Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes

It is shown by isopycnic density gradient centrifugation that the DNAs of the sibling species Drosophila hydei, Drosophila neohydei and Drosophila pseudoneohydei differ regarding the numbers and proportions of satellite DNA bands. An overwhelming proportion of all repetitive nucleotide sequences of the DNA is contained in these satellite fractions. The majority of the satellites are species specific despite the close phylogenetic and cytological relationship between the three species studied. — By in situ hybridization experiments it is demonstrated that the various satellite sequences occupy different positions within the chromosomes. All types of localization patterns, from a wide spread occurrence in all chromosomes to an apparent restriction to kinetochore regions of single chromosomes, have been observed. Main band DNA, on the other hand, in its hybridization behavior reflects the DNA distribution according to the banding pattern in giant chromosomes. Generally satellite sequences seem to be included in -heterochromatic chromosome regions but no relation to the heterochromatin of the Y-chromosome was found. — Renaturation studies support various evidence that satellite sequences occur in tandemly repetitious units. At least some of this repetitious material seems to be linked to non-satellite DNA sequences or to DNA of other satellites.     

Nucleotide sequences of HS-α satellite DNA from kangaroo rat dipodomys ordii and characterization of similar sequences in other rodents

The most common repeated nucleotide sequences of the highly repetitive satellite HS-α fraction from kangaroo rat Dipodomys ordii was determined using ribosubstitution methods. This sequence was α nucleotides long and represented about 25% of the total HS-α satellite DNA, while the remaining DNA was composed of sequence variants related to the most common sequence. The sequences of the commonest of these variants are reported. Furthermore, the most common repeated sequence was identical to that reported for the α satellite of guinea pig Cavia porcellus. The α satellites of guinea pig, Cavia porcellus, pocket gopher, Thomomys bottae and antelope ground squirrel, Ammospermophilus leucurus, are shown to have sequences in common with the kangaroo rat. This implies that the simplest repeated sequences of mammalian satellite DNAs may persist over much longer evolutionary times than previously thought.Attempts to explain the very rapid quantitative changes in satellites whose sequence is strongly conserved have led us to consider that they might have a role in sympatric speciation. Among the novel features of the model presented is that fluctuations in satellites could be due to “speciation genes.” Such genes would confer a strong selective advantage in certain situations, and could explain the many puzzling instances in which large numbers of new related species have appeared over a short evolutionary span.     

Construction of cDNA libraries for highly efficient DNA sequencing from the 3' end of expressed genes

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5' ends of cDNA clones. Obtaining high-quality DNA sequences from the 3' ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3'-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3' mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3' ends of 98% of all attempted clones.     

Regional organization of eukaryotic DNA sequences as studied by the formation of folded rings

The observed frequency of folded rings has been determined as a function of fragment length and degree of resection for DNA from mouse and Necturus. The thermal stability of the ring closure and the kinetics of ring formation have been studied. As seen in the case of Drosophila DNA, mouse and Necturus DNA display a decreasing frequency of folded rings as fragment length increases. We interpret this to mean that repetitious sequences of a given type are clustered into many thousands of characteristic regions, called g-regions. The present paper focuses on the interior organization of g-regions. Variations of two competing models may be entertained: “tandem repetition” and “intermittent repetition”. If the g-regions were composed of exact, tandemly-repeating sequences, all observations can be easily explained. In order to maintain the idea that the g-regions contain repetitious blocks located at regular, or irregular intervals, one must suppose that such repetitious blocks are long (>200 nucleotide pairs), not internally repetitious, and represent perhaps 80% of the nucleotides in the g-region. Such a sequence can be thought of as a fractional-tandem repeat. For example: HIJXXXABC … HIJXXXABC … HIJXXX, where the X's stand for nucleotides composing sequences that are unrelated to each other, and the letters (ABC … HIJ) represent nucleotides in the non-internally-repetitive repeating sequence. We feel that debate cart now be profitably devoted to the question of whether approximately 80 or 100% of the tandemly-repetitious unit is in fact tandem.     

Nucleotide sequence of mouse satellite DNA.

