DNA distribution and intrachromosomal organization of moderately repetitive sequences

The DNA content of individual subregions along the 4th chromosome of Drosophila hydei has been measured. 51% of the subregions have a DNA content averaging 0.7-0.8 pg; 31% a mean DNA content of 0.25-0.35 pg and 18% a mean DNA content of 1.7 pg. Moreover the structural chromosomal distribution of moderately repetitive DNA is not random since the specific activity of the chromosomal segments in terms of those sequences is not the same. 9% of the subregions are very poor in repetitive sequences and 18% rich in repetitive DNA while being very poor in DNA.     

Molecular dating of the diversification of Phyllostominae bats based on nuclear and mitochondrial DNA sequences

Times of divergence among the three tribes included within the subfamily Phyllostominae were estimated using a Bayesian approach to infer dates of divergence based on mitochondrial and nuclear sequence data. The subfamily Phyllostominae is particularly attractive for such analysis, as it is one of the few groups of bats to have fossil specimens. Our molecular time analyses suggest that diversification among tribes and genera of phyllostomine bats occurred during the Early to Mid-Miocene, and was coincident with diversification events in two co distributed taxa: Caviomorph rodents and New World monkeys.     

DNA polymerase-catalyzed elongation of repetitive hexanucleotide sequences: application to creation of repetitive DNA libraries

We demonstrate the elongation of various hexanucleotide sequences with thermophilic DNA polymerase, under isothermal or thermal cyclic reaction conditions. We prepared 10 types of double repeat hexanucleotide duplexes with various GC compositions containing between 0 and 6 GC nucleotides per repeat and incubated these duplexes with thermophilic Taq DNA polymerase and dNTPs at various temperatures. All of the model repetitive short duplexes were elongated under the isothermal incubation conditions, although there were some differences in the elongation efficiencies derived from the GC composition in the repetitive sequences. It was also found that all of the model repetitive duplexes were extended more effectively by a 3-step thermal cyclic reaction involving denaturation, annealing, and extension. On the basis of this technique, we prepared a glutamate-encoding short repetitive duplex and created long repetitive DNAs under isothermal and thermal cyclic reaction conditions. DNA sequencing analysis of the cloned repetitive DNA revealed that well-ordered long repetitive DNAs of various chain lengths were created by this DNA polymerase-catalyzed ligation method, and these were easily cloned into vectors by the TA-cloning method. This method could be useful for obtaining DNAs encoding arbitrary long repetitive amino acid sequences more effectively than the conventional T4 ligase-catalyzed ligation method.     

Molecular and genomic organization of clusters of repetitive DNA sequences in Caenorhabditis elegans.

Repetitive sequences in Caenorhabditis elegans are interspersed along the holocentric chromosomes. We have physically mapped some of these repetitive families and found that, although the distribution of members of each family is relatively even along the chromosomes, members of more than one family tend to cluster in some locations. We compared the sequence organization of 11 clusters located at known positions on different chromosomes in the N2 strain. These studies allow a comparison between repetitive elements belonging to the same family that are located on the same or on different chromosomes, providing an important tool in the study of genome turnover and evolution.     

Cytological localization and organization of dispersed middle repetitive DNA sequences of Drosophila subobscura

To study the middle repetitive fraction of the Drosophila subobscura genome, 26 phage clones containing repetitive sequences were examined by Southern DNA blot analysis and by in situ hybridization to polytene chromosomes. These results led to a classification of the clones according to five different types of hybridization patterns. Two types, each containing seven clones, are characterized by hybridization at 100 to 300 sites dispersed over the euchromatic parts of the chromosomes, and in addition by one prominently labelled chromosome band. One of these two classes also showed strong labelling of the chromocentre. The remaining types of hybridization pattern lacked a prominent band but showed hybridization either to the euchromatic regions or to the chromocentre or both. Chromosome A (=X) was the preferred location of prominently labelled bands and it also showed an excess of labelling by some clones. Some of the cloned dispersed sequences were localized cytologically on chromosomes of larvae from crosses between different strains of D. subobscura and between two closely related species, in order to detect heterozygosity at hybridization sites. Comparisons of the chromosomal distribution of labelling sites showed differences in number and location, indicating the possibility of transposition events.     

