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PubMed2003

Mouse telomere analysis using an optimized primed in situ (PRINS) labeling technique.

Telomeres are chromosomal elements composed of variable numbers of a TTAGGG repeated DNA sequence required for genomic stability. Telomeric length is correlated with the number of copies of this repeated DNA sequence and is an important property relevant to telomeric function. Recently, it has been demonstrated that the length of the shortest telomere, not average telomeric length, is important for cell viability and chromosomal stability. Consequently, assays permitting assessment of telomeric length are important for the analysis of genomic instability disorders. The length of individual telomeres can be analyzed using the primed in situ (PRINS) labeling reaction, which produces a labeled copy of the telomeric DNA repeats in situ. In this study, we tested different variables to optimize the PRINS reaction to enable it to be applied to the detection of mouse telomeric DNA and the study of telomeric length. The specificity, efficiency and uniformity of staining were evaluated using digital fluorescence microscopy. Labeling efficiency is dependent upon the conditions used to denature the telomeric DNA and reaction duration. Staining uniformity is increased at higher annealing and elongation temperatures as well as when a fluorescently labeled nucleotide is incorporated during the elongation step. Our results also indicate that chromosomal background staining is observed when a fluorochrome-labeled nucleotide is used as opposed to a hapten-labeled nucleotide. From this study, we conclude that an optimized PRINS technique can be reliably employed to analyze mouse telomeres and, compared with the FISH (fluorescence in situ hybridization) technique, presents advantages including greater cost efficiency and reduced processing time. These advantages may encourage wider use of the PRINS technique for quantitative evaluation of the length of individual telomeres in situ.

Animals↗
PubMed1978

Hybridization properties of human X-chromosomal RNA transcripts from murine--human hybrids.

A human--mouse hybrid cell line which has retained the human X chromosome and a fragment of chromosome 2 contains RNA sequences transcribed from human DNA. Nuclear RNA enriched for human sequences was prepared from this hybrid cell line by a multistep hybridization procedure to mouse and human DNA immoblized on nitrocellulose filters. The properties of this RNA were analyzed by RNA--DNA hybridization techniques. The results indicate that we have prepared a RNA fraction enriched for sequences homologous to repeated DNA sequences of the human X chromosome.

Animals↗
PubMed1991

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗
PubMed1981

Discrete-length repeated sequences in eukaryotic genomes.

Two of the four repeated DNA sequences near the 5' end of the silk fibroin gene hybridize with discrete-length families of repeated DNA. These two families comprise 0.5% of the animal's genome. A repeated sequence with a conserved length has also been found in the short class of moderately repeated sequences in the sea urchin. The discrete length, interspersion, and sequence fidelity of these moderately repeated sequences suggests that each has been multiplied as a discrete unit. Thus, transposition mechanisms may be responsible for the multiplication and dispersion of a large class of repeated sequences in phylogenetically diverse eukaryotic genomes. The repeat we have studied in most detail differs from previously described eukaryotic transposable elements: it is much shorter (1300 base pairs) and does not have terminal repetitions detectable by DNA hybridization. A simple technique for identifying such discrete-length repeated sequences is described.

Animals↗
PubMed2003

Effects of sequence on repeat expansion during DNA replication.

Small DNA repeat tracts are located throughout the human genome. The tracts are unstable, and expansions of certain repeat sequences cause neuromuscular disease. DNA expansions appear to be associated with lagging-strand DNA synthesis and DNA repair. At some sites of repeat expansion, e.g. the myotonic dystrophy type 2 (DM2) tetranucleotide repeat expansion site, more than one repeat tract with similar sequences lie side by side. Only one of the DM2 repeat tracts, however, is found to expand. Thus, DNA base sequence is a possible factor in repeat tract expansion. Here we determined the expansion potential, during DNA replication by human DNA polymerase beta, of several tetranucleotide repeat tracts in which the repeat units varied by one or more bases. The results show that subtle changes, such as switching T for C in a tetranucleotide repeat, can have dramatic consequences on the ability of the nascent-strand repeat tract to expand during DNA replication. We also determined the relative stabilities of self-annealed 100mer repeats by melting-curve analysis. The relative stabilities did not correlate with the relative potentials of the analogous repeats for expansion during DNA replication, suggesting that hairpin formation is not required for expansion during DNA replication.

Base Sequence↗
PubMed1993

[Repeating sequences, organizing the telomeric region of chromosomes from the eukaryotic genome].

