Structural transition in inactive Balbiani ring chromatin of Chironomus during micrococcus nuclease digestion

We have analysed by micrococcus nuclease digestion the chromatin structure of genes in the Balbiani ring (BR) regions of a Chironomus cell line. Gel electrophoresis of the DNA fragments reveals a repeating structure which consists of two repeat sizes, a long repeat seen in the large fragments and a small repeat seen in the small fragments. The two repeats hardly overlap, except in a narrow transition zone which is at a different fragment size in the BR 2.2 and the BR 2.1 gene. The sizes of the large repeats fit the repeat of the underlying DNA sequence. The short repeats are between 170 and 180 bp, and after H1 depletion the short repeat in the BR 2.2 gene is 160 bp. Our most favoured interpretation of these data is that in intact chromatin the nucleosomes in the BR genes are phased with respect to the repeating DNA sequence, whereas micrococcus nuclease digestion leads to loss of a nucleosome-positioning constraint and hence to rearrangement of the nucleosomes. Our results imply a possible artefact of nuclease digestion of chromatin, which has to be taken into account in mapping nucleosome positions.     

Relationship between micro- and minisatellites in the human genome

Micro- and minisatellites constitute an essential part of DNA with a low sequence complexity and carry several important functions. A search for tandem repeats in the human genome with a length of a repeat unit of up to 70 bp, including repeats with a great number of nucleotide substitutions, has been performed using the TaadeaSWAN program. It was shown that, for a considerable number of minisatellites with the length of the repeating unit of less than 25 nt, a shorter repeating motif can be distinguished in the sequence of this repeat, which often is similar to the sequence of minisatellites widely occurring in the human genome. A model of hierarchic origination of minisatellites in the human genome is suggested.     

The bovine genome contains polymorphic microsatellites

Dinucleotide repeats constitute so-called microsatellites of the human and other eukaryotic genomes. Microsatellite polymorphisms can be identified through the amplification of the microsatellite DNA using the polymerase chain reaction (PCR), followed by resolution of the amplified DNA fragments on a polyacrylamide sequencing gel. We performed a preliminary sequence database search to identify bovine sequences containing (CA)n, (AC)n, (GT)n, or (TG)n blocks, with n greater than or equal to 6. This search yielded 10 sequences containing one or two of the specified repeat blocks and often additional dinucleotide repeat blocks. One of the microsatellite-containing regions has been sequenced twice from independent clones and the reported sequences showed variation in the number of repeats. PCR-amplified fragments of another sequence, the gene for steroid 21-hydroxylase, ranged from 186 to 216 nucleotides in 43 unrelated animals. The database search, as well as the hypervariable microsatellite in the bovine steroid 21-hydroxylase gene, indicates that dinucleotide blocks may be an abundant source of DNA polymorphism in cattle.     

Length and sequence heterogeneity of the histone gene repeat unit of the sea urchin, S. purpuratus

Histone gene repeats in S. purpuratus are shown to be of variable length and sequence. Two recombinant plasmids containing the full-length 6.3 kb histone repeat unit are found to differer in length at two sites in the repeating structure and in the occurrence of two restriction enzyme recognition sites. Variation in repeat length is also demonstrated in the unfractionated DNA of five sea urchins and in a sample of DNA enriched for histone gene sequences by density gradient methods. The repeats in each individual are of a very limited number of major classes, which may differ from one another in overall length or in distribution and presence of particular restriction enzyme sites. Variations are found to occur at many regions of the repeat; some have been mapped specifically to spacer regions. Repeats may differ dramatically from individual to individual since there is no one type of repeat class common to all, although the absolute length differences of the repeats that are found are small.     

A helical string of alternately connected three-helix bundles for the cell wall-associated adhesion protein Ebh from Staphylococcus aureus

The 1.1 MDa cell-wall-associated adhesion protein of staphylococci, Ebh, consists of several distinct regions, including a large central region with 52 imperfect repeats of 126 amino acid residues. We investigated the structure of this giant molecule by X-ray crystallography, circular dichroism (CD) spectrometry, and small-angle X-ray scattering (SAXS). The crystal structure of two repeats showed that each repeat consists of two distinct three-helix bundles, and two such repeats are connected along the long axis, resulting in a rod-like structure that is 120 A in length. CD and SAXS analyses of the samples with longer repeats suggested that each repeat has an identical structure, and that such repeats are connected tandemly to form a rod-like structure in solution, the length of which increased proportionately with the number of repeating units. On the basis of these results, it was proposed that Ebh is a 320 nm rod-like molecule with some plasticity at module junctions.     

Subtelomeric regions of yeast chromosomes contain a 36 base-pair tandemly repeated sequence.