Temporal variation in the organization of a Neotropical assemblage of leaf-nosed bats (Chiroptera: Phyllostomidae)

In the present study, I described the organization of a Neotropical bat assemblage, and tested whether this organization was variable in time. In an Atlantic Forest reserve in southeastern Brazil bats were captured monthly with mist nets over 4 years, and individuals were classified into guilds. I analyzed only leaf-nosed bats, and observed that guilds of fruit-eating bats dominated the assemblage. This pattern was repeated across months and years. However, among frugivores, canopy and understory guilds peaked during different months, but in both cases during the rainy season, while variation among habitat-opportunistic species was not explained by rainfall. The most reliable ecological service delivered by phyllostomid bats in the area is seed dispersal, although other services may be also important in particular seasons. My results suggest that the observed patterns of temporal species turnover are related to the abundance of preferred food items.     

High-resolution organization of mouse telomeric and pericentromeric DNA

We studied the organization of telomeric, major and minor satellite DNA sequences located in the pericentromeric regions of mouse telocentric and Robertsonian metacentric chromosomes by high-resolution fluorescence in situ hybridization. Molecular data have already proved that in telocentrics, from the physical chromosome end, telomeric sequences are followed by minor and then by major satellite DNA. We showed that the three families of repetitive DNA are organized as uninterrupted long-range cluster repeats and that there is no intermingling between telomeric and minor satellite DNA or between the major and the minor tandem repeats or with non-satellite DNA. The pericentromeric region of metacentric chromosomes consists of a small block of minor satellite DNA sandwiched between two blocks of major satellite DNA.     

A repetitive DNA element,associated with telomeric sequences in Drosophila melanogaster,contains open reading frames

He-T sequences are a complex repetitive family of DNA sequences in Drosophila that are associated with telomeric regions, pericentromeric heterochromatin, and the Y chromosome. A component of the He-T family containing open reading frames (ORFs) is described. These ORF-containing elements within the He-T family are designated T-elements, since hybridization in situ with the polytene salivary gland chromosomes results in detectable signal exclusively at the chromosome tips. One T-element that has been sequenced includes ORFs of 1,428 and 1,614 bp. The ORFs are overlapping but one nucleotide out of frame with respect to each other. The longer ORF contains cysteine-histidine motifs strongly resembling nucleic acid binding domains of gag-like proteins, and the overall organization of the T-element ORFs is reminiscent of LINE elements. The T-elements are transcribed and appear to be conserved in Drosophila species related to D. melanogaster. The results suggest that T-elements may play a role in the structure and/or function of telomeres.by W. Hennig     

Structural genes adjacent to interspersed repetitive DNA sequences

The observation that repetitive and single copy sequences are interspersed in animal DNAs has suggested that repetitive sequences are adjacent to single copy structural gene sequences. To test this concept, single copy DNA sequences contiguous to interspersed repetitive sequences were prepared from sea urchin DNA by hydroxyapatite fractionation (repeat-contiguous DNA fraction). These single copy sequences included about one third of the total nonrepetitive sequence in the genome as determined by the amounts recovered during the hydroxyapatite fractionation and by reassociation kinetics. ³H-labeled mRNA from sea urchin gastrula was prepared by puromycin release from polysomes and used in DNA-driven hybridization reactions. The kinetics of mRNA hybridization reactions with excess whole DNA were carefully measured, and the rate of hybridization was found to be 3–5 times slower than the corresponding single copy DNA driver reassociation rate. The mRNA hybridized with excess repeat-contiguous DNA with similar kinetics relative to the driver DNA. At completion 80% of that mRNA hybridizable with whole DNA (approximately 65%) had reacted with the repeat-contiguous DNA fraction (50%). This result shows that 80–100% of the mRNA molecules present in sea urchin embryos are transcribed from single copy DNA sequences adjacent to interspersed repetitive sequences in the genome.     