The telomeric regions of the eukaryotic genome are composed of different types of the repeated DNA sequences. Extended subtelomeric repeats (STR) are located close to the terminating repeats (TR) at the extreme ends of the chromosomes. The first part of the review is devoted to the TR sequences. All of them are the simple satellite-similar tandem repeats (T/A)1-8 G1-8. TR, as a substrate of specific telomere elongating enzyme telomerase, controls the whole replication of the chromosomal DNAs. Information accumulated up to this time is sufficient to ascertain the principal uniformity of the structure, the functions and organization of TR in all eukaryotic genomes. The second part of the review is devoted to the STR sequences. The STRs are prolonged repetitive sequences localized between the TR cluster and non-repetitive genes. The STRs have been identified in rye, yeast, Trypanosoma, Drosophila, Chironomus, murine and human genomes. It can be seen from the analysis of experimental results that the STRs of different organisms may have general properties. The information about the functions of STRs is now being obtained for only the STR of Drosophila. The STR is the complex repetitive family located both in telomeres and in heterochromatin. The sequences from the family were recently shown to be capable of translocation and curing destroyed chromosomal ends. According to the latest information, the STR sequences are organized in two different types of clusters in the Drosophila genome. The types have different organization and different genome localization. Is the organization of both telomeric and heterochromatin regions of the Drosophila genome from the same structural elements common for higher eukaryotes or it is the characteristic feature of the Drosophila genome only, is to be cleared.

Animals↗
PubMed1976

Conservation of repeated DNA base sequences in Crustacea: a molecular approach to decapod phylogeny.

Analysis of data obtained from molecular hybridization of 3H-labeled repetitious DNA has been utilized to reconstruct the broad outlines of phylogenetic relationships among decapod Crustacea. This molecular reconstruction agrees reasonably well with the paleontological record, and with other schemes obtained by comparative morphological and serological approaches. Preliminary evidence is in line with the hypothesis that continuous addition of new repeated sequence families to the genome over long periods of time may in part account for the correlation observed between percent repetitious DNA hybridized and divergence time. It is tentatively concluded that a core of DNA base sequence homology has been highly conserved throughout the evolution of the Crustacea. Demonstration of inter-species sequence homology has important implications to models which relegate a genetic regulatory function to repeated DNAs.

Animals↗
PubMed1984

Isolation and characterization of six different chicken actin genes.

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Actins↗
PubMed1978

Analysis of plant genomes. V. Comparative study of molecular properties of DNAs of seven Allium species.

The genomes of seven plant species belonging to the genus Allium and exhibiting a threefold variation in their nuclear DNA content were analyzed by studying their reassociation kinetics, equilibrium centrifugation behavior in neutral CsCl gradients, and melting properties. The reassociation kinetics experiments revealed the presence of 44-65% repeated DNA sequences. A comparison between DNA contents and the proportion of repeated DNA sequences indicated that, in Allium, increase in the genome size is not exclusively due to variations in the proportions of repetitive DNA. The total DNA as well as the various repetitive DNA fractions in all the Allium species examined exhibited, in spite of a few differences, a gross similarity in their behavior in neutral CsCl gradients and in their melting properties.

Base Sequence↗
PubMed1995

Concerted evolution of repetitive DNA sequences in eukaryotes.

A large fraction, sometimes the largest fraction, of a eukaryotic genome consists of repeated DNA sequences. Copy numbers range from several thousand to millions per diploid genome. All classes of repetitive DNA sequences examined to date exhibit apparently general, but little studied, patterns of "concerted evolution." Historically, concerted evolution has been defined as the nonindependent evolution of repetitive DNA sequences, resulting in a sequence similarity of repeating units that is greater within than among species. This intraspecific homogenization of repetitive sequence arrays is said to take place via the poorly understood mechanisms of "molecular drive." The evolutionary population dynamics of molecular drive remains largely unstudied in natural populations, and thus the potential significance of these evolutionary dynamics for population differentiation is unknown. This review attempts to demonstrate the potential importance of the mechanisms responsible for concerted evolution in the differentiation of populations. It contends that any natural grouping that is characterized by reproductive isolation and limited gene flow is capable of exhibiting concerted evolution of repetitive DNA arrays. Such effects are known to occur in protein and RNA-coding repetitive sequences, as well as in so-called "junk DNA," and thus have important implications for the differentiation and discrimination of natural populations.

Amphibians↗
PubMed1982

Characterization of a cloned repetitive DNA sequence concentrated on the human X chromosome.

A tandemly repeated DNA sequence organized predominantly, if not entirely, in a specific manner on the human X chromosome has been cloned in pBR322 and characterized. The sequence was detected as a 2-kilobase band in ethidium bromide-stained agarose gels of BamHI-digested total human nuclear DNA. Although in situ hybridization of the cloned sequence to human metaphase chromosomes showed a single major site of hybridization at the centromere region of the X chromosome and minor sites of hybridization at several autosomal centromeres, Southern blot analysis of restricted total human DNA indicated that the cloned probe is related to other repeated DNAs, particularly the human alphoid DNAs. Restriction enzyme analysis of the cloned fragment revealed an internal repeat structure based upon multiples of 170 base pairs, confirming this relatedness. All available data, however, suggest that the 2-kilobase spacing of BamHI sites within the repeat may be specific to the X chromosome.

Cells, Cultured↗
PubMed1991

Recognition of a chromosome truncation site associated with alpha-thalassaemia by human telomerase.

