Identification of a Novel Subgroup of Koala Retrovirus from Koalas in Japanese Zoos

We identified a new subgroup of koala retrovirus (KoRV), named KoRV-J, which utilizes thiamine transport protein 1 as a receptor instead of the Pit-1 receptor used by KoRV (KoRV-A). By subgroup-specific PCR, KoRV-J and KoRV-A were detected in 67.5 and 100% of koalas originating from koalas from northern Australia, respectively. Altogether, our results indicate that the invasion of the koala population by KoRV-J may have occurred more recently than invasion by KoRV-A.     

Evolution of Repeated Sequence Arrays in the D-Loop Region of Bat Mitochondrial DNA

Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near the tRNA-Pro gene are present in all vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from 15% to 63%. Fewer repeats among heteroplasmic than homoplasmic individuals in a species with up to nine repeats indicates selection may act against long arrays. A lower limit of two repeats and more repeats among heteroplasmic than homoplasmic individuals in two species with few repeats suggests length mutations are biased. Significant regressions of heteroplasmy, θ and π, on repeat number further suggest that repeat duplication rate increases with repeat number. Comparison of vespertilionine bat consensus repeats to mammal control region sequences revealed that tandem repeats of similar size, sequence and number also occur in shrews, cats and bighorn sheep. The presence of two conserved protein-binding sequences in all repeat units indicates that convergent evolution has occurred by duplication of functional units. We speculate that D-loop region tandem repeats may provide signal redundancy and a primitive repair mechanism in the event of somatic mutations to these binding sites.     

Tnat1 and Tnat2 from Arabidopsis thaliana: novel transposable elements with tandem repeat sequences.

A computer-aided homology search of databases found that the nucleotide sequences flanking ATLN44, a non-LTR retrotransposon (LINE) from Arabidopsis thaliana, are repeated in the A. thaliana genome. These sequences are homologous to flanking sequences of 664 bp with terminal inverted repeat sequences of about 70 bp. The 664-bp sequence and most of the 14 homologues identified were flanked by direct repeat sequences of 9 bp. These findings indicate that the repeated sequence, named Tnat1, is a transposable element that duplicates a 9-bp sequence at the target site on transposition and that ATLN44 is inserted in one Tnat1 member. Interestingly, all of the Tnat1 members had tandem repeats comprised of several units of a 60-bp sequence, the number of repeats differing among Tnat1 members. Of the Tnat1 members identified, one was inserted into another sequence repeated in the A. thaliana genome: that sequence is about 770 bp long and has terminal inverted repeat sequences of about 110 bp. The sequence is flanked by direct repeats of a 9-bp sequence, indicating that it is another transposable element, named Tnat2, from A. thaliana. Moreover, Tnat2 members had a tandem repeat about 240 bp long. Tnat1 and Tnat2 with tandem repeats in their internal regions show no homology to each other or to any of the elements identified previously; therefore they appear to be novel transposable elements.     

The complete mitochondrial genome of Sasakia funebris (Leech) (Lepidoptera: Nymphalidae) and comparison with other Apaturinae insects

Sasakia funebris, a member of the lepidopteran family, Nymphalidae (superfamily Papilionoidea) is a rare species and is found only in some areas of South China. In this study, the 15,233 bp long complete mitochondrial genome of S. funebris was determined, and harbors the gene arrangement identical to all other sequenced lepidopteran insects. The nucleotide composition of the genome is highly A + T biased, accounting for 81.2%. All protein-coding genes (PCGs) start with typical ATN codons, except for COI which begins with the CGA codon. All tRNAs have a typical clover-leaf secondary structure, except for tRNA^Ser(AGN), the dihydrouridine (DHU) arm of which forms a simple loop. The S. funebris A + T-rich region of 370 bp contains several features common to the Lepidoptera insects, including the motif ATAGA followed by a 19 bp poly-T stretch, and two tandem repeats consisting of 18 bp repeat units and 14 bp repeat units. The phylogenetic analyses of Apaturinae based on mitogenome sequences showed: (S. funebris + Sasakia charonda) + (Apatura metis + Apatura ilia). This result is consistent with the morphological classification.     