Comparative phylogeography of short-tailed bats (Carollia: Phyllostomidae)

This is the first study of comparative phylogeography involving closely related species of Neotropical bats of the family Phyllostomidae. We compared patterns of geographical variation within the five species of fruit-eating bats currently recognized in the genus Carollia using the complete mitochondrial cytochrome-b gene. Our results suggest that the combined effect of the uplift of the Andes and the Panamanian land bridge has been as important for bats as for terrestrial mammals in shaping present-day biodiversity in the New World tropics. Species in this genus can be arranged in two highly supported clades, with a deep subdivision within each that corresponds well to differences across the Andes. We found three congruent phylogeographical patterns across species in this genus. First, the closer relationship between samples from western Ecuador and those from Central America, compared with populations east of the Andes in C. brevicauda, C. castanea and C. perspicillata. Second, the likelihood of a similar timing in South America for the arrival and diversification of C. brevicauda and C. perspicillata from their Central America ancestors. Third, the expansion of C. perspicillata and C. sowelli into northwestern Central America in the relatively recent past. Using a molecular clock, with rates ranging from 2.3 to 5% per 10(6) years, diversification within Carollia would have occurred over the last 1-4.5 Myr. These estimates agree well with the last rise of the Northern Andes and the Panama isthmus.     

Chromosomal evolution of neotropical cichlids: the role of repetitive DNA sequences in the organization and structure of karyotype

Cichlids are important in the aquaculture and ornamental fish trade and are considered models for evolutionary biology. However, most studies of cichlids have investigated African species, and the South American cichlids remain poorly characterized. Studies in neotropical regions have focused almost exclusively on classical cytogenetic approaches without investigating physical chromosomal mapping of specific sequences. The aim of the present study is to investigate the genomic organization of species belonging to different tribes of the subfamily Cichlinae (Cichla monoculus, Astronotus ocellatus, Geophagus proximus, Acaronia nassa, Bujurquina peregrinabunda, Hoplarchus psittacus, Hypselecara coryphaenoides, Hypselecara temporalis, Caquetaia spectabilis, Uaru amphiacanthoides, Pterophyllum leopoldi, Pterophyllum scalare, and Symphysodon discus) and reexamine the karyotypic evolutionary patterns proposed for this group. Variations in some cytogenetic markers were observed, although no trends were found in terms of the increase, decrease, or maintenance of the basal diploid chromosome number 2n = 48 in the tribes. Several species were observed to have 18S rDNA genetic duplications, as well as multiple rDNA loci. In most of the taxa analyzed, the 5S rDNA was located in the interstitial region of a pair of homologous chromosomes, although variations from this pattern were observed. Interstitial telomere sites were also observed and appear to be involved in chromosomal rearrangement events and the accumulation of repeat-rich satellite DNA sequences. Our data demonstrated the karyotypic diversity that exists among neotropical cichlids, suggesting that most of this diversity is due to the repetitive sequences present in heterochromatic regions and that repeat sequences have greatly influenced the karyotypic evolution of these fishes.     

Differential maintenance of DNA sequences in telomeric and centromeric heterochromatin

Repeated DNA in heterochromatin presents enormous difficulties for whole-genome sequencing; hence, sequence organization in a significant portion of the genomes of multicellular organisms is relatively unknown. Two sequenced BACs now allow us to compare telomeric retrotransposon arrays from Drosophila melanogaster telomeres with an array of telomeric retrotransposons that transposed into the centromeric region of the Y chromosome >13 MYA, providing a unique opportunity to compare the structural evolution of this retrotransposon in two contexts. We find that these retrotransposon arrays, both heterochromatic, are maintained quite differently, resulting in sequence organizations that apparently reflect different roles in the two chromosomal environments. The telomere array has grown only by transposition of new elements to the chromosome end; the centromeric array instead has grown by repeated amplifications of segments of the original telomere array. Many elements in the telomere have been variably 5'-truncated apparently by gradual erosion and irregular deletions of the chromosome end; however, a significant fraction (4 and possibly 5 or 6 of 15 elements examined) remain complete and capable of further retrotransposition. In contrast, each element in the centromere region has lost ≥ 40% of its sequence by internal, rather than terminal, deletions, and no element retains a significant part of the original coding region. Thus the centromeric array has been restructured to resemble the highly repetitive satellite sequences typical of centromeres in multicellular organisms, whereas, over a similar or longer time period, the telomere array has maintained its ability to provide retrotransposons competent to extend telomere ends.     