Telomeres define the ends of chromosomes; they consist of short tandemly repeated DNA sequences loosely conserved in eukaryotes (G1-8(T/A)1-4). Telomerase is a ribonucleoprotein which, in vitro, recognizes a single-stranded G-rich telomere primer and adds multiple telomeric repeats to its 3' end by using a template in the RNA moiety. In conjunction with other components, telomerase may balance the loss of telomeric repeats due to DNA replication. Another role of telomerase may be the de novo formation of telomeres. In eukaryotes like Tetrahymena, this process is an integral part of the formation of macronuclear chromosomes. In other eukaryotes this process stabilizes broken chromosomes. A case of human alpha-thalassaemia is caused by a truncation of chromosome 16 that has been healed by the addition of telomeric repeats (TTAGGG)n. Using an in vitro assay, I show here that human telomerase correctly recognizes the chromosome 16 breakpoint sequence and adds (TTAGGG)n repeats. The DNA sequence requirements are minimal and seem to define two modes of DNA recognition by telomerase.

Base Composition↗
PubMed1996

HMG box proteins interact with multiple tandemly repeated (GCC)n (GGC)m DNA sequences.

A number of tandemly repeated DNA sequences have the ability to form hairpin structures by forming non-standard base pairs. When (GCC)15 and (GGC)15 strands are annealed together, the expected duplex is the only product. However, when (GCC)15 is annealed with (GCC)10, depending on the relative concentrations, up to five complexes can be detected in native gels. Three of these species are susceptible to limited digestion by Exo VII, suggesting they are duplexes containing single stranded tails. The remaining two bands are resistant to the enzyme, and have low mobility on native gels, consistent with branched structures. The latter complexes bind HMG box proteins, members of a highly abundant class of non-histone proteins of the nucleus. These proteins, modeled in this study by the second box fragment from rat HMG1, HMGb, interact strongly with branched or chemically modified DNA, relative to normal duplexes. The expansion of triplet repeats in genomic DNA is associated with tumor formation as well with a variety of heritable neurologiocal disorders. It is our thesis that the stability of branched intermediate structures that arise in replication of these sequences and promote expansion can be influenced directly by the presence of two highly abundant proteins in the cell nucleus: the HMG box proteins, HMG1/2, and the histone H1, which associates with HMG1/2.

Bacterial Proteins↗
PubMed1997

[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.

Adenine↗
PubMed2000

Physical map of a conditionally dispensable chromosome in Nectria haematococca mating population VI and location of chromosome breakpoints.

Certain isolates of the plant pathogenic fungus Nectria haematococca mating population (MP) VI contain a 1.6-Mb conditionally dispensable (CD) chromosome carrying the phytoalexin detoxification genes MAK1 and PDA6-1. This chromosome is structurally unstable during sexual reproduction. As a first step in our analysis of the mechanisms underlying this chromosomal instability, hybridization between overlapping cosmid clones was used to construct a map of the MAK1 PDA6-1 chromosome. The map consists of 33 probes that are linked by 199 cosmid clones. The polymerase chain reaction and Southern analysis of N. haematococca MP VI DNA digested with infrequently cutting restriction enzymes were used to close gaps and order the hybridization-derived contigs. Hybridization to a probe extended from telomeric repeats was used to anchor the ends of the map to the actual chromosome ends. The resulting map is estimated to cover 95% of the MAK1 PDA6-1 chromosome and is composed of two ordered contigs. Thirty-eight percent of the clones in the minimal map are known to contain repeated DNA sequences. Three dispersed repeats were cloned during map construction; each is present in five to seven copies on the chromosome. The cosmid clones representing the map were probed with deleted forms of the CD chromosome and the results were integrated into the map. This allowed the identification of chromosome breakpoints and deletions.

Ascomycota↗
PubMed2005

Exact tandem repeats analyzer (E-TRA): a new program for DNA sequence mining.

Exact Tandem Repeats Analyzer 1.0 (E-TRA) combines sequence motif searches with keywords such as 'organs', 'tissues', 'cell lines' and 'development stages' for finding simple exact tandem repeats as well as non-simple repeats. E-TRA has several advanced repeat search parameters/options compared to other repeat finder programs as it not only accepts GenBank, FASTA and expressed sequence tags (EST) sequence files, but also does analysis of multiple files with multiple sequences. The minimum and maximum tandem repeat motif lengths that E-TRA finds vary from one to one thousand. Advanced user defined parameters/options let the researchers use different minimum motif repeats search criteria for varying motif lengths simultaneously. One of the most interesting features of genomes is the presence of relatively short tandem repeats (TRs). These repeated DNA sequences are found in both prokaryotes and eukaryotes, distributed almost at random throughout the genome. Some of the tandem repeats play important roles in the regulation of gene expression whereas others do not have any known biological function as yet. Nevertheless, they have proven to be very beneficial in DNA profiling and genetic linkage analysis studies. To demonstrate the use of E-TRA, we used 5,465,605 human EST sequences derived from 18,814,550 GenBank EST sequences. Our results indicated that 12.44% (679,800) of the human EST sequences contained simple and non-simple repeat string patterns varying from one to 126 nucleotides in length. The results also revealed that human organs, tissues, cell lines and different developmental stages differed in number of repeats as well as repeat composition, indicating that the distribution of expressed tandem repeats among tissues or organs are not random, thus differing from the un-transcribed repeats found in genomes.

Cells, Cultured↗