Comparison of 18FDG-PET with 131iodine and 99mTc-sestamibi scintigraphy in differentiated thyroid cancer

Based on sequence analyses of 17 complete centromeric DNA monomers from ten different deer species, a model is proposed for the genesis, evolution, and genomic organization of cervid satellite I DNA. All cervid satellite I DNA arose from the initial amplification of a 31-bp DNA sequence. These 31-bp subrepeats were organized in a hierarchical fashion as 0.8-kb monomers in plesiometacarpalia deer and 1-kb monomers in telemetacarpalia deer. The higher-order repeat nature of cervid centromeric satellite DNA monomers accounts for their high intragenomic and intraspecific sequence conservation. Such high intraspecific sequence conservation validates the use of a single cervid satellite I DNA monomer from each deer species for interspecific sequence comparisons to elucidate phylogenetic relationships. Also, a specific 0.18-kb tandem duplication was observed in all 1-kb monomers, implying that 1-kb cervid satellite I DNA monomers arose from an unequal crossover event between two similar 0.8-kb ancestral DNA sequences.     

A family of highly repetitive DNAs from "ginbuna" (Carassius auratus langsdorfi) genome common to Carassius auratus populations

A family of highly repetitive DNAs (Hi-a) in the genome from a population of the crucian carp, tentatively identified as the ginbuna (Carassius auratus langsdorfi), was isolated from the HindIII digests and characterized. The Hi-a monomer (268 or 269 bp) was AT-rich (64.9%) with internal repetitive oligomers. The nucleotide similarity among monomers within the same individual was 88-98%, whose sequence alterations occurred mostly at restricted sites. Hybridization analyses revealed that the Hi-a family was organized into tandem array(s) representing satellite DNAs, and that its variants that share certain restriction sites of the repeat unit were dispersed into the tandem array(s) of Hi-a DNAs at varying periodicities. The lack of sequences homologous to the ginbuna Hi-a in the genomes from cyprinid fishes other than gengorobuna and goldfish suggests that Hi-a DNAs are specifically present in the species C. auratus. The genome of goldfish differed somewhat in the quantity or the genomic organization of Hi-a DNAs from that of the ginbuna.     

Nucleotide sequence and genomic organization of repeating units of the AT-rich family or repeats in the diploid wheat Triticum monococcum L

In diploid wheat Triticum monococcum L., a novel family of AT-rich repeated DNA sequences arranged in clusters of tandem arrays of 560-bp fragments is described. The nucleotide sequence of a 556-bp repeat unit was determined. The basic structural unit of this repeated DNA family contains subrepeats with the main motif AAACATTGTTAC. This type of repetitive DNA sequence could evolve via tandem amplification of short subrepeats with some subsequent divergence, and/or via recombination with unique sequences. Structural organization of homologous sequences, revealed by blot hybridization in other representatives of the Triticeae tribe, suggest that this family of repeated DNA is genome-specific.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

The rRNA-encoding DNA array has an altered structure in topoisomerase I mutants of Saccharomyces cerevisiae

All the chromosomes from isogenic TOP1 and top1 strains have similar mobility on pulsed-field gels except for chromosome XII, which fails to migrate into the gels in top1 mutants. Chromosome XII contains the tandem repeats of rRNA-encoding DNA (rDNA). When a segment of chromosome XII containing only rDNA is transferred to chromosome III by a recombination event, chromosome III fails to enter a pulsed-field gel in extracts from top1 strains, indicating that the aberrant migration of chromosome XII in top1 mutants is caused by the presence of rDNA. Failure of chromosome XII to migrate into a pulsed-field gel occurs only in preparations from exponentially growing top1 cultures and not in preparations from stationary-phase top1 cultures. rDNA from a top1 strain does enter the gel if it is cut with an enzyme (Pst I) that cuts the tandem rDNA array into single 9-kb repeat units, indicating that more than a single repeat unit is required to maintain the aberrant structure.     

An 85-kb tandem triplication in the slow Wallerian degeneration (Wlds) mouse

Wallerian degeneration is the degeneration of the distal stump of an injured axon. It normally occurs over a time course of around 24 hr but it is delayed in the slow Wallerian degeneration mutant mouse (C57BL/Wlds) for up to 3 weeks. The gene, which protects from rapid Wallerian degeneration, Wld, previously has been mapped to distal chromosome 4. This paper reports the fine genetic mapping of the Wld locus, the generation of a 1.4-Mb bacterial artificial chromosome and P1 artificial chromosome contig, and the identification of an 85-kb tandem triplication mapping within the candidate region. The mutation is unique to C57BL/Wlds among 36 strains tested and therefore is a strong candidate for the mutation that leads to delayed Wallerian degeneration. There are very few reports of tandem triplications in a vertebrate and no evidence for a mutation mechanism so this unusual mutation was characterized in more detail. Sequence analysis of the boundaries of the repeat unit revealed a minisatellite array at the distal boundary and a matching 8-bp sequence at the proximal boundary. This finding suggests that recombination between short homologous sequences ("illegitimate" or "nonhomologous" recombination) was involved in the rearrangement. In addition, a duplication allele was identified in two Wlds mice, indicating some instability in the repeat copy number and suggesting that the triplication arose from a duplication by unequal crossing over.     