The nucleotide sequence of uncloned mouse satellite DNA has been determined by analyzing Sau96I restriction fragments that correspond to the repeat unit of the satellite DNA. An unambiguous sequence of 234 bp has been obtained. The sequence of the first 250 bases from dimeric satellite fragments present in Sau96I limit digests corresponds almost exactly to two tandemly arranged monomer sequences including a complete Sau96I site in the center. This is in agreement with the hypothesis that a low level of divergence which cannot be detected in sequence analyses of uncloned DNA is responsible for the appearance of dimeric fragments. Most of the sequence of the 5% fraction of Sau96 monomers that are susceptible to TaqI has also been determined and has been found to agree completely with the prototype sequence. The monomer sequence is internally repetitious being composed of eight diverged subrepeats. The divergence pattern has interesting implications for theories on the evolution of mouse satellite DNA.     

Localisation of satellite DNA in the microchromosomes of the Japanese quail by in situ hybridization

In situ hybridisation of radioactive complementary RNA has been used to localise the G-C rich repetitious DNA satellite in chromosomes of the Japanese quail. The satellite sequences are located predominantly in the microchromosomes. No cross hybridisation is found with duck or chicken microchromosomes. The relationship between repetitious DNA, heterochromatin and nucleolus-organisation is discussed.     

Chromosomal localizations by in situ hybridization of the repetitious human DNA families and evidence of their satellite DNA equivalents

Four of the five major repetitious human DNA families, have been mapped by the in situ hybridization technique at their T_OPT values. Two of the lighter density DNA families have autoradiographic grain patterns over heterochromatic chromosomal regions that resemble those of known satellite DNAs. The two heaviest density DNA families have autoradiographic grain patterns of middle repetitious DNAs, with all chromosomes showing labelling. Some evidence suggests that one of these DNA families is concentrated in certain chromosomal regions. Both DNA families exhibit biphasic T_OPT curves. The presence of two thermal stability classes of hybrids suggests sequence interspersion. By co-enrichment studies in Ag⁺-Cs₂SO₄ gradients, evidence suggests the origin of the three lightest density renaturated human DNA families to be satellites I, II and III.     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.     

HIVbase: a PC/Windows-based software offering storage and querying power for locally held HIV-1 genetic, experimental and clinical data

BACKGROUND: Human immunodeficiency virus (HIV) research involves ongoing, repetitious sequencing of the HIV genome and the massive accumulation of associated investigational data. As a result, the storage of annotated DNA and/or protein sequences, as well as information retrieval, have become increasingly difficult tasks, with scientists extracting less information from their collected data than they should. Objectives: Our objective was to design and develop a software package to aid researchers in the storage, analysis and exploration of their HIV-associated data. RESULTS: HIVbase contains familiar, easy-to-use interfaces and functionality for integrating many types of disparate data. The software contains tools that allow for the mass import of raw genetic data, eliminate repetitious sequence translations, have the ability to identify automatically and store HIV regions of interest from nucleic acid or protein sequences, allow for the export of data in commonly used analysis-ready formats, and for unique querying approaches.     

Conformations of satellite DNAs.

X-ray fiber diffraction studies of satellite DNAs from Gecarcinus lateralis, Drosophila virilis and Mus musculus, all of which have highly repetitious base sequences but with different degrees of sequence complexity, reveal only classical polynucleotide duplex structures in contrast to some highly repetitious synthetic DNAs.     

Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment

Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next‐generation sequencing (NGS)‐based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide‐induced mutant rice population (F₂). The integration of multiplex, semi‐nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t‐test against position‐prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls.     

