The new RENT family of repetitive elements in Nicotiana species harbors gene regulatory elements related to the tCUP cryptic promoter.

The tCUP cryptic constitutive promoter was discovered in the tobacco genome by T-DNA (transfer DNA) tagging with a promoterless GUS-nos gene. Here, we show that the portion of the tCUP sequence containing a variety of cryptic gene regulatory elements is related to a new family of moderately repetitive sequences (10(2) copies), the RENT (repetitive element from Nicotiana tabacum) family. The RENT family is found only in certain Nicotiana species. Five RENT elements were cloned and sequenced. The RENT elements are a minimum of 5 kb in length and share 80-90% sequence similarity throughout their length. The 5' termini are the same in the isolated RENT family members and are characterized by a conserved border sequence (TGTTGA(T or C)ACCCAATTTT(T or C)). The 3' ends of RENT sequence similarity vary in location and sequence. The tCUP cryptic promoter originated from a unique truncated RENT element that interrupts a phytochelatin synthase-like gene that may have undergone rearrangements prior to or resulting from T-DNA insertion. No evidence was found for expressed coding regions within the RENT elements; however, like the cryptic gene regulatory elements within the tCUP sequence, the isolated RENT elements possess promoter activity and translational enhancer activity.     

Fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting reveal host-specific genetic diversity of Vibrio halioticoli-like strains isolated from the gut of Japanese abalone

When analyzed by fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting, a total of 47 Vibrio halioticoli strains isolated from four Japanese abalone species and one turban shell species formed three clusters that roughly reflect the different species of host abalone from which they were isolated. The V. halioticoli isolates from turban shells were distributed evenly among the clusters. Representative isolates from two clusters were deemed separate species or subspecies by DNA-DNA hybridization.     

Fluorescent Amplified Fragment Length Polymorphism and Repetitive Extragenic Palindrome-PCR Fingerprinting Reveal Host-Specific Genetic Diversity of Vibrio halioticoli-Like Strains Isolated from the Gut of Japanese Abalone

When analyzed by fluorescent amplified fragment length polymorphism and repetitive extragenic palindrome-PCR fingerprinting, a total of 47 Vibrio halioticoli strains isolated from four Japanese abalone species and one turban shell species formed three clusters that roughly reflect the different species of host abalone from which they were isolated. The V. halioticoli isolates from turban shells were distributed evenly among the clusters. Representative isolates from two clusters were deemed separate species or subspecies by DNA-DNA hybridization.     

Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence

A repeating element of DNA has been isolated and sequenced from the genome of Bordetella pertussis. Restriction map analysis of this element shows single internal ClaI, SphI, BstEII and SalI sites. Over 40 DNA fragments are seen in ClaI digests of B. pertussis genomic DNA to which the repetitive DNA sequence hybridizes. Sequence analysis of the repeat reveals that it has properties consistent with bacterial insertion sequence (IS) elements. These properties include its length of 1053 bp, multiple copy number and presence of 28 bp of near-perfect inverted repeats at its termini. Unlike most IS elements, the presence of this element in the B. pertussis genome is not associated with a short duplication in the target DNA sequence. This repeating element is not found in the genomes of B. parapertussis or B. bronchiseptica. Analysis of a DNA fragment adjacent to one copy of the repetitive DNA sequence has identified a different repeating element which is found in nine copies in B. parapertussis and four copies in B. pertussis, suggesting that there may be other repeating DNA elements in the different Bordetella species. Computer analysis of the B. pertussis repetitive DNA element has revealed no significant nucleotide homology between it and any other bacterial transposable elements, suggesting that this repetitive sequence is specific for B. pertussis.     

Mer22-related sequence elements form pericentric repetitive DNA families in primates

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.     

Repetitive DNA and Feulgen hydrolysis kinetics in three species of Bufo.

Two North American species of the genus Bufo (Bufo cognatus and Bufo boreas, 2n = 22) and one African species (Bufo regularis, 2n = 20) were analyzed with respect to their repetitive DNA fractions and the behaviour of their chromatin to the acid hydrolysis at different times. The mean melting point of the total isolated DNA decreased from 89 degrees C to 87 degrees C with a genome size increase from 4.4 to 7.5 pg. The differences in genome size can only partly be explained on the basis of repetitive DNA fractions (renaturing up to Cot 10 in 0.12 M phosphate buffer). Several fractions in this repetitive range behave independently in the three species and the spectrum of repetitive fractions in the African Bufo regularis differs distinctly from those of the American toads. When fixed chromatin of these species in histochemical preparations is hydrolyzed with 5N HCl during the Feulgen reaction, the kinetics of depurination are equal in all species, while hydrolytic DNA breakdown proceeds distinctly more slowly in Bufo reularis as compared to the other species.     

Sequence organization of porcine DNA.

