repeat units, followed by assessing their potential for use as genetic markers when mapped to a rice recombinant inbred line (RIL) population. Sites of segregating minisatellite loci were mapped onto 11 of the 12 rice RIL linkage maps. The implications for the use of rice minisatellite core sequences as genetic markers on linkage maps in rice are discussed. Received: 1 March 1999 / Accepted: 22 June 1999     

Organization of the 1.9-kb repeat unit RCE1 in the centromeric region of rice chromosomes

This paper presents the first report on the structure of a 14-kb centromere sequence in a cereal genome that includes 1.9-kb direct repeats. The cereal centromeric sequence (CCS1) conserved in some Gramineae species contains a 17-bp motif similar to the CENP-B box, which serves as the binding site for the centromere-specific protein CENP-B in human. To isolate centromeric units from rice (Oryza sativa L.), we performed PCR using the CENP-B box-like sequences (CBLS) as primers. A 264-bp clone was amplified by this method, and called RCS1516. It appeared to be a novel member of the CCS1 family, sharing about 60% identity with the CCS1 sequences of other cereals. Then, a 14-kb genomic clone, λRCB11, carrying the RCS1516 sequence was isolated and sequenced. It was found to contain three copies of a 1.9-kb direct repeat, RCE1, separated by 5.1- and 1.7-kb. A 300-bp sequence at the 3′ end of RCE1 is highly conserved in all three copies (>90%) and is almost identical to the RCS1516 sequence including the CBLS motif. The copy number of RCE1 was estimated to range from 10² to 10³ in the haploid genome of rice. Cloned RCE1 units were used for fluorescent in situ hybridization (FISH) analysis, and signals were observed on almost every primary constriction of rice chromosomes. Thus it was concluded that RCE1 is a significant component of the rice centromere. The λRCB11 clone contained at least four A/T-rich regions, which are candidate for matrix attachment regions (MARs), in the sequences between the RCE1 repeats. Other elements that are homologous to the short centromeric repetitive sequences pSau3A9 and pRG5, detected in both sorghum and rice, were also found in the clone. Received: 9 June 1998 / Accepted: 16 September 1998     

Cloning a selected fragment from a human DNA ''fingerprint'': isolation of an extremely polymorphic minisatellite.

A large hypervariable DNA fragment from a human DNA fingerprint was purified by preparative gel electrophoresis and molecular cloning. The cloned fragment contained a 6.3 kb long minisatellite consisting of multiple copies of a 37 bp repeat unit. Each repeat contained an 11 bp copy of the "core" sequences, a putative recombination signal in human DNA. The cloned minisatellite hybridized to a single locus in the human genome. This locus is extremely polymorphic, with at least 77 different alleles containing 14 to 525 repeat units per allele being resolved in a sample of 79 individuals. All alleles except the shortest are rare and the resulting heterozygosity is very high (approximately 97%). Cloned minisatellites should therefore provide a panel of extremely informative locus-specific probes ideal for linkage analysis in man.     

The ingi and RIME non-LTR retrotransposons are not randomly distributed in the genome of Trypanosoma brucei

The ingi (long and autonomous) and RIME (short and nonautonomous) non--long-terminal repeat retrotransposons are the most abundant mobile elements characterized to date in the genome of the African trypanosome Trypanosoma brucei. These retrotransposons were thought to be randomly distributed, but a detailed and comprehensive analysis of their genomic distribution had not been performed until now. To address this question, we analyzed the ingi/RIME sequences and flanking sequences from the ongoing T. brucei genome sequencing project (TREU927/4 strain). Among the 81 ingi/RIME elements analyzed, 60% are complete, and 7% of the ingi elements (approximately 15 copies per haploid genome) appear to encode for their own transposition. The size of the direct repeat flanking the ingi/RIME retrotransposons is conserved (i.e., 12-bp), and a strong 11-bp consensus pattern precedes the 5'-direct repeat. The presence of a consensus pattern upstream of the retroelements was confirmed by the analysis of the base occurrence in 294 GSS containing 5'-adjacent ingi/RIME sequences. The conserved sequence is present upstream of ingis and RIMEs, suggesting that ingi-encoded enzymatic activities are used for retrotransposition of RIMEs, which are short nonautonomous retroelements. In conclusion, the ingi and RIME retroelements are not randomly distributed in the genome of T. brucei and are preceded by a conserved sequence, which may be the recognition site of the ingi-encoded endonuclease.     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

Structure of an unusual sea urchin U1 RNA gene cluster

Genomic clones containing multiple copies of the Lytechinus variegatus U1 gene have been isolated from a gene library in the phage lambda EMBL3. These clones contain both types of U1 RNA gene repeats interspersed in the same 15-kb fragment. In addition, about 1/3 of the repeat units contain a 260-bp insert 460 bp prior to the first nucleotide of the U1 RNA sequence. The inserted sequence is abundant in the sea urchin genome as judged by Southern blots of genomic DNA. There are no repeated sequences flanking the insert. The insert occurs at the same position in the highly conserved 5'-flanking region at which a deletion has previously been reported.     