De novo assembly and characterization of the complete chloroplast genome of radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) is an edible root vegetable crop that is cultivated worldwide and whose genome has been sequenced. Here we report the complete nucleotide sequence of the radish cultivar WK10039 chloroplast (cp) genome, along with a de novo assembly strategy using whole genome shotgun sequence reads obtained by next generation sequencing. The radish cp genome is 153,368 bp in length and has a typical quadripartite structure, composed of a pair of inverted repeat regions (26,217 bp each), a large single copy region (83,170 bp), and a small single copy region (17,764 bp). The radish cp genome contains 87 predicted protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Sequence analysis revealed the presence of 91 simple sequence repeats (SSRs) in the radish cp genome.     

Molecular characterization of a family of tandemly repeated DNA sequences,TR-1, in heterochromatic knobs of maize and its relatives

Two families of tandem repeats, 180-bp and TR-1, have been found in the knobs of maize. In this study, we isolated 59 clones belonging to the TR-1 family from maize and teosinte. Southern hybridization and sequence analysis revealed that members of this family are composed of three basic sequences, A (67 bp); B (184 bp) or its variants B' (184 bp), 2/3B (115 bp), 2/3B' (115 bp); and C (108 bp), which are arranged in various combinations to produce repeat units that are multiples of approximately 180 bp. The molecular structure of TR-1 elements suggests that: (1) the B component may evolve from the 180-bp knob repeat as a result of mutations during evolution; (2) B' may originate from B through lateral amplification accompanied by base-pair changes; (3) C plus A may be a single sequence that is added to B and B', probably via nonhomologous recombination; and (4) 69 bp at the 3' end of B or B', and the entire sequence of C can be removed from the elements by an unknown mechanism. Sequence comparisons showed partial homologies between TR-1 elements and two centromeric sequences (B repeats) of the supernumerary B chromosome. This result, together with the finding of other investigators that the B repeat is also fragmentarily homologous to the 180-bp repeat, suggests that the B repeat is derived from knob repeats in A chromosomes, which subsequently become structurally modified. Fluorescence in situ hybridization localized the B repeat to the B centromere and the 180-bp and TR-1 repeats to the proximal heterochromatin knob on the B chromosome.     

Evolution of Tandemly Repeated Sequences: What Happens at the End of an Array?

Tandemly repeated sequences are a major component of the eukaryotic genome. Although the general characteristics of tandem repeats have been well documented, the processes involved in their origin and maintenance remain unknown. In this study, a region on the paternal sex ratio (PSR) chromosome was analyzed to investigate the mechanisms of tandem repeat evolution. The region contains a junction between a tandem array of PSR2 repeats and a copy of the retrotransposon NATE, with other dispersed repeats (putative mobile elements) on the other side of the element. Little similarity was detected between the sequence of PSR2 and the region of NATE flanking the array, indicating that the PSR2 repeat did not originate from the underlying NATE sequence. However, a short region of sequence similarity (11/15 bp) and an inverted region of sequence identity (8 bp) are present on either side of the junction. These short sequences may have facilitated nonhomologous recombination between NATE and PSR2, resulting in the formation of the junction. Adjacent to the junction, the three most terminal repeats in the PSR2 array exhibited a higher sequence divergence relative to internal repeats, which is consistent with a theoretical prediction of the unequal exchange model for tandem repeat evolution. Other NATE insertion sites were characterized which show proximity to both tandem repeats and complex DNAs containing additional dispersed repeats. An ``accretion model' is proposed to account for this association by the accumulation of mobile elements at the ends of tandem arrays and into ``islands' within arrays. Mobile elements inserting into arrays will tend to migrate into islands and to array ends, due to the turnover in the number of intervening repeats. Received: 18 August 1997 / Accepted: 18 September 1998     

The microsatellites and minisatellites in the genome of Fenneropenaeus chinensis.