We have determined the nucleotide sequence of a region of DNA derived from the end of one chromosome of the yeast, Saccharomyces cerevisiae. Inspection of the sequence reveals the presence of 12 tandem direct repeats, each 36 nucleotides long and having nearly identical sequence. Each 36 base-pair repeat can be further subdivided into three tandem sub-repeats of a similar 12 base-pair sequence. Analysis of total genomic yeast DNA from several strains by Southern hybridization suggests that the number of tandem 36 base-pair repeat units may vary from approximately 8 to 25 among different telomeric regions. Differences in the number of repeats may have arisen by unequal crossing over between them. Furthermore, the finding that the pattern of bases at multiple variable positions within the repeat unit is not random suggests that these regions may undergo gene conversion events that render them homogeneous.     

Mouse genomic DNA sequences homologous to sea urchin TU elements are genetically stable polydispersed repeats useful for analysis of multiple RFLPs.

Segments of the murine genome that hybridize to the inverted repeat regions of the transposable TU elements of sea urchins include tandem repeats of a sequence (CTCC) that encodes the recognition site for the restriction enzyme Mnl1, as do the analogous polypurine/polypyrimidine (pPu/pPy) stretches in humans. The Mnl1-sensitive repeats, which exist as a microsatellite sequence 200-300 bp in length, lack the terminal dyad symmetry characteristic of the TU elements and are structurally and functionally distinct from these elements. DNA fragments containing these repeat units that are isolated from different generations of isogenic (or congenic) mice or from different tissues of genetically identical individuals are indistinguishable by RFLP analysis; however, they show restriction fragment length polymorphism in different strains. This polymorphism appears to reflect DNA sequence changes occurring at sites flanking the repeats rather than variability in the number of repeats. Their genetic stability and occurrence in a wide variety of animal species make the Mnl1 repeats useful in studying genetic variation that has occurred over an evolutionary time scale of greater duration than can be examined conveniently by VNTR analysis.     

Sequence analysis of trinucleotide repeat microsatellites from an enrichment library of the equine genome.

Microsatellites are useful tools for the construction of a linkage map and parentage testing of equines, but only a limited number of equine microsatellites have been elucidated. Thus, we constructed the equine genomic library enriched for DNA fragments containing (CAG)n repeats. The enriched method includes hybridization-capture of repeat regions using biotin-conjugated oligonucleotides, nucleotide substrate-biased polymerase reaction with the oligonucleotides and subsequent PCR amplification, because these procedures are useful for the cloning of less abundant trinucleotide microsatellites. Microsatellites containing (CAG)n repeats were obtained at the ratio of one per 3-4 clones, indicating an enrichment value about 10(4)-fold, resulting in less time consumption and less cost for cloning. In this study, 66 different microsatellites, (CAG)n repeats, were identified. The number of complete simple CAG repeats in our clones ranged 4-33, with an average repeat length of 8.8 units. The microsatellites were useful as sequence-tagged site (STS) markers. In addition, some clones containing (CAG)n repeats showed homology to human (CAG)n-containing genes, which have been previously mapped. These results indicate that the clones might be a useful tool for chromosome comparison between equines and humans.     

Minisatellite polymorphism as a tool to distinguish closely related Lactococcus lactis strains

Genome plasticity is considered as a means for bacteria to adapt to their environment. Plasticity in tandem repeat sequences on bacterial genomes has been recently exploited to trace the epidemiology of pathogens. Here, we examine the utility of minisatellite (i.e., a repeat unit of six nucleotides or more) typing in non-pathogenic food bacteria of the species Lactococcus lactis. Thirty-four minisatellites identified on the sequenced L. lactis ssp. lactis strain IL1403 genome were first analyzed in 10 closely related ssp. lactis strains, as determined by randomly amplified polymorphic DNA (RAPD). The selected tandem repeats varied in length, percent identity between repeats, and locations. We showed that: (i) the greatest polymorphism was in orfs encoding exported proteins or in intergenic regions; (ii) two thirds of minisatellites were little- or non-variable, despite as much as 90% identity between tandem repeats; and (iii) dendrograms based on either RAPD or minisatellite analyses were similar. Seven minisatellites identified in this study are potentially useful for lactococcal typing. We then asked whether tandem repeats in L. lactis were stable upon very long-term (up to two years) storage. Despite large rearrangements previously reported in derivative strains, just one of 10 minisatellites tested underwent an alteration, suggesting that tandem repeat rearrangements probably occur during active DNA replication. We conclude that multiple locus minisatellite analysis can be a valuable tool to follow lactococcal strain diversity.     

Genotyping of white spot syndrome virus prevalent in shrimp farms of India

DNA extracts from white spot syndrome virus (WSSV) that had infected post-larvae and juveniles of cultured shrimp, wild shrimp and crabs, which had been collected from different hatcheries and farms located along both the east and west coasts of India, revealed considerable variation in several previously identified WSSV DNA repeat regions. These include the 54 bp repeat in ORF 94, the 69 bp repeat in ORF 125 and the compound 45 and 57 bp repeat region in ORF 75. In ORF 94, 13 genotypes were observed with the number of repeats ranging from 2 to 16 units. While 7 repeat units were commonly observed (11.3%), no samples with 11 or 15 repeat units were found. In ORF 125, 11 types were found, with repeats ranging from 2 to 14 units. The most prevalent genotype displayed 4 repeat units (47.1%); no samples with 6 or 13 repeats were observed. The compound repeat region of ORF 75 displayed 6 different patterns of repeats. Samples with the same repeat pattern in one ORF did not always show identical repeat patterns in one or both of the other repeat regions. These data suggest that combined analysis of all 3 variable loci could be used to differentiate and characterize specific WSSV strains. For general epidemiological studies, the best marker with maximum variation is ORF 94, followed by ORF 125 and ORF 75. The 3 repeat regions above were used to compare WSSV genotypes from disease outbreaks on 3 sets of farms from different locations in the state of Andhra Pradesh. The genotypes within each farm set were almost identical, but differed between farm sets, suggesting that WSSV transmission occurred directly through virus carriers or water exchange between adjacent farms at each location. These findings show that genotyping can be a useful epidemiological tool for tracing the movement of WSSV within infected populations.     

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.     

Highly discriminatory typing method for Listeria monocytogenes using polymorphic tandem repeat regions

Tandem repeats (TR), which are repetitive nucleotide sequences in DNA, are polymorphic both in repeat number and sequence. In this study, we developed a new typing method, multilocus TR sequence analysis (MLTSA), for the foodborne pathogen Listeria monocytogenes using sequence polymorphisms in three tandem repeat regions. The obtained dendrogram clustered L. monocytogenes strains of lineage I and lineage II separately, and formed three groups within the lineage I cluster, each of which included one of the three major L. monocytogenes epidemic clones (ECI, ECIa, and ECII). These results were consistent with a previously established virulence-gene-based MLST method. In comparison, our method grouped some epidemiologically related isolates together, which virulence-gene-based MLST did not. Moreover, our method, using three tandem repeat regions, showed a higher discriminatory power than the MLST method, which uses six virulence gene regions. This MLTSA approach using sequence polymorphisms in TR regions could be a useful tool in the epidemiological study of L. monocytogenes.     

The intragenomic polymorphism of a partially inverted repeat (PIR) in Gallus gallus domesticus, potential role of inverted repeats in satellite DNAs evolution

We report here the molecular characterization of the basic repeating unit of a novel repetitive family, partially inverted repeat (PIR), previously identified from chicken genome. This repetitive DNA family shares a close evolutionary relationship with XhoI/EcoRI repeats and chicken nuclear-membrane-associated (CNM) repeat. Sequence analyses reveal the 1430 bp basic repeating unit can be divided into two regions: the central region ( approximately 1000 bp) and the flanking region ( approximately 430 bp). Within the central region, a pair of repeats (86 bp) flanks the central core ( approximately 828 bp) in inversed orientation. Due to the tandem array feature shared by the repeating units, the inverted repeats fall between the central core and flanking region. Southern blot analyses further reveal the intragenomic polymorphism of PIR, and the molecular size of repeating units ranges from 1.1 kb to 1.6 kb. The identified monomer variants may result from multiple crossing-over events, implying the potential roles of inverted repeats in satellite DNAs variation.     

青鱼微卫星标记的开发与特性分析

青鱼(Mylopharyngodon piceus)是中国最为重要的淡水养殖鱼类。开发青鱼的微卫星标记能为青鱼的遗传多样性分析提供更多工具。本研究使用磁珠富集法,利用生物素探针(CA)10和(GACA)6,富集得到青鱼基因组微卫星片段,进一步通过设计微卫星引物检验其在青鱼原种群体中的有效性和多态性水平。结果显示,所构建文库中849个克隆含有微卫星序列,通过利用PCR技术在吴江原种青鱼36个个体中进行多态性筛选,获得了25个多态性微卫星位点。其平均等位基因数(Na)和有效等位基因数(Ne)分别为7.08和3.526,平均观测杂合度(Ho)和期望杂合度(He)分别为0.602和0.619,平均多态信息含量(PIC)为0.568。其中,Mp23、Mp27和Mp35这3个位点极显著偏离哈迪-温伯格平衡(P 0.01)。本研究开发的微卫星标记能为青鱼种质资源的评价和保护等研究提供工具。     