The sequence organization of porcine DNA isolated from thyroid has been analyzed by hydroxylapatite (HAP) chromatography. The reassociation of 0.4 kilobase (Kb) DNA fragments shows, besides the presence of 5% inverted repeat sequences (foldback DNA), that 45% of the genome is represented by high (10%) and intermediate (35%) repetitive components, whereas the remaining 50% is unique sequences. 30% of the unique sequences consists of 1,000 nucleotide fragments interspersed with repetitive elements 400 nucleotides in length. The remaining 20% is longer unique sequences (10,000 nucleotides) apparently not linked to repetitive elements.     

Extreme length and length variation in the first ribosomal internal transcribed spacer of ladybird beetles (Coleoptera: Coccinellidae)

DNA sequences of the first ribosomal internal transcribed spacer (ITS1) were isolated from 10 ladybird beetle species (Coleoptera: Coccinellidae) representing four subfamilies (Coccinellinae, Chilocorinae, Scymninae, and Coccidulinae). The spacers ranged in length from 791 to 2,572 bp, thereby including one of the longest ITS1s and exhibiting one of the most extreme cases of ITS1 size variation in eukaryotes recorded to date. The causes of length variation were therefore analyzed. Almost no putatively homologous sequence similarities were identified for the taxa included. The only exception was for the subfamily Coccinellinae, which yielded sequence similarities in six regions of approximately 550 nucleotide positions, primarily at the 5' and 3' ends of ITS1. The majority of differences in ITS1 length between taxa could be attributed to the presence of repetitive elements with comparatively long repeat units. Repetition arose several times independently and was confined to the middle of the spacer which, in contrast to the 5' and 3' ends, had not been inferred in previous studies to be subject to functional constraints. These elements were characterized by high rates of evolutionary change, most likely as a result of high substitution rates in combination with inefficient homogenization across repeats. The repeated origin and subsequent divergence of "long" repetitive elements should thus be assumed to be an important factor in the evolution of coccinellid ITS1.     

A short interspersed repetitive element found near some mouse structural genes.

We have isolated and characterized a family of interspersed repetitive elements which make up about 1% of the mouse genome. The elements represent a group of homologous but non-identical units about 400 bp in length. Individual members of the family show considerable divergence from one another. The spacial relationships between members of the family and a number of other identified mouse sequences including structural genes have been determined; these elements are found on the 5' as well as 3' sides of various genes at distances ranging from less than 1 to 7.5 kilobases (Kb). The sequences are present in the DNA of all species of Mus. Related sequences are present in the rat genome at a repetition frequency similar to that in the mouse genome. A partial sequence of one member of the family is presented.     

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG) _n , (AG/TC) _n or (AAG/TTC) _n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars. Received: 1 November 1999 / Accepted: 14 April 2000     

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis,Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41-42 bp CNM repeat and the XHOI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.     

眼斑拟石首鱼重复DNA序列的染色体定位

针对眼斑拟石首鱼Sciaenops ocellatus染色体标记匮乏的问题, 利用荧光原位杂交(FISH)定位了眼斑拟石首鱼的18S rDNA、5S rDNA和端粒序列。结果显示, 眼斑拟石首鱼的核型公式为2n=48t; 仅有1对18S rDNA位点, 位于第1对染色体的次缢痕部位; 有2对5S rDNA位点, FISH信号强度不等, 强信号位于第8对染色体的近着丝粒端, 弱信号位于第3对染色体的臂间。端粒FISH信号出现于所有染色体的两端, 但表现出染色体两端信号不平衡的特点, 着丝粒端FISH信号明显强于远端信号。这一特点为判定染色体的方向提供了便利。结合其他石首鱼的核型数据可以推断, 2n=48t的核型及单对近着丝粒分布的18S rDNA位点是石首鱼的共同祖征; 在石首鱼进化过程中, 曾发生活跃但不影响宏观核型的小规模重排。研究结果丰富了眼斑拟石首鱼染色体的辨识标记, 并为研究石首鱼染色体进化提供了基础数据。     

Cytogenetic and random ampliied polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of S?o Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus.     

Chromosomal studies on five species of the genus Leptodactylus Fitzinger, 1826 (Amphibia, Anura) using differential staining

Cytogenetic studies were carried out on five species of Leptodactylus, namely L. fuscus, L. notoaktites, L. labyrinthicus, L. ocellatus, and L. podicipinus, after standard staining, Ag-NOR and C-banding as well as BrdU incorporation for three of them. The species had 2n = 22 chromosomes and two basic karyotype patterns. Chromosome 8 was a marker bearing a secondary constriction. In all species, this secondary constriction corresponded to the Ag-NOR site. The species had centromeric C-bands in all chromosomes of the complement, but some interstitial or telomeric bands seemed to differentiate some karyotypes, either at the species or the population level. In L. ocellatus, the C-banding pattern confirmed the occurrence of a heteromorphic pericentric inversion in chromosome 8 in specimens from one of the populations. The BrdU incorporation technique showed no detectable difference in the replication patterns of the major bands in the chromosomes of L. notoaktites, L. labyrinthicus, and L. ocellatus.     