Tetrad analysis shows that gene conversion is the major mechanism involved in mutation at the human minisatellite MS1 integrated in Saccharomyces cerevisiae

Minisatellites are arrays of tandemly repeated DNA sequences which occur at thousands of locations in the human genome. They are frequently hypervariable with respect to allele length as a result of high rates of complex and incompletely understood recombination-based germline mutation events that alter the repeat copy number. MS1 is one of the most variable minisatellites so far isolated from the human genome. We have integrated MS1, flanked by synthetic markers, in the vicinity of a hot spot for meiotic double-strand breaks upstream of the LEU2 locus in chromosome III of Saccharomyces cerevisiae. Here we present the first tetrad analysis of mutations at a human minisatellite locus. The data showed that mutant alleles occur as single mutants in one of the spores in a tetrad, also when the mutant structure was the result of a combination of intra- and inter-allelic rearrangements. The conversional transfer of repeat units from one allele to the other was associated with flanking marker conversion which always involved the same flank of the minisatellite. The results demonstrate that conversion is the predominant mechanism by which minisatellite alleles mutate to new lengths, and also support the assumption that cis-acting elements are involved in the regulation of the mutational process in humans.     

The minisatellite MSB1, in the fungus Botrytis cinerea,probably mutates by slippage

A minisatellite was identified in the intron of the ATP synthase of the filamentous fungus Botrytis cinerea, and it was named MSB1. This is the second fungal minisatellite described to date. Its 37-bp repeat unit is AT-rich, and it is found at only one locus in the genome. The introns of 47 isolates of Botrytis species were sequenced. The number of tandem repeats varied only from 5 to 11, but there were many repeat variants. The structure of MSB1 is peculiar: the variants are in the same physical order in all individuals, and this order follows the most parsimonious tree. These original characteristics, together with a total lack of recombination between alleles of the flanking regions, suggest that MSB1 probably mutates by slippage. MSB1 was found in the intron of the ATP synthase of all of the Botrytis species analyzed, but the repeat unit was not found in any other genus examined, including Sclerotinia, which is the genus closest to Botrytis.     

Survey and analysis of microsatellites in the silkworm, Bombyx mori: frequency, distribution, mutations, marker potential and their conservation in heterologous species

We studied microsatellite frequency and distribution in 21.76-Mb random genomic sequences, 0.67-Mb BAC sequences from the Z chromosome, and 6.3-Mb EST sequences of Bombyx mori. We mined microsatellites of >/=15 bases of mononucleotide repeats and >/=5 repeat units of other classes of repeats. We estimated that microsatellites account for 0.31% of the genome of B. mori. Microsatellite tracts of A, AT, and ATT were the most abundant whereas their number drastically decreased as the length of the repeat motif increased. In general, tri- and hexanucleotide repeats were overrepresented in the transcribed sequences except TAA, GTA, and TGA, which were in excess in genomic sequences. The Z chromosome sequences contained shorter repeat types than the rest of the chromosomes in addition to a higher abundance of AT-rich repeats. Our results showed that base composition of the flanking sequence has an influence on the origin and evolution of microsatellites. Transitions/transversions were high in microsatellites of ESTs, whereas the genomic sequence had an equal number of substitutions and indels. The average heterozygosity value for 23 polymorphic microsatellite loci surveyed in 13 diverse silkmoth strains having 2-14 alleles was 0.54. Only 36 (18.2%) of 198 microsatellite loci were polymorphic between the two divergent silkworm populations and 10 (5%) loci revealed null alleles. The microsatellite map generated using these polymorphic markers resulted in 8 linkage groups. B. mori microsatellite loci were the most conserved in its immediate ancestor, B. mandarina, followed by the wild saturniid silkmoth, Antheraea assama.     

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.     

ISH51: a large, degenerate family of insertion sequence-like elements in the genome of the archaebacterium, Halobacterium volcanii.

We describe a new family of repetitive elements in the genome of the archaebacterium Halobacterium volcanii. There are some 20-30 copies of this element, which we designate ISH51. Sequenced copies show typical insertion sequence characteristics (terminal inverted repeats, direct flanking repeats of "target site" DNA). However, members of the ISH51 family are highly heterogeneous, showing on average only 85% primary sequence homology; and some genomic copies appear to be severely truncated. Some ISH51 elements are clustered together in regions of relatively AT-rich DNA. There are at least five such AT-rich "islands" in the H. volcanii genome. Repetitive sequences homologous to ISH51 are found in the genomes of most Halobacterium and Halococcus species.