Through two-time sequencing randomly in Fenneropenaeus chinensis, 2,597,000 bp cumulative length random genomic sequences about occupying 1.23 per thousand of the entire genome are obtained, in which the length of the first time sequencing is 884,000 bp, by cutting the genome DNA with Sau3AI enzyme, and the second is 1,713,000 bp by breaking the genome DNA with the physical method, ultrasonic. Using tandem repeat finder (TRF) soft to analyze the sequences, 4,588 tandem repeats are found, in which the number of microsatellites (1-6 bp) is 3,888, and 700 for minisatellites ( >or= 7 bp). The cumulative length of repeats is 305,555 bp, accounting for 11.72% of total cumulative sequence length, in which the cumulative length of microsatellites is 232,979 bp, accounting for 8.97% of total sequence length, and greater than those of other organisms, such as human and mosquito, etc. The dinucleotide repeat type is dominant in which the dominant repeat class is AT. The second abundant repeat type is trinucleotide, of which the dominant repeat class is AAT. Interestingly, of all of repeat types, the repeat numbers and repeat classes of primer number repeat types, such as pentanucleotide, heptanucleotide, elevennucleotide, etc. are less than those of repeat types beside them. The phenomena may involve the genesis and the evolution of microsatellites and minisatellites.     

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A + T content (84.76%) and strong C skew (− 0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644 bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58 bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.     

Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins

Full-consensus designed ankyrin repeat proteins were designed with one to six identical repeats flanked by capping repeats. These proteins express well in Escherichia coli as soluble monomers. Compared to our previously described designed ankyrin repeat protein library, randomized positions have now been fixed according to sequence statistics and structural considerations. Their stability increases with length and is even higher than that of library members, and those with more than three internal repeats are resistant to denaturation by boiling or guanidine hydrochloride. Full denaturation requires their heating in 5 M guanidine hydrochloride. The folding and unfolding kinetics of the proteins with up to three internal repeats were analyzed, as the other proteins could not be denatured. Folding is monophasic, with a rate that is nearly identical for all proteins (∼ 400-800 s^− 1), indicating that essentially the same transition state must be crossed, possibly the folding of a single repeat. In contrast, the unfolding rate decreases by a factor of about 10⁴ with increasing repeat number, directly reflecting thermodynamic stability in these extraordinarily slow denaturation rates. The number of unfolding phases also increases with repeat number. We analyzed the folding thermodynamics and kinetics both by classical two-state and three-state cooperative models and by an Ising-like model, where repeats are considered as two-state folding units that can be stabilized by interacting with their folded nearest neighbors. This Ising model globally describes both equilibrium and kinetic data very well and allows for a detailed explanation of the ankyrin repeat protein folding mechanism.     

Nucleotide sequence, genomic organization and evolution of a major repetitive DNA family in tilapia (Oreochromis mossambicus/hornorum).

A highly repetitive DNA sequence from tilapia (Oreochromis mossambicus/hornorum) has been cloned and sequenced. It is a tandemly arrayed sequence of 237 bp and constitutes 7% of the fish genome. The copy number of the repeat is approximately 3 x 10(5) per haploid genome. DNA sequence analysis of 7 cloned repeats revealed a high degree of conservation of the monomeric unit. Within the monomeric unit, a 9 bp AT rich motif is regularly spaced approximately 30 bp apart and may represent the progenitor of the amplified sequence. One cloned repeat, Ti-14, contained a 30 bp deletion at a position flanked by a 7 bp direct repeat. The Ti-14 sequence appears to have been amplified independently of the major 237 bp tandem array. A higher-order repeat unit, defined by longer-range periodicities revealed by restriction endonuclease digestion, is further imposed on the tandem array.     

Isolation of CAG/CTG repeats from within the chromosome 2p21-p24 locus for autosomal dominant spastic paraplegia (SPG4) by YAC fragmentation