Studies of the CAG repeat in the Machado-Joseph disease gene in Taiwan

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by cerebellar ataxia and pyramidal signs associated in varying degrees with a dystonic-rigid extrapyramidal syndrome or peripheral amyotrophy. Unstable CAG trinucleotide repeat expansion in the MJD gene on the long arm of chromosome 14 has been identified as the pathological mutation for MJD. While investigating the distribution of CAG repeat lengths of the MJD gene in Taiwan’s population, we have identified 18 MJD-affected patients and 12 at-risk individuals in seven families. In addition, we have analyzed the range of CAG repeat lengths in 96 control individuals. The CAG repeat number ranged from 13 to 44 in the controls and 72–85 in the affected and at- risk individuals. Our results indicated that the CAG repeat number was inversely correlated with the age of onset. The differences in CAG repeat length between parent and child and between siblings are greater with paternal transmission than maternal transmission. Our data show a tendency towards the phenomenon of anticipation in the MJD families but do not support unidirectional expansion of CAG repeats during transmission. We also demonstrated that PCR amplification of the CAG repeats in the MJD gene from villous DNA was possible and might prove useful as a diagnostic tool for affected families in the future. Received: 4 December 1996 / Accepted: 5 March 1997     

Sequence homologies of diverse length tandem repetitions near ends of vaccinia virus genome suggest unequal crossing over.

The 180,000 base pair (bp), covalently closed, linear duplex DNA genome of vaccinia virus contains a 10,000 bp inverted terminal repetition within which are one set of 13 and one set of 18 tandem 70 bp repeating units. A 967 bp segment containing the innermost 70 bp repeat and an adjacent region notable for a scarcity of restriction endonuclease sites has been sequenced. This was facilitated by the cloning of TaqI and partial TaqI fragments in pBR322. We found that the innermost 70 bp repeat overlaps one of two adjacent 125 bp repeats, following which are eight repeats of 54 bp, parts of 54 bp and 70 bp repeats, and four consecutive 6 to 7 bp repeats. The 70, 125, and 54 bp repeating units have extensive sequence homologies and redundancies that suggest evolution by unequal crossing over. Schemes whereby unequal crossovers of 54 bp repeats lead to a recombinant segment 86% homologous to the 125 bp repeat and unequal crossovers of 125 bp repeats lead to a recombinant segment 94% homologous to the 70 bp repeat were considered. This propensity for sequence divergence should provide a useful marker for comparing the relatedness of poxviruses.     

Repetitive sequences of the sea urchin genome: Distribution of members of specific repetitive families

Three repetitive sequence families from the sea urchin genome were studied, each defined by homology with a specific cloned probe one to a few hundred nucleotides long. Recombinant λ-sea urchin DNA libraries were screened with these probes, and individual recombinants were selected that include genomic members of these families. Restriction mapping, gel blot, and kinetic analyses were carried out to determine the organization of each repeat family. Sequence elements belonging to the first of the three repeat families were found to be embedded in longer repeat sequences. These repeat sequences frequently occur in small clusters. Members of the second repeat family are also found in a long repetitive sequence environment, but these repeats usually occur singly in any given region of the DNA. The sequences of the third repeat are only 200 to 300 nucleotides long, and are generally terminated by single copy DNA, though a few examples were found associated with other repeats. These three repeat sequence families constitute sets of homologous sequence elements that relate distant regions of the DNA.     

High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand

White spot syndrome virus (WSSV) presently causes the most serious losses to shrimp farmers worldwide. Earlier reports of high DNA sequence homology among isolates from widely separated geographical regions suggested that a single virus was the cause. However, we have found surprisingly high variation in the number of 54 bp DNA repeats in ORF94 (GenBank AF369029) from 55 shrimp ponds (65 shrimp samples) experiencing WSSV outbreaks in Thailand in 2000 and 2002. These were detected by PCR amplification using primers ORF94-F and ORF94-R flanking the repeat region. Altogether, 12 different repeat groups were found (from 6 to 20 repeats) with 8 repeats being most frequent (about 32%). Extracts prepared from individual shrimp in the same outbreak pond belonged to the same repeat group while those collected at the same time from separate WSSV outbreak ponds, or from the same ponds at different times, usually belonged to different repeat groups. This suggested that different outbreaks were caused by different WSSV isolates. In contrast to the highly variable numbers of repeats, sequence variation within the repeat region was confined to either T or G at Position 36. These variations may be useful for epidemiological studies on the local and global movement of WSSV, since there is high variation in the number of repeats (good for local studies) but little sequence change (good for global studies).