Physical mapping of 5S and 18S-25S rDNA and repetitive DNA sequences in Aegilops umbellulata.

An accurate physical map of the location of the 5S and the 18S-5.8S-25S rRNA genes and a repetitive DNA sequence has been produced on Aegilops umbellulata Zhuk., (2n = 2x = 14) chromosomes by in situ hybridization. Chromosome morphology together with the hybridization pattern of pSc119.2, a DNA sequence from rye, allowed identification and discrimination of different chromosomes; pSc119.2 hybridizes with all Ae. umbellulata chromosomes at the telomeres, except for the short arm of chromosome 6U, and shows intercalary sites on the long arms of chromosomes 6U and 7U. The 5S and 18S-25S rDNA have been mapped physically only on the short arms of chromosomes 1U and 5U. On chromosome 1U the order of the genes is 5S rDNA subterminal and 18S-25S rDNA more proximal, while on chromosome 5U the position of the genes is reversed. The relative order of the genes, together with the hybridization pattern of the pSc119.2, is useful in identifying whole chromosomes or chromosome segments from Ae. umbellulata in recombinant or addition lines with wheat. The data help link the physical organization of chromosomes to the genetic map. Other members of the Triticeae vary in the presence and order of the 5S and 18S-25S rDNA sequences on groups 1 and 5, indicating multiple and complex evolutionary rearrangements of the chromosome arms.     

Origin-dependent initiation of DNA replication within telomeric sequences

Replication of telomeres requires the action of telomerase, the semi-conservative replication machinery and the stabilization of the replication fork during passage through telomeric DNA. Whether vertebrate telomeres support initiation of replication has not been experimentally addressed. Using Xenopus cell free extracts we established a system to study replication initiation within linear telomeric DNA substrates. We show binding of TRF2 to telomeric DNA, indicating that exogenous DNA exclusively composed of telomeric repeats is recognized by shelterin components. Interaction with telomere binding proteins is not sufficient to prevent a DNA damage response. Notably, we observe regulated assembly of the pre-replicative complex proteins ORC2, MCM6 and Cdc6 to telomeric DNA. Most importantly, we detect origin-dependent replication of telomeric substrates under conditions that inhibit checkpoint activation. These results indicate that pre-replicative complexes assemble within telomeric DNA and can be converted into functional origins.     

How bats achieve a small C-value: frequency of repetitive DNA in Macrotus

Bats possess a genome approximately 50–87% the size of other eutherian mammals. We document that the events that have achieved or maintained a small genome size in the Mexican leaf-nosed bat Macrotus waterhousii have resulted in a lower copy number of interspersed and tandemly repetitive elements. These conclusions are based on examination of 1726 randomly chosen recombinant cosmids, with an average insert size of 35.7 kb and representing 2.6% of the haploid genome of M. waterhousii. Probes representative of microsatellites [(GT)_n, (CT)_n, (AT)_n, (GC)_n] and a tandem repeat (rDNA) were used to estimate frequency of repetitive elements in the M. waterhousii genome. Of the four dinucleotides, (GT)_n was present in 33.5% of the clones, (CT)_n was present in 31.0% of the clones, and (AT)_n and (GC)_n were not represented in any of the clones examined. The 28S rDNA and a repetitive element from M. californicus were found in three and four clones, respectively. The dinucleotides (GT)_n and (CT)_n occurred together in the same clone more frequently than expected from chance. Although our data do not allow us to empirically test which mechanisms are maintaining copy number of repetitive DNA in the bat genome, the nonrandom association of these different families of repetitive DNA may provide insight into a mechanism that proportionately reduces diverse families of repetitive DNA that are known to be amplified by very different mechanisms.     

Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro

Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes. We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands. Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures. Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase. Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.