DNA sequence organization in the genome of Nicotiana tabacum

The genome of Nicotiana tabacum was investigated by DNA/DNA reassociation for its spectrum of DNA repetition components and pattern of DNA sequence organization. The reassociation of 300 nucleotide DNA fragments analyzed by hydroxyapatite chromatography reveals the presence of three major classes of DNA differing in reiteration frequency. Each class of DNA was isolated and characterized with respect to kinetic homogeneity and thermal properties on melting. These measurements demonstrate that the genome of N. tabacum has a 1C DNA content of 1.65 pg and that DNA sequences are represented an average of 12,400, 252, and 1 times each. — The organization of the DNA sequences in the N. tabacum genome was determined from the reassociation kinetics of long DNA fragments as well as S1 nuclease resistance and hyperchromicity measurements on DNA fragments after annealing to C₀t values at which only repetitive DNA sequences will reassociate. At least 55% of the total DNA sequences are organized in a short period interspersion pattern consisting of an alternation of single copy sequences, averaging 1400 nucleotides, with short repetitive elements approximately 300 nucleotides in length. Another 25% of the genome contains long repetitive DNA sequences having a minimal genomic length of 1500 nucleotides. These repetitive DNA sequences are much less divergent than the short interspersed DNA sequence elements. These results indicate that the pattern of DNA sequence organization in the tobacco genome bears remarkable similarity to that found in the genomes of most animal species investigated to date.     

Age-independent telomere length predicts fitness in two bird species

Telomeres are dynamic DNA-protein structures that form protective caps at the ends of eukaryotic chromosomes. Although initial telomere length is partly genetically determined, subsequent accelerated telomere shortening has been linked to elevated levels of oxidative stress. Recent studies show that short telomere length alone is insufficient to induce cellular senescence; advanced attrition of these repetitive DNA sequences does, however, reflect ageing processes. Furthermore, telomeres vary widely in length between individuals of the same age, suggesting that individuals differ in their exposure or response to telomere-shortening stress factors. Here, we show that residual telomere length predicts fitness components in two phylogenetically distant bird species: longevity in sand martins, Riparia riparia, and lifetime reproductive success in dunlins, Calidris alpina. Our results therefore imply that individuals with longer than expected telomeres for their age are of higher quality.     

Algivore or Phototroph? Plakobranchus ocellatus (Gastropoda) Continuously Acquires Kleptoplasts and Nutrition from Multiple Algal Species in Nature

The sea slug Plakobranchus ocellatus (Sacoglossa, Gastropoda) retains photosynthetically active chloroplasts from ingested algae (functional kleptoplasts) in the epithelial cells of its digestive gland for up to 10 months. While its feeding behavior has not been observed in natural habitats, two hypotheses have been proposed: 1) adult P. ocellatus uses kleptoplasts to obtain photosynthates and nutritionally behaves as a photoautotroph without replenishing the kleptoplasts; or 2) it behaves as a mixotroph (photoautotroph and herbivorous consumer) and replenishes kleptoplasts continually or periodically. To address the question of which hypothesis is more likely, we examined the source algae for kleptoplasts and temporal changes in kleptoplast composition and nutritional contribution. By characterizing the temporal diversity of P. ocellatus kleptoplasts using rbcL sequences, we found that P. ocellatus harvests kleptoplasts from at least 8 different siphonous green algal species, that kleptoplasts from more than one species are present in each individual sea slug, and that the kleptoplast composition differs temporally. These results suggest that wild P. ocellatus often feed on multiple species of siphonous algae from which they continually obtain fresh chloroplasts. By estimating the trophic position of wild and starved P. ocellatus using the stable nitrogen isotopic composition of amino acids, we showed that despite the abundance of kleptoplasts, their photosynthates do not contribute greatly to the nutrition of wild P. ocellatus, but that kleptoplast photosynthates form a significant source of nutrition for starved sea slugs. The herbivorous nature of wild P. ocellatus is consistent with insights from molecular analyses indicating that kleptoplasts are frequently replenished from ingested algae, leading to the conclusion that natural populations of P. ocellatus do not rely on photosynthesis but mainly on the digestion of ingested algae.     

Evaluation of Morphological Variation in the Lichen Diploschistes ocellatus (Ascomycota, Ostropales): Evidence from Nuclear rDNA ITS Sequence Data

Abstract: A morphologically distinct population (var. almeriensis ) of the lichen species Diploschistes ocellatus from southern Spain is examined using morphological and molecular methods. While the morphological examination revealed some unique features of var. almeriensis, the ITS sequence data did not support a distinction from D. ocellatus s. str. The Kishino-Hasegawa test did not rule out trees distinguishing between the two morphotypes, although spectral analytical data support an inclusion of var. almeriensis in D. ocellatus. The influence of potentially erronously aligned ambiguous sites is evaluated by analysis of different alignments. However, all alignments agree in placing all specimens of D. ocellatus together and, consequently, in the consensus tree D. ocellatus s. lat. forms a monophyletic clade with high bootstrap support.     

Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.