Isolation and characterization of a satellite DNA family in the Saccharum complex

EaCIR1, a 371-bp Erianthus-specific satellite DNA sequence, was cloned from TaqI restricted genomic DNA after agarose-gel electrophoresis. This sequence has 77% homology with a 365-bp satellite of Helictotrichon convolutum and 72% homology with a 353-bp tandem repeat sequence from Oryza sativa. PCR primers defined in the conserved regions of these repetitive sequences were used to isolate other satellite DNAs in different representatives of the Saccharum complex: SoCIR1 in Saccharum officinarum, SrCIR1 in Saccharum robustum, SsCIR1 and SsCIR2 in Saccharum spontaneum, and MsCIR1 in Miscanthus sinensis. EaCIR1 and SoCIR1 were localized to subtelomeric regions of the chromosomes by fluorescence in situ hybridization. Southern hybridization experiments, using two representatives of this repeat sequence family as probes, illustrated contrasting species-specificity and demonstrated the existence of similar repetitive elements in sorghum and maize.     

Characteristics of two salmonid repetitive DNA families in rainbow trout, Oncorhynchus mykiss

DNA sequence and genomic location of two repetitive DNA families in rainbow trout (Oncorhynchus mykiss) were investigated to develop molecular markers for chromosome identification. DNA fragments with sequences similar to the tandem and interspersed elements described in other salmonids were isolated. One clone showed differential hybridization to 12 pairs of chromosomes and should be a useful marker for physical mapping.     

Mobile genetic elements, chiasmata, and the unique organization of beta-heterochromatin

Beta-heterochromatin in Drosophila and the Syrian hamster share a similar DNA organization, few unique sequences, and scrambled repeats of mobile elements without tandem repetition. DNA in alpha-heterochromatin is tandemly repetitious, and we now show that the repeat unit can either contain or lack a mobile element. The tandem repeat organization of alpha-heterochromatin is presumably due to a concertina-like mechanism of unequal exchange between repeat units. Although both heterochromatin types are late replicating and can incorporate mobile retroposons, the sequence distinction between the two heterochromatins appears to be due to a property conferred by chiasmata upon the process of homologous recombination in beta-heterochromatin but not in alpha-heterochromatin. Chiasmata seem to suppress the concertina mechanism of unequal exchange and impart to beta-heterochromatin its nontandem, scrambled repeat organization.     

Chromosome-specific molecular organization of maize (Zea mays L.) centromeric regions

A set of oat-maize chromosome addition lines with individual maize (Zea mays L.) chromosomes present in plants with a complete oat (Avena sativa L.) chromosome complement provides a unique opportunity to analyze the organization of centromeric regions of each maize chromosome. A DNA sequence, MCS1a, described previously as a maize centromere-associated sequence, was used as a probe to isolate cosmid clones from a genomic library made of DNA purified from a maize chromosome 9 addition line. Analysis of six cosmid clones containing centromeric DNA segments revealed a complex organization. The MCS1a sequence was found to comprise a portion of the long terminal repeats of a retrotransposon-like repeated element, termed CentA. Two of the six cosmid clones contained regions composed of a newly identified family of tandem repeats, termed CentC. Copies of CentA and tandem arrays of CentC are interspersed with other repetitive elements, including the previously identified maize retroelements Huck and Prem2. Fluorescence in situ hybridization revealed that CentC and CentA elements are limited to the centromeric region of each maize chromosome. The retroelements Huck and Prem2 are dispersed along all maize chromosomes, although Huck elements are present in an increased concentration around centromeric regions. Significant variation in the size of the blocks of CentC and in the copy number of CentA elements, as well as restriction fragment length variations were detected within the centromeric region of each maize chromosome studied. The different proportions and arrangements of these elements and likely others provide each centromeric region with a unique overall structure.     

A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

The penicillin gene cluster is amplified in tandem repeats linked by conserved hexanucleotide sequences

The penicillin biosynthetic genes (pcbAB, pcbC, penDE) of Penicillium chrysogenum AS-P-78 were located in a 106.5-kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild-type strains P. chrysogenum NRRL 1951 and Penicillium notatum ATCC 9478 (Fleming's isolate) contain a single copy of the 106.5-kb region. This region was bordered by the same TTTACA hexanucleotide found between tandem repeats in strain AS-P-78. A penicillin overproducer strain, P. chrysogenum E1, contains a large number of copies in tandem of a 57.9-kb DNA fragment, linked by the same hexanucleotide or its reverse complementary TGTAAA sequence. The deletion mutant P. chrysogenum npe10 showed a deletion of 57.9 kb that corresponds exactly to the DNA fragment that is amplified in E1. The conserved hexanucleotide sequence was reconstituted at the deletion site. The amplification has occurred within a single chromosome (chromosome I). The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.     

Molecular origin of the mosaic sequence arrangements of higher primate alpha-globin duplication units

The human adult alpha-globin locus consists of three pairs of homology blocks (X, Y, and Z) interspersed with three nonhomology blocks (I, II, and III), and three Alu family repeats, Alu1, Alu2, and Alu3. It has been suggested that an ancient primate alpha-globin-containing unit was ancestral to the X, Y, and Z and the Alu1/Alu2 repeats. However, the evolutionary origin of the three nonhomologous blocks has remained obscure. We have now analyzed the sequence organization of the entire adult alpha-globin locus of gibbon (Hylobates lar). DNA segments homologous to human block I occur in both duplication units of the gibbon alpha-globin locus. Detailed interspecies sequence comparisons suggest that nonhomologous blocks I and II, as well as another sequence, IV, were all part of the ancestral alpha-globin-containing unit prior to its tandem duplication. However, sometime thereafter, block I was deleted from the human alpha1-globin-containing unit, and block II was also deleted from the alpha2-globin-containing unit in both human and gibbon. These were probably independent events both mediated by independent illegitimate recombination processes. Interestingly, the end points of these deletions coincide with potential insertion sites of Alu family repeats. These results suggest that the shaping of DNA segments in eukaryotic genomes involved the retroposition of repetitive DNA elements in conjunction with simple DNA recombination processes.     

Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements

Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase- and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE- and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.     

Heteroplasmy, length and sequence variation in the mtDNA control regions of three percid fish species (Perca fluviatilis, Acerina cernua, Stizostedion lucioperca)

The nucleotide sequence of the control region and flanking tRNA genes of perch (Perca fluviatilis) mtDNA was determined. The organization of this region is similar to that of other vertebrates. A tandem array of 10-bp repeats, associated with length variation and heteroplasmy was observed in the 5' end. While the location of the array corresponds to that reported in other species, the length of the repeated unit is shorter than previously observed for tandem repeats in this region. The repeated sequence was highly similar to the Mt5 element which has been shown to specifically bind a putative D-loop DNA termination protein. Of 149 perch analyzed, 74% showed length variation heteroplasmy. Single-cell PCR on oocytes suggested that the high level of heteroplasmy is passively maintained by maternal transmission. The array was also observed in the two other percid species, ruffe (Acerina cernua) and zander (Stizostedion lucioperca). The array and the associated length variation heteroplasmy are therefore likely to be general features of percid mtDNAs. Among the perch repeats, the mutation pattern is consistent with unidirectional slippage, and statistical analyses supported the notion that the various haplotypes are associated with different levels of heteroplasmy. The variation in array length among and within species is ascribed to differences in predicted stability of secondary structures made between repeat units.     

Isolation and chromosomal localization of a germ line-specific highly repetitive DNA family in Acricotopus lucidus (Diptera, Chironomidae)

In the chironomid Acricotopus lucidus, parts of the genome, the germ line-limited chromosomes, are eliminated from the future soma cells during early cleavage divisions. A highly repetitive, germ line-specific DNA sequence family was isolated, cloned and sequenced. The monomers of the tandemly repeated sequences range in size from 175 to 184 bp. Analysis of sequence variation allowed the further classification of the germ line-restricted repetitive DNA into two related subfamilies, A and B. Fluorescence in situ hybridization to gonial metaphases demonstrated that the sequence family is highly specific for the paracentromeric heterochromatin of the germ line-limited chromosomes. Restriction analysis of genomic soma DNA of A. lucidus revealed another tandem repetitive DNA sequence family with monomers of about 175 bp in length. These DNA elements are found only in the centromeric regions of all soma chromosomes and one exceptional germ line-limited chromosome by in situ hybridization to polytene soma chromosomes and gonial metaphase chromosomes. The sequences described here may be involved in recognition, distinction and behavior of soma and germ line-limited chromosomes during the complex chromosome cycle in A. lucidus and may be useful for the genetic and cytological analysis of the processes of elimination of the germ line-limited chromosomes in the soma and germ line.     

Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication

We previously reported on the generation of approximately 50-kb size defective genomes (DGs) which appeared to retain less than 2.2% of the standard Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA between 85.0 and 87.2 MU while the rest of the virus DNA had been largely deleted (Lee and Krell, J. Virol., 66:4339-4347, 1992). To investigate these presumably repeated sequences further, we cloned and analyzed the most abundant hypermolar 1.80-kb Xhol DNA fragment as well as a minor but also supermolar 1.74-kb Xhol fragment of the DGs. These two DNA segments collectively covered 2371-bp of the standard AcMNPV DNA with a 1174-bp overlap around the Xhol site at 85.9 MU. Analysis of DGs by two-dimensional gel electrophoresis indicated that the 1.80- and 1.74-kb Xhol fragments (and most other novel Xhol fragments of the DG) were organized as tandem repeats in the DGs. We identified, in the DG population, small supercoiled DNA molecules approximately 1.0- to 8.2-kb (and possibly up to around 50 kb, the size of the major defective DNA species) in size and which contained the same DNA sequence as that of the major 1.80-kb repeat in the DGs. Furthermore, these cloned repeat sequences, represented by pLK1.80 and pLK1.74 showed AcMNPV infection-dependent autonomous replication, suggesting that an origin of DNA replication might reside within the HindIII to EcoRI segment (85.1 to 86.6 MU) of the HindIII-K fragment.     

Sex identification of turkey embryos using a multiplex polymerase chain reaction

A considerable portion of the W chromosome in Gallinaceous birds consists of tandem repetitive DNA. In the turkey, a 0.4-kb PstI element is repeated about 10,000 times in the female diploid genome but is undetectable as such a unit in males. In this study a multiplex polymerase chain reaction was developed to identify the sex of turkeys based upon the PstI repeat. The technique utilized two pairs of primers, the first pair was designed to amplify a region of the PstI repetitive element, resulting in the production of a 177-bp fragment in females. The other pair was designed to amplify a region of the adenosine triphosphate (ATP) synthase gene, present in both males and females. The simultaneous use of all four primers in the same reaction resulted in the coamplification of a 177-bp and a 250-bp fragment in females and a 250-bp fragment in males. This technique was used to verify the sex of 45 adults of known sex and to identify the sex of 74 embryos from Day 5 to hatch. This procedure is rapid and permits the sexing of many embryos in a short time. The ability to sex early embryos can facilitate studies on avian sex determination.     

Unequal cross-over is involved in human alpha satellite DNA rearrangements on a border of the satellite domain

It can be invoked from the theory of tandem repeat homogenization that DNA on a satellite/non-satellite border may carry sequence marks of molecular processes basic to satellite evolution. We have sequenced a continuous 17-kb alpha satellite fragment bordering the non-satellite in human chromosome 21, which is devoid of higher-order repeated structure, contains multiple rearrangements, and exhibits higher divergence of monomers towards the border, indicating the lack of efficient homogenization. Remarkably, monomers have been found with mutually supplementary deletions matching each other as reciprocal products of unequal recombination, which provide evidence for unequal cross-over as a mechanism generating deletions in satellite DNA.     

Transferable Streptomyces DNA amplification and coamplification of foreign DNA sequences

The 8.8-kb amplifiable unit of DNA of Streptomyces achromogenes subsp. rubradiris, AUD-Sar 1, which carries 0.8-kb terminal direct repeats and a spectinomycin resistance determinant, can mediate high-level amplification of an AUD-Sar 1-derived 8.0-kb DNA sequence not only in S. achromogenes but also in the heterologous host Streptomyces lividans. This was seen upon introduction of AUD-Sar 1 into chloramphenicol-sensitive strains of S. lividans via the temperature-sensitive (39 degrees C) plasmid pMT660, which contains the thiostrepton resistance gene tsr. Following the cultivation of transformants at 39 degrees C on media containing spectinomycin, a number of strains which were unable to grow on thiostrepton and which carried the amplified 8.0-kb DNA sequence as arrays of 200 to 300 copies of tandem 8.0-kb repeats were found. Chloramphenicol-resistant strains of S. lividans did not yield amplified sequences under similar conditions. Studies with plasmids carrying inserted antibiotic resistance genes at two sites of AUD-Sar 1 yielded coamplified sequences which contain the inserted DNA. Transformation with a plasmid carrying a 1.0-kb deletion in AUD-Sar 1 followed by growth under similar conditions yielded a 7.0-kb repeated DNA sequence. Southern analysis revealed the absence of vector sequences located on the right side of AUD-Sar 1 in the input plasmids in all examined DNA samples of amplified strains. In contrast, a majority of the samples revealed the presence at unit copy level of AUD-Sar 1 left-adjacent sequences which are part of the input plasmids and in several samples the presence of certain vector sequences located near them. The results suggest input plasmid integration into the S. lividans chromosome prior to the generation of the amplified sequences and the deletion of AUD-Sar 1 adjacent sequences.