Structure and evolution of primate cytochrome c oxidase subunit II gene

The sequence of cytochrome oxidase subunit II (COII) mRNA from the cynomolgus macaque has been determined. Availability of the sequence from a non-human primate has allowed examination of the evolution of the COII gene and protein along the primate lineage. Comparison with existing protein and DNA sequences, combined with estimates of divergence derived from calculations designed to compensate for multiple mutation and reversion events, indicates that although the rate of fixation of nucleotide substitutions at silent sites is somewhat lower in primates than non-primates, the rate of fixation at replacement sites is 4-5-fold higher. The data also suggest that the rate of divergence at replacement sites along the primate lineage has not been uniform, but has decreased 2-2.5-fold since the higher primate branch point, in the absence of a comparable change in the rate substitution at silent sites. Both primate mRNAs differ from their non-primate homologues in having 3'-untranslated regions of 20-25 nucleotides. Examination of the monkey and human untranslated sequences suggests that these regions have evolved by duplication events occurring in both cases within 2-3 nucleotides following the translational stop codon. The primate mRNAs are also exceptional in that both can form stable stem and loop structures immediately preceding the postulated duplication site that may have played a role in facilitating the mutational events involved. Comparison of the human and monkey protein sequences has revealed regions conserved in primates that are significantly more hydrophobic than their non-primate counterparts. The possible effects of these alterations on the interaction between COII and cytochrome c are discussed.     

Essential elements in the coding region of mRNA for translation of ColE2 Rep protein

Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine-Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses have shown that there are sequences important for the Rep translation in the regions upstream the GA cluster sequence and downstream the initiation codon. To further define regions important for translation of the Rep mRNA, a set of the ColE2 rep genes bearing single-base substitution mutations in the coding region near the initiation codon was generated and their translation activities examined. We showed that translation of the Rep mRNA was reduced by some of these mutations in a region ranging at least 70 nucleotides from the initiation codon in the coding region, indicating the presence of translation enhancer(s) outside the translation initiation region which is covered by the ribosome bound to the initiation codon. Some of them seem to be essential and specific for translation of the ColE2 Rep mRNA due to the absence of a canonical SD sequence.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Molecular structure and flanking nucleotide sequences of the natural chicken ovomucoid gene

Five independent clones containing the natural chicken ovomucoid gene have been isolated from a chicken gene library. One of these clones, CL21, contains the complete ovomucoid gene and includes more than 3 kb of DNA sequences flanking both termini of the gene. Restriction endonuclease mapping, electron microscopy and direct DNA sequencing analyses of this clone have revealed that the ovomucoid gene is 5.6 kb long and codes for a messenger RNA of 821 nucleotides. The structural gene sequence coding Ifor the mature messenger RNA is split into at least eight segments by a minimum of seven intervening sequences of various sizes. The shortest structural gene segment is only 20 nucleotides long. All seven intervening sequences are located within the peptide coding region of the gene, and the sequences at the 5' and 3' untranslated regions of the mRNA are not interrupted by intervening sequences. The DNA sequences of the regions flanking the 5' and 3' termini of the gene have been determined. Thirty nucleotides before the start of the messenger RNA coding sequence is the heptanucleotide TATATAT, which is also present in a similar location relative to the chicken ovalbumin gene and other unique sequence eucaryotic genes. This sequence resembles that of the Pribnow box in procaryotic genes where a promoter function has been implicated. Seven nucleotides past the 3' end of the gene is the tetranucleotide TTGT, a sequence found to be present at identical locations as either TTTT or TTGT in other eucaryotic genes that have been sequenced. These conserved DNA sequences flanking eucaryotic genes may serve some regulator function in the expression of these genes.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Evaluation of intra- and interspecific divergence of satellite DNA sequences by nucleotide frequency calculation and pairwise sequence comparison

Satellite DNA sequences are known to be highly variable and to have been subjected to concerted evolution that homogenizes member sequences within species. We have analyzed the mode of evolution of satellite DNA sequences in four fishes from the genusDiplodus by calculating the nucleotide frequency of the sequence array and the phylogenetic distances between member sequences. Calculation of nucleotide frequency and pairwise sequence comparison enabled us to characterize the divergence among member sequences in this satellite DNA family. The results suggest that the evolutionary rate of satellite DNA inD. bellottii is about two-fold greater than the average of the other three fishes, and that the sequence homogenization event occurred inD. puntazzo more recently than in the others. The procedures described here are effective to characterize mode of evolution of satellite DNA. Published: March 4, 2003