Pure autosomal dominant spastic paraplegia (SPG) is a genetically heterogeneous neurodegenerative disorder of the central nervous system clinically characterized by progressive spasticity mainly affecting the lower limbs. Three distinct loci have been mapped to chromosomes 14q (SPG3), 2p (SPG4) and 15q (SPG6). In particular, SPG4 families show striking intrafamilial variability suggestive of anticipation and evidence has been provided that CAG/CTG repeat expansions may be involved. To isolate CAG/CTG repeat containing sequences from within the SPG4 candidate region, a novel approach was developed. Fragmentation vectors were assembled allowing direct fragmentation of yeast artificial chromosomes (YACs) with a short (> or = 21 bp) CAG/CTG sequence as the target site for homologous recombination. We used the CAG/CTG YAC fragmentation vectors to isolate CAG/CTG containing sequences from four YACs spanning the SPG4 candidate region between D2S400 and D2S367. A total of four CAG/CTG containing sequences were isolated of which three were novel. However, none of the four CAG/CTG repeats showed expanded alleles in two Belgian SPG4 families. In addition, we showed that the CAG/CTG alleles detected by the repeat expansion detection (RED) method could be fully explained by two polymorphic nonpathogenic CAG/CTG repeats on chromosomes 17 and 18, respectively. Also, the RED expansions in six SPG families could not be explained by amplification of the CAG/CTG repeats at the SPG4 locus. Together, our data do not support the hypothesis of a CAG/CTG repeat expansion as the molecular mechanism underlying SPG4 pathology.     

Conservation and antigenicity of N-terminal sequences of GP185 from different Plasmodium falciparum isolates

Complementary DNA (cDNA) clones for GP185, a major antigenically diverse glycoprotein of Plasmodium falciparum, were isolated from a cDNA library of the Honduras I/CDC (Honduras I) isolate, and 1052 bp were sequenced. The expression of cDNA fragments in Escherichia coli using the vector pCQV2 allowed verification of the reading frame. This GP185 cDNA sequence, like the cDNA sequence for a homologous gene of the K1 isolate [Hall et al., Nature 311 (1984) 379-382], codes for a polypeptide which is truncated due to multiple, in-frame stop codons. This polypeptide corresponds to the N-terminal 15% of the proposed coding region of the GP185 gene [Holder et al., Nature 317 (1985) 270-273]. Comparison of the nucleotide sequences for the GP185 gene of Honduras I and five other isolates indicated that there are two areas of conserved DNA sequence, one of 310 bp (beginning 181 bp upstream from the proposed initiation codon) and the other of greater than or equal to 360 bp (located entirely within the coding region), separated by a region encoding isolate-specific tandem amino acid repeats. Rat antiserum was raised to a fusion protein derived from the conserved regions and the intervening repeat region of this Honduras I protein. This antiserum bound GP185 on immunoblots of the homologous Honduras I isolate and the heterologous K1 isolate, which has different tandem repeats. Serum from owl monkeys and humans previously infected with P. falciparum reacted with the fusion protein on immunoblots demonstrating that determinants in the N-terminal 15% of GP185 were immunogenic in infected individuals and suggesting that some of these sites are conserved among isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

The segment inversion site of herpes simplex virus type 1 adopts a novel DNA structure

The 12-base pair (bp) tandem direct repeat sequences (DR2) at the joint region (a sequence) of herpes simplex virus type 1 (strain F) adopt a new type of DNA conformation under the influence of negative supercoiling. The novel conformation is dependent on the number of the DR2 repeats; the 19 mer (228 bp total) and the 14 mer (168 bp) readily form the alternate structure whereas pentamer, trimer, and dimer repeats show somewhat different properties. S1 and P1 nuclease studies reveal that the new conformation has a major structural aberration at its center and conformational periodicities which are not identical on the complementary strands. Also, the effect of salt and pH, the location of reaction with bromo- and chloroacetaldehyde, the type of sequence (direct repeat) involved, and the nature and extent of supercoil-induced relaxations demonstrate that this structure differs from previously recognized conformations including left-handed Z helices, cruciforms, bent DNA, and slipped structures. We propose the existence of a novel conformation, anisomorphic DNA, with different structures on the complementary strands which elicit a structural aberration at the physical center of the tandem sequences. Since the oligopurine X oligopyrimidine sequence may be inherently inflexible, this supercoil-induced structural change and the physical stress on these inserts in recombinant plasmids tend to deform (crack) the DR2 sequences at their centers. Possible roles for anisomorphic DNA in the functions of this segment of intense biological activity are proposed.     

A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis

The mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU-VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T_m value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p = 0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T_m value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently.